CZ20004147A3 - Vaccine - Google Patents

Abstract

There is disclosed the use of: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GM1-binding activity, or an agent other than VtxB having Gb3-binding activity; or (iii) an agent having an effect on intracellular signalling events mediated by GM1-binding or Gb3 binding; as an immunomodulator for a vaccine against infectious diseases.

Description

Technical field of reagents. Furthermore, the invention relates to an immunomodulator comprising an antigen and a method of vaccination

The invention relates to an immunomodulator that can be used in a vaccine for use against a number of infectious vaccine compositions, preferably in combination using a vaccine composition.

BACKGROUND OF THE INVENTION

Cholera toxin (Ctx) and its closely related heat-labile enterotoxin of E. coli (Etx) are potent immunogens and mucosal adjuvants. Their inherent toxicity renders them unsuitable for human use. For example, although Ctx is the most commonly used mucosal adjuvant in experimental animals, it is not suitable for use in humans because it has diarrhea-inducing properties. Efforts have been made to separate toxicity from adjuvant activity, for example by using the Ctx and Etx components as a substitute for holotoxins alone. E. coli (Vtx) verotoxin is another known bacterial toxin.

Ctx and Etx are heterohexameric proteins that show enzymatically active subunit A and pentameric subunit B. Ctx and Etx are known to bind GM1-ganglioside (GM1), a glycosphingolipid found on the surface of mammalian cells. Vtx binds to Gb3, which is similar to the type of receptor for GM1.

In the case of vaccine development, the toxicity problem has to be addressed, where the adjuvant activity of non-toxic B subunits has been investigated previously. A number of reports describe experiments in which commercial CtxB or EtxB products are used. These compositions are inevitably contaminated with small but substantial amounts of the active toxin. Thus, the biologically adjuvant activity derived from subunit B is not separable from the adjuvant activity of the whole toxin (Wu and Russell (1993) Infection and Immunity 61: 314-322, US-5182109). A subsequent study using recombinant CtxB (rCtxB) suggests that CtxB is a weak mucosal adjuvant and only the addition of natural holotoxin can elicit a strong response (Tamura et al., (1994) Vaccine 12: 419-426). Other studies suggest that rCtxB lacks ADP-ribosylating and cAMP stimulating holotoxin activities and that as an adjuvant mechanism is associated with these properties, CtxB is not suitable for use as an adjuvant (Vajdy and Lycke (1992) Immunology 75: 488-492, Lycke et al. al (1992) Eur.

J. Immunol. 22: 2277-2281, Douce et al., (1997) Infection and

Immunity 65: 2821-2828).

In another study, intranasal administration of ovalbumin using rCtxB as an adjuvant results in a weak antibody response. The non-toxic Ctx derivative with mutation in subunit A also results in poor antigen response, while the presence of active subunit A dramatically increases adjuvant activity, suggesting that the active subunit A is essential (Douce et al., (1997) Infection and Immunity 65: 2821 -2828).

It has also been shown that rCtxB and rEtxB can be used to promote tolerance to heterogeneous antigens (Sun et al (1994) Proc.

Nati. Acad. Sci . 91: 4610-4614; Sun et al (1996) Why. Nati. Acad. Sci. 93: 7196-7201, Bergerot et al (1997) Why. Nati. Acad. Sci. 94: 4610-4614, Williams et al (1997) Why. Nati. Acad. Sci. 94: 5290-5295), which suggests that these molecules j sou suitable for use as adjuvant.

SUMMARY OF THE INVENTION

Despite the knowledge in the art that CtxB and EtxB exhibit a weak adjuvant enhancing effect and may actually act as a tolerogen, the use of rEtxB (which contains no

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IV residual holotoxin or subunit A) in an intranasal vaccine suitable for HSV in a mouse model and surprisingly it was found that this toxin is able to stimulate a protective immune response to the viral vaccine. Specifically, it was found that:

(i) agents such as EtxB and CtxB stimulate a high degree of local (mucosal) antibody production (although immunization using rEtxB stimulated a lower degree of total serum antibody production compared to the Ctx / CtxB combination); ii) antibody production is unbalanced towards antibodies that do not fix complement, especially on

S-IgA and IgG1, agents such as EtxB and CtxB also stimulate local and systemic T cell proliferative responses, agents such as CtxB and EtxB tend to shift the immune response from the Th1-associated response to the Th2-associated response.

v) when agents such as CtxB and EtxB are used as immunomodulators, some of the serious effects of a Th2-related response such as IgE generation can be avoided, rEtxB is a more potent immunomodulator compared to rCtxB, agents such as EtxB and CtxB are capable of altering the manner in which antigen-presenting cells absorb and process antigen, thereby increasing antigen persistence; (viii) when an agent such as EtxB and CtxB is coupled to the antigen, it is possible to change the antigen processing pathway by changing binding to and ix) VtxB exhibits similar immunomodulatory effects on the leukocyte population in vitro with those exhibiting EtxB and CtxB.

vi

Vil,

These important discoveries underlie various aspects of the invention and allow inventors to predict that they are pure. EtxB, CtxB and VtxB, as well as other agents, are capable of binding to GM1 or Gb3 or mimicking the effects of binding. These agents can be used in a vaccine as immunomodulators in the prophylactic and therapeutic vaccination against HSV-1 infection, as well as other infections whose prevention or treatment results from immunomodulation of the types listed above.

Stimulation of immune response

EtxB, CtxB, VtxB and other agents capable of binding or mimicking the effects of binding to GM1 or Gb3 are capable of acting as immunomodulators and stimulating specific immune responses to antigen immunization.

With respect to a first aspect of the invention, the invention describes the use of:

(i) a non-toxin-free EtxB, CtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1 binding activity, or an agent other than VtxB that exhibits Gb3 binding activity, or (iii) agents that exhibit an intracellular effect signaling events mediated by GM1 binding or Gb3 binding, as an immunomodulator suitable for an infectious disease vaccine.

According to a second aspect of the invention, the invention provides a vaccine composition suitable for use against an infectious disease, wherein the infectious disease is caused by an infectious agent, wherein the vaccine composition comprises an antigenic determinant and an immunomodulator selected from the group consisting of:

(i) an entire toxin without EtxB, CtxB or VtxB, (ii) a non-EtxB or CtxB agent that exhibits GM1-binding activity or an agent,

which is not VtxB and which exhibits Gb3-binding activity, or (iii) an agent having an effect on intracellular signaling events mediated by binding to GM1 or Gb3, wherein said antigenic determinant is an antigenic determinant of said infectious agent.

The antigen and the immunomodulator may bind. For example, they may be linked covalently or genetically to form a single active agent. In a specific embodiment of the invention, the antigen and the immunomodulator may be chemically combined. The antigen and the immunomodulator can be chemically coupled using a heterobifunctional crosslinker. In most applications of said aspect of the invention, separate administration (in which the antigen and immunomodulator is not coupled) is preferred, as this allows separate administration of the different portions.

The invention further provides a kit suitable for vaccinating a mammal, such as a human or animal, against an infectious disease, comprising:

(a) one of the following:

(i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that has GM1 binding activity or an agent other than VtxB that exhibits Gb3 binding activity, or (iii) an agent having an effect on intracellular signaling events mediated by binding to GM1 or Gb3; and

b) an antigenic determinant that is an antigenic determinant of an infectious disease that is suitable for co-administration with said vaccine immunomodulator.

· · · · · · · · · · · · · · · · · · · · · · · · · · · ·

The vaccine composition and kit of the invention may be used in a prophylactic and therapeutic vaccine method, wherein the "prophylactic vaccine is administered to healthy individuals to prevent disease development and" the therapeutic vaccine is administered to individuals with existing infection to reduce or minimize infection or eliminate the immunopathological phenomena of the disease.

