MXPA01005398A - Peptide-based vaccine for influenza - Google Patents

Abstract

A human synthetic peptide-based influenza vaccine for intranasal administration comprises a mixture of flagella containing at least four epitopes of influenza virus reactive with human cells, each expressed individually in Salmonella flagellin, said influenza virus epitopes being selected from the group consisting of:(i) one B-cell hemagglutinin (HA) epitope;(ii) one T-helper hemagglutinin (HA) or nucleo-protein (NP) epitope that can bind to many HLA molecules;and (iii) at least two cytotoxic lymphocyte (CTL) nucleoprotein (NP) ormatrix protein (M) epitopes that are restricted to the most prevalent HLA molecules in different human populations.

Description

AN INFLUENZA VACCINE BASED ON PEPT1DO FIELD OF THE INVENTION The present invention relates to influenza vaccines, and particularly to peptide-based vaccines comprising conserved epitopes of both B lymphocytes and T lymphocytes recognized by HLA prevalent in humans.

Ab Abbreviations: antibodies; CTL: cytotoxic T-lymphocytes; EID: ineffective dose in the egg; HA: haemagglutinin; HAU: hemagglutination unit; i.n .: intranasal; i.p .: intraperitoneal; NP: nucleoprotein; PMBC: peripheral blood mononuclear cells; TT: tetanus toxoid.

BACKGROUND OF THE INVENTION Influenza is a public health concern, which results in an economic burden, morbidity and even mortality. Influenza infection can result in a variety of disease states, with a range of subclinical infection through middle respiratory tract infection and tracheobronchitis to severe occasionally lethal viral pneumonia. The reasons for this broad spectrum of severity are explained by the site of infection and the immune status of the host. The most important characteristic of influenza, from the immunological point of view are the rapid, unpredictable changes of surface glycoproteins, hemagglutinin and neuraminidase, referred to as antigenic changes and abatement. These changes lead to the emergence of new strains of influenza, which enable the virus to escape from the immune system and are the cause of almost all annual epidemics (Laver et al., 1980 and 1980a, Webster, 1982). Immunization to the influenza virus is limited by this marked antigenic variation of the virus and by the restriction of the infection to the respiratory mucous membranes. The currently available and permitted influenza vaccine is based either on the complete inactive virus, or on the viral surface glycoproteins. These influenza vaccines do not induce complete, long-term and cross-strain immunity. The influenza virus comprises two surface antigens: neuraminidase (NA) and hemagglutinin (HA), which carry out gradual changes (change and drift), leading to high antigenic variations in influenza. HA is a strong immunogen and is the most significant antigen to define the serological specificity of the different strains of virus. The HA molecule (75-80 kD) comprises a plurality of antigenic determinants, several of which are in the regions that carry out sequence changes in different strains (determinants specific for strains) and others in regions that are common to many molecules HA (common determinants). E.U.A. No. 4,474,757 describes a synthetic vaccine against a plurality of different influenza viruses comprising a suitable macromolecular vehicle having attached to it a peptide which is an antigenic fragment of HA which is common to a plurality of different viral strains of influenza. One of the common determinants described is the epitope HA 91-108 which is conserved in all strains of H3 influenza subtypes. The nucleoprotein (NP) is located in the viral nucleus and is one of the antigens specific to the group which distinguish between influenza A, B and C virus, unlike HA, NP is one of the most conserved viral proteins , being 94% conserved in all influenza A viruses. The antibodies specific for NP of influenza A virus do not have neutralizing activity of the virus, but NP is an important target for cytotoxic T lymphocytes (CTL) which they are cross-reactive with all viruses A (Townsend and Skehel, 1984). CTL recognizes synthetic short peptides that correspond to the linear regions of the NP molecule of influenza (Townsend et al., 1985 and 1986). PCT International Publication WO 93/20846 describes a recombinant synthetic vaccine against a plurality of viral strains other than influenza comprising at least one chimeric protein comprising the amino acid sequence of flagellin and at least one amino acid sequence of an epitope of HA or NP of the influenza virus or an aggregate of said chimeric protein. Following this method, it was found that a synthetic recombinant anti-influenza vaccine based on three epitopes was highly efficient in mice. This vaccine includes HA 91-108, HA a B cell epitope from HA which is conserved in all H3 strains and produces neutralizing anti-influenza antibodies, together with the epitopes of the T helper and CTL from NP (NP 55-69 and NP 147-158, respectively), which induces immune responses restricted to MHC. Each of these epitopes is expressed in the flagellin of the vaccine strain for Salmonella. The isolated flagella were administered intranasally to mice, resulting in protection against viral infection (Levi and Arnon, 1996).

BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention, the epitopes of the influenza peptide reactive with human cells were expressed in Salmonella flagellin and tested for their effectiveness in a human / mouse chimera in which human PBMC was functionally grafted. Virus clearance after challenge and resistance to lethal infection were found only in the vaccinated mice and translation of human antibodies specific for viruses was also high in this group. The FACS analyzes showed that the majority of the human cells in the transplanted mice were CD8 + and CD4 +, indicating that the protection was mediated mainly by the cellular immune response. The present invention thus relates to a synthetic influenza vaccine based on human peptide for intranasal administration comprising a mixture of flagella containing at least four epitopes of the influenza virus each expressed individually in the flagellin of Salmonella, said epitopes of the Influenza viruses are reactive with human cells and are selected from the group consisting of: (i) a B cell hemagglutinin epitope; (HA) an epitope of hemagglutinin (ii) of the helper T or nucleoprotein (HA) that can bind to many (NP) HLA molecules; and (iii) at least two epitopes of cytotoxic lymphocyte, (CTL) nucleoprotein (NP), or matrix protein (M) that are restricted to the most prevalent HLA molecules in different human populations. The preferred B cell epitope HA is the hemagglutinin epitope of influenza virus 91-108 [HA 91-108] of the sequence: Ser-Lys-Ala-Phe-Ser-Asn-Cys-Tyr-Pro-Tyr -Asp-Val-Pro-Asp-Tyr-Ala-Ser-Leu The preferred epitopes of the T helper are the hemagglutinin epitope of influenza virus 307-319 [HA 307-319] of the sequence: Pro-Lys- Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr and the epitope of HA of 306-324 [HA 306-324] of the sequence: Cys-Pro-Lys-Tyr-Val Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr-Gly-Met-Arg-Asn-Val The epitopes of the cytotoxic T-lymphocytes (CTL) used in the vaccine of the invention will change according to the type of population, mainly Caucasian or non-Caucasian (of Asian or African origin). For Caucasian populations, the preferred CTL epitopes are the nucleoprotein epitope (NP) of influenza virus 335-350 [NP 335-350] of the sequence: Ser-Ala-Ala-Phe-Glu-Asp-Leu-Arg Val-Leu-Ser-Phe-lle-Arg-Gly-Tyr and the NP epitope of 380-393 [380-393] of the sequence: Glu-Leu-Arg-Ser-Arg-Tyr-Trp-Ala-lle Arg-Thr-Arg-Ser-Gly In a preferred embodiment of the invention, the intranasal influenza vaccine consists of a mixture of the four epitopes of the influenza virus: hemagglutinin epitopes HA91-108 and HA307-319, and epitopes of nucleoprotein NP335-350 and NP380-393, expressed individually in flagellin of Salmonella. For non-Caucasian populations, other CTL epitopes can be used. The present invention also relates to the use of a mixture of flagella containing at least four epitopes of the influenza virus each individually expressed in Salmonella flageiin, as described above, for the preparation of a synthetic human influenza vaccine for administration intranasal The present invention further relates to a method for inducing a human immune response and conferring protection in humans against influenza virus, which comprises administering to human individuals a synthetic influenza vaccine based on peptides comprising a mixture of flagella, as described above BRIEF DESCRIPTION OF THE DRAWINGS In the following legends, "tetraconstruction" means a mixture of flagella that expresses the four influenza epitopes HA91-108, HA307-319, NP335-350 and NP380-393, respectively. Figs. 1A-1 B describe typical FACS histograms of human lung lymphocytes in radiated human / mouse chimeras immunized with tetraconstruction. Samples were taken 7 days after immunization. The cells were separated on ficoll gradient and stained with anti-CD45 together with anti-CD3 (figure 1A) or together with anti-CD19 (figure 1 B), conjugated to the respective fluorescent dye. Histograms show that after Immunization the majority of human cells are T cells and almost no B cells can be detected. Fig. 2: Lung homogenates from immunized and non-immunized mice as well as groups of mice no transplants were analyzed for the virus titer five days after the viral test. The mice were immunized with the tetraconstruction (left block) or with native flagellin that does not express the influenza epitopes (middle block). Another control group did not receive PBMC but was immunized with the tetraconstruction (right block). The figure presents the average data of 7 repeated experiments, in which each group consisted of 6-animals. In each experiment, a different donor was used. Fig. 3: Production of human antibodies (total amount of IgG, IgM and IgA), in radiated human / mouse chimeras (6-8 animals per group in 7 repeated experiments, different donors were used in each experiment) immunized with tetraconstruction ( left column) or with native flagellin that does not express the influenza epitopes (middle column). Another control group did not receive FBMC, but was immunized with the tetraconstruction (right column). The serum samples were diluted 1: 10, the lung samples were diluted 1: 60. The production of Ac in the group that was transplanted and vaccinated with the tetraconstruction (left column) was significantly higher than in the other control groups. Fig. 4: Percentage of survival of human / mouse radiation chimeras from the lethal test after intranasal vaccination with tetraconstruction. Mice (5-10 animals per group in two repeated experiments, different donors were used in each experiment) were transplanted with PBMC on day 0, vaccinated on day 9 and tested on day 7 afterwards. Vaccination with tetraconstruction (black circles), with native flagellin (hollow circles) or non-transplanted mice that were vaccinated with tetraconstruction (squares).

