CN1694725A - Use of peptides for early detection of mycobacterial disease - Google Patents

Abstract

Several protein and glycoprotein antigens secreted by M. tuberculosis have been identified as "early" Mtb antigens based on the presence of early antibodies in Mtb-infected individuals prior to the development of clinically diagnosed disease. Epitope-containing peptide fragments of these early Mtb antigens were identified, especially the 88 kDa secreted protein, GlcB (SEQ ID NO: 106) and the Mtb antigen MPT51 (SEQ ID NO: 107). These peptides and mutants thereof, peptide multimers comprising two or more repeating fragments of one or more peptides, fusion polypeptides comprising early Mtb antigenic proteins, peptides or both can be used for the detection of early TB in a host rapid immunoassay. A preferred immunoassay is the detection of antibodies in the urine of the host. The invention also provides antigen composition, kit and method for detecting early Mtb antibody. Antigenic proteins and peptides can also be used in vaccine compositions.

Description

Use of peptides for early detection of mycobacterial disease

technical field

The present invention belongs to the field of microbiology and medicine, and relates to a method for rapid and early detection of human mycobacterial diseases, which is based on the detection of antibodies against particularly "early" mycobacterial protein antigens and their reactive epitopes, said Such use of antigens and their epitopes has not been found before. The detection of such antibodies relies on selected mycobacterial proteins, their peptides or fusion polypeptides (peptide multimers, polymeric proteins), thus enabling the diagnosis of TB earlier than at present. The present invention also provides a surrogate marker for screening TB susceptible populations, especially patients infected with human immunodeficiency virus (HIV).

Background technique

In 1995, the World Health Organization predicted that there would be about 90 million new cases of tuberculosis (TB) during the ensuing decade, resulting in about 30 million deaths (Raviglione, MC et al., 1995, JAMA.273: 220 -226). Expansion of HIV patients with a high probability of TB infection leads to worldwide re-emergence of TB (Raviglione, MC et al., 1992, Bull WHO 70:515-526; Harries A.D., 1990, Lancet.335:387-390 ). This trend has fueled interest in improving TB vaccines, diagnostics, drugs, and drug delivery methods. Immunodeficiency caused by HIV increases the chance of reactivating latent TB, increases susceptibility to primary disease, and accelerates disease progression (Raviglione et al., 1992, supra; 1995, supra; Shafer RW et al. al., 1996, Clin.Infect.Dis.22:683-704; Barnes PF et al., 1991, N.Engt.J Med.324:1644-1650; Selwyn PA et al., 1989, N.Engl.J . Med. 320:545550).

The importance of cellular immunity in the prevention and treatment of TB has been known. Much work in this area has focused on identifying the antigens of the causative bacterium, Mycobacterium tuberculosis (M. tuberculosis; also abbreviated "Mtb" in the present The role of cell populations in host-pathogen interactions (Andersen, Petal., 1992, Scafzd. J. Immuf2ol. 36: 823-831; Havlir, DV et al., 1991, Infect. Immun. 59: 665-670; Orme, I Metal., 1993, J. Ilifect. Dis. 167:1481-1497).

The detected delayed hypersensitivity of the skin immune response against a purified protein derivative of Mtb (abbreviated "PPD") is currently the only available marker for detecting the latency of infection with Mtb. However, the sensitivity of the PPD skin test was significantly reduced during HIV infection (Raviglione et al., 1992, supra, 1995, supra; Graham NMH et al., 1991, JAMA267:369-373; Huebner RE et al., 1994 , Cli71.Infect.Dis.19:26-32; Huebner RE et al., 1992, JAMA 267:409-410; Caiaffa WT et al., 1995, Arch.Intern.Med.155:2111-2117). Furthermore, inoculation with the closely related mycobacterium Bacillus Calmette-Guerin (BCG) or previous exposure to other mycobacteria can lead to false positive results in the PPD skin assay. Not only does the PPD reaction fail to distinguish active, subclinical disease from the latent period of infection, but the time between a positive skin test and development of clinical disease can be months to years (Selwyn PA et al., supra).

Because of the susceptibility of immunocompromised individuals to TB infection, the US Centers for Disease Control and Prevention recommends prophylactic isoniazid therapy for all HIV seropositive (HIV ⁺ ), PPD positive (PPD ⁺ ) individuals. However, the optimal timing of treatment with this therapy is unclear and ideally should coincide with the replication of already latent bacteria. Unnecessary treatment must be avoided due to the severe toxic side effects of long-term isoniazid treatment (Shafer et al., supra). Whether this treatment leads to the development of drug-resistant strains is unclear. In developing countries, preventive therapy is severely limited by the large number of PPD ⁺ individuals who lack adequate medical-social and financial resources. High-risk populations have also been found in the United States, mainly intravenous drug users, the homeless, incarcerated, and slum dwellers (Fitzgerald, JM et al., 1991, Chest 100:191-200; Graham et al., supra ; Friedman, LNet al., 1996, NewEzlgl.J.Med.334:828-833) and family members in contact with TB patients. Therefore, it is urgent to find other surrogate markers for early detection and timely treatment of active, subclinical TB in these high-risk populations.

Antibody responses to TB have been studied for decades, primarily for the purpose of developing serodiagnostic tests. Although some seroreactive antigens/epitopes have been identified, there has been little research interest in the antibody response to Mtb due to the lack of facile techniques for detecting the corresponding antibodies. Studies with crude antigen preparations have revealed that healthy individuals possess antibodies capable of cross-reacting with several mycobacterial antigens, which may result from exposure to commensal and environmental bacteria and from vaccination. (Bardana, EJ et al., 1973, Clin. Exp. Immunol. 13: 65-77; Das, S et al., 1992, Clin. Exp. Immunol 89: 402-406; Del Giudice, G et al., 1993, JImmunol.150:2025-2032; Grange, JM, 1984, Adv.Tuberc.Res.21:1-78; Havlir, DV et al., supra; Ivanyi, J et al., 1989, Brit.Med. Bull. 44: 635-649; Verbon, A et al., 1990, J. Gen. Microbiol. 136: 955-964). Several mycobacterial antigens have been isolated and characterized (Young, DB et al., 1992, Mol. Microbiol. 6:133-145), including 71 kDa DnaK, 65 kDa GroEL, 47 kDa elongation factor tu, 44kDa PstA homologue, 40kDa L-alanine dehydrogenase, 38kDa PhoS, 23kDa superoxide dismutase, 23kDa outer membrane protein, 12kDa thioredoxin, and 14kDa GroES. Most of these antigens have significant homology to similar proteins in other mycobacterial and non-mycobacterial prokaryotes (Andersen, AB et al., 1992, Infect. Immun. 60:2317-2323; Andersen, ABet al., 1989, Infect.Immun.57:2481-2488; Braibant, M et al., 1994, Infect.Irnmu71.62:849-854; Carlin, N et al., 1992, Infect.Iminun.60: 3136-3142; Garsia, RJ et al., 1989, Infect.Immun.57:204-212; Hirschfield, GR et al., 1990, J.BacterioL 172:1001013; Shinnick, TM et al., 1989, Nucl.Acids Res.17:1254; Shinnick, TM et al., 1988, Infect.Immun.56:446-451; Wieles, B et al., 1995, Infect.Immun.63:4946-4948; Young, DB et al., supra; Zhang, Y et al., 1991, Mol. MicrobioL 5:381-391). Therefore, most individuals (healthy or patients) have antibodies against conserved regional epitopes of these antigens. These antibodies caused the uninformative (potentially misleading) cross-reactivity observed on crude Mtb antigen preparations (Davenport, MP et al., 1992, Infect. Immun. 60:1170-1177; Grandia , AA et al., 1991, Immunobiol.182:127-134; Meeker, HC et al., 1989, Infect.Immun.57:3689-3694; Thole, J et al., 1987, Infect.Immun.55: 1466-1475).

Because this cross-reactive antibody would mask the specificity of the Mtb antigen, some purified antigens such as 38kDa PhoS, 30/31kDa "antigen 85" (discussed in detail below), 19kDa lipoprotein, 14kDa GroES and Lipoarabinomannose (LAM) (Daniel, T et al., 1985 Chest.88:388-392; Drowart, L et al., 1991, Chest.100:685-687; Jacket, PSet al., 1988, J. Clin.Microbiol.26:2313-2318; Ma, Y et al., 1986, Am Rev RespirDis 134:1273-1275; Sada, E et al., 1990, J.Clin.Microbiol.28:2587-2590; Sada , ED et al., 1990, J.Infect.Dis.162:928-931; Van Vooren, JP et al., 1991, J.Clin.Microbiol.29:2348-2350). Notably, the choice of which antigen to test is primarily determined by (a) availability, (b) immunodominance in the animal immune response, or (c) ease of its biochemical purification. These criteria do not take into account the reactivity to antigens to which humans naturally develop an immune response to mycobacterial disease. For a period of time, the use of the 38kDa antigen provided extremely high serum sensitivity and specificity (Daniel, TM et al., 1987, Am Rev Respir Dis 135:1137-1151; Harboe, M et al., 1992, J. Infect. Dis. 766: 874-884; Ivanyi, J et al., 1989, supra). However, the presence of anti-38kDa antibodies was primarily associated with post-treatment, late and recurrent TB in contrast to antibodies to the antigens discovered by the inventors (Bothamley, GH et al., 1992, Thorax. 47:270-275 ; Daniel et al., supra Ma et al., supra.).

A convention for naming mycobacterial proteins is to use the MPB and MPT numbers. MPB is used to represent the protein purified from M.bovis BCG, and the numbers behind it represent the relative mobility of the protein in 7.7% polyacrylamide gel under the condition of pH9.5. MPT indicates protein isolated from Mtb. In previous protein assays of the present invention, there was no difference between the N-terminal amino acid sequences of these two mycobacteria.

Wiker and colleagues have studied a series of Mtb secretory protein families, which includes a complex of three proteins 85A, 85B and 85C (also called "85 complex" or "85cx") (Wiker, H.G.etal., 1992, Scafzd. J. Immunol. 36:307-319; Wiker, H.G. et al., 1992, Microbiol. Rev. 56:648-661). Active Mtb-related components were also obtained from the secretion. Although complex 85 is associated with the bacterial surface, this complex is also thought to be a major component of secreted proteins in mycobacterial cultures. In most SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, 85A and 85C were not well depolymerized, however, three separate bands were produced in isoelectric focusing.

Genes encoding six secreted proteins have been cloned: 85A, 85B, 85C, "Antigen 78" (usually referred to as a 38 kDa protein), MPB64 and MPB70. Three separate genes encoding 85A, B and C located at isolated sites in the mycobacterial chromosome (Content, J. et al., 1991, Infect. Immun. 59:3205-3212). The gene encoding the antigen MPT-32 (a 45/47kDa secreted antigen complex) has been cloned, sequenced and expressed (Laqueyrerie, A. et al., 1995, Infec. Immuii..63:4003-4010 ), and was designated as the apa gene. Further elucidation of the biochemical and immunochemical properties of the Mtb protein and glycoprotein is required, which is of great importance in serodiagnosis.

The following table shows the molecular weights of antigen 85 complexed with two other antigens (in SDS-PAGE) as described by Wiker and colleagues, along with other designations:

Ag85A = MPT44 = 31 kDa

Ag85B=MPT59=30KDa

Ag85C=MPT45=31.5kDa

MPT64 = 26 kDa

MPT51 = 27kDa

Ag78----=38kDa

MPT32=45/47kDa (38/42kDa considered by the inventors)

The Wiker group studied 5 Cross-reactivity between two active secreted Mtb proteins. mAb HBT4 reacts with MPT51 protein.

The amino acid sequences listed in the table below show the homology of the 85A, 85B, 85C, 1 and MPT64 fragments. The uppermost number corresponds to the portion of the sequence shown. The N-terminal sequences of the isolated proteins were determined and aligned by visual inspection. The sequence of positions 66 to 91 of MPT64 was deduced from the cloned gene.

1 5 10 15 20 25 30 35 serial number

85A(1-39)FSRPGLPVEYLQVPS PSMGRDIKVQFQSGGANSP ALYLL 1

85B(1-39)FSRPGLPVEYLQVPS PSMGRDIKVQFQSGGNNSP AVYLL 2

85C(1-37)FSRPGLPVEYLQVPSA SMGRDIKVQFQGGG PHAVYLL 3

MPT51(1-32) APYENLMVPS PSMGRDIPVAFLAGG PHAVYLL 4

MPT64(66-91) APYE LNITSATYQS AIPPRG TQAWL 5

The N-terminal sequence of MPT51 has 72% homology compared to the sequence of each Ag85 component (when the P at position 2 of the three Ag85 components aligns with the P at position 7).

Studies in TB patients have shown a sensitivity of approximately 50% for detecting antibodies to the Ag85 complex. With regard to specificity, the components of Ag85 are highly cross-reactive, so positive responses were expected (and found) in healthy controls, especially those in geographic areas with high exposure to atypical mycobacteria crowd. The type of control group largely determines the degree of specificity. Notably, conventional BCG vaccination does not appear to elicit a significant antibody response, and interestingly, antibodies to mycobacterial antigens increased when anti-TB chemotherapy was administered. Several studies have verified the presence of antibodies to different Mtb antigens in TB sera or in the sera of patients with other diseases. See, such as Espitia, Cet et al., 1989, Clin Exp Immunol 77:373-377; Van Vooren, JP et al., 1991, J Clin.Microbiol.29:2348-2350; Wiker etc. (supra) C.Espitia et al., 1995, Infect.Immun.63:580-584, using anti-M.bovis protein rabbit polyclonal antiserum and anti-Mtb antigen mAb, found the interaction between Mtb 50/55kDa protein and M.bovis BCG 45/47kDa antigen sex. Both antigens are secreted glycoproteins. The N-terminal sequences and the overall amino acid content of these proteins are very similar. 2D gel electrophoresis showed that Mtb50/55kDa antigen has at least 7 different components. Mtb 50/55 kDa purified protein was found in the serum of 70% of pulmonary TB patients (n=77) in solid phase immunoassay. The N-terminus of the Mtb 41kDa antigen, MPT32, is very similar to the N-terminus of the 50/55kDa and 45-47kDa proteins. Based on these observations, the authors speculated that these antigens have diagnostic potential, however, the use of these antigens as early TB diagnostic reagents has neither been analyzed nor even suggested.

Importantly, the analysis of antibodies at different disease stages is still deficient in the art, which is one of the main objectives of the present invention. So far, with the possible exception of MPT32 (which will be described here), no antigen has emerged as a suitable candidate for a diagnostic test in the early stages of TB. Because the antigen/epitope detected in human natural infection and disease development is significantly different from the antigen/epitope in passive immunization of inoculated animal vaccines (Bothamley, G.etal., 1988, Eur.J.Clin.Microbiol. Infect.Dis.7:639-645; Calle, J.et al., 1992, J.Immunol.149:2695-2701; Hartskeerl, R.A.et al., 1990, Infect.Immun.58:2821-2827; Laal , S.etal., 1991, Proc.Natl.Acad.Sci.USA.88:1054-1058; Meeker, H.C.etal., 1989, Infect.Immun.57:3689-3694; Verbon, A., 1994, Trop .Geog.Med.46:275-279), so it is urgent to select antigens that can stimulate the human immune system. This will require the identification of useful protein antigens and peptide epitopes for early diagnostic testing of TB and for vaccine preparation.

TB in people living with HIV

Although the literature on HIV-uninfected TB-infected individuals is extensive, reports on antibody responses to Mtb in HIV/TB patients are sparse and controversial. Farber, C. et al., 1990, J. Infect. Dis, 162: 279-280, reported that 7 of 8 HIV/TB patients had antibodies to p32 antigen (same as 85A). Da Costa, C. et al., 1993, Clin. Exp. Immunol. 91: 25-29, reported the presence of anti-lipoarabinomannose (LAM) antibodies in 35% of such patients. Barer, L. et al., 1992, Tuber. Lung. Dis. 73: 187-191, reported the presence of anti-PPD antibodies in 36% of HIV/TB patients. Martin-Casabona, N. et al., 1992, J. Clin. Microbiol. 30: 1089-1093, reported that 73% of such patients had anti-sulfolipid (SLIV) antibodies. In addition, van Vooren, P. et al., 1988, Tubercle. 69: 303-305, reported the detection of anti-p32 antibodies in an HIV-TB patient several months before clinical manifestations of TB. And for Ag60 antibody (Saltini C.et al., 1993, Am Rev Respir Dis 145:1409-1414; van derWerf, T.S.et al., 1992.Med Microbiol Immunol 181:71-76) and Ag85B antibody (McDonough, J.A. et al., 1992, J Lab.Clin.Med.120:318-322) were not detected in these patients.

Therefore, there is a particular need for a method that can detect early TB infection in HIV patients, as these populations are among the most susceptible to TB infection in the world.

antibodies in urine

Many laboratories have reported the presence of antibodies in the urine, mainly against infectious agents. For example, Takahashi S; et al. (Clin Diagn Lablmnlunol, 1998, 5: 24-27) found rubella virus antibodies in urine and serum samples of healthy individuals who had been vaccinated against rubella. Shutov AM et al. Arkh (RUSSIA) 1996, 68: 35-37 detected antibodies to the virus causing hemorrhagic fever with renal syndrome (HFRS) in urine and concluded that the detection of Virus antibodies can be used in early diagnosis. Vereta LA; et al. (Vopr Virusol (RUSSIA) 1993, 38: 18-21) used a commercial diagnostic indirect immunofluorescence assay for the detection of hantavirus antibodies in urine samples from HFRS patients. Koopmans M et al. (J Med Virol, 1995, 46:321-328) revealed the presence of antibodies to human cytomegalovirus in urine samples by ELISA and immunoblotting. Zhang X et al. (J Med Virol, 1994, 44: 187-191) used commercial immunoassay technology to detect hepatitis C virus (HCV) antibody in urine. The same team (Constantine NT et al., Am J Clin Pathol, 1994, 101:157-161) detected HIV antibodies in urine. Perry KR et al., J Med Virol 1992, 38:265-270, Detection of IgG and IgM antibodies to hepatitis A and hepatitis B core antigens in urine samples.

Some Japanese researchers (Hashida S et al., J Clin Lab Anal, 1994, 8: 237-246; Hashinaka K et al., J Clin Microbiol 1994, 32: 819-22; Hashida Set al., J Clira Lab Anal 1994 , 8: 149-156 Hashida S et al., J Clin LabAnal1994, 8: 86-95) using a hypersensitive enzyme immunoassay technique (immune complex transfer enzyme immunoassay technique) and recombinant protein as an antigen by detecting urine IgG antibodies to HIV-1 in the sample can be used to diagnose HIV-1 infection in asymptomatic carriers. They report improved sensitivity by using concentrated urine samples and using different recombinant HIV antigens to allow detection of conjugated enzyme activity for longer periods of time.

Urnovitz HB et al., (Lancet Dec 11 1993, 342:1458-9), found that 7 individuals who were negative for HIV-1 antibodies in the licensed serum EIA test were negative in the urine EIA and western blot (WB) test. Positive. Connell JA et al., JMed Virol 1993, 41:159-64, describe a rapid, simple and robust IgG-capture enzyme-linked immunosorbent assay (GACELISA), which is suitable for the detection of anti-HIV1 and Anti-HIV2 antibodies. An early study by this laboratory (Connell JA et al., Lancet, 1990, 335: 1366-1369) described the detection of anti-HIV antibodies in urine by GACELISA. Gershy-Damet GM et al. Trans R Soc Trop MedHyg 1992, 86: 670-671, Successful diagnosis of HIV-1 and HIV-2 in urine in Africa using assays for unprocessed saliva and urine samples. They found that the assay was as accurate for serum as conventional EIAs under the same conditions.

In a study by Dr. A. Friedman-Kien and his colleagues, paired urine and serum samples from the same HIV-infected individuals were tested for hepatitis B surface antigen (HBs), hepatitis B core antigen (HBc), CMV and HIV antibody (CaoY et al., 1989, AIDS. Res. Hum. Retrovir. 5: 311). All infected individuals with anti-HIV antibodies in their serum also had anti-HIV antibodies in their urine; there was no such correlation for HBs and CMV antibodies. Anti-HIV antibodies in urine belong to the IgG class, and gp160 and gp120 are the best understood proteins. Based on these observations, a method for diagnosing HIV-1 using urine was developed.

Given the prevalence of TB among HIV-infected persons, especially in developing countries, and the risk and cost of collecting blood/serum for serodiagnosis, the inventors evaluated the presence of anti-mycobacterial antibodies in the urine of TB patients. The inventors concluded that Mtb infects the mucosal surface of the lung, which can induce antibody production in the mucosal tissue, resulting in the presence of antibodies in the urine. The positive results of these studies are as follows. The use of urine as a test sample would be welcomed by public health officials and industry.

The above citations are not intended to be an admission that any one is pertinent prior art. All descriptions of the date or content in the literature are based on the information available to the applicant only, and cannot confirm the correctness of the date and content.

Summary of the invention

The present inventors systematically analyzed the reactivity of serum and urine of TB patients with Mtb antigens to elucidate the main targets of human antibody responses in the early stages of infection pathogenesis. The inventor observed that the patient's serum was initially adsorbed with E. coli antigen, which successfully reduced the interference of cross-reactive antibodies, thus obtaining a new method for serological research. Antibody-recognized Mtb antigens in most patients early in disease development can be identified by immunoadsorbed sera. These antigens are therefore also useful tools in methods of diagnosing TB. A particularly prominent antigen among these is the high molecular weight secreted protein 88kDa or 85kDa (as the case will be described below). These proteins were referred to as "88 kDa proteins" and, as it was later discovered, were the product of the glcB gene, also known as GlcB (see below).

In addition to being used for early diagnosis of mycobacterial disease in patients before the disease develops into radiographic or bacteriological symptoms, the present invention also provides for the first time a surrogate marker for use in a low-cost screening method on high-risk populations with TB things. This application was discovered by using the method described in this invention to analyze the antibody response of HIV-infected TB patients (HIV/TB) to Mtb secreted antigens at various stages of disease development. Most HIV/TB patients have detectable antibodies to Mtb secreted antigens months or even years before the onset of clinically active tuberculosis. These patients are referred to as "HIV/pre-TB". However, HIV/TB patients have significantly lower levels of antibodies compared to TB patients who are not infected with HIV (referred to as "non-HIV/TB"), suggesting that antibodies are specific for only a limited number of Mtb antigen classes. Antibodies to the 88 kDa antigen described above are present in about 75% of HIV/pre-TB sera patients who eventually develop clinical TB. HIV/TB patients who failed to form anti-Mtb antibodies did not differ in lymphocyte profile from those who were positive. These findings led to the development of serum-surrogate markers of active preclinical TB for use in HIV-infected individuals as well as any high-risk population. Therefore, the present invention provides a new method for early detection of Mtb infection in an immune population. Utilizing this discovery could make a remarkable contribution to the early detection of tuberculosis disease, thereby speeding up treatment.

The present invention aims to provide an antigenic composition for early detection of Mycobacterium tuberculosis disease or infection, or for immune application of Mycobacterium tuberculosis infection, which includes

(a) a peptide selected from the following sequences (1) CGTDGAEKGPTYNKVRGDK (SEQ ID NO: 108); (2) KIGIMDEERRTTVNLKAC (SEQ ID NO: 109); (3) ELAWAPDEIREEVDNNC (SEQ ID NO: 110); (4) HRRRREFKARAAEKPAPSDRAG (SEQ ID NO: 111); (5) ARDELQAQIDKWHRRR (SEQ ID NO: 112); (6) LNRDRNYTAPGGGQ (SEQ ID NO: 113); (7) GAPQLGRWKWHDPWV (SEQ ID NO: 114) (8) VGNLRIARVLYDF Sequence ID No.: 117); (9) QAQIDKWHRRRVI (Sequence Identification Number: 126); (10) WHRRRVIEPIDMD (Sequence Identification Number: 127); (11) IEPIDMDAYRQFL (Sequence Identification Number: 128); (12) ITTTAGPQLVVPV (Sequence Identification Number: 134); (13) PQLVVPVLNARFA (SEQ ID NO: 135); (14) VLNARFALNAANA (SEQ ID NO: 136); (15) ALNAANARWGSLY (SEQ ID NO: 137); (16) ARWGSLYDALYGT (SEQ ID NO: 138) (17) SVLLINHGLHIEI (SEQ ID NO: 154); (18) HGLHIEILIDPES (SEQ ID NO: 155); (19) GGQFTLPGRSLMF (SEQ ID NO: 170); (20) FVRNVGHLMTNDA (SEQ ID NO: 172); ( 21) DRVVFINTGFLDR (SEQ ID NO: 191); (22) NCQSILGYVVRWV (SEQ ID NO: 216); and (23) GYVVRWVDQGVGC (SEQ ID NO: 217);

The prerequisite is that the composition is not a full-length protein with the sequence ID number: 106 or the sequence ID number: 107;

(b) a mutant or functional derivative of the peptide in (a) that retains reactivity with antibodies specific for GlcB or MPT51; or

(c) A combination of any two or more of peptides (1)-(23) in (a) or a mutant or functional derivative of (b).

In one embodiment, the above-mentioned antigenic composition is a fusion polypeptide comprising:

(a) one or more of peptides (1)-(23) or variants thereof linked to (b)

(b) One or more proteins selected from the sequence of SEQ ID NO: 106, SEQ ID NO: 107 and other early Mtb antigens.

Wherein, the fusion polypeptide includes one or more optional linkers, which link any two or more proteins or peptides.

Also include a kind of antigen composition as above, it is:

(a) A peptide polymer having the general formula

P ¹ _n

Wherein P ¹ is any one of peptides (1)-(23) or alternative mutants thereof, n=2-8,

(b) A peptide polymer having the general formula

(P ¹ -X _m ) _n -p ²

wherein, P ¹ and p ² are any one of peptides (1)-(23) or conservative substitution mutants thereof, and wherein

(i) ^P1 and ^p2 may be the same or different, each of ^P1 - _Xm may be a different ^peptide or an immediate mutant thereof; and

(ii) X is (A) C ₁ -C ₅ alkyl, C ₁ -C ₅ alkenyl, C ₁ -C ₅ alkynyl, C ₁ -C ₅ polyether containing up to 4 oxygen atoms, wherein, m =0 or 1, n=1-7; or (B) Gly _z wherein, z=1-6, and

Wherein the peptide polymer reacts with the specific antibody of GlcB or MPT51 protein.

The present invention also aims to provide the above antigenic composition, which is a recombinant peptide polymer with the general formula:

(P ¹ -Gly _z ) _n -p ²

wherein, P ¹ and p ² are any one of peptides (1)-(23) or conservative substitution mutants thereof, and wherein

(a) ^P1 and ^p2 can be the same or different, and each ^P1 in ^P1 - _Xm can be a different peptide or its immediate mutant;

(b) n=1-100, and z=0-6, and

Among them, the peptide multimer reacts with the specific antibody of GlcB or MPT51 protein.

An antigenic composition for early detection of Mycobacterium tuberculosis disease or infection, comprising one or more peptide mixtures or linked into peptide polymers or fusion proteins, the one or more peptides have early tuberculosis A fragment sequence of a mycobacterial antigen or a derivative of the antigen, said antigen having the following characteristics:

(i) react with antibodies in a tuberculosis patient at an earlier stage than positive saliva smear and lung cavity disease, and

(ii) Not reactive with sera from healthy controls or healthy persons with latent inactive tuberculosis The composition does not contain other M. tuberculosis proteins which are not early M. tuberculosis antigens.

The present invention also provides a method of using the above antigen composition. A preferred method for early detection of mycobacterial disease or mycobacterial infection in a subject comprises: determining whether the specificity of the above-mentioned peptide or mutant, fusion protein or peptide multimer is present in a sample of biological fluid from a suspected patient with active TB Antibodies, where the presence of antibodies indicates the presence of a disease or infection.

In the method described above, biological fluid samples are preferably taken from persons with active TB symptoms, but before they can be identified as late-stage TB symptoms manifested by (a) acid-fast bacilli on smears of saliva or other lung-associated fluids Positive, (b) lung cavity disease, or (a) and (b).

Typically, the method includes the step of obtaining a sample of biological fluid from the subject prior to testing.

The above methods preferably include removing antibodies specific to cross-reactive epitopes or antigens present in proteins of Mycobacterium tuberculosis and other species from the sample prior to testing, eg, immunoadsorbing the sample with E. coli antigen.

The above method may further comprise testing the sample for the presence of specific antibodies to one or more other early antigens of Mycobacterium tuberculosis selected from the group consisting of:

(a) Mycobacterium tuberculosis protein GlcB protein, which has the sequence identification number: 106 amino acids;

(b) Mycobacterium tuberculosis MPT51, which has the sequence identification number: 107 amino acids;

(c) a protein characterized as Mycobacterium tuberculosis antigen 85C;

(d) a glycoprotein characterized as the Mycobacterium tuberculosis antigen MPT32; and

(e) comprising one or more fusion proteins of (a)-(d).

Preferred subjects in the above methods are human beings, such as people infected with HIV-1 or high-risk groups of tuberculosis.

In a preferred embodiment of the above method, the biological fluid sample is serum, urine or saliva.

The method may further comprise testing a sample of the subject's saliva or other bodily fluid for mycobacteria.

The present invention also aims to provide a test kit for early detection of mycobacterial tuberculosis, which comprises:

(a) an antigenic composition as described above, and

(b) Necessary reagents for detecting antibodies bound to the peptide.

The kit may also include one or more early antigens of Mycobacterium tuberculosis, for example, an antigen selected from:

(a) Mycobacterium tuberculosis protein GlcB protein, which has the sequence identification number: 106 amino acids;

(b) Mycobacterium tuberculosis MPT51, which has the sequence identification number: 107 amino acids;

(c) a protein characterized by Mycobacterium tuberculosis antigen 85C;

(d) a glycoprotein characterized by the Mycobacterium tuberculosis antigen MPT32; and

(e) A fusion protein comprising one or more of (a)-(d).

