EA004794B1 - Vaccine - Google Patents

Abstract

1. The use of EtxB, CtxB or VtxB free from whole toxin as an immunomodulator for a vaccine against infectious diseases. 2. The use according to claim 1, wherein the immunomodulator is EtxB free from whole toxin. 3. The use according to claim 1, wherein the infectious disease is one for which the infectious agent is a member of the herpes virus family. 4. The use according to claim 3, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected from the group consisting of HSV-1, HSV-2, EBV, VZV, CMV, HHV-6, HHV-7 and HHV-8. 5. The use according to claim 4, wherein the infectious agent is selected from the group consisting of HSV-1, HSV-2, CMV or EBV. 6. The 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. 7. The use according to claim 1 or 2, wherein the infectious disease is caused by an infectious agent, and the infectious agent is a parainfluenza virus. 8. The 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. 9. The 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. 10. The use according to claim 9, wherein the infectious agent is selected from the group consisting of hepatitis A, B, C and D viruses. 11. The use according to claim 10, wherein the infectious agent is a hepatitis A virus or a hepatitis C virus. 12. The use according to claim 1 or 2, wherein the infectious disease is meningitis. 13. The use according to claim 12, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected form the group consisting of Neisseria meningitidis, Haemophilus influenzae type B and Streptococcus pneumoniae. 14. The use according to claim 1 or 2, wherein the infectious disease is pneumonia or a respiratory tract infection. 15. The 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 pneumoniae, Legonella pneumophila and Mycobacterium tuberculosis. 16. The use according to claim 1 or 2, wherein the infectious disease is a sexually-transmitted disease. 17. The 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. 18. The use according to claim 1 or 2, wherein the infectious disease is a gastrointestinal disease. 19. The 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, enterohaemorrhagic and enteroaggregative E. coli, rotavirus, Salmonella enteritidis, Salmonella typhi, Helicobacter pylori, Bacillus cereus, Campylobacter jejuni and Vibrio cholerae. 20. The use according to claim 1 or 2, wherein the infectious disease is a superficial infection. 21. The use according to claim 20, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected from the group consisting of Staphylococcus aureus, Streptococcus pyogenes and Streptococcus mutans. 22. The use according to claim 1 or 2, wherein the infectious disease is a parasitic disease. 23. The use according to 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 for use against an infectious disease, which infectious disease is caused by an infectious agent, wherein the vaccine composition comprises an antigenic determinant and an immunomodulator selected from EtxB, CtxB or VtxB free from whole toxin, wherein said antigenic determinant is an antigenic determinant of said infectious agent. 25. A vaccine composition according to claim 24 in which the infectious disease is HSV-1 infection and wherein the antigenic determinant is an antigenic determinant of HSV-1. 26. A vaccine composition according to claim 24 or 25 in which the immunomodulator is EtxB free from whole toxin. 27. A vaccine composition according to claim 24, 25 or 26 in which the immunomodulator and the antigenic determinant are separate moieties. 28. A vaccine composition according to claim 24, 25 or 26 in which the immunomodulator and the antigenic determinant are linked by a bifunctional crosslinking reagent. 29. A kit for vaccination of a mammalian subject against an infectious disease, which kit comprises: a) a vaccine immunomodulator selected from EtxB, CtxB or VtxB free from whole toxin, and b) an antigenic determinant of the infectious disease, for coadministration with the said vaccine immunomodulator. 30. A method of preventing or treating a disease in a host, which method comprises the step of inoculating said host with a vaccine comprising at least one antigenic determinant and an immunomodulator, where the immunomodulator is EtxB, CtxB or VtxB free from whole toxin. 31. The use of EtxB, CtxB or VtxB free from whole toxin to upregulate the production of antibodies at mucosal surfaces. 32. The use of EtxB, CtxB or VtxB free from whole toxin as an immunomodulator in a vaccine, to prolong antigen presentation and give sustained immunological memory in a mammalian subject. 33. A vaccine composition for use against an infectious disease, which infectious disease is caused by an infectious agent, which vaccine comprises an antigenic determinant and a immunomodulator selected from EtxB, CtxB or VtxB free from whole toxin, wherein said antigenic determinant is an antigenic determinant of said infectious agent and wherein the immunomodulator prolongs presentation of the antigenic determinant and gives sustained immunological memory. 34. The use of EtxB, CtxB or VtxB free from whole toxin in a conjugate with antigen or antigenic determinant to target the delivery or said antigen or antigenic determinant to the cytosol or nucleus of an antigen presenting cell. 35. The use of EtxB, CtxB or VtxB free from whole toxin in a conjugate with antigen or antigenic determinant to upregulate the presentation of said antigenic determinant, or an antigenic determinant derived from said antigen, by MHC class I molecules. 36. A vaccine composition which comprises a) EtxB, CtxB, and b) an EBV antigen, for use in the treatment and/or prevention of EBV associated diseases. 37. A therapeutic composition which comprises EtxB, CtxB for use in the treatment of EBV-associated diseases.

Description

The present invention relates to an immunomodulator for use in a vaccine that is intended for use against a wide range of infectious agents. In addition, the present invention relates to a vaccine composition comprising an immunomodulator, preferably in combination with an antigen, and a vaccination method using said vaccine composition.

Cholera toxin (C! X) and its thermolabile enterotoxin E. soi (Είχ) are strong immunogens and mucosa-specific adjuvants. However, their own toxicity makes them unsuitable for human use. For example, although Ox is the most widely used mucosa-specific adjuvant in experimental animals, it is not suitable for use in humans because of its pronounced ability to cause diarrhea. Attempts have been made to separate their toxicity from activity as adjuvants, for example, using the components Oh and Είχ to replace their own holotoxins. Verotoxin E. soy (U1x) is another known bacterial toxin.

Ox and Είχ are heterohexameric proteins consisting of enzymatically active subunit A and pentamer subunit B. It is known that Ox and Είχ are associated with 6M1-ganglioside (6M1), glycosphingolipid, which is ubiquitously present on the surface of mammalian cells. Vίx binds to CH3, which is a similar type of receptor for 6M1.

In order to solve the toxicity problem in vaccine development, the activity of nontoxic subunits B as an adjuvant was previously studied. However, many publications have described experiments that used marketed drugs Ox and Εί \. These drugs are inevitably contaminated with a small, but biologically significant, amount of active toxin, with the result that the inherent activity of the B subunit as an adjuvant cannot be separated from the activity as an adjuvant of the complete toxin (XYi and Bi55e1, 1HGs1yup aib 1tiyu 61: 314-322 (1993), υδ-5182109). Subsequent studies using the OhBB recombinant subunit (hOxB) suggested that OhB is ineffective as a mucosal-specific adjuvant and only the addition of native holotoxin can cause strong concomitant responses (Tatiga et al., Wassche 12: 419-426 (1994) ). Other studies have suggested that TOXB lacks the ability to ADFribosylation and cAMP-stimulation characteristic of holotoxin, and since the mechanism of action of adjuvants is associated with these abilities, OXB cannot be used as an adjuvant , Col. 75: 488-492 (1992), Lauske et al., Ειπ. 1. 1 22% 2277-2281 (1992), Voise et al., 1Ges! And Aib 1: 65: 2821-2828 (1997)).

In another study, it was found that intranasal administration of ovalbumin using gOXB as an adjuvant resulted in low humoral immune responses. The non-toxic Ox derivative with a mutation in subunit A also led to weak responses to associated antigens, while the presence of the active subunit A greatly increased the activity of the adjuvant, which suggests that the active subunit A is crucial (Voise et al. (1997) , above).

It was also found that tOxB and hb1xB can be used to increase tolerance to heterologous antibodies (8ii et al., Proc. Ian Asab. Δοΐ. 91: 4610-4614 (1994), 8ii, et al., Proc. 11. Asab. 8сг 93: 7196-7201 (1996), Vetdetoy et al., Proc. No. 11. Asab. 8sk 94: 4610-4614 (1997), Ubyato et al., Proc. No. 1. Asab. 8сг 94: 5290-5295 (1997), these data suggest that these molecules cannot be used as adjuvants.

The basis of the present invention

Although according to the current state of the art, OhB and ΕίxB are bad adjuvants and can actually act as tolerogens, this study explores the use of hb1xB (containing neither holotoxin residue nor subunit A) in the intranasal vaccine for herpes simplex virus (H8U) when simulated in mice, and it was unexpectedly found that he has the ability to stimulate protective immune responses to control infection with a virus. In particular, when creating the present invention found that

I) agents such as ΕίxB and OHB stimulate high levels of local production (in the mucosa) of antibodies (although immunization with hb1xB stimulated the production of lower total serum antibody titers than immunization with the combination of Ox / OHB);

II) the distribution of the obtained antibodies is shifted to non-complementing antibodies, first of all, 8-1dL and 1§61;

Iii) agents such as ΕίχВ and ОВВ also stimulate local and systemic T-cell proliferative responses;

PG) agents, such as OHB and ΕίχВ, tend to shift the immune response from Ty1-related response to Ty2-related response;

Y) when agents, such as OVB and ВχB, are used as immunomodulators, some of the adverse effects of Ty2-related responses, such as the formation of 1dE, are eliminated;

Vi) τΕΐχΒ is a more effective immunomodulator than γΟχΒ;

VII) agents, such as ΕίχΒ and ΟχΒ, have the ability to change the way in which an antigen-presenting cell internalizes and processes the antigen, which leads to an increase in the persistence of the antigen;

VIII) if an agent, such as ΕΐχΒ and ΟχΒ, is associated with an antigen, then it becomes possible to change the way the antigen is processed by changing the connection with the immunomodulator; and

Ix) νΐχΒ exhibits similar ΕίχΒ and ΟχΒ immunomodulating properties with respect to the коη νίίτο leukocyte populations.