After day 40, the survival rate remained the same and all the mice that survived eventually recovered. Fig. 5: The body weight of the mice that survived, which is indicative of the severity of the disease and the potential for a recovery process. The irradiated human / mouse chimeras (5-10 animals per group in two experiments per group, different donors were used in each experiment) were transplanted with PBMC at day 0, vaccinated intranasally on day 9 and tested intranasally 7 days later with a lethal dose of the virus. The mice vaccinated with the tetraconstruction (black circles) lost less weight and recovered faster than the other groups. The control groups consisted of transplanted mice that were administered with native flagellin (hollow circles) or non-transplanted mice that were vaccinated with the tetraconstruction (squares), after 40 days, all the mice that survived slowly recovered and gained weight. Fig. 6: Protective vaccination of irradiated human / mouse chimeras transplanted with PBMC and immunized intranasally with tetraconstruction. Each group of human / mouse chimeras (5-10 animals per group in two repeated experiments, different donors were used in each experiment) was transplanted with PBMC obtained by leucoapheresis from a donor that was infected 7 days after immunization with a of the three influenza strains: A / PR.8 / 34 (H1 N1), AJapanese / 57 (H2N2) or A / exas / 1/77 (H3N2). Both the transplanted mice (left column) and the non-transplanted mice (right column) were vaccinated with the tetraconstruction. However, only the transplanted mice were able to resist the infection and the virus titer in their lungs was significantly lower. Fig.7: Human serum antibodies against influenza virus following the immunization of irradiated lethally irradiated human / mouse chimeras (5-10 per group) radioprotected with 3x106 SCID of bone marrow (BM) and transplants with 70x106 of human PBMC. All groups were immunized with tetraconstruction and then tested with sublethal doses of the H1N1 strain (black diamonds) or H2N2 (black circles) or H3N2 (black squares). The control group consisted of mice that were given irradiated SCID that did not receive PBMC and were immunized with the same vaccine before the test with H1 N1 (empty diamonds) or H2N2 (empty circles) or H3N2 (empty squares).

