UA98612C2 - Immunogenic composition useful for human individual comprising an antigen or antigenic preparation in combination with an adjuvant comprising qs21 in the form of a liposome and 3d-mpl - Google Patents

Abstract

The present invention provides a human dose of an immunogenic composition comprising an antigen or antigenic preparation, in combination with an adjuvant which adjuvant comprises an immunologically active saponin fraction derived from the bark of Quillaja Saponaria Molina presented in the form of a liposome and a lipopolysaccharide wherein said saponin fraction and said lipopolysaccharide are both present in said human dose at a level of below 30 μg. The present invention further provides an adjuvant composition in a human dose suitable volume comprising between 1 and 30 μg of a lipopolysaccharide and between 1 and 30 μg of an immunologically active saponin fraction presented in the form of a liposome.

Description

This invention relates to improved vaccine compositions, methods of their production and their use in medicine. In particular, the invention considers adjuvant vaccine compositions, where the adjuvant is a liposomal preparation consisting of saponin and lipopolysaccharide.

The present invention further relates to influenza vaccine compositions and vaccination regimens for immunization against influenza.

New compositions or vaccines with improved immunogenicity are always needed. As one strategy, adjuvants are used to try to improve the immune response to any particular antigen.

Lipopolysaccharides (LIPS) are the main surface molecule and are found exclusively in the outer layer of the outer membrane of gram-negative bacteria. LPS prevent the destruction of bacteria with the help of serum complement and phagocytic cells, they are involved in growth with the formation of colonies. Lipopolysaccharides are a group of structurally related complex molecules with a size of approximately 10,000 Da and consist of three covalently linked areas: () a chain of O-specific polysaccharide (O-antigen) in the outer area; (iii) the main oligosaccharide of the central part; (ii) lipid A - as the innermost region serving as a hydrophobic anchor, it contains glucosamine disaccharide units that carry long-chain fatty acids.

It has been demonstrated that the biological activity of lipopolysaccharides, such as lethal toxicity, pyrogenicity and adjuvantity, is related to the lipid A fragment. In contrast, immunogenicity is related to the O-specific component of the polysaccharide (O-antigen). Both lipopolysaccharides and lipid A have long been known for their strong adjuvant effects, but the high toxicity of these molecules prevented their use in the manufacture of vaccine compositions.

Therefore, significant efforts have been made to reduce the toxicity of LPS or lipid A while preserving their adjuvant properties.

Mutant ZaiitopeiPa tippezoia K595 was isolated in 1966 from the culture of the parent (smooth) strain (Igideghii; ei a. 1966 App. MM Asai. 5si. 133:349-374). Selected colonies were screened for their susceptibility to lysis by a group of phages, and only those colonies that showed a narrow range of susceptibility (susceptible to only one or two phages) were selected for further study.

This attempt led to the isolation of a deep rough (deer goshudi) mutant strain, which is defective from the point of view of biosynthesis of lipopolysaccharides, it was designated as 5. tippezoia 595.

Compared to other LPS, the lipopolysaccharides produced by the 5. tippezoia K595 mutant have a relatively simple structure: (i) they do not contain the O-specific region, which is the feature responsible for the variability from the wild-type smooth phenotype to the rough phenotype of the mutant and leads to loss of virulence; (i) the main region is very short, this property increases the sensitivity of the strain to a variety of chemicals; (iii) fragment of lipid A is extensively acylated and contains up to 7 fatty acids.

4-monophosphoryl lipid A (MPI), which can be obtained by acid hydrolysis of LPS extracted from a deep rough mutant strain of gram-negative bacteria, retains the adjuvant properties of lipopolysaccharide while exhibiting toxicity, which is reduced by a factor of more than 1000 (with lethal dose data on chicken embryos) (doppzop ei aiY. 1987 Veu. Ipiesi. 20 biv. 9 Burri:5512-

Z516). Lipopolysaccharide is usually refluxed in solutions of a mineral acid of moderate strength (eg, 0.1 M HCl) for a period of approximately 30 minutes. This method leads to dephosphorylation in position 1 and cleavage of the carboxyl group in position 6", resulting in MRI.

Lipid A of 3-O-deacylated monophosphoryl (30O-MRI), which can be obtained by hydrolysis

MRI. in weakly alkaline conditions, has additionally reduced toxicity while maintaining adjuvant properties, see 054.912.094 (KiBry IttypospetisaI5). Alkaline hydrolysis is usually carried out in an organic solvent, such as a chloroform/methanol mixture, by saturating an aqueous solution of a weak base, for example, a 0.5 M solution of sodium carbonate at pH 10.5.

Further information on obtaining C30O-MRI can be obtained, for example, from O54,912,094 and

MO02/078637 (Sogiha Sogrogaiop).

Quilai saponins are a mixture of triterpene glycosides extracted from the bark of the soap tree (Ociiazda zaropagia). Raw saponins are widely used as adjuvants in veterinary medicine.

OiiY-A is a partially purified aqueous extract of quilai saponin material. O521 is a

A purified non-toxic fraction of OtsiY-A was produced, and the method of its production is disclosed (as OA21) in the patent

US Mo 5,057,540.

As an example, influenza vaccines and human wart virus (HPV) vaccines are formulated with adjuvants.

Influenza viruses are one of the most widespread viruses in the world, affecting both humans and livestock. Influenza causes significant economic losses, morbidity, and even mortality.

The influenza virus is an enveloped RNA virus with a particle size of approximately 125 nm in diameter.

It consists mainly of an inner nucleocapsid or ribonucleic acid (RNA) core bound to a nucleoprotein, surrounded by a viral envelope with a lipid bilayer structure and outer glycoproteins. The inner layer of the viral envelope consists mainly of matrix proteins, and the outer layer consists mostly of lipid material obtained from the host. The influenza virus consists of two surface antigens, glycoprotein neuraminidase (MA) and hemagglutinin (HA), which appear as 10-12 nm long peaks on the surface of the particles. These surface proteins, especially hemagglutinin, determine the antigenic specificity of influenza subtypes.

These surface antigens are progressively, sometimes quickly, subject to some changes, which leads to antigenic variations of influenza. These antigenic changes, called "variability," are unpredictable and can have a dramatic effect from an immunological standpoint, as they eventually lead to the emergence of new strains of influenza that enable the virus to evade the immune system, causing well-known, almost annual epidemics.

The World Health Organization, in cooperation with national health committees and vaccine manufacturers, determine the influenza virus strains that must be included in the flu vaccine each season.

HA is the most important antigen in determining the serological specificity of different strains of influenza. This 75-80 kDa protein contains numerous antigenic determinants, some of which are located in regions subject to sequential changes in different strains (strain-specific determinants), and others in regions that are common to many NA molecules (common determinants). .

Influenza viruses cause epidemics almost every winter, with the prevalence of infection with type A or B virus up to 4,095 within six weeks. Influenza virus infection leads to various pathological conditions, from subclinical infection due to mild upper respiratory tract infection to severe viral pneumonia. Typical influenza epidemics cause an increase in the incidence of pneumonia and lower respiratory tract infections, as evidenced by increased hospitalizations or deaths. The severity of the disease, first of all, is determined by the age of the host, his immune status and the location of the infection.

Elderly people aged 65 and over are particularly vulnerable, accounting for 80-90% of all influenza-related deaths in developed countries. People with underlying chronic conditions are also more likely to develop complications. Babies as young as a few months old can also suffer severe illness. Thus, these groups are especially in need of protection. In addition to these risk groups, health ministries also recommend vaccination of healthy adults who are in contact with the elderly.

Vaccination plays a critical role in controlling annual influenza epidemics. Currently available influenza vaccines are either inactivated or live attenuated influenza vaccines.

Inactivated influenza vaccines consist of three possible forms of antigen preparation: whole inactivated virus, subvirion, where purified virus particles are destroyed by detergents or other reagents in order to solubilize the lipid envelope (the so-called "split" vaccine), or purified HA and MA (subunit vaccine). These inactivated vaccines are administered intramuscularly (i/m) or intranasally (i/n).

Vaccines against influenza of all types are usually trivalent vaccines. They generally contain antigens derived from two strains of the influenza A virus and one strain of the influenza B virus.

A standard dose for injections of 0.5 ml in most cases contains 15 μg of the antigenic component of hemagglutinin from each strain, as evidenced by the data of simple radial immunodiffusion (RAI) (9.M. Tesppidie Tog Te azzau oi ipisepga paetaddishipip apiidep: adariaynop og roiepsu deiieptipayop oi ipasiimaiied u/poie migiv apa zybypii massipev. .). Vioi stage (1977) 237-247; 9. M. Mooai ei ai., Ipiegtaiyopa! soParogaiime zishau oyi zipdie gadiai ayiviop apa

Ititipoe!esigtorpogeviv Tesppidnev og She azzau oi Nkaetadadishipip apiidep oi ipipeplga migiv. U. Vioi. fuck 9 (1981) 317-330).

Influenza vaccines currently available are considered safe for all age groups (Oye et al. 1999, Massipe, 17, 3094-3101). However, there have been several reports that current flu vaccines are ineffective in children under two years of age. In addition, vaccine efficacy rates for preventing typical confirmed influenza disease have been reported to be 23-72 95 for the elderly, significantly lower than the 60-90 95 efficacy rates reported for young adults (Somayeghi, 1994, U. At. May. Avzos, 21, 166-1665; Sgo55, 1995, App Ipiet. Mea. 123, 523-527). It has been demonstrated that the effectiveness of the influenza vaccine is correlated with the serum titers of antibodies to the inhibition of hemagglutination (NI) of the viral strain, and several studies have found that older adults show lower NI titers after influenza immunization than younger people (Mygazko, 2002, Ehregitepiai! degopiiodu, 37, 427-439).

Thus, there is currently a need for new vaccines with improved immunogenicity.

Antigenic vaccine formulation with strong adjuvants is a possible approach to enhance immune responses to subviral antigens.

The ME59-adjuvanted subunit influenza vaccine in the form of an oil-in-water emulsion is commercially available and has demonstrated the ability to induce a higher antibody titer than a vaccine derived from an unadjuvanted subunit vaccine (Oye 20 Bopayo ei ai!. 1999, Massipe, 17, 3094-3101). However, in a later publication, the same vaccine did not demonstrate an improved profile compared to a split vaccine without adjuvant (Ried-Wagyega et al., 2004, Massipe 23, 283-289).

By their origin, during the interpandemic periods, influenza viruses circulate similarly to the viruses of the previous epidemic. Viruses spread among people with varying levels of immune status due to infections that people have been exposed to earlier in their lives. Such circulation over a period, usually 2-3 years, gives rise to new strains that have changed enough to cause an epidemic again in the general population, this process is called "antigenic variability". "Variants of variability" can have different impacts in different populations, regions, countries, or continents in any one year, although over several years their overall impact is often the same.

In other words, influenza pandemics occur when a new influenza virus appears, against which the human population lacks immunity. Typical influenza epidemics cause an increase in the incidence of pneumonia and lower respiratory tract disease, as evidenced by increased rates of hospitalization or mortality. These complications are more likely to occur in the elderly or those with underlying chronic conditions, but infants as young as a few months old can also experience severe disease.

At unpredictable intervals, new influenza viruses appear with the main surface antigen, hemagglutinin, but with a completely different subtype than the strains that circulated the previous season. The resulting antigens can change by 20-50 95 in relation to the corresponding protein of the strains that were distributed among people until that moment. This can lead to the virus seeping through "herd immunity" and causing pandemics. This phenomenon is called "antigenic variability". It is believed that, at least in the past, pandemics occurred when influenza viruses from different species, such as avian or swine influenza viruses, crossed the species barrier. If such viruses have the potential to spread from person to person, they can spread globally over a period of months to a year, resulting in a pandemic. For example, in 1957 (the Asian influenza pandemic), viruses of the subtype

H2M2 has been replaced by NIMI viruses that have been circulating in the human population since at least 1918, when this virus was first isolated. H2NA and M2MA underwent antigenic variability in the period 1957-1968 until, with the appearance of a new subtype of influenza, NA was replaced in 1968 (Hong Kong influenza pandemic) by NZM2, after which M2MA continued to act together with NZNA (McKayita et al. , 1991, Eridetio). Intesi. 106, 383-395).

Features of the influenza virus strain that give it the potential to cause a pandemic outbreak are as follows: it contains a new hemagglutinin compared to the hemagglutinin in existing strains, which may or may not be accompanied by a change in the neuraminidase subtype; it can be transmitted horizontally in the human population; and it is pathogenic to humans. A new hemagglutinin may be one that has not been detected in the human population for a significant period of time, probably several decades, such as H2. Or it could be a hemagglutinin that did not previously exist in the human population, such as H5, HO, H7 or Hb found in birds. In any case, the majority, or at least a large proportion, or even the entire population has not previously encountered the antigen and is immunologically unprepared for it.

Papillomaviruses are small DNA tumor viruses that are very species-specific. To date, more than 100 individual human papillomavirus (HPV) genotypes have been described. HRMs are generally either specific for the skin (eg, HRU-1 and -2) or for mucous membranes (eg,

NRU-6 and -11) and usually cause benign tumors (warts) that do not go away for several months or years. Such benign tumors can be painful for a person who has such a tumor, but in general, with a few exceptions, these tumors are not life-threatening. Some NRUs are also associated with cancer. The most pronounced positive correlation between HRM and cancer in humans exists between HRU-16 and HRU-18 and cervical carcinoma. Cervical cancer is the most common malignancy in developing countries, with approximately 500,000 new cases occurring worldwide annually. At this time, from a technical point of view, it is possible to actively fight primary HRM-16 infections, and even diagnose cancer containing HRM-16, using vaccines. For an overview of the prospects of prophylactic and therapeutic vaccination against NRMU-16, see in Sazop.), Siip. Ittipoipeg. 1994; 1(4) 293-306 apa Nadepezeye M.BE., Iptesiop5 ip Medisipe 1997 14(7) 555-556, 559-564.

Although slight variations occur, all described NRU genomes contain at least eight early genes, from E! to E8, and two late genes 1 and 12. In addition, the upstream moving regulatory region contains regulatory sequences that apparently control most of the transcriptional events of the NRU genome.

Vaccines based on HRM 11 are described in UMO94/00152, UMO94/20137, UMO93/02184 and

UMO94/05792. Such a vaccine may contain antigen 1 as a monomer, capsomer, or virus-like particle. Methods of obtaining MIR are well known in this field and include disassembly approaches-

reassembly of MIR in order to ensure increased homogeneity, for example, as described in

M/09913056 and 056245568. Such particles may additionally consist of proteins 12. Vaccines based on 12 are described, for example, in M/O93/00436. Other HRM vaccine approaches are based on early proteins such as E7: or fusion proteins such as 1 2-E7.

There is still a need for improved vaccines, especially in the case of influenza and in specific influenza pandemics, as well as for the elderly population, or in the case of NRU vaccines.

Adjuvants containing combinations of lipopolysaccharide and quilai saponins have been considered previously, for example in EPOb71948. This patent demonstrated strong synergy when combining lipopolysaccharide (30O-MRI) with quilai saponin (20521). It has now been found that good adjuvant properties can be provided by combinations of lipopolysaccharide and quilai saponin as immunostimulatory agents in the adjuvant composition, even when the immunostimulators are present in low human dose amounts.

In the first aspect of this invention, an immunogenic composition consisting of an antigen or its antigenic preparation, in combination with an adjuvant composition containing an immunologically active fraction of saponin from the bark of Oysha|a Zaropagia Moiip, presented in the form of a liposome and lipopolysaccharide, is proposed.

In the second aspect of this invention, an immunogenic composition consisting of an influenza virus or its antigenic preparation in combination with a saponin adjuvant presented in the form of a liposome is proposed. In a specific variant of this aspect, the immunogenic composition additionally contains a derivative of lipid A, for example, ZO-MRI.

In accordance with the present invention, the saponin adjuvant in the form of a liposome contains the active fraction of saponin from the bark of OshiPazda Zaropagia Moiipa, such as 0521, and a sterol, for example, cholesterol, in a saponin/sterol ratio of 1:1 to 1:100 w/w.

In particular, the specified immunogenic composition contains an antigen with a CO4 T-cell epitope. Alternatively, the specified immunogenic composition contains an antigen with a B-cell epitope.

The invention also relates to the use of an influenza virus or its antigenic preparation and an adjuvant containing an immunologically active fraction of saponin from the bark of Oysha|a Zzaropagia Moiipa, presented in the form of a liposome and lipopolysaccharide, in the production of an immunogenic composition for the prevention of influenza virus infection and/or disease.

The invention also relates to the use of the human papilloma virus antigen or antigens or its antigenic preparation and adjuvant containing the immunologically active fraction of saponin from the bark of Ocyya/|a

Zaropagia Moiip, presented in liposome and lipopolysaccharide form, in the manufacture of an immunogenic composition for the prevention of human papillomavirus infection and/or disease. The invention also relates to the use of cytomegalovirus antigen or antigens or its antigen preparation and adjuvant containing an immunologically active fraction of saponin from the bark of Oyyar baropagia Moitpa, presented in the form of a liposome and lipopolysaccharide, in the production of an immunogenic composition for the prevention of cytomegalovirus infection and/or disease.

The invention also relates to the use of the determined Bigeriosossi5 rpeshtopiae antigen or antigens or its antigenic preparation and adjuvant containing an immunologically active fraction of saponin from the bark of Oshisha) Zaropagia Moiip, presented in the form of a liposome and lipopolysaccharide, in the production of an immunogenic composition, to prevent infection with Bigeriosossi5 rpeshtopiae and/or disease.

The invention also relates to the use of the Riakhtoditst TaiIsiragit antigen or antigens or its antigen preparation and an adjuvant containing an immunologically active fraction of saponin from the bark of Ocyya/|a

Zaropagia Moiip, presented in the form of liposome and lipopolysaccharide, in the production of an immunogenic composition for the prevention of infection with Riaztodaischt haIsiragitis and/or malaria disease.

The invention also relates to the use of the varicella virus antigen or antigens or its antigenic preparation and adjuvant containing the immunologically active fraction of saponin from the bark of Ocyya/|a

Zaropagia Moiip, presented in liposome and lipopolysaccharide form, in the manufacture of an immunogenic composition for the prevention of varicella virus infection and/or disease.

In another aspect, the use of (a) an antigen or its antigen preparation and (B) an adjuvant, as defined above, in the production of an immunogenic composition for stimulating at least one, or at least two, or all of the following phenomena in humans is proposed: (i) improved immune response of T-cells cp4 against the indicated antigen or its antigenic preparation, (ii) improved humoral immune response against the indicated antigen or its antigenic preparation, (ii) improved reaction of B-lymphocytes against the indicated antigen or its antigenic preparation.

In particular, said antigen is an influenza virus, MMR, cytomegalovirus, varicella zoster virus (VVV), Zygeriosossi5 rpestopiae antigen, or malaria, or an antigen preparation thereof, and said human is an individual or population at risk, such as an adult at high risk of the disease , an elderly person or an infant. In a specific version, the use of an antigen or its antigenic preparation and adjuvant, as defined in this description, is proposed in the manufacture of an immunogenic composition for vaccinating a person, in particular, the elderly, against the pathogen from which the antigen in the immunogenic composition is obtained. Specifically, the indicated antigen is influenza virus, human papilloma virus, cytomegalovirus, chicken pox virus, bigeriosossi5 rpeshtopiae, Rhizotodiitis ragazi(e), antigen or antigens or antigen preparations.

Also proposed is a method of vaccination that includes the delivery of an antigen or an antigenic composition, in particular, an influenza virus or an influenza virus, a cytomegalovirus, a varicella virus, a Zigeriosossi5 rpeshtopiae, a Riaetodisht ragazie, or an antigen preparation and an adjuvant thereof, as defined above, to an individual or a population in need of this antigen.

In a particular embodiment, the immunogenic composition can stimulate an enhanced CO4 T-cell immune response against said antigen or an antigen preparation thereof, and in particular, it can additionally stimulate either a humoral immune response or an enhanced B-lymphocyte response or both compared to the response occurring with non-adjuvanted antigen or antigen composition. Specifically, the indicated immune response of CO4 T cells includes stimulation of the cross-reactive reaction of CO4 T-helpers. A specifically indicated humoral immune response includes stimulation of a cross-reactive reaction.

In a further embodiment, a method or application, as defined above, of protection against infection or disease caused by a pathogen, which is a variant of the pathogen from which the antigen in the immunogenic composition is derived, is provided. In another variant, the proposed method or application, as defined above, of protection against infections or disease caused by a pathogen that contains an antigen that is a variant of the specified antigen in an immunogenic composition. In a specific variant, it is proposed to use an antigen, in particular influenza or NRU, or its antigenic preparation in the production of an immunogenic composition for re-vaccination of people who received a previous vaccination with an immunogenic composition containing an antigen, in particular influenza or

NRU, or its antigen preparation in combination with an adjuvant, as described in this invention.

In a specific variant, the composition used for repeated vaccination may additionally contain an adjuvant. In another specific embodiment, the immunogenic composition for re-vaccination contains an antigen that consists of widespread CO4 T-cell epitopes and the antigen or antigenic composition used in the previous vaccination. In particular, the immunogenic composition for re-vaccination contains an influenza virus or its antigenic preparation, which consists of widespread T-cell epitopes of CO and the influenza virus or antigenic preparation of the virus used for the first vaccination.

In one aspect, revaccination is administered to subjects who received an influenza vaccine in a previous season. Usually, re-vaccination is carried out at least 6 months after the first vaccination, preferably after 8-14 months, more preferably approximately after 10-12 months. In another aspect, subjects are revaccinated after receiving a composition comprising an influenza virus or an antigen preparation thereof, wherein at least one strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak.

In a further aspect of the present invention, the use of an influenza virus or its antigen preparation from a first influenza strain in the production of an immunogenic composition as defined in the present invention for protection against influenza infections caused by a variant influenza strain is proposed.

The invention also relates to a method of vaccination that includes the delivery of an influenza virus or its antigen preparation and an adjuvant as defined in this invention.

In another aspect, a method of vaccinating a person or a population at risk, such as adults at high risk of disease or the elderly, is provided, said method comprising administering an immunogenic composition against influenza comprising an influenza virus or an antigenic preparation thereof in combination with an ad' juvant, as defined in the present invention.

In yet another variant of the invention, a method of re-vaccinating people who have received a previous vaccination with an immunogenic composition of influenza, consisting of an antigen of influenza or its antigenic preparation from at least one strain of the influenza virus in combination with an adjuvant, as defined in this invention, and specified the method includes the introduction of an immunogenic composition containing the influenza virus or its adjuvant or non-adjuvant antigen preparation to the indicated person. The present invention also relates to a method of obtaining an immunogenic composition, which contains a combination of a saponin adjuvant in the form of a liposome with an influenza virus or its antigen preparation and optionally with 30O-MRI.

Other aspects and advantages of the present invention are described further in the following detailed description of preferred embodiments thereof.

Figure 1 is a schematic representation of MRI acquisition.

Figure 2 - humoral reaction against different strains of influenza after immunization of ferrets with experimental formulations: test for inhibition of hemagglutination (ZM / - ISO95) before and after heterologous start of vaccination (NIMI A/5(osKpoYit/24/90), after immunization (NIMI A/ Meme/

SaIedopia/20/99, НЗМ2 А/Rapata/2007/99 and В/5papdopad/7/97) and after a heterologous provaccination sample (НЗМ2 ALLuotipad/3/2003).

Figure C - study on ferrets: titer of viruses in nasal washes after provaccination test (Day 42).

Figure 4 - study on mice: humoral response against three strains of influenza vaccine after immunization of mice with experimental drugs: test for inhibition of hemagglutination (SMT -/- IS95) on the 21st day after immunization (NIMI A/Mem/CaIedopia/20/99, НЗМ2 ALLuotipa/3/2003 and

In/Lapazy/10/2003).

Figure 5 - study on mice: intermediate immune response of cells: influenza-specific T-cell responses to CO4 on the 7th day after immunization.

Figure 6 - mouse study: media for CO4 - pool strain (all doubles) - Day 0 and Day 21.

Figure 7 - OMT at 0 and 21 days for NI antibodies.

Figure 8: Incidence of local and general symptoms in humans (total and associated with level 3) reported during the follow-up period of 7 days after immunization with adjuvants against influenza virus, comparison of adjuvants with two different concentrations immunostimulants.

Figure 9: humoral reactions to HRM 16 and 18 11 in mice after immunization with adjuvant formulations of NRU, comparison of adjuvants with two different concentrations of immunostimulators.

Figure 10: Cell-mediated immune response in mice: intracellular staining of cytokine - M P16 and 18 СО4- T cells after immunization with adjuvant formulations of NRU, comparison of adjuvants with two different concentrations of immunostimulators.

Figure 11: production of specific B-lymphocytes after immunization with adjuvant formulations

NRU, comparison of adjuvants with two different concentrations of immunostimulants.

Figure 12: Preclinical comparison of adjuvant vaccines against 5. rpestopiae in mice, comparison of adjuvants with two different concentrations of immunostimulators.

Figure 13: EHIZBA titer of anti-d9B guinea pigs after immunization with adjuvant vaccine sb, comparison of adjuvants with two different concentrations of immunostimulators.

Figure 14: titer of neutralizing anti-CMV antibodies in guinea pigs after immunization with adjuvant vaccine Ob, comparison of adjuvants with two different concentrations of immunostimulators.

Figure 15: EHIZA titer of anti-98 mice after immunization with adjuvanted vaccine 98.

Figure 16: titer of neutralizing anti-CMV antibodies of mice immunized with adjuvant vaccine 98.

Figure 17: mouse study: cell-mediated specific immunity of CO4- and Sov against CMV after re-stimulation with peptide pool 98 (7 days after the second immunization).

Figure 18: studies on mice: cell-mediated specific immunity of CO4 against

CMV after re-stimulation with two different dosages of the 9B peptide pool (21st day after the second immunization).

Figure 19: studies on mice: cell-mediated specific immunity SOvzh against

CMV after re-stimulation with two different dosages of the 9B peptide pool (21st day after the second immunization).

Figure 20: Geometric mean antibody titer (GMT) against C5R periporozoid protein after immunization with adjuvant vaccine KT5.5 in mice, comparison of adjuvants with two different concentrations of immunostimulators.

Figure 21: Geometric mean antibody titer (GMT) against hepatitis B surface antigen (HB) after immunization with KT5.5 adjuvanted vaccine in mice, comparison of adjuvants with two different concentrations of immunostimulants.

Figure 22: expression of I-2 and/or gamma-interferon ex mimo T-cells CO4 and CO8, specific for C39R, after immunization with adjuvant KT5.5 immunogenic composition, comparison of adjuvants with two different concentrations of immunostimulators.

Figure 23: expression of II-2 and/or gamma-interferon ex mimo T-cells CO4 and CO8, specific for HB, after immunization with adjuvant CT5.5 immunogenic composition, comparison of adjuvants with two different concentrations of immunostimulators.

Figure 24: humoral responses in mice after immunization with adjuvant trivalent split influenza vaccine (A/Memz/Caledopia-AL/uotype, B//iapdose), immunostimulants in two different concentrations.

Figure 25: Cell-mediated immune response in mice after immunization with an adjuvanted trivalent influenza vaccine (A/Mem CaEdopia-ALL/uotype, B//iapdzy), immunostimulators in two different concentrations.

Figure 26: preclinical results of studies on mice comparing MAM 9E vaccine adjuvant with

ABO1B or ABO1E.

Figure 27: titer of viruses in nasal washes after the start of administration and carrying out a preventive test with influenza virus antigens - simple or adjuvant immunization with A/Meu/ Saiedopia. - ALuotipo,

V/Liapdzy with the help of adjuvant compositions containing immunostimulants in two different concentrations in ferrets.

Figure 28: Body temperature in ferrets after the beginning of administration and carrying out a prophylactic test with influenza antigens. Immunization with a simple or adjuvanted vaccine A/Mem/Sayiedopia"-Aluuotipod,

V/Liapdzy with the help of adjuvant compositions containing immunostimulants in two different concentrations.

Figure 29: anti-NO titer for A strains of the trivalent vaccine formulation after immunization and provacation test with influenza antigen preparations. Simple or adjuvant immunization

A/Mem/ Saiedopia"-AL//uotipu, V/Lapdzy with the help of adjuvant compositions containing immunostimulants in two different concentrations.

Figure 30: anti-NI titer for the B/diapdza strain and the variable strain used for provacation after immunization and provacation with influenza antigen preparations.

Immunization with a simple or adjuvant vaccine A/Meu/ CaIedopia.-AL/L/uotipdo, B/Liapdzy with the help of adjuvant compositions containing immunostimulants in two different concentrations.

The inventors of the present invention have discovered that an adjuvant composition comprising a saponin presented in liposome form and a lipopolysaccharide, wherein each immunostimulant is present in an amount of mcg or less per human dose, can improve immune responses to an antigenic preparation and at the same time possess lower reactogenicity than some of the previously known formulations, where immunostimulants were present in higher amounts per human dose.

The inventors of the present invention have also discovered that an anti-influenza formulation consisting of an influenza virus or its antigen preparation together with an adjuvant containing a saponin presented in the form of a liposome, and optionally additionally with a derivative of lipid A, for example, C30O-MRI ., could improve the immune response of CO4 T cells against the indicated antigen or antigenic composition in comparison with the formulation obtained with a non-adjuvanted virus or its antigen preparation. Formulations with a saponin adjuvant presented in the form of a liposome are mainly used to stimulate CO4 T-cell responses against influenza, which can detect influenza epitopes represented by MHC class II molecules. In this application, it was found that it is effective to direct the system of cell-mediated immunity in order to increase the reactivity against homologous and variable strains of influenza (compared to vaccination and infection).

It is in a specific embodiment of the present invention that the compositions for use according to the present invention may be able to provide a person with better seroprotection against influenza after repeated vaccination, as determined by the number of cases of correlates of protection against influenza in humans. In addition, in another specific embodiment, the indicated composition for use according to the present invention can also induce a stronger B-lymphocyte response after the first vaccination of a human patient, and a more pronounced humoral response after repeated vaccination in comparison with a non-adjuvanted composition.

Adjuvant compositions against influenza according to the present invention have several advantages: 1) Superior immunogenicity: they will restore the weak immune response in the elderly (over 50 years, usually over 65 years) to the levels observed in young people (antibody and/ or T cell responses); 2) Predominant cross-protection profile: increased cross-protection against variant (variable) influenza strains; 3) They would also allow the use of a reduced dose of antigen for a similar response, thus guaranteeing increased capacity in case of urgent need (e.g., pandemic).

In another aspect of the invention, the inventors have discovered that an adjuvant composition as defined herein exhibits immunogenicity results in terms of both antibody production and influenza-specific post-vaccination CO4 rates that are equivalent to, or sometimes greater than those generated with a non-adjuvanted vaccine. This effect is particularly important for the elderly population and can be achieved with an adjuvant as defined herein and a lower dose of immunostimulants. In addition, a trend was demonstrated where the symptoms of reactogenicity were higher in the group that received the adjuvanted vaccine with the highest concentration of immunostimulants, compared to the group that received the adjuvanted vaccine, where the immunostimulants were contained in a lower concentration.

These data can be applied to other forms of the same antigens, as well as to other antigens.

Saponin adjuvant

The adjuvant composition of this invention contains a saponin adjuvant presented in the form of a liposome.

A particularly suitable saponin for use in the present invention is Option A and its derivatives. Options

A is a saponin preparation extracted from the South American tree Ocia)a bZaropagia Moiipa, it was first described by Oaezdaaga ei ai, in 1974 ("Zaropipa adiimapie", AgsPim. Tig die desatie

Migiviogzepipa, Moi. 44, brgipdeg Mepad, Vepip, p243-254), as having adjuvant activity.

Purified fragments of OC A were extracted by HPLC, retaining adjuvant activity without the toxicity associated with OC A (EP 0 362 278), such as 057 and 0521 (also known as CA7 and

OSA21). 05-21 is a natural saponin from the bark of Ocypa|a zaropagia Moiip, which stimulates ХОв8 cytotoxic T-cells (STI), TA1 cells and a dominant IdgA antibody response, and it is a preferred saponin in the context of this invention.

In the corresponding form of the present invention, the saponin adjuvant within the immunogenic composition is a derivative of Zaropagia toilipidia A, preferably of the immunologically active fraction Oci A, for example,

O517 or 0521, suitable is 02521. In one variant, the compositions of this invention contain an immunologically active saponin fraction, substantially in pure form. Preferably, the compositions of this invention contain 2521 in substantially pure form, ie, 0521 has a purity of at least 90 95, for example, at least 95 95, or at least 98 95.

In a specific embodiment, 0521 is present in a less reactogenic composition where it is quenched by an exogenous sterol, such as cholesterol. There are several specific forms of less reactogenic compositions where 0521 is quenched by exogenous cholesterol. In a specific embodiment, the saponin/sterol is in the form of a liposomal structure (MO 96/33739, Example 1). In this embodiment, the liposomes accordingly contain a neutral lipid, e.g., phosphatidylcholine, which is accordingly non-crystalline at room temperature, e.g., egg yolk phosphatidylcholine, dioleoylphosphatidylcholine (BOC), or dilaurylphosphatidylcholine. Liposomes can also contain a charged lipid, which increases the stability of the liposomal structure 0521 for liposomes consisting of saturated lipids. In these cases, the amount of charged lipid is, respectively, 1-2095 (mass), preferably 5-10 905. The ratio of sterols to phospholipid is 1-50 90 (mol/mol), respectively 20-25 95.