Agents such as EtxB have the capacity to alter the nature of the immune response after infection occurs. A therapeutic vaccine (i.e., a vaccine where it is not required to contain an antigen) containing such an agent may find particular use in circumstances where the immune response has failed to combat infection. The vaccine may be particularly useful in the treatment of a chronic disease, for example a disease in which the causative agent is selected from the group consisting of herpes virus, hepatitis virus, HIV, TB and parasites.

The invention further provides a method of preventing or treating a host disease, said method comprising inoculating said host with a vaccine comprising at least one antigenic determinant and an immunomodulator, wherein the immunomodulator is (i) an EtxB, CtxB or VtxB without a whole toxin; or CtxB which exhibits GM1 binding capacity or an agent other than VtxB and exhibits Gb3 binding capacity, or (iii) an agent that exhibits an effect on intracellular signaling events mediated by binding to GM1 and Gb3.

The vaccine can be packaged to be suitable for co-administration and can be administered in a variety of ways, such as intranasal, oral, intravaginal, urethral or ocular administration. Intranasal immunization is currently preferred.

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When the vaccine is administered intranasally, it can be administered as an aerosol or in liquid form.

The antigenic determinant and immunomodulator may be administered to the subject as a single dose or multiple doses.

The immunomodulator, vaccine, kit and method of the invention can be used against diseases wherein the infectious agent is a member of the herpes virus family. The infectious agent may be selected from the group consisting of HSV-1, HSV-2, EBV, VZV, CMV, HHV-β, HHV-7 and HHV-8. The infectious agent is HSV-1, HSV-2, CMV or EBV.

In this embodiment of the invention, the antigenic determinant of the product of the mid-early, early or late gene (e.g., surface glycoprotein) of herpes virus. If the infectious agent is HSV-1 or HSV-2, then the antigenic determinant may be an antigenic determinant of a gene product selected from the following group: gD, gB, gC or latency associated transcript (LAT).

If the infectious agent is EBV, then the antigenic determinant can be an antigenic determinant of gp340 or gp350 or a latent protein (for example, EBNA 1, 2, 3A, 3B, 3C and -LP, LMP-1, -2A and 2B or EBER).

In another embodiment of the invention, the vaccine, kit and method are used against a disease wherein the infectious agent is an influenza virus.

In this embodiment, the antigenic determinant is a viral envelope protein (e.g., hemagglutinin and neuraminidase) or an internal protein (e.g., a nuclear protein).

In this embodiment, the immunomodulator, vaccine, kit and method are used against a disease wherein the infectious agent is parainfluenza virus.

In another embodiment of the invention, the immunomodulator, vaccine, kit and method have been used for diseases wherein the infectious agent is a respiratory syncytial virus.

In another embodiment of the invention, the immunomodulator, vaccine, kit and method have been used in a disease wherein the infectious agent is hepatitis virus. For example, the infectious agent is selected from the group consisting of hepatitis A, B, C, and D. The infectious agent may in particular be hepatitis A or C.

In another embodiment of the invention, the immunomodulator, vaccine, kit and method are used in the case of meningitis, wherein the infectious agent is selected from the group consisting of Neisseria meningitidis, Haemophilus influenzae type B and Streptococcus pneumoniae.

In another embodiment of the invention, the immunomodulator, vaccine, kit and method have been used against pneumonia or respiratory infection wherein the infectious agent is selected from the group consisting of Streptococcus pneumoniae, Legonella pneumophila and Mycobacterium tuberculosis.

In another embodiment of the invention, the immunomodulator, vaccine, kit and method have been used against venereal diseases wherein the infectious agent is selected from the group consisting of Neisseria gonnorheae, HIV-1, HIV-2 and Chlamydial trachomatis.

In another embodiment of the invention, the immunomodulator, vaccine, kit and method have been used in diseases of the gastrointestinal tract wherein the infectious agent is selected from the group consisting of enteropathogenic, enterotoxic and enteroinvasive E. coli, rotavirus, Salmonella enteritis, Salmonella typhi, Helicobacter pylori, Bacillus cereus, Campylobacter jejuni and Vibrio cholerae.

If the infectious agent is selected from the group consisting of enteropathogenic, enterotoxic, enteroinvasive, enterohemorrhagic and entero-aggregating E. coli, then the antigenic determinant may be an antigenic determinant of a bacterial toxin or an adhesion factor.

In another embodiment of the invention, the immunomodulator, vaccine, kit and method are used in a superficial infection wherein the infectious agent is selected from the group consisting of Staphylococcus aureus, Streptococcus pyogenes and Streptococcus mutans.

In another embodiment of the invention, the immunomodulator, vaccine, kit and method have been used in diseases caused by parasites wherein the infectious agent is selected from the group consisting of malaria, Trypanasoma spp., Toxoplasma gondii, Leishmania donovani and Oncocerca spp.

Stimulation of mucosal immune response

EtxB, CtxB, VtxB and other agents capable of binding or mimicking the effects of binding to GM1 or Gb3 are capable of specifically positively regulating the production of mucosal antibodies.

The vaccine immunomodulator, vaccine composition and kit of the invention are particularly effective in diseases where protection against the disease or treatment is affected by the in vivo mucosal immune response. For example, in a disease in which the infectious agent binds to the mucosa or colonizes it during infection. Examples of such diseases are those caused by viruses (HIV, HSV, EBV, CMV, influenza virus, measles virus, mumps virus, rotavirus, etc.) or bacteria (such as E. coli, Salmonella, Shigella, Chlamydia, N. gonnorhoea, T. pallidium, a species of Streptococcus, which include those which cause tooth decay) or are caused by parasites.

In a preferred embodiment of the invention, a vaccine against HSV-1 infection comprising at least one HSV-1 antigenic determinant and an immunomodulator is used, wherein the immunomodulator is:

(and) EtxB, CtxB without whole toxin, (ii) agent, other than EtxB or CtxB a show e GM1 binding activity, or agent, which is other than VtxB and shows Gb-3 custody activity or (iii) agent showing an effect on intracellular signaling phenomena mediated GMl by binding or Gb3 by binding.

Preferably, the immunomodulator is EtxB.

In a preferred embodiment of the invention, a kit suitable for vaccination of mammals against HSV-1 is used, comprising:

(a) a vaccine immunomodulator, which is:

EtxB, CtxB or VtxB bez whole toxin, agent, which is different than EtxB or CtxB a reports GMl custody Activity or agent, which is other than VtxB and shows Gb-3 custody activity or

(iii) an agent having an effect on intracellular signaling events mediated by GM1 binding or Gb3 binding; and (b) at least one HSV-1 antigenic determinant suitable for co-administration with said vaccine immunomodulator.

The invention further provides the use of (i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1 binding activity, or an agent other than VtxB that exhibits Gb-3 binding activity, or 4 4 4 444

(Iii) agents exhibiting an effect on intracellular signaling events mediated by GM1 binding or Gb3 binding in the positive regulation of mucosal surface production of antibodies. Production of serum antibodies that do not fix complement can also be regulated in a negative manner. It is preferred that S-IgA is produced in accordance with the method of the invention.

The immunomodulator, vaccine, kit and method of the invention may be used in conjunction with one or more antigenic determinants.

Negative regulation of pathological components of immune response.

