KR20070067719A - Immunomodulators for Vaccines - Google Patents

Abstract

Used as an immunomodulator for vaccines against infectious diseases

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

The use of is disclosed.

Description

Immune modulator for vaccines {IMMUNOMODULATORS FOR VACCINES}

1: Shows the stimulation of total Ig and IgA in serum (MS) and IgA in ocular lavage fluid (EW) of mice immunized with HSV-1 glycoprotein / rEtxB.

Figure 2: T cell proliferation of (mesenteric lymph nodes) MLN or (uterine lymph nodes) CLN leukocytes of mice immunized with HSV-1 / rEtxB.

Figure 3: T cell proliferation in MLN and CLN cells of mice immunized nasal with HSV-1 Gp in the presence of 1-20 μg of EtxB.

Figure 4 shows the levels of anti-HSV-1 serum Ig in mice administered three HSV-1 glycoproteins at 10 day intervals with various amounts of rEtxB or rCtxB as adjuvants.

5: Reduction of virus shedding, clinical disease and latentness in mice immunized with HSV-1 / rEtxB.

Figure 6: Ig isotype distribution in MS infected with HSV-1 or immunized with HSV-1 Gp in the presence of EtxB or CtxB as an immunomodulator.

Figure 7 shows the distribution of the Ig subclass when HSV-1 Gp was administered nasal with rEtxB or rCtxB as an immunomodulator.

8 shows the immunogenic effect of different amounts of rErxB or rCtxB on the level of HSV-1 specific IgA in ocular lavage fluid administered with HSV-1 glycoprotein.

9: Serum immunoglobulin response of mice immunized with HSV-1 glycoprotein (gp) or mock glycoprotein alone or in the presence of adjuvant.

10: Mucosal IgA in ocular lavage of mice immunized with HSV-1 glycoprotein or mock glycoprotein alone or in the presence of adjuvant.

Figure 11: Mucosal IgA in vaginal lavage of mice immunized with HSV-1 glycoprotein or mock glycoprotein alone or in the presence of adjuvant.

Figure 12 shows the levels of HSV-1-specific immunoglobulins in the serum of mice immunized with HSV-1 glycoprotein at different doses of rEtxB as adjuvant.

Figure 13: IgA levels in ocular lavage of mice immunized with HSV-1 glycoproteins with varying concentrations of rEtxB.

14: Shows the level of IgA in the vaginal lavage fluid of mice immunized with HSV-1 glycoproteins with varying concentrations of rEtxB.

Figure 15: IgG subclass distribution of serum antibody response to HSV-1 after nasal immunization with Ctx / CtxB or rEtxB or ocular infection with HSV-1.

Figure 16: Cytokine production from culture of lymph node cells infected with HSV-1 by eye scar or immunized with nasal administration of HSV-1 glycoprotein with Ctx / CtxB or rEtxB as adjuvant. Indicates.

Figure 17: Shows the level of protection against ocular HSV-1 infection in mice nasal immunized with a mixture of HSV-1 or mimic glycoproteins in the presence of rEtxB as an immunomodulator.

The present invention relates to immunomodulators for vaccines used against a specific range of infectious agents. The present invention further relates to a vaccine composition comprising an immunomodulator, preferably an immunomodulator together with an antigen, and a method of vaccination with the vaccine composition.

Cholera toxin (Ctx) and closely related E. coli heat-labile enterotoxins (Etx) are potential immunogens and mucosal adjuvants. However, due to their inherent toxicity, they are not suitable for human use. For example, although Ctx is the most commonly used mucosal adjuvant for laboratory animals, it is not suitable for use in the human body because of their potential diarrhea-inducing properties. Attempts have been made to separate the toxicity from adjuvant activity, for example by substituting holotoxin itself with components of Ctx and Etx. E. coli verotoxin (Vtx) is another known bacterial toxin.

Ctx and Etx are heterohexameric proteins composed of enzymatically active A subunits and pentamer B subunits. CtxB and EtxB are known to bind to GM1-ganglioside (GM1), a glycosphingolipid found everywhere on the surface of mammalian cells. Vtx binds to Gb3, a similar type of receptor for GM1.

In an attempt to address the problem of toxins for vaccine development, adjuvant activity of non-toxic B subunits has already been studied. However, many reports describe experiments in which conventional methods of making CtxB or EtxB have been used. Inevitably, these preparations are contaminated with small but biologically significant amounts of active toxins, and the adjuvant activity contributing to the B subunit is indistinguishable from the adjuvant activity of whole toxins (Wu and Russell (1993) Infection and Immunity 61: 314-322, US-5182109. Subsequent studies with recombinant CtxB (rCtxB) suggest that CtxB is a poor mucosal adjuvant and that only the addition of the original holotoxin causes a strong bystander response (Tamura et al. (1994) Vaccine 12 419-426). In another study, rCtxB lacked ADP-ribose activity and cAMP-stimulatory activity of protoxins and, moreover, adjuvant mechanisms are associated with this ability, suggesting that CtxB is inappropriate for use as adjuvant (Vajdy and Lycke). (1992) Immunology 75: 488-492, Lycke et al. (1992) Eur. J. Immunol. 22: 2277-2281, Douce et al. (1997) Infection and Immunity 65: 2821-2828).

In another study, nasal administration of ovalbumin with rCtxB as an adjuvant caused a poor antibody response. In addition, non-toxic derivatives of Ctx with mutations in the A subunit caused a weak response to the Bystander antigen, whereas the presence of the active A subunit dramatically increased the adjuvant activity, which required the active A subunit to be essential. (Douce et al. (1997)).

It also discloses that rCtxB and rEtxB can be used to promote resistance to heterologous antigens, suggesting that these molecules are inappropriate for use as adjuvants (Sun et al. (1994) Proc. Natl. Acad. Sci. 91: 4610-4614. , Sun et al. (1996) Proc. Natl. Acad. Sci. 93: 7196-7201, Bergerot et al. (1997) Proc. Natl. Acad. Sci. 94: 4610-4614, Williams et al. (1997) Proc. Natl. Acad. Sci. 94: 5290-5295).

Although CtxB and EtxB teach in the art that they have poor adjuvant performance and can actually act as tolerogens, the present inventors have used a rat model of rEtxB (thus residual holotoxin or A) as a nasal vaccine against HSV. No subunits) and unexpectedly stimulated a protective immune response to the administration of the virus:

In particular, the present inventors

i) Agents such as EtxB and CtxB stimulate high levels of local (mucosal) antibody production (although immunization with rEtxB stimulated lower levels of overall antibody production than Ctx / CtxB combinations);

ii) the distribution of the resulting antibodies is distributed towards non-complement fixing antibodies, in particular S-IgA and IgG1;

iii) agents such as EtxB and CtxB also stimulate local and systemic T-cell proliferative responses;

iv) agents such as CtxB and EtxB tend to convert immune responses from Th1-related responses to Th2-related responses;

v) when drugs such as CtxB and EtxB are used as immunomodulators, some deleterious effects of Th2-related reactions such as IgE production are avoided;

vi) rEtxB is an more effective immunomodulator than rCtxB;

vii) agents such as EtxB and CtxB can transform the way antigen presenting cells refractory and process antigens, increasing antigen persistence;

viii) if agents such as EtxB and CtxB are bound to the antigen, the process route of the antigen can be transformed by changing the binding to the immunomodulatory agent; And

ix) VtxB exhibits immunomodulatory effects in the in vitro leukocyte population similar to those acted by EtxB and CtxB,

Found.