Suitable sterols include p-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. In one particular embodiment, the adjuvant composition comprises cholesterol as a sterol. These sterols are well known in the art, for example cholesterol described in MegskK Ipaeh, 11th edition, p. 341 as a naturally occurring sterol found in animal fat.

Adjuvant compositions of this invention containing 0521 and sterol, especially cholesterol, demonstrate reduced reactogenicity compared to compositions where sterol is absent, while maintaining the adjuvant effect. The study of reactogenicity can be evaluated in accordance with the methods disclosed in UMO 96/33739. According to this invention, sterol means an exogenous sterol, that is, a sterol that is not endogenous to the organism from which the antigen preparation is obtained, but is added to the antigen preparation or later, at the time of preparation of the formulation.

Of course, the indicated sterol can be added during the subsequent manufacture of the formulation of the antigenic preparation with a saponin adjuvant, using, for example, saponin in a form extinguished by sterol. Accordingly, the exogenous sterol is associated with a saponin adjuvant, as described in

MO 96/33739.

If the active saponin fraction is 0521, the ratio of 0521:sterol will typically be present in an amount on the order of 1:100 to 1:1 (w/w), preferably 1:10 to 1:1 (w/w), and preferably 1:5 to 1:1 (wt). Accordingly, additional sterol is present, the O521/sterol ratio is at least 1:2 (w/w). In one embodiment, the O521/sterol ratio is 1:5 (w/w). Sterol, respectively, is cholesterol.

Other suitable saponins are derived from the plants Aezscia5Ppyrrosabsiapitus (horse chestnut) or suorpia5igiPpisht (duck perecotipole). Other saponins that have been reported in the literature include eskin, which is described in MegsKk Ipaech (12th ed.: article 3737) as a mixture of saponins found in the seeds of the horse chestnut, the Latin name of Aeustia5 Pyrrosasiapyth. Its extraction by means of chromatography and purification is described (Riedieg, AggpeitSchei-Rog5sp. 4, 213 (1953)), as well as with the help of ion-exchange resins (Egbhipud eii aI., USA 3,238,190). Fractions of esquin were purified and shown to be biologically active substances (Huo5piKau/a Mei ai. (Speth Riagt Wii (ToKuso) 1996; Aug, 44(8): 1454-1464)). Sapoalbin from surzorpya b5igshpisht (B. Mosichep ei ai., 1968, 9. Rpagt, Ve!d., 42, 213-226) is also described, for example, in relation to the production of IZSOM.

A key aspect of this invention is the fact that the immunologically active saponin, which is preferably 0521, can be used in smaller amounts than previously considered suitable, less than mcg, for example, from 1 to 30 mcg per dose of the immunogenic composition for humans.

Thus, the present invention provides a dose of an immunogenic composition for humans containing an immunologically active saponin, preferably 0521, at a level of 30 μg or less, for example, from 1 to 30 μg.

In one embodiment, the immunogenic composition is in a volume suitable for a human dose, the human dose of the immunogenic composition contains 2521 at a level of about 25 mcg, for example 20-30 mcg, preferably 21 to 29 mcg or 22 to 28 mcg, or 23 to 27 mcg, or 24 to 26 mcg, or 25 mcg.

In another embodiment, the human dose of the immunogenic composition contains 0521 at a level of about 10 μg, for example, 5-15 μg, preferably 6 to 14 μg, for example, 7 to 13 μg or 8 to 12 μg or 9 to 11 μg, or 10 mcg.

In a further embodiment, the human dose of the immunogenic composition contains 2521 at a level of about 5 μg, for example, from 1 to 9 μg, or from 2 to 8 μg, or, respectively, from 3 to 7 μg, or from 4 to 6 μg, or 5

ICG.

A corresponding number of 0521 is, for example, any of 1,2,3,4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 μg (w/v) per dose of the immunogenic composition for humans.

The term "human dose" refers to a dose that is a volume suitable for use in humans. Usually this dose is in the range from 0.3 to 1.5 ml. In one embodiment, the human dose is 0.5 ml. In a further variant, the dose for a person is higher than 0.5 ml, for example, 0.6; 0.7; 0.8; 0.9 or 1 ml. In a further embodiment, the human dose is between 1 ml and 1.5 ml. This invention is characterized by the fact that each human dose contains 30 μg or less, for example, from 1 to 30 μg 20521.

The invention additionally provides an adjuvant composition containing 30 μg or less, for example, from 1 to 30 μg of 2521. Of course, such an adjuvant composition will be contained in an appropriate human dose volume. If an adjuvant in liquid form is to be combined with a liquid form of an antigenic composition, then the adjuvant composition will be in an appropriate human dose volume that is approximately one-half of the intended final human dose volume, e.g., 360 µl of volume for an estimated human dose of 0.7 ml, or 250 µl of volume for an estimated human dose of 0.5 ml. The adjuvant composition is diluted when combined with the antigenic composition in order to obtain the final dose of the vaccine for humans. The final volume of such a dose will usually vary depending on the initial volume of adjuvant composition and the volume of antigenic composition added to the adjuvant composition. Alternatively, the liquid adjuvant is used to restore the composition of the lyophilized antigen. In this variant, the corresponding human dose volume of the adjuvant composition is approximately equal to the final human dose volume. The liquid adjuvant composition is added to the tube containing the lyophilized antigen composition. The final dose for a person can vary from 0.5 to 1.5 ml. In a specific embodiment, the human dose is 0.5 mL, in said embodiment, the vaccine composition of the present invention will contain a level of 0521 30 mcg or less, e.g., 1 to 30 mcg per 0.5 mL human dose, in addition, in this Alternatively, the adjuvant composition of the present invention will contain a level of 0521 30 μg or less, for example, from 1 to 30 μg per 250 μl of the adjuvant composition, or per 500 μl of the adjuvant composition, depending on whether the adjuvant will be combined juvant composition with liquid, or with a composition of lyophilized antigen, respectively.

Specifically, when combining with the influenza antigen, the amount of 0521 can be used, for example, 1-100 μg (wt/vol.) per dose of the composition, preferably 10-50 μg (wt/vol.) per dose of the composition. A corresponding number of 0521 is, for example, any of 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , Ab, 47, 48, 49 or 50 μg (w/v) per dose of the composition. More preferably, the amount of 0521 varies from 25 to 75 μg (w/v) per dose of the composition. Of course, the dosage of the composition will vary from about 0.5 ml to about 1 ml. A typical vaccine dose is 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1 mL. In a preferred embodiment, the final concentration of 2521 50 μg is contained in a milliliter of the vaccine composition, or μg per 0.5 ml dose of the vaccine. In other preferred embodiments, a final concentration of 2521 35.7 μg or 71.4 μg is contained in a milliliter of the vaccine composition. Specifically, a vaccine dose volume of 0.5 ml contains 25 μg or 50 μg of 0521 per dose.

A dose of 0521, accordingly, can enhance the immune response to an antigen in humans. In particular, a suitable amount of 2521 is one that improves the immunological potential of the composition compared to a non-adjuvanted composition, or compared to an adjuvant composition with a different amount of 0521, being accepted according to the reactogenicity profile.

Adjuvant 30O-MRI.

The composition additionally contains an adjuvant, which is a lipopolysaccharide, respectively a non-toxic derivative of lipid A, especially lipid A monophosphoryl or, more preferably, lipid A C-deacylated monophosphoryl (30-MRI).

Z0O-MRI. sold under the name MRI. SiIakhoztiyKiipe Viioodisaiye M.A. and is denoted in this document as MRI. or 30O-MRI.,, see, for example, US patents MoMo 436,727; 4,877,611; 4,866,034 and 4,912,094. First of all, 30O-MR'I. gives reactions of СО4- T cells with IEM-9 phenotype (Tp1). C3O-MRI. can be obtained using the methods disclosed in SV 2 220 211 A. From a chemical point of view, it is a mixture of lipid A 3-deacylated monophosphoryl with 3, 4, 5 or b acylated chains.

Preferably, in the compositions of this invention, a small particle of 30O-MRI is used. A small particle of Z30O-MRI has such a size that it can be sterile filtered through a filter with a hole size of 0.22 μm. Such drugs are described in UMO 94/21292.

A key aspect of this invention is the fact that the lipopolysaccharide, which is preferably 30-MRI., can be used in smaller amounts than previously considered suitable, less than 30 μg, for example, from 1 to 30 μg per dose of an immunogenic composition for humans.

Thus, the present invention provides a dose of an immunogenic composition for humans containing lipopolysaccharide, preferably 30O-MRI, at a level of 30 μg or less, for example, from 1 to 30 μg.

In one embodiment, a dose of the immunogenic composition for humans contains 30O-MRI. at a level of about 25 μg, for example, 20-30 μg, preferably from 21 to 29 μg, or from 22 to 28 μg, or from 23 to 27 μg, or from 24 to 26 μg, or 25 μg. In another variant, a dose of the immunogenic composition for humans contains 30-

MRI. at a level of about 10 μg, for example, from 5 to 15 μg, preferably from 6 to 14 μg, for example, from 7 to 13 μg or from 8 to 12 μg or from 9 to 11 μg or 10 μg.

In a further variant, the human dose of the immunogenic composition contains at a level of approximately 5 μg, for example between 1 and 9 μg, or between 2 and 8 μg, or, respectively, between 3 and 7 μg or 4 and 6 μg, or 5 μg.

With the corresponding amount of Z30O-MRI. is, for example, any 31,2, 3,4,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30 μg (w/v) per dose of the immunogenic composition for humans.

In one embodiment, the dose volume for a person is 0.5 ml. In a further embodiment, the immunogenic composition is in a volume suitable for a human dose, the volume of which is greater than 0.5 ml, for example, 0.6; 0.7; 0.8; 0.9 or 1 ml. In a further embodiment, the human dose is between 1 ml and 1.5 ml. The present invention is characterized by the fact that each human dose contains 30 μg or less, for example, from 1 to 30 μg of 3Z0O-MRI.

The invention additionally provides an adjuvant composition containing 30 μg or less, for example, from 1 to 30 μg of C30O-MRI. Of course, such an adjuvant composition will be contained in the appropriate dose volume for a person. If an adjuvant in liquid form is to be combined with a liquid form of an antigenic composition, then the adjuvant composition will be contained in an appropriate human dose volume that is approximately half of the intended final human dose volume, e.g. 360 μl of volume for an estimated human dose of 0.7 ml, or 250 μl of volume for an estimated human dose of 0.5 ml. The adjuvant composition is diluted when combined with the antigenic composition in order to obtain the final dose of the immunogenic composition for humans. The final volume of such a dose will usually vary depending on the initial volume of adjuvant composition and the volume of antigenic composition added to the adjuvant composition. In an alternative version, the liquid adjuvant composition is used to restore the composition of the lyophilized antigen. In this variant, the corresponding human dose volume of the adjuvant composition is approximately equal to the final human dose volume. The liquid adjuvant composition is added to the tube containing the lyophilized antigen composition. The volume of the final dose for a person can vary from 0.5 to 1.5 ml. In a specific version, the dose volume for a person is 0.5 ml, in this version, the vaccine composition of this invention will contain an amount of 30-

MRI. 30 μg or less, for example, from 1 to 30 μg per human dose of 0.5 ml, in addition, in this embodiment, the adjuvant composition of the present invention will contain an amount of ZO-MRI of 30 μg or less, for example, from 1 to 30 μg per 250 μl of the adjuvant composition, or per 500 μl of the adjuvant composition, depending on whether the adjuvant composition will be combined with a liquid or with a lyophilized antigen composition, respectively.

If the immunogenic composition contains an influenza virus or its antigen preparation, then the adjuvant composition, which consists of a saponin presented in the form of a liposome, and, optionally, additionally with a derivative of lipid A, especially lipid A monophosphoryl or, more preferably, 3O- MRI. In this variant, the amount of ZO-MRI can be used, for example, 1-100 μg (wt/vol.) per dose of the composition, preferably 10-50 μg (wt/vol.) per dose of the composition. With the corresponding amount of 30O-MRI. is, for example, any 3z1,2,3,4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, Ab, 47, 48, 49 or 50 μg (w/v) per dose of the composition. More preferably, the number of 3O-MRI. varies from 25 to 75 μg (w/v) per dose of the composition.

Of course, the dose volume of the composition will vary from about 0.5 ml to about 1 ml. A typical vaccine dose is 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1 mL. In one embodiment, the final concentration of Z0O-MRI is 50 μg contained in a milliliter of the vaccine composition, or 25 μg per 0.5 ml dose of the vaccine. In another variant, the final concentration is 30O-MRI. 35.7 mcg or 71.4 mcg is contained in a milliliter of the vaccine composition. Specifically, a vaccine dose volume of 0.5 ml contains 25 μg or 50 μg of 30O-MRI. per dose

The dose of C30O-MRI, accordingly, can enhance the immune response to an antigen in a person. In particular, with the corresponding number of ZO-MRI. is one that improves the immunological potential of the composition compared to a non-adjuvanted composition, or compared to an adjuvant composition containing a different amount

MRI'., being accepted by the profile of reactogenicity.

Suitable compositions of the present invention are compositions where the liposomes are first prepared without MRI (as described in MO 96/33739) and then MRI is added, preferably in the form of small particles less than 100 nm in size or particles that can be sterile filtered through a filter with with a particle size of 0.22 μm. Thus, MRI. not contained in the vesicle envelope (known as outer MR). Compositions where MR'I. contained within the vesicle envelope (known as internal MRI is also considered an aspect of the present invention. The antigen may be contained within the vesicle envelope or contained outside the vesicle envelope. Preferably, soluble antigens are outside the envelope, while hydrophobic or lipid antigens may be contained inside or outside shells

In one variant, the adjuvant composition of this invention contains both lipopolysaccharide and immunologically active saponin. In a specific embodiment of this invention, lipopolysaccharide is

300-MRI, and immunologically active saponin is 0521. In another variant of this invention, the adjuvant composition consists mainly of lipopolysaccharide and immunologically active saponin in a liposomal formulation. Accordingly, in one form of this variant, the adjuvant composition consists mainly of 30O-MRI. and 0521, it does not necessarily contain a sterol, which is preferably cholesterol.

In another variant of the present invention, the adjuvant composition contains lipopolysaccharide and immunologically active saponin in a liposomal formulation in combination with one or more additional immunostimulants or adjuvants. Accordingly, in one form of this variant, the lipopolysaccharide is 30-MRI, and the immunologically active saponin is 0521.

In a specific embodiment, 20521 and 30-MRI are present in the same final concentration per dose of the human immunogenic composition. In one aspect of this embodiment, a human dose of the immunogenic composition contains a final level of 25 μg of ZO-MRI. and 25 μg of 0521. In another variant, a dose of an immunogenic composition for humans contains a final amount of 10 μg of both MRI, and 0521. In another specific variant, an adjuvant composition is proposed, which has a volume of 250 μl and contains an amount of 25 μg of ZO- MRI. and 25 mcg of 0521, or 10 mcg of each: MRGH. and 0521.

Antigens that can be used with the adjuvant compositions of the present invention include viral, parasitic, bacterial, or tumor-associated antigens, for example, an influenza virus or an antigenic preparation thereof for use in accordance with the present invention, which may be a cleaved influenza virus or an antigenic preparation of a cleaved virus Alternatively, the influenza preparation may contain a different type of influenza antigen, such as an inactivated whole virus or purified HA and MA (subunit vaccine), or influenza virus. In another embodiment, the influenza virus may be a live attenuated influenza vaccine.

The split influenza virus or the antigenic preparation of the split virus for use according to this invention is preferably a preparation of an inactivated virus, where the virus particles are destroyed by detergents or other reagents in order to solubilize the lipid membrane.

Cleaved virus or antigenic preparations of cleaved virus are prepared, respectively, by fragmentation of intact influenza virus, both infectious and inactivated, with solubilizing concentrations of organic solvents or detergents and subsequent removal of all or most of the solubilizing agent and some or most of the viral lipid material.

By a cleaved antigenic preparation of the virus is meant a cleaved preparation of the virus that can be purified to some extent compared to the cleaved virus while retaining most of the antigenic properties of the components of the cleaved virus. For example, when produced in an egg, the cleaved virus may be purified from contaminating egg proteins, or when produced in cell culture, the cleaved virus may be purified from host cell impurities. An antigenic preparation of a split virus may contain split antigenic components of the virus of more than one virus strain. Vaccines containing split virus (called "split influenza vaccine") or antigen preparations of split virus usually contain residual matrix proteins and nucleoproteins, and sometimes lipids, as well as membrane proteins. Such split virus vaccines will usually contain most or all of the structural proteins of the virus, although not necessarily in the same proportions in which they are found in the intact virus.

Alternatively, the influenza virus may be in the form of a whole virus vaccine. This fact may be an advantage over a split virus vaccine for a pandemic situation, as it avoids the uncertainty of whether a split virus vaccine can be successfully produced for a new strain of influenza virus. For some strains, the common detergents used to obtain split virus can damage the virus and render it unusable. Although there is always the possibility of using different detergents and/or developing a different method to produce a split vaccine, this will require some time, which may not be available in a pandemic situation. In addition, for a greater degree of confidence in the whole virus approach, there is also a greater vaccine production capacity than for split virus, as significant amounts of antigen are lost during the additional purification steps required to produce a suitable split vaccine.

In another embodiment, the influenza virus preparation is in the form of a purified subunit influenza vaccine. Subunit influenza vaccines usually contain two major envelope proteins, HA and MA, and may have an additional advantage over whole-virion vaccines in that they are generally less reactogenic, especially in people vaccinated at a young age. Subunit vaccines can be prepared either recombinantly or by purification from fragments of the destroyed virus.

In another embodiment, the drug against the influenza virus is in the form of a virosome. Virosomes are spherical, unilamellar vesicles that store functional viral envelope glycoproteins

NA and MA in the authentic conformation inserted into the bilayer membrane of virosome phospholipids.

The indicated influenza virus or its antigen preparation can be obtained from eggs or from cell culture.

For example, the antigen of the influenza virus or its antigen preparation according to the present invention can be obtained using a conventional method on the eggs of chicken embryos, growing the influenza virus in eggs and purifying the resulting allantoic fluid. Viruses in eggs can accumulate in large quantities for a short time. Alternatively, they can be obtained by any of the methods of the new generation, using a cell or a cell culture for the purpose of growing the virus or the surface antigens of the expressed recombinant influenza virus. Suitable cell substrates for growing the virus include, for example, canine kidney cells such as MSCs or clone cells

MOSC, MOSC-like cells, monkey kidney cells such as ASMK cells, including Mego cells, suitable porcine cell lines, or any other mammalian cell type suitable for producing influenza virus for vaccine purposes. Suitable cell substrates also include human cells, for example, MKSO-5 cells. Suitable cell substrates are not limited to cell lines; for example, primary cells such as chicken embryo fibroblasts and avian cell lines are also used for vaccine production.

The antigen of the influenza virus or its antigenic preparation can be obtained by any of a number of commercially used methods, for example, the method of split influenza virus described in the patent Mo 0O 300 833 and BO 211 444, incorporated herein by reference. Traditionally, cleaved influenza virus has been prepared using a solvent/detergent treatment such as tri-n-butyl phosphate or diethyl ether in combination with Tween M (known as Tween ether digestion), and this method is still used in some production facilities Other agents currently in use for cleavage include detergents or proteolytic enzymes or bile salts, such as sodium deoxycholate, as described in Me BO 155,875, incorporated herein by reference. Detergents that can be used as cleavage agents , include cationic detergents such as cetyltrimethylammonium bromide (TAB), other ionic detergents such as lauryl sulfate, taurodeoxycholate, or nonionic detergents such as those described above, including

Triton X-100 (for example, in the method described by Opa et al., 2000, Bioiodisai5 28, 95-103) and Triton M-101, or a combination of any two or more detergents.

The method of obtaining a split vaccine may include a number of different filtration and/or other separation steps, for example ultracentrifugation, ultrafiltration, zonal centrifugation and chromatography (eg, ion exchange) in various combinations, and optionally, an inactivation step, for example with heat, formaldehyde or p-propiolactone or UV, which can be performed before or after cleavage. The cleavage method can be carried out as a batch, continuous or semi-continuous process. A preferred method of cleavage and purification for a cleaved immunogenic composition is described in UMO 02/097072.

Preferred antigen preparations of the split influenza vaccine of the present invention contain a residual amount of Tween 80 and/or Triton X-100 remaining from the production process, although they may be added or their concentrations adjusted after the production of the split antigen. The advantage is the presence of both Twin 80 and Triton X-100. Preferred intervals for the final concentrations of these nonionic surfactants in the vaccine dose are the following: Tween 80 0.01-1 95, more preferably, approximately 0.1 95 (w/v), Triton X-100-0.001-0, 1 95 (w/v), more preferably, 0.005-0.02 95 (w/v).

In a specific embodiment, the final concentration for Tween 80 is in the range of 0.025 95 to 0.09 95 (w/v). In another specific embodiment, the antigen is administered as a 2-order concentrated mixture that has a Tween 80 concentration in the range of 0.025 95 to 0.295 (w/v) and which must be diluted two-fold to obtain the final adjuvant formulation (or add buffer in the control recipe).

In another specific embodiment, the final concentration of Triton X-100 is in the range of 0.004 95 to 0.017 95 (w/v). In another specific embodiment, the antigen is administered as a 2-fold concentrated mixture containing a concentration of Triton X-100 ranging from 0.005 95 to 0.034 95 (w/v) and which must be diluted two-fold to obtain the final adjuvant formulation ( or add buffer to the control formulation).

Preferably, the anti-influenza drug is made in the presence of a low level of thimerosal, or preferably in the absence of thiomersal. Preferably, the resulting anti-influenza drug is stable in the absence of organomercury preservatives, in particular, this drug does not contain residual thiomersal. In particular, the influenza virus preparation contains a hemagglutinin antigen stabilized in the absence of thimerosal, or it is at low levels of thimerosal (usually mcg/µml or less). Specifically, the stabilization of the influenza B strain is carried out with the help of an alpha-tocopherol derivative, for example, alpha-tocopherol succinate (also known as vitamin E succinate, i.e. ME5). Such preparations and methods of their preparation are disclosed in UMO 02/097072.

A preferred formulation contains three inactivated split virion antigens prepared from seasonal influenza strains recommended by the World Health Organization.

Preferably, the influenza virus or its antigen preparation and the adjuvant according to the present invention are contained in the same container. This approach is called the "one tube approach". Preferably, the test tube is a pre-filled syringe. Alternatively, the influenza virus or its antigenic preparation and adjuvant according to the present invention are contained in separate containers or tubes, they are mixed immediately before use or when the subject is administered. This approach is called the "two-tube approach". For example, if the vaccine is a two-component vaccine for a total dose volume of 0.7 ml, then the concentrated antigens (eg, concentrated trivalent inactivated split virion antigens) are presented in a single tube (335 μl) (antigen container) and a prefilled syringe , contains adjuvant (360 μl) (adjuvant container). During the injection, the contents of the tube containing the concentrated trivalent inactivated antigens of the cleaved virion are withdrawn from the tube using a syringe containing the adjuvant after gently shaking the syringe. Before the injection, the used needle is replaced with a needle for intramuscular injection, and the volume is brought up to 530 μl. In this example, one dose of the candidate reconstructed adjuvanted influenza vaccine corresponds to 530 μl.

In one aspect of the invention, where there is a multivalent composition, at least one strain of influenza in said multivalent immunogenic composition, as defined herein, is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak. Such a strain may also be referred to as a "pandemic strain" in the text below. In particular, when the vaccine is a multivalent vaccine, such as a bivalent, or trivalent or quadrivalent vaccine, then at least one strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak. Suitable strains include, but are not limited to, NMI, NOM2, H7M7, and namMe.

The specified influenza virus or its antigen preparation is preferably multivalent, for example bivalent or trivalent or tetravalent. Preferably, the influenza virus or its antigen preparation is trivalent or tetravalent and contains antigens of three different strains of influenza, with at least one strain associated with a pandemic outbreak or having the potential to be associated with a pandemic outbreak.

Features of the influenza virus strain that give it the potential to cause a pandemic outbreak are as follows: it contains a new hemagglutinin compared to the hemagglutinin of existing strains; it can be transmitted horizontally in the human population; and it IS pathogenic for humans.

A new hemagglutinin may be one that has not been detected in the human population for a significant period of time, probably several decades, such as H2. Or it could be a hemagglutinin that did not previously exist in the human population, such as H5, HO, H7 or Hb found in birds. In any case, the majority, or at least a large part, or even the entire population, has not encountered the antigen before and is immunologically unprepared for it.

Certain groups of people are usually at increased risk of becoming infected with influenza in the event of a pandemic. The elderly, patients with chronic diseases, and young children are particularly vulnerable, but many young and apparently healthy people are also at risk. In the case of H2 influenza, a portion of the population born after 1968 is at increased risk of influenza infection. For these groups, it is important to be effectively protected, as soon as possible and in a simple way.

Another group of people who are in the zone of increased risk are travelers. Today, people travel more than ever before, and the regions where most new viruses appear are China and

Southeast Asia has become a popular travel destination in recent years. This change in travel routes allows new viruses to spread around the world in weeks instead of months or years.

Thus, for these groups of people, there is a specific need for vaccination to protect against influenza in a pandemic or potentially pandemic situation. Suitable strains for this purpose include, but are not limited to: Н5МИ1, НОМ2, Н7М7 and Н2М2.

Optionally, the composition may contain more than three valencies, for example, three non-pandemic strains plus a pandemic strain. Alternatively, the composition may contain three pandemic strains.

Mostly, the composition contains three pandemic strains.

Other examples of antigens for the immunogenic composition of this invention are streptococcal antigens, such as antigens of group A streptococcus or group B streptococcus, but most preferably, Zygeriosossi5 rpeshtopiae. Most preferably, at least one protein and/or at least one saccharide antigen is used. At least one protein antigen(s) of bigeriosossi5 rpeshtopiae is most preferably selected from the group consisting of: pneumolysin, ReparA or its transmembrane deletion variants, RersC or its transmembrane deletion variants, PzaA or its transmembrane deletion variants, glyceraldehyde-3-phosphate dehydrogenase, СbBrA or its transmembrane deletion variants, RIMA, RO, RIV, RICHE, ZrzaA, I MIV, GG ms, GUMA, 5p125, 5p101, 5p128, 5p130 and Zp133, or its immunologically functional equivalent (e.g., fusion domains of the above-mentioned proteins, e.g. fusion proteins RISHUE, described in M/001/98334 and UMO 03/54007).

Certain compositions are described in UMO 00/56359, UMO 02/22167 and UMO 02/22168 (incorporated herein by reference).

The antigen may contain capsular saccharide antigens (preferably conjugated with a carrier protein), where saccharides (most preferably polysaccharide) are obtained from at least four pneumococcal serotypes. In one embodiment, the four serotypes include 568, 14, 19E and 23PE. In a further variant, the composition includes at least 7 serotypes, for example, serotypes derived from serotypes 4, 68, 9M,

14, 18C, 19, and 23B. In a further embodiment, the composition includes at least 10 serotypes, for example, in one embodiment, the composition contains a capsular saccharide derived from serotypes 1, 4, 5, 68, 7E, 9M, 14, 18C, 19E and 23E (predominantly conjugated with carrier protein). In another embodiment, the immunogenic composition contains a capsular saccharide derived from serotypes 1, 3, 4, 5, 6B, 7E, 9M, 14, 18C, 192 and 23E. In a preferred embodiment of the present invention, at least 13 saccharide antigens are included (mostly, 'jugated with a carrier protein), although further saccharide antigens, for example, are 23-valent (such as serotypes 1, 2, 3, 4, 5, 68, 7P, 8, 9M, 9M, 10A, 114A, 12E, 14 , B, 17, 18C, 19A, 19E, 20, 22, 23E and ZZE) are also considered in the invention.

Although the above-mentioned saccharides are preferred in the longer, natural form of the polysaccharide, it should be understood that polysaccharides reduced in size can also be used because they retain immunogenicity (see, for example, EP 497524 and 497525), if there is such a need in connection with binding with a carrier protein.

In order to prevent/reduce the intensity of pneumonia in the elderly population (155 years) and otitis media in infants (under 18 months of age) and children (usually 18 months to 5 years of age), a preferred embodiment of the present invention will be the combination of the polyvalent saccharide Zigeriosossi5 rpeshitopia as described in this description, with a Zigeriosossi5 rpeshtopia protein preferably selected from the group of proteins listed above. A combination of pneumococcal proteins can also be suitably used as described below.

Pneumococcal proteins

Antigens of Zigeriosossi5 rpeshtopia are preferably selected from the group consisting of: a protein of the family of polyhistidine triads (RYX), a protein of the family of Lith (Gu), choline-binding protein, proteins containing motif I of РХТО (where X is any amino acid ), proteins containing a signal sequence

Type HER YXXC (where X is any amino acid) and proteins containing a Type I signal sequence motif. Preferred examples within the indicated categories (or motifs) are the following proteins (abbreviations or immunologically functional equivalents thereof):

The RII (polyhistidine triad) family includes RA, RIV, RO and RNE proteins. The family is characterized by a lipidation sequence, two domains separated by a proline-rich region, and several histidine triads possibly involved in metal or nucleoside binding or in enzymatic activity, (3-5) coiled-coil regions, a conserved M-terminus, and a heterogeneous With the end It is present in all tested strains of pneumococci. Homologous proteins are also found in other streptococci and Meiszegia. Predominant members of this family include RA, RYIV and

RIO. More preferably, it consists of RA or RP. However, it is understood that the terms PE, B, and E refer to proteins containing the sequences disclosed in the citations below, as well as naturally occurring variants (and artificial variants) thereof that exhibit sequence homology identical to the referenced proteins. at least 90 95. Preferably they are at least 95 95 identical, most preferably 97 95 identical.

Regarding RPY proteins, RIPA is disclosed in UUO 98/18930, and the same applies to 5p36. As defined above, it is a protein from the family of polyhistidine triads and contains a signal motif of the HER YHCS type.

RTU is disclosed in MO 00/37105, and it is also designated as 5roz360О0. As defined above, it is also a protein from the family of polyhistidine triads and contains a signal motif type I ХХХС. RIV is disclosed in UMO 00/37105, and it is also designated as 5rozZ6B. Another member of the RPIV family is a NW-degrading polypeptide, as disclosed in UMO 00/17370. This protein also belongs to the family of polyhistidine triads and contains a signal motif of the type IP ХХС. The preferred immunologically functional equivalent is protein 5p42, disclosed in UMO 98/18930. The RYIV fragment (approximately 79 kDa) was discovered in MUO99/15675, which is also considered a member of the RPYH family. RIMCHE is disclosed in UUO00/30299 and is designated as ВМН-3. zrp5A is a choline-binding protein (CBP), disclosed in UMO 98/39450.

The I ui family is a membrane-associated protein associated with cell lysis. The M-Terminal domain includes a choline-binding domain(s), however, the GG ui family does not exhibit all of the features found in the choline-binding protein (CbP) family, as noted below, and is thus considered for purposes of of this invention, the I ui family differs from the Sbrr family. Unlike the Cbr family, the C-terminal domain contains the catalytic domain of the Gui protein family. The family consists of I UA, B and C.

Regarding the Gui family, GUSTA is disclosed in Copaa ei aiYi., Eig U Viospet, 164:621-624 (1987). GUuiV is disclosed in M/O 98/18930, and it is also designated as 5p46. GuiS is also disclosed in M/O 98/18930 and is also designated as 5reo1. The predominant member of this family is I MS.

Another preferred option is a truncation of the Gu family (especially GUCA), where "Gu" as defined above and "truncation" mean proteins that lack 50 95 or more of the choline-binding site. Preferably, such proteins completely lack a choline-binding site. 5p125 is an example of a pneumococcal surface protein with the cell wall anchoring motif I РХТО (where X is any amino acid). Any protein of a given class of pneumococcal surface protein with a given motif is found to be suitable within the context of the present invention, and thus is considered another protein of the present invention. Actually, 5p125 is directly disclosed in the Ministry of Defense

98/18930, it is also known as 2trV - zinc metalloproteinase. 5p101 disclosed in UMO 98/067 34 (where it is indicated by reference 2 u85993). It is characterized by a signal sequence of type I. zr1iZ3 is disclosed in MO 98/06734 (where it is designated by the reference Zh u85992). It is also characterized by a type I signal sequence. 5p128 and 5p130 are disclosed in MO 00/76540.

The proteins used in the present invention are preferably selected from the group of RP, RA, and RICHE or a combination of 2 or all three of these proteins (ie, РАНО, АЖЕ, О-Е or АО-Е).