It has also been found that when using pure EtxB as an immunomodulator as described, the devastating effects of a Th2-associated response, such as the generation of a large number of potentially pathological IgE, were avoided. Despite this, the immune response elicited by the use of EtxB (or CtxB or VtxB) as an immunomodulator appears to be useful for inducing Th2-associated cytokines. In other words, EtxB (or CtxB) induces a shift from a Th1 type response to a Th2 type response. This makes it possible to predict that pure EtxB, CtxB or VtxB as well as other agents capable of binding or mimicking the effects of binding to GM1 or Gb3 will be able to negatively regulate the pathological components of the immune response associated with Th1 and Th2 activation.

The invention further provides the use of (i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1 binding activity, or an agent other than VtxB that exhibits Gb-3 binding activity, or (iii) agents exhibiting an effect on intracellular signaling events mediated by GM1 binding or Gb3 binding and in the negative regulation of the pathological components of the Th2-associated immune response. The pathological components of the Th1-associated immune response can also be negatively regulated.

EtxB and CtxB are known to bind to GM1 and to induce differential effects on the lymphocyte population, including specific CD8 + T cell depletion and associated B cell activation (WO 97/0204). Because EtxB and CtxB are considered to alter the immune response balance such that inflammatory Th1-associated responses are negatively regulated while Th2-associated responses are positively regulated. The Th1 response involves secretion of ylFN by activated cells, resulting in activation of macrophages and delaying hypersensitivity-type responses. Such responses can be an important pathological phenomenon during infections with a number of pathogens. Th2 responses involve activation of T cells to produce cytokines such as IL-4, 11-5, 11-10 and are known to promote secretion of large quantities of antibodies, particularly IgA.

It has then surprisingly been found that when EtxB is used as the immunomodulator as described, the deleterious effects of a Th2-associated response, such as the generation of a large amount of potentially pathological IgE, are prevented. Therefore, EtxB and CtxB are able to negatively regulate the pathological components associated with Th1 and Th2 activation. Such responses vary to achieve high production of non-complement-fixing serum antibodies and IgA production on the mucosal surface.

The use of an agent in accordance with the invention is particularly useful in the therapeutic vaccination of diseases in which immunopathological mechanisms are involved. Examples of such diseases are HSV-1, HSV-2, TB and HIV.

Agents such as EtxB can be used simultaneously as an immunomodulator and therapeutic agent. For example, in diseases where immunopatholigic mechanisms are involved, 0 0 0 0 0 0

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The use of agents included in the vaccine, such as EtxB or CtxB, may not only reduce infection but also eliminate pathological disease processes. Thus, the immunomodulatory agent acts both prophylactically and therapeutically. Examples of infections where vaccination in this manner is therefore likely to be particularly significant include diseases caused by herpes simplex virus, digestive and respiratory pathogens.

Immunomodulation of antigen processing

(a) extended presentation

It has also been found that when EtxB (or CtxB or VtxB) is used as an immunomodulator, the uptake of the antigen and the way it is processed will change. The presence of subunit B causes prolonged presentation, presumably by altering the pathway within the antigen presenting cells so that antigen decomposition is delayed and therefore persists for an extended period of time. This feature of antigen-associated subunit B presentation means that vaccines containing an agent of the invention will increase antigen resistance and this results in sustained immunological memory.

The invention further describes the use of:

(i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1-binding activity or an agent other than VtxB that exhibits Gb3-binding activity, or (iii) agents exhibiting an effect on intracellular signaling events mediated by binding to GM1 or Gb3 as an immunomodulator in a vaccine to prolong antigen presentation and to generate sustained immunological memory in a mammal.

The invention provides a vaccine composition suitable for use against an infectious disease comprising an antigenic determinant and an immunomodulator selected from (i) a non-toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1 a binding activity or agent other than VtxB that exhibits Gb3-binding activity, or (iii) an agent having an effect on intracellular signaling events mediated by binding to GM1 or Gb3, wherein said antigenic determinant is an antigenic determinant of said infectious disease and wherein the immunomodulator prolongs the presence of an antigenic determinant and gives rise to permanent immunological memory.

(b) intracellular targeting of the antigen to the MHC-I-associated pathway; or

MHC-II

As mentioned above, the immunomodulator and antigen may be genetically or covalently linked in a therapeutic or prophylactic vaccine to form a single active agent. It has been found that it is possible to direct the antigen to different parts of the cells and therefore it is possible to achieve different pathways of antigen presentation by changing the antigen binding to the immunomodulator.

By combining an antigen or antigenic determinant with an immunomodulator in a particular pathway, it is possible to achieve translocation of the antigen across the endosomal membrane into the cytosol. It has been predicted that this will increase the deposition of antigenic peptides on MHC class I molecules. The use of an antigen-immunomodulator conjugate may therefore be used to specifically increase cytotoxic C cell (CTL) activation. Induction of CTL is advantageous in the prevention and treatment of a number of diseases, particularly those caused by viruses, intracellular bacteria and parasites.

Alternatively, the antigen-immunomodulator conjugate can be selected such that the antigen is introduced into the nucleus.

The invention further provides a conjugate comprising an antigen or antigenic determinant and an immunomodulator selected from (i) a non-toxin-free EtxB, CtxB or VtxB, (ii) an agent that is other EtxB or CtxB that exhibits GM1-binding activity or an agent that is other than VtxB and which exhibits Gb3-binding activity; or (iii) an agent having an effect on intracellular signaling events mediated by binding to GM1 or Gb3.

illness means

The invention further provides a vaccine composition which is suitable for use against an infectious disease, wherein the infectious is caused by an infectious agent. The vaccine comprises an antigen or antigenic determinant conjugate and an immunomodulator selected from

A toxin-free EtxB, CtxB or VtxB, an agent other than EtxB or CtxB that exhibits GM1-binding activity or an agent other than VtxB that exhibits Gb3-binding activity or an agent exhibiting (i) (ii) ( iii) an effect on intracellular signaling events mediated by binding to GM1 or Gb3, wherein said antigen or antigenic determinant is an antigen or antigenic determinant of said infectious agent.

The antigen or antigenic determinant is associated with the immunomodulator in a variety of ways, including genetic linkage or chemical conjugation. In a preferred embodiment of the invention, the conjugate is a fusion protein prepared by genetic binding of an antigen or antigenic determinant 0 0 0 0 0 0 0 0 0 0 0 000 0 ··· · ··· with immunomodulator. Preferably, the antigen or antigenic determinant is genetically linked to the C-terminus of the immunomodulator. In a second preferred embodiment of the invention, the antigen or antigenic determinant is chemically linked to an immunomodulator. It is preferred that the antigen or antigenic determinants be coupled to an immunomodulator using a dual function crosslinking agent, such as a heterobifunctional crosslinker. More preferably, the crosslinker is byl-γ (-maleimido-butyroxyl) succinimide ester (GMBS) or N-succinimidyl- (3-pyridyl-dithio) propionate (SPDP). The vaccine composition can be administered by a variety of routes, such as intranasal, oral, intravaginal, urethral or ocular. Intranasal immunization is preferred.

The invention further provides the use of (i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1-binding activity or an agent other than VtxB that exhibits Gb3-binding activity or (iii) an agent having an effect on intracellular signaling events mediated by binding to GM1 or Gb3, in conjugate with an antigen or antigenic determinant to introduce said antigen or antigenic determinant into the cytosol or nucleus of the antigen presenting cell.

The invention discloses the use of (i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1-binding activity or an agent other than VtxB that exhibits Gb3-binding activity, or ♦ · · φ · · * · · · · · · · · · · · φ φ · · · · · · · · (iii) an agent having an effect on intracellular signaling events mediated by binding to GM1 or Gb3, in conjugate with an antigen or antigenic determinant, to enhance the presentation of said antigenic determinant or antigenic determinants derived from said antigen by MHC class I molecules.