DETAILED DESCRIPTION OF THE INVENTION The concept of peptide-based vaccine has several advantages over traditional vaccines, including safety considerations, the relatively long shelf life, the ability to direct the immune response towards specific epitopes that are neither suppressive nor hazardous for the host and the possibility for prepare multi-pathogenic vaccines. The efficacy of a peptide vaccine is highly dependent on the exact identification of the immunogenic epitopes that confer protection as well as the efficient presentation of these epitopes to the immune system. The idea of a peptide vaccine for influenza which includes both epitopes of B cells and T cells was previously tested in the mouse model, and it has been shown that said "vaccine" could induce the specific local response in the lungs leading to the protection of mice immunized for viral tests (Arnon and Levi, 1996), in the mouse model used there it was shown that the epitope of the B cell does indeed induce a high production of Ac, while the epitope of the T helper produces specific lymphocyte proliferation and the CTI epitope was important for cytotoxic activity against infected cells. However, efficient protection was achieved only when the mice were immunized with a mixture of the three epitopes (Levi and Arnon, 1996). In accordance with the present invention, for the purpose of human use, the appropriate epitopes have to be selected because the epitopes of the T cell are restricted by MHC. First, the inventors have identified that at least four epitopes of influenza are necessary for human use: an epitope HA of the B cell, an epitope HA or NP of the helper T that can bind to many HLA molecules, and at least two CTL epitopes NP or matrix that are restricted to the most prevalent HLA molecules in different populations. In accordance with the invention, a preferred influenza epitope of P cell is HA 91-108. Preferred influenza epitopes of T helper are HA 307-319 and HA 306-324 (Rothbard), 1988), but NP 206-229 can also be used (Brett, 1991). The CTL epitopes of influenza are different in Caucasian populations, originated in Asia or originated in Africa. For the Caucasian population, the preferred CTL epitopes of influenza are NP335-350 and NP380-393 (Dyer and Middleton, 1993; Gulukota and DeLisi, 1996), and are restricted to the most prevalent HLA molecules in the Caucasian population. Other epitopes of influenza that can be used according to the invention for the Caucasian population are the nucleoprotein epitopes: NP305-313 (DiBrino, 1993); NP384-394 (Kvist, 1991); NP89-101 (Cerundolo, 1991); NP91-99 (Silver et al, 1993); NP380-388 (Suhrbier, 1993); NP44-52 and NP265-273 (DiBrino, 1994), and NP365-380 (Townsend, 1986); and the epitopes (M) of the matrix proteins M2-22, M2-12, M3-11, M3-12, M41-51, M50-59, M51-59, M134-142, M145-155, M164-172 , M164-173 (all described by Nijman, 1993); M17-31, M55-73, M57-68 (Carreno, 1992); M27-35, M232-240 (DiBrino, 1993). For non-Caucasian populations, the CTL epitopes of influenza that can be used are HA458-467 of the sequence Asn-Val-Lys-Asn-Leu-Tyr-Glu-Lys-Val-Lys (NVKNLYEKVK), a CTL epitope for the allele A11 with high frequency in the Japanese, Chinese, Thai and Indian populations (J. Immunol., 1997), 159 (10): 4753-61); M59-68 and M60-68 of the sequences lle-Leu-Gly-Phe-Val-Phe-Leu-Thr-Val (ILGFVFTLTV) and Leu-Gly-Phe-Val-Phe-Leu-Thr-Val (LGFVFTLTV), respectively, two CTL epitopes for HLA-B51 with high frequency in the Thai population (Eur. J. Immunol., 1994, 24 (3): 777-80); and M128-135 of the Ala-Cys-Ser-Met-Gly-Leu-lle-Tyr sequence (ACSMGLIY), a CTL epitope for the B35 allele with high frequency in the West Negroid African population (Eur. J. Immunol. 1996, 26 (2): 335-39). Since the peptides are usually poor immunogens, the effectiveness of the peptide-based vaccine depends on the proper presentation of the epitopes to the immune system. Influenza epitopes are expressed in the flagellin gene of the Salmonella vaccine strain, which provides both vehicle and adjuvant functions. After the breaking of the flagella from the bacteria and other purification steps, the fine suspension of the flagella was used for vaccination. All immunizations were carried out with a mixture of the four epitopes: HA91-108, ha307-319, NP335-350 and NP308-393, which are expressed in Salmonella flagellin, in the absence of any adjuvant. The mixture of these four epitopes is referred to as "tetraconstruction" through the specification. The three epitopes of the T cell used in the vaccine of the present invention were selected because of their specific recognition by HLA prevalent in the Caucasian population, and were included in the vaccine together with the B cell epitope HA 91-108. In order to overcome the problem of antigenic variation of the virus, all of these epitopes are derived from regions conserved in the viral proteins and can therefore induce cross-strain protection. The two CTL epitopes from the internal nucleoprotein are recognized by HLA prevalent in the Caucasian population: the NP 335-350 epitope is restricted to A2, A3, Aw68.1 and the B37 HLA haplotypes, and the epitope NP 380-393 is restricted to the B8 and B27 HLA haplotypes. The epitope of the T helper from hemagglutinin, HA 307-319, is a "universal" epitope restricted to most MHC class II molecules, including DR1, DR2, DR4, DR5, DR7, DR9, DR52A, and others. These epitopes of the T cell, together with the epitope HA 91-108 of the B cell, were individually expressed in flageiin and the resulting mixture of flagella was used without any adjuvant for intranasal vaccination of radiated human / mouse chimeras, thus inducing a response human immune and conferring protection. The vaccinated mice were also protected from a lethal infection and their recovery was faster. To evaluate the capacity of said tetraconstruction to act as a vaccine and stimulate a response of the human immune system, a humanized mouse model was used. The observation that human PBMC can be transferred adoptively i.p. Within mouse SCID and that the grafted cells survive for an extensive period of time producing high levels of human Ig, it has offered many new possibilities in clinical immunological research (reviewed in Mosier, 1991). In particular, many researchers have used this model to study the ability of grafted lymphocytes to generate primary and secondary humoral responses in humans, and for viral research studies.