Other pneumococcal protein antigens that may be included are one or more antigens from the group consisting of: pneumolysin (also referred to as Riu; preferably neutralized by chemical treatment or mutation) (IMO 96/05859, UMO 90/06951, MO 99/038841,

PzaA and its transmembrane deletion variants (Veggu 4 Rayop, Iptesi Ittip 1996 30 Res; 64(12):5255- 62), P5rA and its transmembrane deletion variants (USA 5804193, UMO 92/14488, UMO 99/53940), Rohrs and its transmembrane deletion variants (MO 97/09994, UMUO 99/53940), a member of the choline-binding protein (Cpp) family (for example, CppA and its transmembrane deletion variants (M/O 97/41151; UMO 99/51266)) , glyceraldehyde-3-phosphate dehydrogenases (Ipiesi. Ettyp. 1996 64:3544), H5P70 (UMO 96/40928), RerA (Zapspe;-Veayu eyi am. REM5 MisgorioY Gay 1998, 164:207-14), M similar proteins (Patent application 5B, MO EP 0837130) and adhesin 18627 (Patent application 5B, MO EP 0834568).

The present invention also includes immunologically functional equivalents or truncations of such proteins (as defined above). Regarding the choline-binding protein family, members of this family were originally identified as pneumococcal proteins that could be purified by choline affinity chromatography. All choline-binding proteins are non-covalently bound to phosphorylcholine fragments of cell wall teichoic acid and membrane-associated lipoteichoic acid. From a structural point of view, they contain several common areas throughout the family, although the true nature of the proteins (amino acid sequence, length, etc.) can vary. In general, choline-binding proteins consist of an M-terminal region (M), conserved repeat regions (K1 and/or K2), a proline-rich region (P), and a conserved choline-binding region (C), which consists of multiple repeats that make up about one half of the protein. In this application, the term "choline-binding protein (CbP) family" means selected from the group consisting of choline-binding proteins, as identified in M/O 97/41151, PHOCA, ZrzA, PorC, CpRA, CppO and S.r.o. SrpA was disclosed in UMO 97/41151. СрО and СБро opened in UMO 00/29434. RerS is disclosed in UMO 97/09994.

РЬса opened in UMO 98/21337. Preferably, choline-binding proteins are selected from the group consisting of

SrrA, RBSA, 5r5A and Roros.

If Sbr is another protein used, it may be a truncation of Sbr, with "Sbr" being defined above, and "truncations" include proteins that lack 50 95 or more of the choline-binding site (C). Mostly, such proteins lack a complete choline-binding site. More preferably, such truncated proteins lack (Her) the choline-binding region and (Her) also half of the M-terminal part of the protein, but at least one repetitive region remains (K1 or

K2). Even more preferably, the truncation contains 2 repeat regions (K1 and K2). Examples of such preferred variants are MEixHK2, KIhK2, MKI1xHK2R and KIhKKagR, as illustrated in

UMMO99/51266 or UMMO99/51188, however, other choline-binding proteins that lack such a repeat region are also contemplated in the context of this invention. In the composition according to the present invention, Sbr Shortenings and chimeric proteins (or fusions) can also be used. It mainly consists of

МАХК2 (or КхХН2, or МХХХКагR, or КхХК2Р) Cp and C-terminal parts (Siept, that is, those that do not contain choline-binding domains) GG ui (for example, GuiSsStegt or 5re1 Siept). More preferably, Svrp is selected from the group consisting of SppA, ROSA, 5p5A and RepC. Even more preferably, it is CppA. Preferably, GI ui is | MS (it is also designated as 5ro1).

ReparA or PsaA truncations that lack the choline-binding domain (C) and are expressed as a protein fused to Gui can also be used. Mostly I ui is I us.

It is possible to combine different pneumococcal proteins according to the present invention in the pneumococcal composition.

A preferred combination of proteins according to the present invention is selected from 2 or more (3 or 4) different categories, for example, proteins containing a signal sequence motif of type II and CHS (where X is any amino acid, for example, the family of polyhistidine triads (RP) ...

X is any amino acid, for example, 5p128, Zr130), toxins (for example, RIU), etc.

Preferred examples within the specified categories (or motifs) are the proteins mentioned above or their immunologically functional equivalents. Toxin y-RIY, toxin y-Sbr, RIP-Sr and toxin y-RI-Sr represent the preferred combinations of the category.

Preferred suitable combinations include, but are not limited to, RP --MKIxHK2, ROZMAH K2- 5re1 Siept chimeric or fusion proteins, RIOJRIU, RIPOzhZZrI128, RIIIU»RzZach, RIYUR»5rA, RIPAZHMA HK,

RTIA-MAikhHA2-5reOi Stegt chimeric or fusion proteins, RITAZHRIU, RIAzhZzr128, RIMAzhRzaA, RA RegrA,

MAThA2n y, MAikhA2aARzRA, MAikhHA2aRzaA, MAikhA2--5ri28, ВХхВ2nН у, /ВХА2aRzrA,

KIhK2zhRzad. Predominantly MKIxHK2 (or KI1xK2) originates from CppA or RersC. More preferably, it comes from SrpA.

A particularly preferred combination of pneumococcal proteins includes Riu (or truncation, or its immunologically functional equivalent) z-PO (or truncation, or its immunologically functional equivalent) optionally with MKIxHK2 (or KIxHK2, or MKIxHK2R, or KihkKaR).

Preferably, MKIxHK2 (or KI1xHK2, or MAKihXK2P, or KIxHK2P) originates from CBrA or P5rs. It mostly comes from Sat.

The antigen can be a pneumococcal saccharide conjugate consisting of polysaccharide antigens obtained from at least four serotypes, preferably at least seven serotypes, more preferably at least ten serotypes, and at least one, but preferably 2, 3, or 4 , proteins zigeriosossi5 rpeshtopiae, preferably selected from the group of proteins described above. Preferably, one of the proteins is RP (or its immunologically functional equivalent) and/or Riu (or its immunologically functional equivalent).

The problem with the polysaccharide approach to vaccination is that reg 5e polysaccharides are weak immunogens. In order to overcome it, saccharides can be conjugated with carrier proteins that provide T-cell help. Thus, preferably, the saccharides used in the present invention bind to a similar carrier protein. Examples of such carriers that are currently commonly used for the preparation of saccharide immunogens include diphtheria and tetanus toxoids (OT, OT SKM197, and TT, respectively), Keupoie I ytrei hemocyanin (CIN), OMRS from M. teppoiyi5, and a purified tuberculin protein derivative ( RPO).

The preferred carrier for pneumococcal saccharide, based on immunogenic compositions (or vaccines), is the protein Yu Naeptorpyi5 ipitseplaee (ER 59461 O-B), or its fragments.

Fragments suitable for use include fragments that include T-helper epitopes.

In particular, the Y protein fragment will preferably contain the M-terminal 1/3 of the protein. Carrier protein O is suitable as a carrier in compositions where multiple pneumococcal saccharide antigens are conjugated. One or more pneumococcal saccharides in combination may be suitably conjugated to protein 0.

The next preferred carrier for the pneumococcal saccharide is directly the pneumococcal protein (as defined above in the section "Pneumococcal proteins according to the present invention").

The saccharide can be linked to the carrier protein in any known manner (for example,

U.S. Patent 4,372,945 and Aptogei et al., U.S. Patent 4,474,757). Conjugation is mainly carried out

SOAR (M/O 95/08348).

Preferably, the ratio of saccharide to protein conjugates (w/w) is from 0.3:1 to 1:1, more preferably from 0.6:1 to 0.8:1, and even more preferably from about 0 ,7:1.

Particularly preferred compositions according to the present invention contain one or more conjugated pneumococcal saccharide and one or more pneumococcal protein according to the present invention. In addition, pneumococcal saccharides and proteins can be stably stored as solid components adsorbed on aluminum phosphate in liquid form.

In another aspect of this invention, the vaccine composition may contain human cytomegalovirus (HCMV) antigen. HPV is a human DNA virus that belongs to the herpesvirus family and is a leading cause of birth defects in infants and causes serious medical problems in patients at risk for immune system disorders. Clinical disease causes a variety of symptoms, including dysentery, hepatitis, pneumonitis, and infectious mononucleosis.

In one variant, the NSMM antigen is a fusion protein or its immunogenic derivative consisting of a part of the NSMU glycoprotein fused with a part of the NBM glycoprotein. Glycoprotein HCMM is usually an OB, and glycoprotein NOM is usually a dO, in particular, NOM type 2 90 (902). The fusion usually occurs between the amino acid of the M-terminal part of the protein 908 NSMM and the amino acid of the C-terminal part of the protein 900 NBU. Both the NSMU OV protein and the 9800 NZM protein components of the fusion protein may lack the membrane anchor domain.

Part of the protein 968 HCMM may contain the non-cleavable form of 08 NOMU. This can be achieved by changing one or more amino acids in the cleavage site of the protein, for example, replacing Agd458 and

Agd459 on Sim and Tyig, respectively. Part of the NZM protein may contain the signal sequence 902 (amino acids 1-25) and optionally amino acids 26-52 of dr2 and/or the sequence with 902, which is REOSAG EOREO or its functional equivalents, which may be shorter or longer.

Further sequences with 90 NOM can be added to the fusion protein, for example, in the C-terminus of the ZB protein

TO HIM

In one variant, the fusion protein contains amino acids 1-52 of protein 902 NBU, connected to residues 28-685 of protein 98 NSMU. Such a fusion protein is designated 98685" NSMU. Alternatively, the amino acid sequence REOSAGI EOREO, which is derived from the internal sequence 902, can be incorporated into the C-terminal end of protein 986857 to form a protein designated 9868577

TO HIM These specific fusion proteins are described in more detail in UMO 95/31555.

Another immunogen suitable for use as a HMV vaccine is rrb5, a matrix protein of HMV as described in UMO 94/00150 (Syu oi Nore).

In another variant of this invention, the immunogenic compositions contain an antigen or an antigen preparation,

derived from the human papillomavirus (HPV), which is believed to be responsible for genital warts (HPVb or HRMTI1 and others), and/or the HPV viruses responsible for cervical cancer (HPM16, HRM18 and others).

In one embodiment, the forms of prophylactic or therapeutic compositions against genital warts contain particles 1 or capsomeres and fusion proteins consisting of one or more antigens selected from proteins E1, E2, E5 Eb, E7, 11 and 12 of the human papillomavirus.

In one variant, the forms of the fusion protein are: I 2E7, as disclosed in UMO 96/26277, and the protein

B(1/3)-E7, disclosed in B 9717953.5 (PCT/ЕРО8/05285).

The predominant infection of the cervix is cervical cancer or cervical cancer, the prophylactic or therapeutic composition may contain antigens of cervical cancer 16 or 18. For example, monomers of antigen 11 or 12, or antigens I 1 or

Y2, presented together as virus-like particles (MI P), or one protein 1/1, presented in the structure of MIR or capsomer. Such antigens as virus particles and capsomeres are known as reg 5e.

See, for example, УМО94/00152, МУО94/20137, УМО94/05792, and МОО93/02184.

Additional early proteins can be included individually or as fusion proteins, such as E7, E2 or, preferably, E5, for example; variants that include MI R consisting of 1 E7 fusion proteins (MO 96/11272) are particularly preferred.

In one variant, the antigens of HRU 16 contain early proteins EE or E7, fused with protein-carrier O for the purpose of forming proteins O - fusion of Eb or E7 with HRU 16, or its combination; or combinations of ЕЕ or Е7 with I 2 (ММО 96/26277).

Alternatively, the early Eb and E7 HRM 16 or 18 proteins can be present in a single molecule, preferably in the O-Eb/E7 protein fusion. Such a composition may optionally contain both proteins Eb and proteins E7, or both of HRU 18, preferably in the form of protein O - Eb or protein 0 - E7 of a fusion protein or protein OO Eb/E7 of a fusion protein.

The composition of this invention may additionally contain antigens from other types of NRU, preferably from

NRM 31 or 33.

Oncogenic types of NRU include HRM 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. Thus, the composition of the present invention may contain antigens from one or more of the indicated types of NRU, except

NRM 16 and/or NRU 18.

HRM 11 MI R or capsomeres suitable according to the present invention may contain or consist of 11 full length or immunogenic fragment I 1. If the MI R or capsomer contains or consists of immunogenic fragment 111, then the corresponding immunogenic fragments of HRU 1/1 contain truncations, deletions, substitutions or insertion mutants 1/1. Such immunogenic fragments can accordingly enhance the immune reaction, the specified immune reaction is able to recognize protein 11, for example, virus-like particles from the NRU type, from which protein I 1 was obtained.

Corresponding immunogenic fragments 1 contain truncated proteins 1. In one aspect, the truncation removes the nuclear localization signal. In another aspect, the truncation is a truncation of the C-terminus. In another aspect of the C-terminal truncation, less than 50 amino acids are removed, such as less than 40 amino acids. If 1 originates from HRM16, then in another aspect of C-terminal shortening, 34 amino acids are removed from HRM16 11. If 11 originates from HRM18, then in a further aspect of C-terminal shortening, 35 amino acids are removed from HRM18 AND 1.

The corresponding truncated sequences 1 for HRM16 and 18 are given in MO 06/114312. Sequence

NRMI16 can also be the one disclosed in UMO9405792 or Sh56649167, for example, it is shortened accordingly. Appropriate truncations were made at a position equivalent to that described above, as judged by sequence comparison.

An alternative sequence of HRM18 is disclosed in M/O9629413, which may be shortened accordingly. Appropriate truncations were made at a position equivalent to that described above, as judged by sequence comparison.

Other sequences of HRM16 and HRU18 are well known in the art and may be suitable for use in the present invention.

If protein 1 originates from another type of HRM, C-terminal truncations similar to those made for HRM16 and HRM18 can be used, based on DNA or protein sequence alignment. The corresponding truncated proteins Y1 NRMUZ1 and 45 are given in MO 06/114312.

Appropriate truncations, e.g., HRM 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, can also be made, in one aspect by removing the equivalent C-terminal portions of protein I 1 from those , which are described above, which is estimated by comparing the sequences.

Protein I 1 or an immunogenic fragment according to the present invention may optionally have the form of a fusion protein, for example, a fusion protein of I 1 with I 2 or an early protein.

NRU protein 1, respectively, has the form of a capsomer or a virus-like particle (MI R). In one aspect, virus-like particles, HRMs can be used in the present invention. Virus-like particles, NRMs, and methods of producing MI R are well known in the art. MI R is usually constructed from

It is not necessarily from structural proteins 2 of this virus, see example M/O9420137, 055985610,

МО9611272, 0О5659950881, О5бЗ36177881, ЕР 595935. Any suitable MI R NRU that offers cross-protection such as! 1 or I 1-41 2 MI R.

Accordingly, the virus-like particle is only I 1-MI R.

The composition according to the present invention may contain a combination of НРМ1Е6 11 MI R and НРМ18 11 MI R, or a combination of НРМ 1 MI R with НРМИ16, 18, 31 and 45, or larger combinations, and consists of НРМУ16 and 18 or

NRMI6, 18, 31 and 45 1 1 MI R, or large combinations, and I1 is optionally shortened as described in this description.

In a specific embodiment of the present invention, one or more additional antigens of the cancer-causing types of NRUs are used with antigens HRM16 and/or 18, and the antigens are selected from the following types of NRUs: NRUs 31, 45, 33, 58, and 52. As described herein , the antigen may in each case be 111, for example, in the form of MIR or capsomeres 11. Thus, HRM antigens for use in the given compositions, methods and applications described in this description may contain or consist of 1 MI R or capsomeres with HRM16, 18, 31, 45, 33, 58 and 52. 11 MIR can be only I 1-MI R or in combination with another antigen, for example, 312 in I 1-4 2 MI R. Protein 1 can be shortened accordingly , as described in this description.

MIR formation can be assessed using standard techniques such as electron microscopy and dynamic laser light scattering.

A virus-like particle may consist of full-length protein I1. In one aspect, protein 11, which is used to generate MI P, is a truncated protein 11, as described above.

Virus-like particles can be produced in any suitable cell substrate, such as yeast cells or insect cells, for example in a bacillus virus expression system, while methods for producing MIRs are well known in the art, for example from references M/09913056, US 641694581, 626176581 and 56245568 of this specification, the entire contents of which are incorporated by reference.

A virus-like particle can be obtained using disassembly-reassembly methods, which can give more stable and/or homogeneous virus-like particles of the papillomavirus. For example, MoSagpypu ei ai), 1998 "Otsapiiyaime Oizazzetriu apa Keazzetbriu oi Nytap

Raryotamigikh Ture 11 Migiv Ke Rapyisiev ip Myto" U. Migoiodu 72(1):33-41, describes the disassembly and reassembly of recombinant virus-like particles 11 HRM11, purified from insect cells, in order to obtain a homogeneous preparation MR. In UUMO9913056 and 0О56245568 methods of disassembly-reassembly for the production of virus-like particles of NRU are also described.

In one aspect, the NRU MI R is obtained as described in UUMO9913056 or 56245568.

The compositions of the present invention may contain antigens or antigenic preparations derived from parasites that cause malaria, such as Riaztodisht Taisiragit or Riaztodisht mimah.

For example, possible antigens originating from Riazitodisht Taisiragit contain nacocoloporozoid protein (C5 protein), ET515 M5RI, M5RZ, I ZA, I ZAZ, AMA! and TEAR. KT5 is a hybrid protein consisting essentially of the entire C-terminal part of the nacocoloporozoid protein (C5) P.

Taisiragit, linked through four amino acids of the rg52 part of the hepatitis B surface antigen to the surface (5) antigen of the hepatitis B virus. Its complete structure is disclosed in the international patent application MO PCT/ЕРО2/02591, published under the number UMO 93/10152 from the priority patent application

Great Britain Me9124390.7. When expressed in yeast, ET5 is produced as a lipoprotein particle, and when co-expressed with antigen 5 from HBMU, it produces a mixed particle known as KT5.5. Antigens

TKAR are described in international patent application PCT/ЗВ89/00895, published under number UMO 90/01496. A preferred variant of this invention is a vaccine against malaria, where the antigen preparation contains a combination of KT5.5 and TKAR antigens. Other symplast antigens, which are likely candidates for components of a multi-stage vaccine against malaria, are R. Tasiragit

M5RI, AMAT, M5RZ, EVA, SI OKR, KARY, KAR2, sequestrin, RIEMRI, RIZ32, I ZA1, I ZAZ, 5TAVR,

ZAI ZA, RIEKHRI, Riiz25, Ri28, RE527/25, RI16, Ri548/45, RI5230 and their analogues in Riaztodisht 5 years. One embodiment of the present invention is a composition comprising a KT5.5 or C5 protein or a fragment thereof, such as the C5 portion of KT5.5, in combination with one or more additional malarial antigens that may be selected, for example, from the group consisting of MOP1, M5RZ, AMAT, I CJSC or I 5AZ. Possible antigens from P mimach include nacocoloporozoid protein (SB protein) and Oin antigen binding protein and its fragments, for example, RUKII (see, for example, M/O02/12292).

Other possible antigens that can be used in the immunogenic compositions of the present invention include:

Streptococcal antigens, such as group A streptococcal or group B streptococcal antigens, antigens respectively derived from HIV-1 (e.g. antigen E4 or fragments thereof, or dad or fragments thereof, e.g. raa4, yai, pei, dr120 or dr160 or fragments any of them), human herpes virus such as 90 or its derivatives, or immediate early protein, for example, ISR27 with NZMI or H5eM2, cytomegalovirus (especially human evzr virus) (for example, OB or its derivatives), rotavirus (incl. live attenuated viruses), Epstein-Barr virus (e.g., dr350 or its derivatives), varicella virus (e.g., varicella, II, and IE63), or a hepatitis virus such as hepatitis B virus (e.g., hepatitis B surface antigen or its derivative ), hepatitis A virus, hepatitis C virus, and hepatitis E virus, or from other viral pathogens, such as paramyxoviruses: respiratory syncytial virus (eg, E, M, and OS proteins or their derivatives), parainfluenza virus, measles virus, mumps virus, human papillomaviruses and (eg, NRUb, 11, 16, 18), flaviviruses (eg, yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus) or influenza virus (intact live or inactivated virus, cleaved influenza virus grown in eggs or cells

MOSC, or intact influenza virosomes (as described by K. SiIshsK, Massipe, 1992, 10, 915-920) or their purified or recombinant proteins, for example NA, MP, MA, or M proteins, or combinations thereof), or obtained from bacterial pathogens such as Mei55egia 5pp, including M. dopogpea and M. teppoiai5 (for example, capsular saccharides and their conjugates, transferrin-binding proteins, lactoferrin-binding proteins, RIIS, adhesins); 5. ruodepez (for example, M proteins or their fragments, C5A protease, lipoteichoic acids), 5. adaiascae, 5. tshape5; N. disgeui; Mogaheia 5rr, including M. saiagnaii5, also known as

Vgappategpya saigagtnaiy5 (for example, high and low molecular weight adhesins and invasins;

VogaeieMya 5rr, including B. repivs5i5 (for example, pertactin, pertussis toxin or its derivatives, fibrous hemagglutinin, adenylate cyclase, fimbriae), B. ragaregiib55ie and B. Bgopsizeriisa;

Musobasietsut 5 years, including M. (shbegstiov5i5 (for example, EBATb, antigen 85A, -B or -C), M. Bomiv,

M. Iergaye, M. amisht, M. ragagshbBegsitsio5i5, M. 5ztedtaiyi5; And ediopeia 5yr, including GG. rpeshtorpiia;

E5cPegispia 5pp, including enterotoxic E. soya (e.g., colonization factors, heat-labile toxin or its derivatives, heat-stable toxin or its derivatives), enterohemorrhagic E. soya, enteropathogenic E. soya (e.g., toxin-like Shiga toxin or its derivatives); Mibgio 5pp, including U. spoiegae (for example, cholera toxin or its derivatives); ZpidePa 5rr, including 5. 5oppei, 5. duzepiegiae, 5. Pehpegtii; Hegwipia 5rr, including U. epiegosoyisa (for example, Mor protein), U. rebviiv, U. rzepdoishregsytsio5i5; Cytobacilli 5ppr, including S. pipi (eg, toxins, adhesins, and invasins) and S. soii; ZaItopeia 5 years, including 5. iurpi, 5. ragagurpi, 5. spoiIegaezitsi5, 5. epiegyaii5; And ibegia 5 years, including GI. toposuiodepev; Neiisobrasieg zrr, including N. ruiogi (for example, urease, catalase, vacuolation toxin); Rhoendotopes 5rr, including R. ayeegidiposa; arpuiosossiv5 5yr., including 5. atsgei5, 5. eridegtidie; Epiegosossib5 5yr., including E. Taesaiy5, E. Taesisht; Siozygidiigp 5yr., including S. lyeiapi (for example, tetanus toxin and its derivative), S. Broishipit (for example, botulism toxin and its derivative), S. ainisii (for example, clostridial toxins A or B and their derivatives); Vasiyy5 5yr., including B. apiygasiye (for example, botulism toxin and its derivatives); Sogupebrasiegisht 5yr., including S. airpipegiae (for example, diphtheria toxin and its derivatives); Vogteiia 5rr., including V. riggegegi (e.g. O5rA, OzrS, OBrA, ObBrV), V. dagipii (e.g. O5rA, O5rS, YuBrA, ObBrV), V. aieiyi (e.g. O5rA, OzrS, OBrA, ObBrVI), V. apaegsopia (for example, O5rA, OzrS, OBrA, OBrV), V.

Pegtviya; Epigiispia 5yr., including E. edii and the agent of human granulocytic ehrlichiosis; Kiskeyvia 5pp., including K. hiskeyvii, Spiatuadia 5pp., including S. igaspotaiy5 (eg, MOMP, heparin-binding proteins), S. rpestopiae (eg, MOMP, heparin-binding proteins), S. rechassi; And erioz5riga 5yr., including /G. ipieItodape; Tgeropeta 5rr., including T. raiiidyt (for example, rare proteins of the outer membrane), T. depiisoia, T. puoduzepiegpaye; or obtained from parasites such as Riakhtodisht 5rr., including R. TaIsiragit; Tochoriazeta 5yr., including T. dopaiiyi (for example, 5АС2, ЗАСЗ, T934);

Epiatoera 5yr., including E. Piziouis; Vabrezia 5yr., including V. tisgoii; Tgturapozota 5yr., including T. sgy7i; Siagaia 5 years, including the village. Iatrbia; And ebzptapia 5yr., including /. taogh

Rpesthosuziiy5 5yr., including R. sagipii; Tgispotopazh 5yr., including T. madipaiy5; Zspizoviota 5rr., including 5. tapzopi, or obtained from yeasts such as Sapaida 5rr., including S. aiBisapv;

Stguriosossiz5 5yr., including S. peoiogtapv.

Other preferred specific antigens for M. ishbegsco5 and 5 are, for example, TO Ka12, T NO,

T Naz5, Tr38-1. Ega 14, ORM, MTI, M5G., tTTs2 and pTOS1 (UMO 99/51748), MIB72E and M72. Proteins for M. yshregsko5i5 also include fusion proteins and their variants, where at least two, preferably three polypeptides

M. ishyuregsytso5 and 5 are merged into a larger protein. Preferred fusions include Ca12-TNF-NKaz5, Ega14-ORM-MTI,

ORM-MTI-M5I, Ega14-ORM-MTI-M5I-tTOb2, Ega14-ORU-MTI-M5I, ORM-MTOE-M5I-tTSS2, ТНно-

ORMU-MTI (MO 99/51748). A specific sequence of Ca12-Trp9-CaZ35 that can be mentioned is defined as

ZEO IU MO: 6 MMO2006/117240 together with variants in which 5eg 704 of the indicated sequence is mutated, but not to serine, for example, to Aa, but its derivatives are included in the Ni end of the M-terminus of the corresponding length (for example, ZEO IUO MO: 2 or 4 M/O2006/117240)".

Examples of antigens for Spiathuaia 5rr., such as S. igaspotaiii5, selected from ST858, ST89, ST875,

MOMR, ST622, RitrO, RitrO and their fragments, ZUMIV and any of their immunogenic fragments (for example, RtrOra and Rtrora) and their combinations. Preferred combinations of antigens include CT858,

STO89 and ST875. Specific sequences and combinations that can be used are described in

UMO2006/104890. Preferred bacterial compositions contain antigens derived from Naethorpine5 5pp., including H. ipsepsiae type B (e.g., PEP and its conjugates), atypical H. ipsepsiae, e.g.

OMPO2b, high molecular weight adhesins, P5, Pb, YO protein and lipoprotein 0, fimbrin and fimbrin-derived peptides (US 5,843,464) or multiple copy variants or their fusion proteins.

Hepatitis B surface antigen derivatives are well known in the art and include a set of antigens

Rheb51, Rheb52 5, described in European patent applications EP-A-414 374; 578, and ER 198-474. In one preferred aspect of the vaccine formulation, the antigens of the invention contain HIV-1 antigen, dr120,

especially when expressed in CHO cells. In another embodiment, the antigen compositions of the invention contain 9021 as defined above.

The compositions may also contain an antitumor antigen and be suitable for immunotherapeutic treatment of cancers. For example, the antigen may be tumor rejection antigens such as those used in prostate, breast, rectal, lung, pancreatic, kidney, or melanoma cancer. Examples of antigens include

MACE 1, C and MACE 4 or other MACE antigens as disclosed in M/099/40188, REAME, VACE, I ade (also known as MM Eo5 1) BACE and NACE (UMO 99/53061) or CACE (KobBbip5 and KazhaKati, 1996,

Sitepi Oripiop5 ip Ittypoioadu 8, pp. 628-636; Map dep Eupaee ei ai., Ipiegpaiopa! Choshttai oi Siipisa! 8

And Arogaiogu Kezeagsp (introduced in 1997); Soiteaiie ei ai. (1997), dUoigpa! Oi She Mayopai! Sapseg

Ip5yshe 89, p293. Indeed, these antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer.

MACE antigens for use in the present invention can be expressed as a fusion protein with an expression enhancer or with an immunological fusion partner. In particular, the MACE protein can be fused to protein O from Neatorpiysi5 ipicep7ae B or its lipidated derivative. In particular, the fusion partner may contain the first 1/3 of protein 0. Such constructs are disclosed in UMO99/40188.

Other tumor-specific antigens include, but are not limited to, KBA (CA733) tumor-specific gangliosides, for example, from 5M2 and OM3Z or their conjugates with carrier proteins; or the specified antigen can be a peptide hormone, for example, full-length gonadotropin-releasing hormone (SZpKN, UMO 95/20600), a long peptide of 10 short amino acids, suitable for the treatment of many types of cancer or in immune castration.

Prostate antigens such as prostate-specific antigen (RBA), RBESA (РМАБ 95(4) 1735-1740 1998), ROMA or an antigen known as prostate are preferably used.

Prostase is a prostate-specific protease (trypsin-like) 254 amino acids long with a conserved catalytic triad H-0-5 of serine protease and a prepeptide sequence of the amino-terminus, which indicates a potential secretory function (R. Meibop, and bap, S.

Eegdizop, R. Mov, V. Seyipav, I. Nosa 5 K. Mapa, "MoiIesshciag siopipa apa sNagasiyegizayop oi rgoviaze, ap apagodep-gediiaieai zegipe rgoivaze ulIy rgoviaie gezigisied ehrgezviop, Ip Rgos. Mai." Asad. 5si. OBA (1999) ) 96, 3114-3119). The predicted glycosylate site is described. The predicted structure is highly similar to other known serine proteases, demonstrating that the mature polypeptide consists of a single domain. The mature protein is 224 amino acids long, with one A2 epitope processed naturally.

The sequence of nucleotides of prostase and the established sequence of the polypeptide and homologues are disclosed in Eegdychzop ei ai. (Rhos. Mai!. Asai. Zsi. OBA 1999, 96, 3114-3119) and in international patent applications

UMO 98/12302 (as well as corresponding US Patent 5,955,306), MUMO 98/20117 (as well as corresponding US Patents 5,840,871 and 5,786,148) (prostate-specific kallikrein) and M/O 00/04149 (P7OZR).

The present invention provides compositions that contain fusion products of the prostate protein based on the prostate protein and its fragments and homologues ("derivatives"). Such derivatives are suitable for use in therapeutic vaccine formulations useful in the treatment of prostate tumors. Typically, the fragment will contain at least 20, preferably 50, more preferably 100 contiguous amino acids, as disclosed in the patent and patent applications referenced above.

Another predominant prostate antigen is known as P5O15, 5BEO IU MO: 113 M/O98/37814.

Immunogenic fragments and parts thereof contain at least 20, preferably 50, more preferably 100 contiguous amino acids, as disclosed in the patent application referred to above. For example, see

RBI108 (UMO 98/50567). Other prostate-specific antigens are known from M/098/37418 and UMO/004149. ANOTHER is ZTEAR RMAZ 96 14523 14528 7-12 1999.

Other tumor-associated antigens useful in the context of the present invention include: Rishi-1 in

VioI. Snet 274 (22) 15633-15645, 1999, НА5Н-1, НазН-2, Stiryu (Ziotope ейи а! Виоезавз 199, 21 61-70, US patent 5654140), cryptin - US patent 5 981 215. In addition, antigens , especially relevant for cancer therapy, also contain tyrosinase and survivin.

Mucin is derived from peptides such as Mys, see, for example, US Patents 5,744,144, 5,827,666 UMO 8805054, 4,963,484. Specifically considered Mys 1, obtained from peptides that contain at least one repeated unit of the Mys 1 peptide, preferably at least two such repeats, which are recognized by the 5M3 antibody (US 6,054,438). Another mucin derived from peptides contains a peptide from Mys 5.

The antigen according to the present invention can be breast cancer antigens, such as peg 2 Men, mammaglobin (US Patent 5668267) or antigens disclosed in UMO /00 52165, UUO99/33869,

UO99/19479, UMO 98/45328. Pepg 2 Mei antigens are disclosed in US Patent 5,801,005. Mostly Peg 2

Mey contain the complete extracellular domain (consisting of approximately 1-645 amino acids) or fragments thereof and at least the immunogenic part or the complete intracellular domain of approximately 580 amino acids of the C-terminus. In particular, the intracellular part should contain the phosphorylation domain or its fragments. Such structures are disclosed in MUMO 00/44899. A particularly preferred construct is known as ЕСО, РО the second is known as ЕСО РО, cf. M/O/00/44899. For purposes of this description, Ppeg 2 May may be derived from rat, mouse, or human.

Compositions may contain antigens associated with mechanisms of tumor support (eg, angiogenesis, tumor invasion), for example, ee 2, MESE.

It is assumed that in the compositions according to the present invention, it is possible to use antigens of Woiteiia 5r.

For example, antigens may contain nucleic acid, pathogen-derived antigen, or antigen preparations, recombinantly produced protein, or peptides and chimeric fusion proteins. In particular, the antigen is O5pA. O5pA can be a complete mature protein in an effective lipidated form of the host cell (E. soybean), has the name (Giro-O5pA) or a non-lipidated derivative. Such non-lipidated derivatives consist of the non-lipidated M51-O5pA fusion protein, which contains the first 81 amino acids of the M-terminus of the unstructured protein (M51) of the influenza virus, and the complete O5pA protein, while the other, MOP-O5pA, is a non-lipidated form of O5pA that carries additional amino acids of the M-end. The compositions of this invention can be used for the prevention or therapy of allergies. Such vaccines will contain antigens, specific (e.g., UOeg) and non-specific allergens (e.g., peptides derived from human DE, including, but not limited to, Stanworth decapeptide (EP 0 477 231 VI1)).