Brief description of the drawings

Figure 1 shows the sera of the following groups: TB ^neg HIV ^neg PPD ⁺ control group (O); TB ^neg HIV ^neg PPD ^neg control group (_), TB ^neg , HIV ⁺ , asymptomatic control group (△); and TB patients (•) Reactivity with LAM-free broth filtrate protein (LFCFP) of Mtb H ₃₇ Rv before and after adsorption with E. coli lysates. Each value is the OD value of the test, the mean of which is the horizontal dash shown in the graph.

Figure 2 shows the sera of the following groups: non-HIV, PPD skin test positive (PPD ⁺ ) healthy controls (non-HIV/PPD), non-HIV TB patients (non-HIV/TB) and HIV-infected TB patients Reactivity of all LFCFPs (HIV/pre-TB, HIV/stage-TB and HIV/late-TB) with Mtb. The cut-off is determined by the mean optical density (OD) ± 3 standard deviations, and the cut-off is obtained from the serum of the healthy control group.

Figure 3 is a graph showing late (black bars) and early (white bars) TB patient sera for Mycobacterium tuberculosis LFCFP, purified Ag85C or three fractions (fractions) rich in three early antigens ( 13, 10 and 15) for reactivity (see brackets).

Figure 4 is a graph showing the reactivity of late (black bars) and early (white bars) TB patient sera to M. tuberculosis LFCFP, purified Ag85C or purified MPT32.

Figure 5 is a graph showing the reactivity of urine and serum from advanced TB patients with LFCF and MP32 proteins. Results are expressed as percentage of positive samples.

Description of the preferred embodiment

In the following description, reference will be made to methodological literature known in the fields of immunology, cell biology and molecular biology. Publications and other illustrative material covering such known methods are incorporated herein by reference. Standard literature works addressing the general principles of immunology include A.K. Abbas et al., Cellular and Molecular Immunology (Fourth Ed.), W.B. Saunders Co., Philadelphia, 2000; C.A. Janeway et al., In2munobiology. The Immune System in Healthla and Disease, Fourth ., Garland Publishing Co., NewYork, 1999; Roitt, I. et al., Immunology, (current ed.) C.V. Mosby Co., St.Louis, MO (1999); Klein, J., Immunology, Blackwell Scientific Publications, Inc., Cambridge, MA, (1990). Monoclonal antibodies (mAbs) and their preparation and use are described in: Kohler and Milstein, Nature 256: 495-497 (1975); U.S. Patent No. 4,376, 110; Hartlow, E. et al., Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988); Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyzes, Plenum Press, New York, NY (1980); H. Zola et al., in Monoclonal Hyquebridoma Technibodies: and Applications, CRCPress, 1982)). Immunoassay methods are described in: IColigan, J.E.et al., eds., Current Protocols in Immunology, Wiley-Interscience, New York 1991 (or currentedition); Butt, W.R. (ed.) Practical Immunoassay : The State of the Art, Dekker, New York, 1984; Bizollon, Ch.A., ed., Monoclonal Antibodies and New Trends ill Initiiunoassays, Elsevier, New York, 1984; Butler, J.E., ELISA (Chapter 29), In: van Oss, C.J.etal., (eds), IMMUNOCHEMISTRY, Marcel Dekker, Inc., New York, 1994, pp.759-803; Butler, J.E. (ed.), Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton , 1991; Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986; Work, T.S.et al., Laboratory Techniques and Biochemistfy in Molecular Biology, NY1 Company, NY8, Northing Holland) (Chapter by T. Chard).

The present invention provides a diagnostic immunoassay to detect and/or quantify antibodies specific to mycobacterial antigens, especially early in the pathogenic development of mycobacterial infection and before clinical characterization. Based on this detection, it may be possible to detect TB earlier than before so that an appropriate treatment plan can be established. The best antigen available prior to the present invention for TB serodiagnosis was the 38 kDa secreted protein, also known as Ag78 (see above). However, the present invention enables serological response detection in subjects lacking detectable antibodies to the 38 kDa antigen.

The immunoassay approach is based on the inventors' discovery that certain Mtb antigens induce human responses earlier than other antigens, which induce antibodies only after clinical disease has developed. In immunocompromised HIV-infected patients, antibody responses to some of these antigens can be detected before clinical manifestations of TB appear. Five secreted proteins have been identified as early antigens of diagnostic value. In particular a preferred early antigen is the 88 kDa secreted protein Mtb GlcB, which is preferably enriched semi-purified (at least 50% pure) or highly purified (at least 95% pure, preferably at least 99% pure).

The present invention also provides peptides with epitopes from GlcB and MPT51, which react with TB serum, and these peptides are used for early diagnosis in the form of peptides (single peptides or mixtures thereof), peptide multimers (synthetic or recombinant) comprising one or more different epitope-containing peptides, or fusion polypeptides comprising at least two full-length early antigen proteins, and may also comprise other peptides based on the same Mtb protein or other Mtb proteins gauge.

The method of the present invention is further based on the inventor's concept that before analyzing the antigenic reactivity and specificity of the serum of patients infected with Mtb by crude or semi-purified antigen preparations, first clear the specific antibodies of the cross-reactive antigen ( It is not Mtb specific) is important. However, once purified antigen is obtained or epitope-specific competing EIAs are established based on the present invention (see, e.g., Wilkins, E. et al., 1991, Eur. R Clin. Microbiol. Infect. Dis. 10:559- 563), this earlier step of adsorption should be eliminated.

The present invention relates to (1) Mtb infection or pathogenicity, or infected persons or patients, (2) antibody response to Mtb antigen, (3) Mtb antigen itself or (4) diagnostic test, wherein the terms "early stage" and "late stage" are used ” are defined according to the stage of TB development. Early and late (or late) TB are given their definitions in the table below.

Thus, early TB subjects are either asymptomatic or more typically have one or more "classic symptoms" (eg, fever, cough, weight loss). In the early stages of TB, Mycobacterium tuberculosis is too rare to be detected in smears of saliva or other body fluids, mainly those associated with the lungs (eg, bronchoalveolar lavage, bronchoalveolar lavage) for acid-fast bacilli fluid, pleural effusion). However, Mtb bacilli were present in these subjects and these bacilli were culturable, ie Mtb bacilli could grow in cultured fluids such as body fluids. Finally, subjects with early-stage TB may present with radiographic findings of pulmonary lesions, such as lung infiltrates that have not yet formed a cavity. Any antibodies formed at an early stage are called "early antibodies", and any Mtb antigens recognized by these antibodies are called "early antigens". The fact that antibodies are called "early stage" does not mean that antibodies are not present in later stages of TB. Such antibodies are predicted to be present during development from early TB to mature stages. Early TB 1. Saliva smear, bronchial washing fluid, bronchoalveolar lavage fluid, and pleural effusion were negative for acid-fast bacilli 2. The acid-fast bacillus test of the direct culture fluid of saliva, bronchial washing fluid, bronchoalveolar lavage fluid and pleural effusion was positive 3. Normal chest X-ray or lung infiltration 4. Typical symptoms (fever, cough, loss of appetite, weight loss) late/mature TB 1. The acid-fast bacillus test of saliva smear, bronchial washing fluid, bronchoalveolar lavage fluid, and pleural effusion is positive (may be accompanied by hemoptysis) 2. The acid-fast bacillus test of the direct culture fluid of saliva, bronchial washing fluid, bronchoalveolar lavage fluid and pleural effusion was positive 3. Chest X-ray shows cavitary lesions in the lungs 4. Typical symptoms (as above)

Thus, the term "advanced" or "advanced" characterizes a subject with overt clinical disease and more advanced cavitary disease in the lungs. In advanced TB, Mtb bacteria can not only be cultured from saliva and/or other above-mentioned body fluid smears, but also exist in large quantities in the above-mentioned liquid smears, and can be detected in acid-fast bacilli tests. "Late TB" or "late mycobacterial disease" is used interchangeably with "late TB" or "late mycobacterial disease". Antibodies (including pulmonary cavitary lesions) that appear for the first time after the onset of diagnosed clinical symptoms and other characteristic symptoms are late antibodies, and antigens recognized by late antibodies (not recognized by early antibodies) are late antigens.

To be beneficial according to the present invention, early diagnostic tests must enable rapid diagnosis of Mtb disease at an earlier stage than traditional clinical diagnostic methods, such as radiological examination, bacterial smear and culture or Other experimental methods prior to the present invention. (A positive culture is the final confirmatory test, but it takes two weeks or more).

The immunoassay method generally includes culturing a biological fluid, preferably serum or urine of a suspected TB patient, and reagents comprising one or more early Mtb antigens in the biological fluid. Depending on the peptide unit containing the epitope, these antigens can be combined as mixtures or as aggregated proteins or peptide multimers. The samples are then assayed for antibodies that bind to mycobacterial antigens. The so-called "biological fluid" refers to any fluid from normal or diseased patients that may contain antibodies, such as blood, serum, plasma, lymph, urine, saliva, sputum, tears, cerebrospinal lavage fluid, pleural effusion, bile , ascites, pus and the like. Within the meaning of the term as used in the present invention, it also includes tissue extracts, or culture fluids in which cells or tissues from a subject have been cultured.

Mycobacterial Antigen Composition

The mycobacterial antigenic composition or preparation of the invention may be a composition of one or more isolated secreted proteins or peptides of Mycobacterium tuberculosis. As noted above, the composition may be prepared as a mixture or as a polymeric protein or peptide multimer.

The antigenic composition may be a fully purified or recombinantly obtained preparation of one or more M. tuberculosis proteins or epitope-bearing peptides thereof. In addition, the antigenic composition may also be a partially purified or fully purified preparation containing one or more M. tuberculosis epitopes that bind to antibodies in TB patients. These epitopes may be peptide fragments of the early antigen protein, or other "functional derivatives" of M. tuberculosis proteins or peptides as described below.

"Functional derivative" refers to a "fragment", "mutant", "analogue" or "chemical derivative" of the early antigen protein, as defined below. Functional derivatives retain at least part of the protein's functions, mainly the ability to bind to early antibodies, so that they can be used in the present invention. "Fragment" refers to a segment of any molecule, ie, a shorter peptide. "Mutant" refers to a molecule that is substantially similar to the whole protein or a fragment thereof. Mutant peptides can be directly synthesized by traditional chemical methods, or prepared by recombinant means. A "chemical derivative" of an antigenic protein or peptide includes other chemical moieties that differ from the normal parts of the native protein (or peptide fragment). Covalent modification of peptides is also included within the scope of the present invention. This modification can be accomplished by reacting targeted amino acid residues of the peptide with organic derivatizing reagents capable of reacting with selected side chains or terminal residues.

The four proteins or glycoproteins identified in the Mtb culture filtrate are preferably early Mtb antigens (or sources of antigenic peptides) of the present invention. Therefore, although these proteins are secreted, they are also present in Bacillus cell preparations. Therefore, these early antigens are not intended to be limited to secreted protein forms. The protein features are as follows:

(1) 88kDa protein (GlcB)

The protein is an Mtb secreted protein isolated from the culture filtrate by the present inventors, with a molecular weight of 88 kDa and an isoelectric point of about pH 5.2. The protein has a measured molecular weight of 82-85 kDa in the laboratory of one co-inventor (88 kDa in the laboratory of another co-inventor) and a PI of 5.12-5.19. This protein was initially thought to react with mAb IT-42 and mAb IT-57, but a second protein in the molecular weight range, catalase/peroxidase (katG gene product), was later found to react with these mAbs. The Th 88 kDa protein is the major antigenic component of Fraction 15 (Example I) and Fraction 14 (Example II). This protein corresponds to the protein spot indicated in Ref. No. 124 in the two-dimensional gel (see US Patent 6,245,331 and WO98/29132 (published 09 July 1998), both of which are hereby cited in their entirety; see also below Tables 4 and 6. Thus, despite small differences in molecular weight measurements, they are still one protein (although different isomers may exist).

As described in Example IV, the sequence of the protein was identified by the inventors based on the amino acid composition, which was obtained after the application claiming priority on December 31, 1996 and application number US 60/034,003 Mtb gene sequence related. This protein is the product of the Mtb glcB gene, which encodes malate synthase and is referred to as the GlcB protein. The amino acid sequence (SEQ ID: 106) of the protein is as follows:

MTDRVSVGNL RIARVLYDFV NNEALPGTDI DPDSFWAGVD KVVADLTPQN QALLNARDEL

QAQIDKWHRR RVIEPIDMDA YRQFLTEIGY LLPEPDDFTI TTSGVDAEIT TTAGPQLVVP

VLNARFALNA ANARWGSLYD ALYGTDVIPE TDGAEKGPTY NKVRGDKVIA YARKFLDDSV

PLSSGSFGDA TGFTVQDGQL VVALPDKSTG LANPGQFAGY TGAAESPTSV LLINHGLHIE

ILIDPESQVG TTDRAGVKDV ILESAITTIM DFEDSVAAVD AADKVLGYRN WLGLNKGDLA

AAVDKDGTAF LRVLNRDRNY TAPGGGQFTL PGRSLMFVRN VGHLMTNDAI VDTDGSEVFE

GIMDALFTGL IAIHGLKASD VNGPLINSRT GSIYIVKPKM HGPAEVAFTC ELFSRVEDVL

GLPQNTMKIG IMDEERRTTV NLKACIKAAA DRVVFINTGF LDRTGDEIHT SMEAGPMVRK

GTMKSQPWIL AYEDHNVDAG LAAGFSGRAQ VGKGMWTMTE LMADMVETKI AQPRAGASTA

WVPSPTAATL HALHYHQVDV AAVQQGLAGK RRATIEQLLT IPLAKELAWA PDEIREEVDN

NCQSILGYVV RWVDQGVGCS KVPDIHDVAL MEDRATLRIS SQLLANWLRH GVITSADVRA

SLERMAPLVD RQNAGDVAYR PMAPNFDDSI AFLAAQELIL SGAQQPNGYT EPILHRRRRE

FKARAAEKPA PSDRAGDDAA R

Following the inventors' discovery of the 88kDa protein and its use as an early antigen (see US 6,245,331 and WO 98/29132), Hendrickson RC et al., J Cliva Microbiol 38: 2354-2356 (2000) identified by serum protein analysis combined with tandem mass spectrometry A protein called Mtb81 was identified and sequenced, which can be used to diagnose TB, especially in patients co-infected with HIV. Antibodies to recombinant Mtb81 were detected by ELISA in 25/27 TB patients (92%) who were HIV seropositive, and also in 38/67 individuals (57%) who were only TB positive. No response was observed in 11/11 individuals (100%) who were HIV seropositive only. Mtb81 positivity was not detected in the sera of both PPD negative (0/29) and healthy subjects (0/45). Only 2/57 PPD-positive individuals were positive for Mtb81. Anti-Mtb81 reactivity was detected in the sera of individuals who were TB smear positive and HIV seropositive, but who were TB negative for another antigen, as were sera from individuals with lung cancer and pneumonia. In this group, Mtb81 reacted with 26/37 HIV ⁺ /TB ⁺ sera (70%) and 2/37 (5%) with the 38-kDa antigen. As concluded earlier by the present inventors, the use of Mtb81 as a complementary antigen in TB serodiagnosis holds great promise.

(2) Antigen 85C

This is a Mtb secreted protein with a molecular weight of about 31 kDa and an isoelectric point of about pH 5.17. This protein reacts with mAb IT-49, which was also designated MPT45. Ag85C corresponds to the protein spot marked by Ref. No. 119 in Table 4 or Table 6.

(3)) MPT51

The molecular weight of the Mtb secreted protein is about 27kDa, the isoelectric point is about 5.91, the amino acid sequence identification number is 107, and the sequence is:

APYENLMVPS PSMGRDIPVA FLAGGPHAVY LLDAFNAGPD VSNWVTAGNA MNTLAGKGIS

VVAPAGGAYS MYFNWEQDGS KQWDTFLSAE LPDWLAANRG AAQGGYGAMA LAAFHPDRFG

FAGSMSGFLY PSNTTTNGAI AAGMQQFGGV DTNGMWGAPQ LGRWKWHDPW VHASLAQNN

TRVWVWSPTN PGASDPAAMI GQTAEAMGNS RMFYNQYRSV GGHNGHFDFP ASGDNGWGSW

APQLGAMSGD IVGAIR

The full-length nucleotide and amino acid sequence of MPT51 is available in GenBank since 1997. (GenBank accession number CAA05211: MPT51 [Mycobacterium tuberculosis] submitted 17-OCT-1997 by T. Oettinger). The published GenBank sequence includes the full-length gene, therefore, the amino acid sequence includes a 33 residue signal sequence that is cleaved from the protein prior to secretion. Therefore, the final protein product is that shown in SEQ ID NO:107.

MPT51 reacts with mAb IT-52. This protein corresponds to the protein spot indicated by Ref. No. 170 of the two-dimensional gel, not presented in the present invention (summarized in Tables 4 and 6).

(4) MPT32

The glycoprotein has a molecular weight (bimodal) of 38 and 42 kDa (42/45 kDa according to Espitia et al. (supra)) and an isoelectric point of approximately pH 4.51. It reacts with polyclonal anti-MPT32 antiserum. This protein is the major antigenic component of fraction 13 (see Example). MPT32 corresponds to the protein spot indicated by Ref. No. 14 in Tables 4 and 6.

Another protein, called "49 kDa protein", has a molecular weight of about 49 kDa and an isoelectric point of about pH 5.1. This protein reacted with mAb IT-58, corresponding to the protein spot indicated by Ref. No. 82 in Tables 4 and 6.

Mycobacterium Peptides and Functional Derivatives

The present invention also provides peptides of early antigen Mtb proteins GlcB and MPT51. Such peptides can also be used in diagnostic and vaccine compositions. As shown in Example IX, preferred peptides that are predicted and do react with TB sera include, but are not limited to:

(1) CG TDGAEKGPTYNKVRGDK, corresponding to GlcB residues 151-167 of N-terminal plus CG (SEQ ID NO: 108);

(2) KIGIMDEERRTTVNLKAC, corresponding to GlcB residues 428-445 (SEQ ID NO: 109);

(3) ELAWAPDEIREEVDNNC, corresponding to GlcB residues 586-603 (SEQ ID NO: 110);

(4) LHRRRREFKARAAEKPAPSDRAG, corresponding to GlcB residues 715-736 (SEQ ID NO: 111);

(5) ARDELQAQIDKWHRRR, corresponding to GlcB residues 56-71 (SEQ ID NO: 112);

(6) LNRDRNYTAPGGGQ, corresponding to GlcB residues 314-327 (SEQ ID NO: 113);

(7) GAPQLGRWKWHDPWV, corresponding to MPT51 residues 167-181 (SEQ ID NO: 114);

Analysis of overlapping 13-mer peptides in TB sera revealed that the amino acids in the following 16 peptides were immediately adjacent to residues of GlcB (SEQ ID NO: 106), and these peptides included or were part of the serum-reactive GlcB epitope:

(8) LRIARVLYDF (Sequence ID: 117);

(9) QAQIDKWHRRRVI (Serial Identification Number: 126);

(10) WHRRRVIEPIDMD sequence identification number: 127);

(11) IEPIDMDAYRQFL (Serial Identification Number: 128);

(12) ITTTAGPQLVVPV (Serial Identification Number: 134);

(13) PQLVVPVLNARFA (Sequence Identification Number: 135);

(14) VLNARFALNAANA (Sequence Identification Number: 136);

(15) ALNAANARWGSLY (SEQ ID NO: 137);

(16) ARWGSLYDALYGT (Serial Identification Number: 138);

(17) SVLLINHGLHIEI (Sequence Identification Number: 154);

(18) HGLHIEILIDPES (Serial Identification Number: 155);

(19) GGQFTLPGRSLMF (Sequence ID: 170);

(20) FVRNVGHLMTNDA (Sequence Identification Number: 172);

(21) DRVVFINTGFLDR (Serial Identification Number: 191);

(22) NCQSILGYVVRWV (Serial Identification Number: 216); and

(23) GYVVRWVDQGVGC Sequence ID: 217).

Peptides comprising antibody epitopes should be at least about 5 amino acids. A T cell epitope is preferably about 10-15 amino acids. Accordingly, the present invention includes peptides having about 5-30 residues, having the native Mtb early antigen protein sequence or homologues, substitution mutants, addition mutants or deletion mutants.

When a peptide is administered to a subject, especially in embodiments of the invention as a vaccine, the peptide may be blocked at its N- and C-termini respectively with acyl (abbreviated Ac) and amino (abbreviated Am) groups, e.g. N-terminal acetyl (CH ₃ CO-) and C-terminal amino (-NH ₂ ).

The N-terminus is blocked with a wide range of functional groups, preferably connected to the terminal amino group, which can be: methyl carboxyl; alkanoyl with 1-10 carbon atoms, such as acetyl, propionyl, butyryl; with 1-10 carbons Atomic alkenoyl, such as 3-hexenoyl; Alkynoyl with 1-10 carbon atoms, such as 5-hexynoyl; Aroyl, such as benzoyl or 1-naphthoyl; Heteroaroyl, such as 3 - pyrroloyl or 4-quinoline formyl; alkylsulfonyl, such as methanesulfonyl; arylsulfonyl, such as benzenesulfonyl or sulfonyl; heteroarylsulfonyl, such as pyridine-4-sulfonyl; having Substituted alkanoyl having 1-10 carbon atoms, such as 4-aminobutyryl; substituted alkenoyl having 1-10 carbon atoms, such as 6-hydroxy-3-hexenoyl; having 1-10 carbon atoms Substituted alkynoyl, such as 3-hydroxy-5-hexynoyl; Substituted aroyl, such as 4-chlorobenzoyl or 8-hydroxy-2-naphthoyl; Substituted heteroaroyl, such as 2,4 -Dioxy-1,2,3,4-tetrahydro-3-methyl-6-quinazolinecarbonyl; substituted alkylsulfonyl, such as 2-aminoethanesulfonyl; substituted arylsulfonyl, Such as 5-dimethylamino-1-naphthalenesulfonyl; substituted heteroarylsulfonyl, such as 1-methoxy-6-isoquinolinesulfonyl; carbamoyl or thiocarbamoyl; substituted aminomethyl Acyl (R'-NH-CO) or substituted thiocarbamoyl (R'-NH-CS), where R' is alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl , substituted alkenyl, substituted alkynyl, substituted aryl, or substituted heteroaryl; substituted carbamoyl (R'-NH-CO) and substituted thiocarbamoyl (R'-NH- CS), wherein R' is alkanoyl, alkenoyl, alkynoyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkynoyl, substituted aroyl, or substituted heteroaroyl, All groups as defined above.

The C-terminally blocked functional group can be either an amide or an ester linkage to the terminal carboxyl group. The blocking group is to provide an amide bond of NR ¹ R ² , wherein R ¹ and R ² can be independently selected from the following groups: hydrogen; alkyl, preferably having 1-10 carbon atoms, such as methyl, ethyl, Isopropyl; alkenyl, preferably having 1-10 carbon atoms, such as prop-2-ene; alkynyl, preferably having 1-10 carbon atoms, such as prop-2-yne; having 1-10 carbon atoms Substituted alkyl groups such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, haloalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl , alkanoylalkyl, carboxyalkyl, carbamoylalkyl; substituted alkenyl having 1-10 carbon atoms, such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, Haloalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl; having 1-10 carbon atoms Substituted alkynyl groups such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, haloalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylamino Alkynyl, alkanoylalkynyl, carboxylkynyl, carbamoylalkynyl; aralkyl having 1-10 carbon atoms, such as phenacylmethyl or 2-benzoylethyl; aryl, such as benzene base or 1-naphthyl; heteroaryl, such as 4-quinolyl; alkanoyl with 1-10 carbon atoms, such as acetyl or butyryl; aroyl, such as benzoyl; heteroaroyl, such as 3 - quinolinoyl; OR' or NR'R", wherein R' and R" are independently hydrogen, alkyl, aryl, heteroaryl, acyl, aroyl, sulfonyl, sulfinyl, or SO ₂ - R_ or SO-R_, wherein R" is a substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl, or alkynyl group.

The blocking functional group that provides an ester linkage is OR, where R can be: alkoxy; aryloxy; heteroaryloxy; aralkoxy; heteroaralkoxy; substituted alkoxy; substituted aryloxy ; substituted heteroaryloxy; substituted aralkoxy; or substituted heteroaryloxy.

Selecting an appropriate blocking group can increase other activities of the peptide. For example, attachment of a sulfhydryl group to the N- or C-terminal cap allows attachment of the derivatized peptide to other molecules.

Preparation of peptides and derivatives

General chemical synthesis steps

The peptides of the invention can be prepared using recombinant DNA techniques. However, some shorter peptides can be prepared by solid phase synthesis as described in Merrifield, J. Amer. Chem. Soc., 85:2149-54 (1963), or other methods well known in the art. efficient chemical synthesis method. Solid-phase peptide synthesis can start from the C-terminus of the peptide by coupling the protected a-amino acid to an appropriate resin. This starting material can be prepared by linking a-amino protected amino acid with chloromethylated resin or methylol resin through ester bond, or with BHA resin or MBHA resin through amide bond. Such methods are well known in the art as disclosed in U.S. 5,994,309 (issued 11/30/1999), incorporated herein by reference.

Amino acid substitution and addition mutants

The present invention also includes peptides in which at least one amino acid residue, and preferably only one residue, has been removed and a different residue has been inserted in that position compared to the native Mtb sequence. For protein chemistry and structure, see Schulz, G.E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1979, and Creighton, T.E., Proteins: Structure and Molecular Principles, W.H. Freeman & Co., San Francisco, 1984 , all of which are hereby cited as references. The type of substitutions made on the peptide molecules of the invention are conservative substitutions, wherein one of the following groups may be substituted:

1. Small molecule aliphatic, non-polar or slightly polar residues: such as Ala, Ser, Thr, Gly;

2. Polar, negatively charged residues and their amides: such as Asp, Asn, Glu, Gln;

3. Polar, positively charged residues: eg, His, Arg, Lys;

Due to its unusual geometry, Pro greatly confines peptide chains. Significant changes in functional properties by selection of less conservative substitutions, if not within the range of the above group, is selection outside the range (or two other amino acid groups not listed), which is It will significantly change its effectiveness in maintaining (a) the peptide backbone structure of the surrogate part, (b) the charge or hydrophobicity of the target molecule, or (c) the steric hindrance of the side chain. Preferred substitutes according to the invention are those which do not undergo a fundamental change in the molecular characteristics of the peptide. Even when it is difficult to foresee the definite effect of the substitute, those skilled in the art can evaluate its effect by conventional screening test method, said test method is preferably the biological test method described in the present invention, wherein preferably antiserum, antiserum Pool, or serum test for monoclonal antibodies. Changes in peptide properties including redox or thermal stability, hydrophobicity, propensity to proteolytic degradation or tendency to aggregate with a carrier (or form multimers) are tested by methods well known in the art.

The increase mutant of Mtb peptide preferably includes 1-4 amino acids, may also include X amino acids, and may be added at the N-terminus, C-terminus or both ends at the same time. Amino acids added to the basic peptide unit do not affect the maintenance of the biological reactivity of the peptide, ie antigenicity (recognizable by antibodies or T lymphocytes) or immunogenicity when used as a vaccine.

Peptides used as vaccines are preferably mutants with enhanced stability and/or immunogenicity using conventional protein engineering methods. In one embodiment, the stability is improved by introducing one or more Cys residues at appropriate positions, wherein a disulfide bond is formed between two Cys residues to increase the stability. Another approach is to introduce residues to form alpha helices at positions that do not interfere with the immunological activity of the peptide, such as the N- and C-termini.

For peptides or polypeptides having n residues, n-5 amino acids can be substituted, provided that the immunoreactivity with early Mtb antibodies is not lost.

Chemical derivatives of peptides are also included. Lysine groups and amino terminal residues can be derivatized with succinic acid or other carboxylic anhydrides. Derivatization of cyclic carboxylic anhydrides has the effect of reversing the charge of lysine residues. Other suitable reagents for derivatizing α-amino residues include imidates such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea ; 2,4-pentanedione; and the reaction with glyoxylic acid catalyzed by transferase.

Carboxy side groups, aspartyl or glutamyl, can be selectively modified by reaction with a carbodiimide (R-N=C=N-R'), which can be, for example, 1-cyclohexyl-3 -(2-morpholinyl-(4-ethyl))carbodiimide or 1-ethyl-3-(4-azonium-4,4-dimethylpentyl)carbodiimide. Aspartyl or glutamyl groups can be further converted to asparaginyl and glutaminyl residues by reaction with amino groups.

Other modifications include hydroxylation of proline and lysine, hydroxyl phosphorylation of seryl or threonyl residues, methylation of lysine amino groups (Creighton, supra, pp.79-86), N - Acetylation of the terminal amino group and amidation of the C-terminal carboxyl group.

Polypeptides and Fusion Peptides (Polymeric Proteins)

The present invention also includes long-chain peptides or polypeptides, wherein the sequence of Mtb early antigen peptide or its substitute or increased mutant or its chemical derivatives is repeated two to 100 times, with or without spacers or links in between body. Such molecules are known in the art as multimers, concatemers, or aggregated proteins with multiple epitopes, and these terms are used interchangeably, and refer primarily to peptide multimers in the present invention. When produced recombinantly, they may also be referred to as fusion polypeptides or fusion proteins.

Peptide multimer refers to "P" in the following formula: (PX _m ) _n -P, where m=0 or 1, n=1-100. X is a spacer containing 1-20 glycines or a chemical crosslinker. Therefore, when m=0, there is no spacer between the peptides. When n=1, the multimer is a dimer, etc.

These multimers can be composed of any antigenic peptides or mutants mentioned in the present invention. Furthermore, a peptide multimer may include various combinations of peptide monomers (monomers may be native sequences or mutants thereof). Thus a multimer may comprise several repeats of a first peptide sequence, followed by one or more repeats of a second peptide and so on. Such multimeric peptides can be obtained by chemical peptide synthesis, recombinant DNA techniques or a combination, such as chemical linking of recombinantly produced multimers.

When prepared by chemical synthesis, the multimer preferably has 2-12 repeat units of the core peptide sequence, more preferably 2-8 repeat units, and the total number of amino acids in the multimer should not exceed about 110 residues (or equivalents thereof, when linkers or spacers are included).