These important discoveries are the basis of the various objects of the present invention and allow applicants to assume that pure ΕίχΒ, ΟχΒ and νίχΒ, as well as other agents that have the ability to bind or mimic the effect of binding to CM1 or Cb3, can be valuable as immunomodulators for use in vaccines intended for prophylactic and therapeutic vaccination against Η§ν-1 infection, as well as other infections, the prevention or treatment of which is facilitated by the use of immunomod of the above types.

Stimulation of immune responses

ΕίχΒ, ΟχΒ, νίχΒ and other agents that have the ability to bind or mimic the effect of binding to CM1 or CH3 can act as immunomodulators and stimulate specific immune responses in response to a control infection with an antigen.

According to a first aspect of the present invention, the use of (I) ΕίχΒ, ΟχΒ, or νίχΒ without complete toxin is proposed;

(Ii) an agent other than ΕίχΒ or ΟχΒ, possessing CM 1-binding activity, or an agent other than νίχΒ, possessing CH3-binding activity; or (III) an agent affecting intracellular signals mediated by the binding of CM1 or the binding of CH3;

as an immunomodulator of vaccines for infectious diseases.

According to a second aspect of the present invention, a vaccine composition for an infectious disease is proposed, where the infectious disease is caused by an infectious agent, the vaccine composition comprising an antigenic determinant and an immunomodulator selected from the group comprising (i) ΕίχΒ, ΟχΒ or νίχΒ, which do not contain complete toxin;

(Ii) an agent other than ΕίχΒ or ΟχΒ, possessing CM1-binding activity, or an agent other than νίχΒ, possessing CH3-binding activity; or (iii) an agent that affects intracellular signals mediated by the binding of CM1 or the binding of CH3; moreover, the antigenic determinant is an antigenic determinant of the specified infectious agent.

An antigen and an immunomodulator can be linked, for example, covalently or can be linked genetically to form a single effective agent. According to a particular embodiment of the present invention, the antigen and immunomodulator can be chemically conjugated. For example, an antigen and an immunomodulator can be chemically conjugated using heterobifunctional cross-linking reagents. For the practical implementation of this object of the invention, it is preferable to separate administration (in which the antigen and immunomodulator are not related), since it allows separate administration of different fragments.

According to a third aspect of the present invention, there is provided a kit for vaccinating a mammal, such as a human or other mammal that is an object of veterinary medicine, from an infectious disease, which includes

a) one of the following agents:

(I) ΕίχΒ, ΟχΒ, or νίχΒ, not containing complete toxin;

(Ii) an agent other than ΕίχΒ or ΟχΒ, possessing CM1-binding activity, or an agent other than νίχΒ, possessing CH3-binding activity; or (iii) an agent that affects intracellular signals mediated by the binding of CM1 or the binding of CH3, and

b) an antigenic determinant, which is an antigenic determinant of an infectious disease, intended to be co-administered with an immunomodulator for a vaccine.

The composition of the vaccine according to the second object of the invention and the set according to the third object of the invention can be used in a method of prophylactic or therapeutic vaccination, where the prophylactic vaccine is administered to untreated individuals in order to prevent the development of the disease, and the therapeutic vaccine is administered to individuals already infected with the infection, in order to reduce or minimize infection, or in order to avoid the immunopathological effects of the disease.

Agents such as ΕίχΒ have the ability to change the nature of the immune response if an infection is already occurring. Including such an agent, a therapeutic vaccine (i.e., a vaccine that should not contain an antigen) may find particular use in cases where the immune response is not sufficient to defeat the infection. This use may be particularly valuable in the treatment of a chronic disease, for example, a disease whose pathogens are selected from the group comprising herpes viruses, hepatitis viruses, HIV, TB (tuberculosis bacillus) and parasites.

According to a fourth aspect of the present invention, there is provided a method for preventing or treating a disease in a host, comprising the step of inoculating that host with a vaccine comprising at least one of the antigenic determinants and an immunomodulator, the immunomodulator being (I) ΕίχΒ, ΟχΒ, or νίχΒ that does not contain full toxin;

(Ii) an agent other than ΕίχΒ or ΟχΒ, possessing CM1-binding activity, or an agent other than νίχΒ, possessing CH3-binding activity; or (iii) an agent that affects intracellular signals mediated by the binding of CM1 or the binding of CH3.

The vaccine can be packaged for co-administration and can be administered in many different ways, such as intranasal, oral, intravaginal, urethral or ocular administration. Intranasal immunization is preferred. When the vaccine is administered intranasally, it can be administered as an aerosol or in liquid form.

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

According to the first embodiment, the immunomodulator according to the first object of the invention, the vaccine according to the second object of the invention, the set according to the third object of the invention and the method according to the fourth object of the invention are used to combat the disease, the infectious agent of which is a member of the herpes virus family. For example, an infectious agent can be selected from the group including Η§ν-1, Η§ν-2, ΕΒν (Epstein-Barr virus), νζν (varicella zoster virus), SMU (cytomegalovirus), ν-6 (hepatovirus human), ΗΗν-7 and ΗΗν-8. In particular, infectious agents can represent Η§ν-1, Η§ν-2, SMU, or ΕΒν.

According to this first embodiment, the antigenic determinant is preferably an antigenic determinant of the earliest, early or late gene product (for example, surface glycoprotein) of the herpes virus.

If the infectious agent is Η§ν-1 or Η§ν-2, the antigenic determinant may be the antigenic determinant of a gene product selected from the following group: §E. §Β. §Η or dC or latency-related transcript (LAT).

If the infectious agent is ΕΒν, the antigenic determinant may be the antigenic determinant dr340 or dr350 or latent protein (for example, ΕΒΝΑ-1, 2, 3А, 3В, 3С and ЛР, ЛМР-1, 2А and 2В or).

According to the second embodiment, the immunomodulator according to the first object of the invention, the vaccine according to the second object of the invention, the set according to the third object of the invention and the method according to the fourth object of the invention are used to combat the disease, the infectious agent of which is an influenza virus.

According to this second embodiment, the antigenic determinant is preferably an antigenic determinant of a virus envelope protein (for example, hemagglutinin and neuraminidase) or an internal protein (for example, nucleoprotein).

According to the third embodiment, the immunomodulator according to the first object of the invention, the vaccine according to the second object of the invention, the set according to the third object of the invention and the method according to the fourth object of the invention are used to combat the disease, the infectious agent of which is parainfluenza virus.

According to the fourth embodiment, the immunomodulator according to the first object of the invention, the vaccine according to the second object of the invention, the set according to the third object of the invention and the method according to the fourth object of the invention are used to combat the disease, the infectious agent of which is a respiratory syncytic virus.

According to the fifth embodiment, the immunomodulator according to the first object of the invention, the vaccine according to the second object of the invention, the set according to the third object of the invention and the method according to the fourth object of the invention are used to combat the disease, the infectious agent of which is a hepatitis virus. For example, the infectious agent may be selected from the group consisting of hepatitis A, B, C and Ό viruses. Especially preferred infectious agents may be pathogens of hepatitis A or C.

According to the sixth embodiment, the immunomodulator according to the first aspect of the invention, the vaccine according to the second aspect of the invention, the set according to the third aspect of the invention and the method according to the fourth aspect of the invention are used in the case of meningitis. According to the sixth embodiment, the infectious agent can be selected from the group consisting of No. 188spa teshpdShbsch Ηαοιηορίιίΐιικ 1nPiephaea type B and §1ter1osossi5 répitošae.

According to the seventh variant of the implementation of the immunomodulator according to the first object of the invention, the vaccine according to the second object of the invention, the set according to the third object of the invention and

Ί The method according to the fourth aspect of the invention is applied in the case of pneumonia or respiratory tract infection. According to the seventh embodiment, the infectious agent can be selected from the group consisting of 81grssoscoviyspionaea, Bestocienna rjitoriotya, and Musobastepsinus females.

According to the eighth variant of the implementation of the immunomodulator according to the first object of the invention, the vaccine according to the second object of the invention, the set according to the third object of the invention and the method according to the fourth object of the invention is used in the case of a disease that is sexually transmitted. According to the eighth embodiment, the infectious agent may be selected from the group consisting of No. 1 of the cctope, HIV-1, HIV-2 and Sy1atub1a yasiotayk.