The compositions of the present invention can also be used for the prevention or therapy of other than allergies, chronic disorders, cancer or infectious diseases. Such chronic disorders are diseases such as atherosclerosis and Alzheimer's disease.

The compositions of the present invention are particularly suitable for the immunotherapeutic treatment of diseases such as chronic conditions and cancers, as well as for the therapy of chronic infections. Accordingly, the compositions of the present invention are particularly suitable for the immunotherapy of infectious diseases, for example, tuberculosis (TB), AIDS and hepatitis B virus (HBV) infections.

Also, in the context of AIDS, a method of treating a person who is vulnerable or suffering from AIDS is proposed. The method consists in administering the vaccine of the present invention to the patient, thus reducing the predicted drop in the number of CO4- T cells caused by subsequent HIV infection or slowing or stopping the drop in CO4- T cells in a person who is already infected with HIV.

Other antigens contain rather bacterial (mostly capsular) saccharides than the indicated pneumococcal (or in addition to them) antigens described above. Polysaccharide antigens are usually stored in the form of a liquid mass adsorbed on aluminum phosphate, and thus, to obtain vaccine compositions according to the present invention, the indicated liquid mass is mixed extemporaneously with the adjuvant according to the present invention. Preferably, other bacterial saccharides are selected from the group consisting of: capsular saccharide of serogroup A of M. tepidoi5 (MepA), capsular saccharide of serogroup C of M. tepidoi5 (Meps), capsular saccharide of serogroup MU of M. tepidoi5 (Mep), capsular saccharide of serogroup M/ -135 M. tepipoiidis (MepuUM), capsular saccharide of streptococcal group I group B, capsular saccharide of streptococcal group II of group B, capsular saccharide of streptococcal group PI of group B, capsular saccharide of streptococcal group IM of group B, capsular saccharide of streptococcal group M of group B, capsular saccharide 5iarpuisossi5 aigei5 type 5, capsular saccharide eiarpuisossi5 acgei5 type 8, Mi saccharide from ZaiYitopeiPa iurpi, lipopolysaccharide M. tepipoai5, lipopolysaccharide M. saiagtnaii and lipopolysaccharide N. ipisep7ae. By lipopolysaccharide is meant both natural lipopolysaccharide (or lipo-oligosaccharide) and lipopolysaccharide where the lipid A portion has been neutralized by any of a number of known methods (see, for example,

UMO 97/18837 or UMO 98/33923), or any molecule containing an O-polysaccharide derived from said lipopolysaccharide. Lipopolysaccharide of M. tepipoiidie refers to one or more of the 12 known immunotypes (1,12, 13,14, 15,16,17, 18,19, 110, 111 or 112). Particularly preferred combinations are compositions containing: 1) conjugated Nib, conjugated MepA and conjugated Meps; 2) conjugated Nir, conjugated Mepu and conjugated Meps; 3) conjugated

NiB and conjugated MepS; and 4) conjugated MepA, conjugated MepC, conjugated Mepu, and conjugated Mepuu-135. The amount of P5 in each of the above-mentioned conjugates can be 5 or 10 μg of each per 0.5 ml dose for a person. Preferably Ni, MepA, MepsS, MepuUu-135 and Mepu are TT conjugates.

A problem associated with the polysaccharide approach to vaccination is that reg 5e polysaccharides are poor immunogens. In order to overcome it, the saccharides of this invention can be conjugated with carrier proteins that provide T-cell help. Thus, the saccharides used in the present invention preferably bind to a similar carrier protein.

Examples of such carriers, which are currently commonly used for the preparation of saccharide immunogens, include diphtheria and tetanus toxoids (OT, OT SKMI197 and TT, respectively), fissurellic hemocyanin (KIN), protein O Naetorpyiz5 ipicheplaye (ER 59461 O-B), OMRS M. tepipoiiai and purified tuberculin protein derivative (TPO).

The saccharide can be linked to the carrier protein using a known method (for example, GH

U.S. Patent 4,372,945 and U.S. Patent 4,474,757). Preferably, conjugation is carried out

SOAR (M/O 95/08348).

Preferably the protein saccharide (wt) ratio for the conjugates is from 0.3:1 to 1:1, more preferably from 0.6:1 to 0.8:1, and even more preferably from about 0.7:1 .

Combinations of antigens that provide protection against pneumococcal and other pathogens are included in the present invention. Many pediatric vaccines are currently supplied as combination vaccines to reduce the number of injections a child must receive. Thus, pediatric vaccines can be formulated with other antigens of other pathogens and pneumococcal vaccines of the present invention. For example, the vaccine preparation technology of the present invention can include (or be administered separately but at the same time) a well-known "triovalent" combination vaccine containing diphtheria toxoid (OT), tetanus toxoid (TT) and pertussis components | usually neutralized pertussis toxoid ( RT) and/or fibrous hemagglutinin (ENA), optionally with pertactin (PEM) and/or agglutinin 1-2), for example, the vaccine IMEAMKIH-YUOTra "m (ZtiyKiipeVespat Viiodisa!5), which contains antigens OT, TT, RT, ENA and REM, or a whole-cell component of whooping cough, for example, such as is present on the market Tgyapgih"m, ZtpyyKiIipeVespat Viodisa!v5.

The combined vaccine may also contain another antigen, for example, hepatitis B surface antigen (HB5AO), polio virus antigens (for example, inactivated trivalent polio virus -

IRM), proteins of the outer shell of Mogaheiia saiyatnpaiih, atypical proteins of Naetorpin5 ipYaseplae, proteins of the outer shell of M. tepipodyaiv5 V.

Examples of preferred antigens of the Mogheya protein antigens that can be included in a combination vaccine (especially for prevention of otitis media) are: OMR106 (MO 97/41731 (Apieh) 8 MO 96/34960 (RMSO)); OMR21; ГРА and/or I Br (МО 98/55606 (РМО)|; ТрА and/or ТрвВ (МО 97/13785 and

MO 97/32980 (RMO)|; SorV (Neitipep ME, ei ai. (1993) Iptesi. Ittyp. 61:2003-20101; O5rA1 and/or

O5rAg (MO 93/03761 (University of Texas)Y; OtrSO; Nazk (PCT/ER99/03824); RIO (PCT/ER99/03823);

OMRV5 (PCT/EROO/O1468); IroOb (Great Britain 9917977.2); Iro10 (Great Britain 9918208.1); iro11 (Great Britain 9918302.2); Iro18 (Great Britain 9918038.2); Rb (PCT/ЕРО9/03038); 015 (PCT/ER99/03822); AutryA! (PCT/ERO9/06781); NIUZ (РСТ/ЕРО9/03257); and OtrE. Examples of atypical Naethorpein5 antigens that can be included in a combination vaccine (especially to prevent otitis media) include: fimbrin protein KUSA 5766608-Opio biale Vesvagsi

Eoshtsypaaiyyop)| and its fusion product, containing peptides I, for example, peptide fusions I B1 (); USA 5843464 (051) or UMO 99/64067|. OMR2b6 (MO 97/01638 (Sogies5))Y; Rb (ER 281673 (State

New York University); ТрА and/or ТрВ; Nia; Her; Nip47; NO; Ntm/1; Ntm/2; Ntm/3; Ntm/4; Nar; 015 (MO 94/12641); protein O (ER 594610); P2; and P5 (MO 94/26304).

Other contemplated combinations are pneumococcal saccharide and protein of the present invention in combination with viral antigens, for example against influenza (attenuated, cleaved or subunit

For example, surface glycoprotein neuraminidase (MA) and hemagglutinin (HA), see e.g.

SNa!Ioirka I. eyi ai, Yeig. Udoshgpai Siip. Misgobio!. Iptesi beat 1996. e.g., NM and RE proteins, see Sections above), with varicella (e.g., (attenuated, glycoprotein I-M, etc.), and any (or all) component(s) of MME (measles, mumps, rubella) A preferred pediatric combination vaccine contemplated by the present invention for the complete treatment or prevention of otitis media contains: one or more saccharide antigens of Bigheriosossi5 rpeshtopiae (preferably conjugated to protein 0), one or more pneumococcal proteins (preferably proteins described above) and one or more surface-reactive antigens Mogaheilia sagagnaii" and/or atypical Naethorpyia5 ipiseplae. The protein JO can be suitably used as a carrier protein for pneumococcal saccharide (as mentioned above) because it is an immunogen capable of producing protection of intermediate B-cells against atypical N. ipitseplae (piNi). An antigens of Mogaheia sagittaii" or atypical Naetorpiyia ipiiseplai can be included in the vaccine in subunit form or can be added as antigens present on the surface of outer membrane vesicles obtained from bacteria.

Immunogenic properties of the immunogenic composition used for vaccination according to the present invention

In the present invention, the immunogenic composition is preferably able to stimulate an improved CO4 T-cell immune response against at least one antigenic component or antigenic composition compared to the CO4 T-cell immune response obtained using the corresponding non-adjuvanted composition, i.e. it does not contain an exogenous adjuvant yuvantu (in this description it also means "simple composition"). In a specific embodiment, where the immunogenic composition is an influenza composition, and where the influenza vaccine preparation consists of several influenza strains, one of which is a pandemic strain, an enhanced CO4 T-cell immune response directed against a pandemic influenza strain is indicated.

By "improved CO4 T-cell immune response" is meant that a more pronounced CO4 response is obtained in a mammal after administration of an adjuvant immunogenic composition than after administration of the same composition without an adjuvant. For example, a more pronounced response of CO4 T cells is obtained in a human patient upon administration of an immunogenic composition containing the influenza virus or its antigenic preparation together with an adjuvant according to the present invention, compared to the response induced after the administration of a non-adjuvanted immunogenic composition. containing the influenza virus or its antigen preparation. Such a formulation would be suitably used to stimulate a CO4 T-cell response against influenza, which is capable of detecting influenza epitopes represented by class I molecules! Ministry of Emergency Situations

In particular, but not exclusively, the indicated enhanced CO4 T-cell immune response is obtained in an immunologically naïve patient, that is, a patient who is seronegative for the indicated influenza virus or antigen. Such seronegativity may be the result of the specified patient never being exposed to a similar virus or antigen (a so-called "naïve" patient) or, alternatively, not reacting to the specified antigen once encountered. In a specific aspect, said CO4 T-cell immune response is obtained from a subject at risk of an immune system disorder, for example, an elderly person, usually 65 years of age or older, or a young adult, younger than 65 years of age, at high risk of developing a pathological condition ("high-risk adult"), or a child under two years of age.

Improved immune response of CO4 T cells can be assessed by measuring the number of cells producing any of the following cytokines: - cells producing at least two different cytokines (CO401., 1-2, TEM-u, TMEos); - cells producing at least CO401. and another cytokine (I--2, TMEos, IEM-u); - cells producing at least II--2 and another cytokine (СО40И, TMEо, IEM-у); - cells producing at least IEM and another cytokine (1-2, TMEs, СО401); - cells producing at least TMEs: and another cytokine (1-2, СО401., TEM-u).

An improved T-cell immune response to CO4 will occur if cells producing any of the above-mentioned cytokines are present in greater numbers after administration of an adjuvant composition compared to administration of a non-adjuvant composition. At least one, preferably two, of the five conditions mentioned in this description above will usually occur. In a particular embodiment, cells producing all four cytokines will be present in greater numbers in the adjuvanted vaccine group compared to the non-adjuvanted vaccine group.

The improved CO4 T-cell immune response provided by the adjuvant composition against influenza according to the present invention can ideally be obtained after a single administration. A single-dose approach would be extremely appropriate, for example, in the case of a rapid clinical outbreak of an epidemic situation. In certain circumstances, especially for the elderly population, or in the case of children (less than 9 years of age) who are being vaccinated against influenza for the first time, or in the case of pandemics, it may be preferable to administer two doses of the same formulation for a given season. A second dose of said single composition (still considered a "first vaccination composition") can be administered during the primary immune response and scheduled accordingly. Typically, a second dose of the composition is administered several weeks or about one month later, such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks, after the first dose to help strengthen the immune system of individuals who are immune or weakly susceptible to the vaccine.

In another embodiment, administration of said immunogenic composition stimulates an improved B-lymphocyte response in patients administered an adjuvant immunogenic composition compared to a B-lymphocyte response induced in humans immunized with a non-adjuvanted composition. An improved B-lymphocyte response may mean an increased incidence of peripheral blood B-lymphocytes capable of differentiating to plasma cells that secrete antibodies to the antigen encountered, as measured by the stimulation of migo differentiation.

In another embodiment, administration of said immunogenic composition stimulates an improved humoral response in patients administered the adjuvant immunogenic composition compared to the humoral response induced in humans immunized with a non-adjuvanted composition. Said humoral immune response can be measured according to any of the techniques detailed in Example I, particularly Sections 1.1 (1.1.1), 1.2 (1.2.1) and 1.3 (1.3.5.2). If the immunogenic composition is an influenza composition, a specifically indicated humoral reaction against homologous and heterologous strains is obtained. Specifically, the indicated heterologous humoral immune response refers to the humoral response between influenza strains and is referred to as a "cross-reactive" humoral immune response.

This "cross-reactive" humoral immune response includes stimulation of the response against the influenza strain, which is a variant (drift) of the influenza strain used for vaccination.

An example of such a reaction is illustrated in example 1.3.1 and in fig. 2.

In a specific variant, administration of the indicated adjuvant immunogenic composition stimulates at least two of the following reactions: () improved immune reaction of CO4 T cells, (ii) improved reaction of B-lymphocytes, (ii) improved humoral reaction against at least one antigen component( iv) or antigenic composition in comparison with any immune response obtained with the help of a corresponding non-adjuvanted composition, that is, does not contain an exogenous adjuvant (in this description it also means "simple composition").

In another specific embodiment, vaccination with a composition for the first vaccination is adjuvant, does not affect the CO8 response that would be measured.

It is in a specific embodiment of the present invention that the composition containing the influenza virus or its antigen preparation, made with a saponin adjuvant presented in the form of a liposome, in particular, saponin 2521 in its quenched form with cholesterol, is effective from the point of view of stimulating T-cell responses in a population at risk of immune system disorders. In one variant, the indicated adjuvant is additionally a 30O-MRI adjuvant. In particular, the administration of a single dose of an immunogenic composition for the first vaccination as described in the present invention is able to provide better seroprotection, according to the correlation of protection for influenza vaccines, after re-vaccination against influenza in the elderly population than vaccination with a non-adjuvanted influenza vaccine. The claimed adjuvant formulation can also stimulate an improved CO4 T-cell immune response against the influenza virus compared to the immune response obtained when the non-adjuvanted formulation is administered. The indicated data may be associated with increased sensitivity to vaccination or antigenic manifestation of the infection of miv - a-miz influenza. In addition, they can also be associated with cross-reactivity, that is, a higher ability to respond to strains of influenza variants. Such an improved response may be particularly beneficial for populations at increased risk of immune system disorders, such as the elderly (65 years and older) population, particularly the elderly population at high risk. This response can ultimately lead to a reduction in overall disease incidence and mortality rates, as well as prevention of emergency calls to the hospital for pneumonia and other flu-like illnesses. Such a reaction may also be predominant in the pediatric population (under 5 years, predominantly under 2 years). In addition, it may induce a CO4 T-cell response that is more persistent over time, eg, still present one year after the first vaccination compared to a response induced by a non-adjuvanted formulation.

In a specific aspect, the CO4 T-cell immune response, e.g., an enhanced c0p4 T-cell immune response obtained in a non-naïve subject, includes stimulation of a cross-reactive T-helper response

CO4. In particular, the number of CO4 cross-reactive T-cells is increased. By "cross-reactive" CO4 reaction is meant T-cell CO4, aimed at distinguishing epitopes between influenza strains.

Of course, available influenza vaccines are effective only against infection with influenza virus strains that contain hemagglutinin with similar antigenic properties. When the infectious (circulating) influenza virus undergoes minor changes (for example, point mutation or accumulation of point mutations resulting in amino acid changes, for example in surface glycoproteins, particularly hemagglutinin (a variant of the antigenic variability of the virus strain), the vaccine can still provide some protection, although it may provide only limited protection because newly generated variants may evade immunity stimulated by previous influenza infection or vaccination.Antigenic variability is responsible for annual epidemics that occur during interpandemic periods (MLIeu 5 5Kepei, 1987, App. Neu. Viospet. 56, 365-394).Stimulation of CO4 cross-reactive T cells provides an additional advantage of the composition of the present invention in that it may also provide cross-protection, in other words, protection against heterologous infections, i.e. infections caused by a circulating influenza strain that is a variant (for example, a drift) of the influenza strain contained in the immunogenic k opposition Such stimulation may be preferable if the circulating strain is difficult to reproduce in eggs or grow in cell culture, considering the use of a variable strain as a working alternative. It may also be preferred when the subject receives a second vaccination several months or a year after the first vaccination, and the influenza strain used in the immunogenic composition for the second immunization is a variant strain of the strain used in the composition used for the first vaccination.

Thus, as defined herein, an adjuvant immunogenic composition against influenza has a greater ability to stimulate seroprotection and CO4 cross-reactive T cells in vaccinated elderly. This characteristic may be associated with a greater ability to react against the strain variant of this strain, which is present in the immunogenic composition. This property can become an important advantage in the case of a pandemic situation. For example, a polyvalent influenza immunogenic composition containing any one or more of H5, H2, HO, H7, or Hb strains may provide greater potency against a pandemic variant, i.e., a mutated strain, of said pandemic strain(s) both by subsequent vaccination, as well as in the case of infection with the specified variable strain.

Detection of CO4 cross-reactive T cells after vaccination with influenza vaccine

CO4 T cells that can recognize both homologous and variable strains of influenza are referred to herein as "cross-reactive." Adjuvant compositions against influenza, as described in this description, are capable of demonstrating heterosubtypic cross-reactivity, as there is significant cross-reactivity against variable strains of influenza. As indicated above, it has been found that the immunizing capacity of a pandemic vaccine preparation effective against variable pandemic strains may be an important property in the event of pandemics.

To confirm the above-mentioned observations, CO4 T-cell epitopes have been identified in humans, which contain different strains of influenza (SeiYideg Seyi ai. 1998, Ipi Ittipoi. 10(2):211-22; Seideg SM ei ai. 1996 U Mikoi.

70(7):4787-90; Seideg SM ei ai. 1995 in Migoi. 1995 69(12):7497-506).

In a particular embodiment, the adjuvant composition may provide the additional benefit of providing better protection against circulating strains that have undergone a major change (such as genetic recombination, for example, between two different species) in hemagglutinin (antigenic shift) against which currently available vaccines are not effective.

Revaccination and composition used for revaccination (revaccination composition)

In one embodiment, the present invention provides for the use of an influenza virus or its antigen preparation in the production of an immunogenic composition for re-vaccination of people who have received a previous vaccination with an immunogenic composition, as stated in this description.

In one aspect of the present invention, the use of an influenza virus or an antigenic preparation thereof from a first pandemic influenza strain in the manufacture of an adjuvant immunogenic composition as defined herein to protect against influenza infections caused by an influenza strain that is a variant of said first influenza strain .

In another aspect, the present invention provides the use of an influenza virus or an antigen preparation thereof in the manufacture of an immunogenic influenza composition for revaccination of humans who have received prior vaccination with an adjuvant composition against influenza as claimed herein, or an adjuvant composition against influenza containing variant influenza strain, and the adjuvant is as defined herein.

In another aspect, the present invention provides a method of vaccinating a population of people or a person against one strain of an influenza virus after repeated vaccination of said person or population against a strain of a variant influenza virus, said method comprising administering to said person(s) a first composition containing an influenza virus or an antigen preparation thereof from the first strain of the influenza virus and an adjuvant, as defined in this description, and (ii) the second immunogenic composition containing a variant strain of the influenza virus of the indicated first strain of the influenza virus. In a specific embodiment, said first strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak. In another specific embodiment, said variant strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak. In particular, re-vaccination is carried out with a composition of influenza, which contains at least one strain, which is a circulating pandemic strain. Both the main composition and the composition for revaccination can be polyvalent, that is, it can contain at least two strains of the influenza virus. If the composition(s) are polyvalent, at least one strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak. Usually, repeated vaccination is carried out at least b months after the first vaccination(s), preferably after 8-14 months, more preferably after about 10-12 months.

An immunogenic composition for repeated vaccination (composition for revaccination) can contain any type of antigenic preparation, both inactivated and live attenuated. It may contain the same type of antigen preparation, for example, split influenza virus or its antigen preparation, whole virion, or purified HA and MA (subunit) vaccine, as the immunogenic composition used for the first vaccination. Alternatively, the composition for revaccination may contain a different type of influenza antigen than that used in the first vaccination. The split virus is mainly used. The composition for revaccination can be adjuvant or non-adjuvant.

The non-adjuvant composition for revaccination can be RiItschagih "M/a-Kihe/pPiv5ri, which is administered intramuscularly. The formulation contains three antigens of inactivated cleaved o cline, made from strains recommended by the World Health Organization for the relevant influenza season .

The composition for revaccination can be adjuvant or non-adjuvant. In a specific variant, the composition for revaccination contains a clinical adjuvant, which is a saponin, as defined in this description.

In a specific embodiment, the immunogenic composition for revaccination (also referred to herein as “revaccination composition”) contains an influenza virus or an antigenic preparation thereof, which consists of widespread CO4 T-cell epitopes and an influenza virus or antigenic preparation thereof, which has been used for the first vaccination. A common CO4 T-cell epitope is intended to designate peptides/sequences/epitopes from different antigens that can be recognized by the same CO4 cell (see examples of described epitopes in: szeideg Seyi ai!. 1998, Ipi Ittipo). 19(2):211-22; Sevideg SM ei ai. 1996 in Migoi. 70(7):4787-90; SeyiYideg SM ei ai. 1995 .) Migoi. 1995 69(12):7497-506).

In an embodiment of the present invention, the revaccination composition is a monovalent influenza composition containing an influenza strain that is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak. In particular, the specified strain in the composition for revaccination is a circulating pandemic strain. Suitable strains include, but are not limited to:

НОМ, НеОМ2, Н7М?7 and Н2М2. The specified strain can be the same as that or one of those present in the composition used for the first vaccination. Alternatively, the specified strain may be a variant strain, i.e., a variable strain of the given strain present in the composition used for the first vaccination.

In another specific embodiment, the composition for revaccination is a polyvalent influenza vaccine. In particular, if the booster composition is a polyvalent vaccine, for example, a bivalent, trivalent or quadrivalent vaccine, then at least one strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak. In a specific embodiment, two or more strains in the composition for revaccination are pandemic strains. In another specific embodiment, at least one pandemic strain in the revaccination composition has the same type as one or more strains present in the composition used for the first vaccination. Alternatively, at least one strain may be a variant strain, i.e. a variable strain, such as at least one pandemic strain present in the composition used for the first vaccination. In particular, at least one strain in the composition for revaccination is a circulating pandemic strain. The composition for revaccination can be adjuvant or non-adjuvant.

Usually, the composition for revaccination, if used, is administered in the next flu season, for example, approximately one year after the administration of the first immunogenic composition. The composition for revaccination can also be administered every subsequent year (third, fourth, fifth vaccination, and so on). The composition for revaccination can be the same as the composition used for the first vaccination. Accordingly, the composition for revaccination contains the influenza virus or its antigen preparation, which is a variant strain of the influenza virus that was used for the first vaccination. In particular, influenza virus strains or their antigen preparations are selected according to the reference material distributed by the World Health Organization in such a way that they are adapted to the influenza strain circulating in the year of repeated vaccination.

The influenza antigen or antigenic composition used in revaccination preferably contains an adjuvant, respectively, as described above. The adjuvant can be a saponin presented in the form of a liposome, as described in this description above, which is preferred, but does not necessarily contain an additional adjuvant, such as 30-MRI.

In one aspect, revaccination induces any, preferably two or all of the following: (ii) an improved CO4 response against an influenza virus or an antigen preparation thereof, or (ii) an improved B-lymphocyte response, or (ii) an improved humoral response compared to an equivalent reaction induced after the first vaccination with an influenza virus without an adjuvant or its antigen preparation. Preferably, the immunological response(s) induced after re-vaccination with an adjuvant influenza virus or an antigen preparation thereof, as defined herein, is more pronounced than the corresponding response induced after re-vaccination with a non-adjuvanted composition. Predominantly, the immunological responses induced after repeated vaccination with non-adjuvanted predominantly split influenza virus are more pronounced in the population receiving the first vaccination with the adjuvanted predominantly split influenza composition than the corresponding response in the population receiving the first vaccination with non-adjuvanted , mainly a split composition against the flu.

In a particular embodiment, revaccination of subjects with a booster composition comprising an influenza virus and a saponin adjuvant in liposome form, as defined hereinabove, demonstrates higher antibody titers than the corresponding values in a group of subjects vaccinated for the first time with non-adjuvanted composition and who were revaccinated with a non-adjuvanted composition. The effect of an adjuvant to enhance the response to revaccination in the form of antibodies is particularly important in the elderly population, which is known to have a weak response to vaccination or infections caused by the influenza virus. The advantage associated with the adjuvant composition was also indicated in terms of improved T-cell response to OWL after re-vaccination.

In particular, the adjuvant composition of the present invention is able to stimulate better cross-reactivity against the changing strain (influenza strain from the following influenza season) compared to the protection provided by the control vaccine. The indicated cross-reactivity demonstrated a higher persistence compared to that obtained with the introduction of a non-adjuvant formulation. The effect of an adjuvant in the form of strengthening cross-reactivity against a variable strain is important in the case of a pandemic situation.

In a further embodiment of the present invention, a vaccination scheme is contemplated, in which the first vaccination is carried out with an influenza composition, preferably a cleaved influenza composition consisting of at least one influenza strain that could potentially cause a pandemic outbreak, and a second vaccination is carried out with a circulating strain, such as a pandemic strain, as well as the classical strain.

CO4 epitope in NA

This antigenic variability is mainly located in the epitope regions of the viral surface proteins hemagglutinin (HA) and neuraminidase (MA). Any difference in CO4 and B cell epitopes used by different influenza virus strains to invade the host's adaptive immune response is known to play and play a major role in influenza vaccination. CO4 T-cell epitopes, which contain different strains of influenza, have been identified in humans (see, for example: seiYideg Sei ai. 1998, Ipi Ittypo!).

190(2):211-22; Seideg SM ei aiYi. 1996 9 Migoi. 70(7):4787-90; apa Seideg SM ei aiYi. 1995 9 Migoi. 1995 69(12):7497-506).

In a specific variant, repeated vaccination is carried out using a composition for revaccination, which contains the influenza virus or its antigenic preparation, in which there are widespread epitopes of T-cells CO4 with the antigen of the influenza virus or its antigenic preparation used for the first vaccination. Thus, the present invention relates to the use of an immunogenic composition containing a pandemic influenza virus or its antigenic preparation and a saponin in the form of a liposome, in particular 0521 in its neutralized form with cholesterol, optionally with 30-MRI, in the production of a component of the first vaccination of a multidose vaccine, in addition, the multi-dose vaccine contains as a revaccination dose the influenza virus or its antigenic preparation, in which there are widespread epitopes of T-cells CO4 with the antigen of the pandemic influenza virus or its antigenic preparation of the virus in the dose administered during the first vaccination.

Means of vaccination

The immunogenic compositions of the present invention can be administered by any suitable route, such as intradermally, transmucosally, for example, intranasally, orally, intramuscularly, or subcutaneously. Other ways of drug delivery are well known in this field.

For the immunogenic adjuvant composition, the intramuscular method of drug administration is preferable.

Intradermal administration is another suitable method. Any suitable intradermal delivery device can be used, such as the short needle devices described in US Patents 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, 5,417,662. Intradermal vaccines can also be administered using devices that limit the effective length of needle penetration into the skin, such as those described in M/O99/34850 and EP1092444, incorporated herein by reference, as well as functional equivalents thereof. Also suitable are needleless injectors, which deliver liquid vaccines into the skin through a liquid needleless injector or through a needle that pierces the stratum corneum (5igaisht sogpesht) and produces a stream that reaches the dermis. Безголкові інжектори описані, наприклад, в патентах США 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335, 5,503,627, 5,064,413, 5,520, 639, 4,596,556, 4,790,824, 4,941,880, 4,940,460, УМО 97/37705 and UMO 97/13537. Also suitable are ballistic powder/particle delivery devices that use compressed gas to accelerate the passage of the powder vaccine through the outer layers of the skin to the dermis. In addition, it is possible to use ordinary syringes in the classical method of intradermal administration by Manta (tapioih).

Another suitable method of drug administration is subcutaneous. You can use any suitable device for subcutaneous injection, for example, a classic needle. A needleless injector such as that described in UMO 01/05453, MO 01/05452, MO 01/05451, MO 01/32243, MO 01/41840, MO 01/41839, MO 01/47585, MO 01/ 56637, MO 01/58512, MO 01/64269, MO 01/78810, UMO 01/91835, UMO 01/97884, MO 02/09796, UMO 02/34317. More preferably, the indicated device is filled in advance with a liquid preparation of the vaccine.

Alternatively, the vaccine is administered intranasally. Usually, the vaccine is injected locally into the nasopharynx, preferably without inhalation into the lungs. It is preferable to use a device for intranasal administration, which delivers the vaccine preparation to the nasopharynx without or substantially without entering the lungs.

Preferred devices for intranasal administration of vaccines according to the present invention are sprays. Suitable commercially available nasal sprays include Assy5rgau "m (Vesiop RiskKip5op).

Nebulizers produce very fine sprays that can easily be inhaled into the lungs and thus do not reach the nasal mucosa effectively. Thus, atomizers are not preferred.

Preferred sprays for intranasal administration are devices for which the operation of the device does not depend on the pressure applied by the consumer. These devices are known as threshold pressure devices. Fluid is released from the nozzle only when pressure is applied to the wedge. These devices facilitate the delivery of sprays with regular droplet sizes. Threshold pressure devices suitable for use according to the present invention are known in the art and are described, for example, in MO 91/13281 and EP 311 863 B and EP 516 636, which are incorporated herein by reference. Such devices are commercially available from Rtiesheg trnN and are also described in Wotteg, KK Rpaptaseciisa! Thesppoiodus

Epigoras, Zeri 1999.

Preferred intranasal devices produce droplets (sizes measured using water as the liquid) in the range of 1 to 200 microns, preferably 10 to 120 microns. With a size of less than 10 μm, there is a risk of inhalation, so the presence of no more than about 5 95 droplets of size less than 10 μm is desirable. Droplets larger than 120 µm do not atomize as well as smaller droplets, so it is desirable to have no more than about 5 95 droplets larger than 120 µm.

Vidose delivery is a further preferred feature of the intranasal delivery system for the use of vaccines according to the present invention. Vi-dose devices contain two subdoses of a single dose of vaccine, one subdose for administration in each nostril. Usually, two subdoses are present in a single chamber, and the design of the device allows effective delivery of one subdose at a time.

Alternatively, a single-dose device for administering vaccines according to the present invention can be used.

Alternatively, an epidermal or transdermal method of vaccination is also contemplated by the present invention.

In a specific aspect of the present invention, the adjuvant immunogenic composition for the first administration can be administered intramuscularly, and the adjuvant or non-adjuvant revaccination composition can be administered by another method, for example, intradermally, subcutaneously, or intranasally. In another specific embodiment, the immunogenic composition containing specifically the influenza virus antigen or its antigen preparation for the first administration may contain a standard amount

NA 15 μg per strain of influenza, and the composition for revaccination against influenza may contain a low dose of NA, that is, below 15 kg, depending on the method of administration, it can be administered in a smaller volume.

Populations for vaccination

The target population for vaccination may be individuals at risk of immune system disorders. People at risk of immune system disorders are generally less able to respond to an antigen, particularly the influenza antigen, compared to healthy adults.

Preferably, the target population is a population that is not targeted against influenza, or is naïve (eg, a pandemic strain of miv a mi5), or has not previously responded to influenza infection or vaccination. Preferably, the target population consists of elderly people, respectively, 65 years of age, and this population also includes younger adults at high risk of disease (ie, 18 to 64 years of age), such as people who work in health care settings, or young people with risk factors such as cardiovascular and pulmonary disease or diabetes.

Another target population consists of children 6 months of age and older, especially children 6-23 months of age who have been hospitalized with a relatively high incidence of influenza. Predominantly, the target population is elderly people over 65 years of age.

Vaccination schedules, dosages and additional efficacy criteria

In most cases, suitable immunogenic compositions of the present invention are a standard injectable dose of 0.5 mL, and if the influenza composition contains 15 μg of hemagglutinin antigenic component from all or each influenza strain, as measured by single radial immunodiffusion (RDI) ( In U.M. Umos ei ai.: 9. Vioiyi. apa. 5 (1977) 237-247; 93. M. Mooa ei ai., 9.

Vioi Yeyapa 9 (1981) 317-330). Accordingly, the volume of the vaccine dose will be from 0.5 ml to 1 ml, in particular, the standard is 0.5 ml or 0.7 ml of the vaccine dose volume. For influenza vaccines, a slight correction of the dose volume will usually be made depending on the HA concentration in the original whole sample.

