WO1996015451A1 - Methods of monitoring immune responses to hiv during treatment of secondary infections - Google Patents

Abstract

The present invention provides methods of monitoring an immune response against a first infectious agent, typically a virus. The methods comprise administering a pharmaceutical composition which inhibits growth of a second infectious agent to a patient infected by the first infectious agent and the second infectious agent (usually a bacterium), wherein the second infectious agent induces a predominantly humoral response. A decrease or an increase in a humoral response against the first infectious agent is then detected, thereby detecting a shift towards a cell mediated response against the first infectious agent.

Description

METHODSOFMONITORINGIMMUNERESPONSESTOHIVDURINGTREATMENTOF SECONDARY INFECTIONS

BACKGROUND OF THE INVENTION The present invention relates to therapeutic strategies in treating microbial infections. In particular, the invention relates to redirecting the immune response in virally infected individuals from a predominantly antibody response to a protective cell-mediated response.

One approach for the treatment or prevention of various viral infections is to design therapeutic strategies that effectively harness the body's own immune defense against the virus. The immune response can be broadly categorized into two different types: 1) antibody or humoral response and 2) cell-mediated response. In general, the humoral immune response (HI) is typically associated with immunity to extracellular pathogens, e.g. bacteria, while the cell- mediated immunity (CMI) is closely associated with intracellular pathogens such as viruses.

In many infections, cell-free pathogens or toxins (for example, influenza, polio, and rabies viruses; pneu ococcus bacteria; diphtheria; and tetanus toxins) can be effectively neutralized in the circulation or extracellular fluid by the humoral arm of the immune system through antibodies that bind to the pathogens or toxins and thereby lead to their inactivation or destruction. However, for many intracellular pathogens, such as viruses, cell-mediated immunity (CMI) protects against invaders. In fact, a limited infection ensues when such pathogens induce stable cell- mediated immunity. On the other hand, chronic, progressive, or fatal disease will occur if an antibody is produced and the cell-mediated response declines.

Despite tremendous efforts, the prior art has failed to develop effective treatments for many viral infections. The present invention solves these and other problems in the art.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and 1C show the results of CMI assays in which peripheral blood ononuclear cells (PBMCs) from two patients (1030 and 1030A) were stimulated with recall antigens (flu and tet) , alloantigens, and phytohemagglutinin (PHA) .

Figures IB and ID show the results of CMI assays in which PBMCs from the same two patients were stimulated with HIV envelope peptides.

Figures 2A and 2C show the results of CMI assays in which PBMCs from two patients (1054 and 1054A) were stimulated with recall antigens (flu and tet) , alloantigens, and PHA. Figures 2B and 2D show the results of CMI assays in which PBMCs from the same two patients were stimulated with HIV envelope peptides.

Figures 3A and 3C show the results of CMI assays in which PBMCs from two patients (2045 and 2045A) were stimulated with recall antigens (flu and tet) , alloantigens, and PHA.

Figures 3B and 3D show the results of CMI assays in which PBMCs from the same two patients were stimulated with HIV envelope peptides.

Figures 4A and 4C show the results of CMI assays in which PBMCs from two patients (1055 and 1055A) were stimulated with recall antigens (flu and tet) , alloantigens, and PHA.

Figures B and 4D show the results of CMI assays in which PBMCs from the same two patients were stimulated with HIV envelope peptides.

SUMMARY OF THE INVENTION The present invention provides methods of monitoring an immune response against a first infectious agent, typically a virus. The methods comprise administering a pharmaceutical composition which inhibits growth of a second infectious agent to a patient infected by the first infectious agent and the second infectious agent (usually a bacterium) , wherein the second infectious agent induces a predominantly humoral response. A decrease or an increase in a humoral response against the first infectious agent is then detected, thereby detecting a shift towards a cell mediated response against the first infectious agent. Typically, the humoral response against the first or second infectious agent is measured by detecting antibodies to the first infectious agent in a sample, usually a urine sample, from the patient. The results of these assays can be confirmed by detecting an increase or decrease in a cell mediated immune response against the first infectious agent.

If the first infectious agent is human immunodeficiency virus (HIV) , antibodies are preferably detected using a recombinantly expressed HIV protein, e.g., gpl60 expressed in insect cells. The bacterium is usually one associated with sexually transmitted diseases, e.g., Chlamydia trachomatis or Neiεseria gonorrhoeae . The pharmaceutical composition used to treat the second infection will usually be an antibiotic.

The present invention further provides methods of monitoring an infection in an individual lacking detectable serum antibodies against an agent responsible for the infection. These methods comprise monitoring antibodies to the agent in a urine sample from the individual until antibodies to the agent are not detectable. In these methods the agent responsible for the infection may be a bacterium associated with sexually transmitted disease such as Chlamydia trachomatis or may be HIV. Where antibodies to HIV are detected with a recombinantly expressed HIV protein such as gplβO expressed in insect cells may be used. The results of the methods are confirmed by detecting a cell mediated immune response against the agent.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides methods for redirecting an immune response to enhance responses effective in treating viral infections. The invention is based in part on the discovery that directing an immune response from a primarily humoral response towards a predominantly cell mediated response can effectively control viral infection, in particular HIV infection.

There are two types of T lymphocytes: T killer cells/cytotoxic T lymphocytes (CTLs) and T helper cells. Cell-mediated responses involve the activation of CTLs that react with antigens displayed on the surface of host cells and kill the infected host cell. T helper cells, on the other hand, release various lymphokines that activate other lymphocytes (e.g., B cells or CTLs) to fight an infection. T helper cells can be further categorized into two subsets termed T helper 1 (Thl) and T helper 2 (Th2) based on their different functions and the cytokines they produce. Though originally defined in the mouse, these T helper subsets have been identified in humans as well (G.F. Del Prete et al. (1991), J. Clin . Invest tJ8.:346; S. Romangnani, (1991) Immunol. Today 12.:256; and H. Yssel et al. (1991) J. Exp . Wed., 174:593.. In the following discussion, human helper T cells are referred to simply as Thl and Th2 cells. One of skill will recognize, however, that human helper T cell subsets are more properly referred to as Thl-like and Th2-like cells.