According to the ninth embodiment, the immunomodulator according to the first object of the invention, the vaccine according to the second object of the invention, the set according to the third object of the invention and the method according to the fourth object of the invention are used in case of a disease of the gastrointestinal tract. According to the ninth embodiment, the infectious agent can be selected from the group including enteropathogenic, enterotoxigenic and enteroinvasive E. soi, rotaviruses, 8a1toye 11a ee1e1b1k, 8a1toie1a11yiYy, Neysobeni1tru11, VasShik segeik, Ipatrya irah, Neysobrytruppot1, VasSheik seyik, Ipatrya irah, Neysobirtii ruchetot1, VasSheik seyikik, Iryna, Iryna, ZaVi, Sasha, rotaviruses

If the infectious agent is selected from the group including enteropathogenic, enterotoxigenic, enteroinvasive, enterohemorrhagic and enteroaggregating E. soi, then the antigenic determinant may be an antigenic determinant of bacterial toxin or adhesion factor.

According to the tenth embodiment, the immunomodulator according to the first object of the invention, the vaccine according to the second object of the invention, the set according to the third object of the invention and the method according to the fourth object of the invention are used in the case of a shallow infection. According to the tenth embodiment, the infectious agent may be selected from the group consisting of 81studyosossik aigeik, 81ger1osossik ruodeik and 81ger1osossiki ai1k.

According to the eleventh variant of the implementation of the immunomodulator according to the first object of the invention, the vaccine according to the second object of the invention, the set according to the third object of the invention and the method according to the fourth object of the invention is used in case of parasitic disease. According to the eleventh embodiment, the infectious agent can be selected from the group including Tguraiakota crr., Tokor1akta doibi, Le ^ ytasha bashiouat and Oisosegsa crr.

Stimulation of immune responses in the mucosa

E1xB, C1xB and Y1xB and other agents that have the ability to bind or mimic the effect of binding to CM1 or

CH3, can carry out a specific up-regulation of the production of antibodies in the mucous membrane.

The immunomodulator of the vaccine according to the first object of the invention, the composition of the vaccine according to the second object of the invention, the set according to the third object of the invention is particularly effective in relation to diseases in which protection against infection or treatment of the w uo is carried out, causing an immune response in the mucous membrane; for example, in relation to diseases in which, during infection, an infectious agent binds to the mucosa, forms colonies in it, or penetrates the mucosa. Examples of such diseases include diseases that are caused by viruses (HIV, H8U, EVI, SMU, influenza virus, measles virus, mumps virus, rotavirus, etc.), diseases caused by bacteria (E. soi, 8151toye, 8eda, Sy1atub1a , N. doeiogoea, T. rasbshts, Types of Tyrosus, including species that cause dental caries), and diseases caused by parasites.

According to a preferred embodiment of the second object of the present invention, a vaccine against H8U1 infection is proposed, which includes at least one antigenic determinant H8U-1 and an immunomodulator, the immunomodulator being (I) Е1хВ, С1хВ or У1хВ that does not contain complete toxin;

(Ii) an agent other than E1xB or C1xB, possessing CM1-binding activity, or an agent other than Y1xB, possessing CH3-binding activity; or (iii) an agent that affects intracellular signals mediated by the binding of CM1 or the binding of CH3.

Preferably, the immunomodulator is E1xB. According to a preferred embodiment of the third object of the present invention, a kit has been proposed for vaccinating a mammal against H8U-1, including

a) an immunomodulator of the vaccine, which is (I) E1xB, C1xB or U1xB, which does not contain complete toxin;

(Ii) an agent other than E1xB or C1xB, possessing CM1-binding activity, or an agent other than Y1xB, possessing CH3-binding activity; or (iii) an agent that affects intracellular signals mediated by the binding of CM1 or the binding of CH3, and

b) at least one antigenic determinant H8U-1 for co-administration with an immunomodulator of the vaccine.

The fifth object of the invention is the use of (I) ΕΐχΒ, ΟΐχΒ or ΥΐχΒ. not containing complete toxin;

(Ii) an agent other than ΕΐχΒ or ΟχΒ with CM1-binding activity, or an agent other than νΐχΒ with CH3-binding activity; or (III) an agent that affects intracellular signals mediated by CM1 binding or CH3 binding for up-regulation of antibody production on the mucosal surface. The production of non-complement-binding serum antibodies may also be upregulated. Preferably, according to the fifth aspect of the invention, 84 dA is obtained.

According to this fifth aspect, the agent can be used in combination with one or more antigenic (s) derminant (s).

Down regulation of pathological components of immune responses

The invention also establishes that when pure ΕΐχΒ is used as an immunomodulator according to the described method, this avoids the harmful effects associated with Ty2-cell responses, such as obtaining high titers of potentially pathological deE. Despite this, an immune response triggered by the use of χΒ (or ΟχΒ, or νΐχΒ) as an immunomodulator is likely to be preferable from the point of view of induction of Unbound Cytokines. In other words, ΕΐχΒ (or ΟχΒ) induces a shift in responses from the TP1 cell response to the T2 cell response. This allows applicants to assume that ΕΐχΒ, ΟχΒ, or νΐχΒ, as well as other agents that have the ability to bind or mimic the effect of binding to CM1 or Cb3, may be able to down-regulate the pathological components of the immune response associated with both activation of TY, and with T112 activation.

According to the sixth object of the invention proposed the use of (I) ΕΐχΒ, ΟχΒ or νΐχΒ, not containing complete toxin;

(Ii) an agent other than ΕΐχΒ or ΟχΒ, possessing CM 1-binding activity, or an agent other than νΐχΒ, possessing CH3-binding activity; or (III) an agent that affects intracellular signals mediated by the binding of CM1 or the binding of CH3 for the down regulation of the pathological components of T112-related immune responses. Pathological components of T111-associated immune responses can also be downregulated.

It is known that ΕΐχΒ and ΟχΒ bind to CM1 and induce various effects on lymphocyte populations, including the specific depletion of SE8 + T cells and the associated activation of B cells (AO 97/02045). Hence, it can be assumed that Εΐ Β and Ο Β Β alter the balance of immune responses in such a way that the inflammatory reactions associated with T111 undergo down-regulation, and the reactions associated with T112 undergo up-regulation. Ty1-cell reactions include the secretion of γ-ΣΕΝ (γ-interferon) by T-cell activation, leading to the activation of macrophages and the reaction of delayed hypersensitivity. Such reactions can be an important cause of pathology in infections caused by various pathogens. T112-cell reactions include T-cell activation in relation to the production of cytokines, such as ^ -4, ^ -5, W-Yu, and it is known that they enhance the secretion of high antibody titers, primarily ^ L.

With the creation of the invention, it has been surprisingly found that when ΕΐχΒ is used as an immunomodulator according to the described method, this avoids the harmful effects associated with Ty2-cell responses, such as obtaining high titers of potentially pathological ^ Ε. Thus, ΕΐχΒ and ΟχΒ are able to downregulate the pathological components of the immune response associated with both T111 activation and T112 activation. Such reactions preferably modulate the production of high titers of non-complementing serum antibodies and the production of secretory! DA on mucosal surfaces.

The use of an agent according to the sixth aspect of the invention is especially important for therapeutic vaccination in diseases involving immunopathological mechanisms. Examples of such diseases are diseases caused by Η8ν-1, Ηδν-2, TB and HIV.

The first and sixth objects of the invention can be combined. In other words, agents such as ΕΐχΒ can be used simultaneously as an immunomodulator and as a therapeutic agent. For example, in diseases involving immunopathological mechanisms, the use of agents included in a vaccine, such as ΕΐχΒ or ΟΐχΒ, can serve not only to limit infection, but also to eliminate pathological disease processes. Thus, an immunomodulatory agent can act as both a prophylactic and a therapeutic agent. Examples of infections in which vaccination with such a method is likely to be particularly important include diseases that are caused by the herpes virus family, the pathogens of the gastrointestinal tract and the respiratory tract.

Immunomodulation pathway processing antigen

a) Prolonged presentation.

With the invention, it was also found that when ΕΐχΒ (or ΟΐχΒ or νΐχΒ) is used as an immunomodulator, the internalization of the antigen and the way it is processed changes. The presence of subunit B causes a prolonged presentation, probably by changing the directional migration of the antigen inside the antigen-presenting cell, with the result that the decomposition of the antigen slows down and, therefore, it persists for longer periods of time. This feature of the B-subunit associated with the presentation of the antigen means that vaccines comprising the agent of the present invention should increase the persistence of the antigen and provide a longer immunological memory.

According to the seventh aspect of the present invention, the use of (I) ΕίχΒ, ΟχΒ, or νίχΒ without complete toxin is proposed;

(Ii) an agent other than ΕίχΒ or ΟχΒ, possessing CM1-binding activity, or an agent other than νίχ, possessing CH3-binding activity; or (III) an agent that affects intracellular signals mediated by CM1 binding or CH3 binding, as an immunomodulator in a vaccine for prolonged antigen presentation and for providing a longer immunological memory in a mammal.