Preferred synthetic chemical peptide multimers have the general formula; P ¹ _n

Wherein, P ¹ is a natural Mtb peptide or its substitute or its increased mutant, and n=2-8, wherein the peptide alone or in the form of a multimer has the necessary immunoreactivity.

In another embodiment, a preferred synthetic chemical peptide multimer has the general formula: (P ¹ -X _m ) _n -P ² , wherein P ¹ and P ² are Mtb peptides or increased mutants thereof, wherein

(a) ^P1 and ^P2 can be the same or different; moreover, each ^P1 in the multimer can be a different peptide (or mutant) from its neighbors;

(b) X is C ₁ -C ₅ alkyl, C ₁ -C ₅ alkenyl, C ₁ -C ₅ alkynyl, C ₁ -C ₅ polyether containing up to 4 oxygens, wherein m=0 or 1, n=1-7; X can also be Gly _z where z=1-6, and wherein the peptide alone or in the form of a polymer has immunological activity with anti-Mtb antibody, preferably early antibody.

When prepared by recombinant method, the spacer is _Glyz , as mentioned above, where z=1-6, and the multimer can have the repeating number of the core peptide sequence allowed by the expression system, such as 2-100 repeating units. Preferred recombinantly produced peptide multimers have the general formula:

(P ¹ -Gly _z ) _n -P ²

in:

(a) P ¹ and P ² are Mtb peptides as described above or a substitute or increased mutant thereof, wherein P ¹ and P ² may be the same or different; and, each P ¹ in the multimer may be the same as Peptides (or variants) that differ from adjacent peptides.

Wherein, n=1-100, z=0-6;

Wherein the peptides alone or in aggregate form have the desired immunoreactivity.

In the aforementioned peptide multimer, P ¹ and P ² are preferably selected from any of the following sequences: the sequence identification numbers are 108-114; 117; 126-128, 134-138, 154, 155, 170, 172, 191, 216, and 217.

The multimer is optionally blocked at its N- and C-terminus.

It is understood that such multimers can be constructed from any of the peptides or mutants described in the present invention. While it is preferred that the increased mutant monomers in the multimer possess the biological activity as described above, this is not required as long as the monomers are capable of imparting such activity to the multimer.

The present invention includes a fusion polypeptide, which may include a linear polymer composed of two or more repeating units of the above-mentioned peptide monomers. The peptide monomers may be directly connected at each end point, or there may be a linker between the repeating units of the monomers. And further fused into another polypeptide sequence to enhance the activity of the antigenic peptide.

The multimers and fusion polypeptides of the present invention can therefore also include multiple epitopes from the same or different Mtb proteins that do not occur together, ie in adjacent structures in the native Mtb protein.

The present invention also includes polymeric or fusion proteins that incorporate longer polypeptides, even full-length Mtb proteins, such as GlcB, MPT51 and other early Mtb antigens described in the present invention, such as those of GlcB and MPT51, in multiple combinations. Fusion or fusion of these two with another early antigen protein or proteins. These full-length proteins can be combined with shorter epitope-bearing Mtb peptides or mutants thereof or with peptide multimers (same- or different multimers) to form polymeric proteins. Such fusion proteins optionally include spacers or linkers between some or all of the protein or peptide units.

Peptides and multimers can be chemically linked to form multimers and larger aggregates. Preferred linking polymers include Cys and are linked by disulfide bond formation between -SH residues to form branched and linear peptides or polypeptides.

In addition to the linkers described above, the multimers and fusion polypeptides of the invention may include enzymatically cleavable linkers. Preferred enzymes are metalloprotealse, urokinase, cathepsin, plasmin or thrombin. Preferred linkers contain the sequence VPRGSD (SEQ ID NO: 115) or DDKDWH (SEQ ID NO: 238).

These peptides may be fused to other proteins in the form of fusion polypeptides comprising one or more peptide monomers or mixtures of these peptides, and the other proteins may be, for example, carrier molecules or promote immunogenicity when used as vaccine compositions of protein.

Other compositions within the scope of the present invention are the aforementioned peptides, multimers or fusion polypeptides immobilized on a solid support or carrier for use in immunoassays. "Solid support" refers to any support capable of binding antigens or antibodies. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and artificially modified celluloses, polyacrylamides, polyvinylidene difluoride, agaroses such as Sepharose ^_ and magnetic beads. The support material can have any possible structural shape, as long as the peptide or polypeptide immobilized on it can bind to its target molecule, such as an antibody. Thus, support configurations include particles, beads, wells and impermeable strips and films, the inner surfaces of reaction vessels such as test tubes or microtiter plates, the outer surfaces of rods and the like. Many other suitable carriers for binding the peptide will be available to those skilled in the art, or will be able to be ascertained by routine experimentation.

The kits of the invention described in detail below may include one or more different peptide compositions.

immune test

In a preferred embodiment, the mycobacterial antigenic composition is contacted with a solid support or carrier, such as nitrocellulose or polystyrene, so that the antigenic composition is adsorbed and immobilized on the solid support. The immobilized antigen is then reacted with a sample of biological fluid to test for the presence or absence of anti-Mtb antibodies, any antibodies in the sample will bind to the immobilized antigen. The antibody-bound immobilizer is washed with a suitable buffer prior to the introduction of a detectably labeled conjugate for the determination of the antibody. The conjugate binds to the immobilized antibody. Detection markers are a means of detecting immobilized antibodies.

The preferred conjugates in this assay are anti-immunoglobulin antibodies ("secondary antibodies") of different species. Detection of human antibodies may therefore employ a detectably labeled goat anti-human immunoglobulin "secondary" antibody. The solid support can then be washed a second time with buffer to remove unbound antibody. The amount of bound label on the solid support can be determined by conventional methods appropriate to the type of label (see below).

Such "secondary antibodies" can be specific antibodies to epitopes of specific human immunoglobulin isotypes, such as IgM, IgG ₁ , IgG _2a , IgA and their analogs, thereby enabling specificity of mycobacterial antigens in the sample. Antibody isotypes were detected. Alternatively, the second antibody may be specific for the idiotype of the anti-Mtb antibody in the sample.

Alternative conjugates of the detection sample antibody, other known conjugates to human immunoglobulins can also be used. Examples are staphylococcal immunoglobulin-binding proteins, the best known of which is protein A. There is also staphylococcal protein G, or a recombinant fusion protein between proteins A and G. Protein G (streptococcal groups G and C) binds to the Fc portion of the Ig molecule and binds to the IgG Fab fragment on the VH ₃ region. Protein C of Peptococcus magnus binds to the Fab region of the immunoglobulin molecule. Also included are any other proteins that bind microbial immunoglobulins, such as Streptococcus (eg, Langone, JJ, Adv. ImmunoL 32:157 (1982)).

In another embodiment of the present invention, the biological fluid that may contain Mtb antigen-specific antibodies is associated with a solid support or carrier capable of immobilizing soluble proteins. The support is then treated with a mycobacterial antigen reagent, which is a detectable marker, after which the support is washed with a suitable buffer. Bound antigen is then determined by measuring an immobilized detectable label. If the mycobacterial antigen reagent is not a directly detectable label, a second reagent, which contains a detectable labeled conjugate of the Mtb antigen, usually a secondary anti-Mtb antibody such as a murine mAb, is combined with the immobilized antigen. Combine. The solid support is then washed a second time with buffer to remove unbound antibody. The amount of bound label on the above-mentioned solid support is then determined by conventional methods.

"Solid phase support" refers to any support capable of binding to protein antigens or antibodies or other conjugates described in the present invention. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, polyvinylidene difluoride, dextran, nylon, magnetic beads, amylases, natural and artificially modified celluloses, polyacrylamides, agar Sugar and magnetite. According to the invention, the nature of the carrier is either partially soluble or insoluble. The support material can be in any possible structural shape as long as it is capable of binding the antigen or antibody. Thus, the shape of the support may be spherical, such as a bead, or cylindrical, such as the inner surface of a test tube, or the outer surface of a rod. Surfaces can also be flat, such as paper, test strips, etc. Preferred supports include polystyrene beads, 96-well polystyrene microplates and test strips, all of which are well known in the art. Those skilled in the art will know of many other suitable carriers for binding the antibody or antigen, or will be able to ascertain some by routine experimentation.

Using any of the assay methods described in the present invention, those skilled in the art can determine the operable optimum test conditions for each test through routine experiments. Moreover, other steps such as washing, stirring, shaking, filtering, and the like may also be added to the test according to specific operational requirements.

A preferred immunoassay for the detection of antibodies specific for mycobacterial antigens in the present invention is enzyme-linked immunosorbent assay (ELISA), or more commonly termed enzyme immunoassay (EIA). In this assay, the detectable label associated with the bound antibody or bound antigenic reagent is an enzyme. When in contact with a substrate, the enzyme will react with it to generate a chemical moiety that can be detected by methods such as spectrometry, fluorometry or visual observation. Enzymes used in the present invention for measurable labeling reagents include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, hydrogen peroxide Enzymes, malate dehydrogenase, staphylococcal nuclease, asparaginase, delta-5-pime isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase , glucose-6-phosphate dehydrogenase, glucoamylase and cholinesterase. For a description of the EIA procedure, see Voller, A. et al., J. Clin. Pathol. 31: 507-520 (1978); Butler, J. E., Meth. Enzymol. 73: 482-523 (1981); Maggio, E. ( ed.), Enzyme Immunoassay, CRC Press, Boca Raton, 1980; Butler, JE, In: Structure of Antigens, Vol.1 (Van Regenmortel, M., CRC Press, Boca Raton, 1992, pp.209-259; Butler, J.E. , In: van Oss, CJ et al., (eds), Immunochemistry, Marcel Dekker, Inc., New York, 1994, pp.759-803; Butler, J.E. (ed.), Immunochemist7-y of Solid-Phase Immunoassay , CRCPress, Boca Raton, 1991).

In another example, the detectable label may be a radiolabel, and the method is a radioimmunoassay (RIA), which is a method well known in the art. See, e.g., Yalow, R. et al., Nature 184:1648 (1959); Work, TS, et al., Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, NY, 1978, which are hereby incorporated by reference literature. Radioisotopes can be detected by gamma counters, scintillation counters, or autoradiography. Particularly suitable radioisotopes for the present invention are ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H and ¹⁴ C.

Antigen or antibody reagents can also be labeled with fluorophores. Fluorescently labeled antibodies are exposed to light of appropriate wavelengths, and the antibodies are detected by the fluorescence of the fluorophore. The most commonly used fluorophores are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, or fluorescent metals such as ¹⁵² Eu or other lanthanides element. These metals are bound to the antibody using metal chelators.

Antigens or antibodies used in the present invention may also be labeled for detection by binding to chemiluminescent compounds. The chemiluminescent-labeled antibody or antigen is then detected by measuring the light generated during the chemical reaction. Chemiluminescent labels can be luminescent amines, isoluminescent amines, theromaticacridinium esters, imidazoles, acridinium salts and oxalates. Likewise, bioluminescent compounds such as bioluminescent proteins can also be used as labels for antigen or antibody reagents. Binding was detected by measuring luminescence. Bioluminescent compounds include luciferin, luciferase, and luminescent proteins.

Detectable labeling reagents for the present invention can be detected by a scintillation counter, for example, where the detectable label is a radioactive gamma emitter; or by a fluorometer, where the label is a fluorophore. When enzyme labeling is used, it can be determined by colorimetric measurement of the colored product produced by the conversion of the chromogenic substrate caused by the enzyme. It can also be determined by visual color comparison of an appropriate standard or control with the product of the enzymatic reaction.

Immunoassays of the invention may be "double layer" or "sandwich" methods. The antibody-containing fluid to be detected can be brought into contact with the solid support. After the mycobacterial antigen is added, a certain amount of soluble and detectable labeled antibody is added to form a tertiary complex, namely, solid-phase antibody, antigen and labeled antibody, and then the complex is measured and/or quantitatively determined. Sandwich assays are described in Wide, Radioimmune Assay Method, Kirkham et al., Eds., E. & S. Livingstone, Edinburgh, 1970, pp 199-206.

Methods other than RIA and EIA include various types of agglutination assays, including direct and indirect methods, which are well known in the art. In these test methods, agglutination of particles containing an antigen (native or chemically linked) indicates the presence of the corresponding antibody. Any particle including latex, charcoal, kaolinite or bentonite, as well as microbial cells or erythrocytes, can serve as an agglutinable carrier (Mochida, US 4,308,026; Gupta et al., J. Immunol. Meth. 80:177-187 (1985); Castelan et al., J.Clin.Pathol.21:638(1968); Singer et al., Amer.J.Med.(Dec.1956,888; Molinaro, US 4,130,634).Traditional particle agglutination or erythrocyte agglutination test speed Faster, but less sensitive than RIA and EIA.However, the agglutination test method has the advantage of requiring less experimental environmental conditions, which is applicable in less developed countries.

In addition to detecting antibodies, the present invention also provides methods for determining and enumerating cells that secrete antibodies specific for a mycobacterial antigen. For example, various plaque or spot assays can be used, in which a sample containing lymphocytes, such as peripheral blood lymphocytes, is mixed with an antigen-containing reagent. When the antibody-secreting cells in the sample secrete antibodies and the antibodies react with the antigen, the number of antibody-secreting cells (or plaques forming cells) can be determined. The antigen may be bound to indicator particles, such as red blood cells, preferably sheep red blood cells, arranged in a layer. When individual cells secrete antibodies, the antibodies bind to surrounding red blood cells that have antigens. Antibody-bound red blood cells are lysed by the addition of complement components, resulting in "holes" or "plaques" in the red blood cell layer. Each plaque corresponds to an antibody-secreting cell. In another embodiment, a sample of cells containing secreted antibodies is added to a surface covered with an antigen-containing reagent, such as a mycobacterial antigen alone or bound to bovine serum albumin, the Antigen reagents are attached to polystyrene. After cells secrete antibodies that bind to the immobilized antigen, the cells are then gently washed away. Bound antibodies can be made to appear as colored "spots" around where the cells were originally present, using modified EIA methods or other staining methods well known in the art. (See, for example, Sedgwick, JD etc., J Immunol.Meth.57:301-309 (1983); Logtenberg, T. etc., Immunol.Lett.9:343-347 (1985); Walker, A.G. etc., J. Immunol. Meth. 104:281-283 (1987).

The present invention also aims to provide a kit or reagent system for implementing the above method. Such kits include reagent kits that include the essential reagents necessary for the methods of the invention. Reagent systems come in individual commercial packages, which may be compositions or mixtures (with compatibility between the reagents), in a shape suitable for a test device, or more typically, a test kit. An assay kit is a packaged combination of one or more containers, devices, or the like containing the necessary reagents, and generally includes instructions for assay procedures. The kit can also include containers of material that can be used for storage, use, or both. The kits of the invention may include configurations and compositions for carrying out the various test formats described above.

For example, a kit for determining the presence of anti-Mtb early antibodies comprises one or more early Mtb antigens, which may be in immobilizable form or have been immobilized on a solid support, and an anti-Mtb antigen capable of recognizing anti-Mtb Detectable labeled conjugates of early antibody samples, such as labeled anti-human Ig or anti-human Fab antibodies. Kits for determining the presence of early Mtb antigens comprise an immobilizable or immobilized "capture" antibody reactive with one or more epitopes of the Mtb antigen, and a detectably labeled second ("detection") antibody reactive with another epitope of the Mtb antigen recognized by the capture antibody. Kits may also include any commonly used labels or detectable labels, such as radioisotopes, enzymes, chromophores or fluorophores. The kit may also include reagents capable of precipitating immune complexes.

The kits of the present invention may also include auxiliary chemical reagents, such as buffers, and solution components for binding the antigen to the antibody.

The invention also provides a method for identifying, isolating and characterizing the early Mtb antigen. For example, an adsorbed patient serum or pool of serum containing antibodies to one or more antigens can be used in the initial stages of antigen preparation and purification, as well as in the cloning of protein antigens. The antiserum can be further adsorbed to a Mtb or other mycobacterial agent to render it functionally monospecific or polyspecific. This "enriched" antiserum can be used in conjunction with standard biochemical purification techniques to detect the presence or absence of antigen in fractions of the purification process. Antisera may also be used in an immobilized form as an immunosorbent in the affinity purification of antigens according to standard methods in the art. In addition, antisera can also be used in expression cloning methods to detect the presence or absence of antigens in colonies or in plaques of phage expressing antigens.

Once the antigen has been purified, e.g., by using patient early antibodies that have been determined to be specific for the antigen of interest, the resulting antigen can be used for immunization of animals to prepare high titer antisera or preferably to obtain the antigen specific mAbs. Such animal antisera or mAbs can be used as a surrogate for patient antisera, or in combination with experimental body fluid samples in competitive immunoassays. Antisera or mAbs can thus be used to prepare or purify antigens, or to detect antigens in immunoassays, for example as conjugates (capture antibodies or detection antibodies) in immunoassay sandwich methods.

The present invention provides an immunoassay for detecting the presence or absence of Mtb early antigens in body fluids or bacterial cultures of body fluids from persons suspected of Mtb infection. A sensitive immunoassay such as EIA direct sandwich method or competitive EIA method can detect Mtb protein (early antigen) on the order of picograms. The competitive assay can detect the specific epitope of the Mtb antigen without purification of the antigen. This assay can detect Mtb in patients earlier than standard bacterial assays (ie, the appearance of colonies on agar). Thus, this method can provide a basis for the decision to initiate clinical treatment hours or days after the antigen has been detected in body fluids. This method has significant advantages over the traditional 2-4 weeks (or longer) required to culture Mtb microorganisms from patient samples. The earlier the stage of infection, the lower the titer value of Mtb in the body fluid, the more favorable the method of the present invention compares with the traditional diagnostic method. Many immunoassays for various Mtb antigens are known in the art and could serve as the basis for the early antigen assays of the present invention (Wilkins et al., supra; Verbon, 1994, supra; Benjamin, RG et al., 1984, J. Med.Micro.18; 309-318; Yanea, MA et al., 1986, J.Clin.Microbiol.23:822-825; Ma et al., supra; Daniel et al., 1986, 1987, supra; Watt.G et al., 1988, J. Infec. Dis. 158:681-686; Wadee, AA et al., 1990, J. Clin. Microbiol. 23:2786-2791). For an example of competitive EIA for the Mtb antigen, see Jackett et al., supra.

In a preferred immunoassay sandwich, human antisera (or pools) or mAbs, preferably murine, are used as capture antibodies and are immobilized on a solid support, preferably a microplate. A test antigen preparation, such as Mtb culture supernatant or extract, is added to the immobilized antibody. After washing, a second "detection" antibody, such as a specific murine mAb specific for the same antigen or preferably specific for a different epitope of the same protein, is bound in the presence of a certain amount of mAb directed against the specific epitope, preferably a murine mAb. Sexual mAbs. Detection mAbs can be enzyme-conjugated. In addition, second-step reagents such as mouse immunoglobulin-specific enzyme-labeled antibodies can be used to detect the immobilized antigen.

The present invention isolates a Mtb antigen, which is then used to prepare one or more epitope-specific mAbs, preferably in mice. These putative early Mtb-specific mAbs were screened with patient sera known to be reactive with early antigens. The murine mAbs prepared in this way were then used in a highly sensitive epitope-specific competitive immunoassay for early stage TB. Thus, the presence or absence of antibodies specific for early epitopes of Mtb in patient samples was detected by the ability to compete with known mAbs for binding to purified early antigens. This test method requires less mycobacterial preparation than pure because the mAb provides the specificity necessary to detect patient antibodies against the epitope for which the mAb is specific under competitive assay conditions. sex.

In addition to detecting early Mtb antigens or early antibodies, the present invention also provides a method for detecting immune complexes containing early Mtb antigens by using the above-mentioned EIA method. Circulating immune complexes are considered to have diagnostic value for TB. (See, eg, Mehta, PK et al., 1989, Med. Microbiol. Immunol. 178; 229-233; Radhakrishnan, VV et al., 1992, J. Med. Microbiol. 36: 128-131). Methods for testing immune complexes are well known in the art. The complex can be decomposed under acidic conditions, and the resulting antigen and antibody can be detected by immunoassay. See, eg, Bollinger, RC et al., 1992, J. Infec. Dis. 165;913-916. The immune complexes can be analyzed directly or resolved by precipitation by known methods such as polyethylene glycol precipitation.

The purified Mtb early antigen as described above is preferably produced by recombinant methods. See Example IV. Traditional bacterial expression systems utilize Gram-negative bacteria such as E.coli or Salmonella species. But such a system is not suitable for the preparation of Mtb antigen (Burlein, JE, In: Tuberculosis; Pathogenesis, Protection and Control, B. Bloom, ed., Amer Soc Microbiol, Washington, DC, 1994, pp.239-252). Rather, it is preferred to utilize a homologous mycobacterial host for the recombinant production of the early Mtb antigenic protein or glycoprotein. Control methods and gene expression methods are described in Burlein, supra. Expression in mycobacterial hosts, especially M. bovis (strain BCG) or M. smegmatis is well known in the art. Two examples, one is a mycobacterial gene (Rouse, DA et al., 1996, Microbiol.22:583-592) and the other is a non-mycobacterial gene, such as the HIV-1 gene (Winter, N et al., 1992, Vaccines 92, Cold Spring Harbor Press, pp.373-378) are expressed in mycobacterial hosts, which are hereby cited as examples in the art. The preceding three references are incorporated herein by reference in their entirety.

Antibody testing in urine

The present invention also provides a diagnostic method for TB in urine, which can be used as an independent diagnostic test, or as an adjunct method to the aforementioned serum diagnostic method. The method enables the operator to (1) determine the presence of anti-mycobacterial antibodies in the urine of early-stage TB patients (non-cavitary, smear-negative) and HIV-infected TB patients; (2) determine the specific mycobacterial Characterization of antigens, such as mycobacterial antigens in the culture filtrate, which have stable and strong reactivity with antibodies in urine; and (3) obtaining antigens that can be recognized by antibodies in urine.

Only 50% of TB cases are diagnosed in smear-positive (=advanced) cases, whereas relatively early-stage TB patients are usually smear-negative. Moreover, in HIV-endemic developing countries, the number and proportion of HIV-infected TB patients is increasing.

Serum and urine samples were obtained from non-cavitary and/or smear-negative, culture-positive TB patients and HIV-infected TB patients. Serum and urine samples from PPD-positive people and PPD-negative healthy people, HIV-infected people without tuberculosis, or people in close contact with TB patients can be used as negative controls.

The reactivity of serum samples with culture filtrate proteins and purified antigens of Mycobacterium tuberculosis (MPT32, Ag85C and 88Kda as described) is preferably determined by ELISA. All sera are preferably depleted of cross-reactive antibodies prior to ELISA testing.

Preferred assay methods are described below and are not intended to be limited to the specific steps (or sequence of steps), conditions, quantities of reagents and materials.

Briefly, wells on an ELISA plate were coated with 200 μl of E. coli lysate (500 μg/ml suspension) (Immulon 2, Dynex, Chantilly VA.) and wells were blocked with 5% bovine serum albumin (BSA) . Serum samples (diluted 1:10 with PBS-Tween-20) were adsorbed 8 times with E. coli lysate. The adsorbed sera were then used in ELISA assays.

50 μl of antigen, suspended in coating buffer at 2 μg/ml (except for all culture filtrate proteins used at 5 μg/ml), was bound overnight to the wells of the ELISA plate. After washing 3 times with PBS (phosphate buffered saline), the wells were blocked with 7.5% FBS (fetal bovine serum, Hyclone, Logan, UT.) and 2.5% BSA in PBS, and kept at 37° C. for 2.5 hours. 50 μl of serum samples were added to each well, and the samples were diluted in a predetermined ratio (1:1000 for culture filtrate protein, 1:50 for Ag85C, 1:150 for MPT32, and 1:200 for 88kDa antigen). At 37°C, the antigen-antibody binding is up to 90 minutes. The plate was washed 6 times with PBS-Tween-20 (0.05%), and 50 μl of alkaline phosphate-conjugated goat anti-human IgG (Zymed, CA) diluted 1:2000 in PBS-Tween-20 was added to each well. After 60 minutes, the plate was washed 6 times with Tris-buffered saline (50 mM Tris, 150 mM NaCl) and developed with a Gibco BRL amplification system (Life Technologies, Gaithersburg, MD). The reaction was stopped by adding 50 μl of 0.3M _H2SO4 and the absorbance _was read at 490 nm. The cut-off point of ELISA detection is the mean absorbance (optical density OD) + 3 standard deviations (SD) of the negative control group, wherein the control group includes PPD positive and PPD negative healthy subjects.

The reactivity of urine samples to various antigens was initially determined on undiluted urine samples as described above. For the ELISA determination of urine samples, the results obtained by the inventors (see Example VII) showed that the preferred concentration of culture filtrate protein was 4 μg/ml suspension in 125 μl per well, and 2 μg/ml suspension in 125 μl per well for MPT32. ml solution. Urine was left overnight in the antigen-coated wells. If smear-negative and HIV-infected patients have urine antibody titers lower than those observed in smear-positive patients, it is necessary to first concentrate the urine sample. When concentrating, preferably use an Amicon concentrator to remove proteins with a molecular weight of 30 kDa. Determine the presence of antibodies to the above antigens in the concentrated urine sample. Optimal conditions for these tests are readily determined. Antibody sensitivity and specificity are readily determined by testing both urine and serum samples with one or more antigens.

As described in Example VII, culture filtrate proteins separated by ELISA and 1D SDS-PAGE showed that the urine antibodies were directed against the same antigens recognized by the serum antibodies, but with lower urine antibody titers. Several proteins were identified based on their reactivity with different anti-mycobacterial monoclonal antibodies, 2D maps of culture filtrate proteins were shown, or peptides were sequenced (as described in the present invention, see also Sonnenberg, M.G. et al. 1997, Infect. Immun. 65:4515).

Based on this map, the inventors obtained 2-D maps of antigens detected by early serum antibodies (from smear-negative patients), and from late-stage, smear-positive HIV-uninfected TB patients and HIV-infected TB patients Recognized by antibodies (described in Examples). Screening was performed to determine whether, in addition to MPT32, Ag85C and the 88 kDa protein, other antigens included in the test were preferred. It is preferable to determine whether anti-MPT51 antibodies are well detected in urine because this protein is highly recognized by serum antibodies in both early and late stages of TB and its identity and sequence are known.

Culture filtrate antigens of Mtb were separated on 2-D gels and transferred to obtain 2-D spots as described below. Briefly, 70 μg of culture filtrate protein was suspended in 30 μl of isoelectric focusing sample buffer (e.g., 9M urea, 2% NP-40, 5% β-mercaptoethanol, and 5% pH3-10 or pH4- 6.5 ampholytes). The ampholyte used in the following examples is referred to as "Apholyte ^™ ", which is a copolymer of glycine, glycylglycine, amine and epichlorohydrin. In the isoelectric focusing step in the 2-D gel analysis of the examples, two ampholytes ^TM (PH3-10 or pH4-6.5) with different pH ranges were used. (As used in the Examples, the ampholyte class numbers for these two osmolytes are 17-0456-01 and 17-0452-01.) The above samples were incubated at 20°C for 3 hours. 25 μl of this preparation was used in 6% polyacrylamide IEF tube gel, wherein the gel contained 5% ampholytes of PH3-10 and PH4-6.5, and the ratio of the two electrolytes was 1:4, at 1kV Focus up to 3 hours. After focusing, the tube gel was soaked in sample transfer buffer for 30 minutes, and then subjected to second dimension electrophoresis with 15% SDS-polyacrylamide gel. Each gel was first run at 20 mA for 0.3 hours and then at 30 mA for 1.8 hours. The isolated proteins were transferred without subsequent blotting. The 2-D blot was washed with PBS and blocked with 5% BSA for 2-2.5 hours. After washing the spots again, the spots were placed in urine samples (either undiluted or concentrated) and shaken overnight. Subsequently, washed with PBS-Tween, the blot was exposed to anti-human IgG conjugated to alkaline phosphatase, and then to the appropriate substrate. Antigens reactive with urine samples were identified based on 2-D plots. Antigens recognized by antibodies in urine samples of smear-negative, non-cavitating (=early) TB patients, smear-positive (=advanced) TB patients and HIV-infected TB patients were identified.

Antigens recognized by antibodies in the urine sample as well as antibodies in the serum are used in preferred diagnostic assays. Preferred antigens are the above-mentioned 88kDa protein Glcβ or MPT51 and their epitopes, which are present in the various peptides mentioned above. DNA encoding these proteins or fragments or variants thereof were cloned and expressed.

As described in the present invention, the Mtb culture filtrate preparation contained >100 different proteins (205 protein spots), most of which were in the 49-76 kDa range and were expressed in low amounts in the culture medium (Sonnenberg et al., supra ). This may be the result of culturing Mtb in a small amount of medium, which avoids the problem of difficult separation from proteins in a large amount of medium (BSA, casein digests, etc.). If the immunoreactive protein is well expressed in the culture filtrate and can be separated on a gel, it can be isolated from PVDF spots and sequenced. Since the entire chromosomal sequence of Mtb is known, the peptide sequence used to identify the protein must be accurate.

The nucleotide sequence (ie, the open reading frame) of the gene encoding the protein then becomes the basis for PCR amplification of the relevant DNA in the chromosomal DNA, and then cloned into an expression vector. Because many culture filtrates have low protein content, another, perhaps more reliable, method can be used, which is to use urine antibodies to immunoscreen an expression library of Mtbs to obtain genes encoding related proteins.

These methods can be used, for example, to clone the MPT51 gene or to identify immunoreactive proteins in the 49-76 kDa region. For the expression of MPT51, the shuttle vector pVV16 is preferably used; this vector has the E. coli origin of replication, the Mycobacterium pAL5000 origin of replication, the hygromycin resistance gene and the hsp60 promoter. Modifications were made at the C-terminus to encode six His residues. This vector can be expressed in E.coli or M.smegmatis. Because mycobacterial proteins expressed in E. coli hosts are often less immunoreactive than the same proteins expressed in mycobacterial hosts, it is preferred to express antigens in M. smegmatis. Methods for gene expression in mycobacterial hosts have been described in detail (Gaoora, PO et al., 1997, Med. Principles Pract. 6:91).