Thl cells produce IL-2, 7-interferon (γ-IFN) , and lymphotoxin (LT) , but not IL-4 (BSF-1) or IL-5. Thl cells mediate helper cell functions associated with CTLs, delayed- type hypersensitivity (DTH) and macrophage activation. IL-2 produced by Thl cells plays a major role in the activation and differentiation of CTL precursors into mature CTL effectors. Th2 cells reciprocally produce IL-4, IL-5, and IL-10, and are more effective at stimulating B cells to produce antibodies. Other lymphokines, including GM-CSF and IL-3, are produced either equivalently by Thl or Th2 cells, or preferentially but not exclusively by one or the other population, (T.R. Mosmann, et al. (1986) J^". Immunol . 136:23481. Some of the lymphokines produced by T helper cells are known to have selective activities on the induction of specific classes or subclasses of Ig response (A. O'Garra et al., (1988) Immunol. Today £:45) For instance, lymphokines secreted by Th2 in particular stimulate IgM, IgGl, IgA and IgE classes. As noted above, CMI is generally more effective in fighting intracellular pathogens such as viruses. Examples of infections in which CMI appears to be more effective in fighting infection include viral infections (e.g., human immunodeficiency virus, Epstein-Barr virus) , spirochetal infection of syphilis, protozoan infections of cutaneous and visceral leishmaniasis.

The induction of protective cell-mediated immune responses and of non-protective antibody responses in some infectious and parasitic diseases can be understood in terms of Thl and Th2 cell subsets. The tendency for either the cell-mediated or the antibody response to predominate in any particular immune response is thought to result from cross- regulation, whereby Thl cells (or other coordinately induced cells) inhibit the induction of Th2 responses, probably by production of IFN-7, and Th2 cells (or other coordinately induced cells) inhibit the generation of Thl responses through production of cytokines such as IL-4 and IL-10.

Infections by different microorganisms lead to either a predominantly humoral or cell mediated immune responses. A high percentage of the cells of the immune system are located in the mucosal tissue which is continually accessed by the associated lymphoid tissue and peripheral circulation. The milieu of infectious agents (e.g., bacteria and viruses) at the mucosal sites can create an environment that influence both cellular and humoral mediated immunity. Conditions occur that elicit cytokines that promote the elimination of one type of organism but fail to resolve a second infection due to the mutually exclusive nature of CMI and HI responses in a given environment. Thus, interactive infections can elicit an immune response which is ineffective in eliminating a secondary infection.

Recent observations suggest that apparently harmless, and possibly protective, encounters with human immunodeficiency virus (HIV) can occur. Some individuals who have been exposed to the virus and are therefore at high risk for HIV infection remain apparently uninfected. They do not have antibodies to HIV in their blood, and neither HIV nor its nucleic acids can be detected in blood samples. (M. Clerici et al. (1992) J . Infect . Dis . 164:178.

Peripheral blood ononuclear cells from seronegative, but HIV-exposed, individuals respond to HIV envelope antigens with a Thl-like response, that is, they produce IL-2. Such HIV-specific, apparently cell-mediated responses have been seen in gay men with known sexual exposure, intravenous drug users, health care workers exposed by accidental needle stick, and newborn infants of HIV- positive mothers. Moreover, as asymptomatic, HIV-seropositive individuals progress towards AIDS, their peripheral blood lymphocytes shift from a Thl-predominant to a Th2-predominant pattern of cytokine production. Thus, in HIV infection, progression towards AIDS is predisposed by a shift in general immunity from a Thl (CMI) phenotype to a Th2 (HI) phenotype. The problem in the art has been identifying ways to effectively redirect the immune response to a cell mediated response driven by Thl cells.

The present invention is based on the observation that secondary stimuli (typically a concomitant infection) in a subject with HIV or other viral infection can misdirect the immune system away from a CMI needed to resolve the HIV infection, to a HI. Immunologic stimuli unrelated to HIV infection that can promote a Th2 > Thl state may place an HIV negative individual who is exposed to the virus at increased risk for infection, or accelerate AIDS progression in an individual who is infected but asymptomatic. A concomitant bacterial infection is an example of a secondary stimulus that can induce a strong Th2 response. Another example of a secondary stimulus capable of inducing strong Th2 responses is Epstein Barr virus (EBV) which secretes BCRF-1, a compound homologous to IL-10. Secretion of BCRF-1 by the virus thus inhibits Thl responses.

Patients infected with HIV through sexual intercourse frequently also present coinfections of various pathogens, particularly microorganisms associated with sexually transmitted diseases such as Chlamydia trachomatis , Neisseria gonorrhoeae , herpes simplex virus, Haemophilus ducreyi, Caly∞natojacteriu-π granulomatis , Ureaplasma urealyticum, Mycoplasma spp. (e . g. M. hominis, M. genital iu , M. pirum, and M . fermentans) , Toxoplas a gondii, Actinomyces israelii, Campylobacter spp. , Treponema pallidu , Trichomonas vaginalis, and Candida albicans . Bacterial antigens present in the genital and urinary tract will induce a humoral response that could ultimately be deleterious if it predominates and reduces cell-mediated responses that are more effective in clearing HIV infections. In these patients, it would be desirable to redirect their immune responses from a Th2 to a Thl mode.

Endogenous retroviruses (ERVs) may be another important factor in the pathogenisis of viral infection and other diseases. In humans and other animals, proviral DNA can in some instances remain in the host germline DNA and be transmitted to subsequent generations as an endogenous retrovirus. After a number of generations the proviral DNA may undergo mutations so that it no longer encodes an infectious retrovirus. Such endogenous retroviruses have been implicated in a number of diseases such as cancer and autoimmunity (see, e.g., Rasmussen et al . Acta Neurol . Scand. 88:190-198 (1993) and Venables et al. Br. J. Rheum . 31:841- 846 (1992)).