These important findings are the basis for various aspects of the present invention and other drugs that we can bind to GM1 or Gb3 as well as pure EtxB, CtxB and VtxB, or that mimic the binding effect to GM1 or Gb3 are HSV. Prophylaxis and treatment against other infections as well as other infections has led to the expectation that the vaccines will be useful as immunomodulators used in vaccines, the prevention and treatment of which is beneficial for immunomodulation of the types listed above.

Stimulation of the immune response

Other agents that can bind to EtxB, CtxB, VtxB and GM1 or Gb3 or mimic the binding effects on GM1 or Gb3 can act as immunomodulators and stimulate specific immune responses to antigenic administration. .

In the first aspect of the present invention,

Used as an immunomodulator for vaccines against infectious diseases

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

The use of is provided.

In the second aspect of the present invention,

Vaccine compositions for use in infectious diseases are provided,

Infectious diseases are caused by infectious agents,

The vaccine composition

Antigenic determinants and

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

Immunomodulators selected from

Including;

Antigen determinants are antigenic determinants of the infectious agent.

Antigens and immunomodulators can be combined, for example, by covalent or genetic binding, to form a single agent. In certain embodiments of the invention, the antigen and immunomodulatory agent may be chemically bound. For example, antigens and immunomodulators can be chemically bound using heterobifunctional crosslinkers. In most of the applications of this aspect of the invention, separate administration (antigen and immunomodulatory agent are not combined as described above) is preferably administered separately as separate administration is possible.

In the third aspect of the present invention,

a) (i) EtxB, CtxB or VtxB without whole toxin;

  (ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

  (iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

One of; And

b) antigenic determinants of infectious disease for co-administration with the vaccine immunomodulatory agent;

Including,

Kits for vaccinating mammals, such as humans or vertebrates, for infectious diseases are provided.

The vaccine composition of the second aspect of the invention and the kit of the third aspect of the invention can be used in a prophylactic or therapeutic vaccination method wherein the "prophylactic vaccine" is administered to an undosed subject to prevent the development of an infection. And a "therapeutic vaccine" is administered to a subject having an infection to reduce or minimize the infection or to eliminate the immunopathological consequences of the disease.

Agents such as EtxB have the ability to change the nature of the immune response that occurred once the infection occurred. Therapeutic vaccines comprising such agents (ie, requiring no antigen) can be used for specific purposes when the immune response fails to eliminate the infection. This application treats chronic diseases such as, for example, diseases caused by causative agents selected from the group consisting of herpes viruses, hepatitis viruses, HIV, TB and parasites. Can be used for specific purposes.

In the fourth aspect of the present invention,

Inoculating the host with a vaccine comprising at least one antigenic determinant and an immunomodulatory agent;

The immunomodulatory agent:

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding; sign

Methods for preventing or treating a disease in a host are provided.

The vaccine may be packaged in co-administration and administered in a number of different routes, such as nasal, oral, vaginal, urethral or ocular administration. Nasal administration is presently preferred. When the vaccine is administered nasal, it can be administered in aerosol or liquid form.

Antigen determinants and immunomodulators may be administered to a subject in a single dose or in multiple doses.

In the first embodiment, the immunomodulator of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the kit of the third aspect of the present invention, and the method of the fourth aspect of the present invention are characterized in that the infectious agent is a herpes virus family. Used for a member of the disease. For example, the infectious agent can be selected from the group consisting of HSV-1, HSV-2, EBV, VZV, CMV, HHV-6, HHV-7 and HHV-8. In particular, the infectious agents may be HSV-1, HSV-2, CMV and EBV.

In this first embodiment, the antigenic determinant is preferably the antigenic determinant of the immediate early, early or late gene product of the herpes virus (such as surface glycoproteins for example).

If the infectious agent is HSV-1 or HSV-2, the antigenic determinant may be the antigenic determinant of a gene product selected from the group gD, gB, gH, gC or latency associated transcript (LAT). have.

If the infectious agent is EBV, the antigenic determinants are antigenic determinants or latent proteins of gp340 or gp350 (e.g., EBNAs 1, 2, 3A, 3B, 3C and -LP, LMP-1, 2A and 2B or EBER) antigenic determinants.

In the second embodiment, the immunomodulator of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the kit of the third aspect of the present invention, and the method of the fourth aspect of the present invention are characterized in that the infecting agent is influenza. It is used against diseases that are viruses.

In this second embodiment, the antigenic determinant is preferably a coat protein of the virus (eg hemagglutinin and neuraminidase) or an internal protein (eg nu It is an antigenic determinant of cleopotein.

In the third embodiment, the immunomodulator of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the kit of the third aspect of the present invention, and the method of the fourth aspect of the present invention, the infection factor is parainfluenza virus ( parainfluenza).

In the fourth embodiment, the immunomodulator of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the kit of the third aspect of the present invention, and the method of the fourth aspect of the present invention, the infectious agent is RS virus (respiratory). syncytial virus).

In the fifth embodiment, the immunomodulator of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the kit of the third aspect of the present invention, and the method of the fourth aspect of the present invention, the infection factor is Hepatitis virus. It is used against diseases that are. For example, the infectious agent may be selected from the group consisting of hepatitis A, B, C and D. In particular, the infectious agent may be Hepatitis A or C.

In the sixth embodiment, the immunomodulator of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the kit of the third aspect of the present invention, and the method of the fourth aspect of the present invention are used for meningitis. do. In this sixth embodiment, the infectious agent is a naked ceria mening GT display (Neisseria meningitidis ), Haemophilus influenzae type B, and Streptococcus pnueumoniae .

In a seventh embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention are used against pneumonia or respiratory infection. do. In this seventh embodiment, the infectious agent is streptococcus pneumoniae (Streptococcus pneumoniae), Lego Nella pneumophila (Legonella pneumophila), and the Mycobacterium It may be selected from the group consisting of Mycobacterium tuberculosis .

In the eighth embodiment, the immunomodulator of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the kit of the third aspect of the present invention and the method of the fourth aspect of the present invention are used for sex-transfer disease. do. In this eighth embodiment, the infectious agent is Neisseria. gonnorheae ) , HIV-1, HIV-2 and Chlamydia trachomatis .

In the ninth embodiment, the immunomodulator of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the kit of the third aspect of the present invention, and the method of the fourth aspect of the present invention are directed to gastrointestinal disease. It is used against. In the ninth embodiment, the infectious agent is enteric pathogenic, gut toxicity and intestinal permeability E. coli, rotavirus, Salmonella Entebbe utility disk (Salmonella enteritidis), Salmonella typhimurium (Salmonella typhi), Helicobacter pylori (Helicobacter pylori), Bacillus cereus ( Bacillus cereus ), Campylobacter jejuni ) and Vibrio cholera ( Vibrio) cholerae ) can be selected from the group consisting of.

If the infectious agent is selected from the group consisting of enteropathogenic, enterotoxicity, enteroinvasiveness, enterohemorrhagic and enterocombination E. coli, the antigenic determinant may be an antigenic determinant of bacterial toxin or adhesion factor. have.