According to the eighth object of the present invention, a vaccine composition for use in relation to an infectious disease is proposed, which includes an antigenic determinant and an immunomodulator selected from (I) ΕίχΒ, ΟχΒ or νίχί that do not contain a complete toxin;

(Ii) an agent other than ΕίχΒ or ΟχΒ, possessing CM1-binding activity, or an agent other than νίχ, possessing CH3-binding activity; or (III) an agent affecting intracellular signals mediated by the binding of CM1 or the binding of CH3;

where the antigenic determinant is an antigenic determinant of a specified infectious disease and where the immunomodulator prolongs the presentation of the antigenic determinant and provides a longer immunological memory.

b) Intracellular directional migration of antigen to the pathway associated with MΗC-I or MHC-P.

As mentioned above, an antigen and an immunomodulator in a therapeutic or prophylactic vaccine can be linked, for example, covalently or genetically, to form one effective agent. With the creation of the invention it has been established that it is possible to direct the antigen to different cell compartments and, as a result, to change the way the antigen is presented by changing the association of the antigen with the immunomodulator.

By implementing a specific association of an antigen or antigenic determinant with an immunomodulator, translocation of the antigen through the endosomal membrane into the cytosol can be facilitated. The inventors have suggested that this should increase the peptide loading of the antigen of class I molecules of the major histocompatibility complex (MHC). Thus, the antigen-immunomodulator conjugate can be used to specifically enhance the activation of cytotoxic T cells (CT). Induction of TSH is important for the prevention and treatment of many diseases, especially those caused by viruses, intracellular bacteria and parasites.

The antigen-immunomodulator conjugate binding can also be selected so that the antigen is delivered to the nucleus.

According to a ninth aspect of the invention, a conjugate is proposed comprising an antigen or antigenic determinant and an immunomodulator selected from (I) ΒχΒ, Οχ or νίχ содержащих that do not contain complete toxin;

(Ii) an agent other than ΕίχΒ or ΟχΒ, possessing CM1-binding activity, or an agent other than νίχ, possessing CH3-binding activity; or (III) an agent affecting intracellular signals mediated by the binding of CM1 or the binding of CH3.

According to the tenth object of the invention, a vaccine composition for use against an infectious disease is proposed, the infectious disease being caused by an infectious agent, wherein the vaccine composition contains a conjugate comprising an antigen or antigenic determinant and an immunomodulator selected from (I) ΕίχΒ, ΟχΟ or νίχί, not containing full toxin;

(Ii) an agent other than ΕίχΒ or ΟχΒ, possessing CM1-binding activity, or an agent other than νίχ, possessing CH3-binding activity; or (III) an agent affecting intracellular signals mediated by the binding of CM1 or the binding of CH3;

moreover, the antigen or antigenic determinant is an antigen or antigenic determinant of the specified infectious agent.

An antigen or antigenic determinant can be linked to an immunomodulator by various methods, including genetic clutch or chemical conjugation. According to the first preferred embodiment, the conjugate is a fusion protein obtained by genetic adherence of an antigen or antigenic determinant with an immunomodulator. Preferably the antigen or antigenic determinant is associated with the C-terminus of the immunomodulator. According to the second preferred embodiment, the antigen or antigenic determinant is chemically conjugated to an immunomodulator. Preferably, the antigen or antigenic determinant is chemically conjugated to an immunomodulator with a bifunctional cross-linking reagent, such as a heterobifunctional cross-linking reagent. More preferably, the cross-linking reagent is a complex YL-y (maleimidobutyroxyl) succinimide ester (CMB8) or N-succinimidyl- (3pyridyldithio) propionate (8PBR).

The vaccine composition can be administered in a variety of different ways, such as intranasal, oral, intravaginal, urethral or ocular administration. Intranasal immunization is preferred.

According to the eleventh aspect of the present invention, the use of (I) ΕΐχΒ, С 1.x V or νΐχΒ that does not contain complete toxin is proposed;

(Ii) an agent other than ΕΐχΒ or ΟχΒ, possessing CM 1-binding activity, or an agent other than νΐχΒ, possessing CH3-binding activity; or (III) an agent affecting intracellular signals mediated by the binding of CM1 or the binding of CH3;

in conjugate with antigen or antigenic determinant for the directional transfer of antigen or antigenic determinant into the cytosol or nucleus of the antigen-presenting cell.

According to the twelfth object of the present invention, the use of (I) ΕΐχΕΐ, ΟχΒ or νΐχΒ without complete toxin is proposed;

(Ii) an agent other than ΕΐχΒ or ΟχΒ, possessing CM1-binding activity, or an agent other than νΐχ, possessing CH3-binding activity; or (III) an agent affecting intracellular signals mediated by the binding of CM1 or the binding of CH3;

in conjugate with antigen or antigenic determinant for up-regulating the presentation of this antigenic determinant or antigenic determinant, obtained from the indicated antigen, with the help of MHC class I molecules.

Preferably, the use of a conjugate according to the twelfth object of the invention is carried out in combination with the use of an agent according to the fifth object of the invention in order to stimulate strong CT-responses and to increase the regulation of the production of antibodies in the mucous membrane. This activity is especially important for the prevention and treatment of viral infections, such as influenza.

ΕΐχΒ is the preferred immunomodulator

As previously assumed, ΕΐχΒ and ΟχΒ have similar properties. However, when creating the present invention, it was found that τΕΐχΒ is a stronger and more effective immunomodulator than τΟχΒ. Thus, the preferred immunomodulator is ΕΐχΒ or agents that mimic the effects of ΕΐχΒ.

EVV

ΕΒν is one of eight known human herpes viruses. Infection usually occurs in early childhood; however, clinical symptoms are usually mild or not detectable at this stage. Primary infection of ΕΒν in later life stages is associated with infectious mononucleosis (MI), which is the second most common juvenile disease in the United States. ΕΒν also has oncogenic potential. There is a strong association between ΕΒν and Burkitt's endemic lymphoma (BL) and undifferentiated nasopharyngeal carcinoma (NFC). In addition, most lymphomas that occur in immunocompromised patients are caused by ΕΒν, and it has been established that there is a connection between certain Hodgkin lymphomas and ΕΒν.

Latently infected ΕΒν cells express a small amount of so-called latent proteins. These include six nuclear proteins (α-1, 2, 3A, 3B, 3C, and LP), three integral membrane proteins (LMP-1, 2Α, and 2B) and two non-polyadenylated viral RNAs () that affect RNA splicing .

The latent membrane protein 1 ΕΒν (LMP-1) is present in the plasma membrane of infected cells. It is also expressed in nasopharyngial carcinomas (NFC) and ΕΒν-positive Hodgkin's lymphomas (BUT), which indicates the role of LMP-1 in the development of these tumors. The LMP-1 gene can alter the phenotype of uninfected cells, causing up-regulation of cell surface activation markers, thereby enhancing cell proliferation. LMP-1 can also alter signal pathways and has anti-apoptotic actions. It is well established that no cellular immune response against this viral antigen has been detected in any healthy carriers or in patients with tumors.

Many animal viruses in the course of evolution have developed mechanisms to avoid detection of the host by the immune system. As a rule, these mechanisms involve interaction with the translocation system of a peptide bound to TAP. It is possible that, in the process of evolution, similar mechanisms developed in order to avoid detection by the immune system, which allows it to maintain persistence in the host. This explains why certain cellular immune responses cannot recognize the latent protein ΕΒν ΕΒΝΑ1, and can explain the lack of such responses in relation to LMP-1.

According to the thirteenth object of the invention proposed composition of the vaccine, which includes

a) one of the following agents:

(I) ΕίχΒ, ΟχΒ, or νίχΒ, not containing complete toxin;

(Ii) an agent other than ΕίχΒ or ΟxΒ, possessing CM1-binding activity, or an agent other than νίχΒ, possessing CH3-binding activity; or (iii) an agent that affects intracellular signals mediated by the binding of CM1 or the binding of CH3, and

b) генν antigen, which is intended for the treatment and / or prevention of связанныхν-related diseases.

The specific composition of the vaccine according to the fourteenth aspect of the invention includes ΕίχΒ, ΟxΒ, or an agent other than ΕίχΒ or ΟχΒ, which has CM 1-binding activity.

According to the fourteenth object of the invention proposed therapeutic composition of the vaccine, which includes (I) ΕίχΒ, ΟxΒ or νίχΒ, not containing complete toxin;

(Ii) an agent other than ΕίχΒ or ΟxΒ, possessing CM1-binding activity, or an agent other than νίχΒ, possessing CH3-binding activity; or (iii) an agent that affects intracellular signals mediated by the binding of CM1 or the binding of CH3;

for use in the treatment of связанныхν-related diseases.

The specific therapeutic composition according to the thirteenth aspect of the invention includes ΕίχΒ, ΟxΒ, or an agent other than ΕίχΒ or ΟχΒ, which has CM 1-binding activity.

Based on the known evidence that ΕίχΒ forms a double cap with LMP-1 and that ΕίχΕί enhances fragmentation of LMP-1, it can theoretically be assumed that χΕί (and other agents of the type типаχΒ that possess CM1-binding activity) can be used to stimulate anti -ΕΒν immune responses. This activity can be used in vaccines for the prevention of diseases associated with иν and in therapeutic treatments for the treatment of such diseases if they have developed.