Recently, Lubin et al., 1994, described a new method that enables human PBMC implants in normal strains of mice followed by a lethal dose-dose irradiation allowing rapid and effective grafting of human cells. As previously reported, in such radiated human / mouse chimeras, a human as well as cellular humoral response (CTL) can be generated by immunization either with external antigens or with allogeneic cells (Marcus et al, 1995, Segal et al, 1996). , giving advantages to this model in comparison with the SCID mouse model previously used by Mosier. Additional advantages of this model is that the dissemination of the grafted lymphocytes is very fast and that both the B and T lymphocytes were found by FACS analysis in significant numbers in the iinfoid tissue within a few days post-transplant (Burakova et al. 1997). For the evaluation of the efficiency of the human influenza vaccine according to the invention, we use this model of radiated human / mouse chimeras. Although the number of human B cells after transplantation was low (Figure 1), the chimeric mouse was able to produce specific antibodies in response to i.p. of antigens. This is consistent with previous findings, which show that by the second week post-transplant, grafted human B and T cells form follicles in the splenic and lymnoid nodules. In addition, its phenotype was that of memory cells, that is, mainly CD45RO positive and CD45RA negative (Burakova et al, 1997).

In accordance with the present invention, the human / mouse radiation chimera was immunized with the tetraconstruction administered by the intranasal route. This is the first report of induction of local immune response in the nasal cavity and lungs after intranasal immunization in the human / mouse radiation chimera. The induction of local immune response in the lungs was demonstrated by the presence of specific anti-influenza antibodies in lung homogenates (Figure 3), by the elevated CD8 + lymphocyte ratio and by viral clearance as a result of immunization with the tetraconstruction (figure 2). The tetraconstruction of flagellin could also protect the mice from the lethal dose test of the virus, which is the ultimate demonstration of the protective effect. Under these conditions, in which the dose tested is orders of magnitude greater than that found in a natural infection, all chimeras were infected regardless of their immune status. However, while none of the immunized mice that were not transplanted with the human lymphocytes survived the infection, and only 50% of the transplanted but not immunized mice survived, the transplanted and immunized group that was completely protected and showed 100% survival (figure 4). Partial protection in unvaccinated mice is probably due to polyclonal stimulation and expansion of memory cells that originate from the donor. This could be due either to prior donor exposure to the antigen or due to cross-reactivity to some degree with other antigens voided, a phenomenon that was previously reported for other antigens (Marcus et al, 1995). However, although such partial protection was observed in fact, a significant difference in the efficiency of the recovery process between the immunized and non-immunized groups was observed as evident both for the survival rate and for the weight loss pattern (FIGS. , 5). Although the HLA phenotypes of the PMBC donors were not determined, all transplanted mice were protected as a result of vaccination, indicating that the epitopes used in the present invention are in fact recognized by a wide variety of HLA molecules. One of the most acute problems related to currently existing influenza vaccines is the narrow range of their specificity and their restricted specific activity of strain. Rapid variation in viral surface glycoproteins leads to the appearance of new strains with high variability in their sero-specificity, and therefore the vaccines that contain the external glycoproteins of some specific strains are limited in their efficiency for these strains. In accordance with the present invention, the inventors also established the cross protection capacity of the tetraconstruction vaccine. All the epitopes that were included in the tetraconstruction are regions conserved in the respective proteins, and consequently, the antibodies against recombinant flagella were able to recognize several strains of influenza (table 1). Consequently, the immunization of the chimeric mice with the epitopes reached the production of specific antibodies and their infection protection with sublethal doses for three different influenza strains, of the specificity H1, H2 or H3 (figure 6). Thus, the results with the tetractruction in accordance with the invention demonstrate the ability of a vaccine based on synthetic peptide to confer protection against the influenza viral test. The recombinant flagellin construct in fact exhibits the epitopes of influenza B and T cells to human immune cells in an efficient manner and induces both humoral and cellular response. Since the epitopes used in the T cell are recognized by a variety of HLA molecules, the vaccine was effective in all experiments in which different donors were used with HLA unknown type, indicating the applicability of this method for a human vaccine in a heterologous population.