In the tenth embodiment, the immunomodulator of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the kit of the third aspect of the present invention, and the method of the fourth aspect of the present invention are directed to a superficial infection. It is used against. In this tenth embodiment, the infectious agent can be selected from the group consisting of Staphylococcus aureus , Streptococcus pyogenes and Streptococcus mutans . have.

In the eleventh embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention are used for parasitic infection. . In this eleventh embodiment, the infectious agent is malaria, Trypanasoma S. spp . ), Toxoplasma gondii ), Leishmania donovani ) and Oncocerca spp. can be selected from the group consisting of.

Stimulation of the mucosal immune response

Other agents that can bind to EtxB, CtxB, VtxB and GM1 or Gb3 or mimic the binding effects on GM1 or Gb3 can specifically upregulate mucosal antibody production.

The vaccine immunomodulator of the first aspect of the present invention, the vaccine composition of the second aspect of the present invention and the kit of the third aspect of the present invention are particularly effective for protecting from infection or treatment in vivo by mucosal immune response. Effective against the disease received. For example, for these diseases infectious agents bind to colonies or enter the mucosa during infection. Examples of such diseases include diseases caused by viruses (HIV, HSV, EBV, CMV, influenza, measles, mumps, rotavirus, etc.), bacteria (E. coli, Salmonella, Shigella, Diseases caused by parasites and diseases caused by chlamydia, N. gonnorhoea, T. palladium, and Streptococcus spp. Include.

In a preferred embodiment of the second aspect of the present invention,

The present invention

Vaccine against HSV-1 infection is provided comprising at least one HSV-1 antigenic determinant and immunomodulatory agent,

The immunomodulatory agent:

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

to be.

Preferably the immunomodulatory agent is EtxB.

In a preferred embodiment of the third aspect of the present invention,

a) (i) EtxB, CtxB or VtxB without whole toxin;

   (ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

   (iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding; sign

Vaccine immunomodulators, and

b) at least one HSV-1 antigenic determinant for co-administration with the vaccine immunomodulatory agent;

Including,

Kits for vaccinating mammals against HSV-1 are provided.

In the fifth aspect of the present invention,

Used to upregulate production of antibodies on mucosal surfaces

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

The use of is provided.

The production of non-complement-binding serum antibodies may also be upregulated. Preferably, S-IgA is produced in accordance with the fifth aspect of the invention.

In this fifth aspect of the invention, the medicament may be used in combination with one or more antigenic determinant (s).

Downregulation of Pathological Components of the Immune Response

We also found that when pure EtxB was used as an immunomodulator in this way, it could potentially prevent the deleterious effects of Th2 related responses such as the production of high levels of pathological IgE. Nevertheless, the immune response induced by the use of EtxB (or CtxB or VtxB) as an immunomodulator is favorable for the induction of Th2-related cytokines. That is, EtxB (or CtxB) induces the transition from Th1-type responses to Th2-type responses. This suggests that the present inventors have found that pathogenetic components of the immune response related to both Th1 activity and Th2 activity are not only pure EtxB, CtxB or VtxB but also other agents that can bind to GM1 or Gb3 or mimic the binding effect on GM1 or Gb3. It was made possible to predict that they could be adjusted down.

In the sixth aspect of the present invention,

Used to downregulate pathological components of Th2-related immune responses

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

The use of is provided.

Pathological components of Th1-related immune responses can also be downregulated.

EtxB and CtxB are known to bind GM1 to induce other effects on the white blood cell population, including specific depletion of CD8 + T cells and associated activation of B cells (WO 97/02045). Thus, EtxB and CtxB are believed to change the balance of the immune response such that the inflammatory Th1 related response is down-regulated while the Th2 related response is upregulated. The Th1 response involves the release of γIFN by activated T-cells leading to macrophage activity and a delayed type of hypersensitivity response. Such a response may be an important cause of pathology during infection by many pathogens. Th2 responses include the activity of T-cells that produce cytokines such as IL-4, IL-5, IL-10 and are known to promote the secretion of high levels of antibodies, particularly high levels of IgA.

It has been surprisingly found that when EtxB is used as an immunomodulator in this way, potentially harmful effects of Th2 related responses, such as the production of high levels of pathological IgE, are prevented. Thus, EtxB and CtxB can downregulate the pathological components of the immune response involved in both Th1 and Th2 activation. Such reactions are favorably regulated to produce high levels of secreted IgA production on non-complement binding serum antibodies and mucosal surfaces.

The use of the medicament according to the sixth aspect of the present invention is particularly useful for therapeutic vaccines for diseases involving immunopathological mechanisms. Examples of such diseases are HSV-1, HSV-2, TB and HIV.

The first and sixth aspects of the present invention can be combined. That is, drugs such as EtxB can be used simultaneously as immunomodulators and therapeutics. For example, in diseases involving immunopathological mechanisms, the use of vaccines incorporating agents such as EtxB or CtxB can not only limit infection but also eliminate pathological disease progression. Thus the immunomodulatory agent acts both prophylactically and therapeutically. Thus, examples of infections in which vaccination is likely to have particular value in this method include those caused by the herpes virus, intestinal pathogens and respiratory pathogens.

Immune Regulation of Antigen Processing Pathways

a) extension of presentation

We also found that when EtxB (or CtxB or VtxB) is used as an immunomodulator, antigen internalization and process pathways are transformed. The presence of B subunits leads to prolonged presentation, which is made possible by transforming antigen pathways within antigen presenting cells, such that antigen degradation is delayed and thus maintained over a longer period of time. This feature of the B-subunit associated with antigen presentation allows the vaccines incorporating the agents according to the invention to increase antigen persistence and support immunological memory.

In the seventh aspect of the present invention,

As an immunomodulator in a vaccine, it is used to prolong antigen presentation and support immunological memory in mammals.

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

 The use of is provided.

In the eighth aspect of the present invention,

Antigenic determinants and

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

Immunomodulators selected from

Including;

The antigenic determinant is an antigenic determinant of an infectious disease, and the immunomodulatory agent extends the presentation of the antigenic determinant and supports immunological memory.

Vaccine compositions for use in infectious diseases are provided.

b) MHC -I or MHC -II of antigens for related pathways Intracellular Targeting

As noted above, antigens and immunomodulators in a therapeutic or prophylactic vaccine can be combined, eg, covalently or genetically, to form a single agent. We have found that by converting the binding of an antigen to an immunomodulatory agent, the antigen can be directed to cells in other compartments and thus to other antigen presentation pathways.

By binding the antigen or antigenic determinant to the immunomodulator in some way, it is possible to promote the translocation of the antigen across the endosomal into the cytoplasm. We expect this to increase the loading of antigenic peptides on MHC class I molecules. Thus, the use of antigen-immunoregulator conjugates can be used to increase the activity of cytotoxic T cells (CTLs), in particular. Induction of CTLs is beneficial for the present invention and for the treatment of many diseases, particularly diseases caused by viruses, intracellular bacteria and parasites.

Binding of the antigen-immunoregulator conjugates may also be chosen such that the antigen is transported into the nucleus.