Endogenous retroviruses are thought to be activated by a number of factors such as immune stimuli, steroid hormones, and chemical carcinogens. For instance, immune responses, especially inflammatory responses, lead to HERV expression (see, e.g., Rasmussen et al . , supra) . Some animals comprise ERVs capable of being expressed as infectious viral particles. Many human endogenous retorviruses (HERVs) are capable of protein expression and polypeptides encoded by HERVs have been isolated from human material.

For instance, U.S.S.N. 08/321,689 shows that the presence of anti-HERV antibodies is correlated with various diseases such as cancers, chronic renal failure, autoimmune diseases, and sexually transmitted diseases. The presence of these antibodies can thus be used as a useful marker for disease. More importantly, inhibition of the expression or activity of the HERV antigens can be used to treat disease. Without wishing to be bound by theory, it is believed that HERVs are improtant element in the pathogenesis of disease. In particular, HERV antigens having immunosuppressive abilities allow certain cells (e .g. , virally infected, neoplastic cells, self-reactive lymphocytes) to escape normal immune surveillance by cytotoxic T cells and other elements of the cell mediated immune system. In the absence of a mechanism for controlling their growth, the cells proliferate and the disease progresses. Polypeptide products of HERV gene expression, however, may have pleiotropic effects. Thus, in addition to immune suppression, the polypeptides may be more directly involved in the etiology of disease. For instance, neurotoxic HERV gene products may be involved in degenerative nervous system diseases. Alternatively, HERV gene expression may be a marker for rearrangement events which cause disease.

As noted above, inflammatory and other responses can lead to HERV expression. Thus, early treatment of bacterial infections which induce such responses can be used to prevent HERV expression and the diseases associated with that expression. Alternatively, as explained more fully below, if HERV expression is already induced in response to particular stimuli, the methods of the invention can be used to direct the patient's immune response towards a CMI response.

A number of HERV antigens have been identified. For instance, immunosuppressive polypeptides having sequences related to a retroviral transmembrane envelope protein from murine and feline leukemia viruses, pl5E, have been detected in human tissues using monoclonal antibodies (Snyder an et al . (Immunology Today 5:240-244 (1984)). A central hydrophilic 26 amino acid region of pl5E is thought to be important to the immunosuppressive properties of this protein (Schmidt et al . Proc . Natl . Acad . Sci . USA 84:7290 (1987)). This region of the protein has some sequence identity with sequences from HTLV, HIV, as well as human cytokines (Foulds et al . Br. J. Cancer 68:610-616 (1993) and Haraguchi et al. J. Leukocyte Biol . 52:469 (1992)). Cianciolo et al . Science 230:453-455 (1985) describe a number of sythentic peptides homologous to retorviral envelope proteins, inluding pl5E (CKS-17 and 4-1) .

As noted above, the methods of the present invention can be used to shift the immune response to a CMI response and thereby inhibit viral replication and/or HERV expression.

The data presented here show that minimizing the contribution of an HI inducing organism, which misdirects the immune system, will allow inibibition of HERV expression or resolution of a viral infection (e.g. HIV infection) through a CMI response. The invention thus provides methods of switching the immune system to a protective cell mediated response by inhibiting a humoral response in interactive infections. In preferred embodiments, a concomitant bacterial infection can be treated with standard therapies to reduce bacterial infection. Such therapies include antibiotics (e.g. , doxycycline, reciphin, tetracycline, erythromycin, amoxycilin, cefotaxime and the like) . EBV and other viral infections can be treated using standard antiviral agents such as acyclovir, interferon a , and ganciclovir. Treatment regimes useful for treating various bacterial and viral infections are well known to the clinician and are described for instance in, Harrison ' s Principles of Internal Medicine , Wilson et al . , eds. 12th ed. (McGraw-Hill New York, 1991, see, e.g., Chapters 85 and 86). Other approaches to inhibit a Th2 driven response is the use of antibodies and other compounds which bind lymphokines secreted by Th2 cells and thus block the inhibitory action of these compounds on Thl cells. For instance the administration of anti-IL-4 or anti-ILlO monoclonal antibodies could be used to induce CMI. Other compounds that could be used to block the action of these lymphokines include solubilized IL-4 or IL-10 receptors.

The invention also provides methods for monitoring the progression of disease or the efficacy of treatment by determining whether a cellular or humoral response is predominant. Typically, this is achieved by determining antibody levels in serum or other bodily fluids such as urine, saliva, cerebrospinal fluid, semen, and the like. In the case of HIV and concomitant bacterial infections, determination of urine antibodies is particularly preferred. A variety of techniques for detecting antibodies can be used (e.g., western blots, enzyme im unoassays) . See , Harlow and Lane Antibodies: A Laboratory Manual (Cold Spring Harbor Publications, N.Y. , 1988) for a description of suitable methods.

In preferred assays, the determination of HIV antibodies is carried out using HIV antigens in which conformational epitopes are preserved. The results presented below demonstrate that in some individuals, western blots give negative results but assays using recombinantly produced proteins (e.g. gpl60) which retain protein conformation are able to detect the presence of HIV antibodies. Thus, these assays are a more sensitive assay for HIV infection and provide a more accurate method to monitor infection.

Methods for the isolation of DNA sequences encoding HIV antigens such as gpl60 and the expression of these sequences in a variety of expression systems is well known to those of skill in the art. One of skill will recognize that the desired polypeptides can be expressed in recombinantly engineered cells such as bacteria, yeast, insect (especially employing baculoviral vectors) , and mammalian cells. As described in detail, below, expression in insect cells is particularly preferred. In brief summary, the expression of natural or synthetic nucleic acids encoding the desired antigenic protein will typically be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible) , followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the polynucleotide sequence encoding the polypeptides. For a general description of the recombinant expression of cloned genes see Sambrook, et al . , Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. A variety of methods for the administration of antibiotics and other antimicrobial agents can be used to redirect the immune response. The particular antibiotic and the method of administration are not critical to the invention. Suitable formulations for use in the present invention are found in Remington ' s Pharmaceutical Sciences , Mack Publishing Company, Philadelphia, PA, 17th ed. (1985) .