EXAMPLES Materials and methods 1. -Mice BALB / c mice (4-8 weeks of age) were obtained from the Olac farms (Bicester, UK), the NOD / SCID mice (4-6 weeks of age) from the Animal Breeding Center of the Weizmann Institute (Rehovot, Israel). All mice were fed sterile food and acidic water containing ciprofloxacin (20 μg / ml). 2. - Conditions of the regimen The BALB / c mice were exposed to a total body lethal irradiation (TBI) of 4 Gy followed by 10 Gy and 3 days later. The radiation source is a 150-A 60Co gamma ray (produced by The Atomic Energy of Canada, Kanata, Ontario). Bone marrow cells from the NOD / SCID mice (4-6 weeks of age) were obtained according to Levite et al., 1991. The irradiated recipient mice were injected with 2-3x10 6 cells from SCID bone marrow (iv in 0.2 ml of phosphate buffered saline (PBS)) one day after irradiation. 3. Preparation and transplantation of human peripheral blood lymphocytes The yellow coat of normal volunteers was layered on Limphoprep solution (Nycomed, Oslo, Norway) and centrifuged at 2000 rpm for 20 min. The interlayers were collected, washed twice, counted and resuspended in PBS pH 7.4, at the desired cell concentration. Human PBMC (70x106 cells in 0.5 ml PBS) were injected i.p. within recipient mice, conditioned as described above. The control mice did not receive human PBMC. "4. Leukapheresis procedure The leucoapheresis was carried out on normal volunteers.The cells were collected by processing 3-4 liters of blood through V50 (USA) for 3-3.5 hours.The leucoapheresis product was centrifuged at 1200 rpm for 10 min and the plasma was removed.