In the ninth aspect of the present invention,

Antigen or antigenic determinant and

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

Immunomodulators selected from

There is provided a conjugate comprising a.

In the tenth aspect of the present invention,

Provided are vaccine compositions for use against infectious diseases caused by infectious agents,

The vaccine composition

Antigen or antigenic determinant and

i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

Immunomodulators selected from

Including;

The antigen or antigenic determinant is the antigen or antigenic determinant of the infectious agent.

An antigen or antigenic determinant can be bound to an immunomodulator by a variety of methods, including genetic or chemical binding. In a first preferred embodiment, the conjugate is a fusion protein formed by genetically binding an antigen or antigenic determinant to an immunomodulator. Preferably the antigen or antigenic determinant is genetically bound to the C-terminus of an immunomodulator. In a second preferred embodiment the antigen or antigenic determinant is chemically bound to an immunomodulatory agent. Preferably the antigen or antigenic determinant is bound to an immunomodulator using a bifunctional cross-linker such as a heterobifunctional cross-linker. More preferably the cross-linking agent is N-γ (-maleimino-butyoxy) -succinimide ester (GMBS) or N-succinimidyl- (3-pyridyl-dithio) -propionate (SPDP) .

The vaccine composition can be administered by a number of different routes, such as nasal, oral, vaginal, urethral or ocular administration. Nasal administration is preferred.

In the eleventh aspect of the present invention,

Used in conjunction with an antigen or antigenic determinant for transporting or targeting the antigen or antigenic determinant to the cytoplasm or nucleus of antigen-expressing cells

(i) EtxB, CtxB or VtxB without whole toxin;

* (ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

The use of is provided.

In the twelfth aspect of the present invention,

Used in conjunction with antigen or antigenic determinants for upregulation of antigenic determinants or antigenic determinants derived from antigens by MHC class I molecules

* (i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

The use of is provided.

Preferably the use of the conjugate of claim 12 of the present invention is used in conjunction with the use of a medicament according to claim 5 of the present invention to stimulate a strong CTL response and to upregulate mucosal antibody production. This action is particularly useful for the prevention and treatment of viral infections, for example influenza.

EtxB Is the preferred immunomodulator

Conventionally, EtxB and CtxB have been considered to have similar characteristics. However, we have found that rEtxB is a more potent and effective immunomodulator than rCtxB. Preferred immunomodulators are therefore agents which have an effect similar to EtxB or EtxB.

EBV

EBV is one of eight known human herpes viruses. Infection usually occurs in early childhood, but usually the clinical symptoms are mild or not detected at this stage. Primary infection by EBV is later associated with infectious mononucleosis in life, which is the second most frequent disease in adolescence in the United States. EBV also has a carcinogenic potential. It is closely related to EBV and endemic Burkitt's lymphoma (BL) and undifferentiated nasopharyngeal carcinoma (NPC). In addition, a large proportion of lymphomas occurring in immuno-deficient patients are caused by EBV and have been shown to be linked between specific Hodgkin's lymphomas and EBV.

Latent EBV-infected cells express a small number of so-called "latent" proteins. These serve as six nuclear proteins (EBNAs 1, 2, 3A, 3B, 3C and -LP), three integral membrane proteins (LMP-1, 2A and 2B) and RNA splicing roles. Two non-polyadenylation virus induced RNAs (EBERs).

EBV latent membrane protein 1 (LMP-1) is present in the plasma membrane of infected cells. It can also be expressed in nasopharyngeal carcinomas (NPCs) and EBV-positive Hodgkin's lymphoma (HD), which plays a role for LMP-1 in the development of such tumors. The LMP-1 gene can transform the phenotype of uninfected cells due to upregulation of cell surface activation markers that promote cell proliferation. LMP-1 can also convert signaling pathways and has anti-apoptotic action. The cellular immune response directed against these viral antigens has not been reliably demonstrated to some extent in healthy carriers or tumor patients.

Many animal viruses include mechanisms for avoiding detection by the host immune system. In general, this mechanism involves interference using a TAP-related peptide translocation system. This allows EBV to also contain similar mechanisms to avoid immune system detection, thus allowing the virus to persist in the host. This explains why specific cellular immune responses are impossible to detect EBV latent protein EBNA1 and may explain the apparent lack of response to LMP1.

In the thirteenth aspect of the present invention,

Used to treat and / or prevent EBV-related diseases

a) (i) EtxB, CtxB or VtxB without whole toxin;

   (ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

   (iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

One of; And

b) EBV antigens;

There is provided a vaccine composition comprising a.

In particular, the vaccine composition of claim 13 of the present invention comprises an agent other than EtxB, CtxB or EtxB or CtxB having GM1-binding activity.

In a fourteenth aspect of the present invention,

Used to treat EBV-related diseases

(i) EtxB, CtxB or VtxB without whole toxin;

(ii) agents other than EtxB or CtxB with GM1-binding activity, or agents other than VtxB with Gb3-binding activity; or

(iii) agents that affect intracellular signaling mediated by GM1-binding or Gb3-binding;

There is provided a therapeutic composition comprising a.

In particular, the therapeutic composition of claim 14 of the present invention comprises an agent other than EtxB, CtxB or EtxB or CtxB having GM1-binding activity.

Cocapting EtxB with LMP1, based on the knowledge that EtxB promotes fragmentation of LMP-1, causes EtxB (and other drugs, such as CtxB with GM1 binding activity) to be anti-EBV. It may be useful to stimulate an immune response. This activity applies to vaccines for preventing EBV-related diseases, and to therapeutic treatments for treating such diseases that have already advanced.

Without being bound by theory, it is believed that when cocapturing EtxB with LMP-1, the antigen is processed by another intracellular route, which allows the antigen to bypass the normal process route blocked by the virus. Thus, the antigen is effectively present on the cell surface. The action of EtxB also allows the epitopes of antigens to be present on the cell surface to be different from the epitopes present if the antigens are processed by conventional routes.

The vaccine of claim 13 of the present invention can be used to inhibit the development of infection by EBV, or EBV-related diseases in EBV-infected individuals. The vaccine may also include a separate adjuvant, or a medicament (such as EtxB or CtxB) that can act as adjuvant by itself.

The specified agents of the fourteenth aspect of the present invention can be used alone (ie, without antigen) in the treatment of EBV-related diseases already advanced in a subject.

Preferred medicaments for use in the 13th and 14th aspects of the present invention are EtxB.

EBV antigens are antigens that can be derived from EBV itself or antigens expressed and elicited by EBV-infected host cells by the action of EBV. Preferably the antigen is an EBV latent membrane protein. More preferred antigens are LMP-1, LMP-2A, LMP-2B, and EBNA-1 as well as antigen fragments thereof. Antigens can be isolated directly from EBV infected cells or prepared by synthetic or recombinant methods.

13 and 14 of the present invention are particularly suitable for the treatment and / or prophylaxis of the following diseases: infectious mononucleosis, Burkitt's lymphoma, Nasofaringian tumor and Hodgkin's lymphoma. These aspects of the invention are particularly suitable for the treatment and / or prophylaxis of Nasofaringian tumors and Hodgkin's lymphomas.

Vaccines or therapeutic compositions according to aspects 13 and 14 of the present invention may be used to prevent or treat the development of EBV-related diseases in a mammalian subject by administering an immunologically effective amount to the subject.