The pharmaceutical compositions can be used for single administrations or a series of administrations and may be intended for parenteral, topical, oral or local administration. In the case of treatments for infection by an organism associated with STDs, the pharmaceutical compositions are preferably administered orally or directly to the genital tract. The amount administered to the patient will vary depending upon what is being administered, the state of the patient and the manner of administration. In therapeutic applications, compositions are administered to a patient already suffering from a disease in an amount sufficient to at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient. For instance, for the treatment of chlamydial infection, antibiotics will generally be administered in the range of about 100 mg to about 500 mg two to four times per day for 7 to 14 days.

The following examples are provided to illustrate, but not limit, the present invention.

EXAMPLE 1 This example presents data from clinical studies showing the antibiotic treatment of secondary bacterial infections in HIV infected individuals and its effect on redirecting the immune response towards a response effective in eliminating an HIV infection. Subjects attending an obstetrics/gynecology clinic were diagnosed for various disorders and treated with antibiotics for bacterial infections. Their urine was tested for antibodies to either C. trachomatis or HIV-1 as described below.

Test for Chlamydia trachomatis Antibodies

Infection by C. trachomatis was determined by testing urine and serum samples for the presence of antibodies to the pathogen using western blots as follows. A. Materials and Methods for preparation of C. trachomatis immunoblots.

1) Preparative gels (polyacrylamide-SDS slab gels; Bio-Rad Mini-Protean II Dual Slab Cell system) each consisting of a 12% T resolving gel and a 4% T stacking gel were prepared according to standard procedures.

2) C. trachomatis LGV2 Strain 434 containing 10¹⁰ elementary bodies/ml [Biodesign, Inc.] were diluted to 0.75 mg/ l (both lots) with 2X Chlamydia Sample Buffer [20% glycerol; 2.5% SDS; 0.1 M Tris-HCl; 5% 2-mercaptoethanol; 0.005% bromophenol blue; and heated at 100C for 10 minutes.

3) Prestained Protein Molecular Weight Standards 14.3K-200K MW (Gibco BRL] mixture of 7 proteins) was diluted to 3.5 mg/ml (0.5 mg/ml each protein) with 2X Chlamydia Sample Buffer (formula cited above in A.2) and heated at 100C for 5 minutes.

4) The preparative gels were each loaded with 100 μg denatured C. trachomatis (preparative lane) and 17.5 μg denatured Gibco BRL standard (reference lane) . These samples were electrophoresed in a discontinuous buffer system [0.025 M Tris; 0.192 M glycine; 0.1% SDS, pH 8.4].

5) Following polyacrylamide gel electrophoresis (PAGE) , the reference lane portion and approximately 2 mm of the preparative lane portion of each preparative gel were separately stained with 0.3% Coo assie brilliant blue R250 (placed on rocking platform for 2 hours, room temperature) and then deβtained with destainer containing 22.5% isopropyl alcohol and 10% glacial acetic acid. 6) Following PAGE, the remaining preparative lane portions of the preparative gels (after A.5) were equilibrated separately in IX blot buffer [0.025 M Tris; 0.192 M glycine; 20% methanol, pH 8.3] for approximately 20 minutes, then transferred for 80-90 minutes (112 volts, 0.4 amperes) to Immobilon-PVDF transfer membranes, pore size 0.45 μ (Millipore) .

7) Following electrophoretic transfer, each transfer membrane was cut into 2 mm strips [yielding 20-25 immunoblots (approx. 4-5 μg C. trachomatis protein per immunoblot) ] and blocked separately with 3% each of bovine, equine and goat serum in TBS containing 0.1% sodium azide (placed on a rocking platform for 1.5 hours, room temperature) . C. trachomatis blots were then stored at 2-8 degrees C in TBS containing 0.1% sodium azide (each set of blots derived from a single transfer membrane) . Following electrophoretic transfer each preparative lane (gel) was stained and destained according to instructions in A.5.

B. Western blot procedures were performed on C. trachomatis immunoblots as briefly outlined below:

1) A purified IgG_x monoclonal antibody to MOMP [Biodesign International (100 μg/ml) ] was used to identify the location of the 39.5K MW MOMP on C. trachomatis immunoblots.

2) Monoclonal antibody to MOMP must be run as necessary with each set of immunoblots (each set of immunoblots derives from a single transfer membrane) . 3) C. trachomatis immunoblots were placed in alternate troughs on a rocking platform at room temperature.

4) C. trachomatis immunoblots were washed (followed by aspiration) with 1 ml TBS/Tween (50mM Tris; 200 mM NaCl; 0.3% Tween-20, pH 7.2 +/- 0.2) for at least 2 minutes 4 times.

5) 0.25 ml Western Blot (WB) Sample Diluent (3% each of bovine, equine and goat serum plus 0.01% each of bovine IgG coated beads, equine IgG coated beads and goat IgG coated beads plus 0.1% NP-40 in TBS containing 0.1% sodium azide) was prepared for dilution of samples.

6) Sample incubation procedure: a. 3 sets of C. trachomatis immunoblots were utilized: [each set derives from gels loaded with C. trachomatis at 100 μg/gel (approximately 4-5 μg/immunoblot) ] . b. Reagent blanks were included with each of the 3 sets of C. trachomatis immunoblots (0.5 ml WB Sample Diluent per trough) . Note that goat anti-human

(GAH) conjugate (see B. 8a) was utilized in connection with these reagent blanks. c. Human positive serum was diluted 1/100 with WB Sample Diluent in a polypropylene tube and transferred to troughs as applicable (0.5 ml per trough) . Note that 1/100 human positive serum was included with each of the 3 sets of C. trachomatis immunoblots. d. Normal mouse serum was diluted 1/200 with WB Sample Diluent in a polypropylene tube and transferred to troughs as applicable (0.5 ml per trough). Note that 1/200 normal mouse serum was included with each of the 3 sets of C. trachomatis immunoblots. e. Monoclonal antibody to C. trachomatis MOMP was diluted 1/5 (20 μg/ml) with WB Sample Diluent in a polypropylene tube and transferred to troughs as applicable (0.5 ml per trough). Note that 20 μg/ml MOMP monoclonal was included with each of the 3 sets of C. trachomatis immunoblots. f. 0.25 ml of urine samples were added to troughs containing 0.25 ml WB Sample Diluent for a 1/2 dilution of each sample, g. Samples were incubated with C . trachomatis immunoblots overnight at room temperature on a rocking platform (15 hours) .