A preferred use of the conjugate of the invention is to be used in combination with an agent of the invention to stimulate a strong CTL response and to positively regulate mucosal antibody production. This activity is particularly useful in the prevention and treatment of viral infections, such as influenza.

EtxB is a preferred immunomodulator

EtxB and CtxB are considered to exhibit similar properties. However, rEtxB was found to be a more potent and efficient modulator compared to rCtxB. Because the preferred immunomodulator is EtxB or an agent that mimics the effects of EtxB.

EBV

EBV is one of eight known human herpes viruses. The infection usually occurs in early childhood, but the clinical symptoms at this stage are usually mild or not detectable. Later in life, primary EBV infection is associated with infectious mononucleosis (IM), the second most common disease in the United States during adolescence. EBV also shows oncogenic potential. There is a strong association between EBV and endemic Burkit's lymphoma (BL) and undifferentiated nasopharyngeal carcinoma (NPC). Also, a large proportion of lymphomas that occur in immunocompromised patients are caused by EBV and there is a relationship between certain Hodgkin's lymphomas and EBV.

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Cells latently infected with EBV express a small amount of so-called "latent proteins". Said latent proteins include six nuclear proteins (EBNA 1, 2, 3A, 3B, 3C a-LP), three integrated membrane proteins (LMP-1, 2A and 2B) and non-polyadenylated virus (EBER) RNA with a certain splice role RNA.

EBV latent membrane protein 1 (LMP-1) is present in the plasma membrane of infected cells. It is also expressed in nasopharyngeal carcinomas (NPCs) and Hodgkin's lymphomas that are EBV positive (HD), indicating the role of LMP-1 in the development of these tumors. The LMP-1 gene can alter the phenotype of uninfected cells, causing positive regulation of activation markers on the cell surface, promoting cell proliferation. LPM-1 can also alter signaling pathways and has anti-apoptotic effects. A cellular immune response directed against this viral antigen is not associated with any healthy vehicle or cancer patients.

Many animal viruses include mechanisms that prevent their detection by the host's immune system. Typically, this mechanism involves interference by a TAP-associated peptide translocation system. EBV is thought to exhibit similar mechanisms to prevent detection by the immune system, allowing it to persist in the host. This explains why certain cellular immune responses are not detectable by the EBV latent EBNA1 protein and the apparent absence of such an immune response against LMP1.

The invention further provides a vaccine composition further comprising

(a) one of the following:

(i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1-binding activity or an agent other than VtxB and which exhibits Gb3-binding activity, or (iii) an agent having an effect on intracellular signaling events mediated by binding to GM1 or Gb3,

(b) an EBV antigen suitable for use in the treatment and / or prevention of EBV-related diseases.

Included in a particular vaccine composition of the invention are EtxB, CtxB or a non-EtxB or CtxB agent that exhibits GM1 binding activity.

The invention further provides a therapeutic composition comprising:

(i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1-binding activity or an agent other than VtxB and which exhibits Gb3-binding activity, or (iii) an agent having an effect on intracellular signaling events mediated by binding to GM1 or Gb3, wherein said therapeutic agent is used in the treatment of a disease associated with EBV.

The invention further provides a therapeutic composition of the invention comprising EtxB, CtxB or an agent that differs from EtxB or CtxB and which exhibits GM1-binding activity.

Based on the knowledge that EtxB associates with LMP1 and that EtxB promotes fragmentation of LMP-1, it has been hypothesized that EtxB (and other CtxB-like agents exhibiting GM1 binding activity) is useful in stimulating an anti-EBV immune response. This activity finds use in vaccines where it prevents EBV-related diseases and provides treatment in the event of disease.

When EtxB associates with LMP-1, it is believed that the antigen is processed by a different intracellular method that allows the antigen to undergo a normal processing that is blocked by the virus. Thus, the antigen is effectively presented on the cell surface. Thus, various epitopes of antigen that are present on the cell surface may cause the effects of EtxB. These epitopes may differ from the epitopes that are present on the cell surface when processed by the conventional route.

The vaccine of the invention may be used to prevent infection caused by EBV or the development of a disease associated with EBV in individuals infected with EBV. The vaccine may also contain a separate adjuvant or agent (such as EtxB or CtxB), which may act as an adjuvant.

The agents of the invention may be used according to the invention alone (i.e., without antigen) in the treatment of a disease associated with EBV if the subject has already become ill.

A preferred agent for use in the present invention is EtxB.

EBV antigen is an antigen derived from EBV itself or an antigen that expresses EBV-infected host cells by EBV. Preferably, the antigen is a latent membrane protein of EBV. LMP-1, LPM-2A, LPM-2B and EBNA-1 antigens, as well as antigenic fragments thereof, are particularly preferred. The antigen may be isolated directly from EBV infected cells or may be prepared by synthetic or recombinant means.

Further, the invention describes the treatment and / or prevention of the following diseases: infectious mononucleosis, Burkitt's lymphoma, nasopharyngeal carcinomas, and Hodgkin's lymphomas. The invention is believed to be useful in the treatment and / or prevention of nasopharyngeal carcinomas and Hodgkin's lymphomas.

The vaccine or therapeutic composition of the invention can be used to prevent the development or treatment of EBV-related diseases in a mammal by administering an immunologically effective amount of a subject.

For example, the mammal may be a healthy individual infected or uninfected with EBV, an immunodeficiency individual, or an individual with an EBV-related disease.

The vaccine may be administered by any suitable route. The agent and antigen can be administered to the mammal together or separately. The agent and antigen may be present separately or may be bound, for example, covalently or genetically to form a single active agent.

suggests receptor

Signaling associated with GM-1 and Gb-3

It is believed that binding of GM1 or Gb3 may result in intracellular signaling directly or indirectly. There has also been evidence that interacts with at least one other involved in intracellular signaling that EtxB is associated with GM1. It may happen that binding of EtxB (or CtxB)

GM1 binding to intracellular signaling. It is known which processes can cause intracellular signaling. This may be the phosphorylation of GM1 or protein. When EtxB / CtxB binds to cell surface GM1, bound GM1 is internalized into vesicles (Williams et al (1999) Immunology GM1 and other glycolipids (such as Gb3) are preferably found in "membrane grafts" Therefore, it is possible that the internalization of GM1, as a result of B-subunit binding, results in the association of such proteins, leading to the mediation of intracellular signaling events.

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Definition

An adjuvant is a compound that nonspecifically increases the immune response to an antigen, thereby differentiating it from a vaccine carrier because of the targeting of the antigens to the desired site. The term "immunomodulator" is used to indicate an agent that acts as an adjuvant to stimulate a certain immune response but which also directs the immune response in a particular direction.

The term "co-administration" is used when the site and time of administration of the antigens and the immunomodulator are such that the necessary immune response is stimulated. While the antigen and immunomodulator may be administered at the same time and at the same site, it may be advantageous to administer the antigen at a different time and / or at a different site compared to the immunomodulator. The antigen and immunomodulator may be administered together with the first step, then the immune response may be enhanced in the second step by administering the antigens alone.

The term "antigenic determinant" refers to a site on antigens that recognize an antibody or T-cell receptor. Preferably, it is a short peptide derived from protein antigens or as glycopeptides and also includes a portion thereof, but the term also includes saccharide antigenic determinants. The term modified amino acid or carbohydrate sequences that stimulate responses that recognize the whole organism.

There are a number of known methods of identifying an infectious agent, the antigenic determinants of which is, if possible, given

Potentially by enhancing immune convalescent patients, or by monitoring the antigen.

protective antigens may identify the responses in infected patients or in infected animals or convalescent immune responses to in vitro preparations

For example

(i) serum samples from infected or convalescent patients or infected or convalescent animals may be tested against whole cell lysates by infectious agents or lysates of cells infected with the reagent by standard Western blotting methods to detect antigen (s) recognized by the immune serum.