The mammalian subject may be, for example, a healthy EBV-infected or uninfected individual, an immunodeficiency individual, or an individual with an EBV-related disease.

The vaccine can be administered by any suitable route. Agents and antigens may be co-administered or administered separately to a mammalian subject. Agents and antigens may be separated or covalently or genetically bound to form a single agent, for example.

GM-1 and Gb3 Related signaling

Without being bound by theory, it is believed that GM1 or Gb3 binding directly or indirectly causes intracellular signaling. We also found evidence suggesting that EtxB interacts with at least one other receptor involved in GM1 related intracellular signaling. Binding of EtxB (or CtxB) to GM1 promotes binding to proteins that cause intracellular signaling. It is not known what specifically triggers signaling. This can be phosphorylation of GM1 or protein. When EtxB / CtxB binds to GM1 on the cell surface, the bound GM1 is internalized in vesicles (Williams et al. (1999) Immunology Today 20; 95-101).

GM1 and other glycolipides (such as Gb3) are also known to preferentially be located in "membrane rafts" known as important protein receptors. Thus, internalization of GM1 as a result of B-subunit binding causes cocapping of such proteins that are triggered to mediate intracellular signaling.

Justice

An adjuvant is a substance that increases the immune response to the antigen non-specifically as distinguished from the target of the vaccine carrier, the antigen for the desired site. The term "immunomodulator" is used herein to refer to an agent that acts like an adjuvant to stimulate a particular immune response but also directs the immune response in a particular direction.

The term "coadministration" is used to mean the site and timing of administration of an antigen and an immunomodulator such as an essential immune response is stimulated. Thus, the antigen and immunomodulatory agent may be administered simultaneously and at the same site, while it may be beneficial to administer the antigen at a different time and / or site than the immunomodulatory agent. For example, the antigen and the immunomodulatory agent may be administered together in the first stage and then the immune response may be enhanced in the second stage in which the antigen is administered alone.

The term “antigen determinant” as used herein refers to a site on an antigen recognized by an antibody or T-cell receptor. Preferably a short peptide derived from a protein antigen or part thereof, but the term also includes glycopeptides and carbohydrate antigen determinants. The term also includes amino acids or carbohydrates of modified sequences that stimulate the response of recognizing the entire organism.

There are a number of known methods for identifying antigenic determinants for a given infectious agent.

For example, potential protective antigens can be identified by raising the immune response in an infected patient or in a recovery phase or in an infected or recovery phase or by monitoring the immune response to an antigen-containing preparation in vitro. have. E.g,

i) Serum samples from infected patients or recovering patients or infected or recovering animals are recognized by immune serum against whole cell lysates of infectious agents or against lysates of cells infected by said infectious agents. Can be screened by Western blotting standard techniques for detecting antigen (s);

ii) Serum samples from infected or recovering patients or infected or recovering animals may be partially or highly purified antigens from infectious agents or against lysates of cells infected by the infectious agents. Screened by ELISA standard techniques used to coat these microtitre wells, or screened by immunoblotting to detect antigen (s) recognized by immune serum Can be;

iii) Serum samples from infected patients or recovering patients or infected or recovering animals can be screened for whole cell lysates derived from recombinant expression systems encoding one or more antigens of interest, and the immune serum Can be screened using ELISA or Western blotting standards to detect antigen (s) recognized by;

iv) Serum samples of infected patients or recovering patients or infected or recovering animals are cloned antigen recognized by said immune serum against an expression library containing genes cloned from the infectious agent of interest. Or screened using colony blot immunodetection to identify the fragment; or

v) PBLs or PBL's of infected or convalescent patients blood, lymph node cells, spleen cells, or lamina propria cells of infected or convalescent animals are derived from infectious agents or lysates of cells infected by said infectious agents. Cultured in vitro in the presence of some or highly purified antigen, or in the presence of a recombinant expression system encoding one or more antigens capable of detecting antigen-specific T-cell proliferative responses.

Alternatively, for example, specifically by signature tagged mutagenesis technology developed by Holden or in vivo, such as IVET (in vivo expression technology) developed by Mekalanos. Detecting gene products essential for the survival of pathogens in vivo by detection of induced gene products or by differential fluorescence induction developed by Falkow to identify gene populations that may be potential protective antigens. It is possible. Using this method the gene product can be screened as described above. The genes can be cloned into expression vectors and the recovered antigens can be included in the vaccine combination with agents that modulate glycosphingolipid-related activity.

Various methods are known for isolating antigens for a given infectious agent.

For example, surface components of an infectious agent comprising one or more latent protective antigens can be extracted using a method capable of recovering the antigen from the infectious agent or from cells infected by the infectious agent. have. This may include the use of cell disruption techniques to lyse cells such as sonication and / or detergent extraction. Centrifugation, ultracentrifugation or precipitation can be used to prepare collected antigens. As an example of such a method, J. Infect. Dis. (Richard et al. (1998), 177; 1451-7) describes antigen preparations containing HSV-1 glycoproteins.

In addition, antigens of infectious agents or antigens of cells infected by the infectious agents are extracted by various methods including, but not limited to, urea extraction, alkali or acid extraction, or detergent extraction, followed by chromatographic separation. Can be. Recovered material in voids or elution peaks containing one or more potential protective antigens may be used in the vaccine combination.

Alternatively, genes encoding one or more latent protective antigens can be cloned into various expression vectors suitable for antigen production. These are, for example, Escherichia coli), Bacillus spp (Bacillus spp.), Vibrio spp (Vibrio spp . ), Sacarromyces cerevisiae , bacterial or eukaryotic expression systems such as mammalian cell lines and insect cell lines. Antigens can be recovered by conventional extraction, separation and / or chromatographic methods.

As used herein, the terms "CtxB", "EtxB" and "VtxB" include molecules in their natural and presynthetic forms. Recombinant forms are particularly preferred. Recombinant forms of the molecule can be produced by inserting a gene or genes encoding the particular polypeptide chain (chains) from which the protein is formed into an appropriate vector and then transfecting into the appropriate host. For example, genes encoding polypeptide chains of the EtxB assembly can be inserted into, for example, plasmid pMM68 and then used to transfect host cells such as Vibrio sp. 60. Proteins are purified and isolated by known methods per se. Mutant genes expressing active mutant CtxB, EtxB or VtxB proteins can be prepared by methods known from wild type genes.

The terms "CtxB", "EtxB" and "VtxB" also contain portions of mutant molecules and other synthetic molecules (CtxB, EtxB or VtxB) that have the ability to bind GM1 or Gb3 or mimic the effects of binding to GM1 or Gb3. It includes).

Agents other than EtxB and CtxB with GM1 binding action and agents other than VtxB with Gb3 binding action include antibodies to which GM1 or Gb3 binds.

For the preparation of antibodies, various hosts, including goats, rabbits, rats, mice, and the like, can be immunized by injecting GM1 or Gb3 or its derivatives or moters. Depending on the host species, various adjuvants may be used to increase the immune response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide and lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpets Surface active substances such as hemocyanin (keyhole limpet hemocyanin) and dinitrophenol. BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human supplements.