Briefly, to clone a specific gene into an expression vector, in-frame fusions are obtained by PCR amplification of the gene of interest using primers containing restriction sites. The PCR product is purified, digested with appropriate restriction enzymes and purified again. The vector DNA is cut with appropriate restriction enzymes and then purified. The PCR product was combined with the vector and electroporated into DH5α and grown overnight in the presence of hygromycin. Several antibiotic-resistant colonies were grown in a small amount of medium, and plasmid DNA was isolated by minipreps. Check the size of the insert in these colonies. The insert of one or more colonies is sequenced.

For electroporation into M. smegmatis, the 7H9 medium was shaken to allow bacterial growth to reach an absorbance of 0.8-1.0. Bacteria were collected, washed twice with ice water, once with ice-cooled 10% glycerol, and suspended therein. Colonies were electroporated into cells with plasmid DNA inserts that had been sequenced. Electroporated cells were grown in 7H9 for 3-4 hours and then plated on phage-containing plates. Several tolerant colonies grew in a small amount of medium for 48-72 hours. M. smegmatis spheroids were sonicated, and lysates were separated by SDS-PAGE and reacted with urine samples containing antibodies to determine the presence or absence of immunoreactive proteins. Colonies expressing the desired protein were expanded and the His-tagged recombinant protein was purified using a commercial nickel-agar column (Qiagen).

Reactivity of recombinant proteins with whole urine samples was assessed by ELISA assay as described above. Combinations of antigens, preferably single epitopes, exhibit the best sensitivity and specificity.

Antibody-positive urine samples were used to screen the Mtb chromosomal DNA expression library for the preparation of one or more proteins in the 49-67 kDa range. A set of urine samples from 10-15 TB patients (the culture filtrate protein of Mtb detected in Western blot - a sample showing strong reactivity) was adsorbed by E. coli lysate, and the expression library was screened with appropriate dilution.

Briefly, E. coli Y1090 infected with appropriate phage plaque-forming units were plated on top agar of LB plates. After 2.5 hours at 42°C, a nitrocellulose filter saturated with isopropyl β-D thiogalactopyranoside (IPTG) was placed on the top of the plate, and placed at 37°C for 2.5 hours. The filters were removed, rinsed thoroughly, and left overnight in the urine sample. After washing again, the filters were placed in a 1:1000 dilution of alkaline phosphatase-conjugated anti-human IgG, followed by the addition of BCIP-NBT substrate. Positive plaques (recombinant phage) were placed on the original plate, cut and screened again, then purified.

Screening of libraries with urine antibodies identified several proteins. In order to identify clones expressing an antigen recognized by the antibodies of the majority of patients, the cloned phages were used to construct lysogenic bacteria in E. coli Y1089. Single colonies of lysogenic bacteria were grown in LB medium at 32°C until the absorbance at 600nm was 0.5. The lysogenic bacteria were induced to express the recombinant protein by raising the temperature to 45° C., and IPTG (10 mM) was added. The culture was incubated at 37°C for an additional 1.5 hours to accumulate the recombinant protein and the bacteriospheres were collected. Bacterial spheres were sonicated in a small volume of PBS, and lysates were separated on 10% SDS-PA gels, followed by electroblotting on nitrocellulose membranes. The signature was tested on urine samples from 20-25 TB patients, and cloned genes encoding highly immunoreactive proteins recognized by all or most of the urine samples were identified. Only the lysate of E. coli Y1089 or the lysate of Y1089 lysogenized with the λgtl 1 vector was used as a control.

The DNA of recombinant clones encoding highly immunoreactive proteins was isolated using the commercial Wizard LambdaPreps DNA purification system (Promega) and digested with EcoRI to obtain insert fragments. The DNA inserted in the clone was subcloned into the pGEMEX-1 vector (Promega) with the same reading frame as λgtl1 at the EcoRI cloning site. The recombinant plasmid (Pgemex plus cloned insert DNA) was transfected into viable E. coli JM 109 cells. Plasmid DNA was isolated with Wizard Plus Minipreps (Promega) and automatically sequenced with primers starting from the SP6 and T3 primer-specific promoters, and the promoters following the primer movement flanked the multiple cloning site of PGEMEX-1. The nucleotide sequence was used in a similar study to the Mtb gene sequence to identify the protein and obtain the sequence of the full gene.

Once the protein was identified and the gene sequence was known, the gene was cloned for expression as described above for cloning the MPT 51 gene.

In summary, combinations of antigens or epitopes have been identified through serological studies, and the above identified and prepared neoantigens/epitopes provide the basis for a sensitive early diagnostic test for TB. If the sensitivity of antibody detection in urine samples is sufficient, blood testing is not necessary. Otherwise, a sensitive diagnostic test can also be obtained using a serum + urine test method. The antigens provide an inexpensive and rapid method of diagnosing TB in a low-cost format (dipping strips or flow cartridges).

vaccine

The foregoing and the following examples prove that the patients infected with Mtb do produce antibodies to the early antigens of the present invention. These antigens prime the immune system and produce an immune response. Accordingly, vaccine compositions and methods of use can be designed to enhance such immunity, preferably eliciting an immune response at a stage where bacterial infection can be prevented or delayed. Vaccine compositions are especially useful for preventing Mtb infection in high risk populations. Vaccine compositions and methods are also useful for veterinary use.

Accordingly, the present invention includes a vaccine composition which immunizes an immunized individual against Mtb infection. An early Mtb antigen, preferably one of the four antigens described in the present invention, or a peptide in the antigen, is used as an active ingredient to prepare a vaccine composition. These four proteins are (a) a 88 kDa protein with a pI value of about 5.2 and a sequence identification number of 106; (b) a protein characterized as the Mtb antigen 85C; (c) a protein characterized as the Mtb antigen MPT51 (SEQ ID number: 107) a protein; and (d) a glycoprotein characterized by the Mtb antigen MPT32. Vaccines may also include one or more proteins of the present invention, their peptides or functional derivatives, or DNA encoding proteins, and pharmaceutically acceptable carriers or vehicles.

Preferred peptides for use in vaccine compositions include the 23 peptides described above for diagnostic compositions, which may be used alone, in combination or aggregated into linear polymers.

In one embodiment, the vaccine comprises a fusion protein or peptide multimer comprising an Mtb early antigen, such as the full length protein and/or one or more of the aforementioned peptides.

The vaccine composition may further include an adjuvant or other immune stimulating agent. The Mtb early antigen protein or its epitope-carrying peptide used in the vaccine is preferably prepared by recombinant, preferably in prokaryotic cells.

Preferred immunogens are full length proteins or longer epitope-bearing Mtb early antigen protein fragments, especially those fragments reactive with early antibodies. If a shorter epitope-bearing fragment, such as a fragment containing 20 amino acids or less, is used as the active ingredient of the vaccine, it is more beneficial to couple the peptide to an immunological carrier to enhance immunity. Such coupling is well known in the art and includes standard chemical coupling techniques using linkers as described in Pierce Chemical Company, Rockford, Illinois. Suitable carriers are proteins such as keyhole limpet hemocyanin (KLH), E. coli pilin protein k99, BSA, or rotavirus VP6 protein.

Another vaccine example is a peptide multimer or fusion protein comprising the Mtb early antigen protein or an epitope-bearing peptide linearly fused into another amino acid sequence. Due to the ease of controlling recombinant substances, multiple copies of selective epitope regions can be included in a single fusion protein molecule. Still other approaches are to "mix and match" several different epitope regions into a single multimer or fusion protein.

Such active ingredients are preferably recombinant products, preferably administered as protein or peptide vaccines. In another embodiment, the vaccine is in the form of a bacterial strain (preferably known as a "vaccine strain") whose genes have been transfected to express proteins or peptides with epitopes. Some known vaccine strains of Salmonella are described below. Salmonella dublin live vaccine strain SL5928 aroA148fliC(i): Tn10 and S. typhimurium LB5000 hsdSB121 leu-3121 (Newton S.M. et al., Science 1989, 244: 70).

Salmonella strains expressing the Mtb protein or fragment of the present invention can be constructed by known methods. Thus, a plasmid encoding a protein or peptide is constructed. The plasmid can first be screened in a suitable host such as E. coli strain MC1061. The purified plasmid was then introduced into S. typhimurium strain LB5000 so that the plasmid DNA was appropriately modified for introduction into the Salmonella vaccine strain. Plasmid DNA isolated from LB5000 was introduced into eg S. dublin strain SL5928 by electroporation. The expressed Mtb protein or fragment encoded by the plasmid in SL5928 can be verified by Western blot test of bacterial lysate and specific antibody of relevant antigen or epitope.

The active ingredient or active ingredient mixture in the protein or peptide vaccine composition can be prepared by the method of traditional vaccine preparation. The active ingredient is usually dissolved or suspended in a pharmaceutically acceptable carrier, such as phosphate buffered saline. The vaccine composition may include an immunostimulatory agent or adjuvant, such as complete or incomplete Freund's adjuvant, aluminum hydroxide, liposomes, beads such as latex or gold beads, ISCOMs and the like. For example, intramuscular or subcutaneous injection of 0.5ml of complete Freund's adjuvant or a synthetic adjuvant with few side effects is preferably used in all initial immunizations; it can also be followed by injection of incomplete Freund's adjuvant as a booster injection. General methods for preparing vaccines are described in Remington's Pharmaceutical Science; Mack Publishing Company Easton, PA (latest edition).

The active protein in the liposomal pharmaceutical composition is dispersed therein or present in microparticles consisting of an aqueous center layer attached to a lipid layer. The active protein is preferably present in or out of the aqueous and lipid layers, or in either case in a heterogeneous system, which is commonly referred to as a liposomal suspension. The hydrophobic or oleaginous layer usually comprises phospholipids such as lecithin and sphingomyelin, a carrier such as cholesterol, more or less ionic surfactants such as diacyl phosphates, stearamide or phosphatidic acid and/or other hydrophobic substances. Adjuvants, including liposomes, are discussed in the following literature, which is hereby incorporated by reference: Gregoriades, G. et al., Immunological Adjuvants and Vaccines, Plenum Press, New York, 1989 Michalek, S.M. et al., "Liposomes as Oral Adiuvants, "Curr. Top. Microbiol. Immunol. 146:51-58 (1989).

Vaccine compositions preferably comprise (1) an effective amount of an active ingredient, i.e. a protein or peptide, and (2) a suitable amount of a carrier molecule, or carrier excipient if desired, (3) preservatives, buffers and the like . Vaccine formulations are described in Voller, A. et al., New Trends and Developments in Vaccines, University Park Press, Baltimore, Maryland (1978).

As with all immunogenic compositions that elicit antibody production, an effective amount of the immunogenic protein or peptide of the invention must be determined empirically. Factors considered include the immunogenicity of the native peptide, whether the peptide is complexed or covalently coupled to an adjuvant or carrier protein or other carrier, and the route of administration of the composition, i.e. intravenous, intramuscular, subcutaneous, etc., and Number of doses administered. These factors are known in the field of vaccines and can be determined by immunological researchers with appropriate experiments.

Routes of vaccine administration are generally known in the art. Systemic administration is usually by the parenteral route, as well as several other known effective routes of administration. For formulation, peptide vaccines can be administered through the mucous membranes in combination with penetrants such as bile salts or fusidic acid, and often a surfactant. Peptides can also be administered subcutaneously. It can also be used as an oral preparation. The dose depends on the mode of administration, the nature of the patient, and the nature of the carrier/adjuvant. Preferably, the effective amount of protein or peptide is about 0.01 μg/kg ^-1 mg/kg body weight. Patients can be systemically immunized by injection or oral administration, such as vaccine strains, with 10 ⁸ -10 ¹⁰ bacteria administered once or multiple times. Typically, multiple administrations are a standard immunization regimen for vaccines, as is standard in the art. For example, the dosing interval of the vaccine from one to four vaccinations may be about 2-6 weeks, preferably monthly intervals.

Vaccination with the vaccine composition produces an immune response, which is an antibody response, a cell-mediated response, or both, which blocks one or more stages of the Mtb bacterial infection cycle, preferably blocking Binding and entry to the host cell stage.

Through the overall description of the present invention, it will be easier to understand the present invention in conjunction with the following examples, which are not intended to limit the present invention.

Example I

Immunological Dominance of High Molecular Weight Antigens in Human Antibody Responses to Mtb Antigens

Materials and methods

The study population consisted of 58 HIV ^neg individuals diagnosed with pulmonary tuberculosis. Of those, 16 were from the infectious disease clinic at the Veterans Affairs Medical Center in New York. All patients were culture positive for Mtb, 9/16 patients were smear negative, 14/16 showed minimal to no radiographic lesions, and all individuals had within 1-2 weeks of initiation of TB chemotherapy or earlier Bleeding symptoms. Eight sera were obtained from Leonid Heifitz and Lory Powell (National Jewish Center, Denver, CO). The other 20 sera were provided by JMPhadtare (Grant Medical College, Bombay, India). Serum samples from 14 cases were provided by S. Singh, Lala Ram Sarup Tuberculosis Hospital, Mehrauli, New Delhi, India. Most of the 42 patients were smear-positive with radiographic features of intermediate-to-advanced lung disease and bleeding within 4-24 weeks of starting chemotherapy. The control group included the following populations:

(a) 16 HIV ^neg , TB ^neg , PPD ⁺ healthy individuals (recent immigrants from endemic countries, or medical staff caring for TB patients at VA medical centers);

(b) 23 HIV ^neg , TB ^neg healthy controls, 7 of them had negative PPD skin test (PPD ^neg ), and the PPD reactivity of the remaining 16 was unknown;

(c) 48 HIV ⁺ , PPD ^? , asymptomatic healthy individual, CD4 cell count > 800/mm ³ .

Group (b) individuals were included because TB is already a major opportunistic disease in HIV-infected populations.

antigen

Antigen reagents are all cell sonicate (CS), all culture filtrate (CF), lipoarabinomannose (LAM), LAM-free culture filtrate protein (LFCFP), all cell wall (CW), SDS-soluble cell wall protein (SCWP), and cell wall core (CWC), all isolated from MtbH37Rv. CS was from Mtb grown in Middlebrook 7H9 broth for 2-3 weeks (Difco Laboratories, Detroit, MI). The bacteria were collected by centrifugation at 1000 rpm for 30 minutes, and then the bacterial pellets were suspended in phosphate buffered saline (PBS) containing PMSF, EDTA and DTT, each with a final concentration of 1 mM. The suspension was cooled in liquid nitrogen, then thawed (several times) to wake up the cell walls, and then sonicated for 20 min at 4°C. The sonicated product was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected.

To obtain the remaining antigens, Mtb was grown in glycerol-alanine-salt medium to the mid-log phase (14 days). Cells were removed by filtration through a 0.22 μm membrane, and the culture supernatant was ultrafiltered using an Amicon instrument (Beverly, MA) with a 10,000 MW filter membrane, and then concentrated. The concentrate (CF) was dialyzed against 100 mM ammonium bicarbonate and dried by lyophilization.

To obtain LFCFP, CF was suspended (7 mg/ml) in a buffer containing 50 mM Tris HCl (pH 7.4), and 150 mM NaCl, followed by the addition of 20% TritonX-114 to a final concentration of 4%. The suspension was shaken overnight at 4°C. The 4% TritonX-114 suspension was heated to 37°C for 40 minutes to form a biphasic system and then centrifuged at 12,000xg. The aqueous phase was extracted twice with 4% TritonX-114 to ensure complete removal of lipoarabinomannose, lipomannose (LM) and phosphatidylinositol mannoside (PIM). The final aqueous phase was deposited with 10 volumes of cold acetone, and the beads were washed several times with cold acetone to remove residual TritonX-114. LAM-free aqueous CFPs were suspended in 100 mM ammonium bicarbonate, divided into aliquots, and dried by lyophilization.

LAM, LM and PIM were extracted from whole cells by mechanical disruption of the bacilli in a Bead Beater (Biospec Products, Bartelsville, OK) containing 4% Triton-X 114 in PBS (pH 7.4). Unbroken cells and cell walls were removed by centrifugation at 12000g, 4°C for 15 minutes. The supernatant was collected to obtain a two-phase system. The resulting detergent phase was back-extracted several times with cold PBS and macromolecules in the final wash phase were precipitated with 10 volumes of cold acetone. The precipitate was collected after centrifugation and air-dried. The precipitate (containing lipopolysaccharide) was suspended in PBS, and the remaining protein was extracted with PBS-saturated phenol. The aqueous phase was collected, and after distilling off the water, the lipopolysaccharide was dried by freeze-drying. LAM was further separated from LM and PIM by size exclusion chromatography as previously described (Chatteriee, D. et al., 1992, J. Biol. Chez. 269:66228-66233).

In order to isolate all the CW, Mtb cells were inactivated by isothermal killing at 80°C for 1 hour, and Mtb was suspended at a concentration of 0.5 g cells/ml in PBS, pH 7.4, 4% Triton X-114, PMSF, in buffer with pepsin inhibitor, EDTA, and deoxyribonuclease. Cells were ground in a strain grinder with 0.1 mm zirconia strain. Lysed cells were first centrifuged at 3000xg for 5 minutes to remove unbroken cells and then centrifuged at 27,000xg for 20 minutes at 4°C. The resulting pellets were washed three times with cold PBS at room temperature. Call the final ball CW.

Wash CW with 2% SDS in PBS, pH 7.4, at room temperature to obtain SCWP. Closely related proteins were isolated by extracting the CW pellet three times with 2% SDS in PBS, pH 7.4, at 55°C. At 55°C, the 2% SDS extract was recovered and the SDS was removed with an Extracti gel column (Pierce, Rockford, IL). The eluate was dialyzed against double distilled water, divided into aliquots, and dried by lyophilization.

CWC (mycolyl-arabinogalactan-peptidoglycan complex) was prepared according to the method described in (Daffe, M. et al., 1990, J; Biol. Chem. 265: 6734-6743) with only a few changes. get. The SDS-insoluble material obtained after SCWP extraction was suspended in PBS, 1% SDS, 0.1mg/ml proteinase K, and incubated at 50°C for 20 hours. The insoluble matter was centrifuged into pellets, washed twice with 2% SDS at 95°C for 1 hour, and then centrifuged to collect the insoluble matter. Wash several times with water and 80% acetone to remove SDS.

LFCFP was separated by size using a preparative SDS-PAGE system (model 491 Prep cell, Bio-Rad, Hercules, CA). CFP (20-25mg) was loaded directly into 30ml 10% preparative polyacrylamide tube gels containing 6% layering gels which were loaded into cast tubes with an internal diameter of 37mm. The running buffer used consisted of 25 mM Tris, pH 8.3, 192 mM Glycine, 0.1% SDS. Proteins were separated by electrophoresis using a gradient increasing wattage method at 8W for 3.13 hours, 12W for 2.5 hours and finally 20W for 11.1 hours. The constant effluent was 5 mM sodium phosphate, pH 6.8, and the protein in the effluent at the bottom of the gel tube was collected. The first 65ml of eluent was blank solution, after that, 80 fractions were collected at a speed of 0.4ml/min, each 4.2ml. The fractions were detected by one-dimensional SDS-PAGE, and the fractions were combined according to the detection results. SDS was removed from the pooled concentrated fractions using an Extracti-gel column (Pierce). The combined fractions were dried and stored frozen until testing.

Adsorption of serum by E.coli sonicate

Cultivate E.coli (Y1090) in Luria-Bertani medium overnight, centrifuge to obtain bacillus pellets, and process them according to the above method for sonicating Mtb bacilli, except that the sonication time is 30 seconds. 200 μl of E. coli lysate at a concentration of 500 μg/ml was suspended in 20 mM carbonate buffer, pH 9.6, and coated in each well of a 2 ^B ELISA plate (Dynatech, Alexandria, VA) and left overnight. Plates were washed and blocked with 5% BSA in PBS (Bovine Serum Albumin, Sigma Enmuno-chemicals, St. Louis) for 90 minutes. Triton X-100 (1% final concentration) was added to each serum sample to inactivate HIV, followed by heating to 55°C for 60 minutes. Samples from HIV-free individuals were processed in the same way to maintain consistency in sample preparation. The serum (20 μl) of each individual was diluted to 200 μl with PBS/Tween20 (0.05%), and added to 96 wells of the culture plate. Diluted serum samples were transferred to E. coli-coated, blocked ELISA plates using a multichannel pipette. Serum samples were reacted with the bound E.coli antigen for 90 minutes, and then transferred to another ELISA plate, which was coated with E.coli and blocked, as above. Serum samples were adsorbed 8 times by E.coli antigen, and then the samples were transferred to 96-well tissue culture plates, and sodium azide (final concentration: 1 mM) was added to each well. The protocol can handle small numbers of multiple samples quickly and efficiently. Absorbed serum samples were to be used within one week.

ELISA analysis of Mtb antigen

50 μl of antigen was suspended in coating buffer at 5 μg/ml (except CS and SCWP, which were 15 μg/ml and 1 Lg/ml, respectively) and bound to wells in ELISA plates overnight. After washing with PBS three times, each well was blocked with PBS containing 7.5% FBS (fetal bovine serum, Hyclone, Logan, UT.) and 2.5% BSA at 37°C for 2.5 hours. Subsequently, the sera were diluted to 1:1000 with PBS/Tween20 (0.05%, PBST) containing 1% FCS and 0.25% BSA, and 50 μl of each serum was added to each well. Antigen-antibody binding was performed at 37°C for 90 minutes, and then the plate was washed 6 times with PBST. 50 [mu]l of alkaline phosphatase-conjugated goat anti-human IgG (Zymed, CA) diluted 1:2000 (same diluent as serum samples) was added to each well. After 60 minutes, the plate was washed 6 times with Tris-buffered saline (50 mM Tris, 150 mM NaCl) and developed with the Gibco BRL amplification system (Life Technologies, Gaithersburg, MD). Stop the reaction with 50 μl _of 0.3M _H2SO4 and read the absorbance of the plate at 490 nm. Optimal antigen and antibody concentrations for each antigen were determined by plate titration with a limited number of control and TB-free sera prior to ELISA testing of total sera.

The ELISA determination of the size of the various levels obtained by preparative polyacrylamide gel electrophoresis is the same as the above method, except that the antigen is added at 2 μg/ml, and the final dilution ratio of the serum test is 1:200. 42 cases of TB serum and 44 cases of no TB control Groups (16 PPD ⁺ ; 7 HIV ^neg , PPD ^neg ; and 21 HIV ⁺ , asymptomatic individuals) were included in these assays.

Characterization of known antigens of mycobacteria in LAM-free CFP separated by size

The following mAbs were obtained from the World Health Organization (with permission from Dr. Thomas M. Shinnick, Centers for Disease Control, Atlanta):

IT-53 IT-13 IT-46 IT-63 IT-61 IT-51 MLO4-A2

IT-45 IT-64 IT-15 IT-49 IT-52 IT-69 SAID2D

IT-42 IT-70 IT-23 IT-48 IT-67 IT-4 CS-01

IT-41 IT-43 IT-62 IT-59 IT-68 IT-1

IT-56 IT-58 IT-47 IT-60 IT-19 IT-20

"IT" is the standard designation for the collection of anti-Mtb antibodies by the World Health Organization. Other names of mAbs, the antigens they recognize and the laboratory source are in Engers, H. et al., 1986, Infect. Immun.51: 718-720; Khanolkar-Young, S. et al., 1992, Infect. Immun.60 : 3925-3925; Young et al., supra, which are hereby incorporated by reference in their entirety. Antiserum MPT32 to the 50/55 kDa antigen was obtained from NIH, Protocol 1-AI-25147. The table below summarizes these antibodies and their reactivity.

In the ELISA assay, the antigen composition of the size class detected by the antibody is similar to the antigen composition used for the reactivity assay of human serum, except that the concentration of the antibody defined above is 50 μl/well recommended by the laboratory. The secondary antibody in the ELISA assay was alkaline phosphatase-conjugated rabbit anti-mouse IgG or goat anti-rabbit IgG (1:2000, Sigma Imnunochemicals), added in an amount of 50 μl/well.

SDS-PAGE and Western blot analysis

All components (LFCFP and its fractions) were separated on a 10% SDS-PA microgel, and the proteins were transferred to nitrocellulose membranes before detection with antibodies. In order to better identify the antigen in the fraction 15 recognized by the test serum in the ELISA, all spots of LFCFP and fractions 10 and 15 were assayed with the following samples:

(a) 6 TB sera, which were positive for LFCFP in the ELISA assay;

(b) 6 TB sera that were negative by ELISA; and

(c) Sera from 6 cases in the PPD ⁺ healthy control group.

All spots were screened for antibody binding using alkaline phosphatase-conjugated rabbit anti-human IgG followed by chromogenic reaction with BCIP/NBT substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD).

Statistical Analysis

Positivity cut-off points in all ELISA assays were set as mean absorbance or optical density (OD) ± 3 standard deviations (SD) (control group). The Wilcoxon signed rank for cohort samples was used to compare the reactivity of sera before and after adsorption. The SD of the above two groups were compared by F test. The reactivity of TB sera to LFCFP and the reactivity of TB sera to other antigens were compared using McNemar's paired test. All statistical analyzes were performed with The Graphpad Instat program.

result

A. Efficacy of E.coli lysate adsorption test serum

Evaluation of 38 HIV ^neg (16 PPD ⁺ , 7 PPD ^neg , 15 PPD unknown) tuberculosis-free individuals and 21 HIV-infected asymptomatic individuals before and after removal of cross-reactive antibodies by adsorption with E. coli lysates , Reactivity of sera from 42 TB patients with LFCFP ( FIG. 1 ). There was no difference in reactivity among control serum subgroups. The mean absorbance (OD±SD) of the unadsorbed control serum was 0.316±0.111 and that after adsorption was 0.165±0.05 (Table 1). The reduction in reactivity was statistically significant (p<0.0001). Furthermore, the variance (expressed as SD) after adsorption of the control serum samples was significantly lower (p<0.0001) compared to the SD before adsorption of the same serum (Figure 1, Table 1). The average OD of TB serum before adsorption was 0.911±0.454, and the average OD of the same serum after adsorption was 0.694±0.440 (Figure 1). Although TB serum reactivity after adsorption was also significantly lower (p < 0.0001) compared to serum before adsorption, the SD of TB samples before and after adsorption was similar. Thus, high levels of cross-reactive antibodies present in control and test sera were adsorbed by E. coli lysates. For controls, depletion of these antibodies reduced baseline seroreactivity. However, as expected, despite the reduction in antibody levels, the error for each TB serum was not affected. The SD value exceeding 3 times the mean value of serum in the control group was set as the threshold value of positive reactivity.

Antibodies reactive with LFCFP were detectable in 25/42 (60%) of unadsorbed TB sera (Figure 1). When assayed after adsorption, anti-mycobacterial antibodies were detected in 4/17 (24%) of the remaining, previously seronegative sera, increasing the sensitivity to 69% (Figure 1).

Table 1 Comparison of serum before adsorption and serum adsorbed by E.coli

Serum  Mean O.D.±S.D. P value ^a P ^valueb control before adsorption After adsorption ＜0.0001 ＜0.001 0.316±0.111 0.165±0.050 TB patients 0.911±0.454 0.694±0.440 ＜0.0001 NS

a: Wilcoxon signed-rank test comparing serum before and after adsorption.

b: F-test comparing the standard deviation of serum before and after adsorption. NS; not significant.

These experiments also used the highest O.D. value of the control serum as a cut-off point for analysis, the method can also be found in the literature (Ivanyi et al., 1989, supra). Before adsorption, the O.D.s values of 59 control sera ranged from 0.16 to 0.68 (Fig. 1). 24/42 (57%) TB sera had O.D.s values higher than the highest control sera. After adsorption, the O.D.s values of the same control sera ranged from 0.08-0.25, while 31/42 (74%) of TB sera showed positive antibodies. Thus, antibodies to the Mtb antigen were detected in 7/18 (39%) of the remaining, previously negative sera. Given the increased sensitivity of sera after adsorption, all sera were thereafter adsorbed prior to the various assays.

Example II

Antibodies to the 88kDa secreted antigen of Mycobacterium tuberculosis as a preclinical target for TB in HIV-infected individuals surrogate markers

A. Materials and methods

1. Serum:

The study population included 49 HIV-infected individuals who had developed or manifested TB (HIV/TB) within the last few years from the infectious disease clinic of the V.A. Medical Center in New York. A total of 259 serum samples were obtained from these patients. Including:

(a) 136 samples obtained from 38 patients in several conditions prior to the onset of clinical TB symptoms ("HIV/pre-TB");

(b) 37 samples from 37 patients with clinically and bacteriologically diagnosed TB ("HIV/Stage-TB"), and including several group (a) patients;

(c) 86 serum samples taken from 35 patients within months of starting TB treatment ("HIV/late-TB"). Most of the patients in group (c) were also members of groups (a) and/or (b).

The diagnosis of TB is based on positive Mtb culture fluid.

The sera of 20 non-HIV TB patients (non-HIV/TB) were used as positive controls, of which 19 were smear-positive, and all patients showed intermediate to advanced cavitary imaging symptoms. Sera from 19 non-HIV/PPD skin test positive persons were used as negative controls. In order to eliminate non-specific reactions, the study included: (i) sera from 35 asymptomatic HIV-infected individuals with CD4 cell count > 800; (ii) 48 serum samples from 16 HIV-infected individuals whose blood The broth was positive for Mycobacterium avium ("HIV/MAI"). Among them, 28 HIV/MAI serum samples were collected several months before the emergence of MAI bacteria.

The secreted antigen of Mtb H37Rv (referred to as LAM-free culture filtrate protein (LFCFP)) was prepared as described in Example 1. Antigen mixtures were then separated according to molecular weight using a BioRad 491 PrepCell (Hercules, CA) in 30 ml 10% preparative polyacrylamide tube gels containing 6% stacking gels. Fractions were collected according to molecular weight (as SDS-PAGE fractions) and dried.

LFCFP and its fractions were dissolved on 10% SDS-PA mini-gels and transferred to nitrocellulose membranes before detection using serum. The secondary antibody used was alkaline phosphatase-conjugated rabbit anti-human IgG and the substrate was BCIP/NBT (Kirkegaard and Perry Laboratories, Gaithersburg, MD).