Without going into theory, it can be assumed that when ΕίχΒ forms a double cap with LMP-1, the antigen is processed by another intracellular pathway that allows the antigen to bypass the normal processing path that is blocked by the virus, resulting in the antigen being effectively presented on the cell surface. The action of Είχ can also lead to the fact that epitopes of antigen can be presented on the surface of cells, different from those that are presented if the antigen is processed in the usual way.

A vaccine according to the thirteenth aspect of the invention can be used to prevent ΕΒν infection or the development of связанныхν-related diseases in infected ΕΒν individuals. A vaccine may also include a special adjuvant or agent (such as ΕίχΒ or ΟχΒ), which itself can act as an adjuvant.

The agents specific to the fourteenth object of the present invention can be used separately (i.e., without an antigen) for the treatment of связаннойν-related disease that has already developed in a patient.

The preferred agent for use according to the thirteenth and fourteenth objects of the invention is ΕίχΕί.

ΕΒν antigen is an antigen that can be obtained from ΕΒν itself, or an antigen that must be expressed by an infected ΕΒν host cell under the influence of ΕΒν. Preferably, the antigen is a latent membrane protein ΕΒν. Particularly preferred are the LMP-1, BMR-2A, ^ MP-2Β and ΕΒΝΑ-1 antigens, as well as their antigenic fragments. The antigen can be isolated directly from infected cells or can be obtained synthetically or recombinantly.

The thirteenth and fourteenth objects of the invention are particularly suitable for the treatment and / or prevention of the following diseases: infectious mononucleosis, Burkitt's lymphoma, nasopharyngeal carcinoma, and Hodgkin's lymphoma. These objects of the invention are probably particularly useful for the treatment and / or prevention of nasopharyngeal carcinoma and Hodgkin lymphomas.

The vaccine or therapeutic composition according to the thirteenth and fourteenth objects of the invention can be used to prevent the development or to treat an связаннойν-related disease in a mammalian patient by administering an immunologically effective amount to the patient.

The mammalian patient may be, for example, a healthy infected ΕΒν or non-infected individual, an immunodeficient individual, or an individual with ΕΒν-related disease.

The vaccine may be administered in any acceptable way. The agent and antigen can be administered together or administered separately to a mammalian patient. The agent and antigen can be separately or can be linked, for example, covalently or genetically linked, with the formation of one effective agent.

CM-1 and CH3-associated signals

Without going into theory, it can be assumed that CM-1- and Cb3-binding can directly or indirectly trigger intracellular signals. With the creation of the invention, data have been obtained which suggests that at least one other receptor is involved in the intracellular signaling associated with CM1. It may be that the binding of ΕίχВ (or OhB) with CM1 facilitates binding to the protein that triggers the intracellular signal. It is not yet known what mechanism specifically triggers the intracellular signal, it may be phosphorylation of CM1 or protein. When ΕίχВ / СΐχВ binds CM1 on the cell surface, the associated CM1 is internalized by vesicles (\ UPnatk et al., Iptipokdu Tobau 20: 95-101 (1999)). It is known that CM1 and other glycolipids (such as CH3) are preferably located in membrane clusters in which the receptors of the crucial protein are also found. Hence, it is possible that the internalization of CM1 as a result of B-subunit binding causes the formation of a double cap of such proteins, which leads to the launch of their ability to mediate intracellular signals.

Definitions

An adjuvant is a substance that, unlike the carrier, nonspecifically enhances the immune response to an antigen, the purpose of which is to direct the antigen to the desired site. The term immunomodulator in the context of the present description is used to designate an agent that acts similarly to an adjuvant, stimulating certain immune responses, but which also directs the immune response in a certain direction.

The concept of co-administration is used to indicate that the place and time of administration of the antigen and immunomodulator are those that are necessary to stimulate the immune response. Thus, if an antigen and an immunomodulator can be introduced at the same time and into the same area, this may have advantages over the introduction of the antigen and immunomodulator at different times and / or into different areas. For example, the antigen and immunomodulator can be administered together in the first stage, and then the immune response can be enhanced in the second stage by introducing a single antigen.

The concept of antigenic determinant in the context of the present description refers to a site on an antigen that is recognized by an antibody or T-cell receptor. Preferably, it is a short peptide derived from, or a part of, an antigen protein, however, it is also understood that the term includes glycopeptide and carbohydrate antigenic determinants. The term also includes modified amino acid or carbohydrate sequences that stimulate responses recognized by the whole organism.

There are numerous well-known methods by which you can identify the antigenic determinants of this infectious agent.

For example, potentially protective antigens can be identified by an increase in immune responses in infected or convalescent patients, in infected or convalescent animals, or by monitoring w ν immune responses to antigen containing drugs. For example,

I) serum samples obtained from infected or recovering patients or infected or recovering animals can be screened for all cell lysates of the infectious agent or cell lysates infected with the indicated agent using standard Western blot for detecting antigen (s), recognized (s) immune serum;

Ii) serum samples obtained from infected or convalescent patients or infected or recovering animals can be screened for partially or completely purified antigens of the infectious agent or lysates of cells infected with the specified agent by the standard method in which partially or almost completely purified antigens are used to sensitize microplate wells, or by immunoblotting to detect that (those) antigen (s) that is (are) recognized by the immune serum mended;

Iii) Serum samples obtained from infected or convalescent patients or infected or recovering animals can be screened for all cell lysates obtained from recombinant expression systems encoding one or more antigens of interest, and using standard methods or Western blots. to detect antigen (s) recognized by the immune serum;

PG) Serum samples obtained from infected or recovering patients or infected or recovering animals may be screened for an expression library containing the cloned genes of the infectious agent of interest using colony immunoblot detection to identify which clones express antigens or fragments thereof that are recognized by immune serum; or

ν) ΡΒΩ from the blood of infected or convalescent patients or клетокΩ cells of lymph nodes, spleen cells or cells of the lamina propria of the mucous membrane of infected or recovering patients can be cultivated η νίΐτο in the presence of partially or almost completely purified antigens obtained either from an infectious agent or from cell lysates infected with this agent, either from a recombinant expression system that encodes one or more antigens, in such a way as to detect antigen-specific e proliferative response of T cells.

Alternatively, it is possible to identify the products of genes that play an important role in the survival of огη νίνο pathogens, for example, using the method of mutagenesis using a labeled key (δίβηαΙιΐΌΐΌ-Ιαββοά) developed by Holden (Ηοΐάβη), or by detecting gene products that are specifically induced by ίη νίνο, for example, using a method such as ίνΕΤ (Ίη νίνο Εχρτβδδίοη Τοοίιηοίοβν), developed Mekalanosom (Meca ^ communication) or differential fluorescence induction developed Falkou (Έη11 <ο \ ν), to identify a subset of genes, among which may be potential protective antigens. Using these methods, gene products can be screened as described above. Genes can be cloned into expression vectors, and antigens can be isolated for inclusion in vaccine compositions along with agents that modulate glycosphingolipid-associated activity.

There are numerous methods by which it is possible to isolate antigens of a given infectious agent.

For example, components of the surface of an infectious agent, including one or more potentially protective antigens, can be extracted from the agent or cells infected with the agent using procedures that allow the isolation of antigens. This method may include the use of cell disruption techniques for the purpose of cell lysis, such as ultrasound irradiation and / or detergent extraction. Centrifugation, ultrafiltration, or precipitation may be used to form the antigens formed. The preparation of the antigen containing the δν-1 glycoproteins described in ShbAtY8 et al., Ί. Shesk Όίδ. 177: 14511457 (1998) is an example of such a method.

In addition, the antigens of the infectious agent or cells infected with this agent can be extracted using various techniques, including urea extraction, alkaline or acid extraction or detergent extraction, followed by chromatographic separation, but is not limited to these techniques. Material obtained in the free volume of the chromatographic column or in eluted peaks, which includes one or more potentially protective antigens, may be used in vaccine compositions.

Alternatively, genes encoding one or more potentially protective antigens can be cloned into various expression vectors suitable for producing antigens. These include bacterial or eukaryotic expression systems, such as Εδοίκποίιία οοΐί, Βαοίΐΐηδ 8ρρ., ΝΦπο 8ρρ., 8 satatuseus seguaia, mammalian and insect cell lines. Antigens can be isolated by conventional methods of extraction, separation and / or chromatography.

The designations ΟχΒ, ΕίχΒ and νίχΒ in the context of the present description include natural and recombinant forms of the molecule. Especially preferred is the recombinant form. The recombinant form of the molecule can be obtained according to a method in which a gene or genes encoding a specific polypeptide chain (or chains) that forms a protein is inserted into an acceptable vector and then used to infect an acceptable host. For example, a gene encoding a polypeptide chain, based on which ΕίχΒ is formed, can be inserted, for example, into the plasmid pMM68, which is then used to infect host cells, such as Liu 8ρ.60. Protein is purified and isolated by a well-known method. Mutant genes expressing the active mutant protein ΟχΒ, ΕίχΒ, or νχχ can be obtained from wild-type genes using well-known methods.