Humanized monoclonal antibodies are preferred in the present invention. Monoclonal antibodies can be prepared using any technique provided for production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, hybridomas (1975 Nature 256: 495-497) first described by Koehler and Milstein, human B-cell hybridoma technology (Kosbor et al. (1983), Immunol Today 4:72; Cote et al. (1983) Proc Natl Acad Sci 80: 2026-2030) and EBV-hybridoma technology (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp77-96). do. In addition, techniques developed for the production of “chimeric antibodies” can be used to splice mouse antibody genes into human antibody genes to obtain molecules with appropriate antigen specificity and biological activity (Morrison et al. (1984) Proc Natl Acad Sci 81: 6851-6855; Neuberger et al. (1984) Nature 312: 604-608; Takeda et al. (1985) Nature 314: 452-454). Alternatively, the techniques described for the preparation of single chain antibodies (US Pat. No. 4,946,779) can be applied to prepare target interaction component specific single chain antibodies.

Antibodies can also be produced in vivo by inducing production in leukocyte populations or as described in Orlandi et al. (1989, Proc Natl Acad Sci 86: 3833-3837) and Winter G and Milstein C (1991; Nature 349: 293-299). It can be prepared by screening recombinant immunoglobulin libraries or panels of highly specific binders.

Antibody fragments containing specific binding sites for GM1 or Gb3 can also be generated. For example, such fragments include, but are not limited to, F (ab ') ₂ fragments and disulfide bonds of F (ab') ₂ fragments, which can be produced by pepsin digestion of antibody molecules. It includes Fab fragments that can be produced by reducing them. Alternatively, the Fab expression library can be configured to quickly and easily identify monoclonal Fab fragments with the desired specificity (Huse WD et al. (1989) Science 256: 1275-1281).

Peptide libraries and libraries of organisms can be prepared by combinatorial chemistry and then screened for their GM1 / Bg3 binding capacity. Synthetic compounds, natural products, and potentially other sources of biologically active materials can be screened in a number of ways that are common to those skilled in the art.

GM1 or Gb3 or fragments thereof can be used to screen peptides or molecules by any of a variety of screening techniques. Molecules can be free in solution, attached to a solid support, generated on the cell surface, or located within the cell. Degradation of activity or complex bond formation between GM1 or Gb3 and the drug being tested can be measured.

Another method of measuring the binding of GM1 / Gb3 is to use purified GM1 / Gb3 to coat the microtiter plate. After blocking, the agent is applied to the plate under irradiation and allowed to interact before washing and detecting antibodies specific to the agent. Direct or indirect binding of the antibody with the enzyme or radiolabel allows for subsequent quantification of the binding using colorimetric or radioactivity based methods (ELISA or RIA, respectively).

Another method of measuring the binding of GM1 / Gb3 is to combine the saccharide moiety of GM1 / Gb3 to an appropriate column substrate so that standard affinity chromatography can be performed. Removal of known compounds applied to the column from the dilution can be used as evidence for binding activity, or alternatively, when a mixture of compounds is applied to the column, elution and subsequent analysis are performed with ganglioside binders. The characteristics of can be measured. In the case of proteins, the analysis may include peptide sequencing and tryptic digest mapping, which are compared using available databases. In this case the eluted protein cannot be identified in this way, so standard biochemical assays such as, for example, mass determination by laser desorption mass spectrometry can be used to further characterize the compound. Non-proteins eluted from the GM1-affinity column can be analyzed by HPLC and mass spectrometry of a single uniform peak.

Another method of measuring the ability to bind GM1 / Gb3 and the precise affinity of its interactions is to report plasmon surface resonance as previously reported (Kuziemko et al. (1996) Biochem 35: 6375-6384). Is to use.

Alternatively, phage displays can be used to identify candidate agents to which GM1 or Gb3 binds.

Phage display is a molecular screening method using recombinant bacteriophage. The technique has a gene encoding a suitable ligand (in this case, a candidate agent) capable of reacting with GM1 / Gb3 (or a derivative or equivalent thereof) or a nucleotide sequence (or a derivative or equivalent thereof) encoding the same. Transgenic bacteriophage. Transformed bacteriophages (preferably bound to solid supports) express the appropriate ligands (such as candidate drugs) and display them on their phage coats. An entity or entities (such as cells) with a target molecule that recognizes the candidate agent are isolated and amplified. Successful candidate agents are then characterized. Phage display is advantageous over standard affinity ligand screening techniques. The phage surface represents the candidate agent in three two-dimensional forms that more closely resemble the naturally occurring form. This allows for more specific and higher affinity binding for screening purposes.

Another technique for screening is to provide high yield screening of drugs with appropriate binding affinity for GM1 or Gb3, and the details are based on what is described in WO 84/03564. Similarly, many other small peptide test compounds are synthesized on solid substrates such as plastic pins or some other surface. Peptide test agents are reacted and washed with the target interacting component fragment. The bound target interaction component is then detected by appropriately applying methods well known in the art. Purified target interacting components may also be coated directly onto the plate for use in the aforementioned drug screening techniques. Alternatively, non-neutralized antibodies can be used to capture the peptide and immobilized on a solid support.

In all aspects of the invention, agents having GM1-binding activity or Gb3 binding activity may also cross-link GM1 or Gb3 receptors. EtxB is one such agent capable of cross-linking the GM1 receptor in pentamer form.

There are several ways to identify drugs that are mediated by GM1 / Gb3 binding but which themselves may affect intracellular signaling that does not bind GM1 or Gb3. For example, if the agent upregulates CD25 or MHC class II on B cells, upregulates CD25 of CD8 + T cells, or promotes apoptosis of CD8 + T cells, or by monosite If it is shown to upregulate secretion but the drug does not bind to GM1 or Gb3 (eg, by the binding assay mentioned above), the drug may have a similar effect to the GM1 / Gb3 binding effect. It can be concluded that.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings and the following examples.

Example 1 rEtxB can be used in combination with HSV-1 Gp for immunotherapy.

Rat nasal passages were immunized three times with 10 μg of HSV-1 glycoprotein (Gp) and 10 or 20 μg of rEtxB. Controls were injected with simulated preparations of viral glycoproteins either untreated or derived from HIV-uninfected tissue culture cells. Antibody levels are expressed as% post infection level. Total production of Ig and IgA in serum and IgA in eye washes was stimulated by HSV-1 glycoprotein / rEtxB (see FIG. 1). We also found that doses of rEtxB as low as 0.1 μg were effective to stimulate this response.

In addition, T-lymphocytes extracted from cervical lymph nodes (located at the site of vaccination) and mesenteric lymph nodes (falling from the site of vaccination) from immunized mice were cultured with HSV-1 in vitro. Proliferation was observed in culture in vitro without mock HSV-1 Gp or without antigen (see FIG. 2).

It was demonstrated in FIG. 3 that T lymphocytes proliferated in response to HSV-1 Gp from MLN and CLN of mice immunized with HSV-1 Gp in the presence of varying amounts of EtxB.

4 shows the results of anti-HSV-1 serum Ig generated by administering HSV-1 glycoprotein to the rats every three days and changing the amount of EtxB (or CtxB).