Accordingly, said immunogenic composition contains a low dose of HA antigen, for example, any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 μg of HA per influenza strain.

Preferably, the dose of the vaccine according to this invention, in particular the low dose of the vaccine, can be administered in a smaller volume than conventional split influenza vaccines that are administered by injection, which is usually about 0.5; 0.7 or 1 ml per dose. Small volume doses according to this invention are preferably less than 500 μl, more preferably less than 300 μl, even more preferably no more than about 200 μl or less per dose.

Thus, a preferred dose of a small volume vaccine according to one aspect of the present invention is a dose with a low dose of antigen in a small volume, for example, about 15 μg or about 7.5 μg HA or about 3.0 μg HA (per strain) in a volume of approximately 200 μl.

The anti-influenza drugs of the present invention preferably meet certain international criteria for vaccines.

International standards are used to measure the effectiveness of influenza vaccines.

The official criteria of the European Union for an effective vaccine against influenza are listed below in the table. 1.

Theoretically, in order to meet the requirements of the European Union, the flu vaccine must meet only one of the criteria listed in the table for all flu strains included in the vaccine. Compositions according to the present invention meet at least one such criterion.

However, in practice, at least two or all three criteria should be agreed for all strains, especially for a new vaccine, for example, for a new vaccine designed for a different route of administration. Under certain circumstances, two criteria may be sufficient. For example, it may be acceptable for all strains to meet two of the three specified criteria, while some but not all strains (eg, two of the three strains) meet the third criterion. These requirements are different for adult populations (18-60 years) and elderly populations (> 60 years).

Table 1

11111111 l8-6bbrokv.///// | »boOrokiv./:KO x The seroconversion rate is defined as the percentage of vaccinated people in whom there was an increase in hemagglutinin inhibition (NI) in the serum by at least four orders of magnitude after vaccination, for each vaccine strain. "k The conversion factor is defined as the ordinal increase in the geometric mean value (GMT) of serum NI titers after vaccination, for each vaccine strain. --x The protection rate is defined as the percentage of vaccinated individuals with a serum NI titer equal to or greater than 1:40 after vaccination (for each vaccine strain) and is usually taken as indicating protection.

Another aspect of the present invention provides a method of developing a vaccine against diseases known to be treated by CO4- T-cell activation, which includes 1) selection of an antigen containing CO4- epitopes, and 2) combining said antigen with a saponin ad 'juvant in the form of a liposome, as defined in this description above, and the indicated vaccine when administered to the indicated mammal can stimulate an enhanced reaction of T cells CO4 in the indicated mammal.

The disclosures of all references in this application, including patent applications and issued patents, are accompanied by full references in this description.

For the avoidance of doubt, the terms "comprise", "comprises" and "comprises", as used by the inventors in this specification, may optionally be replaced by the terms "comprise", "compose 3" and "compose 3", respectively, in each case.

The present invention will be further described with reference to the following non-limiting examples:

Example I describes immunological readout methods used in studies on mice, ferrets, and humans.

Example II describes the preparation of liposomal adjuvant MRI /0521.

Example II describes a preclinical evaluation of an adjuvanted and non-adjuvanted influenza vaccine in ferrets.

Example IM demonstrates the preclinical evaluation of an adjuvanted and non-adjuvanted influenza vaccine in naïve and baseline C57VI/6 mice.

Example M describes the comparison of adjuvanted influenza vaccine with ZO-MRI in two different concentrations in mice.

Example MI describes a comparison of adjuvanted influenza vaccine with ZO-MRI at two different concentrations in the elderly.

In Example MI! described the preclinical evaluation of adjuvanted NRU vaccines in mice.

In Example MI! described the preclinical evaluation of adjuvant and non-adjuvant immunogenic compositions with cytomegalovirus.

In Example IX, the preclinical evaluation of the adjuvant composition of the KT5.5 vaccine with ZO-MRI in two different concentrations is described.

Example X describes the clinical evaluation of adjuvant vaccine CT5.5 with ZO-MRI in two different concentrations.

Example I - Immunological methods of reading 11. Methods on mice

I.1.1. Hemagglutination inhibition test

Testing methodology

Antibody titers against hemagglutinin of three influenza virus strains were determined using the hemagglutination inhibition (NI) test. The principle of the NI test is based on the ability of specific antibodies against influenza to inhibit hemagglutination of chicken erythrocytes (RBCs) under the influence of influenza virus hemagglutinin (HA). Heat-inactivated serum samples were first treated with kaolin and chicken erythrocytes to remove non-specific inhibitors. After primary treatment, serum samples, diluted by two orders, were incubated with 4 hemagglutination units of each influenza strain. Next, chicken erythrocytes were added and inhibition of agglutination was evaluated. Titers were expressed as equivalent values of the highest serum dilution in which it completely inhibited hemagglutination. Since the first dilution of the sera was 1:20, the undetected level was estimated as a titer equal to 10.

Statistical analysis

Statistical analysis was performed on NI titers after vaccination with SHOMIZTAT.

The protocol used for the analysis of discrepancies can be briefly described as follows:

- Year data transformation; - the Shapiro-Wilk test (Zparigo-MLIK) for each population (group) to check the normality of the distribution of groups; - Cochran's test (Sospgap) to check the homogeneity of differences between different populations (groups); - two-way analysis of disagreements carried out in groups; - Tukey's test (TyKeu) NZO multiple comparisons.

I.1.2. Intracellular cytokine staining

This technique allows determining the number of antigen-specific T-lymphocytes on the basis of cytokine production: effector T-cells and/or effector T-lymphocytes produce interferon-y and/or main T-lymphocytes produce 1ІІ--2. On the 7th day after immunization, peripheral blood mononuclear cells are collected.

Lymphoid cells re-stimulate IP miigo in the presence of a secretion inhibitor (brefeldin):

Next, the specified cells are processed according to the usual method of immunofluorescence using fluorescent antibodies (204, СО8, IEM-u and 1-2). The results are expressed as the occurrence of cytokine-positive cells within the limits of T-cells СО4/С08. Intracellular staining of T-cell cytokines was performed on day 7 of peripheral blood mononuclear cells after the second immunization. Blood was collected from mice and placed in heparinized KRMInAaa medium. For blood KRMINADAY - diluted suspensions of peripheral blood lymphocytes were divided into layers on a gradient

Matyutai! according to the recommended protocol (centrifugation for 20 minutes at 2500 rpm and room temperature). Mononuclear cells were removed at the border of division, washed 2 times in

ErmitAdia, and suspensions of peripheral blood mononuclear cells were adjusted to 2x105 cells/ml in

ERMI with a content of 5 95 serum of calf embryos.

Antigen stimulation of peripheral blood mononuclear cells was carried out at a final concentration of 1x106 cells/ml (tube for analysis using a cell sorter with fluorescence excitation |РАС5|Й) using trivalent split influenza vaccine on microbeads (5 μg NA/strain) or whole RI (1 μg NA/strain) and then incubated for 2 hours at 37 °C with the addition of anti-CO28 and anti-CO494 (1 μg/ml for both).

Addition of both antibodies, increased proliferation and cytokine production by activated T cells and natural killer cells may provide a common stimulatory signal to induce cytotoxic T lymphocytes.

In addition, peripheral blood mononuclear cells were also stimulated overnight with trivalent split influenza vaccine (30 μg NA/strain) or whole BI (5 μg

HA/strain) - by pulsing bone marrow-derived dendritic cells (1x109 cells/ml), which were prepared by pulsing bone marrow-derived dendritic cells with split influenza vaccine (60 μg HA/strain) or trivalent RI of whole influenza (10 μg NA/strain) for two hours at 37 "C. After the stage of re-stimulation of the antigen, mononuclear cells of peripheral blood are incubated overnight in the presence of brefeldin (1 μg/ml) at 37 "C in order to inhibit cytokine secretion.

Staining of interferon-y (IEM-ULI -2/604/C08) is performed as follows: Cell suspensions are washed, resuspended in 50 μl of a phosphate buffer solution containing 1 95 of calf embryo serum, containing 2 95 Es of blocking reagent (1/50; 2.4052). After incubation for 10 minutes at 4 "С, 50 μl of a mixture of anti-СО4-PE (2/50) and anti-СО8 on Ср (3/50) was added and incubated for 30 minutes at 4 "С. After washing b in a phosphate buffer solution containing 1.95% fetal calf serum, the cells were infiltrated, resuspended in 200 μl of Suoiich-Suiuregt (Kii VO) and incubated for 20 minutes at 4 "C. Then the cells were washed with Regt Umakhp (Kii VO) and resuspended in 50 μl a mixture of anti-IEM-y ARS (1/50) and anti-II-2 RITS (1/50), diluted

Rep Umah 5p. After incubation for a minimum of 2 hours and a maximum of a whole night at 4 "С, the cells were washed with Regt UmMakhpy and resuspended in a phosphate buffer solution containing 1 95 fetal calf serum and 1 96 paraformaldehyde. Sample analysis was performed using

EAS5. Live cells were covered (E5C/55C) and assembled -- 50,000 transformations (lymphocytes) or 35,000 transformations on CO4 T-cells. The percentage content of IEM in z- or I/2 was calculated on СО4-- and СХО8-- closed populations.

I.2. Ways on ferrets

I.2.1. Haemagglutination inhibition test (NO)

Testing methodology

Antibody titers against hemagglutinin of three influenza virus strains were determined during the hemagglutination inhibition (NI) test. The principle of the NI test is based on the ability of specific anti-influenza antibodies to inhibit hemagglutination of chicken erythrocytes (RBCs) by influenza virus hemagglutinin (HA). Serum samples were first treated with 25 95 neuraminidase (COE) solution and heat inactivated to remove non-specific inhibitors. After primary treatment, serum samples, diluted by two orders, were incubated with 4 hemagglutination units of each influenza strain. Next, chicken erythrocytes were added and inhibition of agglutination was assessed using reading gaps. Titers were expressed as equivalent values of the highest serum dilution in which it completely inhibited hemagglutination. Since the first serum dilution was 1:10, the undetected level was scored as a titer of 5.

Statistical analysis

Statistical analysis was performed on NI titers (day 41 before the provacation test using OMIZTAT. The protocol used for the analysis of discrepancies can be briefly described as follows: - Year data transformation; - Shapiro-Wilk (Zparigo-MLIK) test for each populations (groups) to check the normality of the distribution of groups; - Cochran's test (Sospgap) to check the homogeneity of differences between different populations (groups); - one-sided AMOMA interaction test: - Tukey's test (TyKeu) NSO of multiple comparisons.

I.2.2. Nasal washes

Nasal washes were obtained by injecting 5 ml of phosphate buffer solution into both nostrils of awake animals. The inoculum was collected in Petri dishes and placed in sample containers in dry ice.

Determining the titer of viruses in nasal washes

All nasal wash samples were first sterile filtered through Zrip X filters (Soviag) in order to exclude bacterial contamination. Ten-fold serial dilutions of nasal washes of 50 μl were transferred to microtiter plates containing 50 μl of medium (10 wells/dilution). Next, 100 μl of Medline-Derby canine kidney cells (MOSC, 2.4x105 cells/ml) were added to each well and incubated at 35 °C for 5-7 days. After 6-7 days of incubation, the culture medium was carefully removed, and 100 μl of 1/20 M/5T-1 containing medium and incubated for another 18 hours.

The intensity of yellow formazan staining produced by the reduction of M/5T-1 by viable cells, which is proportional to the number of viable cells present in the well at the end of the virus titration assay, is determined by measuring the optical density in each well at the appropriate wavelength (450 nm). The limit indicator is defined as the average value of ОЙ for uninfected control cells - 0.3 ОО (0.3 ОО corresponds to w/- with ZIЮem ОО for uninfected control cells).

A positive result is determined when the OO "limit indicator, and vice versa, a negative result is determined when the OO "limit indicator. Virus propagation titers were determined by "Key and Miepsi" and expressed as 109 TCIUOz/ml.

I.3. Assays to assess the immune response in humans

I.3.1. Analysis of hemagglutination inhibition

The immune response was determined by measuring NI antibodies using the method described by the Collaborating Center for Influenza Problems of the World Health Organization, Centers for Disease Control, Atlanta, USA (1991).

Antibody titers were measured on frozen and thawed serum samples using a standardized and comprehensively validated micromethod using 4 hemagglutination inhibition units (4 IU) of the relevant antigens and 0.595 chicken erythrocyte suspension.

Non-specific serum inhibitors were removed by heat treatment and receptor-depleting enzyme.

The obtained sera were evaluated for NO antibody levels. Starting with an initial dilution of 1:10, serial dilutions (with a factor of 2) were prepared to a final dilution of 1:20480. The end point of the titration was taken as the highest dilution stage that showed complete inhibition (100 95) of hemagglutination. All analyzes were performed in duplicate,

I.3.2. Neuraminidase inhibition assay

The analysis was performed on microtiter plates covered with fetuin. 2 serial dilutions of the antiserum were prepared and mixed with a standardized amount of influenza A virus NZM2,

NIM! or influenza B. The test was based on the biological activity of neuraminidase, which enzymatically releases neuraminic acid from fetuin. After cleavage of the final neuraminic acid, O1-O-galactose-M-acetyl-galactosamine was detected. Horseradish peroxidase (HPR)-labeled peanut agglutinin from Agaspis Ppurodaea, which specifically binds to galactose structures, was added to the wells. The amount of bound agglutinin can be detected and quantitative determination of the reaction of the substrate with tetra-methylbenzidine (TMB). The highest antibody dilution that still inhibits viral neuraminidase activity by at least 50 95 is shown to be the MI titer.

I.3.3. Analysis of neutralizing antibodies

Measurements of neutralizing antibodies were performed on frozen and thawed serum samples.

Neutralization of the virus with the help of antibodies contained in the serum was determined using the microneutralization assay. In the analysis, serum was used without further processing. Each serum sample was analyzed three times. A standardized amount of virus was mixed with serial dilutions of serum and incubated to ensure binding of antibodies to the virus. A cell suspension containing a certain number of MOSC cells was then added to the mixture of virus and antiserum and incubated at 33 C. After the incubation period, virus replication was visualized by hemagglutination of chicken erythrocytes. 50 9o serum neutralization titer was calculated according to the method

Reed and Munch (Keea apa Myepsi).

I.3.4. Cell-mediated specific immunity assessed by flow cytometry of cytokines (SES)

Antigen-specific CO4 and CO8 T-cells of peripheral blood can be re-stimulated by migo to produce 1-2, CO40I, TME-alpha and IEM upon incubation with the appropriate antigen. Thus, antigen-specific CO4 and CO8 T-cells can be enumerated by flow cytometry after conventional labeling of cell phenotype by immunofluorescence, as well as intracellular cytokine production. In this study, influenza vaccine antigen, as well as peptides derived from a specific influenza protein, were used as an antigen to re-stimulate influenza-specific T cells. Results were expressed as the incidence of cytokine(s)-positive CO4 or CO8 T cells within the CO4 or CO8 T cell subpopulation.

I-3.5. Statistical methods

I.3.5.1. Primary Endpoints - Percentage, intensity, and association with vaccination of persistent local and systemic signs and symptoms during the next medical follow-up period for 7 days (ie, day of vaccination and 6 subsequent days) after vaccination and overall. - The percentage content, intensity and connection with vaccination of unstable local and systemic signs and symptoms in the next period of medical observation within 21 days (ie, the day of vaccination and the following days) after vaccination and in general. - Cases of serious side effects throughout the study.

I.3.5.2. Secondary endpoints

For the humoral immune response:

Observed variables: - On days 0 and 21: serum hemagglutination inhibition (NI) and MI antibody titers were analyzed separately against each of the three influenza virus strains presented in this vaccine (anti-NI1MI1, anti-NZM2 and anti-B antibodies). - On days 0 and 21: titers of neutralizing antibodies were analyzed separately against each of the three influenza virus strains presented in this vaccine.

Obtained variables (with a 95 95 confidence interval): - Geometric mean value of serum NO antibody titers (GMT) with a 95 95 confidence interval (95 9o CI) before and after vaccination. - Seroconversion rates" from 95 95 CI on the 21st day. - Conversion factors "" from 95 95 CI on the 21st day. - Seroprotection indicators"" from 95 95 CI on the 21st day. - Geometric mean serum MI antibody titers (with 95 95 confidence intervals) at all time points. "Seroconversion rates were defined as the percentage of vaccinated individuals who had at least a 4-fold increase in serum NO titers on day 21 compared to day 0 for each vaccine strain. The conversion factor was defined as the geometric mean increase in serum NO titers per Day 21 compared to day 0 for each vaccine strain Protection rate is defined as the percentage of vaccinated individuals with a serum NO titer of 40 post-vaccination (for each vaccine strain) that is commonly accepted as vaccination indicating protection.

For a cell-mediated specific immune reaction:

Observed variable:

At 0 and 21 days: frequency of cytokine-positive СО4/С0О8 cells per 105 in different trials.

In each test, the reaction of T-cells СО4/СОО8 is quantified: - Influenza peptide antigen (p) (the exact nature and origin of the indicated antigens must be provided/explained). - Split influenza antigen (51). - Solid influenza antigen (m/u).

Obtained variables:

- cells that produce at least two different cytokines (СО401., 1-2, ТЕМ-у, ТМЕ-с); - cells that produce at least CO40!. and another cytokine (1-2, TME-o, IEM-u); - cells that produce at least 1-2 and other cytokines (CO40, TME-o, IEM-u); - cells that produce at least IEM-u and another cytokine (I--2, TME-o, СО401); - cells that produce at least TME-c and another cytokine (1-2, СО401., TEM-u).

I.3.5.3. Analysis of immunogenicity

Immunogenicity analysis is based on the total vaccinated cohort. The following parameters were calculated for each treatment group (with a 95 95 confidence interval): - Geometric mean titers (CMT) of NI and M1 antibodies on day 0 and 21. - Geometric mean titers (MT) of neutralizing antibodies on day 0 and day 21. - Conversion factors for the 21st day. - Day 21 seroconversion rates (53) are defined as the percentage of vaccinated individuals with at least a 4-fold increase in serum NI titers on day 21 compared to day 0. - Day 21 seroprotection rates are defined as the percentage of vaccinated people with a titer of No serum is 1:40. - The incidence of CO4/CO08 T-lymphocytes secreted in the reaction was summarized (descriptive statistics) for each vaccination group, at each time point (day 0, day 21) and for each antigen (peptide influenza (ri), cleaved influenza ( 57) and solid influenza (UM)). - Descriptive statistics of the individual difference between time points (after-before) corresponding to each vaccination group and each antigen (rii, 51 and m/i) in each of 5 different trials. - A non-parametric test (Kruskall-Wallis test) was used to compare location differences between C groups, and the statistical p-value was calculated for each antigen in each of 5 different tests. All tests showing two-sided P values: 0.05 were considered statistically significant.

Example II - Preparation of liposomal adjuvant MRI /2521

II. From Obtaining a liquid suspension MR.

MRI liquid mass. (abbreviation for 30-MRI, i.e. lipid A of 3-O-desacylated monophosphoryl, used in this document) is prepared from lyophilized powder of 30O-MRI.. Liquid mass of MRG. is a stable concentrated (approximately 1 mg/ml) aqueous dispersion of raw material ready for use in a vaccine formulation or adjuvant formulation. A schematic representation of the production method is shown in Fig. 1.

For the maximum batch size of 12 g, the liquid MRI mass is obtained in sterile glass containers. Dispersion MRI. consists of the following stages: - MRI powder suspension. in water for injections; - disaggregation of any large aggregates by heating (thermal treatment); - reduction of the particle size from 100 nm to 200 nm using micro rarefaction; - pre-filtering on the pre-Zaposieap filter with the size of the holes 0.8/0.65 μm; - sterile filtration at room temperature (Zapobgap R with a hole size of 0.22 μm).

MRI powder. was lyophilized using microdilution, resulting in a stable colloidal dispersion in water (MRI particles with a size acceptable for sterile filtration).

The lyophilized MRI powder is dispersed in water for injection to obtain a 10 mg/ml crude suspension. Next, the suspension is subjected to heat treatment, constantly stirring. After cooling to room temperature, the process of micro-liquefaction begins in order to reduce the size of the particles. Micro rarefaction is carried out using the M11OEN Misgoynpidis5 apparatus, in which the dispersion continuously circulates through the micro-racial interaction chamber, at a certain pressure for the minimum number of passes (number of cycles: Ptip). The micro-dilution rate, which represents the number of cycles, is calculated based on the measured flow rate and dispersion volume.

On this equipment at a given pressure, the resulting flow rate may vary from one interaction chamber to another and during the life cycle for a particular interaction chamber. In this example, the E20U Misgoynchidis5 interaction camera is used. Since the efficiency of microdilution is related to the pressure-flow rate pair, the processing time can vary from batch to batch. The required duration of 1 cycle is calculated based on the flow rate. The flow rate under consideration is the flow rate measured using water for injection immediately prior to the introduction of the MRI into the apparatus. One cycle is defined as the time (in minutes) required for the full volume of MRI. passed through the apparatus once. The time required for n cycles is calculated as follows: n x number of MRIs. for treatment (ml) / flow rate (ml/min)

Thus, the number of cycles is adjusted accordingly. The minimum number of cycles to perform (ptip) is described for the preferred equipment and interaction cameras used. The total number of cycles for circulation is determined by measuring the size of the particles performed after the cycles

Ptip. The particle size limit (DIT) is determined on the basis of historical data. Measurements are carried out using the technique of photon correlation spectroscopy (PC5), and the dit is expressed as a single-modal result (7amegade). With this limit, micro-dilution can be stopped after Ptip cycles. Above this limit, microthinning continues until a satisfactory size reduction is obtained, a maximum of 50 more cycles.

If filtration does not occur immediately after microdilution, then dispersed MRI. stored at a temperature from 42 to 8 "C pending its transfer to the filtration area.

After microdilution, the dispersion is diluted with water for injection and filtered under aseptic conditions through a filter with a hole size of 0.22 μm in laminar flow. The final concentration of MRI. is 1 mg/ml (0.80-1.20 mg/ml). 1.2 Preparation of liposomal adjuvant MRI/0521

This adjuvant, named OR, contains ZO-MRI. and 2521 in quenched form with cholesterol, it was prepared as described in M/O 96/33739, incorporated by reference herein. In particular, the adjuvant ABZOI1 was mainly manufactured as Example 1.1 in M/O 96/33739. ABO1B adjuvant contains: liposomes, which, in turn, consist of dioleoylphosphatidylcholine (PORS), cholesterol and ZO MR.

IU of 1000 μg of SORS, 250 μg of cholesterol and 50 μg of C30O-MRI, each value is given approximately per dose of vaccine), 521 (50 μg per dose), phosphate buffer mass and water brought to a volume of 0.5 ml.

The ABZO1TE adjuvant contains the same ingredients as ABO1B, but at a lower concentration in the amount of 500 μg of roORS, 125 μg of cholesterol, 25 μg of ZO-MRI, and 25 μg of 0521, phosphate Massey buffer and water made up to volume 0, 5 ml.

In the process of producing liposomes containing MRI, RoORS (dioleoylphosphatidylcholine), cholesterol and MRI are dissolved in ethanol. When the solvent evaporates under vacuum, a lipid film is formed. Add a saline solution of phosphate buffer (ZU mM MagHRO), 41 mm

КН2РО», 100 mM Mass) at pH 6.1, and the mixture is subjected to preliminary homogenization, which is followed by high-pressure homogenization at 15,000 psi? (approximately 15-20 cycles). This process leads to the production of liposomes, which are sterile filtered through a filter with a pore size of 0.22 μm in aseptic (class 100) conditions. The sterile product is then distributed into sterile glass containers and stored in a cold room (from 42 to 8 "С).

Liposomes produced in this way contain MRI in the membrane. ("MRI. in variant" UMO 96/33739).

O521 is added to the aqueous solution and brought to the desired concentration.

Example PI - preclinical evaluation of adjuvanted and non-adjuvanted influenza vaccine in ferrets.

Sh.1. Rationale and objectives

Influenza infection in a ferret model closely mimics influenza in humans with respect to both susceptibility to infection and clinical response.

The ferret is an animal extremely susceptible to infection with both influenza A and influenza B viruses without prior adaptation of the virus strains. Thus, this animal is an excellent model system for studies of protection afforded by vaccines administered against influenza. This study investigated the effectiveness of various trivalent split adjuvanted or non-adjuvanted vaccines to reduce disease symptoms (body temperature) and spread of virus in nasal secretions of ferrets challenged with homologous strains.

The purpose of this experiment was to demonstrate the effectiveness of an adjuvanted vaccine against influenza in comparison with a simple (non-adjuvanted) vaccine.

The endpoints of the study were: 1) Primary endpoint: reduction of virus spread in nasal washes after homologous provaccination test; 2) Secondary endpoints: humoral response analysis of NI titers. 1.2. Experiment design

II.2.1. Treatment/group (Table 1)

Female ferrets (Mizhyeia rshogiiv Tygo) at the age of 14-20 weeks were delivered from the kennel MIZAM (Hampshire,

Great Britain). On day 0, ferrets were injected with the heterosubtypic strain of NIM1 A/5(osKPoYt/24/90 (4 HTCIV/mL). On day 21, ferrets received an intramuscular injection of the full human dose (500 μg vaccine dose, 15 μg NA / strain) combinations of NIM1 A/Mem/ SaIedopia/20/99, NZM2 A/Rapata/2007/99 and

V/5papdaopad/7/97. Then, on the 42nd day, the ferrets were given a provaccination test by intranasal administration of the heterosubtypic strain НЗМ2 AL/уОотипд/3/2003 (4.5 I of TSIO5oO/ml).

Table 1

Comments dosage dosage that is provocative

1111111111111prob77 | | 77777777

Full dose for Start of administration

Trivalent and NIMI of the 1st person: 15 μg |V/m; day 21 I/N simple NA/strain (A/5osKOIt/24/90), day (9) - Start of administration

Trivalent / | Full dose for NIM 2 MRI-O521 in humans: 15 μg |V/m; day 21 I/N piposomah NA/strain (M Vioskotuga/ vo); day 6 ferrets/group. In/P - Individual/pool

II.2.2. Obtaining vaccine recipes (Table 2)

Formulation 1: Trivalent split simple (non-adjuvant) formulation: A solution of phosphate buffer concentrated by 10 orders of magnitude (pH 7.4 at a concentration of one order) is added to the water for injections, as well as a mixture containing Tween 80, Triton X- 100 and ME5 (in the amount, taking into account the detergents present in the strains). After mixing for 5 minutes, 15 μg of each of strains NIMI, NZM2 and 17.5 μg of strain B were added, mixing for 10 minutes between each addition.

The formulation is mixed for at least 15 minutes and stored at 4°C unless administered directly.

Formulation 2: Trivalent split adjuvanted influenza vaccine with MRI/O521 in liposomes:

A solution of phosphate buffer concentrated by 10 orders of magnitude (pH 7.4 at a concentration of one order of magnitude) is added to the water for injection, as well as a mixture containing Tween 80, Triton X-100 and MEZ (in the amount, taking into account detergents , present in strains). After mixing for 5 minutes, 15 μg of each of strains NIMI, NZMI2 and 17.5 μg of strain B were added, mixing for 10 minutes between each addition. The recipe is stirred for 15 minutes. Add the previously obtained mixture of the so-called ОО521-MRIip to the recipe, and then mix for at least 15 minutes.

The premix 0O521-MRI p is a mixture of liposomes (prepared from POORS 40 mg/ml, cholesterol 10 mg/ml, MRI 2 mg/ml) and immunostimulant 0521. This premix is incubated for at least 15 minutes before adding its to a trivalent split mixture.

MRI concentration. and 0521 in the final formulation is 50 μg per 500 μl. The recipe is stored at 4 "C, if not administered directly.

Note: Phosphate buffer solution, concentrated by 10 orders, is added to each recipe to achieve isotonicity, and it is concentrated by 1 order in the final volume.

The volume of H2O is calculated in order to achieve the target volume.

Table 2

Final composition of recipes 1 and 2 (recipes prepared with split strains (for 500 μl). Triton

NIM: 15 mcg 1 NZM2: 15 mcg | 375 mcg! 55 mcg / 50 mcg

A: 17.5 mcg

NIM: 15 mcg 2 NZM2: 15 mcg | 375 mcg! 55 mcg / 50 mcg 1 mg 250 mcg | 50 mcg | 50 mcg

A: 17.5 mcg

PI.2.3. Reading (Table 3)

Table From provacation sample anti-NI sho, test on . immunization, after serum 2. - antibodies (NO titers) due to inhibition of hemagglutination of the provaccination sample

PI.3. The results

A schematic representation of the results is shown in fig. 1 and 2.

PI.3.1. Humoral immunity (Fig. 1). Hemagglutination inhibition activity against vaccine strains

NZM2 (vaccine strain A/Rapata/2007/99 and provaccine strain ALL/uotipa/3/2003) was found in the sera of 6 animals per group on day 17 after the start of intranasal heterologous administration on day 21 after immunization and on 13th day after the preliminary test.

Anti-hemagglutinin antibody titers of the three influenza virus strains were determined using the hemagglutination inhibition (HI) test as detailed in Example 1.2.1. The following conclusions were made: - For two strains A/NZMI2 and for all groups, activation of NI titers was observed in all vaccinated groups after immunization. - After immunization with A/Rapata/2007/99 vaccine, statistically significant higher titers were observed

NO anti-A/Rapata/2007/99 when the trivalent split vaccine is adjuvanted with MRI/O521 in liposomes compared to the trivalent split vaccine alone. - After immunization with A/Rapata/2007/99 vaccine, trivalent split adjuvant vaccine with

MRI/O521 in liposomes could significantly increase Ni titers to the heterologous strain

ALMuotipa/3/2003 (cross-reactivity to the provaccination test with this variable strain). - After the provaccination trial with the vaccine ALLuotipd/3/2003, a significant increase in NI anti-AL/uuotipad/3/2003 titers was observed both for the trivalent split simple vaccine and for the trivalent split adjuvant vaccine with MRI /2521 in liposomes. - Statistically significant higher NI titers were observed for strains A/Mem/Caledopia/20/99 and B/Zpapdopod/7/97 when the trivalent split vaccine is adjuvanted with MRI/0521 in liposomes compared to the trivalent split vaccine alone. 1.3.2. The spread of the virus (Fig. 3).

Viral titration of nasal washes was performed on 6 animals per group, as described in detail in example I.2.3. Nasal irrigation was performed by injecting 5 ml of phosphate buffer solution into both nostrils of awake animals. The inoculum was collected in Petri dishes and placed in sample containers at a temperature of -80 "C. - Two days after the provaccination test, a statistically significant decrease in the spread of the virus was observed when the trivalent split-adjuvanted vaccine with MRIG/2521 in liposomes was administered compared to the trivalent split-adjuvanted vaccine simple vaccine. - On the 49th day: (7 days after the provaccination test) no viruses were found in the nasal washes. 1.3.3. Results of the experiment

Greater humoral responses (NI titers) were observed when trivalent split adjuvanted vaccine with MRI /20521 in liposomes was administered compared to trivalent split simple vaccine for all 4 strains.

After immunization with A/Rapata/2007/99 vaccine, only the trivalent split adjuvant vaccine with MRI/9521 in liposomes was able to significantly increase Ni titers to the heterologous strain

ALMuotipa/3/2003 (cross-reactivity to the provaccination test with this strain).

MRI/2521 in liposomal formulations proved to be effective when testing protective efficacy in ferrets (reduction of virus spread after heterologous provacation test). It was found that the observed cross-reactivity after immunization with the trivalent split vaccine with MRI/2521 in liposomes against the variable strain used for the challenge was correlated with the protective effect observed in ferrets.

Example IM - preclinical evaluation of adjuvanted and non-adjuvanted influenza vaccine in naïve and primed C57VI/6 mice.

IM.1. Design and tasks of the experiment

C57VI/6 mice with heterologous strains were used for this experiment.

The aim of this experiment was to compare the humoral (Ni titers) and medial (IC5, intracellular staining of the cytokine) immune responses induced by a commercially available trivalent split vaccine (RiIshyagih"m) (zIahoZtyypKiIpe versus a trivalent subunit vaccine (SpPigop vaccine, Adhirra!"M), and also the response of the media obtained with the indicated vaccines adjuvanted with liposomes containing only ZO-MP', only 00521 (0521 in liposomes, i.e. neutralized 0521) or MPI/0521 in liposomes In an example below in this specification , the formulations were prepared starting from the splitting of the monomass to achieve the same composition as in the Ritsagi vaccine, rather than from commercially available doses of Ritsagi.

IM.1.1. Treatment/groups

Female C57VI/6 mice aged 6-8 weeks were brought from Nagyap Nogei, the Netherlands. On day 0, mice were vaccinated with heterosubtypic strains (5 μg whole inactivated hemagglutinin Nim!

A/Veiipd/262/95, NZM2 A/Rapata/2007/99, V/5papdaopd/7/97). On the 28th day, mice were injected with an intramuscular injection of 1.5 μg of a trivalent split NA vaccine (A/Mem/CaIedopia/20/99,

ALMuotipa/3/2003, V//Lapd5zy/10/2003) simple or adjuvant (see groups in tables 4-6 below).