(ii) serum samples from infected or convalescent patients or infected or convalescent animals may be tested against partially or highly purified antigens derived from an infectious agent or cell lysates infected with that agent, by a standard ELISA method, where partially or highly purified antigens used for coating. wells on microtiter plates or by immunoblotting to detect these antigens that are recognized by immune serum.

(iii) serum samples from infected or convalescent patients or infected or convalescent animals may be tested against whole cell lysates obtained from recombinant expression systems that encode one or more antigens recognized by immune serum; (iv) serum samples from infected or convalescent patients or infected; convalescent animals can be screened against an expression library that contains cloned genes from infectious agents using colony transfer immunodetection to detect those clones that express antigens or fragments thereof by the immune system, or

(v) PBLs from the blood of infected or convalescent patients or PBLs, lymph node cells, spleen cells or lamina propria cells from infected or convalescent animals may be cultured in vitro in the presence of partially or fully purified antigens obtained from either infectious agent or lysates of cells infected with that agent or a recombinant expression system that encodes one or more antigens such that it detects specific T cell proliferative responses.

Alternatively, it is possible to detect gene products that are essential for the survival of pathogens in vitro as shown by the labeled mutagenesis method developed by Holden or by the detection of gene products specifically induced in vivo, such as IVET (in vivo expression technology) developed by Mekalanos, or by differential fluorescence induction, developed by Falkow, identification of a set of genes that include potential protective antigens. Using these methods, gene products can be tested as described above. Genes can be cloned into expression vectors, and antigens are obtained from inclusion in vaccine compounds along with agents that modulate glycosphingolipid-related activity.

There are a number of known methods that allow the isolation of antigens in a given infectious agent.

The surface components of the infectious agent containing one or more potential protective antigens can be extracted from the agent or from the cells infected with the agent using techniques that allow the recovery of the antigens. This may include the use of a cell disruption method, lysing cells such as sonication and / or detergent extraction. Centrifugation, ultrafiltration or precipitation may be used to prepare the collected antigens. Antigen preparations containing HSV-1 glycoproteins are described in Richards et al., (1998) J. Infect. Dis. 177, 1451-7, for examples of such methods.

The antigens of the infectious agent or from cells infected with said agent may be extracted by a variety of methods including extraction from urine, alkaline or acid extraction or detergent extraction and subjected to chromatographic separation. The material obtained in the void space or at the maximum elution containing one or more potential protective antigens may be used with vaccine compounds.

Alternatively, genes that encode one or more potential protective antigens may be cloned into various expression vectors suitable for antigen production. This may include bacterial or eukaryotic expression systems. For example, Escherichia coli, Bacillus spp., Vibrio spp. , Sacarromyces cerevisiae, mammalian and insect cell lines. Antigens may be obtained by conventional extraction, separation and / or chromatographic procedures.

The terms "CtxB," EtxB, and "VtxB" include natural and recombinant forms of the molecules. The recombinant form is particularly preferred. The recombinant form of the molecule can be produced by a method in which the gene or genes encoding the specific polypeptide chain (s) from which the peptide is formed is incorporated into a suitable vector and then used to transfect a suitable host. For example, the gene encoding the polypeptide chain from which EtxB is derived can then be inserted into, for example, plasmid pMN68, which is then used to transfect host cells such as Vibrio sp.60. The protein is then purified and isolated in a number of ways. Mutant genes expressing the active mutant protein CtxB, EtxB or VtxB can be produced by known methods from the wild-type gene.

The terms "CtxB," EtxB, and VtxB also include mutant molecules and other synthetic molecules (comprising portions of CtxB, EtxB, or VtxB) that retain the capacity to mimic the effects of GM1 or Gb3 binding.

• · • ·

Non-EtxB and CtxB agents that retain binding capacity and non-VtxB agents that retain Gb3 binding activity include antibodies that bind GM1 or Gb3.

In the case of antibody production, various hosts including goats, rabbits, rats, mice, etc. can be immunized by injection of GM1 or Gb3 or any derivative thereof, or

homologem. Depending on the specie host may be in order. to enhance the immunological response, use various adjuvants. Such adjuvants include, but are not limited to, mineral gels of

Freunda such as aluminum hydroxide and surfactants,

- such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, hemocyanin of gastropods and dinitrophenol.

BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum can be used as human adjuvants.

According to the invention, humanized monoclonal antibodies are preferred. Monoclonal antibodies can be prepared using a method suitable for the production of antibody molecules by continuous cultivation of cell lines. Methods include, but are not limited to, the hybridoma technique described above (Koehler and Milstein, 1975, Nature 256: 495-497), the human B-cell hybridoma technique (Kosbor et al., (1983) Immunol. Today 4 : 72, Cote et al., (1983) Proc.

Nati. Acad. Sci. 80: 2026-2030) and the EBV-hybridoma technique (Cple et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan

R Liss Inc., Pp. 77-96). In addition, a method developed for producing "chimeric antibodies, splicing mouse antibody genes into human antibody genes to produce a molecule with suitable antigen specificity and biological activity (Morrison et al., (1984) Proc. Natl. Acad. Sci.) Can be used. 81: 6851-6855, Neuberger et al., (1984) Nature 312: 604-608, Takeda et al., (1985) Nature 314: 452-454). Alternatively, the method described for producing single chain antibodies (US Patent No. 4,946,779) can be adapted to produce a target interaction portion of specific single chain antibodies.

Antibodies can be produced by inducing in vivo production in a lymphocyte population or by testing a library of recombinant immunoglobulins or panels of highly specific binding agents as described in Orlandi et al., 1989, Proc. Nati. Acad. Sci 86: 3833-3837 and Winter G and Milstein C., 1991, Nature 3837 and Winter G and Milstein C., (1991),

Nature 349: 293-299.

Antibody fragments can be generated that contain specific binding sites for GM1 or Gb3. For example, such fragments include F (ab ') ₂ fragments, which may be formed by digesting the antibody molecule with pepsin, and Fab fragments, may be formed by reducing the amount of disulfide bridges of F (ab') ₂ fragments. Alternatively, a Fab expression library can be constructed, allowing the monoclonal Fab fragments of the desired specificity to be determined quickly and easily (Huse WD et al., (1989) Science 256: 1275-1281).

Peptide or organic libraries can be prepared by combinatorial chemistry and then tested for their ability to bind GM1 / Gb3. Synthetic compounds, natural products and other sources of potentially biologically active materials can be tested in a variety of ways well known in the art.

GM1 or Gb3, or fragments thereof, can be used to test peptides or molecules by any of a variety of assay techniques. The molecule may be free in solution, fixed on a solid surface, may be formed on a cell surface, or may be located within a cell. Removal of activity or binding complex formation between GM1 or Gb3 is measured.

Another method for determining binding to GM1 / Gb3 is to use purified GM1 / Gb3 to coat microtiter plates. After blocking the platelets, the agent of interest is applied to the plate and allowed to react with specific reagents prior to washing and detection. Conjugation of the antibodies either directly or indirectly with the enzyme or with the radiolabel allows subsequent quantification of binding using either colorimetric or radioactive methods (ELISA or RIA).