All sera were adsorbed on E. coli lysates prior to ELISA testing. Adsorption and ELISA detection were performed as in Example 1.

2. Lymphocyte Staining and Flow Cytometry Analysis

Cells were incubated with Simultest CD3/CD4 and CD3/CD8 (Becton Dickinson Immuno-cytochemistry systems, San Jose, CA) reagents by standard procedures (Gordin F.M. et al., 1994, J.Irfect.Dis.169:893-897). dyeing. Flow cytometric analysis was performed with Becton Dickinson FACScan.

3. Statistical analysis: the operation is the same as before.

B. Results

1. Reactivity of sera from HIV/TB patients to mycobacterial antigens

The reactivity of total LFCFP in 259 sera from 49 HIV/TB patients and Mtb was compared with that of sera from 16 non-HIV/PPD ⁺ individuals (negative controls) and 20 non-HIV/TB patients (positive controls) reactivity for comparison. Each individual sample was tested at least three times for the presence of anti-Mtb antibodies. A representative ELISA assay showing antibody levels for each of these groups is shown in Figure 2. Setting the cut-off point at the mean OD±3SD of 16 sera from non-HIV/PPD ⁺ individuals, LFCFP antibodies were found in 16/20 (80%) of non-HIV/TB sera. As a control, only 9/37 (24%) of HIV/stage-TB sera had such antibody reactivity. However, 17/38 (45%) HIV/TB patients were HIV/pre-TB seropositive and 13/35 (34%) HIV/late-TB seropositive.

In conclusion, HIV ⁺ persons had lower antibody levels in sera than non-HIV persons (in all three groups). Mean OD values for the non-HIV/TB and HIV/stage-TB groups were statistically significant (compared to all sera (p=0.0001), or to antibody-only sera (p=0.0165)). Antibody levels measured by OD were significantly lower in HIV/pre-TB sera than in non-HIV/TB sera (p=0.0001 for all sera; p=0.0007 for antibody-positive sera).

Evaluation of the specificity of anti-Mtb antibody responses in HIV/TB patients. Sera from 35 HIV-infected asymptomatic individuals (CD4 ⁺ cell number >800) and 48 sera from 16 HIV/MAI patients with 19 non-HIV/PPD ⁺ healthy controls and 20 HIV/TB patients Serum was tested together. Using the mean OD±3SD of 19 non-HIV/PPD ⁺ healthy control sera as a cut-off point, 2/35 sera from the HIV- ⁺ group and 7/48 from the HIV/MAI group showed minimal reactivity with Mtb secreted antigens . These results confirmed the specificity of HIV/TB sera reacting with Mtb antigens.

2. Time of Anti-Mtb Antibody Appearance in HIV/TB Patients

Since antibodies to Mtb secreted antigens are present in about half of HIV/pre-TB sera, these antibodies appear earlier than the time TB is clinically established. Anti-Mtb antibodies were measured on sera from 6 antibody-positive patients, 3 antibody-negative HIV/TB patients, and 3 HIV/MAI patients. All six antibody-positive individuals had circulating antibodies at various times in the years preceding clinical symptoms of TB. One of the patients developed anti-Mtb antibodies about 1.5 years before the clinical diagnosis of TB, and the other patient developed anti-Mtb antibodies about 4.5 years before. The remaining 4 patients all had circulating antibodies 5-6 years earlier. In contrast, 3 antibody-negative HIV/TB patients and 3 HIV/MAI patients were consistently negative.

3. Reactivity of HIV/TB Sera with Isolated Secreted Antigens

In order to determine whether the antigenic characteristics (in the preparation of LFCFP) reacted with antibodies of HIV/TB patients were different from those recognized by antibodies of non-HIV/TB patients, nine ELISA ⁺ HIV/TB (two HIV/TB Western blots of LFCFP separated by SDS-PAGE were detected in sera from 7 HIV/pre-TB and 3 non-HIV/TB patients. Results were compared to antibody reactivity of 6 HIV- ⁺ asymptomatic controls and 5 non-HIV/PPD ⁺ healthy controls (ELISA ^neg ). As described in Example I, all sera (healthy and patient) reacted with the 65 kDa and 30-32 kDa antigens. Serum from non-HIV/TB patients reacts with a variety of antigens (about 20) between about 26 kDa and about 115 kDa. Among them, the reactivity with the 38kDa antigen present in a large amount and the 88kDa antigen present in a small amount was the strongest. Reacts with antigens with molecular weights of 32-38, 45-65, 72-78 and 80-115kDa.

In contrast, 8/9 HIV/TB sera did not react with the 38 kDa antigen, but with antigens in the 45-65 kDa range, although this was very low in some patients. Reactivity with the 72-78 kDa antigen was also reduced or completely lost. Reactivity to the 80-115 kDa antigen was present in the sera of two patients, but was significantly reduced in the remaining patients. Most HIV/TB sera appear to be more reactive with the 88 kDa antigen than with other antigens in this molecular weight range. Sera from asymptomatic HIV-infected individuals or PPD ⁺ healthy controls did not show any significant response at the same dilution. Therefore, it can be concluded that fewer types of antigens are recognized by HIV/TB sera than non-HIV/TB sera.

4. Reactivity of HIV/TB serum with size-separated LFCFP

To narrow down the range of antigens recognized by HIV/TB patients in LFCFP, LFCFP was separated based on molecular weight, resulting in 14 overlapping fractions. Western blots were performed on sera collected from 6 ELISA ⁺ non-HIV/TB or 6 HIV/TB patients to identify isolated fractions containing strongly seroreactive proteins. In addition to the 65 kDa and 30-32 kDa antigens that were previously shown (Example 1) to react with all sera (healthy and patient), non-HIV/TB sera also interacted with fractions 6-14 with molecular weights above 30-32 kDa antigenic reaction.

More specifically, antigens of approximately 32-38 kDa in fractions 6, 7 and 8 were also reacted. A distinct 38 kDa band was shown in fractions 9 and 10. In addition, antigens of 45, 50 and 58-60 kDa were also detected in fraction 10. Small amounts of 38 kDa antigen and 30-32 kDa antigen were also found in fractions 11-14, most of the serum reactive proteins were in the 56-68 kDa range in fraction 11 and in the 58-76 kDa range in fraction 12 Within the range of 65-76 kDa in fraction 13 and 65-88 kDa in fraction 14. A distinct 88 kDa band is only visible in fraction 14.

A similar blot was tested with sera from 6 ELISA ⁺ HIV/TB patients (5 HIV/pre-TB and 1 HIV/stage-TB) with weak antigen reactivity in fractions 6-9. According to the test results of HIV/TB individual sera, there was little or no reactivity with the 38 kDa antigen in fractions 9-14. However, antigenic reactivity with the 45, 50 and 58-60 kDa doublet in fraction 10 was discernible, albeit weaker (which had strong reactivity with non-HIV/TB sera), and with fraction 11- Other antigens in 14 were also reactive. Reactivity with the 88 kDa antigen in fraction 14 was strong and clearly discernible.

These results indicate that the antigen reactivity of the fraction 10-14 in HIV/TB sera is better than that of the other fractions. Therefore, among the antigens recognized by non-HIV/TB patients, the antigens recognized by HIV/TB patients are only a subclass. For example, antibodies to the 38kDa antigen were not found in HIV/TB, whereas antibodies to antigens in fractions 10-14, especially the 88kDa antigen, were present even in HIV-infected patients.

5. Reactivity of isolated Mtb antigen fractions with individual sera

In order to precisely determine the Mtb antigen species recognized with high frequency by HIV/TB patients, 145 sera from 42 HIV/TB patients were used to determine the reactivity with fractions 7-14 and whole LFCFP (as a positive control). Since the goal of these studies was to identify Mtb antigens that might be used as subclinical TB surrogate markers or as an adjunct in the diagnosis of TB suspected patients, mainly HIV/pre-TB and HIV/TB sera were used. Sera from 18 non-HIV/PPD ⁺ (negative controls) and 20 non-HIV/TB patients (positive controls) were included.

As shown above (Figure 2), using the mean OD±3SD of non-HIV/PPD ⁺ control sera as cut-off points, 16/20 (80%) of the non-HIV/TB sera had antibodies to all LFCFPs. 50% (21/42) of HIV/TB patients had antibodies to unisolated LFCFP. However, 74% (31/42) of the same patients showed positive reactivity with the antigen of fraction 14 of the isolate. 62% (26/42) of patients were reactive with the antigen of isolate 13 and 38% (16/42) with isolate 12 (although the OD values were lower for the antigens of isolate 12 and 13). About 50-60% of the sera reacted with antigens in fractions 9 and 10, although less reactive than fraction 14 antigen. As shown in Example I, non-HIV/TB patients who reacted with unisolated LFCFP also reacted with isolated fraction 14 antigen.

HIV/TB sera were analyzed for reactivity with unisolated LFCFP and with antigen in fraction 14 by comparing the HIV/pre-TB and HIV/period-TB groups. In contrast, 66% of HIV/stage-TB and 74% of HIV/pre-TB sera had antibodies binding to fraction 14 antigens.

To follow the timing of the emergence of antibodies to fraction 14 antigen, multiple serum samples from individual patients were tested for reactivity with fraction 14 and LFCFP. Antibodies to these antigens are present in the sera of individual patients (antibody positive) years before the onset of clinical TB symptoms. In contrast, multiple serum samples from antibody-negative patients were consistently negative.

6. Cell Morphology of Antibody Positive and Negative HIV/TB Patients

T cell morphology of antibody-positive HIV/TB patients reactive with fraction 14 antigen was compared with antibody-negative T cells, both in HIV/pre-TB and HIV/period-TB stages. There were no significant differences in the two groups of HIV/TB patients.

c. to discuss

The foregoing results demonstrate that antibodies to Mtb secreted antigens are present in about 74% of HIV/TB patients for months to years before clinical TB symptoms appear. Since reactivity is lower in HIV/TB patients than in non-HIV/TB patients, depletion of cross-reactive antibodies allows reactivity not to be masked, so depletion of cross-reactive antibodies prior to testing allows serum samples to detect this An early anti-mycobacterial antibody.

The Mtb antigens that induce antibody production in HIV/TB patients are less diverse than in non-HIV/TB patients: in these HIV/TB patients, antibodies to several antigens with a molecular weight of 32-45 kDa were absent. Antibodies to the strongly seroreactive 38 kDa antigen present in 50-60% of non-HIV/TB TB patients are absent in most HIV/TB patients. (Example I; Danielet al., 1987, supra; Bothamley, 1992, supra; Espitia C. et al., 1989, supra; Verbon A. et al., 1993, Am Rev Respir Dis 148:378-384). More notably, the antigens recognized by the antibodies in the HIV/TB sera were those in fraction 14, which mainly comprised the 88 kDa reactive antigen. Antibodies specific for the 88 kDa antigen were detected in the pre-TB sera of 74% of HIV ⁺ individuals who were about to develop clinical TB.

Example I shows that the 88kDa antigen (GlcB) (present in isolated fraction 15 in Example I and isolated fraction 14 in Example II) is a Mtb secreted antigen that induces antibody production early in disease progression (in non- - HIV TB patients). Therefore, the detection of anti-88kDa antibodies in high-risk HIV-infected individuals can be used as a diagnostic test, and antibodies can be used as a surrogate marker to identify active preclinical TB. Only about 1/3 of HIV/TB patients are PPD ⁺ (Fitzgerald JM et al., ^Chest 100:191-200), a T cell-mediated immune approach, at the time of clinical TB presentation. In contrast, 66% of HIV/TB patients had antibodies to the 88 kDa antigen (GlcB). The use of this new surrogate marker, along with other early antibodies, for the detection of individuals at high risk of developing or active TB would be a major contribution to slowing the global TB epidemic.

In the United States, only about 3% of TB patients are HIV-infected. However, HIV seroprevalence ranges from 17% to 66% in developing countries (Raviglione et al., 1992, supra; Shafe et al., supra). The proportion of HIV patients who are not immune to PPD is large, ranging from 33% in Zaire to more than 90% in Brazil, 43% in early HIV infection and 100% in advanced HIV patients (Raviglione et al. al., 1992, supra).

Delayed hypersensitivity of skin test reactivity is unstable in HIV ⁺ individuals. Because the development of PPD reactivity and the production of anti-mycobacterial antibodies are not necessarily synchronized (Das, S. et al., Clin. Exp. Immunol. 1992; 89: 402-06; Kardjito, T. et al., Tubercle. 1988, 63:269-274; Balestrino, EA et al., Bull.World Health Org.1984, 62:755-761), the simultaneous use of two markers will improve our early detection of such patients and timely establishment of treatment program capabilities.

The results of serodiagnostic tests performed by some investigators in HIV-infected persons are controversial. For example van Vooren et al., supra, report that all Mtb secreted antigens are present for several months in patients who subsequently develop TB. They also reported that 7/8 HIV/TB patients had circulating antibodies to the p32 antigen (Ag85A). In the present study, this antigen was present in 6-9 of the isolated fractions. Indeed, when only antibodies specific to the 38 kDa antigen and the Ag85B antigen were considered, the reactivity between HIV/TB patient sera and these isolated fractions could be due to the presence of this antigen (McDonough et al., supra). DaCosta et al., supra, found anti-LAM antibodies in about 35% of HIV/TB patients, as Barer et al. (supra) used PPD as antigen (Tuber Lung Dis 1992, 73:187-91). These reports were all similar in that antibodies to unisolated LFCFP were detectable in approximately 25% of HIV/stage-TB sera at the onset of clinical symptoms of TB. However, the sera of 66% of such patients reacted with the antigen of fraction 14 of the isolate. McDonough et al. (supra) failed to detect antibodies to Ag85B in the sera of HIV/TB patients, possibly due to the limitation of the number of antigens recognized by HIV/TB patients. The A-60 antigen used by some investigators (Saltini et al., supra; van derWerf et al., supra) has low sensitivity and poor specificity even in non-HIV/TB patients, which are Known populations with higher antibody levels (Charpin D et al., Am Rev Respir Dis 1990, 142: 380-384; Qadri, S. et al., Can J Microbiol 1991, 38: 804-806).

The reason why approximately 25-30% of HIV/TB patients exhibit a lack of antibodies to the 88 kDa antigen GlcB is unclear. In HIV/TB patients, there was no correlation between CD4 ⁺ cell counts and antibody levels. Similarly, there is a lack of correlation between CD4 ⁺ cell counts and delayed hypersensitivity responses (Huebner et al., 1994, supra), suggesting that not only quantitative but also functional differences in CD4 ⁺ cell subsets are critical to the immune response of HIV-infected individuals. Status matters.

The presence of circulating antibodies to Mtb-secreted antigens in HIV/TB patients prior to the development of clinical symptoms suggests that Mtb replicates in vivo before the complete loss of the immune system until clinical disease develops. Epidemiological studies have shown that HIV-infected persons develop rapidly from primary infection to clinical disease (Small, PM et al., NEngl JMed 1993, 328: 1137-1141; Daley, CLetal., NEng JMed 1992, 326: 231-235 ; Edlin BR et al., NEngl JMed 1992, 326:1514-1521; Coronado VG et al., Jlnfect Dis 1993, 328:1137-1155). It is therefore possible that only patients who reactivate latent TB and establish a secondary immune response have anti-Mtb antibodies. Recent studies (Alland Detal., NEngl JMed 1994, 330: 1710-1716; Small et al., supra) analyzing restriction fragment length polymorphisms (RFLPs) in Mtb strains indicated that about 60-70% of TB cases in New York (and San Francisco) due to reactivation of a latent infection.

Apparently, anti-mycobacterial antibodies in antibody-negative patients may circulate in the form of immune complexes that bind to antigens, thus masking the presence of antibodies in the assay. In at least a subset of patients with this phenomenon, it is recommended to increase the frequency of detection of antibodies in HIV/late-TB sera.

The present results suggest that patients with persistent circulating antibodies to the Mtb 88 kDa antigen, GlcB, can be treated prophylactically against TB, as are PPD ⁺ HIV-infected patients (Shafer, et al., supra; Pape, JW et al., Lancet 1993, 342: 268-272). The patients of the present invention are selected on the basis of clinically confirmed TB patients. Their PPD reactivity is unknown. In HIV-infected individuals, the time from a positive PPD skin allergy test to development of clinical disease ranges from 1 to 7 years (Selwyn et al., supra; Huebner et al., supra). There were no parameters to determine the optimal timing and duration of prophylactic anti-TB therapy. Further analysis of antibody responses in HIV/PPD ⁺ individuals who develop clinical TB disease will allow further exploration of the optimal timing of prophylactic treatment in these individuals.

Example III

Through 2-D polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis and electrospray mass spectrometry Defining Mtb culture filtrate proteins

As mentioned above, Mtb was cultured in vitro to obtain various proteins in the extracellular environment, which are referred to herein as culture filtrate proteins (CFPs). The most notable feature of this part of the protein is immunodominance. CFP is considered to be a major repository of antigens associated with protective immune responses and provides biochemical definition for this fraction of proteins. Recently, it was deduced that the double immune response produced by the inoculation of experimental animals with live virus heat-killed bacilli was due to the secretion of active antigens by live Mtb. The ability of Mtb CFP to induce protective T cell responses also supports this hypothesis. To define the immunologically active components in the isolated fractions, several proteins including 6kDa ESAT6, 24kDa MPT64, Ag85 complex and MPT32 were purified and characterized. Several CFPs elicited strong antibody responses, including MPT32, the 38 kDa PstS homologue, and the 88 kDa protein GlcB. The present inventors found that these antigens and other antigens described in the present invention can be used in the serodiagnosis of early TB.

Among the most extensively characterized Mtb CFPs prior to the present invention, Nagai and colleagues purified 12 major proteins, partially characterized them and provided 2-D PAGE profiles. Several other proteins, primarily defined by mAb reactivity, have been identified in culture filtrate preparations. The culture filtrate includes not only active secreted proteins but also bacterial molecules released into the medium during replication or autolysis. As described by Andersen et al. (supra), culture filtrate protein profiles are highly dependent on culture time. Furthermore, the medium used and the culture method (stationary or shaking) also affect the characteristics of the CFP. Therefore, due to the variable protocols for preparing CFP reagents, its protein composition is understandably difficult to obtain from the existing literature.

In this example, the inventors combined 2-D PAGE, Western blot analysis, N-terminal amino acid sequence analysis and liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) to obtain a detailed separation profile of the culture filtrate proteins , and the partial amino acid sequences of 5 proteins that have not been defined before have been obtained. These proteins are more abundant in the culture filtrate proteins that can be used as early antigens for serum diagnosis of TB.

In addition, the 2-DPAGE patterns of CFP of three Mtb laboratory strains H37Ra, H37Rv and Erdman were analyzed, and there were only minor differences among them. The results described below provide a detailed description of the protein signature and profile of this newly understood immunologically important proteome to which clinically isolated Mtb can be compared. Protein definitions of the major TB early antigens recognized by circulating antibodies in patients during the early stages of TB development are described in Examples V and VIII below.

A. Materials and methods

1. Growth of Mtb and Preparation of Culture Filtrate Protein

Mtb strains H37Rv (ATCC 27294) and H37Ra (ATCC 25177) were obtained from the American Type Culture Collection (Rockville, MD). Mtb strain Erdman (TMC 107) was from the Trudeau Mycobacterial Collection. First, each Mtb strain was inoculated from 1 ml of a frozen stock into 10 ml of glycerol alanine salt (GAS) medium; three identical cultures were prepared for each strain. After inoculation, culture at 37°C for 14 days, accompanied by slight shaking during the culture, after diluting each 10ml of culture solution with 10 times the volume of medium, then take 10ml of culture solution and dilute with 10 times the volume of medium, and do this more than twice . The resulting 1 liter of culture is called pass number four. For each Mtb strain, use 3 L of Dilution No. 4 to inoculate 30 L of GAS medium. After 14 days of culture at 37°C with gentle shaking, the culture supernatant was filtered to remove cells and the CFPs were concentrated as described above. Proteins in concentrated culture filtrates were analyzed by bicinchoninic acid protein quantification.

In order to determine the growth curves of Mtb strains H37Ra, H37Rv and Erdman, the fast-growing Mtb culture solution with an optical density of 0.1 at 600 nm was inoculated into a culture tube (13 by 100 mm) containing 3 ml of GAS medium with 0.05% Tween 80. These culture solutions were cultured at 37° C. under stirring, and the optical density at 600 nm was measured every 12 hours for a total of 22 days.

2. Antibodies

mAbs IT-69(HBT11) and IT-67(L24.b4) were provided by Dr. A.B. Andersen, Statens Seruminstitut, Copenhagen, Denmark. mAb A3h4 was provided by Drs.P.K.Das and A.Rambukana of the University of Amsterdam in the Netherlands, mAbs F126-2 and HYB 76-8 were provided by Dr.A.Kolk of the Royal Tropical Research Institute in Amsterdam, the Netherlands and Dr.I.Rosenkrands of Copenhagen, Denmark, Statens Seruminstitut provided separately. All other mAbs were provided by the WHO Monoclonal Antibody Bank and then deposited by Dr. T. Shinnick, CDC, Atlanta, Georgia. Anti-MPT63 polyclonal serum was provided by Dr.H.Wiker, University of Oslo, Norway. Dr. S. Nagai provided specific polyclonal sera for MPT32, MPT35, MPT46, MPT53 and MPT57.

3. SDS-PAGE and 2-D PAGE of culture filtrate protein

Standard SDS-PAGE analysis was performed under reducing conditions using a gel (7.5 x 10 cm x 0.75 mm) containing 6% stacking gel, 15% resolving gel. Each gel was separated at 10 mA for 15 minutes, followed by 15 mA for 1.5 hours.

The 2-D PAGE protein separations obtained by the O'Farrell method were slightly different from the SDS-PAGE separations. Specifically, 70 μg of CFP was dried and suspended in 30 μl of isoelectric focusing (IEF) sample buffer (9M urea, 2% NP-40, 5% β-mercaptoethanol and 5% ampholyte pH 3-10 ( Pharmacia Biotech, Piscataway, NJ)), and cultivated at 20°C for 3 hours. A portion of 25 μg of protein was separated with a 6% polyacrylamide IEF gel tube (1.5mm by 6.5cm) containing 5% Pharmalytes pH3-10 and 4-6.5 at a ratio of 1:4. 10mM H ₃ PO ₄ and 20 mM NaOH were used as catholyte and anolyte, respectively, at 1 kV, and focused for 3 hours. The gel in the tube was then pipetted into sample transfer buffer for 30 min before loading onto a preparative SDS-polyacrylamide gel (7.5 x 10 cm x 1.5 mm) containing 6% stacking gel , 15% dissolving gel. Each gel was subjected to two-dimensional electrophoresis at 20 mA for 0.3 hours, followed by separation at 30 mA for 1.8 hours. Stain with silver nitrate to visualize the protein.

4. Computer Aided Analysis of 2D Gels

Silver-stained 2-D PAGE gels were photographed with a cooled CCD digital camera and analyzed with MicroScan1000 2-D gel analysis software (Technology Resources, Inc., Nashville, TN). Use the spot filter to locate the position of the protein peak and analyze it. The allowable minimum peak height is 1.0, and the minimum peak area is 2.0.

5. Western blot analysis

Proteins analyzed by 2-D or SDS-PAGE were transferred to nitrocellulose membranes (Schleicherand Schuell, Keene, NH.) and washed with 0.05M Tris-HCl, pH 7.5, 0.15M NaCl, and 0.05% Tween80 (TBST) solution for blocking. These membranes were incubated for 2 hours with specific antibodies diluted to appropriate concentrations in TBST (Table 2). After washing, membranes were incubated for 1 hour with goat anti-mouse or anti-rabbit alkaline phosphatase-conjugated antibody (Sigma) diluted in TBST. The color was developed with the substrates nitrotetrazolium blue and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).

The profile of proteins in 2-D PAGE gels reacted with specific antibodies was stained with 0.1% India ink, which is a secondary dye for total protein after detection by immunoblotting. Alternatively, the Digoxigenin (DIG) Total Protein/Antigen Double Stain Kit (Boehringer Mannheim, Indianapolis, IN) can be used for antibody-reactive proteins that cannot be stained by secondary staining with India ink. Briefly, after electroblotting, membranes were washed three times with 0.05M _K2HPO4 , pH _8.5 . The membrane was mixed with 0.05M K ₂ HPO ₄ solution (pH 8.5 ) contact and incubate the membrane for 1 hour at room temperature to allow all proteins to bind to digoxigenin. The membrane was then blocked with 3% bovine serum albumin in 0.05M Tris-HCl, pH 7.5, 0.15M NaCl (TBS) for 1 hour and then washed with TBS. Incubation with specific antibodies was performed as previously described, and the membrane was then incubated for 1 hour with mouse-anti-DIG-Fab fragments conjugated to alkaline phosphatase diluted 1:2000 in TBS. Wash three times with TBS and detect with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibody. Proteins reactive with specific anti _- Mtb protein antibodies were developed with the substrate 4-(1,4,7,10-tetraoxodecyl)-1-naphthol and 1.8% _H2O2 . BCIP and [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-tetrazolium chloride] as substrates for secondary color development of all proteins labeled with digoxigenin .

6. Amino Acid Sequence Analysis

In order to obtain the N-terminal amino acid sequence of the selected protein, CFPs (200 μg) were separated by 2-D PAGE, and then transferred to a polyvinylidene difluoride membrane (Millipore, Milford, Mass.) % methanol in CAPS buffer for 1 hr. Stain with 0.1% Coomassie brilliant blue in 10% acetic acid and destain with 50% methanol and 10% acetic acid solution. Immobilized proteins were subjected to automated Edman degradation using a gas-phase sequencer with a continuous flow reactor. Phenylthiohydantoin amino acid derivatives were identified by on-line reverse phase chromatography as described previously.

7. LC-MS-MS Analysis

The sequence of the peptide fragments of the screened CFP was determined by LC-MS-MS. CFPs (200 mg) were separated by 2-D PAGE, the gel was stained with 0.1% Coomassie Brilliant Blue, and proteins immobilized on PVDF membranes were destained as described. The desired protein was excised from the gel, washed several times with distilled water to remove residual acetic acid, and then digested with insulin for in-gel protein dissolution. Peptides were eluted from acrylamide and separated by C18 capillary RP-HPLC. The RP-HPLC eluate from the microcapillary was directly introduced into a Finnigan-MAT (San Jose, CA) TSQ-700 three-part quadrupole mass spectrometer. Mass spectrometry and data analysis were as described by Blyn et al.

C. Results

1. Protein definition in the culture filtrate of Mtb H37Rv

Through the efforts of the World Health Organization (WHO) Scientific Working Groups (SWGs) on Immunology of Leprosy (IMMLEP) and Immunology of Combined Diseases (IMMTUB), a large library of mAbs against mycobacterial proteins has been established. This library, as well as mAbs and polyclonal sera not included therein, allowed the identification of known mycobacterial proteins in culture filtrates of Mtb. A detailed study of the literature was performed on mAbs and/or polyclonal sera that had been identified to react with 35 MtbCFPs (Table 2). First, in preparation for the study, western blot analysis was used to determine the presence of these proteins in culture filtrates of H37Rv. The antibodies and sera used in the experiments, except IT-56, all showed reactivity with specific proteins in the preparation. mAb IT-56 is specific for the 65kDa Mtb GroEL homologue; protein is predominantly cytosolic-associated. Furthermore, mAb IT-7 reacts with 14kDa but not 40kDa CFP.

2. 2-D PAGE patterns of known CFPs in Mtb H37Rv

Using 2-D Western blot analysis coupled with secondary staining (India ink or Dig total protein/antigen double staining), proteins reactive with specific mAbs or polyclonal sera were separated on 2-D PAGE of CFP of Mtb H37Rv within the graph display. A total of 32 specific proteins detected by reactive antibodies were separated by 2-D PAGE (Table 2). However, the two antibodies (IT-1 and IT-46), which were reactive in traditional Western blot analysis, failed to detect any protein in the 2-D separation range (Table 2), which may be due to the lack of exposed For linear epitopes, exposure of the epitope usually requires the denaturing conditions used to break down the molecule to occur.

Most antibodies recognize a single protein spot. However, there are also several antibodies (IT-3, IT-4, IT-7, IT-20, IT-23, IT-41, IT-42, IT-44, IT-49, IT-57, IT-58 , IT-61 and MPT32) react with multiple proteins. Five of them, IT-23, IT-42, IT-44, IT-57 and IT-58, reacted with clusters of proteins whose molecular weights were centered at 36 kDa, 85 kDa, 31 kDa, 85 kDa and 50 kDa, respectively. The proteins in each cluster migrated over a narrow PI range, suggesting that the antibody reacts with multiple isoforms of the corresponding protein. The 85 kDa protein cluster (which includes the "88 kDa" GlcB early antigen) is recognized by IT-57 and the most dominant component is recognized by IT-42.

Polyclonal sera against MPT32 recognized 45 and 42 kDa proteins with similar PI values. In determining the position of glycosylation on MPT32 (see above), we observed that this protein is prone to autohydrolysis to form a 42 kDa product. Therefore, the 42 kDa protein detected with anti-MPT32 serum is a breakdown product of the 45 kDa MPT32 glycoprotein. mAb (antibody T-49, specific for the antigen 85 (Ag85) complex), clearly detected the three gene products of the complex (Ag85A, B and C). The region of greatest cross-reactivity with the antibody was located at molecular weights below 16 kDa. The most dominant protein in this region was reacted with mAb IT-3 specific for the 14kDa GroES homologue. This mAb also recognized several neighboring proteins around 14kDa. Interestingly, different proteins of the same protein cluster were associated with anti-MPT57 and Anti-MPT46 polyclonal sera reacted with mAbs IT-4, IT-7, IT-20.