Table 4 (non-adjuvant) - Rishyagih similar contains liposomes liposomes li - 6 dvy in M LMtt | Initial sterol administration 0 with liposomes containing 79 ЯРВ5 77771717

IM.1.2. Obtaining vaccine recipes

Recipe for group 1:

A solution of phosphate buffer concentrated by 10 orders of magnitude (pH 7.4 at a concentration of one order of magnitude) is added to the water for injections, as well as a mixture containing Tween 80, Triton X-100 and MEZ (in the amount, taking into account the detergents present in strains). After mixing for 5 minutes, 15 μg of each of strains NIMI, NZMO2 and 15 μg of strain B were added, with mixing for 10 minutes between each addition. The formulation is mixed for at least 15 minutes and stored at 4°C unless administered directly.

Recipe for group 2:

A solution of phosphate buffer concentrated by 10 orders of magnitude (pH 7.4 at a concentration of one order of magnitude) is added to the water for injections, as well as a mixture containing Tween 80, Triton X-100 and MEZ (in the amount, taking into account detergents , present in strains). After mixing for 5 minutes, 15 μg of each of strains NIMI, NZMO2 and 15 μg of strain B were added, with mixing for 10 minutes between each addition. The recipe is stirred for 15 minutes. Concentrated liposomes containing Z30O-MRI are added. (obtained from SORS 40 mg/ml, cholesterol 10 mg/ml, ZO-MRI 2 mg/ml), in order to achieve the final concentration of MRI. 50 mcg per dose. The formulation is mixed for at least 15 minutes and stored at 4°C unless administered directly.

Recipe for group 3:

A solution of phosphate buffer concentrated by 10 orders of magnitude (pH 7.4 at a concentration of one order of magnitude) is added to the water for injections, as well as a mixture containing Tween 80, Triton X-100 and MEZ (in the amount, taking into account detergents , present in strains). After mixing for 5 minutes, 15 μg of each strain NIMI, NZM2 and 15 μg of strain B were added, mixing for 10 minutes between each addition. The formulation is mixed for 15 minutes. A premix of liposomes (derived from OARS 40 mg/ml, cholesterol 10 mg/ml) and 0521, referred to as "00521", is added to achieve a 0521 concentration of 50 mcg per dose. This preliminary mixture is incubated for at least 15 minutes before adding it to the trivalent split mixture. The formulation is mixed for at least a few minutes and stored at 4°C unless administered directly.

Recipe for group 4:

A solution of phosphate buffer concentrated by 10 orders of magnitude (pH 7.4 at a concentration of one order of magnitude) is added to the water for injections, as well as a mixture containing Tween 80, Triton X-100 and MEZ (in the amount, taking into account detergents , present in strains). After mixing for 5 minutes, 15 μg of each of strains NIMI, NZMO2 and 15 μg of strain B were added, with mixing for 10 minutes between each addition. The recipe is stirred for 15 minutes. Add a mixture of liposomes containing 30-

MRI (prepared from SORS 40 mg/ml, cholesterol 10 mg/ml, ZO-MRI 2 mg/ml) and 92521 in order to achieve concentrations of 0521 and MRI. 50 mcg per dose. This mixture is incubated for at least 15 minutes before adding it to the trivalent split mixture. The so-called recipe

"trivalent cleaved MRI /2521 in liposomes" is mixed for at least 15 minutes and stored at 4 "C, if not administered directly.

Note: In groups 1-4, phosphate buffer solution, concentrated by 10 orders, is added in order to achieve isotonicity, and it is concentrated by 1 order in the final volume. The volume of the NGO is calculated in order to achieve the target volume.

Recipe for group 5:

One dose of Addgirai"M is mixed with an equal volume of phosphate buffer solution with a pH of 7.4. The formulation is mixed for at least 15 minutes and stored at 4"C, unless administered directly.

Recipe for group 6:

Mix a solution of phosphate buffer pH 7.4 and one dose of Adagir! M. Then add liposomes containing ZO-MRI. (obtained from SORS 40 mg/ml, cholesterol 10 mg/ml, C30O-MRI 2 mg/ml), stirring, in order to achieve the equivalent of 50 μg of MRI. per dose The formulation is mixed for at least 15 minutes and stored at 4°C unless administered directly.

Note: Phosphate buffer solution is added to achieve isotonicity in the final volume. Addgira! is half of the volume of the recipe.

Recipe for group 7:

Mix a solution of phosphate buffer pH 7.4 and one dose of Addhir! M. A premix of liposomes (prepared from ORS 40 mg/ml, cholesterol 10 mg/ml) and 0521, so-called "CO521", is then added with mixing to achieve the equivalent of 50 μg of 0521. This premix is incubated for at least 15 minutes before adding it. The formulation is mixed for at least 15 minutes and stored at 4°C unless administered directly.

Note: Phosphate buffer solution is added to achieve isotonicity in the final volume. Addgira! is half of the volume of the recipe.

Recipe for group 8:

Mix a solution of phosphate buffer pH 7.4 and one dose of Addgir!" /ml, cholesterol 10 mg/ml, MRI 2 mg/ml) and immunostimulant 0521. This premix is incubated for at least 15 minutes before it is added to the Addhir/phosphate buffer solution mixture. The amount of MRI and O521 in the formulation is 50 mcg each The formulation is mixed for at least 15 minutes and stored at 4°C unless administered directly.

Note: Phosphate buffer solution is added to achieve isotonicity in the final volume. Addgira! is half of the volume of the recipe.

Table 5

The final composition of formulations 1-4 prepared with split strains (for 1 ml)

NIM: 15 μg 1 NZM2: 15 μg 750 μg 110 μg /100 μg

B: 17.5 μg 2 (identical! identical 1 | 110 μg MO μg/ Yamg | 250 μg | 50 μg/ -

With (identical! identical 1 | 110 µg MO µg/ Yamg | 250 µg | - )/ 5Ω

Table 6

The final composition of recipes 5-8 prepared with Addhira vaccine! M (1 ml) | IdozavaccinmAsdira! |. - HER Her 6 dental 5d 1777 them | 250 meg | 5bmzhe | - 07 Tsidentnykh 5th///////// 1777 img | 250 mg -- | 5b0mk 8 |identical5 | img | 250mke | 5bmkg | 5b0mk

THEM. 1.3. Reading (Table 7)

Table 7 and 21st day after

Anti-NI antibodies (NI titers) and om serum inhibition of immunization (49 day) w hemagglutination of bra, SOV8, 11 -2, IEM-u (RAS) RVI. n staining (35th day) and cytokine

In/F - Individual/pool

IM.2. The results

IM.2.1. Humoral response (NO titers 21 days after immunization).

Humoral reactions according to NI titers - fig. 4.

Hemagglutination inhibition activity against three strains of the vaccine (A/Mem/

SaIedopia/20/99, ALMuotipa/3/2003, V/iapdzy/10/2003) in sera of 8 animals per group on the 21st day after immunization. - Compared to mice immunized with phosphate buffer solution, an increase in NI titers was observed after immunization with all candidate influenza vaccines tested against all three strains (trivalent split vaccine or trivalent subunit vaccine). - For all three strains, statistically significant higher NI titers were observed in mice immunized with trivalent split vaccine adjuvanted with 0O521 alone or MRI/0521 in liposomes compared to mice immunized with trivalent split Ria vaccine alone or liposome adjuvanted , containing only 30O-MRI. The ranking for the humoral response was as follows: (MRI /2521 in liposomes - only 0О521) » (Liposomes containing only 30-

MRI. - simple vaccine) » phosphate buffer solution. - For all three strains, statistically significantly higher NI titers were observed in mice immunized with the trivalent subunit adjuvanted with O521 alone, liposomes containing only ZO-MRI or MRI/O521 in liposomes compared to mice immunized with trivalent cleaved prime vaccine The ranking for the humoral response was as follows: (MRI/2521 in liposomes - only 0BO5B21 - liposomes containing only ZO-MRI) » simple vaccine » phosphate buffer solution. - The trivalent split vaccine and the trivalent subunit induced similar NI titers when the formulations were non-adjuvanted or adjuvanted with only O521 or MPI/9521 in liposomes.

IM.2.2. Cell-mediated immune response (IC5 on day 7 after immunization).

Reactions of the CO4 T cell - Figure 5

RVMSOS of 8 mice per group were collected on the 7th day after immunization and tested in 4 pools of 2 mice/group.

In the conditions of the entire number of CO4 T-cells, specific for the Rii vaccine from a whole virus (expressing II--2, IEM-y and both cytokines): - Regardless of the formulation, identical reactions of CO4- T cells for the trivalent vaccine were observed split vaccine and trivalent subunit vaccine. - More pronounced reactions of СО4.- T cells were observed for trivalent formulations (split or subunit), with adjuvant MRI/O521 in liposomes compared to trivalent formulations (split or subunit) simple or with adjuvant in the form of liposomes, which contain only

Z0O-MRI or only 0O521. - For the cellular reaction induced by the trivalent formulation (cleaved or subunit) there is a synergistic effect of liposomes, which also contain 300-MRI 400521, compared to separately dissolved or liposomes. containing only 3O-MRI. - Ranking for cellular response was as follows: MRI /9521 in liposomes » (liposomes containing only 3ZO-MRI, - only 09521 - simple - phosphate buffer solution).

IM.3. Summary of Results and Conclusions - For all three strains, statistically significantly higher NI titers were observed in mice immunized with trivalent formulations (split or subunit) adjuvanted with 009521 or MRI/9521 alone in liposomes compared to mice immunized with simple trivalent formulations (split or subunit). Obviously, liposomes containing only C30O-MRI induce more pronounced humoral reactions when combined with a trivalent subunit than with a trivalent split vaccine. - Regardless of the formulation, similar CO4 T-cell responses were obtained for the trivalent split vaccine (Rinchagih) and the trivalent subunit vaccine (Adhirrai). - Trivalent formulations (split or subunit) with the adjuvant MRI/0521 in liposomes induced more pronounced reactions of CO4 T cells compared to trivalent formulations (split or subunit) simple or with adjuvant in the form of liposomes containing only

C30-MRI. or 0521 only (00521) in liposomes.

Example M. Preclinical comparison of a vaccine containing a preparation of split influenza antigen with adjuvant 3O-MRI /0521 in a liposomal formulation (3O-MRI. in two different concentrations)

M.1. - mice.

M.1.1. Design and purpose of the experiment.

For this experiment, C57B1/6 mice were used, which were initially injected with heterologous strains. The aim of this experiment was to analyze the humoral immune responses (NI titers) and SMI (IC5, intracellular staining of the cytokine) induced by a commercially available trivalent split vaccine (Rinzagih"M) (with adjuvanted and adjuvant liposome contain two different concentrations of ZO-MRI and 0521.

M.1.2 Treatment/Group

Female C57VI/6 mice, 8 weeks old, were brought from Nagyap Nogei, the Netherlands.

On day 0, mice were vaccinated intranasally with heterosubtypic strains (solid inactivated A/Veipod/262/95, NZM2 A/Rapata/2007/99, B/Zpapaopa/7/97). On the 28th day, mice were injected intramuscularly with a trivalent split vaccine (A/Mem/Cylidopia,

ALLuotipo, B/diapd5y) plain or with two different concentrations of adjuvant in the form of immunostimulants in liposomal formulations (see groups in Table 8 below).

Table 8

Trivalent Start of heterologous administration 1 cleaved Rin-1,5 simple rho, continuous inactivated 5 μg/20 μg/strain/ 50 μl μl intranasally

Trivalent liposomes containing | Start of heterologous injection of 2 cleaved RiIshi-1.5 μg ZO-MRI. 50 mcg of 0.5 |00O, continuous inactivated 5 mcg/20 /strain/50 mcg ml dose mcl intranasally in case of use

With cleaved Rin-1.5 ro, continuous inactivated 5 mcg/20 ml dose. μg/strain/50 μl μl intranasally

Trivalent MRI. and О521 25 mcgna Start of heterologous administration 4 cleaved Rin-1.5 05 ML ro, continuous inactivated 5 mcg/20 ,; doses. μg/strain/50 μl μl intranasally oz SHE myrin" cleaved Rin-1.5 ro, continuous inactivated 5 μg/20 0.5 ml doses and μg/strain/50 μl μl intranasal

Phosphate solution Start of heterologous injection of ro, continuous inactivated 5 μ/20 μl buffer intranasally

Recipes are prepared as in the IM example.

M.1.3 Results.

Humoral reactions according to NI titers - fig. 24.

Hemagglutination inhibition activity against three vaccine strains was detected in the sera of 9 animals per group on the 21st day after immunization. - Compared to mice immunized with phosphate buffer solution, increased NI titers were observed after immunization with all influenza vaccine candidates tested against all three strains. - For all three strains, statistically significant higher NI titers were observed in mice immunized with trivalent split vaccine, adjuvanted and 0521 at any concentration, compared to mice immunized with trivalent single split Ria vaccine (maximum p-value - 0 ,03). - No statistically significant difference was observed between the two liposomal adjuvant groups.

Cell-mediated immune reaction (IS5 on the 7th day after immunization), - fig. 25.

RVMSOS of 9 mice per group were collected on the 7th day after immunization and tested in 3 pools of 3 mice/group.

In the conditions of the total number of T-cells of CO4, specific for the Rii vaccine with a whole virus (express II--2, IRM-u and both cytokines):

As can be seen in fig. 25, the most pronounced specific reactions in the form of IEM for СО4 T cells were obtained after immunization with a trivalent split vaccine with an adjuvant in the form of the highest concentration of immunostimulants. However, the reactions of IEM T cells in the form of I/2 and I 2 were similar for the two concentrations of immunostimulators.

An example of MI. Clinical trial in the population of elderly people older than 65 years with a vaccine containing a preparation with a split influenza antigen with an adjuvant in the form of MRI /20521 in a liposomal formulation (3O-MRI in two different concentrations)

MI.1. Research design and objectives

An open-label, randomized phase ILI study to demonstrate the efficacy in the context of a cell-mediated immune response of the candidate influenza vaccines SiakhoztiiNKiipe Viioodisaiv5 containing different adjuvants administered to the elderly population (65 years and older) compared to RiIpagih" M (known as a-Kih "m in Belgium), which is administered to adults (aged 18-40).

Four parallel groups were formed: - 75 adults (aged 18-40 years) in one control group, who received one dose of Ritsagih "M (Rivagih Group). - 200 elderly people (65 years and older) were randomly assigned to in a ratio of 3:32 into three groups: - one group of 75 people who receive an influenza vaccine adjuvanted with ABZO1B; - one group of 75 people who receive an influenza vaccine with an ABO1 E adjuvant; - a control group of the vaccine A swarm of 50 people who receive one dose of RiImchagih "M,

The primary task

The main objective is to demonstrate the effectiveness on the 21st day after vaccination of adjuvanted influenza vaccines administered to the elderly (65 years and older) compared with RiIchagih"M administered to adults (aged 18-40 years) in terms of due to the occurrence of T-lymphocytes CO4, specific for influenza, producing at least two different cytokines (CO401I,, 1-2, TME-os, TEM-u).

Secondary tasks

The secondary tasks are the following: 1) Evaluation of the safety and reactogenicity of vaccination with adjuvant candidate vaccines against influenza within 21 days after intramuscular administration of the vaccine to the elderly (65 years and older). RiIsagih "M is used as a standard. 2) Assessment of the humoral immune response (anti-hemagglutinin titer) 21, 90 and 180 days after vaccination with adjuvant candidate vaccines against influenza. Risagih "M is used as a standard.

Tertiary task

The third task is to assess the cell-mediated immune reaction (production of IEM, HER-2,

CO40Y and TME-o and the reaction of B-lymphocytes) 21, 90 and 180 days after vaccination with adjuvant vaccines against influenza. RiItsagih M is used as a standard.

MI.2. Vaccine composition and administration

Two different adjuvants are used: 1. Liposome-based ABO1B adjuvant containing 50 μg of MRI. and 0521. 2. ABO1E formulation of ABO1B diluted by two orders.

Control: a full dose of RiIshtsagih "M intramuscularly.

All vaccines are intended for intramuscular administration. The strains used in the five vaccines are those recommended by the World Health Organization for the 2005-2006 Northern Hemisphere season, i.e., A/Mem/CaIedopia/20/99 (NIMI), A/Mem/ Mogk/7/2004 (НЗМ2) and

In/Lapazy/10/2003.

The three antigens of the inactivated split virion (monovalent of the in-bulk formulation) used in the formulation of the adjuvanted influenza vaccine candidate are the same as the active ingredients used in the formulation of the commercially available inactivated influenza vaccine

Einagih "m/a-Kih 77--35K Vio with a split virion. they are obtained from viruses grown in eggs. The specified influenza strains are recommended for the 2005/2006 season, as used in the formulation of commercial

Eitsagih "m/a-Kih M for 2005/2006

The strains used in the three vaccines are the strains recommended by the World Health Organization for the 2005-2006 season in the northern hemisphere, i.e. - A/Mem SaIedopia/20/99 (N, M.) IMA-116 ; - A/Mem Hotk/55/2004 (НЗМ2) MUMO Kh-157; - VLLapazy/10/2003.

Similar to Ritsagih "M/a-Kih" M adjuvant vaccine contains 15 μg of hemagglutinin (HA) of each strain of the influenza virus per dose.

MI.2.1. Description of series of adjuvant vaccine AZO1B

The adjuvant vaccine candidate against influenza ABOIV is a 2-component vaccine consisting of concentrated trivalent inactivated antigens of the split virion, placed in a glass ampoule, and the glass ampoule itself containing the adjuvant ABZO1B. During the injection, the contents of the ampoule with the adjuvant are removed and injected into a test tube containing concentrated trivalent inactivated antigens of the split virion. After mixing, the contents are collected in a syringe and the needle is replaced with a needle for intramuscular injection. The used needle is replaced with a needle for intramuscular injection and the volume is adjusted to 1 ml. One dose of reconstituted adjuvant vaccine candidate against influenza ABZO1B corresponds to 1 ml.

The candidate adjuvant vaccine against type ABO1B is a preservative-free vaccine.

MI.2.2. Composition of the adjuvant clinical series ABO1B

One dose of reconstituted adjuvant vaccine against influenza AZOIV corresponds to 1 ml. Its composition is given in the table. 8. It contains 15 μg NA of each strain of influenza virus, as in the registered vaccine

EiItsagih "M/a-KihFf).

Table 8

The composition (influenza and adjuvant components) of the reconstituted adjuvant vaccine candidate against influenza ABOTV

Inactivated cleaved viribiniide ОЙ 77777771 - Liposomes 11111111 Dioleylphosphatidylcholine (PORS)

MI.2.3. The production method of the AbO1B adjuvanted vaccine series

Production of adjuvant vaccine against influenza ABO1B consists of three main stages: - Formulation of trivalent final mass (concentrated 2 times) without adjuvant and filling the container with antigen. - Obtaining ABO1B adjuvant. - Extemporaneous dilution of the adjuvant split vaccine of the AZO1B virus.

Formulation of the trivalent ready-made drug in the form of in-bulk without adjuvant and filling the container with antigen

Volumes of three monovalent preparations in bulk form based on the NA content determined for each of the monovalent preparations in bulk form before preparation of the preparation and for a target volume of 1320 ml. A concentrated saline solution of phosphate buffer is diluted in water for injections

PO» Ma/Ko (80 μg/dose) and a previously prepared mixture of Tween 80, Triton X-100, and c-tocopheryl hydrosuccinate. Next, three concentrated monopreparations in the form of in-bulk (A/Mem/

SaIedopia/20/99 IMA-116, A/Mem/ Moik/55/2004 MUMS Kh-157, V/iapdzy/10/2003) are introduced sequentially to the final solution consisting of a saline solution of phosphate buffer/Tween 80-Triton X -100- solution of o-tocopheryl hydrosuccinate (pH 7.8, 81 mM Mass, 1.56 mM KSI, 4.79 mM MagHPO, 0.87 mM

KNeRO", 7.2 mM MangRO", 72.8 mM K»HRO", 750 μg/ml Tween 80, 110 μg/ml Triton X-100 and 100 μg/ml α-tocopheryl hydrosuccinate) in order to obtain a final concentration of 30 μg NA ( NITM1 and NZM2) strains per ml of trivalent final mass (15 μg NA/strain A /500 μl of trivalent final mass) and 35 μg NA of strain B (17.5 μg NA/ strain B /500 μl of final trivalent preparation in the form of in-bulk ). Between the addition of each monovalent drug in the in-bulk form, the mixture is stirred for 10-30 minutes at room temperature. After adding the last monovalent drug in bulk form and stirring for 15-30 minutes, the pH is checked and adjusted to 7.65:0.25 using NSI or Mmaon.

The final trivalent preparation of antigens in the form of in-bulk is filled in aseptic conditions in sterile glass ampoules of type I with a volume of 3 ml (European Pharm.). Each ampoule contains a volume of 600 μl (500 μl and 100 μl of free space).

Obtaining the adjuvant in the form of in-bulk OR1B and filling the container with the adjuvant

ABO1B adjuvant is prepared by mixing two components: 0521 and liposomes containing

MRI. The method of obtaining each of the specified components is given below. 0521 is a triterpene glycoside obtained from the bark of the Ocia zaropagia tree, produced by the Adiyya company

Umogopeziweg, MA, USA (currently named Apiidepis5). 521 is supplied as a lyophilized powder to O5K VioIodisaie. Preparation of 20521 in O5K Vio consists of suspending 0521 powder in water for injection at a concentration of approximately 5 mg/ml, pH adjustment to pH 6.0-0.2, and sterile filtration.

Liquid mass 0521 is stored at -20" C in polyethylene containers.

MRI is 3-O-deacyl-4-monophosphoryl lipid A, obtained by sequential acid and base hydrolysis of lipopolysaccharide from strain Ke595 of Zaitopea tippezoia. It is produced by O5K

Viioiodisai5, Hamilton, Montana. MRI. in bulk form and supplied as a lyophilized triethylamine (TEA) salt.

In the process of producing liposomes containing MRI, SORS (dioleylphosphatidylcholine), cholesterol and

MRI. dissolve in ethanol. A lipid film is formed by evaporating the solvent under vacuum.

A phosphate buffered saline solution containing 9 mM MagHPO, 41 mM KNePoO, 100 mM (pH 6.1) is added, and the mixture is pre-homogenized followed by high-pressure homogenization at 15,000 psi: (-/- 20 cycles). This procedure leads to the production of liposomes, which are sterile filtered through a filter with a pore size of 0.22 μm in an aseptic (class 100) zone. Next, the received sterile product is placed in sterile glass containers and stored in a cold room (2-8 "С).

The resulting sterile mass of liposomes is mixed with a sterile solution of in-bulk 0521. After mixing for 30 minutes, the resulting mixture is added to a mixture of water for injections and phosphate 500 mM, weight 1M, pH 6.1 when diluted 10 times. The amount of phosphate 500 mM, Mass 1M (pH 6.1) when diluted 10 times is calculated in order to achieve isotonicity in the final volume. Check the pH. Then sterile filtration of the adjuvant (0.22 μm) is carried out and it is placed in ampoules under aseptic conditions. Ampoules are stored at a temperature from - 2 to 18 "C.

Solvent OR1 B is an opalescent colorless liquid, free of mechanical inclusions, contained in a sterile glass ampoule type 1. The target filling of each ampoule is 0.7 ml according to the specification (20.5 ml).

Extemporaneous dilution of adjuvant split vaccine of ABO1B virus

During the injection, the contents of the ampoule containing the adjuvant are withdrawn and injected into a test tube containing concentrated trivalent inactivated antigens of the split virion. After mixing, the contents are collected in a syringe and the needle is replaced with a needle for intramuscular injection, and the volume is brought up to 1 ml. One dose of reconstituted adjuvant vaccine - ABO1B influenza candidate corresponds to 1 ml.

MI.2.4. Description of series of adjuvant vaccine ABO1E

The adjuvant vaccine candidate against ABOTE influenza is a three-component vaccine consisting of concentrated trivalent inactivated antigens of the split virion, placed in a glass ampoule, and a glass ampoule containing the AZOV adjuvant, and a glass ampoule containing a solvent ( sodium chloride solution for injections) to dilute ABO1B by two orders of magnitude.

To prepare the adjuvant ABZO1TE, the contents of the ampoule with the solvent are collected in a syringe and injected into a test tube containing the adjuvant AZO1B, followed by mixing. During the injection of 600 μl of adjuvant

ABOTE is drawn into a syringe from an ampoule containing ABOTE and injected into a test tube containing concentrated trivalent inactivated antigens of the cleaved virion. After mixing, the contents are collected in a syringe and the needle is replaced with a needle for intramuscular injection. One dose of reconstituted adjuvant vaccine candidate against influenza ABZO1B corresponds to 1 ml.

Adjuvanted vaccine candidate against influenza ABO1TE is a preservative-free vaccine.

MI.2.5. Composition of the clinical series of adjuvant ABOTE

One dose of reconstituted ABO1E influenza vaccine corresponds to 1 ml. Its composition is given in table. 9. It contains 15 μg NA of each strain of the influenza virus, as in the registered vaccine RiIshtsagih "M/a-

KihF).

Table 9

The composition (influenza and adjuvant components) of the reconstituted adjuvant vaccine candidate against ABOTE influenza

Inactivated split vibrios - LIPOSOMES!

MI.2.6. The method of production of the ABOTE adjuvant vaccine series

The production of adjuvant pre-a influenza ABO1B vaccine consists of three main stages: - Formulation of the final trivalent drug in the form of in-bulk mass (2x concentrated) without adjuvant and filling the container with antigen. - Obtaining ABO1B adjuvant. - Preparation of ABOTE adjuvant after extemporaneous dilution of adjuvant vaccine of split ABO1E virus.

Formulation of the final trivalent drug in the form of in-bulk without adjuvant and filling the container with antigen

See section M.2.3 regarding adjuvant vaccine against influenza ABO18B.

Obtaining an adjuvant in the form of in-bali. A5O1B and filling the container with adjuvant

See section M.2.3 regarding adjuvant vaccine against influenza ABO18B.

Extemporaneous dilution of the adjuvanted vaccine of the cleaved ABOTE virus

To prepare the ABZO1TE adjuvant, the contents of the ampoule with the solvent are collected in a syringe and injected into a test tube containing the A5BO1B adjuvant, followed by mixing. During the injection of 600 μl of adjuvant

ABOTE is drawn into a syringe from an ampoule containing ABOTE and injected into a test tube containing concentrated trivalent inactivated antigens of the cleaved virion. After mixing, the contents are collected in a syringe and the needle is replaced with a needle for intramuscular injection. One dose of reconstituted adjuvant candidate influenza vaccine ABOTE corresponds to 1 ml.

Four scheduled physician visits per subject: on days 0, 21, 90, and 180 from blood sampling at each visit to assess immunogenicity.

Vaccination scheme: one injection of influenza vaccine on day 0.

MI.2.7 - Immunological analyses

Hemagglutination inhibition test

Immune response is determined by measuring hemagglutination inhibitory antibodies (HI) using the method described by the World Health Organization Influenza Collaborating Center, Centers for Disease Control, Atlanta, USA (1991). Antibody titer measurements were performed on frozen and thawed serum samples using a standardized and comprehensively validated micromethod, using 4 hemagglutination inhibition units (4 IU) of the relevant antigens; 0.5 9o suspension of chicken erythrocytes. Non-specific serum inhibitors were removed by heat treatment and receptor-depleting enzyme. The obtained sera were evaluated for NO antibody levels. Starting with an initial dilution of 1:10, serial dilutions (with a factor of 2) were prepared to a final dilution of 1:20480. The end point of the titration was taken as the highest dilution step that showed complete inhibition (100 Fo) of hemagglutination. All analyzes were performed in duplicate.

Flow cytometry of cytokines (SES), which is used to assess the occurrence of cytokine-positive T-cells cp4 or СО8).

Antigen-specific CO4 and CO8 peripheral blood T-cells can be re-stimulated i.p. migo to produce I/-2, CO40Y, TME-alpha and IM upon incubation with their respective antigen. Thus, antigen-specific CO4 and CO8 T-cells can be enumerated by flow cytometry after conventional labeling of cell phenotype by immunofluorescence, as well as intracellular cytokine production. In this study, influenza vaccine antigens will be used as antigens to re-stimulate influenza-specific T cells. Results will be expressed as the incidence of cytokine(s)-positive CO4 or COB8 T cells within the CO4 or COB8 T cell subpopulation.

EPI5ROT, used to assess the incidence of B-lymphocytes

The technology of EyYi5roi for B-cells makes it possible to make a quantitative determination of B-lymphocytes, specific in relation to this antigen. B-lymphocytes can be stimulated to differentiate into plasma cells and migo after cultivation with Cro for 5 days. Antigen-specific plasma cells generated by ip myo can thus be enumerated using the analysis of the B-cell lineage. The extract of plasma cells generated by miigo is incubated on antigen-coated culture plates. Plasma cells specific for the antigen will form antibody/antigen spots that can be detected by a conventional enzyme-linked immunosorbent assay. In this study, influenza vaccine or anti-human immunoglobulin strains are used to coat culture plates to enumerate anti-influenza cells or plasma cells secreting dos, respectively. The results are expressed as the occurrence of antigen-specific plasma within a million plasma cells producing Id.

Research Characteristics of RVMS

Expression of selected surface/activation markers (ie, CO4., CO8, SraBno, SraB5kKA, C028, C027 or some KICs) can be performed. The function of vaccine-stimulated T lymphocytes can be determined by analyzing homing markers (ie, CSK7, CSHOK5), cytokines (T-helper 1 or T-helper 2 cytokines), or by analyzing the expression of factors associated with regulatory functions such as Rohr3, STI A-4, or TOerD. In particular, the population of CO8-CO28- or populations of other regulatory T cells can be analyzed in relation to the humoral responses of B and T cells to the vaccine antigen.

MI.3. Immunogenicity study results

MI.3.1. Media endpoints and results

To characterize the response of media after vaccination with adjuvanted vaccines against influenza, T-lymphocytes COA and CO8 were stimulated repeatedly by ip miigo, using antigens from three strains of the vaccine (used individually or in the fund). Influenza-specific T-lymphocytes cpa/CO08 were enumerated using flow cytometry followed by conventional immunofluorescence labeling of intracellular cytokine production (1-2, IEM-y, TME- and 20401).

Evaluation of the primary endpoint.

On day 21: Media reaction in all subjects in terms of incidence of influenza-specific T-lymphocyte CO4 per 105 in tests producing at least two different cytokines (II - 2, TEM-u, TME-o ii 040).

To assess the reaction of the media, the occurrence of CO4, specific to the flu, is analyzed in the following way:

Using an efficacy approach, the efficacy of at least one adjuvanted influenza vaccine candidate (administered to adults 2 65 years of age—a group termed the elderly or RI 0) compared with the M vaccine (administered to adults 18-40 years - the group that was called the young age group or the UMO group) was reached if the upper limit of the two-sided 98.75 95 confidence interval on the ratio of the geometric mean value (SM) (between the group that received

РНагих "m (18-40 years old) and the adjuvant vaccine-candidate against influenza (group 2 65 years old) from the point of view of occurrence of T-cells CO4 specific for influenza, producing at least two cytokines on the 21st day was below 2.0.

Shev Ahdoroslyi Fr

SMad flu vaccine

On the 21st day after vaccination, computer processing of the 98.75 95 confidence interval of OM ratios was performed using the analysis of covariance model (AMSRMA) on the logarithm transformation of 10 data of incidence rates. The AMSOMA model included vaccine group as a fixed effect (RiIsagih "m (18-40 years) vs adjuvanted influenza vaccine candidate (265 years)) and incidence of previous vaccination as a regressor. OM ratios and their 98.75 95 CIs were obtained as transformation of the difference index of the corresponding group in the model 98.75 95 CI for the adjusted ZM is obtained by exponential transformation of 98.75905 CI for the least square mean value of the group mentioned above in the AMSOMA model.

Results - Inferential analysis (Table 10)

Table 10 presents the corrected SM and ratio of OM (with their 98.75 95 CI) of influenza-specific T-lymphocyte CO4, which produces at least two cytokines (1-2, IEM-u, TME-o; and

СО401И) on the 21st day after re-stimulation of ip migo with "fundamental antigens II". For each adjuvanted influenza vaccine, the upper limit of the bilateral 98.75 956 Ci OM ratio is well below the clinical limit of 2.0. This fact demonstrates the effectiveness of both adjuvant vaccines against influenza, which are administered to the elderly, in comparison with the RiIvaagih "M vaccine, which is administered to adults aged 18 to 40 years, from the point of view of occurrence of CO4 specific to influenza.

Table 10

Adjusted ratio of CM of influenza-specific T-cells CO4 producing at least two cytokines after re-stimulation with stock antigens of the vaccine, day 21 (ATP cohort, immunogenicity test) 077111111111111111111171111111 CM ratio (RiiuUMs/ABOBIV).d/-/: //ZhZ o pimMa | WALLPAPER | 77777777 Я7771717171717171717171719889сС11сС1С 07711111 The ratio of ZMO (RIUMS/AVOYIV).d-/-: o pimMa | WALLPAPER | 77777777 Y7771717171717171717171719889сС11сС1С

Adjusted SM - geometric mean antibody, adjusted for the main titer; M - number of subjects with available results before and after vaccination; 98.8 95 CI-98.8 95 confidence interval for adjusted OM ratio (Apsom model: baseline adjustment); GI. - lower lignite, ОИ. - upper limit; Data source - appendix, table SHA.