Another way of binding GM1 / Gb3 is by attaching the carbohydrate moiety of GM1 / Gb3 to a suitable column matrix so that standard affinity chromatography can be performed. Removal of known compounds applied to the column from the diluent is used as evidence for binding activity, wherein the mixture of compounds is applied to the column. Elution and subsequent analysis determine the properties of the ganglioside-bound reagent. For proteins, the analysis will include peptide sequencing and tryptic digest mapping, followed by comparison with the available database. If the eluted proteins cannot be identified by a standard biochemical analysis method, for example, laser desorption mass spectrophotometer mass determination is used to further characterize the compound. Non-proteinaceous substances that eluted from the GM1-affinity columns will be analyzed by HPLC and mass spectrophotometry of the individual homogeneous peaks.

Another way of determining the ability to bind to GM1 / Gb3 and the exact affinities of interaction will be by using plasmon surface resonance as described in Kuziemko et al., (1996) Biochem. 35: 6375-6384).

Alternatively, phage display may be used to identify a suitable agent that binds to GM1 or Gb3.

Phage display is a molecular testing protocol that uses recombinant bacteriophage. The technology involves transforming a bacteriophage with a gene that encodes a suitable ligand (in this case a suitable candidate as an agent) that is capable of reacting with GM1 / Gb3 (or a derivative or homolog thereof) or a nucleotide sequence (or a derivative or homolog thereof) that encodes the same . The transformed bacteriophage (which preferably binds to a solid surface) expresses a suitable ligand (such as a candidate agent) and appears in the phage coat. Entities (such as cells) carrying target molecules that recognize a candidate agent have been isolated and amplified. The successful agent candidate is then further characterized. Phage display has some advantages over standard ligand affinity testing technology. On the surface of the phage, the candidate agent appears in a three-dimensional configuration that is very close to its natural configuration. This allows specific and high affinity binding for testing.

Another method of testing is high throughput testing, reagents having a suitable binding affinity for GM1 or Gb3 are tested, and the tests are based on the method described in WO 84/03564. A large number of different small peptide test compounds, such as plastic beads or some other surfaces, were synthesized on the solid substrate. The peptide test reagents react with fragments of the target interaction components and are washed. The bound target interaction component is then detected by a suitable adapted method known in the art. The purified target interaction component may also be coated directly on the plates for use in the above-described drug test method. Alternatively, non-neutralizing antibodies can be used to capture the peptide and mobilize it on a solid surface.

An agent that exhibits GM1 or Gb3 binding activity is also capable of cross-linking GM1 or Gb3 receptors. EtxB is one

9 • 9

Such an agent is capable of cross-linking GM1 receptors based on its pentameric form.

There are a number of methods for identifying agents that exhibit an effect on intracellular signaling mediated by GM1 / Gb3 binding but which do not bind to GM1 or Gb3 themselves. For example, if the agent positively regulates CD25 or MHC class II on B cells or positively regulates CD25 or promotes apoptosis of CD8 + T cells or positively regulates 11-10 monocyte secretion, it is unable to bind to GM1 or Gb3 (as evidenced by one of the binding assays, as described above), it can then be concluded that the agent is capable of mimicking the effect of binding to GM1 / Gb3.

Overview of the drawings

Figure 1 shows stimulation of total Ig and IgA in serum (MS) and IgA in eye wash solution (EW) in mice immunized with HSV-1 glycoproteins / rEtxB.

Figure 2 shows the proliferation of MLN or (cervical lymph node) CLN T cells in HSV-1 / rEtxB immunized mice.

Figure 3 shows T cell proliferation of cells from MLN and CLN mice immunized intranasally with HSV-1 Gp in the presence of 1 to 20 μς EtxB.

Figure 4 shows the amount of anti-HSV-1 serum Ig in mice after administration of HSV-1 glycoproteins three times at ten-day intervals with varying amounts of rEtxB or rCtxB as adjuvant.

Figure 5 depicts the reduction of virus production, clinical disease and latency in mice immunized with HSV-1 / rEtxB.

φ φ

Figure β shows the distribution of isotope 1g in MS following HSV-1 infection or HSV-1 immunization

Gp in the presence of EtxB or CtxB as an immunomodulator.

Figure 7 shows the distribution of IgG subclasses after intranasal administration of HSV-1 Gp with either rETxB or rCtxB as an immunomodulator.

Figure 8 shows the immunogenic effect of varying amounts of rEtxB or rCtxB on the amount of HSV-1 specific IgA in the eye wash solution following administration of HSV-1 glycoprotein.

Figure 9 shows the response of serum immunoglobulins after immunization with HSV-1 mice or glycoprotein (gp) or glycoprotein mimics alone in the presence of an adjuvant.

Figure 10 shows mucosal IgA in eye wash solution after intranasal immunization of HSV-1 mice or with glycoprotein mimics alone or in the presence of an adjuvant.

Figure 11 shows mucosal IgA in vaginal wash after intranasal immunization with HSV-1 mice or with glycoprotein (gp) mimics or glycoprotein mimics alone in the presence of an adjuvant.

Figure 12 shows the amount of HSV-1-specific immunoglubulin in the serum of mice immunized with HSV-1 glycoproteins in the presence of various doses of rEtxB as an adjuvant.

Figure 13 shows the amount of IgA in eye wash solution in mice immunized with HSV-1 glycoproteins in the presence of various concentrations of rEtxB.

Figure 14 shows the amount of IgA in the vaginal wash solution in mice immunized with HSV-1 glycoproteins in the presence of various concentrations of rEtxB.

Figure 15 shows the distribution of the IgG subclass response of serum antibodies to HSV-1 after intranasal immunization with Ctx / CtxB or rEtxB or after ocular HSV-1 infection.

Figure 16 depicts the production of cytokines from cultures of lymph node cells that were harvested from mice infected with either HSV-1 via eye scarification or immunized by intranasal administration of HSV-1 glycoproteins Ctx / CtxB or rEtxB as an adjuvant.

Figure 17 shows the degree of protection against ocular HSV-1 infection in mice immunized intranasally with a HSV-1 mixture or glycoproteins in the presence of rEtxB as an immunomodulator.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: rEtxB can be used in conjunction with HSV-1 Gp for immunization purposes.

Mice were immunized intranasally three times with 10 μς of HSV-1 (Gp) glycoproteins and with either 10 or 20 µg rEtxB. Controls were either untreated or replicated viral glycoproteins obtained from tissue cultures of cells not infected with HIV. The amount of antibodies is expressed as a percentage of the amount after infection. Production of total Ig and IgA in serum and IgA in eye wash solution was stimulated with HSV-1 glycoproteins / rEtxB (Figure 1). Doses of rEtxB that are as low as 0.1 gg have been shown to be still effective in stimulating said response.

T-lymphocytes that originate from immunized mice from the cervical lymph nodes (which constitute the vaccine site) and from the mesenteric lymph nodes (which are remote from the vaccine site) proliferate when cultured in vitro

HSV-1, but not when cultured in vitro with a mimic

HSV-1 Gp or without antigen (Figure 2).

T cell proliferation of MLN and CLN mice immunized with HSV-1 Gp and varying amounts of EtxB in the HSV-1 Gp response is shown in Figure 3.

Serum Ig anti-HSV-1 production in mice after administration of HSV-1 glycoproteins at three-day intervals with varying amounts of EtxB (or CtxB) is shown in Figure 4.

Finally, mice immunized with HSV-1 and rEtxB were shown to show increased virus production after scarification of the cornea of HSV-1 (Figure 5a) and reduced local spread (swelling), widening of the trigeminal ganglion (zosteriform infection), widening to the central nervous system ( encephalitis) and latency compared to control (5b).

Example 2: rCtxB and rEtxB act as immunomodulators

When EtxB is used as an immunomodulator, the Ig isotope distribution is asymmetric (Figure 6). The distribution of Ig subclasses varies depending on whether rCtxB or rEtxb is used as the immunomodulator (Figure 7).