3. N-terminal amino acid sequences of the screened CFPs

The N-terminal amino acid sequences or complete gene sequences and functional groups of CFPs of several Mtbs were obtained by mapping with available antibodies. However, this method lacks those proteins that react with IT-42, IT-43, IT-44, IT-45, IT-51, IT-52, IT-53, IT-57, IT-59, IT-69 , as well as information on several dominant proteins that could not be identified by these methods. The most abundant of these proteins (IT-52, IT-57, IT-42, IT-58 and proteins labeled A-K) were subjected to N-terminal amino acid sequencing (Table 3).

Three of these proteins were found to correspond to previously defined products. The N-terminal amino acid sequence of the protein labeled D is identical to Ag85B and C. This result was unexpected since IT-49 did not detect this protein, and N-terminal amino acid analysis confirmed that those proteins that reacted with IT-49 were members of the Ag85 complex. Second, the N-terminal amino acid sequence of the protein labeled E is identical to that of glutamate synthase. The third protein that reacts with IT-52 is the same as MPT51.

However, five of these proteins analyzed were novel proteins. Three of them, the proteins labeled B, C and IT-58, had no apparent homology to any known sequence in mycobacteria or prokaryotes. The protein labeled I has an N-terminal sequence that is 72% identical to the amino-terminus of α-hydroxysteroid dehydrogenase from Eubacteria, and the protein labeled F has an amino acid sequence induced by the open reading frame recognized by the Mtb plasmid MTCY1A11. homology.

Examples I and II demonstrate that the high molecular weight fraction in the CFP of Mtb has an advantage in reactivity with TB patient sera, since this fraction has a reaction product with mAb IT-57, thereby distinguishing it from other natural fractions. In view of this, protein clusters defined by IT-42 and IT-57 (including the 88 kDa protein GlcB) were excised from 2-D polyacrylamide gels, digested with insulin, and the resulting peptides were analyzed by LC-MS-MS analyze. The molecular weights and fragmentation patterns of the 10 peptides obtained by digestion were consistent with those predicted for MtbkatG-encoded catalase/peroxidase insulin fragments (Table 3). It can be seen that the protein that does not react with IT-57 appears to be the katG product. However, the IT57-reactive protein in the 88 kDa protein cluster (LC-MS-MS analysis) did not share sequence homology with the identified Mtb protein.

Table 2: CFPs of Mycobacterium tuberculosis H ₃₇ Rv and reported

Reactivity of specific mAbs and polyclonal antisera Antibody ¹ MW (kDa) Dilution ratio reactivity 1-D 2-D IT-1(F23-49-7)IT-3(SA-12)IT-4(F24-2-3)IT-7(F29-29-7)IT-10(F29-47-3)IT- 12(HYT6)IT-17(D2D)IT-20(WTB68-A1)IT-23(WTB71-H3)IT-40(HAT1)IT-41(HAT3)IT-42(HBT1)IT-43(HBT3) IT-44(HBT7)IT-45(HBT8)IT-46(HBT10)IT-49(HYT27)IT-51(HBT2)IT-52(HBT4)IT-53(HBT5)IT-56(CBA1)IT- 57(CBA4)IT-58(CBA5)IT-59(F67-1)IT-61(F116-5)IT-67(L24.b4)IT-69(HBT11)F126-2A3h4HYB76-8anti-MPT32anti-MPT46anti- MPT53anti-MPT57anti-MPT63-K64 16kDa12kDa16kDa40kDa21kDa17-19kDa23kDa14kDa38kDa71kDa71kDa82kDa56kDa32kDa96kDa40kDa32-33kDa17kDa25kDa96kDa65kDa82kDa47kDa33kDa30(24)kDa24kDa20kDa30kDa27kDa6kDa50kDa10kDa15kDa12kDa18kDa 1:20001:80001:20001:10001:10001:501:80001:2501:2501:501:501:501:501:501:501:501:501:501:501:501:501:501:501:1001: 1001:501:61:1001:501:1001:1001:1001:1001:1001:200 ++++++++++++++++++++-++++++++++++++ -++++++++++++++-++++ND ^* ++++++++++++++

^* ND: not detected

¹ In parentheses are the nomenclature originally used to classify Mab by the World Health Organization

Table 3: Selected CFPs of M. tuberculosis H ₃₇ Rv identified by LC-MS-MS

N-terminal amino acid sequence or peptide fragment protein N-terminal amino acid sequence identification number homology A None ¹ B APPSCAGLD/GCTV 56 C XXAVXVT 57 D. FSRPGLPVEYLQVPSP 58 Mtb antigen 85A and C E. TEKTPDDVFKLADDEKVEYVD 59 Mtb glutamate synthase f XPVM/LVXPGXEXXQDN 60 Mtb cosmid MTCY1A11 G None ¹ h None ¹ I XVYDVIMLTAGP 61 Eubacterial sp.VPI 12708α-Hydroxysterol dehydrogenase J None ¹ K None ¹ IT-43 None ¹ IT-52 APYENLMVP 62 Mtb MPT 51 IT-58 K/NVIRIXGXTD 63 F126-2 None ¹ Drawn endogenous peptide homology IT-42 FAPLNSWPDANASLDK (129-143) 64 Mt. Catalase/Peroxidase EATWLGDER(201-209) 65 DAITSGIEVVWTNTPTK(311-327) 66 SPAGAWQYTAK(346-356) 67 DGAGAGTIPDPFGGPGR(357-373) 68 RWLEHPEELADEFAK(396-410) 69 TLEEIQESFNSAAPGNIK(519-536) 70 AGHNITVPFTPGR (556-569) 71 TDASQEQTDVESFAVLEPK (569-588) 72 GNPLPAEYMLLDK (599-611) 73 ANLLTLSAPEMTVLVGGLR(612-630) 74 VDLVFGSNSELR (692-703) 75 ALVEVYGADDAQPKF (704-718) 76

¹ "None" indicates that the protein is difficult to sequence by Edman degradation

Table 4: Protein spots detected by computer-aided analysis of silver-stained 2D gels Ref#. wxya H37Ra Erdman MW (kDa) pI antibody reactivity Functional Base/Identifier N-terminal sequence ¹ serial identification number 1 1 1 1 22.39 ≥3 2 2 2 2 17.18 ≥3 3 3 3 3 13.72 ≥3 4 4 4 4 11.75 ≥3 5 5 5 5 23.99 3.09 6 6 6 6 16.98 3.45 7 7 7 7 11.75 3.52 HYB76-8 ESAT6 TEQQWDFAGI 77 8 8 8 N M 27.23 3.63 9 9 N M N M 20.30 3.82 10 10 10 10 21.63 4.14 IT-69 11 11 11 11 38.90 4.31 anti-MPT32 MPT32 DPAPAPPVPT 78 12 12 12 12 20.07 4.31 IT-51 13 13 13 13 13.49 4.46 14 14 14 14 42.17 4.51 anti-MPT32 MPT32 DPAPAPPVPT 78 15 15 15 15 31.44 4.53 16 16 16 16 32.36 4.55 17 17 17 17 11.61 4.55 18 18 18 18 35.48 4.62 19 19 19 19 25.85 4.65 20 20 20 20 21.38 4.68 twenty one twenty one twenty one twenty one 19.72 4.69 twenty two twenty two twenty two twenty two 31.44 4.75 IT-44 twenty three twenty three twenty three twenty three 13.57 4.76 twenty four twenty four twenty four twenty four 48.70 4.79 25 25 25 25 32.55 4.79 IT-44 26 26 26 26 15.67 4.79 anti-MPT53 MPT53 DECIQ 79 27 27 27 27 22.26 4.81 28 28 28 28 28.35 4.83 Ref#. wxya H37Ra Erdman MW (kDa) pI antibody reactivity Functional Base/Identifier N-terminal sequence ¹ serial identification number 29 29 29 29 26.15 4.83 IT-67 MPT64 RIKIF 80 30 30 30 30 23.58 4.84 31 31 31 31 16.88 4.84 anti-MPT63 MPT63 AYPITGKLGSELT 81 32 32 32 32 38.02 4.87 33 33 33 33 29.85 4.87 34 34 34 34 19.05 4.88 35 35 35 35 22.26 4.92 36 36 36 36 35.08 4.93 37 37 37 37 31.44 4.93 IT44/F126-2 38 38 38 38 14.45 4.93 anti-MPT57/IT-3 GroES homologue MPT57 MAKVNIKPLE 82 39 39 39 N M 20.87 4.99 40 40 40 40 28.67 5.00 41 41 41 41 18.62 5.00 42 42 42 42 19.50 5.00 43 43 43 43 40.74 5.02 44 44 44 44 29.68 5.02 45 45 45 45 14.96 5.02 IT-3/4/7/20 46 46 46 46 35.48 5.03 IT-23 PstS CGSKPPSPET 83 47 47 47 47 32.36 5.04 48 48 48 48 28.35 5.04 49 49 49 49 26.00 5.04 50 50 50 50 17.78 5.04 51 51 51 51 46.51 5.05 52 52 52 52 35.89 5.06 IT-23 PstS CGSKPPSPET 84 53 53 53 53 60.60 5.06 54 54 54 54 22.78 5.06 55 55 55 55 47.32 5.07 56 56 56 56 20.18 5.07 A 57 57 57 57 31.62 5.08 Ref#. wxya H37Ra Erdman MW (kDa) pI antibody reactivity Functional Base/Identifier N-terminal sequence ¹ serial identification number 5859 5859 5859 5859 18.6229.68 5.085.08 60 60 60 60 14.54 5.09 anti-MPT46IT-3/4/7/20 MPT46 RDSEK 85 61 61 61 61 47.86 5.09 62 62 62 N M 31.26 5.09 63 63 63 63 25.56 5.09 64 64 64 64 13.11 5.09 65 65 65 65 72.86 5.09 IT-40/IT-41 DnaK homologue MARAVGIDLG 86 66 66 66 66 35.69 5.09 IT-23 PstS CGSKPPSPET 87 67 67 67 67 28.84 5.09 68 68 68 68 42.41 5.10 69 69 69 69 30.20 5.10 70 70 70 70 57.54 5.10 71 71 71 N M 31.62 5.10 72 72 72 72 47.86 5.10 73 73 73 73 38.46 5.10 74 74 74 74 25.56 5.10 B APPSCAGLD/GCTV 88 75 75 75 75 22.00 5.10 76 76 76 76 19.61 5.10 IT-12 19kDa lipoprotein CSSNKSTTG 89 77 77 77 77 28.18 5.10 78 78 78 78 79.43 5.10 79 79 79 79 66.83 5.10 IT-41 DnaK homologue MARAVGIDLG 90 80 80 80 80 42.17 5.10 C XXAVXVT 91 81 81 81 81 29.85 5.10 IT-49/IT-61 Antigen 85 B/MPT59 FSRPGLPVEY 92 82 82 82 82 49.55 5.10 IT-58 K/NVIRIXGXTD 93 83 83 83 83 32.17 5.10 84 84 84 84 38.46 5.11 Ref#. wxya H37Ra Erdman MW (kDa) pI antibody reactivity Functional Base/Identifier N-terminal sequence ¹ serial identification number 85 85 85 85 34.47 5.11 86 86 86 86 58.88 5.11 8788 8788 8788 8788 20.8923.04 5.115.11 IT-10 89 89 89 89 24.27 5.11 90 90 90 90 42.17 5.11 91 91 91 91 29.17 5.11 92 92 92 92 69.98 5.11 IT-41 DnaK homologue MARAVGIDLGT 94 93 93 93 93 26.15 5.11 A3h4 94 94 94 94 25.12 5.11 95 95 95 95 27.86 5.11 96 96 96 96 56.23 5.11 97 97 97 97 15.22 5.11 IT-3/7 98 98 98 98 29.17 5.11 99 99 99 99 106.05 5.12 100 100 100 100 93.33 5.12 101 101 101 101 82.22 5.12 102 102 102 102 32.73 5.12 IT-59 103 103 103 103 31.08 5.12 D: Antigen 85 homologue FSRPGLPVEYLQVPSP 95 104 104 104 104 38.90 5.12 105 105 105 105 58.88 5.12 106 106 106 106 44.41 5.12 107 107 107 107 34.67 5.12 108 108 108 N M 26.61 5.12 109 109 109 109 20.54 5.12 110 110 110 110 38.90 5.13 111 111 111 111 104.71 5.13 112 112 112 112 66.83 5.13 113 113 113 113 85.11 5.14 Ref#. wxya H37Ra Erdman MW (kDa) pI antibody reactivity Functional Base/Identifier N-terminal sequence ¹ serial identification number 114 114 114 114 55.59 5.14 E: glutamate synthase TEKTPDDVFKLAKDEKVEYVD 96 115 115 115 115 42.41 5.14 116117 116117 116117 116117 26.4542.17 5.155.17 118 118 118 118 34.28 5.17 119 119 119 119 31.08 5.17 IT-49 Antigen 85C/MPT45 FSRPGLPVEY 97 120 120 120 120 55.59 5.17 E: glutamate synthase TEKTPDDVFKLDEVE/T 98 121 121 121 N M 25.70 5.17 122 122 122 122 45.71 5.18 123 123 N M N M 20.65 5.18 124 124 124 124 85.11 5.19 IT-42/IT-57 Catalase/Peroxidase MPEQHPPITE 99 125 125 125 125 16.03 5.19 126 126 126 126 39.81 5.20 127 127 127 127 36.94 5.21 128 128 128 128 46.24 5.22 129 129 129 129 27.23 5.22 130 130 130 130 51.29 5.22 131 131 131 131 19.61 5.22 132 132 132 132 42.41 5.24 133 133 133 133 38.02 5.24 134 134 134 134 20.89 5.24 135 135 135 135 46.24 5.26 136 136 136 136 35.48 5.26 137 137 137 137 30.73 5.26 138 138 138 N M 13.49 5.27 139 139 139 139 31.62 5.28 140 140 140 140 29.17 5.30 Ref#. wxya H37Ra Erdman MW (kDa) pI antibody reactivity Functional Base/Identifier N-terminal sequence ¹ serial identification number 141 141 141 141 38.46 5.33 142 142 142 142 42.41 5.34 143 143 143 143 33.50 5.34 144 144 144 144 24.97 5.34 f XPVM/LVXPGXEXXQDN 100 145146 145146 145146 145146 22.6550.12 5.345.35 147 147 147 147 26.92 5.37 G 148 148 148 148 15.67 5.37 149 149 149 149 31.44 5.38 IT-49 Antigen 85A/MPT44 FSRPGLPVEY 101 150 150 150 150 69.18 5.39 151 151 N M 151 94.41 5.40 IT-45 152 152 152 152 35.89 5.45 153 153 153 153 21.13 5.47 154 154 154 154 20.07 5.47 h 155 155 155 155 58.88 5.50 IT-43 156 156 156 156 48.70 5.53 157 157 157 157 82.22 5.61 158 158 158 158 53.70 5.61 159 159 159 159 34.67 5.68 160 160 160 160 57.54 5.70 161 161 161 161 79.43 5.74 162 162 162 162 31.99 5.76 163 163 163 163 29.17 5.80 164 164 164 164 27.86 5.80 165 165 165 165 52.48 5.86 166 166 166 166 45.71 5.86 167 167 167 167 33.11 5.86 168 168 168 168 58.88 5.88 Ref#. wxya H37Ra Erdman MW (kDa) pI antibody reactivity Functional Base/Identifier N-terminal sequence ¹ serial identification number 169 169 169 169 25.85 5.88 170 170 170 170 26.92 5.91 IT-52 MPT51 APYENLMVPPS 102 171 171 171 171 22.13 5.93 172 172 172 172 34.67 5.98 173 173 173 173 31.81 5.98 174 174 174 174 56.23 6.02 175176 175176 175176 175176 98.8652.48 6.086.18 IT-53 I XVYDVIMLTAGP 103 177 177 177 177 42.17 6.18 178 178 178 178 26.61 6.33 179 179 179 179 45.19 6.36 180 180 180 180 30.90 6.39 181 181 181 181 34.47 6.42 J 182 182 182 182 24.83 6.42 183 183 183 183 18.20 6.49 184 184 184 184 38.02 6.55 185 185 185 185 41.93 6.73 186 186 186 186 25.41 6.88 187 187 187 187 133.35 7.00 188 188 188 188 30.20 7.17 189 189 189 189 33.50 7.30 190 190 190 190 24.97 7.39 191 191 191 N M 27.38 7.58 192 192 192 192 40.74 8.39 K 193 193 193 193 20.54 9.64 194 194 194 194 41.93 10.33 195 195 195 195 24.97 10.41 196 196 196 196 32.73 10.74 197 197 197 N M 27.23 ≤10 198 198 198 198 50.12 ≤10 Ref#. wxya H37Ra Erdman MW (kDa) pI antibody reactivity Functional Base/Identifier N-terminal sequence ¹ serial identification number 199 199 199 N M 38.90 ≤10 200 200 200 200 29.68 ≤10 201 201 201 201 24.83 ≤10 IT-17/IT-61 superoxide dismutase/MPT58 MAEYTLPDLD 104 202 202 202 N M 60.60 ≤10 203 203 N M N M 42.17 ≤10 204 204 204 N M 48.70 ≤10 205206 205NM 205206 205NM 38.9020.87 ≤104.83 207 N M 207 N M 20.40 4.79 208 N M 208 N M 15.67 5.02 209 N M 209 N M 22.61 5.11 210 N M 210 210 19.05 5.11 211 N M N M 211 38.95 4.93 212 N M N M 212 59.10 5.04 213 N M N M 213 57.54 5.10 214 N M N M 214 25.85 5.22 215 N M N M 215 26.15 5.24 216 N M N M 216 46.24 5.35 217 N M N M 217 48.70 5.40 218 N M N M 218 53.70 5.43 219 N M N M 219 59.10 6.42 220 N M N M 220 15.80 6.90 221 N M N M 221 32.36 9.00 222 N M N M 222 94.35 9.30

¹ The N-terminal sequence determined by the present inventors is in italics. Ref# = spot number on blotted 2D gel 4. Comparison of CFP features of Mtb strains H37Rv, H37Ra and Erdman

Comparative 2-DPAGE analysis of CFPs from Mtb canonical strains (H37Rv, H37Ra and Erdman) was performed to identify possible differences in the content of each protein component. First, 3 independent H37Rv CFPs were pooled and resolved in 2-D PAGE. Silver-stained gels were digitized and data analyzed with Microscan 1000 2-D gel analysis software. A total of 205 H37Rv protein spots were detected, and each protein was numbered sequentially according to PI from acidic to basic and in descending order of molecular weight (Table 4). The CFP-like maps of H37Ra and Erdman strains were obtained by a similar method, and 206 and 203 protein spots were identified respectively (Table 4). Comparing the three maps with 2D main software revealed striking similarities in the composition of the three culture filtrates. The H37Ra and Erdman culture filtrate protein spots that coincided with the H37Rv culture filtrate protein spots were given the same numbering, giving the proteins of the H37Ra or Erdman strains their initial numbering (Table 4). Proteins present in only one or two typical strains were present in lesser amounts in culture filtrates.

c. to discuss

In contrast to the proteins of the cell wall, membrane, and cytoplasm of Mtb, CFPs are well defined in terms of function, immunogenicity, and composition ^*** . However, the total protein has not been analyzed in detail nor given a molecular definition, nor have 2-D PAGE profiles of most CFPs been obtained. Nagai and co-workers identified and displayed profiles by 2-DPAGE of abundant protein filtrates collected after 5 weeks of culture in Sauton's medium. The culture filtrates used in this study come from the medium to late logarithmic growth medium of Mtb typical strains H37Ra, H37Rv, and Erdman, and these proteins are extensively studied and analyzed in detail for the first time.

The 2-D gel-dissolved protein spots in the filtrate CFP of H37Rv, H37Ra and Erdman strains were analyzed by computer, which were 205, 203 and 206 spots, respectively. Of all spots, 37 were identified using mAbs against CFPs and polyclonal sera. Several of these antibodies recognized more than one spot; several were thought to react with multiple isoforms of the same protein or had previously been shown to recognize more than one gene product. The available antibodies were used to obtain maps of 17 proteins for which some or all amino acid sequences have been reported (Table 4).

To define more molecules, N-terminal sequence analysis was performed on some of the abundant products observed on 2-D PAGE.

The protein migrating between Ag85B and Ag85C was analyzed to have 16 residues (FSRPGLPVEYLQVPSP, [SEQ ID NO: 95]) identical to the N-termini of mature Ag85A and Ag85B, and one residue (position 15) of Ag85C different. This protein spot is apparently only a homologue of Ag85A or B. However, its complex lacks reactivity with Ag85-specific mAb (IT-49), its molecular weight is larger than that of Ag85B, and the PI shift is related to that of Ag85A, presumably the product may be changed due to subsequent translation. Alternatively, the protein may be an unrecognized fourth member of the Ag85 complex. However, none of the members of the Ag85 complex appears to be altered in translation in some reports, while others report several bands similar to Ag85C after isoelectric focusing. However, there is no direct evidence to support the existence of a fourth Ag85 product.

After sequencing, the second product was a 25kDa protein with a PI of 5.34. Its N-terminal sequence (XPVM/LVXPGXEXXQDN, [SEQ ID NO: 100]) shows homology with the fragment (DPVLVFPGMEIRQDN, [SEQ ID NO: 105]) in the open reading frame 28c of the Mtb plasmid (cosmid) MTCY1A11 . Analysis of the deduced sequences revealed a signal peptidase I consensus sequence (Ala-Xaa-Ala) and a distinct signal peptide preceding the N-terminus of the 25 kDa protein sequenced above.

N-terminal sequencing of selected CFPs identified three new products:

(1) a protein with 72% identity to the N-terminus of the 42 kDa α-hydroxysterol dehydrogenase of the eubacterium sp.VPI 12708;

(2) the 27 kDa protein previously defined as MPT-51; and

(3)) The 56 kDa protein previously defined as glutamate synthase.

The N-termini of these three proteins did not show significant homology to any known peptides. For these proteins, or other proteins where N-group analysis is difficult, more advanced methods of sequencing the protein (eg, LC-MS-MS) can yield more sequence information.

The protein clusters recognized by mAbs IT-42 and IT-57 were the main content of this study. Proteins with a molecular weight range of 82-85 kDa in one co-inventor's laboratory (or 88 kDa in another inventor's laboratory) had a pi in the range of 5.12-5.19. The results described in Examples I, II and V mean that CFP of about 88 kDa reacts with 70% of TB patient sera and exhibits 100% specificity. Subsequent 2-D profiling and 2-D Western blot analysis revealed a dominant antigen that induces early antibody responses in TB patients, which is the same protein that IT-57 and IT-42 react with. As mentioned above, this antigen refers to the 88 kDa protein GlcB.

Although N-terminal sequencing of the initial protein cluster failed, LC-MS-MS studies indicated the presence of a product, katG catalase/peroxidase, in the protein cluster.

A detailed map of the culture filtrate of H37Rv was obtained by computer-aided analysis, allowing calibration and comparison of CFPs in other strains of Mtb with different properties. However, all detected differences were associated with the proteins observed in small amounts. One explanation for these differences is that the growth characteristics of the three strains differ significantly. Several studies have shown that Mtb culture time has a significant effect on the protein profile released by mycobacteria into the culture supernatant. In particular, studies by Andersen et al. (supra) showed that a small number of well-defined proteins are secreted during the first three days of the culture period, whereas progressively secreted cell wall proteins emerge during logarithmic growth. For isocitrate dehydrogenase and the 65 kDa GroEL homolog detected cytoplasmic protein release was not observed until late log phase.

This extensive study of pathogenic Mtb strains has identified and continues to identify immunologically important proteins and will lead to the discovery of new causative factors leading to improved chemotherapy. Therefore, the present invention not only facilitates a comprehensive understanding of the physiology of Mtb, but also provides a rapid method for early serodiagnosis.

Example IV

Further Characterization of the 88kDa Antigen by Recombinant Approaches

A. Determining the identity of the 88kDa antigen reactive with mAb IT-57

2-D Western blot analysis and 2-D map of Mtb culture filtrate (see: US 6,245,331, 12 June 2001, and WO 98/29132, published 09 July 1998) showed that the 88kDa antigen with serum predominance may be associated with mAbs IT-42 and IT57 (#101, 113, 124) recognized the same protein. To confirm the identity of these antigens, mass spectrometry was performed on peptides obtained from protein clusters reactive with IT-57 and IT-42. The results showed that the protein #124 reactive with both mAbs was KatG catalase/peroxidase. Analysis of peptides from spots #101 and 113 that reacted with only one mAb protein was inconclusive. To obtain proteins reactive with mAb IT-57, about 20,000 phages with λgt11 Mtb expression library were screened by plaque blot with mAb IT-57. The λgt11 clone reactive with mAb IT-57 and encoding a protein with a molecular weight of 88 kDa was designated "λgt11(IT-57)". The λgt11(IT57)-soluble E.coli lysate and LFCFP were separated by SDS-PAGE polyacrylamide containing 10% gel, then transferred to nitrocellulose filters and detected with mAb IT-57. mAb IT-57 recognizes the 88 kDa band in LFCFP and the 88 kDa band in E. coli lysates with λgt11 (IT-57) as the lysogen. There was no mAb-reactive protein in the lysate of E. coli 1089 lysed with wild-type λgt11 as lysogen.

B. Hybridization of clone encoding 88kDa antigen with katG gene

Since spots reactive with mAb IT-57 and mAb IT-42 overlapped each other on the 2-D blot, it was determined whether the 88kDa protein encoded by the λgt11(IT-57) clone was the product of the katG gene, or whether it was a Different proteins with similar molecular weights and PI values are important. Dr. Sheldon Morris provided the MtbkatG gene encoding hydrogen peroxide/peroxidase and cloned into the mycobacterial shuttle vector pMD31. The katG gene was excised from pMD31 with enzymes KpnI and XbaI to obtain a 2.9 kb insert. The about 3.2 kb insert fragment obtained after the DNA in lambda gt11 (IT-57) was digested with EcoRI was hybridized with the katG gene. The 3.2 kb insert from lambda gt11 (IT-57) hybridized to itself and to the unspliced pMD31 vector containing the katG gene and the katG insert DNA itself (2.9 kb). Therefore, the 88 kDa antigen reactive with mAb IT-57 is indeed hydrogen peroxide/peroxidase.

C. Sequence of recombinant 88kDa antigen expressed in E.coli

In order to confirm that the 88kDa protein obtained from λgt11 (IT-57) is indeed hydrogen peroxide/peroxidase, the inserted DNA in the clone was sequenced and found to be consistent with the Mtb katG sequence (accession number X68081) studied by NCBI BLAST There is 99% homology.

D. Reactivity of TB serum with recombinant hydrogen peroxide/peroxidase expressed in E.coli

To determine the reactivity of 88 kDa hydrogen peroxide/peroxidase with TB patient sera, cell lysates of isolated E. coli-λgt11 (IT-57) were tested against those from 6 advanced TB patients and 4 PPD ⁺ healthy The reactivity of the patient's serum. Neither healthy control nor TB sera reacted with the 88 kDa hydrogen peroxide/peroxidase protein. Thus, the 88 kDa hydrogen peroxide/peroxidase protein was not the serum reactive antigen that was later identified as GlcB.

Reactivity of E. tuberculosis serum with the 88 kDa hydrogen peroxide/peroxidase protein expressed in M. bovis BCG

Since TB patients do not react with recombinant hydrogen peroxide/peroxidase expressed in E. coli, the pMD31:MtBkAtG or control pMD31 plasmid (vector control) was transfected into the katG-negative BCG strain 35747 for testing. The crude lysates of LFCFPs, lysed from λgt11 (IT-57), lysed from E. coli 1089 infected with wild-type λgt11, katG-negative BCG strain containing pMD31:MtbkatG, and katG-negative BCG strain containing pMD31 were passed through 10 % SDS-PAGE polyacrylamide separation on the gel. The separated proteins were transferred to nitrocellulose filters and treated with anti-hydrogen peroxide/peroxidase polyclonal serum (from Dr. Clifton Barry, Rocky Mountain Laboratories, NIAID, Hamilton, MT), mAb IT-57, mAb Sera from patients with IT-42 and advanced TB were tested. Anti-hydrogen peroxide/peroxidase polyclonal serum and mAbIT-57 reacted significantly with the 88kDa antigen in LFCFP, Mtb katG containing M.bovis BCG and E.coliλgt11 (IT-57). MAb IT-42 reacted with the same band in LFCFP and Mtb katG BCG, but not with the 88 kDa protein expressed in E. coli. Control bands containing lysates of E. coli1089 (λgt11) or katG-negative M. bovis BCG (pMD31 only) did not react with either mAbs.

In contrast to the results obtained with anti-hydrogen peroxide/peroxidase antibody responses, TB patient sera recognized the 88 kDa antigen in lysates of katG-negative BCG strains. This proves that the serum reactive 88kDa antigen is a new protein that has not been reported before.

F. Reactivity of TB serum with Mtb 88kDa antigen GlcB

To confirm that the 88 kDa antigen present in Mtb is distinct from hydrogen peroxide/peroxidase, a negative strain of katG-Mtb (ATCC 35822) was tested. The lysate in this strain did not react with any anti-hydrogen peroxide/peroxidase antibodies. However, when healthy sera controls and TB patients from all three groups were tested with the same lysate, all sera from Group III and Group IV reacted with the 88 kDa protein.

Amino Acid Sequence Identification of 88kDa Protein GlcB Reactive with Serum

Culture filtrate proteins of the katG-negative strain of Mtb (ATCC 35822) were resolved by 2-D PAGE as described above. A protein spot ("spot 1") corresponding to a serum-reactive 88 kDa protein was isolated from the gel, and the gel was digested with insulin. The resulting tryptic peptides were extracted and separated on a C18 RP-HPLC column, eluting with increasing concentrations of acetonitrile. Peptides eluted by this method were directly introduced into a FinniganLCQ electrospray mass spectrometer. (see Materials and methods section for details). When in the charged state, the molecular weight of each peptide was determined using a high resolution scanning procedure.

The above mass spectrometry data were entered into the computer program of MS-Fit and searched in the Mtb database, thereby identifying the 88kDa protein GlcB. The data entered into the MS-Fit analysis system and the results obtained are shown below.