Results - Descriptive analysis (fig. b)

The main findings were as follows: 1) Before vaccination, the media response is higher in young adults than in the elderly. 2) After vaccination: - a booster effect of the influenza vaccine was observed in the media reaction in young adults (18-40 years); - media response in elderly people who received adjuvant influenza vaccine is comparable to media response in young adults.

The difference in the reactions of CO4 T-lymphocytes for the studied (I/-2, СбО0401Й, TME-о and IEM-у) all cytokines before and after vaccination was significantly higher in adjuvant vaccines compared to RiImtsagih "M" in all trials.

Analysis of the tertiary problem:

To evaluate the tertiary end point, the occurrence of influenza-specific T-lymphocytes СО4/СО8 and B-lymphocytes was measured on days 0, 21, 90 and 180. - Summed incidence of influenza-specific T-lymphocytes СО4/С08 (descriptive statistics) for each vaccinated group on days 0 and 21 for each antigen. - A non-parametric test (Wilcoxon test (UMisohop)) was used to compare the location of the difference between the two groups (adjuvanted influenza vaccine vs. Richagih"M), and the statistical p-value was calculated for each antigen in each different trial. - Descriptive statistics in the individual difference between the responses at day 21/day 0 (before and after vaccination) is calculated for each vaccination group and for each antigen in each different trial - Non-parametric test (Wilcoxon test) is used to compare the individual difference (before and after vaccination), and the statistical p-value will be calculated for each antigen in each different test.

Table 11 presents the p-values from the Wilcoxon test, which are used to compare the difference in the occurrence of CO4 T-lymphocytes specific for influenza.

Results - Evaluation of the tertiary end point (Table 11) The main findings are as follows: - The geometric mean of the incidence of influenza-specific sbra4 T cells before vaccination was similar in all groups of elderly people, but they were higher in adults aged 18 and older up to 40 years. - In elderly people, the incidence of influenza-specific T-lymphocytes CO4 after vaccination (on the 21st day) was significantly higher after vaccination (with adjuvanted vaccines than

Rinagih "m, - After vaccination, the occurrence of T-lymphocytes specific for influenza CO4 remained lower in elderly people vaccinated with adjuvant vaccines ABZO1B or

AZO1E than in adults aged 18 to 40 years, vaccinated with RiIchagih "M, - The incidence of influenza-specific T-cells CO8 of the geometric mean before and after vaccination was basically similar in all groups.

Table 11

Inferential statistics: p-values from Kruskal-Wallis tests for CO4 T cells at each time point (ATP cohort, immunogenicity trial)

Description of the test 11111101 АВОИВпротыгчвСО | OR: countered IO Z

77111111 Day | Day 2! | Deno | Day!

Results - Evaluation of endpoints of the humoral immune response

Observed variables:

At days 0, 21, 90 and 180: serum hemagglutination inhibitory antibody (HI) titers were analyzed separately against each of the three influenza virus strains represented in this vaccine (anti-NIMI, anti-NZM2 and anti-B antibodies).

The limit frequency indicator for NO antibodies against all vaccine antigens was determined in the laboratory before analysis (it is equal to 1:10). A seronegative subject is a subject whose antibody titer is below the threshold frequency. A seropositive subject is a subject whose antibody titer is greater than or equal to the limit frequency indicator. The antibody titer below the cutoff is an arbitrary value of half the cutoff.

Based on NI antibody titers, the following parameters are calculated: - Geometric mean titers (GMT) of NI antibodies on day 0 and 21, calculated using the anti-Iod mean value of Id titer transformations. - Seroconversion factors (SE) at day 21, defined as the fold increase in serum NO CMT at day 21 compared to day 0. - Seroconversion rates (5C) at day 21, defined as the percentage of vaccinated individuals with titres

NO before vaccination "1:10 and titers after vaccination 21:40, or titers before vaccination 21:10 and an increase of at least 4 orders of titers after vaccination. - Seroprotection rates (PE) at day 21, defined as the percentage of vaccinated persons with titers

NO Serum 9:40 p.m. 95 95 SI for OM is obtained within each group separately. First, the 95 95 CI of the mean iodine-converted titer is obtained, assuming that the iodine-converted titers are normally distributed with unknown variance. Next, 9595 SI for SM is obtained by exponential transformation of 95 95 SI for the average value of the iodine-transformed titer.

The absence of a serological result for a specific measurement of antibodies is not replaced. Thus, a subject without a serological result at a given time point is not considered in the analysis of a given test for a given time point.

The results of the humoral immune reaction (Fig. 7 and Table 12)

The geometric mean values of NI antibodies before vaccination for all Z vaccine strains were within the same interval in the 4 treatment groups. After vaccination, there is a clear effect of 2 adjuvants that increase the humoral response in the elderly compared to standard

EiImagih in the same population (Fig. 7, shown on a linear scale, but a similar effect is also evident on a logarithmic scale).

Average geometric values are significantly higher for NIMI than for OR1E; - significantly higher for NZM2 for both adjuvants. - No significant difference was observed in terms of post-vaccination CMT between the two groups of subjects who received adjuvanted vaccines.

Twenty-one days after vaccination, subjects vaccinated with Rivchagih (18-40 years old) had a stronger NO reaction to Mem SaIedopia and VLapdza strains. As shown in Table 12, adjuvanted influenza vaccines exceeded the requirements of the European authorities for the annual registration of split-virion influenza vaccines and "Note for the guide on the harmonization of requirements for influenza vaccines for the immunological assessment of annual strain changes" (SRMR/VUUR/214 /96)| in subjects over 60 years old.

After vaccination, a statistical difference was observed from the point of view of seroprotective indicators of NO antibodies between the group of Rivtsagih (265 years old) and РішШАЗО18В and РішШАБЗО1ТЕ for the A/Н1М1/Mem SaIedopia strain.

For each vaccine strain, the seroprotection rates for the 2 groups of adjuvant influenza vaccine are in the same interval compared to the Rivagih group (18-40 years).

For each strain of vaccines, the seroprotection indicators for the 2 groups of adjuvant influenza vaccine are in the same interval compared to the group of cattle (18-40 years old), except for the Mem SaIedopia strain.

Table 12

Seroprotection rates, seroconversion rates, and conversion factors at 21 days

(ATP cohort, immunogenicity test)

Indicator

The rate of seroconversion (24-Factor

Strains Group M of seroprotection ordinal conversion (TITR NI240) 95 increase) I95 Fo SI| Fo 95 95 SI| to

EU standard (60 years) | | 56095 | 53095 2 -: | 520 (EU standard (for years) | | 57095 | »40956 | 525

A/Mem

Saiedopia (NIMI) d/Mem Woik (nZ3mg)

B/paws (B)

M - total number of subjects; 95 - Percentage content of subjects with a titer on the 21st day within the specified interval; CI - confidence interval.

MI.3.2. Conclusions regarding the study of immunogenicity - The incidence of influenza-specific CO4 before vaccination was significantly lower in the elderly compared to adults aged 18 to 40 years. Post-vaccination incidence (day 21) remained lower in the elderly compared to the young after RiItsagih'm vaccination. On the contrary, demonstrated efficacy in terms of post-vaccination incidence of influenza-specific COA4 after vaccination with adjuvanted vaccines of the elderly compared to vaccination of adults aged 18 to 40 years with Richagih "M, - Regarding the humoral immune response in terms of antibody reactions NO, all influenza vaccines met the requirements of the European authorities regarding the annual registration of inactivated influenza vaccines "Note for the manual on the harmonization of requirements for influenza vaccines for the immunological assessment of annual strain changes" (SRMR/VUUR/214/96). in the elderly, adjuvanted vaccines mediated at least a tendency to achieve a higher humoral immune response to influenza hemagglutinin than RiItsagih "mM. Table 13 shows the significant difference between the humoral immune response against each vaccine strain mediated in the elderly by adjuvanted vaccines compared with M.

RIsagih"M, elderly people vaccinated with adjuvant vaccines, showed a trend of increasing CMT and seroconversion factor after vaccination on the 21st day against the A/Mem/Mogk strain.

Table 13

Influenza strains that demonstrated a significantly higher humoral immune response (based on not exceeding 95 95 CI) in the elderly vaccinated with different adjuvanted vaccines compared to RiIshtsagih in the same population dvav dentin sleep | овннтя | of seroconversion of seroprotection seroconversion of HBV 0 IA/lemuyuk | 0 |A/yuemSaivdopa |: by mass ddeitjo | 0000000 fAMensayyut

CMT after vaccination. - the geometric mean value of the titer after vaccination.

MI.A4 Conclusions regarding the study of reactogenicity

MI.4.1. Registration of adverse effects (AEs)

Persistent symptoms were registered (see table 14), which appeared in the next period of medical observation for 7 days (vaccination day and 6 following days). Unstable symptoms that appeared during the next period of medical observation for 21 days (vaccination day and 2043 subsequent days) were also recorded. The intensity of the following side effects was assessed as described in Table 15.

Table 14

Resistant local/general side effects

Persistent local side effects Persistent general side effects

Pain at the injection site Fatigue

Redness at the injection site Fever

Swelling at the injection site Headache

Hematoma Pain in the muscles 11111111 Doznob 11111111 |Pain in the joints of the hand due to the injection 11111111 |Pain in the joints in other areas

M.V. The temperature was recorded in the evening. If it was necessary to make additional temperature measurements at other times of the day, then the highest temperature was recorded.

Table 15

Severity scale for persistent symptoms in adults

Side effect Intensity scale Parameter 777717171710 7 | absent sp pyrium 11101111 when touched

Pain at the injection site --- when moving the limb prevents normal activity. era registered the largest diameter

Redness at the injection site: surfaces. the largest diameter was registered by Sor Adoo

Swelling at the injection site: the largest diameter was recorded on the surface of the soda

Hematoma at the injection site: surfaces

Fever" 0 registered temperature us "E 77171710 | absent from bi easily transferred

Headache I - interferes with normal activity interferes with normal activity 77171710 | absent easily tolerated

Fatigue I - interferes with normal activity interferes with normal activity 77171710 | missing

Pain in the joints, at the injection site is easily tolerated and in other areas interferes with normal activity interferes with normal activity 77171710 | absent easily tolerated

Pain in the muscles I: interferes with normal activity interferes with normal activity 77171710 | absent easily tolerated

Chills Р --ИЯ-- - interferes with normal activity interferes with normal activity " Fever is defined as an additional temperature of 237.5 70 (99.5 Р).

The maximum intensity of redness/swelling at the injection site was evaluated as follows: 0 is 0 mm; 1 is » 0 - x 20 mm; 2 is » 20 - x 50 mm; C is » 50 mm.

The maximum intensity of fever was assessed in the following way: 1 is » 37.5 - x 38.0 "C; 2 is » 38.0 - x 39.0 "C; Z is » 39.0.

The investigator makes an intensity score for all other side effects, i.e. non-persistent symptoms, including persistent side effects reported throughout the study. The assessment is based on the clinical assessment of the researcher. The intensity of each registered AE is evaluated according to one of the following categories: 1 (mild) - AE that is easily tolerated by the subject, causing minimal discomfort and not interfering with daily activities; 2 (moderate) - AE that causes significant discomfort, interfering with normal daily activities;

C (severe) - AE that interferes with normal, daily activity (in adults/adolescents, such

AE, for example, interferes with attending work/school and requires necessary corrective therapy).

M.1.4.2. Registration of adverse effects (AEs)

In elderly people, the reactogenicity observed when adjuvant vaccines were administered in conditions of both local and general symptoms was higher than when using Ritsagih "M,

Not only the incidence, but also the intensity of symptoms increased after vaccination with adjuvant vaccines (Fig. 8). The C symptom scale showed a tendency to increase in the group that received the adjuvanted vaccine with the highest concentration of immunostimulants (MR, 0521) compared to the group that received the adjuvanted vaccine where the immunostimulants were at a lower concentration. In all cases, however, the symptoms disappeared quickly.

Example MI!: Preclinical evaluation of adjuvanted NRU vaccines in mice.

In this study, a bivalent human papilloma virus (HPV) antigenic composition was used, combining virus-like particles (MIPs) formed from 1 HRM16 and 11 from HRM18 as an antigen. The task of the study was to compare the effectiveness of this antigenic preparation when it is introduced into the recipe with ABO1B and 1/5 dilution of ABO18B, contrasting it with the adjuvant currently used in the O5K vaccine against uterine cancer, ABZO4 (MRI'. on alums) .

MIII.1 - Vaccination

On days 0 and 28, mice (number - 12 per group) were injected with vaccine formulations containing HRM16/18 and 1 (2 mcg or 0.5 mcg of each), obtained from the Ni-5 80/80 process! and the formulation of which includes ABZO4 (50 mcg MRI. made on alum) or ABO1B (50 mcg 0521 - 50 mcg MRI in 0.5 ml) 1/10 and 1/50 of the human dose. Since the study was conducted in mice, 1/10 of the human dose can be taken as equivalent to the human ABO1B formulation, i.e. 50 mcg 2521 and 50 mcg MRI. in 0.5 ml, and 1/50 can be taken as a 1/5 dilution of the ABO1B formulation for humans, i.e. 10 μg of 0521 and 10 μg of MRI in 0.5 ml.

Blood samples were taken on the 14th and 45th day after the administration of a dose of II for the purpose of analyzing the total number of specific antibodies of the anti-II1 type in individual sera. Intracellular staining of cytokines was measured on the 7th and 14th day after the second immunization with PBMC, and on the 45th day after the second immunization, spleen cells were used for measurement. The occurrence of specific B-lymphocytes MI R was measured on the 45th day after the second immunization, using spleen cells.

MIII.2 - Anti-NRM 16/1811 EPIZA

Quantitative determination of anti-NRMU-16b and NRMU-18 1 antibodies was carried out with the help of EGISA, using NRMU-16 and NRMU-18 I 1 as a coating. Antigens were diluted to a final concentration of 0.5 μg/ml in a phosphate buffer solution and adsorbed overnight at 4°C on microtiter plates with 96 wells (Mahizogr Ittiporiaje, Nunk, Denmark). Then the plates were incubated for 1 hour at 37°C. C with a phosphate buffer solution containing 1 95 albumin; cow's serum (saturation buffer). Sera diluted in a buffer containing a phosphate buffer solution of 0.1 95 Tween 20--1 95 bovine serum albumin were added to plates coated with HRM 11 and incubated for 1 hour 30 minutes at 37 °C. The plates were washed four times with phosphate buffer solution, 0.195 Tween 20, and biotin-conjugated anti-mouse Id (Bako,

Great Britain), diluted 1/1000 in saturation buffer, was added to each well and incubated for 1 hour 30 minutes at 37 "C. After the washing step, streptavidin horseradish peroxidase (Rako, Great Britain), diluted 1/3000 in saturation buffer, was added for additional 30 minutes at 37 "C. Plates were washed as above and incubated for 20 minutes at room temperature with a solution of 0.04 95 O-phenylenediamine (Zidta), 0.03 95 Ng2O" in 0.195 Tween 20, 0.05 M citrate buffer, pH 4.5. The reaction was stopped with 2M NaOH and read at 492/620 nm.

EHIZA titers were calculated from the ZoiMahrRho reference (using the four-parameter equation) and expressed in IU/mL.

MIII.Z - Intracellular staining of cytokines (IS5)

Intracellular staining of T-cell cytokines was performed on peripheral blood lymphocytes on the 7th and 14th day after the second immunization. Mononuclear cells of peripheral blood (1 depot/group) or spleen cells (4 depots from organs per group) were selected from mice. Antigen stimulation of spleen cells was carried out at a final concentration of 5 105 cells/ml (microplates with 96 wells) with MI R 16 or 18, (5 μg/ml) and antibody (1 μg/ml) С0494 СО28, and then incubated for C hours at 37 "C. After the antigen re-stimulation stage, cells were incubated overnight at 37 "C in the presence of brefeldin (1 μg/ml) at 37 "C in order to inhibit cytokine secretion. Cell staining was performed in the following way: cell suspensions were washed, resuspended in 50 μl of a phosphate buffer solution containing 195% fetal calf serum containing 295 μl of blocking reagent (1/60; 2.402). After incubation for 10 minutes at 4 "C, 50 μl of the anti-CO4-ARS mixture (1/50 ) and anti-CO8 on Cp (1/50) and incubated for 30 weeks at 4 °C. After washing in a phosphate buffer solution containing 195% fetal calf serum, the cells were permeabilized by resuspending in 200 μl Suiyuh-Suiorept (Kiiyi VO) and incubated for 20 minutes at 4 "S. Next, the cells were washed with Rept UmMazp (Ki VO) and resuspended in 50 μl of a mixture of anti-IEM-y ARS (1/50) and anti-II-2 RITS (1/50), diluted with Rept Umakhp. After incubation for 2 hours at 4 "C, the cells were washed with Regt UmMazhpy and resuspended in a phosphate buffer solution containing 195% fetal calf serum and 195% paraformaldehyde. The samples were analyzed using BAS5. Live cells were sealed (E5C/55542) and assembled - 20,000 transformations (lymphocytes).The percentage content of IEM-y or I 2-- was calculated on СО4- and Сов closed populations.

MII.4 - B-lymphocytes

On the 45th day after the second immunization, the mice were killed, the cells of the spleens were removed with the help of a gradient of lymphopreparation (cedarlan). Then B-cells were resuspended in KRMI 16 medium (Gibko-Sirso) containing additives (sodium pyruvate 1 mM, non-essential amino acids MEM, Rep/5iger, glutamine and p-2 mercaptoethanol), 595 serum of calf embryos, 50 U/ml IPPII-2 (eBioscience - eViozsiepse) and 3 μg/ml Sro. Cells were cultured for five days at a final concentration of 106 cells/ml, in 5 ml on b flat-bottomed wells. After the ethanol activation step, nitrocellulose plates (Mijsgeep-IR; Mijroge) were coated with 10 μg/ml MIR or goat anti-mouse Id (ZAM;

Zidt), diluting 1/200 in phosphate buffer solution. After the saturation stage with complete medium, 100 μg of 2x105 cells/ml were added to plates coated with MI R, and 100 plates of 105 and

Bx105 cells/ml were added to SAM plates. After incubation for 2 hours at 37 °C, the plates were stored overnight at 4 °C. Plates were washed four times with phosphate buffer solution, 0.1 95 Tween 20 and anti-mouse IdVioi diluted 1/200 in phosphate buffer solution containing 1 95 calf serum albumin. 5 95 serum of calf embryos (dilution buffer) was distributed on plates and incubated for 2 hours at 37 "C. After the washing stage, extravidin was added

HER (bidta), diluted 1/550 in dilution buffer, for an additional 1 hour at: 37 "s.

Plates were washed as above and incubated for 10 minutes at room temperature with AES solution (Zidta). The reaction was stopped by carefully rinsing the plates in running water. When the plates were dried, K5400 was read.

II.5 - Statistical analysis

Formulation mean values were compared using one-way analysis of variance (AMOMA 1). The analysis was carried out and the transformed data of I0d910 for the purpose of normalization. If a significant difference was found between the mean values of the method (p-value x 0.05), pairwise comparisons were made between the mean values at the 0.05 significant level (Student's multiple comparison test

Newman-Keuls (5itsdepi-Memtap-Kepiiv)).

MI1.6 - Results

Mice were immunized as described in MII.1 above. The following groups were used: 1/10 of the human dose (equivalent, 50 1/50 of the human dose (equivalent to 10 1/10 of the human dose (equivalent to 50 1/50 of the human dose (equivalent to 10 1/10 of the human dose (equivalent to 50 1/50 of the dose for a person (equivalent to 10 ovo zneuteivitovmy in Mr 1/10 of the dose for a person (equivalent to 50

11111101 dmeemRinabyamlno)////: SU

MI. 6.1 - Humoral reactions

No significant dose interval was observed for the two tested doses of antigens with dilution of the adjuvant or for antibody titers anti-NRMU16-I 1, or for antibody titers anti-NRUT1 8-11 (Fig. 9).

No significant dose interval was observed for the two tested doses of each adjuvant, whatever the antigen dose, for anti-NRMU16-1 antibody titers of 1.

If you look at the anti-HRM18-11 antibody titers, you can see a small increase in the titer for ABO1B (1/10 NO) compared to AZO1B (1/50 NO) as measured on day 14 after immunization AND! (2.5-order dose interval, p-value - 0.0035), however, this interval was observed only for 2 μg of antigen, and not for 0.5 μg of antigen (p-value - 0.0867). By day 45 post-II immunization, no significant dose interval was observed for the two tested doses of each adjuvant, whatever the antigen dose,

MIII.6.2 - Cellular reactions

Intracellular staining of cytokines

No effect of antigen dose interval was observed for the two antigen doses tested, whatever the adjuvant dose was for HRM18-11. Similar occurrences of specific CO4 T-cells were obtained

MI P1I16 with two tested doses of antigens with different doses of adjuvants (Fig. 10)

A small dose effect was observed for ABZO18 (1/10 NO) (2.6 orders of magnitude, p-value - 0.0009 for

NRMUT18-11; 2 orders of p-value - 0.0187 for HRM16-1 1) in comparison with ABO1B (1/50 HO), however, this interval was observed only for 2 μg of antigen, and not for 0.5 μg of antigen.

Specific B-lymphocytes

No effect of antigen dose interval was observed for the two antigen doses tested, whatever the adjuvant dose was for HRU16 or 1811 (Fig. 11).

No effect of adjuvant dose interval was observed for the two adjuvant doses tested, regardless of antigen dose for NRMU17 or 1811.

As can be seen from the results mentioned above, a 1/5 dilution of ABO1B gives a formulation that has equivalent efficacy in immunogenic compositions as ABO1B itself.

Example MI: Preclinical evaluation of adjuvanted vaccines against 5. rpestopiae in mice.

The pneumococcal vaccine used in this study was an 11-valent adjuvanted pneumococcal conjugate vaccine (11 PCM/A5B), consisting of a mixture of 11 pneumococcal polysaccharide conjugates, adjuvanted with either ABOB1B or AZBO1TE. Conjugates consist of serotypes of purified polysaccharides 1, 3, 4, 5, 6B, 7E, 9M, 14, 18C, 19E and 23E. , tetanus antitoxin (TT), or protein O from N. ipysheplaye (PO). Vaccines are presented as a freeze-dried powder that is reconstituted with one of the liquid adjuvants. 11РСМ/А5 is produced as follows:

Activation and attachment conditions are specific for each polysaccharide. The indicated data are shown in the table below. Size-sorted polysaccharide (with the exception of RB55, 6B and 23E) was dissolved in Mass 2M or in water for injections (M/RI). The optimal concentration of polysaccharides was evaluated for all serotypes. All serotypes, except serotype 18C, are conjugated directly to the carrier protein, as described in detail below.

From a 100 mg/ml stock solution in acetylnitrile or in a solution of acetonitrile/water 50 95:50 95 SOAR (ratio of SOAR/RBZ 0.75 mg/mg P5) was added to the polysaccharide solution. After 1.5 minutes, 0.2M-0.3M Mahon was added in order to obtain specific pH activation. Polysaccharide activation was carried out at a given pH for 3 minutes at 25 °C. Purified protein (O or OT protein) (its amount depends on the initial RB/carrier protein ratio) was added to the activated polysaccharide, and the attachment reaction was carried out at a specific pH for up to 2 hours (depending on the serotype), adjusting the pH. In order to quench the unreacted cyanate ether groups, a 2M glycine solution was then added to the mixture. The pH was adjusted to the quenching pH (pH 9.0). The solution was stirred for 30 minutes at 25 "C, and then continuously stirred slowly overnight at 2-8 7.

Obtaining 18C: 18C was connected to the carrier protein through a linker - dihydrozidadipic acid (AOH). Before conjugation, serotype 18C polysaccharide was subjected to microfluidization.

Derivatization of tetanus antitoxin from EBAS

For derivatization of tetanus antitoxin, the purified TT was diluted in 25 mg/ml of 0.2M Mass, and an AUN strip was added to achieve a final concentration of 0.2M. When the dissolution of the pad was complete, the pH was adjusted to 6.2. Then, EVAS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) was added to reach a final concentration of 0.02M, and the mixture was stirred for 1 hour, adjusting the pH. The condensation reaction was stopped by increasing the pH to 9.0 for at least 30 minutes at 25 °C. Next, the derivatized TT was filtered (10 kDa CO membrane) in order to remove residual amounts of AOH and the EOAS reagent

At the end, the TTan mass was sterile filtered before the joining stage and stored at -70 "С.

Chemical addition of TTadn to P5 18C

Details of the conjugation parameters can be found in Table 1.

Dilute 2g of microfluidized polysaccharide at a certain concentration in water and adjust to 2M Masi by adding Masi powder.

A solution of SOAP (100 mg/ml freshly prepared in 50/50 v/v acetonitrile) was added to achieve the appropriate ratio of SOAP/RB.

The pH was raised to the activation pH of 9.0 by adding 0.3M Mahon and stabilized at this pH until adding

Tan.

After 3 minutes, derivatized TPTadn (20 mg/ml in 0.2 M Mass) was added in order to achieve a ratio of 2TTdn/P5, the pH was adjusted to a pH of 9.0. The solution was left for one hour, adjusting the pH.

For the purpose of quenching, a 2M solution of glycine was added to the P/Tan/SOAR mixture.

The pH was adjusted to the quenching pH (pH 9.0).

The solution was stirred for 30 minutes at 25 °C and then left overnight with continuous slow stirring at 2-8 °C.

Purification of conjugates:

Conjugates were purified by gel filtration using a Zzerpasgu column! 50OONEK gel - filtration, which is balanced by 0.15M Mass (550OONEK for 18C) in order to remove small molecules (including LOBAR), and unconjugated polysaccharide and protein. Based on the different sizes of the molecules of the reaction components, RB-PO, PO-TT or RB5B-OT conjugates are eluted first, then free polysaccharide, then free PO or free OT, and finally, OMAR and other salts (Mas, glycine).

Fractions containing conjugates are found using UFgwonm. Fractions are added to the pool according to their Ka, sterile filtered (0.22 μm) and stored at 2-8 7С. We determined the ratio

RoB/protein in conjugate formulations.

Specific conditions of activation/attachment/quenching of polysaccharide 5. Rpestopiaye-Conjugates of protein O/TT/OT Serotype | Jamkfled | Zmkfld | 4 µfld | 5 | 68 | 7ERmkfld

Initial ratios 1.5/1 1/1 1.5/1 1/1 1 12/11

RZ/RO (mass/mab.)

SOAR conc.

RZkonts (m/iml) | 50 | 50 | 45 | 90 | 238

Initial carrier protein/ratio P5 12/11 12/11 2/1 1.5/1 1 (w/w)

Next, 11 conjugates are mixed together, and the final antigen preparation is mixed with the appropriate adjuvant for immunization.

On days 0, 14, and 28, a group of VaIr/s mice, consisting of 40 females at the age of 4 weeks, was injected intramuscularly with 0.1 μg of 11-valent polysaccharide conjugates, which were included in the formulation with

A5BO18B, or ABO1E. Anti-P5 antibodies were dosed using EGI5A in sera collected on day 42.

As can be seen from fig. 12, response comparisons were observed between the diluted ABO1E formulation compared to the ABO1B formulation, with the exception of RBZ 14, in which a greater response was observed in ABO1B, and RB5 19P, in which a greater response was observed in ABO1TE.

Example II: Preclinical evaluation of adjuvant and non-adjuvant immunogenic compositions against cytomegalovirus.

IH.1: Guinea pigs.

THEM 111 Anti-oV EiYiza

Quantitative determination of anti-c60B antibodies was carried out using EGISA, using 98B as a coating antigen. The antigen was diluted to a final concentration of 4 μg/ml in a phosphate buffer solution and 100 μl incubated overnight at 4 °C on microtiter plates with 96 wells. Then the plates were saturated for 1 hour at 37 °C with 200 μl of a phosphate buffer solution containing 195 bovine serum albumin. Two-fold serial dilutions of sera were added (100 μl/well) and incubated for 1 hour 30 minutes at 37 °C. Plates were washed four times with phosphate buffer solution, C 0.1 95 Tween 20, and 100 μl of anti-guinea pig (to horseradish peroxidase (Baco, UK) was added to each well and incubated for 1 hour 30 minutes at 37 °C. The plates were washed four times with phosphate buffer solution, 0.1 95 Tween 20 and once with water. They were then incubated for 20 minutes at 22 "C with 100 μl of a solution of O-phenylenediamine (Zidta) in 0.1 M citrate buffer, pH 4.2. This reaction was stopped with 100 μl of 2M NaOH" and read at 490/620 nm. 5OYMahrRgo.

Titers were expressed in IU/ml.

The statistical analysis was performed on the 14th day after the data of EyIiz vaccination 2 with the use

COMPENSATION The protocol used for the analysis of discrepancies can be briefly described as follows: - data transformation; - the Shapiro-Wilk test (Zparigo-MLIK) for each population (group) to check the normality of the distribution of groups; - Cochran's test (Sospgap) to check the homogeneity of differences between different populations (groups); - analysis of discrepancies on sample data (one-sided); - Tukey test (TyKeu) BUT of multiple comparisons.

IH.1.2 - Analysis of neutralization

Before carrying out this analysis, cells and MKSOS5 (10,000 cells/200 μl MEM medium) were distributed on microplates with 9b wells and incubated for 3 days with CO at 37760.

Two-order dilutions of inactivated sera (30 minutes at 56 "C) were performed and incubated with 100 μl of viral solution (800 IU/ml) for 1 hour at 37 "C. After incubation, 100 μl of the serum/virus mixture was inoculated onto 96-well microplates containing the MKS5 monolayer.

Plates were centrifuged at 2000 revolutions per minute for 1 hour at 35 "C. After incubation overnight at 37 "C, the plates were fixed with 80 95 acetone solution (20 minutes at -20 "C).

The acetone solution was discarded, and SMM-positive cells were determined using specific monoclonal anti-immediate early antigen for 1 hour at 37°C. The plates were washed three times with phosphate buffer solution and biotin-conjugated anti-mouse Id was added to each well and incubated for 1 hour at 37 "С. After the washing step, streptavidin horseradish peroxidase was added for an additional 30 minutes at 37 "C. The plates were washed four times and incubated for 10 minutes with true blue solution. Specific colored signals were recorded by examining them under a microscope. Neutralization titers were expressed as the inverse proportion to the highest serum dilution, which provides a 50 95 reduction in SMM-positive cells compared to the viral control (SMMU "cells without serum).

IIH.1.3 - Immunization protocols

4 groups were immunized. Each group consisted of 8 female Nagiieu Sti:(Pa) guinea pigs aged 5-8 weeks, except for the control group (group 4) containing only 4 subjects. Subjects were vaccinated intramuscularly on days 0 and 28. Serum samples were collected 28 days after the first immunization and 14 days after the second immunization. ELISA assays were performed as described above on serum taken on day 28 after the first immunization and on day 14 after the second immunization. Neutralization assays were performed as described above on day 14 after the second immunization. The groups were as follows: 1 ov mMas/////// 22111191 ABOIV///::/ pi z PUT: ZY PO ETO

The antigen was prepared as described below:

The vaccine antigen is expressed in Chinese hamster ovary (CHO) cells as a 987" truncated chimera containing peptide sequences from glycoprotein 90 of herpes simplex virus (Negrez 5ytrieh) 2 (NBZM2) at its M and C termini. ЗВ" is truncated at domain of its C-terminus, which contains the membrane anchor sequence, and is thus secreted into the culture supernatant.

For the first three groups, 15 μg of 9877 containing 500 μl of phosphate buffer solution, ABO1B or

ABSOTE (prepared as in example II, 2, described above) was administered by intramuscular injection. In group 4, only phosphate buffer solution was injected intramuscularly.

IH.1.4 - Results

As can be seen in fig. 13, significantly higher EGISA anti-9B titers were observed for the two adjuvant groups compared to the simple o9B group (by 8 and 5.5 orders of magnitude higher for 98 and ABZO1B and dr/AZO1E, respectively). Antibody titers after the second immunization dose were slightly higher (1.5 orders of magnitude) in the 9B/A5O18B group compared to the 9B/AZO1E group.

Plural comparison: Tukey - HER

Simple | (81777777 47917 11111111

NITROGEN HER 78 7771777771717111552939.4....ЮЮ.1777Й11111111111Й11

ABOIV | 8 | 56942 | 7 | Sh Y

Prosta «AZOTE - OR1B

Regarding neutralization titers (Fig. 14): - No specific neutralizing antibodies were observed in simple group 98. - Specific neutralizing antibodies were found in both adjuvant groups. - Similar levels of neutralizing antibodies were observed in both adjuvant groups.