7) Following sample incubation, samples were aspirated from the troughs and immunoblots were washed as stated in B.(4). 8) Conjugate preparations: a. Alkaline phosphatase conjugated affinity purified goat anti-human (GAH) IgG/IgM (H+L) [Jackson Laboratories] was diluted 1/2000 with 5% goat serum in TBS containing 0.1% sodium azide. 0.5 ml of

1/2000 GAH conjugate was added to each immunoblot previously incubated with a urine sample, human positive serum sample or a reagent blank. b. Alkaline phosphatase conjugated affinity purified goat anti-mouse (GAM) IgG (H+L) [Jackson

Laboratories] was diluted 1/2000 with 5% goat serum in TBS containing 0.1% sodium azide. 0.5 ml 1/2000 GAM conjugate was added to the immunoblots incubated with the MOMP monoclonal (see B. 6e) or normal mouse serum (see B. 6d) .

9) Immunoblots were incubated with conjugate for 1 hour. Following incubation, conjugate was aspirated from the troughs.

10) Immunoblots were washed two times as stated in B.4. 11) Immunoblots were washed two more times as stated in B.4 but using IX Substrate Buffer for alkaline phosphatase [prepared by diluting 10X Substrate Buffer (Zymed) to IX with high purity water] .

12) 0.5 ml of substrate solution [0.05 mg/ml BCIP plus 0.1% NBT in IX Substrate Buffer for alkaline phosphatase (see

B.13)] was added to each immunoblot.

13) Immunoblots were incubated with substrate solution for 10 minutes.

14) Substrate solution was aspirated and immunoblots were washed (followed by aspiration) with l ml high purity water for at least 2 minutes 4 times. 16) Immunoblot trays were covered and air-dried for a minimum of 1 hour before recording results.

Test for HIV-1 Antibodies

HIV-l infection was determined by the detection of antibodies to the virus in urine and serum, using enzyme immunoassay (EIA) procedures. For confirmation, specimens repeatedly reactive in the EIA were further tested by a western blot procedure. All serum and urine specimens were handled by established, good laboratory working practices and the CDC guidelines for working with HIV material. (Biosafety in Microbiological and Biomedical Laboratories, U.S. Department of Health and Human Services, 1988, Publication No. (NIH) 88-8395) .

1. Enzvme Immunoassay The EIA employed an HIV-1 recombinant envelope glycoprotein, gpl60, expressed in insect cells using standard techniques. Insect cells with appropriate vectors, usually derived from the SF9 baculovirus, are typically used for expressing the glycoprotein, generally as described in WO 92/22654.

The recombinant envelope protein was then adsorbed onto the wells of microwell strip plates and used in assays generally as follows. Sample buffer and urine specimens or urine controls were added to the wells and incubated. Antibodies in the samples were visualized using a conjugate, consisting of alkaline phosphatase chemically bound to goat antihuman immunoglobulin antibodies. P-nitrophenylphosphate (PNPP) , the substrate for the enzyme, was added to all wells and incubated. If antibodies to HIV-1 were present in the sample, the enzyme produced a color change from colorless to yellow. The intensity of the color was determined to be proportional to the amount of HIV-1 antibodies present in the test sample. The reaction was terminated by the addition of a stop solution containing ethylenediaminetetraacetic acid (EDTA) . The absorbance values are determined spectrophotometrically with a plate reader at a wavelength of

405 run.

The following reagents were used. Sample Buffer - goat IgG, bovine IgG, horse IgG, or non- reactive mouse monoclonal antibody 0.01% v/v, 9% serum (3% horse, 3% bovine, 3% goat) in 0.05M Tris-HCl buffer pH 7.2 and 0.15 M sodium chloride. If desired, the immunoglobulins can be coated on glass beads.

Positive Control - Human urine containing antibody to HIV-1 and 0.1% sodium azide as preservative.

Negative Control - Human urine containing 0.1 % sodium azide as preservative. Conjugate Solution - Alkaline phosphatase labeled goat anti¬ human immunoglobulin in Tris buffered saline containing bovine serum albumin and less than 0.1% sodium azide as preservative. Substrate Solution - P-nitrophenylphosphate (PNPP) in diethanolamine buffer, containing magnesium chloride and 0.1% sodium azide as preservative.

Stop Solution - Solution containing ethylenediaminetetraacetic acid (EDTA) .

Wash Solution dOX. - Tris-buffered saline containing NP-40 and 1.0% sodium azide as preservative. Specimens were collected in accordance with approved standards. Specimens excessively contaminated with bacteria, blood, or sediment were not used because of inconsistent test results. The urine samples were refrigerated at 2-8C and were tested as soon after collection as possible. Specimens to which a urine preservative has been added may be stored at room temperature (15-30C) . For instance, urine to which Stabilur urine preservative (R.P. Cargille Laboratories, Inc., Cedar Grove, NJ) has been added can be used successfully in the assay. PROCEDURE

25 μl of Sample Buffer was added to each well. Next, 200 μl of each specimen or control was added to the appropriate wells. 2 positive and 3 negative controls were assayed with each plate or partial plate of test samples. The plates were incubated at 37C ± 1C for 60 minutes.

At the end of the incubation, the solution was completely aspirated from the wells by using a plate washer or a hand held aspirator connected to a vacuum source. The wells were then filled with IX Wash Solution and immediately aspirated. This step was repeated 5 times.

The plates were blotted by inverting on clean absorbent towels and 100 μl of Conjugate Solution was added to each well containing a specimen or control. Each plate was sealed and incubated at 37 ± 1C for 60 minutes.

At the end of the incubation, the Conjugate Solution was completely aspirated from the wells and the wells were washed with the IX Wash Solution. Next, 100 μl of Substrate Solution was added to each well containing a specimen or control. Each plate was covered and incubated at 37 ± 1C for

30 minutes.