The search data entered that matches the MS

Database NCBInr.07.09.99 DNA frame translation: 3

Bacteria Mycobacteria

Protein molecular weight 65000Da to 97000Da

Protein PI 3.0 to 10.0 All

Digests used Insulin Max. #Missed Cleavages: 2

Modifiers of cysteine Acrylamide

N-terminus of peptide Hydrogen (H)

C-terminus of peptide Free acid (OH)

Sample Identification Number Magic Bullet digest

Maximum Hits in report 25

Possible modification types Oxidation of M

The variation range of peptide mass ±40.1Da

Peptide Quality Average

Min. #Peptides to Match 9

Report MOWSE Score Pfa: 0.4

Peptide mass input value (mass error: ±1.5Da) Mass (m/z) Charge (z) Mass (m/z) Charge (z) 527.9 +2 770.0 +2 1054.5 948.1 +2 559.0 +2 961.8 +2 947.5 810.7 +2 553.4 +2 720.5 +2 560.0 +2 740.5 +2 1105.5 1209.0 +2 696.3 +2 640.8 +2 866.0 +2 933.5 +2 1002.3 +2 784.3 +2 904.7 +2 1545.7 +2 820.6 +2 1287.0 +2

Search results matching MS

Sample identification number: Magic Bullet digest

Database searched: NCBInr.07.09.99

Molecular weight searched: (65000-97000Da) 21170 items were selected

PI range: 324311 items

Searched species: Mycobacterium Selected 5990 items

Combining molecular weight, PI and bacterial species to search, 333 items were selected.

There are 80 search results with MS-Fit (the displayed results are the first 10 matching results)

Summary of Results Rank MOWSEScore #(%) The quality of the match is the highest Protein MW(Da)/pI Strains NCBInr.9.7/98Accession# 1 6.59×10 ³ 19/26 (73%) 80403/5.03 Combined mycobacteria 2497795 (Z78020)glcB 2 425 7/26 (26%) 66600/5.66 Combined mycobacteria 3261657 (Z81368)ggtB 3 168 8/26 (30%) 80142/5.09 Leprosy 2578377 (AL008609)_G 4 52.1 10/26 (38%) 77122/5.39 Combined mycobacteria 2501060 (Z95387)thrS 5 37.9 10/26 (38%) 89926/5.6 Combined mycobacteria 1731250 Probably cation-trans ATPase CY3 6 32.2 7/26 (26%) 95574/6.25 Leprosy 2398706 (Z99125) hypo? protein MLCL 6 32.1 7/26 (26%) 95659/6.33 Leprosy 3024896 (U00013)pps? B1496 C2 18 7 27.3 7/26 (26%) 95033/5.37 Combined mycobacteria 3261590 (Z74025)ctpF 8 26.4 8/26 (30%) 65877/5.56 Leprosy 2959407 (AL022118)re? Helicase DnaB 9 21.1 7/26 (26%) 81578/5.52 Combined mycobacteria 1817676 (Z84724)pkn? 9 20.9 9/26 (34%) 85425/5.37 Combined mycobacteria 1781217 (Z83867) Nuo? 10 18.5 7/26 (26%) 95486/6.93 Combined mycobacteria 2276335 (Z97991) hypo? Protein Rv033

Detailed results:

1.19/26 match (73%) 80403.4Da, pI=5.03.Acc#2497795.Mycobacteriumtuberculosis(Z78020)glcB(=GlcB)

The identified protein was GlcB (Z78020) of Mtb, which was considered a malate synthase based on its homology to proteins in other known bacteria. The accession number of this protein in the NCBI gene bank database is CAB01465 (Cole, S.T.et al., Nature 393:537-544 (1998), which describes the complete genome sequence of Mtb). The sequence of this protein is the aforementioned SEQ ID NO: 106.

Example V

Characteristics of the Serum Predominant Antigens of Mycobacterium Tuberculosis

The goals of this study were to identify the antigens recognized by antibodies in TB patients to elucidate the human response to Mtb and to evaluate the potential of these antigens as serodiagnostic candidates. This objective was achieved by immunoblot analysis of Mtb H37Rv secreted antigen after 1D and 2D electrophoretic separation with TB patient sera and healthy control sera (adsorbed by E. coli).

Of the more than 200 Mtb-secreted proteins, only 26 induce antibody production in TB patients. Several of these antigens were determined to have identity based on (a) their reactivity with murine mAbs, (b) N-terminal amino acid sequencing, and (c) liquid chromatography-mass spectrometry (Example III). Twelve of the 26 antigens were recognized by sera from early-stage patients, non-cavitary TB, and advanced-cavitary TB. Of these 12 antigens, five, including the 88 kDa antigen (Example I), MPT32 and Ag85C, reacted significantly with TB sera; the other two antigens have not been identified. The present invention aims at the development of assays for serodiagnosis using antigens that induce antibody production in early and advanced TB patients (as described in the present invention).

Materials and methods

Subjects: (a) patients with advanced TB

The study included serum samples from 33 HIV-negative individuals with confirmed pulmonary tuberculosis (advanced TB). Twenty of these sera were provided by Dr. J. M. Phadtare (see Example I). Nineteen of these patients were smear-positive, all with radiographically documented intermediate to advanced cavitary lesions. All patients had bleeding within 4-24 weeks of starting treatment.

(b) Early TB patients: All 13 TB patients from Manhattan VA Medical Center, New York's Infectious Diseases Center showed positive cultures, 6/13 were smear negative, and 12/13 had minimal or no imaging lesions. These patients bleed before or within 1 to 2 weeks of starting treatment.

(c) Control group: 23 HIV ^neg , TB ^neg , and healthy subjects were used as the control group. 16 of them were PPD ⁺ (skin test) and the remaining 7 were PPD ^neg .

antigen

The log-phase culture filtrate of MtbH37Rv was used as a source of secreted antigen as described in Example I (culture filtrate proteins or CFPs without LAM). There are more than 200 proteins in LFCFP preparations (Example III, supra). After antigens were separated by size, they were loaded into preparative polyacrylamide gel tubes and proteins were separated by electrophoresis using a gradient of increasing watts (model 491 Prep Cell; Bio-Rad, Hercules, CA.). Fractions were pooled, examined by SDS-PAGE and collected according to molecular weight. Contaminated SDS was removed as described above. Each fraction was reacted with human serum and a large plate loaded with murine mAbs directed against the Mtb antigen, see Example I for reactivity. Sera were immunoadsorbed with E. coli lysates as described in Example I. All ELISA tests, as described in Example I, were performed using E. coli lysate immunoabsorbed sera.

One-dimensional (1-D) SDS-PAGE and 2-D PAGE of LFCFPs

Separation of LFCFPs (8 μg/band) was performed on a microgel containing 10% separating gel and 5% stacking gel using a vertical plate (SE 250 Mighty Small II, Hoeffer Scientific, San Francisco, CA.). Gels were stained with silver (Bio-Rad Silver Stain Kit, Hercules, CA) or used for electrophoretic migration in immunoblotting. The separated proteins were transferred to a nitrocellulose membrane at a constant 100V for 1.5 hours. The 2-D PAGE procedure is described in Example III. Proteins resolved by 2-D PAGE were transferred to a nitric acid membrane.

Western blot analysis

1-D and 2-D blots were blocked with phosphate-buffered saline (PBS) containing 3% BSA for 2 hours and washed with PBS/Tween 2% (wash buffer) for 1 hour. The band containing the isolated LFCFPs was reacted with serum (diluted 1:100 in PBS containing 1% BSA) overnight at 4°C. Spots containing 2-D isolated LFCFPs were tested using four different serum pools containing sera whose reactivity with the above antigen preparations had been determined by ELISA. The serum pool consisted of (a) 6 PPD positive healthy control sera that were not specific for any of the antigens (group I), (b) 6 TB patients who were not reactive to all 3 antigen preparations used in the ELISA (Group II), (c) 6 TB patients who reacted with all LFCFPs and 88kDa size preparations, but not with 38kDa antigen preparations (Group III), and (d) 6 TB patients whose Reacted with both 38 and 88 kDa sized antigens (group IV). The spot was reacted with serum or serum pool, then washed with washing buffer for 1.5 hours, and then alkaline phosphatase-coupled anti-human IgG (diluted ratio 1:2000, Zymed, CA.) was added to it, the effect 1.5 hours. Blots were washed for 2 hours and developed with BCIP/NBT substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD).

Table 5: Classification of TB patients reactivity serogroup n ^a smear positive radiographic void CFP without LAM Fraction containing 88kDa antigen Fraction containing 38kDa antigen I twenty three 0 0 0 0 0 II 9 7 5 0 0 0 III 13 9 5 11 13 0 IV 11 10 11 11 11 11

n ^a = number of members per group

result

Reactivity of Sera with Mtb Secreted Antigens

Sera were grouped according to their reactivity with total LFCFPs as determined by ELISA, or by size-separated serum-reactive proteins containing 38 kDa PstS or 88 kDa (Table 5). Group I included sera from 16 PPD ⁺ and 7 PPD ^neg healthy controls that were not positive with any antigen in the ELISA test. Group II included 9 TB patients who were antibody-negative by testing with all three antigen reagents; 5 of these patients were smear-positive and had cavitary lesions. The remaining 4 patients did not have cavitary lesions, but 2 of them were smear positive. Group III included 13 patients with LFCFPs and antibodies containing the 88 kDa antigen isolate, but not antibodies containing the 38 kDa antigen isolate. Five of them were smear positive and had pulmonary cavitation lesions. Four others showed positive smears without any cavitary lesions. The remaining 4 were smear negative and did not have any cavitary lesions. Group IV included 11 patients, all of whom had antibodies to all three antigenic agents. 10/11 were smear positive, and all patients had imaging features of moderate to deep cavitary lesions.

Antigens in LFCFPs recognized by serum

After silver staining, LFCFP separated by SDS-PAGE showed a wide range of proteins from 14 to >112 kDa. The Western blot spots obtained from the isolated LFCFPs were probed with sera from 4 groups (diluted at 1:100). After extensive sera testing, several blots were obtained. When blots were combined to indicate reactivity, not all antigen bands matched exactly. In order to unify the standard, the 65kDa band was used as the control center. In the sera of Group I (PPD ⁺ and PPD ^neg healthy controls), most antigens recognized by sera from 6 PPD ^neg healthy subjects had molecular weights of 26, 30-32 kDa and 65 kDa. The 30-32 and 65kDa antigens were also recognized by sera from 9 PPD ⁺ healthy controls, of which only 3/9 sera recognized the 26kDa antigen, while 1 serum sample recognized the 68kDa antigen.

Group II tuberculosis sera were those that were negative for antibodies to all three antigenic reagents in the ELISA assay. Although there was some variation between TB sera, all sera reacted with the 30-32kDa and 65kDa antigens, and 5/8 contained antibodies reactive with the 26kDa antigen, which were also recognized by the controls. Serum from one patient showed strong reactivity with 46, 55 and 97 kDa antigens. Four sera, including patients with advanced disease, showed weak reactivity with 74, 76, 88, 105 and 112 kDa antigens and 46-55 kDa antigens. Sera from patients with and without cavitary lesions did not show significant differences in reactivity.

Group III patients had antibodies against LFCFPs and 88kDa preparations in ELISA tests. Sera from 10/11 showed moderate reactivity with the 88 kDa antigen GlcB. In addition, these sera also recognized 74, 76, 105, 112 kDa antigens and some antigens in 46-55 kDa. Despite ELISA non-reactivity, 3/11 sera reacted with the 38 kDa antigen. This may indicate binding of the recently described 38 kDa antigen (Bigi, F. et al., 1995, Infect. Immun. 63:2581-2586), which is a distinct antigen from the PstS protein. There was no difference in the type of response between (a) sera from patients without lung cavitary lesions (lanes 25-30) and (b) sera from advanced patients with cavitary lesions (lanes 31-35).

Group IV patients (lanes 36-43), which reacted with all three antigen reagents, reacted strongly with the 38 kDa antigen and recognized the 34 kDa antigen, which was not recognized by any of the Group III sera in the ELISA assay. Apart from these two antigens, the antigens recognized by the Group IV sera were the same as those recognized by the Group III sera, except that the reactivity of the Group IV sera with individual antigens was significantly enhanced. Seven out of eight sera reacted strongly with the 88kDa GlcB antigen.

In conclusion, all antibody-positive TB patients (groups III and IV) reacted predominantly to antigens with a molecular weight >46 kDa. 74, 76, 88, 105, 112 kDa antigens and antigens between 46-55 kDa are frequently targets of human antibody responses. In contrast, the 38kDa and 34kDa antigens were recognized by a more limited group of patients (group IV).

Identification of antigens recognized by TB patient sera

2D-PAGE has strong resolution for complex protein mixtures. By this method, LFCFPs preparations were broken down into about 200 different proteins. 2-D maps of all Mtb CFPs are found in US 6,245,331 and WO 98/29132 (and discussed in Example III). 2D immunoblot of isolated LFCFPs probed with serum pools from groups I-IV. The reactivity of each serum pool was compared to that of the murine mAbs to identify the antigens recognized by TB patient sera (Table 6).

The results of the reactions with the four serum pools are described in Tables 6A-C. The numbering of each antigen is given in Example III. All four serum pools reacted with 4 secreted antigens and 3/4 of the serum pools reacted with the other two secreted antigens (Table 6A).

These six proteins were clearly visible in 2-D blots reacted with sera from healthy controls (Group I). Reactivity with murine mAb IT-49 identified two of them as Ag 85B (#81, 29 kDa) and Ag 85A (#149, 31 kDa). These antigens correspond to the 30-32 kDa doublet in the 1-D immunoblot. The other two antigens with molecular weights of 55 kDa (#114, 120) and 58 kDa (#86, 96, 105), which reacted with all serogroups, did not react with murine mAbs. A previous antigen had been identified as a glutamate synthase by N group analysis (Example III). These antigens may be similar to the 65 kDa antigen in the 1-D blot that reacts with individual sera. Antigens 26kDa (#19, 29) and 46kDa (#51) reacted with control sera (group I) and antibody-positive TB sera (groups III and IV), but not with the pool of antibody-negative TB sera (group II) . The previous antigen (26 kDa, #19, 29) was identified as MPT64 based on reactivity with the murine mAb IT67, while its detection on the 1-D blot by several control sera suggested the likely 26 kDa antigen.

The reactivity of the pool of sera from Group II TB patients lacking ELISA-reactive antibodies to any of the secreted antigens tested is described in Table 6A. This serum pool showed weak reactivity with these four antigens (29, 31, 55, and 58kDa), while the control group (Group I) reacted with these antigens, but this serum pool did not show reactivity with 25/26 (#19, 29) Any reactivity with the 46 kDa (#51) antigen.

A pool of TB patient sera (group III) containing antibodies against the 88 kDa (GlcB) but not the 38 kDa antigen reacted with the secreted antigen on the 2-D blot in 18 cases (Table 6B). Six of these antigens were identical to those recognized by the healthy control serum pool (Group I, Table 6A). Of the remaining 12 antigens, 3 were below 30kDa: one was 26kDa (#170, MPT51), which reacted with mAb IT52, while the other two antigens (28kDa, #77; and 29/30kDa, #69, 59 ) did not react with any of the mAbs tested. In the 30-60 kDa range, strong reactivity with 31 kDa (#119, mAb IT-49, Ag85C) and 38/42 kDa antigens (#11, 14, MPT32) and low reactivity with one isoform of the 35 kDa antigen (#66, IT-23, PstS). The 49 kDa protein (#82) reacted with mAb IT-58. Antigens with molecular weights of 31 kDa (#103), 42 kDa (#68, 80) and 48 kDa (#24) were not recognized by any mAbs. These antigens correspond to bands in the 1-D blot ranging from 30 to 60 kDa. In the 65-100kDa range, an 85kDa protein (#113, 124, IT-42, IT-57) reacted with this serum pool, but the 74 and 76kDa antigens on the 1-D blot were absent, whereas these antigens in 2-D It can be seen on the imprint. The 85 kDa antigen (#113, 124) on the 2-D immunoblot corresponds to the 88 kDa antigen GlcB (Example I and Example III). The antigen was also confirmed by testing the reactivity of the isolated LFCFPs with the mAbs IT-42 and IT-57, and the identification results were both 88kDa bands by reacting with the two antigens. The 104 kDa protein (#111) corresponds to 105 kDa on the 1-D blot. The antigen corresponding to the 112 kDa antigen on the 1-D immunoblot was not seen on the 2-D immunoblot.

Serum pools from Group IV TB patients recognized 11/12 antigens reactive with Group III serum pools (only the 28 kDa antigen was not recognized, #77; Table 6B). However, the group IV serum pools were compared with 26kDa (#170, MPT51), 31kDa (#119, Ag 85C), 35kDa (#66, PstS), 38/42 (#11, 14, MPT32), 49kDa (#82; IT -58), 85 kDa (#113, 124) and 104 kDa (#111) antigens were more reactive than the group III serum pool. The Group III pool showed only weak reactivity with one isoform of the 35 kDa antigen (#66, PstS), whereas the Group IV pool reacted with all four isoforms recognized by mAbIT-23. In addition to the 11 antigens listed that reacted with both Group III and Group IV serum pools (Table 6B), the latter group reacted with an additional 8 antigens (Table 6C). Antigens with a molecular weight below 30 kDa are 13/14 kDa proteins (#23, 38, IT-12 and SA12, GroES). In the 30-38kDa range, the serum pool recognizes four neoantigens with the same 31kDa molecular weight but different pI values: 31kDa (#15, 16, 22, 25), 31kDa (#62), 31kDa (#57) and 31 kDa (#37) and the fifth antigen 38 kDa (#32). Among them, only 31 kDa (#15, 16, 22, 25) reacted with mAb IT-44, while the remaining 4 antigens had not been described before. In the region above 65 kDa, the pool reacted with 66/72 kDa proteins (#65, 79, mAbs IT-40 and IT-41, DnaK) as well as an unidentified 79 kDa antigen (#78).

Overall, of the 26 antigens recognized by TB sera, 6 were reactive with control sera (Table 6A). Twelve of the 26 antigens were recognized by Group III and Group IV sera (Table 6B). Thus, both patients with early, noncavitary TB and patients with advanced cavitary lesions have antibodies to these antigens. Among these 12 antigens, 5 can be clearly recognized, therefore, as described in the present invention, they can be used as preferred antigens in early TB serodiagnostic detection. They are 85kDa/88kDa antigen (#113, 124; Example 1), 38/42 protein (#11, 14, MPT32), 31kDa antigen (#119, Ag 85C), uncharacterized 49kDa antigen (#82; IT-58) and the 26 kDa antigen (#170, IT-52). In contrast, the other eight antigens listed in Table 6C and the 38 kDa protein (#66, PstS; Table 6B) were mainly recognized by patients with advanced TB and, therefore, their value in serodiagnosis was limited.

discuss

Replicating bacteria secrete about 200 proteins, only individual subsets of which are recognized by the immune system of TB patients to produce specific antibodies in the patient's sera. Even within subclasses, some antigens were recognized by both early and late TB patients, while others were only recognized by late stage patients. In view of the fact that the 38kDa PstS protein is the most successful serodiagnostic antibody in the field (Bothamley et al., 1992, supra; Harboe et al., 1992, J: fect. Dis., supra), several of the disclosed antibodies can be detected in vivo. The antigens recognized by patients lacking anti-38kDa antibodies are very important. As described in the present invention, removal of cross-reactive antibodies by immunoadsorption with E. coli antigens in previous examples allowed the identification of Mtb antigens with strongly serum-reactive epitopes. Previously, there was no success in identifying Mtb antigens that induce antibodies in patients. Verbon et al. (supra) found no difference in the reactivity of patient and control sera. Espitia et al. (supra) (also using unabsorbed serum) identified only a 38 kDa PstS protein. This antigen reacts with only 57% of TB sera. Immunoadsorbed serum with E. coli lysates eliminated cross-reactive antibodies that hinder identification of serum-reactive antigens. Furthermore, 2-D analysis and mapping of each antigen allowed accurate identification of the antigen, which appears to be key in a rationally designed serodiagnosis, and at least 5 secreted proteins could serve as useful serodiagnostic reagents. Antibodies to one of these, the 88 kDa antigen GlcB, are present in 80% of late-stage patients and 50% of early-stage TB patients. The 38/42 kDa antigen (#11, 14, MPT32) was also considered to have serodiagnostic potential (Espitia et al., 1995, supra), but not as an early antigen. The remaining 3 antigens, 49kDa (#82; IT-58), 31kDa antigen (#119, Ag85C) and 26kDa (#170, IT-52), had never been used to evaluate the serum reactivity of patients before the present invention.

The inventor's laboratory changed the reference numbers of some spots shown on 2D gels in US 6,245,331 (12 June 2001) and WO98/29132 (published 09 July 1998), changing numbers to letters. See also the publication Samanich, KM et al., J Iyafec. Dis. 178:1534-1538 (1998), incorporated herein in its entirety. The numerical identification and corresponding letter identification of some spots have been given in the legend of Figure 1 of the above-mentioned publication, and are given again below: new number corresponding number new number corresponding number A Na ^* h na B 86, 96, 105 I 111 C 77 J 57 D. 59,69 K 62 E. 103 L 32 f 68,80 m 78 G twenty four

^* Na = no value assigned, the numbering in the table is based on Sonnenberg et al., 1997, supra, as used in the examples of the present invention.

Table 6: Antigens recognized by various serum pools Antigen MW ^a pI number ^b Reactive Amb (anti-recognized antigen) Reactivity with serum pool GrpI II III IV A. Antigens recognized by all 4 serum pools 25/262931465558 4.65-4.835.105.385.055.14-5.175.11-5.12 19, 298114951114, 12086, 96, 105 IT-67(MPT64)IT-49(Ag 85B)IT-49(Ag 85A)noneGlutamate synthaseNONE +++++±±++ NR++++NR±++ ++++++++++++++ ++++++++++++++++++ B. Antigens recognized only by group III and group IV TB patients 262829/30313135(38)＜42484985(88)104 5.915.105.085.125.175.094.31-4.515.104.795.105.14-5.195.13 1707769, 591031196611, 1468, 802482113, 124111 IT-52(MPT51)nonenonenoneIT-49(Ag85C)IT-23(PstS)polyclonal antiserum(MPT32)nonenoneIT-58IT-42, IT-57none NR||||||||||↓ NR||||||||||↓ ++±++++++±++++++++++ +++NR++++++++++++++++++++++ C. Antigens recognized only by group IV TB patients 13/14313131313866/7279 4.76-4.934.53-4.795.095.084.934.875.09-5.105.10 23, 3815, 16, 22, 256257373265, 7978 SA-12, IT-10(GroES)IT-44NONENONENONENONEIT-40,IT-41(DnaK)NONE NR||||||↓ NR||||||↓ NR||||||↓ ++++±++++++++++

^a Molecular weight of antigen (MW), expressed in kDa

^b numbering corresponds to the CFPs 2-D PAGE pattern of Mtb H ₃₇ Rv (Example III) NR: unreacted

In addition to the aforementioned 5 early antigens, seven other antigens also showed reactivity with the group III serum pool:

(1) 28kDa (#77) antigen,

(2) 29/30kDa (#69, 59) antigen,

(3) 31kDa (#103) antigen,

(4) 35kDa (#66, IT-23) antigen

(5) 42kDa (#68,80) antigen

(6) 48kDa (#24) antigen and

(7) 104 kDa (#111) antigen.

Therefore, one or more of these antigens or their epitope-bearing peptides or reactive variants of peptides, combined with one or more of the five early antigens (or their peptides), are used in immunodiagnostic reagents , which can increase the sensitivity of diagnostic tests.

The other three with significantly stronger serum dominant epitopes were based on significantly enhanced reactivity with antibody-positive TB patient sera (Groups III and IV) than with antibody-negative patient sera and control sera (Groups I and II; Table 6A). Antigens are: (a) 55 kDa (#114, 120, glutamate synthase) antigen, (b) 46 kDa protein (#51, IT-58) antigen and (c) 31 kDa (#149, Ag 85A) antigen. The serodiagnostic potential of Ags 85A (#149) and B (#81) was evaluated by isoelectric focusing separation and Western blot analysis by Van Vooren et al. (supra). The 85A component showed reactivity with both TB and non-TB sera, whereas only 71% of TB sera recognized Ag 85B or C. Importantly, no comparative information was provided for early versus late responsiveness.

The results of the present invention reveal strong reactivity of Ag 85A and 85B with patient sera and weak reactivity with control sera, while 85B is more cross-reactive with control sera. Studies of Ag 85 components revealed that the serodiagnostic potential of these antigens lies in their specific epitopes (Wiker et al., 1992, Microbiol. Rev, supra). This result creates an important basis for this conclusion and provides the basis for the identification and detection of this epitope.

Another protein recently evaluated as a serodiagnostic candidate is MPT64 (26 kDa, #19, 29) (Verbon et al., 1993, supra), which was reported to improve sensitivity in approximately 46% of active TB patients . However, 2-D analysis suggested that this protein, despite its strong reactivity with advanced TB patient sera, failed to discriminate between Group III TB sera (without anti-38kDa antibodies) and healthy controls (Group I).

The early antigens identified in the present invention may not be the only early antigens secreted by Mtb as it grows in vivo. These antigens are likely to be recognized as having strong seroreactive epitopes. Several antigens of Mtb were either up-regulated or down-regulated when the microorganisms were grown among macrophages. It is the inventor's view that in vivo, the Mtb microorganism produces the proteins necessary for survival and growth under specific conditions that differ from those necessary for growth in culture media. Notably, several antigens that induced antibodies in early TB patients (based on reactivity with group III sera) suggested a role in initiating disease in vivo. Thus, Ag 85A, Ag 85C and MPT51 all belong to the family of secreted proteins, which bind to fibronectin (Wiker et al., 1992, Scand. J. Imrraunol., supra)). MPT32 is homologous to a fibronectin-binding protein of M. leprae (Schorey, J.S. et al., 1995, Infect. Immun. 63:26522657).

Notably, the 28kDa antigen (#77) reacted with the group III but not the group IV serum pool, suggesting that some antigens are expressed differently at different stages of disease progression (Amara, R.R. et al., 1996, Infect. Immun. 64:3765-3771).

Based on the previous findings, the present inventors identified seroreactive antigens that can be used in diagnostic tests for TB patients at an earlier stage. As expected, these antigens share homology to similar proteins in other mycobacterial species, thus enabling identification of species-specific epitopes for serodiagnostic use.

If the absence of detectable antibody (by ELISA) is due to the antibody forming an immune complex (Grange, supra) in vivo, the present invention provides a method for identifying such a complex.

Given that Mtb secretes large amounts of antigen as it replicates in culture, it is significant that these small amounts of antigen are still reactive with TB patient antibodies. Additional efforts have been made by those skilled in the art to develop serodiagnostic methods utilizing Ag 85A, 85B and 38 (or 35) kDa antigens. The present invention clearly shows that at least 5 other secreted antigens are recognized by the vast majority of TB patients. These antigens are used in the design of serodiagnostic tests for TB, as disclosed in the present invention.

Example VI

Reactivity of TB patient sera with purified antigen and isolated fractions of secreted antigen

The reactivity of patient and control sera containing LFCFP with fractions 10, 13 and 15 and purified antigens Ag85C and MPT32 are summarized in FIGS. 3 , 4 and in Table 7. As discussed in Examples I and II, fraction 10 was enriched for the 38 kDa antigen, fraction 13 was enriched for MPT32, and fraction 15 was enriched for the 88 kDa antigen GlcB, suggesting that all advanced TB patients with LFCFP antibodies could be treated with Ag85C or isolate The antigen in component 15 was detected. Most patients also had antibodies to MPT32 (fraction 13) and the 38 kDa antigen (fraction 10). However, the Ag85C and 88kDa proteins are recognized by the immune system of most patients and produce antibodies.

All early TB patients who reacted with LFCFP also reacted with MPT32, but none with the 38 kDa antigen. In the early TB group, reactivity was higher with purified MPT32 (Figure 4) than with partially purified antigen (fraction 13) (Figure 3).

These results demonstrate that early TB patient sera can react with at least 3 of the 5 early antigens described in the present invention (Example 1). These findings demonstrate that the use of purified early antigens will increase the sensitivity of detection of early TB patients, leading to improved rapid detection methods.

Of these antigens, only MPT32 has been considered in the development of TB serodiagnosis. But none have ever been shown to be reactive with early TB patient sera. Therefore, this is the first time that these antigens have been suggested as a method for diagnosing the early stages of TB, which is especially important in non-immune patients such as those with HIV infection.

Reactivity of individual sera with antigens

Reactivity of any single antigen on the 2-D blot with the pool of sera may appear to be reactive with only some of the individual sera. To confirm the broad reactivity of the antigens recognized by the group III serum pool, individual sera were tested for the presence of antibodies to two antigens recognized by the group III serum pool, Ag 85C which the inventors had purified and MPT32. Reactivity with purified 38 kDa PstS antigen and 88 kDa antigen GlcB (fraction 15) was also tested. More TB patients than those tested above were tested and patients could be classified as cavitary or non-cavitary TB. Sera from 27/34 (79%) of patients with cavitation and 9/20 (45%) of non-cavitation reacted with the 88 kDa antigen (Table 8), 29/34 (85%) of patients with cavitation and 9/20 (45%) %) of non-cavitary patients reacted with Ag 85C (Table 6). Sera from 29/34 (85%) cavitary and 5/20 (25%) non-cavitary patients reacted with MPT32 (Figure 4 and Table 8). In contrast, 18/34 (53%) cavitary and only 1/20 (5%) non-cavitary patients reacted with purified 38 kDa antigen (Table 8).

Analysis of the above results showed that antibodies could be detected from 31/34 (91%) of cavitary and 12/20 (60%) of non-cavitary TB patients, wherein, among the 3 antigens identified One or more of the reactivity is called positive reactivity.

All TB patients were analyzed to determine whether positive smears and detection of antibodies to purified antigens were comparable as diagnostic methods for TB. Table 9 shows that 43/54 (80%) of all TB patients were diagnosed by sputum smear and 43/54 (80%) by ELISA detection.