The designations ΟχΒ, ΕίχΒ and νίχΒ also include mutant molecules and other synthetic molecules (containing parts of ΟχΒ, ΕίχΒ or νίχΒ) that retain the ability to bind to CM1 or Cb3 or the ability to mimic the effects of binding to CM1 or Cb3.

Agents other than ΕίχΒ ΟχΒ, which retain CM1-binding activity, and agents other than νίχΒ, which retain

Cb3-binding activity includes antibodies that bind CM1 or Cb3.

For the production of antibodies, various hosts, including goats, rabbits, rats, mice, etc., can be immunized by injecting CM1 or Cb3, or any derivative or homologue thereof. Depending on the host species, various adjuvants can be used to enhance the immunological response. Such adjuvants include Freund's mineral gels, such as aluminum hydroxide, and surface active substances, such as lysolecithin, pluronium polyols, polyanions, peptides, oil emulsions, hemocyanin, snail lymph and dinitrophenol, but are not limited to them. BSG (Bacillus Calmette-Guerin) and Sogupebastepit ragutst are potentially human-acceptable adjuvants.

Humanized monoclonal antibodies are preferred according to the present invention. Monoclonal antibodies can be obtained using methods that provide antibody molecules using continuous culture of cell lines. These include hybrid methods, first described by Koeger and Myet, ΝαΐΗΓΟ, 256: 495-497 (1975)), methods using human B-cell hybridomas (Kokorog et al., Ittiηo1. Tobau, 4:72 (1983); Cole et al., Proc. Ναΐΐ. Asab. 8c1. 80: 2026–2030 (1983) and the Hybridum method (Cole et al. (1985) Mopos1 Apoboblic apb Supsegh Tieharu, A1p V. Pkk Shs., P. 77- 96), but not limited to them. In addition, methods developed to obtain chimeric antibodies, involving the splicing of mouse antibody genes with human antibody genes to produce a molecule with the required antigen specificity and biological activity (Moglkop et al., Proc. No. 1. Asab. Δοΐ. 81: 6851-6855 (1984); No. Bordeg et al., No. 1, Nig 312: 604-608 (1984); Takeba et al., No. 1, No. 314: 452-454 (1985)). Alternatively, the techniques described for the production of single-chain antibodies (Patent No. 8,446,779) can be adapted to produce single-chain antibodies specific for the component that represents the target for the interaction.

Antibodies can also be obtained by induction of lp in voo in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents, as described in Og1apb1 et al., Proc. №11. Asab. 8SK 86: 3833-3837 (1989) and Alyeg S. and Mbyet S., No. 1 349: 293-299 (1991)).

Antibody fragments can also be obtained that contain the specific antigen-binding sites CM1 or Cb3. For example, such fragments include P (ab ') ₂ fragments, which can be obtained by pepsin cleavage of the antibody molecule, and PAB fragments, which can be obtained by reducing the disulfide bridges

P (ab ') ₂ fragments, but not limited to them.

Alternatively, RaB expression libraries can be designed to quickly and easily identify monoclonal RaB fragments with the required specificity (Nick Α.Ό. et al., Δοΐпепс 256: 1275-1281 (1989)).

Peptide libraries or libraries of organic compounds can be created using combinatorial chemistry and then screened for their ability to bind to CM1 / CH3. Synthetic compounds, natural products and other sources of potentially biologically active substances can be screened in a variety of ways that will be apparent to those skilled in the art.

CM1 or Cb3 or fragments thereof can be used to screen peptides or molecules using any of the many screening methods. A molecule can be a free molecule in solution, bound to a solid support, located on the cell surface or a molecule localized intracellularly. The lack of activity or the formation of bound complexes between CM1 or CH3 and the agent to be tested can be evaluated.

Another way to determine binding to CM1 / CH3 can be based on using purified CM1 / CH3 to sensitize microtiter plates. After the blockade, the test agent is applied to the plate and allowed to interact with it prior to washing and detection with specific antibodies to this agent. Conjugation of antibodies, either directly or indirectly, with an enzyme or a radioactive label allows the binding to be quantified using methods based either on colorimetric analysis or on the assessment of radioactivity (ShZA and RIA, respectively).

Another way to determine binding to CM1 / CH3 can be based on using the binding of a saccharil fragment of CM1 / CH3 to an acceptable column carrier in order to carry out standard affinity chromatography. Removal of known compounds added to a solvent column can be used as evidence of binding activity, or in another embodiment, if mixtures of compounds are added to the column, elution and subsequent analysis may allow the ability of agents to bind ganglioside to be determined. In the case of proteins, the analysis can include peptide sequencing, and mapping after trypsin digestion, and subsequent comparisons with available databases. If the eluted proteins cannot be identified by this method, then standard biochemical analyzes can be used to obtain additional characteristics of the compound, for example, mass determination using laser desorption mass spectrometry. Non-protein products eluted from the OM1 affinity columns can be analyzed by HPLC and mass spectrometry of individual homogeneous peaks.

Another way to determine the ability to bind with OM1 / OB3 and the exact affinity of interaction can be based on the use of plasma surface resonance, previously described by Ci / 1etko et al., Wusset. 35: 6375-6384 (1996)).

Alternatively, a demonstration using phage can be used to identify potentially active agents that bind to CM1 or Cb3.

Phage demonstration is a molecular screening protocol using recombinant bacteriophage. The method involves the transformation of a bacteriophage genome that encodes the corresponding ligand (in this case a potentially active agent) capable of interacting with CM1 / Cb3 (or with their derivative or homolog) or nucleotide sequence (or its derivative or homolog) encoding the ligand. A transformed bacteriophage (which is preferably bound to a solid support) expresses the corresponding ligand (such as a potentially active agent) and demonstrates it on the phage envelope. An element or elements (such as cells) carrying target molecules that recognize a potentially active agent are isolated and amplified. Then, effective, potentially active agents are characterized. Demonstration using phage has advantages over conventional screening methods based on ligand affinity. On the surface of the phage, the potentially active agent is demonstrated in a three-dimensional configuration, which is more consistent with its configuration found in natural conditions. This allows for screening purposes a binding characterized by greater specificity and higher affinity.

Another screening method, providing large-scale screening of agents with suitable binding affinity for CM1 or Cb3, is based on the method described in detail in PP 84/03564. In general, a large number of various small peptide test compounds are synthesized on a solid substrate, such as plastic needles or some other surfaces. The peptide test agents are reacted with components of the fragments that represent the target for the interaction. Then the associated component, which is a target for interaction, is detected using specially adapted methods known in the art. The purified component, which is a target for the interaction, can then also be used to directly sensitize the plates for use in the aforementioned drug screening methods. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize on a solid support.

In accordance with all aspects of the invention, an agent possessing OM 1 binding activity or OH3 binding activity can also have the ability to form cross-links with the OM1 or OH 3 receptors. E1xB is one of such agents, which has the ability to form cross-links with OM1 receptors, which follows from its pentamer form.

Various methods are known for identifying agents that affect intracellular signals mediated by OM1 / Ob3 binding, but which themselves do not bind to OM1 or Ob3. For example, if the agent has the ability to increase the regulation of SE25 or MHC class II on the surface of B-cells, or to increase the regulation of SE25, or enhance apoptosis of SE8 '-T-cells, or to increase the regulation of secretion of Gb-Y monocytes, but in agent if the ability to bind OM1 or Ob3 is not detected (for example, using one of the binding assays described above), then we can conclude that the agent has the ability to mimic the effect of OM1 / Ob3 binding.

The invention is illustrated below with reference to examples and the accompanying drawings, in which is shown in FIG. 1 - stimulation of total! D and [dA in serum (M8) and ^ A in eye washes (E / Y) of mice immunized with the help of glycoproteins H8U-1 / gE1XB; FIG. 2 - T-cell proliferation (mesenteric lymph node) MB-Ν or (cervical lymph node) ΟΕΝ of lymphocytes of mice immunized with glycoprotein H8U-1 / gE1xB; FIG. 3 - T-cell proliferation of cells obtained from MB-Ν and ΟΕΝ of mice immunized intranasally with Op H8U-1 in the presence of 1-20 μg E1xB; FIG. 4 - serum titer to H8U-1 in mice after administration of H8U-1 glycoproteins three times with 10-day intervals with different amounts of gElxB or hFHBB as an adjuvant; FIG. 5 - reducing the spread of the virus, the clinical symptoms of the disease and latency in mice immunized with