Finally, mice immunized with HSV-1 and rEtxB had decreased virus divergence during corneal ganglion treatment with HSV-1 compared to controls (see FIG. 5B), and localized spread (species and lids). Disease), trigeminal ganglion (zoster infection) spread, central nervous system (encephalitis) spread and latent.

Example 2: rCtxB and rEtxB act as immunomodulators

When EtxB was used as an immunomodulator, it deviated from the Ig iso sample distribution (see Figure 6). The Ig subclass distribution differed depending on which type of rCtxB or rEtxB was used as an immunomodulator (see FIG. 7).

Example 3: rEtxB is a more effective immunomodulator than rCtxB.

Upon stimulation with rEtxB / HSV-1 Gp than rCtxB / HSV-1 Gp, it was confirmed that the HSV-specific IgA level was higher (see FIG. 8).

Example 4: (See FIG. 9)

HSV-1 glycoproteins alone, mock preparations of HSV-1 glycoproteins in mice (prepared by taking uninfected tissue culture cells and following the same treatments used to isolate and purify HSV-1 proteins), or various presumptions HSV-1 glycoprotein combined with mucosal adjuvant was immunized three times in the nasal cavity. In each case the dose of HSV-1 glycoprotein was 10 μg per immunization, which was combined with 10 μg of recombination EtxB or CtxB as adjuvant, or mixed in a mixture of 0.5 μg of Ctx and 10 μg of CtxB. Blood samples were collected three weeks after the last immunization and total anti-HSV-1 antibodies were measured by ELISA. The amount of antibody is expressed as% of stimulated level after egg-induced ocular infection with 10 ⁵ pfu HSV-1 strain SC16. The data (shown in Figure 9) showed that the serum antibody response was most stimulated when the antigen was mixed with a mixture of total Ctx and CtxB. However, when rEtxB was used as a supplement, the response was also stimulated to high levels. In contrast, rCtxB was the adjuvant with the weakest effect.

Example 5: (See FIG. 10)

Mice were immunized as described in Example 4. Tear washes were taken over successive days, then these samples were collected and ELISA assays were performed using specific anti-IgA detection antibodies to assess the extent of Secretory IgA production in the eye.

The amount of antibody is expressed as% of stimulated level after egg-induced ocular infection with 10 ⁵ pfu HSV-1 strain SC16. The data clearly demonstrated that high levels of secreted anti-HSV-1 antibodies were produced by immunization in the presence of Ctx / CtxB or EtxB (see FIG. 10). In contrast to serum antibody response assays, there was no difference in the antibody levels in the eyes between animals immunized with Ctx / CtxB or EtxB adjuvant. This is clear evidence that rCtxB is a very poor adjuvant as a serum antibody.

Example 6: (See Figure 11)

Mice were immunized as described in Example 4. Washing fluid was taken from the reproductive tract over successive days and then these samples were collected and ELISA assays performed using specific anti-IgA detection antibodies to assess the extent of Secretory IgA production in the vagina. The antibody amount is shown as the final titer calculated by linear regression analysis. The data clearly demonstrates that high levels of secreted anti-HSV-1 antibodies are generated at the mucosal sites separated by immunization in the presence of Ctx / CtxB or EtxB. Intravaginal immunization in the presence of rEtxB showed the highest antibody levels. Lower levels were obtained after immunization with Ctx / CtxB and very low secretions were obtained when rCtxB was used as an adjuvant.

Example 7: (See FIG. 12)

Mice were immunized three times in the nasal cavity with HSV-1 glycoprotein (10 μg) alone or in the presence of a proportional sensitization dose of rEtxB. Blood was taken three weeks after the last immunization and anti-HSV-1 antibody levels were assessed by ELISA. The amount of antibody is expressed as% of stimulated level after eye infection induced by egg passage with 10 ⁵ pfu HSV-1 strain SC16. The data clearly demonstrates that rEtxB, which elicits antibody responses to heterologous addition antigens, exhibits a dose-dependent phenomenon at an rEtxB dose of approximately 20-50 μg, showing maximum responsiveness. Furthermore, when the rEtxB dose was 20 μg or more, the levels of anti-HSV-1 antibodies stimulated by nasal infections were comparable or higher than those stimulated by live infectious viral infections.

Example 8: (See FIG. 13)

Mice were immunized as described in Example 7. Eye washes were taken over consecutive days, these samples were collected, and ELISA assays were performed using specific anti-IgA detection antibodies to assess the extent of Secretry IgA production in the eye. The amount of antibody is expressed as% stimulated level after eye infection induced by egg passage with 10 ⁵ pfu HSV-1 strain SC16. The data demonstrate that the IgA response in the eye is maximally stimulated when HSV-1 glycoproteins bind at least 20 μg of rEtxB. Nevertheless, the IgA production levels under these doses were lower than those induced during ocular viral infection.

Example 9: (See FIG. 14)

Mice were immunized as described in Example 7. The degree of Secretory IgA was assessed by taking washes from the reproductive tract over successive days, then collecting these samples and performing ELISA analysis using specific anti-IgA detecting antibodies. The antibody amount is shown as the final titer calculated by linear regression analysis. The data showed that an optimal anti-HSV-1 response was stimulated in the vagina when more than 20 μg of rEtxB was used as an adjuvant.

Example 10: (See FIG. 15)

The egg was infected with 10 ⁵ pfu HSV-1 strain SC16 into the cornea of the mouse, or 10 μg of HSV-1 glycoprotein bound to Ctx / CtxB or rEtxB was injected into the nasal cavity three times. Serum was taken three weeks after the last inoculation and ELISA analyzed for the presence of IgG1 and IgG2a against HSV-1. The amount of antibody is shown as the final titer calculated by linear regression analysis (see FIG. 7A). The data clearly showed that the antibody response to HSV-1 is affected by the way in which the antigen's immune system is present. Predominantly infection with HSV-1 activates the Th1 associated antibody product, as characterized by high levels of the complement binding antibody isotype, IgG2a. Infection stimulates the Th2 associated IgG isotype sample, IgG1, at relatively low levels. The shape of the immune response is clearly pronounced when the data is expressed as the IgG1: IgG2a ratio as shown in Figure 7b. The ratio after infection was substantially less than one. Nasal immunization in the presence of Ctx / CtxB as an adjuvant resulted in the predominant release of Th2 associated IgG1. Significant levels of IgG2a suggest that Ctx / CtxB causes activation of Th1 and Th2 cells. At the point where IgGl: IgG2a is approximately 3, all of the above reactions were activated and Th2 was relatively dominant. Interestingly, the response properties for HSV-1 stimulated by rEtxB as an adjuvant are almost exclusively Th2. High levels of IgG1 produced very small amounts of IgG2a. Th2 reactivity was strongly biased at the point where IgG1: IgG2a was approximately 9.