Example 3: rEtxB is a more efficient modulator compared to rCtxB

The amount of HSV specific IgA (Figure 8) is higher after rEtxB / HSV-1 Gp stimulation compared to rCtxB / HSV-1 Gp.

Example 4: (Figure 9)

Mice were immunized three times intranasally with HSV-1 glycoproteins alone, mimicked with HSV1 glycoprotein preparations (prepared by taking uninfected tissue culture cells and subjecting them to the same treatment regimen as those used to isolate and purify HSV-1 protein) or glycoproteins HSV-1 in combination with a variety of apparent mucosal adjuvants. In each case, the dose of HSV-1 glycoproteins was 10 gg for immunization and these were combined with 10 µg of recombinant EtxB or CtxB, which serves as an adjuvant or a mixture of 0.5 µg of Ctx and 10 µg of CtxB. Three weeks after the final immunization, blood samples were collected and total anti-HSV-1 antibodies were measured by ELISA. The amount of antibodies is reported as a percentage of the amount stimulated after ocular infection induced by scarification with 10 ⁵ pfu HSV-1 strain SC16. The data (shown in Figure 9) show that the strongest serum antibody response is stimulated when the antigen is combined with a mixture of whole Ctx and CtxB. However, a strong response is also stimulated when rEtxB is used as an adjuvant. In contrast, rCtxB is a very weak adjuvant.

Example 5 (Figure 10)

Mice were immunized as described in Example 4. Secretory IgA production in the eyes was tested by obtaining solutions by washing the tears from the eyes on consecutive days, and these samples were then collected and examined by ELISA using specific antibodies that detect IgA. The amount of antibodies is expressed as a percentage of the amount stimulated after ocular infection induced by 10 ⁵ pfu HSV-1 strain SC16. The data clearly demonstrate (shown in Figure 10) that high levels of secreted antibodies are produced after immunization in the presence of either Ctx / CtxB or EtxB. In contrast to the results of the serum antibody response analysis, there is no difference in the amount of antibodies in the eyes between animals immunized with Ctx / CtxB or EtxB that serve as adjuvant. As with serum antibodies, there is clear evidence that rCtxB is a very weak adjuvant.

Example 6: (Figure 11)

Mice were immunized as described in the Example

4. The secretory production of IgA in the vagina was tested by harvesting

0 0 0 00 0 0 00

0000 00 0 0 0 0

0 · 000 000

000 000 00 000 000 000 after washing the sex organs on consecutive days, and these samples were then pooled and tested by ELISA using specific anti-IgA detection antibodies. The amount of antibodies is expressed as end titers, which were calculated by linear regression analysis. The data clearly shows that a large number of secreted anti-HSV-1 antibodies are produced at certain mucosal sites after immunization in the presence of either Ctx / CtxB or EtxB. The vagina releases most antibodies after immunization in the presence of rEtxB. Smaller amounts were released after immunization with Ctx / CtxB and very little secretion occurs when rCtxB is used as an adjuvant.

Example 7: (Figure 12)

Mice were immunized three times intranasally with HSV-1 glycoproteins (10 μρ). either alone or in the presence of an increasing dose of rEtxB, which serves as an adjuvant. Three weeks after the final immunization, blood was drawn and the amount of anti-HSV antibodies tested by ELISA. The amount of antibodies was expressed as a percentage of the amount induced after ocular infection induced by scarification of 10 ⁵ pfu HSV-1 strain SC16. The data clearly show that the capacity of rEtxB to elicit an antibody response to heterologous added antigens is a dose-dependent phenomenon, with maximal response occurring at a dose of 20 to 50 μρ rEtxB. Furthermore, it is clear that at a dose of 20 μρ rEtxB, the amount of anti-HSV-1 antibodies stimulated by intranasal infection is comparable to or higher than that induced by live virulent virus infection.

Example 8: (Figure 13)

Mice were immunized as described in Example 7. Secretory IgA production in the eyes was determined by obtaining tear wash solutions on consecutive days and these samples were then pooled and assayed by ELISA using specific anti-IgA detecting antibodies. The amount of antibodies is expressed as a percentage of the amount stimulated after ocular infection induced by scarification of 10 ⁵ pfu HSV-1 strain SC16 as a percentage. The data demonstrate that the maximal IgA response in the eyes is stimulated when applied

HSV-1 glycoproteins in combination with 20 µg rEtxB. At this dose

IgA production is lower than production during viral eye infection.

Example 9: (Figure 14)

Mice were immunized as described in Example 7. Secretory IgA production in the vagina was determined by obtaining solutions after washing the sex organs for consecutive days, and these samples were then pooled and tested by ELISA using specific anti- IgA detecting antibodies. The amount of antibodies is reported as final titers, which are calculated by linear regression analysis. The data show that the optimal anti-HSV-1 response is stimulated in the vagina when 20 µg or more of rEtxB is used as an adjuvant.

Example 10: (Figure 15)

Mice were infected with either 10 ⁵ pfu HSV-1 strain SC16 by corneal scarification or by triple nasal immunization with 10 µg HSV-1 glycoproteins in combination Ctx / CtxB or rEtxB. Three weeks after the final inoculation, serum was collected and analyzed for ELISA against HSV-1 IgG1 by ELISA. The amount of antibodies was expressed as final titers, which were calculated by linear regression analysis (Figure 7a). The data clearly show that the nature of the antibody response to HSV-1 is influenced by the way antigens are presented to the immune system. Preferably, HSV-1 infection activates Th1-associated antibody production as characterized by a high amount of IgG2a complement fixing antibody isotype. The infection stimulates a relatively small amount of the IgG isotype associated with Th2 IgG1. This immune response profile is clearly visible when the data is expressed as an IgG1: IgG2 ratio, as shown in Figure 7b. Intranasal immunization * 0 · * 9 99 after infection

9

99 9 9 · 9 9 in the presence of Ctx / CtxB as an adjuvant allows release of IgG1 associated with Th2. A substantial amount of IgG2 is also produced, indicating that Ctx / CtxB causes Th1 and Th2 cells to be activated. Activation of both responses and relative dominance of Th2 is reflected in an IgG1: IgG2 ratio of approximately 3. Interestingly, the nature of the response to HSV-1 stimulated with rEtxB, which serves as an adjuvant, almost exclusively dominates Th2. High levels of IgG1 are produced with only a small amount of IgG2a. This strong tendency towards the Th2 response is reflected in an IgG1: IgG2 ratio of approximately 9.

Example 11: (Figure 16)

Mice were infected with either 10 ⁵ pfu HSV-1 strain SC16 by corneal scarification or immunized three times intranasally with 10 µg HSV-1 glycoproteins in combination with Ctx / CtxB or rEtxB. Three weeks after the final inoculation, the lymph nodes were removed from the animals and used to form single cell suspensions which were cultured either in the presence of dead HSV-1 or a virus preparation from tissue cultures of cells that were not infected. On days 4 and 7 of culture, samples were taken and cELISA was performed, showing cytokine secretion. The data clearly show that T cells in cultures were able to respond to HSV-1, but not to a virus mimic. The lymph node cells obtained from mice infected with HSV-1 predominantly produce a Th1 -γ-interferon-associated cytokine (γ-IFN). Lymph node cells collected from animals immunized intranasally produced large amounts of cytokines associated with Th2 IL-4 and IL-10. In addition, both Ctx / CtxB and rEtxB resulted in the activation of T cells that secrete γ-IFN upon in vitro stimulation with HSV-1. This indicates that although Th2 cytokine production predominates in response to an adjuvant, Th1 activation also occurs. This finding corresponds to the findings based on an antibody response analysis.