H8U-1 / gE1xB; in FIG. 6 shows the distribution of the isotype 1d in M8 after infection with H8U-1 or immunization with Cp H8U-1 in the presence of Βχ or SGXB as an immunomodulator, FIG. 7 shows the distribution of subclasses of 1d after intranasal administration of Cp H8U-1 using τΕίχΒ or gGHBB as an immunomodulator; FIG. 8 shows the immunogenic effect of various amounts of τΕίχΒ or rsGhV on the titer of H8U-1 specific 1dA in the eye washes after the administration of glycoproteins H8U-1; FIG. 9 shows the response of serum immunoglobulin after immunization of mice with H8U-1 or glycoproteins (other) imitators (Tosk) individually or in the presence of an adjuvant; FIG. 10 — mucous 1dA titer in eye washings after intranasal immunization of mice with H8U-1 or glycoprotein imitators individually or in the presence of an adjuvant; FIG. 11 - mucosal 1dA titer in vaginal washes after intranasal immunization of mice with H8U-1 or glycoprotein imitators (others) individually or in the presence of an adjuvant; FIG. 12 - titers of H5> U-1-specific immunoglobulin in the serum of mice immunized with glycoproteins H8U-1 in the presence of various doses of τΕίχ ад as an adjuvant; FIG. 13 - titers of 1dA in the eye washes of mice immunized with the help of glycoproteins Н8У-1 in the presence of various concentrations of τΕίχ; FIG. 14 shows the 1dA titers in vaginal washes of mice immunized with the help of glycoproteins H8U-1 in the presence of various concentrations of τΕίχ фиг; FIG. 15 shows the distribution of subclasses of 1 dC in the case of a serum humoral immune response to H8U-1 after intranasal immunization with /χ / ΒχΒ or τΒχΒ or ocular infection with H8U-1; FIG. 16 shows cytokine production in cell cultures of lymph nodes taken from mice that were either infected with H8U-1 by ocular scarification, or immunized with intranasal glycoprotein H8U-1 using Οίχ / ΟίχΒ or τΕίχΒ as an adjuvant, and FIG. 17 - the level of protection against ocular infection of H8U-1 for mice immunized by intranasal administration of a mixture of H8U-1 or glycoprotein imitators in the presence of τΕίχΒ as an immunomodulator.

Example 1. τΕίχΒ can be used in combination with others H8U-1 for immunization.

Mice were immunized intranasally three times, each time using 10 µg of glycoprotein H8U-1 (Cp) in the presence of 10 or 20 µg τΕίχΒ. The controls were either untreated animals or animals that were given a viral glycoprotein preparation (simulator) derived from a culture of uninfected HIV tissue cells. Antibody titers were expressed as percent titers after infection. The production of total 1d and 1dA in serum and 1dA in eye washes was stimulated with glycoproteins Н§V-1 / ^ ΕίxΒ (Fig. 1). With the invention, it was also found that such low doses of τΕίχΒ as 0.1 μg also have efficacy in stimulating such responses.

In addition, proliferation of T lymphocytes was detected in immunized mice obtained from the cervical lymph node (which is located in the vaccination area) and from the mesenteric lymph node (which is located far from the vaccination area) when they were ίη νίΐτο cultured with H8Y-1, but not when they were cultured with a Cp H8U-1 simulator or without an antigen (Fig. 2).

Proliferation in response to the administration of Cp H8U-1 T-lymphocytes from MOI and CEA of mice immunized with Cp H8U-1 and different amounts of ΕίχΒ is shown in FIG. 3

The production of serum 1d to H8U-1 in mice after administration of glycoproteins H8U-1 at 3-day intervals with different amounts of ΕίχΒ (or ΟίχΒ) is shown in FIG. four.

And, finally, it was found that in mice immunized with HVV-1 / ^ ΕίxΒ, compared with the control, the spread of the virus after scarification of the cornea H8-1 (Fig. 5a) and the spread of local symptoms (edema and disease of the eyelid ), spread to the trigeminal ganglion (a form of shingles infection), spread to the central nervous system (encephalitis) and latency (5b).

Example 2. τΟίχΒ and τΕίχΒ act as immunomodulators.

When ΕίχΒ is used as an immunomodulator, there is a shift in the distribution of the 1d isotype (Fig. 6). The distribution of subclasses 1d differs depending on whether τΟχΒ or τΕίχΒ are used as an immunomodulator (Fig. 7).

Example 3. τΕίχΒ is a more efficient immunomodulator than τΟχΒ.

The titers of H8U-1 specific 1 dA (Fig. 8) turn out to be higher after stimulation with τΕίχр / Cf H8U-1 than after stimulation with τΟίχΟί Cf H8U-1.

Example 4. (Fig. 9).

Mice were immunized three times intranasally with H8U-1 glycoproteins, the H8U-1 glycoprotein simulator (obtained by using culture of cells of noninfected tissue and exposing them to the same treatment regimes used for isolating and purifying H8U-1 proteins) or Ηδν-1 glycoproteins in combination with various supposed mucosa-specific adjuvants. In each case, the dose of Ηδν-1 glycoproteins was 10 µg per immunization, and they were combined with 10 µg of recombinant ΕίχΒ or ΟχΒ as an adjuvant or with a mixture of 0.5 µg χ and 10 µg ΟxΒ. Three weeks after the final immunization, blood samples were taken and the total titer of antibodies to Ηδν-1 was determined using ΕΜδΑ. The amount of antibodies was expressed as the percentage of antibody titers stimulated by an ocular infection, which was caused by scarification using 10 ⁵ PFU Ηδν-1 strain 8С16. The data (shown in Fig. 9) suggests that the strongest humoral immune response was stimulated when the antigen was combined with a mixture of Οχ and ΟxΒ. However, a high level of response was also stimulated when τΕίχΒ was used as an adjuvant. In contrast, ιΌ.χΒ manifested itself as a weak adjuvant.

Example 5. (Fig. 10).

Mice were immunized according to example 4.

The production of secretory ^ A in the eye was evaluated using tears washes for several consecutive days, and then these samples were combined and analyzed using ΕΜ8Α using specific identifying antibodies to%. The amount of antibodies was expressed as the percentage of antibody titers stimulated by an ocular infection, which was caused by scarification using 10 ⁵ PFU Ηδν-1 strain 8С16. The data (shown in FIG. 10) clearly shows that high levels of secreted antibodies to Ηδν-1 were produced after immunization in the presence of either Οx / ΟxΒ or ΕίχΒ. In contrast to the results of analyzes of serum humoral immune responses, no differences were found in antibody titers obtained in the eyes between animals immunized with both Οx / ΟxΒ and ΕίχΒ as adjuvants. Just as for serum antibody, clear evidence was obtained that γΟχΒ is a very weak adjuvant.

Example 6. (Fig. 11).

Mice were immunized according to example 4.

Vaginal secretory production was assessed using swabs obtained from the reproductive system for several consecutive days, and then these samples were pooled and analyzed using ΕΟ8Α using specific identifying antibodies to%. The amounts of antibodies were expressed as final titers, which were calculated using the linear regression method. The data clearly shows that high levels of secreted antibodies to Ηδν1 were produced in remote regions of the mucosa after immunization in the presence of either Ώχ ^^ or ΕίχΒ. In the vagina, the highest antibody titers obtained after immunization in the presence of ΕίχΒ. Low titers were obtained after immunization in the presence of Οx / ΟxΒ, and very little secretion was provoked when τΟχΒ was used as an adjuvant.

Example 7. (Fig. 12).

Mice were immunized three times intranasally, either using only Ηδν-1 glycoproteins (10 μg), or in the presence of increasing concentrations of τΕίχΒ as an adjuvant. Three weeks after the last immunization, blood samples were taken and the total titer of antibodies to Ηδν-1 was determined using ΕΟ8Α. The amount of antibodies was expressed as the percentage of antibody titers stimulated by an ocular infection, which was caused by scarification using 10 ⁵ PFU Ηδν-1 strain δΟ6. The data clearly indicate that the ability of τΕίχΒ to trigger humoral immune responses to the addition of heterologous antigens is a dose-dependent phenomenon, with maximum reactivity occurring at a dose of τΕίχΒ of about 20-50 μg. In addition, it is clear that when using doses of ΕίχΒ 20 µg and higher, the titer of antibodies to Ηδν-1, stimulated by intranasal infection, is comparable to or exceeds the titer that is stimulated by infection with a live virulent virus.

Example 8. (Fig. 13).

Mice were immunized according to Example 7. The secretory production in the eye was assessed using a wash of tears obtained over several consecutive days, and then these samples were pooled and analyzed using ΕΟδ специф using specific identifying antibodies to%. The amount of antibodies was expressed as the percentage of antibody titers stimulated by an ocular infection, which was caused by scarification using 10 ⁵ PFU Ηδν-1 strain δί.Ί6. Evidence suggests that the strongest I I responses in the eye were stimulated when the glycoproteins Ηδν1 were used in combination with a dose of τΕίχΒ 20 μg or higher. However, when using such a dose, the titers obtained were lower than those initiated by the infection of the eye with a virus.

Example 9. (Fig. 14).

Mice were immunized according to example 7.

Vaginal secretory production was assessed using swabs obtained from the reproductive system for several consecutive days, and then these samples were pooled and analyzed using ΕΜδΑ using specific identifying antibodies to%. The amounts of antibodies were expressed as final titers, which were calculated using the linear regression method. Evidence suggests that optimal humoral immune responses to H5U-1 are stimulated in the vagina when doses are used as an adjuvant [RLhV 20 µg or higher.

Example 10. (Fig. 15).