Table 7

Patient reactivity to purified or isolated Mtb antigens

Purified antigen or antigen fraction (with diluted serum) Ag85C (1:100) MPT32 (1:150) LFCFP (1:1000) F13(MPT32) ¹ 1/200 F15 (88kDa) 1:200/400 F10 (38kDa) (1:200) subjects All TB Late TB Early TBPPD ⁺ HCPPD ^neg HCHIV ⁺ HCHIV ⁺ TB (pre or at) 36/5072% 28/5254% 30/4272% 19/50 38% 29/4269% 16/4238% 25/2889% 19/2868% 23/2882% 17/2861% 22/2878% 16/2857% 11/2250% 9/2438% 7/1450% 2/1414% 7/1450% 0/140% 0/180% 0/210% 0/160% 0/160% 0/160% 0/160% 0/130% 0/130% 0/160% 0/160% 1/166% 0/160% 0/390% 0/340% 0/210% 0/160% 0/210% 0/210% 16/5231% ND 23/5046% ND 37/5271% 34/52 ^** 65%

The definition of subjects is given in Examples I and II. HC = healthy control. HIV ⁺ TB patients included those diagnosed before (pre) or at (at) TB diagnosis. ^** OD value boundary line.

Not all smear-positive patients had detectable antibodies, nor did all antibody-positive patients have positive smears, and the combination of smear and ELISA diagnosed 50/54 (93%) of TB patients.

Dividing TB patients into cavitary and noncavitary TB patients, 97% (33/34) of cavitary and 45% (9/20) of noncavitary TB patients could be detected by smear. The sensitivity of antibody detection was 91% (31/34) and 60% (12/20), respectively.

Therefore, by combining these two methods, the sensitivity is increased to 100% for cavitary TB and 80% for noncavitary TB (16/20). These results indicate that the greatest sensitivity for TB diagnosis can be obtained by the simultaneous application of sputum smear and ELISA to detect antibodies reactive with the antigen.

Table 8. Reactivity of sera with different Mycobacterium tuberculosis antigens Sensitivity (%) Specificity (%) antigen All TB (n=54) Voidness (n=34) Non-cavitation (n=20) (n=83) 88kDa (GlcB) 70 79 45 100 Ag85C 70 85 45 100 MPT32 63 85 25 98 38kDa 35 53 5 100

Table 9. Diagnosis of tuberculosis

Number of patients (%) patient N Smear ⁺ Ab ⁺ Smear ⁺ /Ab ⁺ tuberculosis 54 43 (80%) 43 (80%) 50 (93%) cavitary TB 34 33 (97%) 31 (91%) 34 (100%) non-cavitating TB 20 9 (45%) 12 (60%) 16 (80%)

^* 8/12 smear-negative patients were antibody positive.

Example VII

Anti-mycobacterial antibodies in urine

subjects

Serum and urine samples were obtained from 23 smear-positive, untreated (=advanced) TB patients attending the TB clinic of LRS Hospital, New Delhi, India for tuberculosis and allied diseases. Forty-one serum and 24 urine samples from PPD-positive and PPD-negative healthy subjects were used as negative controls.

Determination of anti-mycobacterial antibodies:

The reactivity of serum samples with Mtb culture filtrate proteins was determined by ELISA as described above. To determine the presence of anti-mycobacterial antibodies in urine samples, ELISA plates were coated with 125 μl of 4 μg/ml Mtb culture filtrate protein suspension overnight at 4°C. The next morning, the plate was washed with PBS and 125 μl of urine sample was added to each well. After 90 minutes, the plates were washed with PBS-Tween and bound antibody was determined using anti-human IgG-alkaline phosphatase conjugate and enzyme substrate.

Binding with a specificity of over 98%, 78% of (advanced) TB patient sera and 56% of urine samples had antibodies capable of binding the antigen in Mtb culture filtrate proteins (Fig. 5, left). If the presence of antibodies in only one of at least two body fluids (serum and/or urine) is considered, 92% of patients had anti-mycobacterial antibodies. The same serum and urine samples were evaluated for reactivity with purified MPT32 (Figure 5, right), which is one of the preferred antigens for serodiagnostic detection of early infection. Antibodies against MPT 32 were present in both samples of 14/23 (61%) patients. Again, 83% of patients had anti-MPT 32 antibodies when only considering the presence of anti-MPT 32 antibodies in at least one of the two body fluids.

To determine the culture filtrate antigens recognized by these urinary antibodies, culture filtrate proteins were separated on 10% SDS polyacrylamide gels followed by Western blot analysis. Spots were probed with serum and urine from 2 advanced TB patients and 2 PPD ⁺ healthy controls. Serum and urine samples from healthy controls showed cross-reactivity with 30-32 and 65 kDa proteins. As expected, serum samples (tested at a 1:100 dilution) from smear-positive TB patients (=advanced stage) reacted strongly with several protein bands between 20-120 kDa and with the 88 kDa (GIcB) band responses can also be clearly identified. Reactivity with MPT 51, MPT 32 and Ag 85C was however difficult to discern on the 1-D imprint due to the presence of several other proteins of similar molecular weight.

Urine samples from TB patients (assayed undiluted) reacted with some antigens of similar characteristics, but fewer wells reacted. The 88 kDa protein GlcB was recognized by both urine samples, but reactivity with MPT 32, Ag 85C and MPT 51 could not be confirmed on 1-D blots. However, ELISA results showed the presence of anti-MPT 32 antibodies in the urine sample. This shows that the antibodies in the urine sample and the antibodies in the serum are directed against the same antigen, but the antibody titer in the urine sample is lower. These results show that:

(1) Anti-mycobacterial antibodies are present in the urine of a considerable proportion of smear-positive (advanced) TB patients, therefore, the antibodies can be used as the basis for the urine diagnosis of TB.

(2) The detection of anti-mycobacterial antibodies in both urine and serum significantly increases the diagnostic detection rate. The use of two body fluids increases the sensitivity, which is important for the diagnosis of smear-negative (=early) early TB patients, where the sensitivity of antibody detection is 50% with the current serodiagnosis, and the sensitivity of the current method for HIV-infected TB patients was 66%.

(3) The antigens used in urine or urine/serum diagnostic detection of TB are the same as the Mtb antigens (and other antigens) identified in the sera described in the present invention, including full-length proteins, aggregated proteins, peptides and peptide multimers.

Example VIII

Anti-mycobacterial antibodies in non-human mammals

To determine which antigens in mycobacterial culture filtrates are recognized by non-human mammals with TB, the following experiments were performed. Sera were obtained from 2 guinea pigs infected with 4-5 colonies forming disseminated virulent Mtb. Guinea pigs infected as described above develop TB within 14-15 weeks. Infected animals bled at week 15 post-infection and sera were probed for Western blot analysis of culture filtrates separated by 12% SDS-PAGE. Sera from 2 uninfected guinea pigs were cultured as negative controls. Sera from 2 human TB patients were cultured for comparison. Sera from healthy subjects cross-reacted with antigens 65kDa and 30-32kDa in Mtb culture filtrate. Sera from Mtb-infected guinea pigs showed strong reactivity with several antigens in the culture filtrate that were not recognized by control sera. Thus, sera from animals with tuberculosis showed strong reactivity with the 88 kDa protein GlcB, the 42-45 kDa protein, the 38 kDa protein and several other protein bands. This same antigenic signature is recognized by sera from human TB patients.

These results show that TB-affected mammalian sera recognize the same antigenic signatures as those recognized by human TB patients. Thus, the proteins and peptides defined in the present invention that can be used for serodiagnostic TB or TB vaccines in humans can also serve as the basis for TB serodiagnostic tests and vaccines in other mammals. The invention can also be used in the veterinary medical system.

Example IX

Serum reactive peptide of early Mtb protein antigen

Firstly, a study was designed to predict and detect epitopes of Mtb 88kDa GlcB protein and MPT51 protein, which should be antigenic and react with the early antibodies of Mtb-infected persons, so that they can replace whole Natural (or recombinant) molecules are grown for use in early TB diagnostic tests. Amino acid sequences were analyzed using several different algorithms and methods, which can also be used in the same way to detect other Mtb proteins widely (PCGENE, Hydropathy, etc.).

The Hopp-Woods method is described in Hopp, TP & Woods, KR, Proc Natl Acad Sci USA, 1981, 78: 3824-3828; Hopp & Woods, Mollmmunol, 1983 20: 483-489. The method is to determine the protein epitope by analyzing the amino acid sequence to find the largest local hydrophilic site. The method is to assign a numerical value (hydrophilic value) to each amino acid, and then repeatedly average the hydrophilic values of the amino acids of the peptide chain. The site of highest local average hydrophilicity was always located within or adjacent to the epitope. The predicted success rate is determined by the average group length, and the results obtained by the average hexapeptide are better. The method was originally designed to use the large amount of immunochemical information obtained to predict epitopes using 12 proteins. The 1983 publication described a computational method for predicting protein epitope sites requiring only amino acid sequence information. This method was used to predict the major epitopes of the hepatitis B surface antigen, which was adapted for a stand-alone computer and written in the BASIC language so that researchers with limited computer experience and/or knowledge could obtain this information. This document also describes a method for identifying antigenic sites in multiple homologous series of proteins using influenza hemagglutinin.

Another method for analyzing molecular "flexibility" is the Karplus-Stultz method (Stultz CM, Karplus M., Proteins 2000 40:258-289). The method is based on the Dynamic Ligand Design (DLD) algorithm, an automated method for the formation of new ligands. The algorithm links small functional groups that are energetically dominant sites located at the binding site of the target molecule. These positions and orientations of small functional groups can be determined using multi-copy simultaneous search methods (or experimental data). A new simulated annealing scheme can be used to optimize the pseudo-potential of the ligand on the binding site.

The method of Jameson and colleagues was used to predict the antigenic region of the molecule. Jameson BA & WolfH, Comput Appl Biosci 19884:181-186 introduced a computer algorithm to predict topological features of proteins composed of primary amino acid sequences. The computer program calculates surface accessibility parameters and combines these values with domain framework flexibility parameters to predict secondary structure. The output of the algorithm, the antigenic index, is used to form a linear surface profile characteristic of the protein. Since most, if not all, antigenic sites are located on the surface of exposed regions of the protein, this procedure provides a reliable method for predicting potential antigenic determinants. Applying the method to well-characterized proteins yielded strong correlations between predicted antigenic indices and known structural and biological data. The publication Wolf, H. et al., ComputAppl Biosci 1988, 4: 187-19 describes a complete set of procedures for amino acid sequence analysis. For proteins whose sequence is known, the exact three-dimensional structure is rarely known. Therefore, it is preferable to use primary amino acids to directly predict important structural parameters. The authors introduce a broad, user-defined method for the analysis of amino acid sequence information, which is based on published algorithms, designed to standard protein databases, calculated affinity/hydrophobicity, surface probability, and flexibility values to obtain predicted binary level structure. Data is output in an easy-to-read graph, and several parameters may be multi-layered in a single graph to simplify data interpretation. The suite of methods includes new algorithms for predicting potential antigenic sites. Therefore, the software package provides an advantageous tool for analyzing amino acid sequences for the analysis of antigenic sites. These algorithms are given functional notations in the UWGCG (Univ of Wisconsin Genetics Computer Group) program collection.

For the present invention, the foregoing analysis was performed in collaboration with Macromolecular Resources, Inc. (Sigma/Genosys), which also synthesized the following peptides (95% pure). Biotin-linked peptides representing several epitopes were synthesized and their reactivity with sera from TB patients, PPD positive and PPD negative healthy controls was evaluated. Each peptide was recognized by antibodies from different patients.

Table 10: Identification of peptides with early TB immunoreactive epitopes in the 88kDa protein Peptide of 88KDa protein ^* Sequence ID: 106Genbank#CAB01465 serial identification number The proportion of serum that reacted TB control 5274 CG TDGAEKGPTYNKVRGDKaa 151-167 ^* 108 25/57 1/40 5275 KIGIMDEERRTTVNLKACaa 428-445 109 4/24 1/24 5276 ELAWAPDEIREEVDNNCaa 586-603 110 21/57 1/40 5277 LHRRRREFKARAAEKPAPSDRAGaa 715-736 111 3/24 1/24 5936 ARDELQAQIDKWHRRRaa 56-71 112 22/57 1/40 5937 LNRDRNYTAPGGGQaa 314-327 113 14/57 0/24 Peptide from MPT51 SEQ ID NO: 107 (Genbank #CAA05211) 5939 GAPQLGRWKWHDPWVaa 167-181 114 10/57 1/40.

a PPD ⁺ TB patient serum

b PPD ⁺ control serum

c A similar study was performed with MPT51. For MPT51

^* Added N-terminal C and G (underlined); not part of native protein sequence. Amino acid numbering (aa 51.., etc.) indicates the corresponding position of the peptide in the full-length sequence.

Use the following method. 50 [mu]l of biotin-conjugated peptide in blocking buffer (7.5% fetal bovine serum, 2.5% BSA) was pipetted into each well of a streptavidin-coated microtiter plate. 50 μl of serum was added to each well and incubated for 1 hour at room temperature. Plates were washed 4 times with 0.05% PBST. Add 100 μl of anti-human antibody and incubate for 1 hour at room temperature. Plates were then washed 6 times with Tris buffer, and substrates and amplification reagents were added. The absorbance of the chromogenic reaction product was read at 490 nm on a microplate reader.

The results are shown in Table 10, where the peptides for which responses were observed are reported. Reactivity was assigned as a fraction showing a positive reaction (absorbance > 2.5 standard deviations over negative control serum) with TB isolates. Serum reactivity of peptides at different dilutions was determined; responses were detectable at 1/5 and 1/20 dilutions, but generally better responses were observed at 1/10 dilutions.

Example X

Identification of Peptides Carrying Epitopes in Mtb Protein GlcB Using SPOTs Technology

Additional epitopes on the GlcB protein (=88 kDa protein) were identified using the SPOTS epitope mapping technique (Sigma Genosys). For technical details, see Sigma GenosysCustora SPOTs Techfzical Manual, v.l.l (available at http://www.genosys.com).

Following the inventors' instructions, Sigma Genosys synthesized a custom library of 13-mer peptides with seven amino acid overlaps that were covalently bound to derivatized cellulose membranes. The sequences of 122 peptides formed all the proteins shown in Table 11. The membrane was used in a colorimetric assay for immunoreactivity. After free immobilized peptides are reconstituted, a second and subsequent antibody or antiserum is used. Thus, different patterns of peptides based on the same peptide substrate were obtained, which were used in quantitative and qualitative comparisons.

To visualize the spectra, the membrane was rinsed briefly with methanol, followed by three washes (10 min each) with Tris-buffered saline. Block overnight at room temperature with casein-containing blocking reagent provided by Sigma Genosys. After blocking, the membrane was washed with Tris-buffered saline containing Tween-20 (0.05%) and exposed to a 1:100 diluted pool of serum from 6 confirmed, smear-positive TB patients.

To identify reactive peptides, overlapping peptide pools were incubated with serum pools for 4 hours, washed and probed with a Ia:200 dilution of β-galactosidase-conjugated anti-human IgG. After incubation, the membrane was washed and contacted with the substrate of β-galactosidase (X-gal in N,N'-dimethylformamide (DMF)). Membranes can be reused after being thoroughly washed with deionized water (30 min, 3 changes) by placing the membrane in DMF and then washing with buffer A (urea 48% w/v, SDS, 1% w/v, and β-mercaptoethanol, pure reagent diluted 1/1000) and buffer B (50% ethanol/10% acetic acid (v/v)) for extraction. Previously deposited substrate was removed, the membrane blocked again and probed with sera from 6 healthy individuals with positive PPD skin tests. This serum pool was also diluted 1:100. And these 2 identical serum pools (TB patients and healthy people) were tested at double concentration (ie 1:50 dilution). As a control, membranes were probed with [beta]-galactosidase-conjugated anti-human IgG (not previously exposed to human serum) to identify peptides that bound non-specifically to the secondary antibody (or enzyme).

Based on the reactivity of 122 overlapping peptides (SEQ ID NO: 116-237) with serum pools, the following peptides were identified as strong immunogens in TB patients: Sequence ID No: 117; Sequence ID No: 126; Sequence ID No: 127 ;Serial ID: 128; Serial ID: 134; Serial ID: 135; Serial ID: 136; Serial ID: 137; Serial ID: 138; Serial ID: 154; Serial ID: 155; Identification Number: 170; Serial Identification Number: 172; Serial Identification Number: 191; Serial Identification Number: 216; and Serial Identification Number: 217.

These sequences included clearly recognized epitopes (2-5x) that were more strongly developed with the TB serum pool than with the control serum. These sequences are beyond the epitopes previously identified by computer-based algorithms and were found to be immunogenic in patients (see Example IX).

All documents cited above are hereby incorporated by reference whether or not expressly cited.

After the above detailed description of the present invention, it can be understood that without departing from the spirit and scope of the present invention, those skilled in the art can achieve the same purpose within a wide range of equivalent parameters, concentrations and conditions without appropriate experiments.

Table 11: Analysis of GlcB overlapping peptide sequences ^* sequence serial identification number sequence serial identification number sequence serial identification number sequence serial identification number 1. MTDRVSVGNLRIA 116 32. FGDATGFTVQDGQ 147 62. IHGLKASDVNGPL 178 92. LHYHQVDVAAVQQ 208 2. VGNLRIARVLYDF 117 33.FTVQDGQLVVALP 148 63. SDVNGPLINSRTG 179 93. DVAAVQQGLAGKR 209 3. ARVLYDFVNNEAL 118 34. QLVVALPDKSTGL 149 64. LINSRTGSIYIVK 180 94. QGLAGKRRATIEQ 210 4. FVNNEAALPGTDID 119 35. PDKSTGLANPGQF 150 65. GSIYIVKPKMHGP 181 95. RRATIEQLLTIPL 211 5. LPGTDIDPDSFWA 120 36. LANPGQFAGYTGA 151 66. KPKMHGPAEVAFT 182 96. QLLTIPLAKELAW 212 6. DPDSFWAGVDKVV 121 37. FAGYTGAAESPTS 152 67. PAEVAFTCELFSR 183 97. LAKE LA WAP DEIR 213 7. AGVDKVVADLTPQ 122 38. AAESPTSVLLINH 153 68. TCELFSRVEDVLG 184 98. WAPDE IREEVDNN 214 8. VADLTPQNQALLN 123 39. SVLLINHGLHIEI 154 69. RVEDVLGLPQNTM 185 99. REEVDNNCQSILG 215 9. QNQALLNARDELQ 124 40. HGLHIEILIDPES 155 70. GLPQNTMKIGIMD 186 100.NCQSILGYVVRWV 216 10. NARDELQAQIDKW 125 41. ILIDPESQVGTTD 156 71. MKIGINDEERRTT 187 101. GYVVRWVDQGVGC 217 11. QAQIDKWHRRRVI 126 42. SQVGTTDRAGVKD 157 72. DEERRTTVNLKAC 188 102. VDQGVGCSKVPDI 218 12. WHRRRVIEPIDMD 127 43. DRAGVKDVILESA 158 73. TVNLKACIKAAAAD 189 103. CSKVPDIHDVALM 219 13. IEPIDMDAYRQFL 128 44. DVILESAITTIMD 159 74. CIKAAADRVVFIN 190 104. IHD VALMEDRATL 220 14. DAYRQFLTEIGYL 129 45. AITTIMDFEDSVA 160 75. DRVVFINTGFLDR 191 105. MEDRAT LRIS SQL 221 15. LTEIGYLLPEPDD 130 46. DFEDSVAAVDAAD 161 76. NTGFLDRTGDEIH 192 106. LRIS SQL LAN WLR 222 16. LLPEPDDDFTITTS 131 47. AAVDAADKVLGYR 162 77. RTG DEIHT SMEAG 193 107. LLANWLRHGVITS 223 17. DFTITTSGVDAEI 132 48. DKVLGYRNWLGLN 163 78. HTSMEAGPMVRKG 194 108. RHGVITSADVRAS 224 18. SGVDAEITTTAGP 133 49. RNWLGLNKGDLAA 164 79. GPMVRKGTMKSQP 195 109. SADVRASLERMAP 225 19. ITTTAGPQLVVPV 134 50. NKGDLAAAVDKDG 165 80. GTMKSQPWILAYE 196 110. SLERMAPLVDRQN 226 20. PQLVVPVLNARFA 135 51. AAVDKDGTAFLRV 166 81. PWILAYEDHNVDA 197 111. PLVDRQNAGDVAY 227 21. VLNARFALNAANA 136 52. GTAFLRVLNRDRN 167 82. EDHNVDAGLAAGF 198 112. NAGDVAYRPMAPN 228 22. ALNAANARWGSLY 137 53. VLNRDRNYTAPGG 168 83. AGLAAGFSGRAQV 199 113. YRPNAPNFDDSIA 229 23. ARWGSLYDALYGT 138 54. NYTAPGGGQFTLP 169 84. FSGRAQVGKGNWT 200 114. NFDDSIAFLAAQE 230 24. YDALYGTD VIPET 139 55. GGQFTLPGRSLMF 170 85. VGKGMWTMTELMA 201 115. AFLAAQELILSGA 231 25. TDVIPETDGAEKG 140 56. PGRSLMFVRNVGH 171 86. TMTELMADMVETK 202 116. ELILSGAQQPNGY 232 26. TDGAEKGPTYNKV 141 57. FVRNVGHLMTNDA 172 87. ADMVETKIAQPRA 203 117. AQQPNGYTEPILH 233 27. GPTYNKVRGDKVI 142 58. HLMTNDAIVDTDG 173 88. KIAQ PRAGASTAW 204 118. YTEPILHRRREF 234 28. VRGDKVIAYARKF 143 59. AIVDTDGSEVFEG 174 89. AGASTAWVPSPTA 205 119. HRRRREFKARAAE 235 29. IA YARK FLD SVP 144 60. GSEVFEGIMDALF 175 90. WVPSPTAATLHAL 206 120. FKARAAEKPAPSD 236 30. FLDDSVPLSSGSF 145 61. GIM DAL FT GLIAI 176 91. AATLHALHYHQVD 207 121. EKPAPSDRAGDDA 237 31. PLSSGSFGDATGF 146 62. FTGLIAIHGLKAS 177

^* Serum-reactive peptides are marked in bold and larger font

Claims (21)

1. antigen composition, said composition is used for the earlier detection of Mycobacterium tuberculosis disease or infection or is used for the host immune of tuberculosis mycobacterium, and compositions comprises:

(a) a kind of peptide, it is selected from:

(1) CGTDGAEKGPTYNKVRGDK, (sequence recognition number: 108);

(2) KIGIMDEERRTTVNLKAC, (sequence recognition number: 109);

(3) ELAWAPDEIREEVDNNC, (sequence recognition number: 110);

(4) LHRRRREFKARAAEKPAPSDRAG, (sequence recognition number: 111);

(5) ARDELQAQIDKWHRRR, (sequence recognition number: 112);

(6) LNRDRNYTAPGGGQ, (sequence recognition number: 113);

(7) GAPQLGRWKWHDPWV, (sequence recognition number: 114);

(8) VGNLRIARVLYDF (sequence recognition number: 117);

(9) QAQIDKWHRRRVI (sequence recognition number: 126);

(10) WHRRRVIEPIDMD sequence recognition number: 127);

(11) IEPIDMDAYRQFL (sequence recognition number: 128);

(12) ITTTAGPQLVVPV (sequence recognition number: 134);

(13) PQLVVPVLNARFA (sequence recognition number: 135);

(14) VLNARFALNAANA (sequence recognition number: 136);

(15) ALNAANARWGSLY (sequence recognition number: 137);

(16) ARWGSLYDALYGT (sequence recognition number: 138);

(17) SVLLINHGLHIEI (sequence recognition number: 154);

(18) HGLHIEILIDPES (sequence recognition number: 155);

(19) GGQFTLPGRSLMF (sequence recognition number: 170);

(20) FVRNVGHLMTNDA (sequence recognition number: 172);

(21) DRVVFINTGFLDR (sequence recognition number: 191);

(22) NCQSILGYVVRWV (sequence recognition number: 216); And

(23) GYVVRWVDQGVGC (sequence recognition number: 217);

Precondition is that described compositions is not to have the full length protein that sequence recognition number is 106 or 107 sequence.

(b) mutant or the functional derivatives of peptide in a kind of (a), it has the reactivity with described GlcB or MPT51 specific antibody; Or

(c) mutant of the peptide of (1)-(23) or described (b) or two or more mixture in the functional derivatives in any described (a).

2. according to the antigen composition of claim 1, it is a fused polypeptide, comprising:

(a) one or more described peptide (1)-(23) or described mutants, it links to each other with (b)

(b) one or more protein are selected from sequence recognition number 106, sequence recognition number: 107 and another kind of early stage Mtb antigen;

Wherein fused polypeptide comprises an optional connector or a plurality of connector, and this connector is used to connect any two or more described protein or peptides.

3. according to the antigen composition of claim 1, it is;

(a) peptide multimer has general formula; P ¹ _n

Wherein, P ¹Be any or its alternative mutant in peptide (1)-(23), and n=2-8,

(b) peptide multimer has general formula: (P ¹-X _m) _n-P ²

Wherein, P ¹And P ²Be the alternative mutant of any or its conservative in peptide (1)-(23), wherein

(i) P ¹And P ²Can be identical or different; And, P ¹-X _mEach P in the structure ¹Can be peptide or the mutant different with adjacent peptide;

(ii) X is

(A) C ₁-C ₅Alkyl, C ₁-C ₅Thiazolinyl, C ₁-C ₅Alkynyl, contain the C of 4 oxygen at most ₁-C ₅Polyethers, m=0 or 1 wherein, n=1-7; Or

(B) Glyz z=1-6 wherein, and

Wherein peptide multimer and described GlcB or the proteinic specific antibody of MPT51 react.

4. according to the antigen composition of claim 1, it has general formula for the reorganization peptide multimer

(P ¹-Gly _z) _n-P ²

Wherein, P ¹And P ²Be the alternative mutant of any or its conservative in peptide (1)-(23), and

(a) P ¹And P ²Can be identical or different; And, P ¹-Gly _zEach P in the structure ¹Can be to be adjacent different peptide of peptide or mutant;

(b) n=1-100, z=0-6; And

Wherein peptide multimer and described GlcB or the proteinic specific antibody of MPT51 react.

5. antigen composition that is used for the earlier detection of Mycobacterium tuberculosis disease or infection, it comprises one or more peptides, exist or be connected on peptide multimer or the fused protein with form of mixtures, described one or more peptides are derivants of early stage antigen of mycobacterium tuberculosis fragment sequence or have this antigen fragment sequence, and have following feature:

(i) react with lunger's internal antibody, the residing stage of this patient is early than the beginning of the expectorant smear positive and pulmonary cavitary pathological changes;

(ii) the serum with normal healthy controls person or preclinical inactive tuberculosis healthy person does not react; Described compositions significantly is different from other mycobacterium tuberculosis protein matter, and these protein do not have the feature of above-mentioned described antigen of mycobacterium tuberculosis.

6. earlier detection method of the experimenter being carried out mycobacteria property disease or infection, the biological fluid sample that comprises the doubtful person of detected activity phase TB, to detect the specific antibody that whether has arbitrary antigen composition among the claim 1-5, the existence of wherein said antibody shows to have described disease or infection.

7. according to the method for claim 6, wherein, described antigen composition is the fused protein of claim 2.

8. according to the method for claim 6, wherein, described antigen composition is the peptide multimer of claim 3 or 4.

9. according to the arbitrary method among the claim 6-8, it further comprised the step that obtains the biological fluid sample from the experimenter before described detection.

10. according to arbitrary method among the claim 6-9, wherein said biological fluid sample is taken from and is had active stage pulmonary tuberculosis symptom but among the experimenter before pulmonary tuberculosis symptom in late period occurring, described late period, pulmonary tuberculosis was identified the acid-fast bacilli smear positive of (a) expectorant or the relevant liquid of other lung by following method, (b) empty pneumonopathy becomes, or (a) and (b).

11., before described detection step, also comprise the cross reactivity epi-position or the antigenic specific antibody that are present in mycobacterium tuberculosis and other mushroom removed from sample according to the arbitrary method among the claim 6-10.

12. according to the method for claim 11, wherein said removing is with E.coli antigen described sample to be carried out immunoadsorption.

13. according to the arbitrary method among the claim 6-12, it comprises further whether the described sample of detection exists the specific antibody of one or more other mycobacterium tuberculosis early antigen, and described antigen is selected from:

(a) mycobacterium tuberculosis protein GlcB albumen, it has sequence recognition number: 106 aminoacid sequence;

(b) antigen of mycobacterium tuberculosis MPT51 has sequence recognition number: 107 aminoacid sequence;

(c) a kind of feature is with the protein of antigen of mycobacterium tuberculosis 85C;

(d) a kind of feature is with the glycoprotein of antigen of mycobacterium tuberculosis MPT32; And

(e) comprise the fusion rotein of one or more (a)-(d).

14. according to the arbitrary method among the claim 6-13, wherein said experimenter is human.

15. according to the method for claim 14, wherein said experimenter infects to be had HIV-1 or is phthisical high-risk colony.

16. according to the arbitrary method among the claim 6-15, wherein said biological fluid sample is serum, urine or saliva.

17. according to the method for claim 16, wherein said biological fluid sample is urine.

18. according to the arbitrary method among the claim 6-17, it further is included in the sample of described experimenter's expectorant or other body fluid and detects mycobacteria.

19. a test kit that is used for the mycobacterium tuberculosis earlier detection comprises:

(a) any antigen composition among the claim 1-5, with

(b) be used to detect the necessary reagent of the antibody that combines with described peptide.

19. the test kit of claim 18, it further comprises the early antigen of one or more mycobacterium tuberculosis.

20. the test kit of claim 19, wherein said one or more early antigen are selected from:

(a) mycobacterium tuberculosis protein GlcB albumen has sequence recognition number: 106 aminoacid sequence;

(b) mycobacterium tuberculosis MPT51, it has sequence recognition number: 107 aminoacid sequence;

(c) with the identical protein of antigen of mycobacterium tuberculosis 85C feature;

(d) with the identical protein of antigen of mycobacterium tuberculosis MPT32 feature; And

(e) comprise the fusion rotein of one or more (a)-(d).

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