Example 11: (See Figure 16)

The cornea of the mouse was infected by 10 ⁵ pfu HSV-1 strain SC16 by egg ganglia or 10 μg of HSV-1 glycoprotein bound to Ctx / CtxB or rEtxB was immunized three times in the nasal cavity. Three weeks after the last inoculation, the lymph nodes were removed from the animals and used to prepare single cell suspensions cultured in the presence of lethal HSV-1 or in the presence of virus mock preparations from uninfected tissue culture cells. On day 4-7 of the culture, cell samples were removed and cELISA assay was performed to measure secretion of cytokines. The data clearly showed that T-cells in culture responded to HSV-1, but not to simulated virus preparations. Lymph node cells harvested from mice infected with HSV-1 predominantly produced Th1-associated cytokines γ-interferon (γ-IFN). Lymph node cells taken from nasal immunized animals produced high levels of Th2-associated cytokines, IL-4 and IL-10. In addition, both Ctx / CtxB and rEtxB activated T-cells that secrete γIFN upon in vitro stimulation with HSV-1. This indicates that even though the response to these adjuvants predominantly produces Th2 cytokines, they also cause some Th1 activation. This finding is consistent with the antibody response assay.

E. coli heat-labile enterotoxin B subunit (EtxB), cholera toxin B subunit (CtxB), E. coli verotoxin B subunit (VtxB) and GM1 or Gb3 or bind to GM1 or The use of other agents as immunomodulators that can mimic the binding effect on Gb3 increases the activity of cytotoxic T cells (CTLs) and thus, in many diseases, particularly viruses, intracellular bacteria and parasites. It is beneficial for the treatment of diseases caused by.

Claims (33)

For vaccines against infectious diseases that induce conversion of EtxB (enterotoxin B), CtxB (cholera toxin B) or VtxB (verotoxin B) without whole toxin into immune responses favorable to the production of anti-inflammatory cytokines How to use as an immunomodulator. The method of claim 1, wherein the immunomodulatory agent is EtxB free from whole toxin. The method of claim 1 or 2, wherein the infectious disease is for an infectious agent that is a member of the herpes virus family. The method of claim 3, wherein the infectious disease is caused by an infectious agent, the infectious agent is Herpes simplex virus (HSV) -1, HSV-2, Epistein-Barr virus (EBV), Varicella Zoster Virus (VZV), CMV ( Cytomegalovirus), human herpesvirus (HHV) -6, HHV-7 and HHV-8. 5. The method of claim 4, wherein said infectious agent is selected from the group consisting of HSV-1, HSV-2, CMV or EBV. The method of claim 1 or 2, wherein the infectious disease is caused by an infectious agent, and the infectious agent is an influenza virus. The method of claim 1 or 2, wherein the infectious disease is caused by an infectious agent, and the infectious agent is a parainfluenza virus. The method of claim 1 or 2, wherein the infectious disease is caused by an infectious agent, and the infectious agent is a respiratory virus. The method of claim 1 or 2, wherein the infectious disease is caused by an infectious agent, and the infectious agent is hepatitis virus. 10. The method of claim 9, wherein said infectious agent is selected from the group consisting of hepatitis A, B, C and D viruses. The method of claim 10, wherein the infectious agent is hepatitis A virus or hepatitis C virus. The method of claim 1 or 2, wherein the infectious disease is meningitis. The method of claim 12, wherein the infectious disease is caused by an infectious agent, and the infectious agent is Neisseria mening GT display (Neisseria meningitidis ), Haemophilus influenza type B, and Streptococcus pueumoniae . The method of claim 1 or 2, wherein the infectious disease is pneumonia or respiratory tract infection. 15. The method of claim 14 wherein the infectious disease is caused by an infectious agent, and the infectious agent is streptococcus pneumoniae in (Streptococcus pneumoniae), resorcinol Nella pneumophila (Legonella pneumophila ) and Mycobacterium Method selected from the group consisting of Mycobacterium tuberculosis . The method of claim 1 or 2, wherein the infectious disease is a sexually-transmitted disease. The method of claim 16, wherein the infectious disease is caused by an infectious agent, the infectious agent is Neisseria gonnorheae ) , HIV-1, HIV-2 and Chlamydia trachomatis . The method of claim 1 or 2, wherein the infectious disease is an enteric disease. 19. The method of claim 18, wherein the infectious disease is caused by an infectious agent, the infectious agent is enteropathogenic, enterotoxic, enteric invasive, enterohemorrhagic and enterocollective E. coli, rotavirus, Salmonella enteritidis , Salmonella typhimurium (Salmonella typhi), Helicobacter pylori (Helicobacter pylori), Bacillus cereus (Bacillus cereus), Campylobacter claim Juni (Campylobacter jejuni ) and Vibrio cholera ( Vibrio) cholerae ), characterized in that it is selected from the group consisting of. The method of claim 1 or 2, wherein the infectious disease is caused by an infectious agent, the infectious agent is Staphylococcus aureus , Streptococcus pyogenes and Streptococcus mutans. ( Streptococcus mutans ). The method of claim 1 or 2, wherein the infectious disease is a parasitic disease. The method of claim 21, wherein the infectious disease is caused by an infectious agent, the infectious agent is Malaya, tripanazo spp ( Trypanasoma spp . ), Toxoplasma gondii ), Leishmania donovani ) and oncocerca spp ( Oncocerca spp . Method selected from the group consisting of). Antigenic determinants that are antigenic determinants of infectious agents, and Immunomodulators selected from EtxB, CtxB or VtxB without whole toxin, Including; A vaccine composition for use in an infectious disease caused by an infectious agent that induces a shift to an immune response favorable to the production of anti-inflammatory cytokines. The vaccine composition of claim 23, wherein the infectious disease is HSV-1 infection and the antigenic determinant is an antigenic determinant of HSV-1. The vaccine composition according to claim 23 or 24, wherein the immunomodulatory agent is EtxB free of protoxins. The vaccine composition of claim 23 or 24, wherein the immunomodulator and antigenic determinant are separate moities. a) one of EtxB, CtxB or VtxB without whole toxin, and b) antigenic determinants of infectious disease for co-administration with vaccine immunomodulators; Including; A kit for vaccinating mammals against infectious diseases that induces a shift to an immune response that is favorable for the production of anti-inflammatory cytokines. Inoculating the host with a vaccine comprising at least one antigenic determinant and an immunomodulatory agent; The immunomodulatory agent: EtxB, CtxB or VtxB without whole toxin, A method of preventing or treating a disease in an animal host, except for humans, which induce a transition to an immune response that is favorable for the production of anti-inflammatory cytokines. A method of using EtxB, CtxB or VtxB without whole toxin to upregulate the production of antibodies on mucosal surfaces and induce a shift to an immune response that is favorable for the production of anti-inflammatory cytokines. As an immunomodulator in vaccines, EtxB, CtxB free of whole toxin to prolong antigen presentation in mammals, support immunological memory, and induce conversion to immune responses favorable to the production of anti-inflammatory cytokines Or how to use VtxB. Antigenic determinants, and Immunomodulators selected from EtxB, CtxB or VtxB without whole toxin, Including; The antigenic determinant is an antigenic determinant of an infectious disease, and the immunomodulatory agent extends the presentation of the antigenic determinant and supports immunological memory and induces a transition to an immune response that is favorable for the production of anti-inflammatory cytokines. Vaccine compositions used against infectious diseases caused by infectious agents. Used to treat EBV-related diseases a) EtxB or CtxB, and b) EBV antigen A vaccine composition comprising; inducing a conversion to an immune response favorable to the production of anti-inflammatory cytokines. A therapeutic composition comprising EtxB or CtxB that is used in the treatment of EBV-related diseases and induces a transition to an immune response that is favorable for the production of anti-inflammatory cytokines.

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