At the end of the incubation, 50 μl of Stop Solution was added to each well containing a specimen or control. A microplate reader was used to read the plates at 405 run.

Plates were read within 30 minutes of adding the Stop

Solution.

The absorbance value of each Negative Control well was preferably not greater than 0.200. One Negative Control value was discarded if outside this range and the mean calculated by adding the two remaining absorbance values and dividing by 2. If two or more Negative Control values were greater than 0.200, the assay was determined to be invalid and was repeated. Each Positive Control had an absorbance value equal to or greater than 0.900. No Positive Control value was discarded. If any Positive Control value was less than 0.900 the assay was determined to be invalid and was repeated. The cutoff value for determining a sample was positive was 0.180 plus the mean absorbance of the Negative

Controls. Samples with absorbance values greater than or equal to the cutoff value were considered initially reactive.

All initially reactive samples were retested in duplicate using another aliquot of the original specimen. If after repeat testing, the absorbance values of both duplicate samples were less than the cutoff value, then the original specimen was considered to be a non-repeatable reactive sample and negative for antibodies. If after repeat testing, the absorbance value of one or both of the duplicates was equal to or greater than the cutoff value, the sample was considered repeatably reactive (RR) , and potentially positive for antibodies to HIV-1. 2. Western Blot

Bio-Rad Novapath HIV-1 Immunoblot Test (Bio-Rad, Inc. Richmond, California) was used generally according to manufacturer's instructions. In the assays, a test batch was defined as a set of no more than 10 or 30 immunoblot strips depending on the kit size utilized. A non-reactive serum kit control (NR) and 2 levels of reactive serum kit controls (Rl and R2) were included with each serum test batch.

Procedures for testing human serum or plasma were according to manufacturer's instructions. The methods were modified as follows, the working wash/diluent was prepared as needed at IX the day of use. In addition, the steps which succeed aspiration of the final water rinse from the troughs were as follows. The trays were covered with paper towels and strips were allowed to air-dry before recording results (strips generally require overnight air-drying) . When strips were dry, results were recorded on a Serum Western Slot Test Results chart according to the instructions.

For urine samples, a Negative (non-reactive) Urine Control, 2 levels of reactive urine controls, a non-reactive serum kit control (NR) and a weak reactive serum kit control (R2) were included with each urine test batch.

The manufacturer's instructions were followed with the following modifications. 2 ml Working Wash/Diluent was added to troughs to which serum kit controls were added. 1 ml Working Wash/Diluent was added to troughs to which the Negative Urine Control, Positive Urine Control or the urine test samples were added. 1.8 ml Working Wash/Diluent was added to the trough to which the Low Positive Urine control was added. The trays were covered and placed on a rocker for 5 minutes before adding patient or control samples. Trays were rocked at an angle of approximately 7 degrees at 15-18 cycles per minute. After rocking, 20 μl of NR (nonreactive) serum kit control was added to the small segment formed by the two ridges at the numbered end of a trough containing 2 ml of Working Wash/Diluent for a 1/100 dilution.

Using the same procedure, the following steps were solutions were added: 20 μl of R2 (weak reactive) serum kit control was added to a separate trough containing 2 ml of Working Wash/Diluent for a 1/100 dilution. 1 ml of Negative Urine Control was added to a trough containing l ml of Working Wash/Diluent for a 1/2 dilution, l ml of Positive Urine Control was added to a separate trough containing 1 ml of

Working Wash/Diluent for a 1/2 dilution. 0.2 ml of the Low Positive Urine Control was added to a trough containing 1.8 ml of Working Wash/Diluent for a 1/50 dilution. 1 ml of urine test samples was added to troughs containing 1 ml of Working Wash/Diluent for a 1/2 dilution of each urine test sample. The trays were covered and incubated on rockers overnight (18-22 hours) at room temperature (15-30C) . After incubation, the troughs were aspirated and 5 ml Working Wash/Diluent was added to each trough. The trays were recovered and incubated on rockers for 10 minutes at room temperature. The aspiration and incubation steps were repeated.

Troughs were then aspirated completely and 2 ml of Anti-Human IgG Enzyme Conjugate was added to each trough and incubated on rockers for 30 minutes at room temperature.

Troughs were aspirated again and 5 ml Working Wash/Diluent was added to each trough. Trays were incubated on rockers for another 10 minutes at room temperature. The aspiration and incubation steps were repeated. Troughs were aspirated completely and 2 ml Substrate

Solution was added to each trough and incubated on rockers for 10 minutes at room temperature. Troughs were aspirated, approximately 5 ml high purity water was added to each trough, and the trays were incubated on rockers for 5 minutes at room temperature. The aspiration and incubation steps were repeated.

The troughs were aspirated and the trays were covered with paper towels and strips were allowed to air-dry before recording results (strips generally require overnight air- drying) .

For quality control, the Negative (non-reactive) Urine Control must not exhibit any bands or background. The Low Positive Urine Control must exhibit the following band profile: The gpl20 band must be distinct but significantly less reactive than the gpl60 band. ENVELOPE bands must be typical in appearance, i.e. diffuse. The immunoblot must also exhibit at least 1 band representing gene products from each of the POLYMERASE (p65, p51, p32) and GAG (p55, p24, plβ) regions. These bands must exhibit reactivity equivalent to or exceeding the reactivity of the gpl20 band.

The Positive Urine Control typically exhibited reactivity exceeding that of the Low Positive Urine Control at the gpl60, gpl20, p65, p55, p51, gp41-43, p32, p24 and pl8 band positions. It also exhibited various non-specific bands. Serum kit controls NR and R2 were included with urine test batches as an additional check of test kit functionality. The NR serum kit control typically exhibited various immunoreactive bands and/or non-specific bands. Generally, the reactivity of these bands was weaker than that associated with the gpl20 band of immunoblots incubated with Low Positive Urine Controls. ENVELOPE region bands (gpl60, gpl20, gp41-43) , if present, were atypical in appearance (thin lines) . The NR serum kit control typically exhibited no bands on immunoblots. The R2 serum kit control typically exhibited reactivity at the gpl60, gpl20, p65, p55, p51, gp41-43, p32, p24, and pl8 band positions. These bands generally exceeded the reactivity associated with the gpl20 band of immunoblots incubated with Low Positive Urine Controls. The R2 serum kit control also exhibited some degree of reactivity at the pl5 band position as well as various non-specific bands.