THEM.2 - Mice

IH.2.1-EGI5A anti 98

Quantitative determination of anti-c60B antibodies was carried out using EGISA, using 98B as a coating antigen. The antigen was diluted to a final concentration of 1 μg/ml in a phosphate buffer solution and 100 μl was incubated overnight at 4 °C on microtiter plates with 96 wells. Then the plates were saturated for 1 hour at 37 °C with 200 μl of a phosphate buffer solution containing 195 bovine serum albumin. Two-fold serial dilutions of sera were added (100 μl/well) and incubated for 1 hour 30 minutes at 37 °C. The plates were washed four times with phosphate buffer solution, 0.1 95 Tween 20, and 100 μl of horseradish peroxidase streptavidin was added to each well for an additional 30 minutes at 37 "С. Tablets were washed four times with phosphate buffer solution, 0.1 95 Tween 20 and once with water. Then they were incubated for 10 minutes at 22 "C with 100 μl of 75 95 solution of tetra-methyl-benzidine in 0.1 M citrate buffer, pH 5.8.

This reaction was stopped with 100 μl of 0.4 M H25O" and read at 450/620 nm. Eiizz titers were determined by interpolation of OO indicators from the ZOYMahrRho standard. Titers were expressed in IU/ml.

The statistical analysis was performed on the 14th day after the data of EyIiz vaccination 2 with the use

COMPENSATION The protocol used for the analysis of discrepancies can be briefly described as follows: - data transformation; - the Shapiro-Wilk test (Zparigo-MMIK) for each population (group) to check the normality of the distribution of groups; - Cochran's test (Sospgap) to check the homogeneity of differences between different populations (groups); - analysis of discrepancies on sample data (one-sided); - Tukey's test (TyKeu) NZO multiple comparisons.

IH.2.2 - Analysis of neutralization

Before carrying out this analysis, MKSO5 cells (10,000 cells/200 μl of MEM medium) were distributed on microplates with 96 wells and incubated for 3 days with 595 °C at 37 °C. Two-fold dilutions (60 μl) of inactivated sera were performed (30 minutes at 56 C) was incubated with 60 μl of virus solution (800 IU/ml) for 1 hour at 37 °C. After incubation, 100 μl of the serum/virus mixture was inoculated onto 96-well microplates containing MKO5 cells. The plates were centrifuged at 2000 revolutions per minute for 1 hour at 35 "С. After overnight incubation at 37 "C, the tablets were fixed with 80 9o acetone solution (20 minutes at -20 "C). The acetone solution was discarded, and positive SMM cells were determined using specific monoclonal anti-immediate early antigen I (I-I) for 1 hour at 37 °C. The plates were washed three times: with phosphate buffer solution and biotin-conjugated anti-mouse Id was added into each well and incubated for 1 hour at 37°C. After the washing step, streptavidin horseradish peroxidase was added for an additional 30 minutes at 37°C. The plates were washed four times and incubated for 10 minutes with true blue solution. Specific colored signals were recorded by examining them under Neutralization titers were expressed as inversely proportional to the highest dilution of serum giving a 5095 reduction of positive SMM cells compared to the viral control (SMMU "cells without serum).

IIH.2.3 - Intracellular cytokine staining

Intracellular staining of T-cell cytokines was performed on peripheral blood lymphocytes on the 7th and 21st days after the second immunization. Peripheral blood lymphocytes were collected from mice (1 depot per group). Antigen stimulation of ip migo lymphocytes (final concentration 1057 cells/ml) was carried out either with a peptide pool including the SMU sequence or with protein 98. The peripheral blood lymphocyte/antigen mixture was incubated for 2 hours at 37 °C. The cells were then incubated overnight in in the presence of brefeldin (1 μg/ml) at 37 "С in order to inhibit cytokine secretion.

Staining of cells is carried out in the following way: cell suspensions were washed, re-suspended in 50 μl of a phosphate buffer solution containing 1 96 of calf embryo serum containing 2 95 Eu of blocking reagent. After incubation for 10 minutes at 4 "С, 50 μl of a mixture of anti-СО4 PE and anti-СО8 was added to Ср and incubated for 30 minutes at 4 "С. After the stage of washing in a phosphate buffer solution containing 1 95% fetal calf serum, the cell membranes were permeabilized by re-suspending in 200 μl Suoiich-Suioregpt (Ki VesKiop OisKipbop) and incubated for 20 minutes at 4 "C. Then the cells were washed with Rept Umah5p (Kii VO) and re-suspended in 50 μl of the anti-IEM mixture of ARS zhant-1Y-2 RITS diluted with Regt UmMaz. After incubation for 2 hours at 4 "С, the cells were re-suspended in a phosphate buffer solution containing 1 95 fetal calf serum "from 1 95 paraformaldehyde.

Analysis of samples was performed using EAS5. Live cells were gated (EZS/5552) and assembled for 20,000 transformations. The percentage content of IEM in и- or ІІ/2-- was calculated on СО4- and

Owls in closed populations.

IX.2.4 - Immunization protocols

4 groups were immunized. Each group consisted of 12 female C57VI/6 mice aged 4-10 weeks. now a "ZO PO ho U Z a 111ov1111111111711111111111111111ABOIV///// 811 Gov11111111111111111ABOTE GG (

The antigen was prepared as described below:

The vaccine antigen is expressed in Chinese hamster ovary (CHO) cells as a 98" truncated chimera containing peptide sequences from the glycoprotein dO of herpes simplex virus (Negrez 5itrieh) 2 (H5BM2) at its M and C termini. ЗВ" is truncated at domain of its C-terminus, which contains the membrane anchor sequence, and is thus secreted into the culture supernatant.

For each group at a concentration of 1.5 μg/dose, 9877 consists of 625 μl of phosphate buffer solution, or adjuvant ABO1TIV or ABOTE (prepared as in example II.2, described above, with a concentration of immunostimulants of 100 μl per ml or 50 μl of immunostimulants per ml, respectively). 50 μl (that is, 1/10 of the human dose of 0.5 ml) was administered intramuscularly. One control group of mice was injected with a saline solution. Injections were carried out on day 0 and 28. Serum samples were collected 14 days after the second injections for EI I5A and neutralization assays. RVI were collected on the 7th and 21st days after the second injections at IS5.

IH.2.5 - Results

EGIZA anti-98 titers (Fig. 15).

A very weak to undetectable level of anti-98B antibodies was observed in the non-adjuvant 98 group.

However, a high antibody response (65 and 66 orders of magnitude higher) was observed in both adjuvant groups as in

ABZO1B and ABO1TE, respectively. No statistically significant difference was observed between the ABO1B group and

OR1E.

Plural comparison: Tukey - HER

Simple | (12 77772132 17771111 Her

NITROGEN | 12 i17777771759317..i...i1 11111111

ABOIV | 12 | 39375... |... | She

Simple «А5ЗО1 Е - OR1В.

Anti-SMM neutralization titers (Figure 16).

Significantly higher neutralization titers of anti-9B were observed for the two adjuvant groups compared to the simple 9B group. No significant difference in antibody neutralization titers was observed between formulations

ABO18 and AZO1E.

Cell-mediated immunity.

Due to the very low level of response observed after repeated stimulation of 7 samples after

After immunization, no difference between the groups can be detected and the final response to CO4 and COB8 stimulation cannot be recognized (Fig. 17). These reactions, ranging from weak to undetectable, probably occurred due to a technical problem when obtaining the samples. However, reactions can be seen within 21 days after the second injection. CO4 data (Fig. 18) show no difference after re-stimulation with B (5 μg/ml) or peptides (2 μg/ml or 4 μg/ml). A similar cytokine profile is observed for ABOTE and ABO1B. No final reaction can be observed for CO8 stimulation (Fig. 19).

These experiments demonstrate that for a different antigenic composition and in two different organisms, an adjuvant with lower levels of immunostimulants is as immunologically effective as one with higher levels.

X: Preclinical evaluation of adjuvant vaccine CT5.5

X.1 - Recipe

The antigenic composition, KT5.5, is produced in sagmisiae, and it consists of two proteins, KT5 and 5, which are intracellularly and spontaneously assembled into mixed polymeric structures of solid particles, each of which is estimated to contain an average of 100 polypeptides. KT5 is a 51 kDa hybrid polypeptide chain of 424 amino acids, consisting of 189 aa from the surface antigen of the sporozoite of the ME53Z strain of the malaria parasite R. gaiIsiragit (antigen C5R, a. a. 207-395), fused to the amino end of protein 5 of the hepatitis B virus. 5 is a 24 kDa polypeptide (226 amino acids long) corresponding to the surface antigen of the hepatitis B virus. The lyophilized granule of the antigen contains approximately 50 μg (when developed for introduction into a formulation of 0.5 ml with ABO1B) or 25 μg (when developed for introduction into recipe of 0.5 ml with ABO1TE) antigen.

A5BO1B and ABO1E were prepared by mixing different components (phosphate buffer solution, liposomes,

MRI. and 0521) in the tank, the components were mixed under aseptic conditions. Then the product was sterile filtered before filling into ampoules or syringes. Before use for the purpose of reconstitution of lyophilized granules with antigen, the liquid adjuvant was stored at 2 "С - 48 76.

X.2 - Experiments on mice

Two experiments were performed on mice, the purpose of which was to compare immune reactions specific to KT5.5 induced by KT5.5/ABZO18B in comparison with KT5.5, the formulation of which included ABOTE. In each experiment, C57VI/6 mice (10 mice/group) were immunized intramuscularly three times with an interval of two weeks with 10.5 or 2.5 μg of ET5.5, which contains ABO1TV or ABO1E adjuvants. Two groups, A5 controls, were immunized either with OR13 or only

ABZO1E. Reactions of antibodies specific for HB and C5 were evaluated for each mouse using EGISA 15 days after the third immunization. Geometric mean antibody titers and their 95 95 confidence intervals were calculated for all mice receiving the same treatment in both experiments. Statistical analyzes were performed on pooled data from both experiments to assess adjuvant and antigen dose effects. Specific responses to CO4 and CO8 T cells were measured by flow cytometry 7 days after the second and third immunizations on hemocyte pools taken from 5 mice per group. Thus, we found the indicators for each group in each experiment.

Humoral immune response

As can be seen in fig. 20 and 21, both ABO1IV and ABO1TE adjuvants induce strong comparable antibody responses, however, C5R and HB.

The three-line method of AMOMA on titers of geometric mean values of anti-C5R demonstrated that there are no significant differences between ABOTB and ABO1TE for 5 or 2.5 μg doses of vT5.5.

For a dose of 10 μg, ABO1B adjuvant was found to stimulate higher anti-C5 titers than ABZO1E, and the CMT ratio "ABO1B group/ABO1TE group" was 1.93 (95 95 CI: 1.33-2.79; p - 0.001).

Cell-mediated specific immune reaction

Figures 22 and 23 demonstrate the levels of C5P-specific CO4 and CO8 T cells and

HBs that express II -2 and/or IEM-u.

The C5P-specific CO4 response tends to increase with ABZO1B compared to ABOTE after three immunizations, while the CO8 T-cell response with ABOTE is equivalent or better than with ABOB1B.

The HB-specific CO4 response tended to increase with ABOBIV compared with ABOTE after three immunizations except for the lower dose of CT5.5, in which T cell CO4 levels were equal between the two adjuvants. Reactions of T-cells CO8, specific for HB, induced with the help of KT5.5, in the composition with ABO1TE, are equivalent or better than the reactions induced with the help of KT5.5, in the composition with A5O18B.

It is believed that these differences are within the expected variability of cellular immunology analyses.

Preclinical evaluation of the KT5.5/ABZO1E vaccine in mice revealed an acceptable safety profile similar to that of KT5.5/АЗО18В.

XI: Clinical assessment of KT5.5/ABZO1E.

Formulations were prepared as described in example X above. Sucrose was used as a filler in a lyophilized granule with an antigen. As in example X, the liquid adjuvant is used to restore the lyophilized antigen. ABOTE was prepared as described in example 1I.2 and stored at 2 "C - 48 "C until it is needed for reconstitution.

The IU is currently conducting a randomized, double-blind study of the safety and immunogenicity of KT5.5 with adjuvant ABZOTE phase in children aged 18 months to 4 years living in Gabon. The vaccination scheme is a vaccination scheme for children aged 0, 1, 2 months. For KT5.5/АЗО1Е when 3 doses are administered intramuscularly according to the vaccination scheme in 0, 1, 2-month children aged 18 months to 4 years living in a malaria-endemic zone, the research objectives are as follows:

Primary - safety assessment within one month after administration of dose 3; - determination of the effectiveness of the oil-in-water emulsion of the KT5.5 adjuvanted vaccine in terms of anti-C5 antibody response one month after administration of dose 3.

Secondary - assessment of reactogenicity within one month after administration of dose 3; - determining the effectiveness of the oil-in-water emulsion of the KT5.5 adjuvanted vaccine from the point of view of the anti-HB antibody response one month after the administration of dose 3; - observation of seroprotection against hepatitis B within one month after administration of dose 3; - observation of the anti-C5 reaction within one month after the administration of dose 3.

Tertiary - safety within 1 month C after administration of dose C to 12 months after administration of dose 3; - humoral immune response to antigen C5 within 12 months after administration of dose 3; - humoral immune response to HB antigen within 12 months after administration of dose 3.

Research - assessment of T-cell-mediated immune response to antigen C5 within 12 months after administration of dose 3; - evaluation of the immune response of B-lymphocytes to the C5 antigen within 12 months after the administration of dose 3; - observation of the anti-C5 reaction within one month after the administration of dose C according to the documented status of NVM immunization at screening.

The study enrolled 180 subjects, 90 of whom received an adjuvant vaccine with a pre-patent oil-in-water emulsion adjuvant (referred to as "control" in the tables below), and 90 subjects received an adjuvant vaccine from ABOTE. Healthy male and female children aged 18 months to 4 years were screened. Vaccines were administered by intramuscular injection into the deltoid muscle.

Incidence and nature of symptoms (persistent and non-persistent) reported during the 7-day post-vaccination period (days 0-6) after each dose and overall (Entire vaccinated cohort) 11111111 | Any symptom General symptoms Local symptoms:/ 111111 1959с1| | | |959c1| | | |9595s (|trulaM p (95 (SHO OS (m oka 96 f- OS (m Ty 96 0 s dozai (53-19 oz ee o ze vve i ze ze zve ze

Gr.2 190 | 47,522|41.4|62.9| 90 | 26 |28.9 119.8 39.4 | 90 | C2 35.6 |25.7 46.3)

Gr.2 | 87 | 53 60.9149.9|71.2| 87 | 39 |44.8 341 55.9, 87 | 34 Y39.1|28.8 501

Gr.2 | 85 82 96.5190.0199.3| 85 | 50 |58.8 47.6 69.4 | 85 | 79 192.9|85.9 97.4 dose of vitiation/subjects|Gr.2 | 90 | 85 (94.4|87.5|98.2.| 90 | 70 |77.8|67.8| 85.9 | 90 | 84 |(93.3|86.1197.5

Gr.1-KT5,5.ABO1E; Group 2 - control; GG. - lower limit; Oh - the upper limit.

For each dose and overall/subject:

M - number of subjects with at least one injected dose; n/96 - the number/percentage of subjects with at least one symptom when given any vaccine under investigation.

In general / for the dose:

M « the number of administered doses; n/do- the number / percentage of doses after the manifestation of at least one symptom during the administration of any vaccine under study; 95 95 SI - exact confidence interval, I. - lower limit, ОЙ. - the upper limit.

These data demonstrate that the ABOTE-adjuvanted CT5.5 vaccine provided acceptable reactogenicity results in the pediatric population when compared with the control drug.

Serological responses were measured by evaluating antibody responses to HB and C5R duplications (anti KZ321IK).

Serum for the determination of antibodies was collected at screening, on the 60th and 90th day (at the second vaccination and the third vaccination). Anti-C5 antibody levels were measured by standard EHIZA methodology using the KZ32I EC antigen adsorbed on a standard antibody plate as a control according to laboratory standard operating procedures. The results are recorded in EU/ml.

The antibody specific for the hepatitis B surface antigen was measured using a commercially available EGI5ZA immunoassay (AOBAV EIA test kit from ArBroyu), or an equivalent assay according to the instructions for this assay. The results are recorded in mO/ml.

Indicators of seropositivity and MS for anti-C5 antibodies (All vaccinated cohort) 11111111 b5ESHUML | om Her 11111111 res | 77771 5 of her "Antibody| group| timing M n) 96| AI | ОЙ | value I | Sh1 | Mip | Mach screening Sh|8910100| 00141 03 |09 | 09 | «05 | «05

Gr. 1

Anti-c5 screening Sh|9011/|11|001|60 03 |02|09 | "051 05

Gr.2

FIMOes) | 80 |80|100|95.5| 100 | 164.8 |134.1|202.6| 6.3 |2093.6

Gr. 1-KT5,5.OR1E

Gr. 2 - Control;

OMS - the geometric mean value of the concentration of antibodies, calculated on all subjects;

M - number of subjects with available results; n/9o x number/percentage of subjects with a concentration within the specified interval; 95 95 CI-95 95 confidence interval; And I. - lower limit, AI. - upper limit; MIN/MAX - minimum/maximum.

Indicators of seropositivity and MS for anti-HB antibodies (All vaccinated cohort) 11111111 | as MmMOomMy | // sms Her Her dust I PET et PONYA PON TESTU o THEY PON POLYA "Antibody|group|timing MI p| 96| | OSiMeaning | 0 / Mi | Mach screening 89043 48.3137.6|59.2| 40.8 | 23.3 | 71.4 | "10.0 | 46421.6

Gr. 1

Anti-

HB screening 190037 41,|30,8|52,0)Y 20,0 | 12.8 | 31.0 | "10.0 | 30796.4

Group 2 (FIP(090) 80.80 |100|95.5|100| 19485.0 |13511.3(28099.9| 178.6 (1103974)

Gr. 1-К15,5.АБО1Е;

Gr. 2 - Control;

OMS - the geometric mean value of the concentration of antibodies, calculated on all subjects;

M - number of subjects with available results; n/9о6 x number/percentage of subjects with a concentration within the specified interval; 95 95 CI-95 95 confidence interval; AND! I. - lower limit, SHOI. - upper limit;

MIN/MAX - minimum/maximum.

These data demonstrate that the KT5.5 vaccine formulation with ABO1TE adjuvant produced acceptable humoral immune responses in the pediatric population when compared to a validated control.

Example XI!: Preclinical evaluation of varicella virus with ABO1B in comparison with ABOTE.

The candidate vaccine consists of a shortened MAM envelope protein, 9E, produced in CHO cells.

For this study, C57VI/6 mice (p-48) were first vaccinated with one human dose (HO) of Magiigih (4-4 (09 plaque-forming units/dose), which was administered subcutaneously. Five weeks after the start of vaccination, Magiigih mice were divided into 5 groups of 12 mice each, and on days 0 and 28 they were injected intramuscularly (into the tibia) with 5 μg of only ZE, 5 μg of FECABZOTE" (1/10 NOU) or 5 μg

I9EHABZO18B (1/10 NO). The control group of mice (only with the initial dose) was injected with a saline solution (0.9 95 mass). Immune responses were assessed 14 and/or 30 days after the second vaccination. The levels of all DE specific antibodies and the occurrence (I2/LEM) of cytokine-producing T cells СО4 and СО8 were evaluated.

Reactions specific to the antibodies of JAE:

EZA was developed in order to find and determine the amount of ZE-specific antibodies in the sera of mice, using the ZE protein as a coating antigen. EGIZA titers were determined as the inverse of the serum dilution produced by measuring absorbance (optical density), equal to 50 95 of the maximum absorbance index. EHIZA titers were calculated using regression analysis.

The data demonstrate that ZE ABZOTE and 9E A5BO1B induced similar levels of ZE specific for antibodies (p-value » 0.05). Both formulations stimulated significantly higher responses compared to antigen 9E alone (10-13 orders of magnitude, p-value "0.05) both on the 14th and 30th day after the second vaccination (Fig. 26).

ZE reactions, specific in relation to СО4 and СО8

Cytokine production was assessed in CO4 and CO8 T-cells using the technique of intracellular cytokine staining. From each group, 12 mice were selected for spleen cells 30 days after the second vaccination, and 4 groups were placed in the pools of the spleen. Spleen cells (1 x 105) were incubated for 2 hours in the presence of ZE peptides (63 peptides) covering the whole ZE protein (20aa peptides / 10aa overlay), and then the cells were incubated overnight in the presence of brefeldin. Next, the cells were stained with fluorescent tAbB, specific for the cell surface CO4/CO8, after permeation with intracellular cytokines 1-2 and IEM-u.

As shown in fig. 26, although similar cytokine profiles (P2LEM-y) were stimulated both with Y9E

A5O1B and ZE ABO1TE formulations, the ABO1B formulation stimulated a greater amount of CO4 and CO8 cells producing cytokines (by 2 orders, p»0.05 for CO4, by 3.6 orders, p»0.05 for CO8). Due to the unexpectedly high variability of T cell responses, the possibility of finding a significant difference between adjuvant doses was very limited ("50 95"). It is important that both d9E, which included

A5BO1B or ABO1E stimulated CO4 T-cells, producing cytokine, with a significantly higher value (by 13.3 orders, p«e0.05) compared to only ZE. The higher number of different POP cells was also stimulated by 9E with ABO1TV or ABO1E (by 3.8 orders of magnitude, p«e0.05) in the composition of the formulation compared to only the OE antigen.

Example XII: Preclinical evaluation of ABO1B versus ABO1TE in a ferret model infected with influenza.

Materials and methods

Female ferrets (Mizhyeia rshogiiv Tygo) at the age of 14-20 weeks were delivered from the kennel MIZAM (Hampshire,

Great Britain). On day 0, the heterosubtypic NIMI strain was administered intranasally to ferrets at a dose of 250 μl of A/5iosKpoYpt/24/90 (4 hours of TCIOvo/ml). On day 21, ferrets were injected intramuscularly with a full human dose (1000 μl vaccine dose, 15 μg HA per strain A) in combination with H1M1

A/Memzh/ SaiIedopia/20/99 (15 μg/ml), NZM2 Alluotipad/3/2003 (15 μg/ml) and B/) Lapd5y/10/2003 (17.5 μg/ml). Then, on the 42nd day, ferrets were given a provaccinal test intranasally with 250 μl of the heterosubtypic strain M/p.A/MU7/55/04 (4.5 units of TSIUso/ml).

Vaccinations on day 21 were carried out either with a simple trivalent formulation ("simple" in the tables below), or with a trivalent adjuvant formulation with ABO1TV ("ABO1B" in the tables below), or

ABO1TE ("ABO1TE" in the tables below). Formulations were prepared as described in Example C above.

Body temperature monitoring:

Visual temperature determinations were monitored during the challenge period and evaluated using telemetry implants that recorded each individual animal's temperature every 15 minutes before and after challenge. All implants were checked and updated, and recalibrated with O5I before placement in the abdominal cavity. All animals were placed individually in one cage during the specified measurements.

Temperatures were recorded every 15 minutes for 6 days before the start of vaccination and for 4 days after the start of vaccination, as well as on the 3rd day before the provaccination test and for 7 days after it.

Hemagglutination inhibition test (NO).

Testing methodology

Antibody titers against hemagglutinin of three influenza virus strains were determined using the hemagglutination inhibition (NI) test. The principle of the NI test is based on the ability of specific antibodies against influenza to inhibit hemagglutination of chicken erythrocytes (RBCs) under the influence of influenza virus hemagglutinin (HA). Serum samples were first treated with 25 95 neuraminidase (COE) solution and heat inactivated to remove non-specific inhibitors. After primary treatment, serum samples, diluted by two orders, were incubated with 4 hemagglutination units of each influenza strain. Next, chicken erythrocytes were added and inhibition of agglutination was assessed using reading gaps. Titers were expressed as equivalent values of the highest serum dilution in which it completely inhibited hemagglutination. Since the first serum dilution was 1:10, the undetected level was scored as a titer of 5.

Statistical analysis

Statistical analysis was performed on NI titers (day 41 before the provacation test using OMIZTAT. The protocol used for the analysis of discrepancies can be briefly described as follows: - Year data transformation; - Shapiro-Wilk (Zparigo-MLIK) test for each populations (groups) to check the normality of the distribution of groups; - Cochran's test (Sospgap) to check the homogeneity of differences between different populations (groups); - one-way analysis of differences carried out in groups; - Tukey's test (TyKeu) NSO of multiple comparisons.

Determining the titer of viruses in nasal washes

All nasal wash samples were first sterile filtered through Zrip X filters (Soviag) in order to exclude bacterial contamination. Ten-fold serial dilutions of nasal washes of 50 μl were transferred to microtiter plates containing 50 μl of medium (10 wells/dilution). Next, 100 μl of Medline-Derby canine kidney cells (MOSC, 2.4x105 cells/ml) were added to each well and incubated at 35 °C for 6-7 days. After 6-7 days of incubation, the culture medium was carefully removed, and 100 µL of 1/20 M/5T-1 containing medium and incubated for another 18 hours The intensity of the yellow formazan dye produced upon reduction of M/5T-1 by viable cells, which is proportional to the number of viable cells present in the well at the end of the assay titration of the virus is determined by measuring the optical density in each well at the appropriate wavelength (450 nm). The limit value is determined as the average value of OO for uninfected control cells - 0.3 00 (0.3 OO corresponds to h/- Z BISHЄmM

OO for uninfected control cells). A positive result is determined when the OO "limit indicator, and vice versa, a negative result is determined when the OO "limit indicator. Viral spread titers were determined by "Key and Miepsi" and expressed as 1 od TCI/ml.

Lymphoproliferation analysis.

PBMCs were collected by density gradient centrifugation (20 minutes at 2500 rpm and 4 °C) in Risoya mammalian lympholyte solution Sedapape. PBMCs were resuspended in 5 ml of culture medium (KRMI/AAa at 4 °C) and 10 95 normal serum ferret. Supplements consisted of 100 mM sodium pyruvate non-essential amino acids MEM, penicillin/streptamycin, glutamine, and 1000x concentrated D-2 mercaptoethanol. Freshly isolated PBMCs were directly used in i.p. migo proliferation assays. Cells were plated in 96-well flat-bottomed tissue culture plates at 2x105 cells/wells and grown in culture with different concentrations of antigen (from 0.1 to 1 μg HA of whole inactivated virus) for a period of time from 44 to 96 hours, and then labeled with a pulse with 0.5 μCi ("NI thymidine. The introduction of the radiolabel was assessed by 4-16 hours using p-emission spectroscopy.

The results

Viral load in nasal washes after a provaccination test.

Nasal washes were collected 2 days before the start of vaccination (start of vaccination - day 0), 1, 2 and 7 days after the start of vaccination, as well as 4 days before the provaccination test (provaccination test - day 42) and during a period of 7 days after the preliminary test. Grool| 2 | 0 | i | ze | "7 | 39 | 42| 43 | 44 | 45 | 47 | 49

Simple | 0.82 | 1.84,535/| 1.85 08 | /1.82)|5771|444| 1.97 | 09

ABOTE | 0082 | |g2gyl1|583| 1651 08 | /|162|493)|415| 24 | 0.65

WIPO | 081 | |2GB|538| pe |082| |1.74| 25 | 1.89 | 1,350 09

Spread of the virus after the start of vaccination.

In all ferrets, the peak of virus spread was observed 2 days after the start of vaccination. 7 days after the start of vaccination, only residual viral load was observed in all groups.

The spread of the virus after the provacation test.

The peak of the spread of the virus was observed 24 hours after the provacation test.

Viral titration 3 days after challenge showed high viral titers (no protection) in ferrets immunized with trivalent split simple vaccine. Less reduction in virus shedding was observed in ferrets immunized with trivalent split vaccine with

AZO1E than trivalent split adjuvant vaccine with ABO1B.

Temperature monitoring:

Body temperature was monitored for 6 days before the start of vaccination (start of vaccination - day 0) and for 4 days after the start of vaccination, as well as from the 3rd day before the provaccination test and for 7 days after it (provaccination test - day 42). Measurements were taken every minute and the average value was calculated at noon for each group. The results can be seen in fig. 28.

After starting vaccination.

Body temperature recorded before, during and after the start of vaccination showed an increase in temperature in all groups.

After the preliminary test.

Interpretation of body temperature monitoring is difficult. A slight increase in body temperature was observed after challenge in ferrets immunized with trivalent split simple vaccine and trivalent split ABOTE vaccine, but not with trivalent split ABO1B vaccine. The indicators given below were obtained in a number of ferrets with an elevated temperature »0.4 76.

An increase in temperature after a preliminary test

Trivalent simple vaccine: 5/8 (--0.4, 0.4, 40.5, 0.7, -0.8)

Trivalent OR1TV 0/8

Trivalent ABOTE 6/8 (--0.4, -0.4, --0.5, --0.5, --0.9,--1.6).

This reading is less powerful than other readings used in ferrets.

Haemagglutination inhibition test (NO)

Serum samples were collected 4 days before vaccination, 17 days after vaccination, 21 days after immunization and 13 days after challenge. The results can be seen in fig. 29 and 30. For all three vaccine strains, statistically significant higher NI titers were observed in ferrets immunized with trivalent split adjuvanted vaccine with ABO1B or

ABZO1E in comparison with the trivalent split simple vaccine. No difference was observed between the two adjuvant groups. Compared with other groups, statistically significant higher titers of NI cross-reactivity were observed in A/Mem/MogkK NZM2 (provaccination test strain) after immunization of ferrets with trivalent split adjuvant vaccines with ABO18B.

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Claims (26)

1. An immunogenic composition in a volume acceptable for a human dose comprising an antigen or an antigen preparation in combination with an adjuvant, wherein the adjuvant comprises 0521 presented in liposome form and 30-MRI, wherein 0521 and indicated 30-MRI, are both present in the indicated human dose at the level of 9 to 30 μg. 2. The immunogenic composition according to item I, where the specified adjuvant composition additionally includes a sterol, and the saponin/sterol ratio is in the range from 1:1 to 1:100 wt./wt. 3. Immunogenic composition according to claim 2, where the indicated sterol is cholesterol. 4. Immunogenic composition according to any of items 1-3, where the ratio 0521:30-MRI is 1.1. 5. Immunogenic composition according to any of the previous items, where 30-MRI is indicated. is present in the amount of 25 mcg. 6. Immunogenic composition according to any of the previous items, where the indicated 0521 is present in an amount of 25 μg. 7. The immunogenic composition according to any of the previous items, where the indicated volume, acceptable for a human dose, is from 0.5 to 1.5 ml. 8. The immunogenic composition according to any of items 1-7, where the indicated antigen or antigenic preparation is derived from the varicella virus (VVV). 9, Immunogenic composition according to claim 8, where the specified antigen or antigen preparation derived from M/U is a shortened protein of the ULU shell, ev. 10. The immunogenic composition according to any of items 1-7, where the specified antigen or antigenic preparation is derived from RiIaztodiyt GaIstragit. 11. Immunogenic composition according to any of items 1-7, where the indicated antigen comes from M. Shshbegsiov185. 12. The immunogenic composition according to claim 11, where the specified antigen or antigenic preparation derived from Musobasiegisht shregsco5i5 is selected from ТКа12, ТНО, ТКаз35, ТЗ38-1, Eta 14, ОРУ, МТИ, МБ, тТТС2 and ЯТССИ1. 13. The immunogenic composition according to claim 11, where the indicated antigen or antigenic preparation derived from Musobasiegiit Shshregsitsiovi5 is Ka-12-TpPO-Ka35 or its variant, in which 5eg/04 of the sequence is mutated to another amino acid other than serine, or its derivatives containing an M-terminal histidine tag of acceptable length. 14. Immunogenic composition according to claim 11, where the antigen is M72. 15. Immunogenic composition according to any of items 1-7, where the antigen is derived from NSMU. 16. Immunogenic composition according to any of items 1-7, where the antigen is derived from NIU-1. 17. Immunogenic composition according to any of items 1-7, where the antigen is derived from SpPatuvia 5 years 18. The immunogenic composition according to claim 17, where the specified antigen or antigenic preparation derived from SpPatucdia 5yr. is that of Spatuydia (gaspotaiv 19. The immunogenic composition according to any one of claims 17 or 18, where the specified antigen or antigenic preparation derived from SPiatudia 5rr. is selected from ST858, STO89, ST875, MOMR, STe22, PtrO, Ptro, 5U/IV and immunogenic fragments of any of them and their combinations. 20. The immunogenic composition according to claim 19, where the indicated antigen or antigenic preparation derived from Spiatuan 5yr is a combination of ST858, STOV89 and ST875. 21. A human dose of the immunogenic composition according to any of items 1-20 for use in medicine. 22. An adjuvant composition in a volume acceptable for use in a human dose of an immunogenic composition consisting of 9-30 μg of 30-MRI and 9-30 μg of 0521 presented in the form of a liposome. 23. Adjuvant composition according to claim 22, where 30-MRI is specified. is present in the amount of 25 mcg. 24. The adjuvant composition according to claim 22, where the indicated 30-MRI is present in the amount of 10 μg. 25. Adjuvant composition according to claim 22 or claim 23, where the specified 0521 is present in the amount of 25 μg. 26. An adjuvant composition according to any one of claims 22 or 24, wherein said 0521 is present in an amount of 10 μg.