Claims (37)

Patient Claims Infectious disease is selected from the group Use of (i) a non-toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1-binding activity or an agent other than VtxB that exhibits Gb3-binding activity or (iii) an agent that exhibits an effect on intracellular signaling mediated by binding to GM1 or Gb3 as an immunomodulator for an infectious disease vaccine. Use according to claim 1, wherein the immunomodulator is EtxB which does not contain the entire toxin. Use according to claim 1 or 2, wherein the infectious disease is a disease wherein the infectious agent is a member of the herpes virus family. Use according to claim 3, wherein the infectious agent is caused by HSV-1, HSV-2, EBV, VZV, CMV, HHV-β, HHV-7 and HHV-8. Use according to claim 4, wherein the infectious agent is selected from the group consisting of HSV-1, HSV-2, CMV or EBV. Use according to claim 1 or 2, wherein the infectious disease is caused by an infectious agent and the infectious agent is an influenza virus. Use according to claim 1 or 2, wherein the infectious disease is caused by an infectious agent and is parainfluenza virus. Use according to claim 1 or 2, wherein the infectious disease is caused by an infectious agent and the infectious agent is a respiratory syncytial virus. Use according to claim 1 or 2, wherein the infectious disease is caused by an infectious agent and the infectious agent is a hepatitis virus. The use of claim 9, wherein the infectious agent is selected from the group consisting of hepatitis A, B, C and D. agent ge virus Use according to claim 10, wherein the infectious hepatitis A or C. Use according to claim 1 or 2, wherein the infectious disease is meningitis. Use according to claim 12, wherein the infectious disease is caused by an infectious agent and the infectious agent is selected from the group consisting of Neisseria meningitidis, Haemophilus influenzae type B and Streptococcus pneumoníae. Use according to claim 1 or 2, wherein the infectious disease is pneumonia or respiratory tract infection. Use according to claim 14, wherein the infectious disease is caused by an infectious agent and the infectious agent is selected from the group consisting of Streptococcus pneumoníae, Legonella pneumophilia and Mycobacterium tuberculosis. Use according to claim 1 or 2, wherein the infectious disease is a sexually transmitted disease. Use according to claim 16, wherein the infectious disease is caused by an infectious agent and the infectious agent is selected from the group consisting of Neisseria gonnorheae, HIV-1, HIV-2 and Chlamydia trachomatis. The use of claim 1 or 2, wherein the infectious disease is a gastrointestinal disease. Use according to claim 18, wherein the infectious disease is caused by an infectious agent and the infectious agent is selected from the group consisting of enteropathogenic, enterotoxigenic, enteroinvasive, enterohemorrhagic and enterocumulative E. coli, rotavirus, Salmonella enteritidis, Salmonella typhi, Helicobacter pylori, Bacillus cereus , Campylobacter jejuni and Vibrio cholerae. Use according to claim 1 or 2, wherein the infectious disease is a superficial infection. The use of claim 20, wherein the infectious disease is caused by an infectious agent and the infectious agent is selected from aureus, a group comprising Staphylococcus pyogenes and Streptococcus mutans. The use of claim 1 or 2, wherein the infectious disease is a parasitic disease. The use of claim 22, wherein the infectious disease is caused by an infectious agent and the infectious agent is selected from the group consisting of malaria, Trypanasoma spp., Toxoplasma gondii, Leishmania donovani and Oncocerca spp. 24. A vaccine composition suitable for use against a disease caused by the vaccine composition comprising an immunomodulator selected from (i) a non-toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB and which exhibits GM1-binding activity, or an agent other than VtxB and which exhibits Gb3-binding activity, or (iii) an agent that has an effect on intracellular signaling events mediated by binding to GM1 or Gb3, wherein said antigenic determinant is an antigenic determinant said antigenic agent. 25. The vaccine composition of claim 24, wherein the infectious disease is a HSV-1 infection and the antigenic determinant is an HSV-1 antigenic determinant. The vaccine composition of claim 24 or 25, wherein the immunomodulator is EtxB free of whole toxin. The vaccine composition of claim 24, 25 or 26, wherein the immunomodulator and antigenic determinant are separate portions. 28. The vaccine composition of claim 24, 25 or 26, wherein: Streptococcus to an infectious infectious agent, wherein the antigenic determinant and the immunomodulator antigenic determinant are linked by a dual function crosslinker. 29. A kit suitable for vaccinating a mammal against an infectious disease, comprising (a) one of the following: (i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1-binding activity, or an agent other than VtxB that exhibits Gb3-binding activity, or (iii) an agent that exhibits an effect on intracellular signaling mediated by binding to GM1 or Gb3; and b) an antigenic determinant which is an antigenic determinant of an infectious disease and is suitable for co-administration with said vaccine immunomodulator. 30. A method of preventing and treating a host disease comprising inoculating said host with a vaccine comprising at least one antigenic determinant and an immunomodulator which is: (i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1-binding activity, or an agent other than VtxB that exhibits Gb3-binding activity, or (iii) ) an agent that exhibits an effect on intracellular signaling mediated by binding to GM1 or Gb3. 31. Use (and) EtxB, CtxB or VtxB without all over toxin, (ii) agents that Yippee other than EtxB or CtxB and which exhibits GM1-binding activity, or agents other than VtxB and which exhibits Gb3-binding activity; or (iii) agents that exhibit an effect on intracellular signaling events mediated by binding to GM1 or Gb3 to positively regulate mucosal surface antibody production. • · · · · · · · · · · Use of (i) a non-toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1-binding activity or an agent other than VtxB that exhibits Gb3-binding activity or (iii) agents that exhibit an effect on intracellular signaling events mediated by binding to GM1 or Gb3 and as an immunomodulator in a vaccine to prolong antigen presentation in a mammal and generate sustained immunological memory. 33. A vaccine composition, characterized in that it is suitable for use against an infectious disease caused by an infectious agent, wherein the vaccine comprises an antigenic determinant and an immunomodulator selected from (i) EtxB, CtxB or VtxB without the entire toxin; which is other than EtxB or CtxB and which exhibits GM1-binding activity, or an agent other than VtxB and which exhibits Gb3-binding activity, or (iii) an agent that has an effect on intracellular signaling events mediated by binding to GM1 or Gb3, wherein said antigenic determinant is an antigenic determinant of said infectious agent, and wherein the immunomodulator extends the presentation of the antigenic determinant and generates a persistent immunological memory. Use of (i) EtxB, CtxB or VtxB without the entire toxin, (ii). agents other than EtxB or CtxB that exhibit GM1-binding activity, or agents other than VtxB that exhibit Gb3-binding activity, or (iii) agents that exhibit an effect on intracellular signaling events mediated by binding to GM1, or Gb3, in an antigen or antigenic determinant conjugate for target introduction of said antigens or antigenic determinants into the cytosol or into the nucleus of the antigen presenting cell. Use of (i) a toxin-free EtxB, CtxB or VtxB, (ii) an agent other than EtxB or CtxB that exhibits GM1-binding activity or an agent other than VtxB that exhibits Gb3-binding activity or (iii) agents that exhibit an effect on intracellular signaling events mediated by binding to GM1 or Gb3 in conjugate with an antigen or antigenic determinant to positively regulate the presentation of said antigenic determinant or antigenic determinants derived from said antigens by MHC class I molecules. 36. A vaccine composition comprising (a) EtxB, CtxB or a non-EtxB or Ctx reagent that exhibits GM1 binding capacity, and b) an EBV antigen that is suitable for use in the treatment and / or prevention of EBV-related diseases 37. A therapeutic composition comprising EtxB, CtxB or an agent other than EtxB or CtxB that exhibits GM1-binding capacity and is suitable for use in the treatment of EBV-related diseases.