The results of urine samples were interpreted and recorded as follows. A positive test result is indicated by reactivity to gplβo only and/or reactivity to any two of the following products: p24; gp41-43; gpl20/gpl60 or gpl60 only. The absence of a band is indicated by the notation NR on the Urine Western Blot Test Results chart. The presence of a band is indicated by the notation R on the Urine Western Blot Test Results. A negative test result is indicated by the absence of any bands on an immunoblot strip. An indeterminate test result is indicated by any pattern of one or more bands that does not meet the positive criteria.

Test for cell-mediated immunity to HIV-1

Peripheral blood ononuclear cells (PBMC) from subjects were tested for IL-2 response to phytohemagglutinin (PHA) , recall antigens, alloantigens and HIV-1 env peptides using the method described by Clerici et al. (1989) Nature 339:383. Briefly, PBMC were either unstimulated, or stimulated with influenza A virus, tetanus toxoid, HLA alloantigens, PHA, or five HIV-1 env peptides (Tl, T2, T_H4.1, P18IIIB and P18MN) previously shown to be recognized by T lymphocytes of HIV-1⁺ individuals and HIV-1^" (seronegative) individuals exposed to HIV-1. Peptides Tl and T2 correspond to residues 428-443 and 112-124 of gpl20 ( IIIB isolate) , respectively. TH4.1 (referred to as T4 in the tables) corresponds to residues 834-848 of gpl60. Plδlllb (referred to as 3b in the tables) corresponds to residues 315-329 of gpl60 (Illb isolate) . P18MN (referred to as mn in the tables) corresponds to residues 315-329 of gpl60 (mn isolate) . The IL-2 produced after 7 days of culture was measured in a bioassay using the IL-2-dependent CTLL cell line. The supernatants were used at 1:2, 1:4 and 1:8 dilutions in the assays. Twenty four hours later the stimulated CTLL cultures were pulsed with [³H]thymidine. Thymidine incorporation was measured 18 hours later and is expressed in counts per minute.

Results

Tables 1-4 and Figures 1-4 present the results of antibiotic treatment of four HIV infected individuals and their partners. In each case, treatment with antibiotics lead to resolution of the concomitant Chlamydia infection and a reduction of anti-Chlamydia antibodies in both urine and serum. Following this pattern, antibodies to HIV also dropped indicating a switch from a predominantly humoral immune response against HIV to a predominantly cell mediated response. These results were confirmed by the assays for CMI which showed a strong cell mediated response against various HIV antigens. The results are discussed in more detail below.

The tables present data for each individual tested. Each patient was tested for urine antibodies to Chlamydia trachomatis (CT) by Western Blot (column 1) . The second column identifies what bands the urine antibodies recognized in the Western Blot. Columns 3 and 4 show the results of CT Western Blot tests in serum. Column 5 shows the results of the urine HIV enzyme immunoassays (EIA) , described above. Column 6 shows the results of urine HIV Western Blot tests. Column 7 and 8 show the results for the same two tests in serum. The final two columns identify the diagnosis and treatment, respectively, for each patient.

The figures show the results of the CMI assays, by which the above assays were confirmed. The first and third panels (Figures 1A, 2A, 3A, 4A, and IC, 2C, 3C, and 4C) show the results of assays in which the T cells were stimulated with recall antigens (flu and tet) alloantigens (allo) and PHA. The second and fourth panels (Figures IB, 2B, 3B, 4B, and ID, 2D, 3D, and 4D) show the results of assays in which the T cells were stimulated with the HIV envelope peptides identified above. Each bar represents dilutions of the supernatants at 1:2, 1:4, and 1:8 respectively. The units on the y axis are counts per minute.

Table 1 shows the results of these assays in a patient 1030A and partner 1030 following treatment with doxycycline. Table l shows that patient 1030A was originally not reactive (NR) as measured in the urine HIV EIA, but was positive in the serum Western Blot and indeterminate in the urine Western Blot. After treatment with doxycycline, this patient was negative in all assays on 6/29/93. On 7/8/93 and 7/13/93 positive or indeterminate results were obtained, but returned negative readings were found on subsequent assays. The partner 1030 showed a similar pattern. Figures 1A through D show the results of the CMI assays, which were used to confirm the results shown in Table 1. Patient 1030A showed strong response to recall antigens as well as alloantigens (Figure 1A) . Figure IB shows the cell mediated response to the various HIV envelope peptides and shows a strong response to peptide T4. The partner 1030 Figures (IC and D) showed a similarly strong response to peptide T4.

Table 2 and Figures 2A-D show results for patient 1054 and partner 1054A. Patient 1054 started on 12/7 as positive in the CT urine western blot and repeatedly reactive (RR) in the urine HIV EIA. After treatment with reciphin and doxycycline, this patient was negative and not reactive in all assays. The partner 1054A, was positive for CT antibodies but did not show reactivity in any of the HIV assays. Figures 2A and B show that patient 1054 had strong CMI response against recall antigens and alloantigens but showed no response against any HIV peptides (Figure 2B) , indicating that the virus had apparently been cleared. Table 3 and Figures 3A through D show results of assays for patient 2045 and partner 2045A. 2054 was positive or indeterminate in all assays on 6/18/93. After treatment, by 7/21/93, urine HIV EIA was not reactive (NR) and the HIV Western Blot test was also negative. The CT urine Western Blot, however, was positive. The 1045A was positive only for CT antibodies. In addition, the urine EIA for HIV was positive, whereas the urine Western Blot and serum assays were all negative. These results show the ability of the urine HIV EIA to detect antibodies not detectable in serum. The CMI assays showed that the T4 peptide was recognized by T cells in both patients.