Mice were infected with either 10 ⁵ pfu H5U-1 strain 5O16 by scarification of the cornea, or they were immunized three times intranasally with 10 μg of glycoprotein H5U-1 in combination with Ox / OxB or hBGHV. Three weeks after the last inoculation, serum samples were taken and analyzed with the aid of the presence of IdC2a to H5U-1. The amounts of antibodies were expressed as final titers, which were calculated using the linear regression method (Fig. 7a). The data clearly show that the nature of the humoral immune response to H5U-1 is influenced by the way in which the antigen is presented to the immune system. Infection with H5U-1 mainly activates antibody production associated with Ty1, which is characterized by high titers of the complement-binding antibody isotype [dC2a. Infection stimulates relatively low titers of the TY2 isotype CA, i.e. CEE This immune response profile is clearly visible when the data is expressed as the IdC1: IdC2a ratio, as shown in FIG. 7b. The ratio after infection is significantly lower.

1. Intranasal immunization in the presence of an adjuvant Ox / OHB triggers the release of mainly those associated with Ty2 CEE. High enough titers of ^ C2a are also obtained, which suggests that Ox / OxB promotes the activation of Ty1 and Ty2cells. The activation of both responses and the relative dominance of Ty2 is reflected by the IdC1: IdC2a ratio, which is about 3. It is interesting to note that the nature of the response to Н5У-1 stimulated by hN1xB as an adjuvant is almost completely Ty2-dominant. High titers and only very small amounts of ^ C2a were obtained. This strong shift in relation to the reactivity of Ty2 reflects the value of the ratio IdC1: IdC2a, which is approximately 9.

Example 11. (Fig. 16).

Mice were infected with either 10 ⁵ pfu H5U-1 strain 5O16 by scarification of the cornea, or they were immunized three times intranasally with 10 μg of glycoprotein H5U-1 in combination with Ox / OxB or [OxB. 3 weeks after the last inoculation, the lymph nodes were removed from the animals and used to obtain suspensions of individual cells that were cultured either in the presence of killed H5U-1 or a viral preparation-simulator from a culture of uninfected tissue. On 4-7 days of cultivation, cell samples were isolated and subjected to analysis of ^ ^ to detect the secretion of cytokines. The data clearly indicate that the T cells in the cultures had the ability to respond to H5U-1, but practically did not respond to viral simulators. Cells of lymph nodes obtained from mice that were infected with H5U-1, mainly produced the cytokine γ-interferon (y-SHY) associated with Ty1. Lymph node cells derived from mice that were immunized intranasally, produced high titers of Ty2-associated cytokines No. 4 and Yu. In addition, both Ox / OxB and ZhxH resulted in the activation of T-cells that secreted γ-Σ при, when stimulated with 1 and νίΙΐΌ Н5У-1. This suggests that although the response to these adjuvants is mainly aimed at the production of Ty2-cytokines, some activation of Ty1 also takes place. These data are consistent with the results obtained in the analysis of humoral immune responses.

Claims (37)

CLAIM 1. The use of ΕΐχB, OhB or YίχB, not containing a complete toxin, as an immunomodulator in vaccines for infectious diseases. 2. The use according to claim 1, where the immunomodulator is ΕΐχB, not containing a complete toxin. 3. The use according to claim 1 or 2, where the infectious disease is a disease, the infectious agent of which is a representative of the herpes virus family. 4. The use according to claim 3, where the infectious disease is caused by an infectious agent and the infectious agent is selected from the group comprising H5U-1, H5U-2, ΕBU (Epstein-Barr virus), U2U (chickenpox virus), WMD (cytomegalovirus), NNU-6 (human herpes virus), NNU-7 and NNU-8. 5. The use according to claim 4, where the infectious agent is selected from the group comprising H5U1, H5U-2, WMD or ΕBU. 6. The use according to claim 1 or 2, where the infectious disease is caused by an infectious agent and the infectious agent is an influenza virus. 7. The use according to claim 1 or 2, where the infectious disease is caused by an infectious agent and the infectious agent is a parainfluenza virus. 8. The use according to claim 1 or 2, where the infectious disease is caused by an infectious agent and the infectious agent is a respiratory syncytial virus. 9. The use according to claim 1 or 2, where the infectious disease is caused by an infectious agent and the infectious agent is a hepatitis virus. 10. The use according to claim 9, where the infectious agent is selected from the group comprising hepatitis A, B, C and Ό viruses. 11. The use of claim 10, where the infectious agent is a hepatitis A virus or hepatitis C. 12. The use according to claim 1 or 2, where the infectious disease is meningitis. 13. The 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 NZZSPA TESHIDSHBSCH, Naetory L1 and 8 tPyspas type B and 81Gerlososperidae. 14. The use according to claim 1 or 2, where the infectious disease is pneumonia or an infection of the respiratory tract. 15. The application of clause 14, where the infectious disease is caused by an infectious agent and the infectious agent is selected from the group comprising 81 Reality at the Readers 1a and Musoaclischt Schlose10818. 16. The use according to claim 1 or 2, where the infectious disease is a disease that is sexually transmitted. 17. The use according to clause 16, where the infectious disease is caused by an infectious agent and the infectious agent is selected from the group consisting of No. 188epa doppogneNeea. HIV-1, HIV2 and Syatul 1gas1yuta8. 18. The use according to claim 1 or 2, where the infectious disease is a disease of the gastrointestinal tract. 19. The 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 enteroaggregating E. co11, rotaviruses, 8a1toe11a ei1e8eu8e1e1e1e1e1e1e1e1e1e1e1e1e1e1e1e1e1e1e1e1e1e1e1e1eu , Satru1bac1eg) e) ish and Ulbu with Ho1ege. 20. The use according to claim 1 or 2, where the infectious disease is a shallow infection. 21. The use according to claim 20, where the infectious disease is caused by an infectious agent and the infectious agent is selected from the group consisting of 81 arHu1ossoss8 aigey8, 81ger1ossoss8 rhode8 and 81ger1ossoss8 ty1ai18. 22. The use according to claim 1 or 2, where the infectious disease is a parasitic disease. 23. The use according to claim 22, wherein the infectious disease is caused by an infectious agent and the infectious agent is selected from the group consisting of malaria, Tguraia8ota 8 pp., Tohor1a8ta doibi, Ee18tasha boiouat and Oisosegs 8rp. 24. A vaccine composition for immunoprophylaxis of an infectious disease, wherein the infectious disease is caused by an infectious agent and where the vaccine composition comprises an antigenic determinant and an immunomodulator selected from E1xB, C (xB or U1xB not containing a complete toxin, the antigenic determinant being an antigenic determinant of this infectious agent. 25. The vaccine composition according to paragraph 24, where the infectious disease is an H8U-1 infection and where the antigenic determinant is an antigenic determinant of H8U1. 26. The vaccine composition according to paragraph 24 or 25, where the immunomodulator is an E1xB containing no complete toxin. 27. The vaccine composition according to paragraphs 24, 25 or 26, where the immunomodulator and antigenic determinant are different fragments. 28. The vaccine composition according to paragraphs 24, 25 or 26, where the immunomodulator and antigenic determinant are associated with a bifunctional cross-linking reagent. 29. A kit for vaccinating a mammal against an infectious disease, including a) a vaccine immunomodulator selected from E1xB, C (xB or U1xB not containing a complete toxin, and b) the antigenic determinant of an infectious disease for co-administration with a specified immunomodulator of the vaccine. 30. A method for preventing or treating a disease in a host, comprising the step of inoculating the host with a vaccine that includes at least one antigenic determinant and an immunomodulator, wherein the immunomodulator is E1xB, C (xB or UXxB not containing a complete toxin. 31. The use of Е1хВ, С (хВ or УГхВ, not containing a complete toxin, for up-regulation of antibody production on the surfaces of mucous membranes. 32. The use of EGxB, C (xB or UGxB not containing a complete toxin as an immunomodulator in a vaccine to prolong the presentation of antigen and achieve a longer immunological memory in a mammal). 33. The composition of the vaccine used for immunoprophylaxis in relation to an infectious disease, where the infectious disease is caused by an infectious agent, where the vaccine contains an antigenic determinant and an immunomodulator selected from ExxB, C (xB or U1xB not containing a complete toxin, and the antigenic determinant is an antigenic determinant the specified infectious agent and immunomodulator prolongs the presentation of the antigenic determinant and provides a longer immunological memory. 34. The use of EGxB, C (xB or UGxB not containing a complete toxin in combination with an antigen or antigenic determinant for the directed transfer of antigen or antigenic determinant into the cytosol or nucleus of an antigen presenting cell. 35. The use of ΕΐχΒ, CXB or U1xB not containing a complete toxin in combination with an antigen or antigenic determinant for up-regulation of the presentation of the indicated antigenic determinant or antigenic determinant obtained from the indicated antigen by class I MHC molecules. 36. A vaccine composition comprising a) ΕΐχΒ, ΟχΒ, and b) ΕΒν antigen intended for the treatment and / or prevention of ΕΒν-related diseases. 37. A therapeutic composition comprising ΕίχΒ, ΟχΒ, intended for the treatment of связанных related diseases.