Table 4 shows the results of assays from patient 1055 and partner 1055A. Again the patient 1055 was positive or indeterminate in all assays but was negative for both CT antibodies and HIV antibodies after treatment. Example 2 This example provides data showing that presence of anti-HERV antibodies in urine is correlated with development of symptoms of HIV infection. Thus, these data indicate that controlling stimuli that lead to HERV expression (e.g., bacterial infections that induce inflammatory responses) can be used to inhibit disease progression.

The EIAs described below were carried out generally as described above. The HERV antigens 4-1 and CKS-17 are described in Cianciolo et al . Science 230:453-455 (1985). The results are presented in Table 5.

Urine Antibodies to HIV-1 and Endogenous Retroviral Envelope Peptides in Healthy and HIV-1 Infected Subjects

#Positive Subjects¹ (% Positive) Category Sample Size HIV-1 4-1 CKS-17

Healthy² 7 (O%) 0 (O%) 0 (O%)

HIV - Asymptomatic³ 9 9 (100%) 0 (O%) 0 (O%) HIV - Symptomatic⁴ 10 10(100%) 7 (70%) 1 (10%)

AIDS* 41 41 (100%) 15 (37%) 4 (10%)

¹EIA was run in duplicate on all urine samples. Criteria for positive was one or more urines from a given subject demonstrating an OD reading above the cutoff.

²Subjects testing Western blot confirmed negative for serum HIV-l antibodies. Consecutive voids were taken on all 7 subjects. The minimum was 4 and the maximum was 9 for a total of 49 voids tested. All 49 were urine antibody negative for HIV-l, 4-1 and CKS-17.

³Subjects testing Western blot positive for serum HIV-l antibodies but without clinical signs or symptoms of the disease. Some subjects may present with persistent generalized lymphadenopathy or minor skin lesions. Consecutive voids were taken on all 9 subjects. The minimum was 4 and the maximum was 12 for a total of 76 voids tested. All 49 were urine antibody positive for HIV-l and urine antibody negative for 4-1 and CKS-17.

Subjects testing positive for serum HIV-l antibodies and presenting with diseases or conditions associated with HIV infection. Symptoms may include fever, weight loss, diarrhea, night sweats, fatigue, oral thrush, hairy leukoplakia, idiopathic thrombocytopenic purpura and viral infections such as herpes zoster. Consecutive voids were taken on all 10 subjects. The minimum was 8 and the maximum was 15.

⁵Subjects testing positive for serum HIV-l antibodies and presenting with specific opportunistic infections, the most common being PCP, or certain types of malignant diseases, most commonly Kaposi's sarcoma (CDC, MMWR Vol 41/No. RR-17, 1993). Only one void was collected for each subject.

The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, and patent applications cited in herein are hereby incorporated by reference.

TABLE 1

TABLE 2

TABLE 3

TABLE 4

Claims

WHAT IS CLAIMED IS:

1. A method of monitoring an immune response against a first infectious agent, the method comprising the steps of: administering a pharmaceutical composition which inhibits growth of a second infectious agent to a patient infected by the first infectious agent and the second infectious agent, wherein the second infectious agent induces a predominantly humoral response; and detecting a decrease or an increase in a humoral response against the first or second infectious agent.

2. The method of claim 1, wherein a decrease in the humoral response against the first infectious agent is detected, thereby detecting a shift towards a cell mediated response against the first infectious agent.

3. The method of claim 1, wherein the step of detecting the humoral response against the first infectious agent is carried out by detecting antibodies to the first infectious agent in a sample from the patient.

4. The method of claim 3, further comprising detecting an increase or decrease in a cell mediated immune response against the first infectious agent.

5. The method of claim 3, further comprising detecting antibodies to the second infectious agent in a second sample from the patient.

6. The method of claim 5, wherein the second sample from the patient is a urine sample.

7. The method of claim 3, wherein the first infectious agent is a virus.

8. The method of claim 7, wherein the virus is HIV.

9. The method of claim 8, antibodies to HIV are detected using a recombinantly expressed HIV protein.

10. The method of claim 9, wherein the HIV protein is gplβo.

11. The method of claim 9, wherein the HIV protein is expressed in insect cells.

12. The method of claim 9, wherein the sample from the patient is a urine sample.

13. The method of claim 1, wherein the second infectious agent is a bacterium.

1 . The method of claim 13, wherein the bacterium is associated with a sexually transmitted disease.

15. The method of claim 14, wherein the bacterium is Chlamydia trachomatis .

16. The method of claim 14, wherein the bacterium is Neissaria gonorrhooae .

17. The method of claim 13, wherein the pharmaceutical composition is an antibiotic.

18. λ method of monitoring an immune response against HIV, the method comprising: administering a pharmaceutical composition which inhibits growth of a bacterium associated with a sexually transmitted disease to a patient infected by HIV and the bacterium, wherein the bacterium induces a predominantly humoral response; detecting an increase or decrease in antibodies to HIV in a urine sample from the patient; and detecting an increase or decrease in a cell mediated immune response against HIV in the patient.

19. A method of monitoring an infection in an individual lacking detectable serum antibodies against an agent responsible for the infection, the method comprising the steps of: (a) detecting antibodies to the agent in a urine sample from the individual; and

(b) repeating step (a) until antibodies to the agent are not detectable.

20. The method of claim 19, wherein the agent responsible for the infection is Chlamydia trachomatis .

21. The method of claim 19, wherein the agent responsible for the infection is HIV.

22. The method of claim 21, wherein antibodies to HIV are detected using a recombinantly expressed HIV protein.

23. The method of claim 22, wherein the HIV protein is gpl60.

24. The method of claim 22, wherein the HIV protein is expressed in insect cells.

25. The method of claim 19, further comprising the step of detecting a cell mediated immune response against the agent.

26. The method of claim 19, further comprising detecting the presence of antibodies to a second infectious agent.

27. The method of claim 26, wherein the second infectious agent is a bacterium.

28. The method of claim 27, wherein the bacterium is associated with a sexually transmitted disease.

29. The method of claim 28, wherein the bacterium is Chlamydia trachomatis .

30. The method of claim 27, further comprising administering to the patient a pharmaceutical composition which inhibits the growth of the bacterium.