. - Chimeric flagellin The oligonucleotides corresponding to the designated influenza epitopes, designated NP335-350 (SAAFEDLRVLSFIRGY), NP380-393 (ELRSRYWAIRTRSG) and two hemagglutinin peptides of subtype H3: HA91-108 (SKAFSNCYPYDVPDYASL) and HA307-319 (PKYVKQNTLKLAT) they were synthesized in a 380B Applied Biosystems DNA synthesizer, with additional GAT sequence at the 3 'of each oligonucleotide in order to preserve the EcoRV restriction site, as described (Levi and Arnon, 1996). The synthetic oligonucleotides were inserted into the EcoRV site of plasmid pLS408 and eventually transformed into a live flagellin negative vaccine strain (an Aro A mutant) of Salmonella dublin SL5928 by transduction, using phage P22HT105 / 1 int. Finally, the flagella were purified after the acid break and a fine suspension was used for immunization (Levi and Arnon, 1996). 6. Preparation of Recombinant Bacteria The construction of the expression vector pLS408 is described by Newton et al., 1989, incorporated herein by reference. The synthesized oligonucleotides were inserted into the EcoRV site of plasmid pLS408, and transformed into competent E. coli JM101 cells. The colonies containing the recombinant plasmid were selected when tested with one of the oligonucleotides labeled with 32p-ATP. The plasmids from the positive colonies were purified and the orientation of the insert was determined using restriction analysis. The desired plasmids were used to transform competent Salmonella typhimurium LB5000 cells (a negative restrictive, a non-flagellated, non-flagellated modification) (Bullas and Ryu, 1983, incorporated herein by reference in its entirety) and transferred to a live flagellin negative vaccine strain ( a Mutant Aro A) of Salmonella dublin SL5928 by transduction using phage P22HT105 / 1 int (Orbach and Jackson, 1982, and Schmieger, 1972, both fully incorporated herein by reference). Transformed S. dublin was selected for ampicillin resistance, motility under light microscopy and growth in semisolid LB agar plates, supplemented with oxoid nutrients from # 2 broth. The selected clones were grown overnight in two liters of LB ampicillin medium and the flagellin was purified by acidic cleavage, in accordance with the technique described by Ibrahim et al., 1985, incorporated herein by reference. 7. - Isolation of flagella Flagella were isolated in accordance with Ibrahim et al., 1985: Bacterial cells from a culture grown overnight in LB / ampicillin medium were concentrated and suspended in a small volume of PBS. The pH was reduced with 1 M HCl to 2.0 M and the suspension was incubated at room temperature for 30 minutes with gentle agitation. The bare cells were removed by centrifugation at 5000 rpm for 15 minutes and the pH readjusted to 7.4. Then the flagella were precipitated with (NH4) 2S04 (35% w / v) and kept overnight at 4 ° C. The pellet obtained after centrifugation at 10,000 for 10 minutes at 4 ° C was dissolved in PBS, dialysed against a large volume of PBS at 4 ° C and any precipitate formed was discarded. The resulting protein was stored at -20 ° C. These resulting flagella are an aggregate of the flagellin protein and can be used as such for a vaccine. The presence of the chimeric flagellin HA and NP protein epitopes of the invention are shown in FIG. 2 after SDS-PAGE of flagella. 8. Immunization and infection of chimeric animals On the ninth day after transplantation of PBMC, the human / mouse chimeras were immunized once, intranasally with a mixture of 25 μg of each hybrid flagellin construct in a total volume of 50 μl PBS or, in the control group, with 75 μG of native flagella. This amount was predetermined as the optimal dose in a preliminary experiment in BALB / c mice. The infection of mice was carried out 7 days later when inoculated intranasally with the allantoic fluid of infection, 50 μ 10 4 HAU virus per mouse.In both immunization and infection, the mouse was under light anesthesia of ether.The chimeras were sacrificed on the fifth day after infection, his lungs were removed for viral titration. 9. FACS analysis of PBMC donors and human cell grafts in chimeric mice For the evaluation of human cell grafts in human / mouse chimeras, mice with human lymphocyte grafts were sacrificed 27-29 days after PBMC transplantation. The lymphocytes from the lung homogenates as well as the peritoneal washings were separated on a ficoll-paque gradient (Pharmacia Biotech AB, Upsala, Sweden) and incubated for 30 minutes on ice with a mixture of appropriate fluorescently labeled monoclonal antibodies. After washing, double fluorescent analysis of the human antigens was carried out with a FACScan analyzer (Beckton-Dickinson, CA). The following antibodies were used which recognize the specific molecules of human surface: anti-CD45-phycoerythrin (PE) (clone HI30) of Pharmigen; chlorophyll protein anti-CD3-peridinin chlorophyll protein (PerCP) (clone SK7); and anti-CD19-FITC (clone 4G7) (Beckton-Dickinson, CA).

. Determination of human immunoglobulin. Total human Ig was quantified in serum samples from interspersed ELISA using goat F (ab) 2 - purified from antihuman Ig (G + M + A) (Sigma) as the capture agent and peroxidase conjugate purified from Goat anti-human Ig (G + M + A) (Sigma) as the detection reagent. The human serum immunoglobulin of known concentration was used as the standard. The ELISA was carried out as described by Marcus et al., 1995. 11. Determination of human-specific immunoglobulins for influenza Lung and serum homogenates were tested for specific human anti-influenza antibodies, the virus (100 HAU / ml) was adsorbed on ELISA plates and the blocking was carried out with 1% albumin bovine serum (BSA) in PBS. The Ig, rabbit anti-human was conjugated with horseradish peroxidase (Sigma) and used as second antibodies. Plates were read at 414 nm following the addition of the substrate (ABTS). 12. Influenza virus Influenza strains A / PR / 8/34 (H1N1) were used, A Japanese / 57 (H2N2) and A / Texas / 1/77 (H3N2). Virus amounts were measured in hemagglutination units (HAU). For immunization, the inactive virus was used (ATexas / 1/77), purified by sucrose gradient. The growth and purification of virus was in accordance with standard methods (Barret and Inglis, 1985). For the titration, the lung samples were homogenized in PBS containing 0.1% BSA and centrifuged in order to remove remnants. The titrations of the viruses were determined by complete egg titration methods (Barret and Inglis, 1985). The titer was calculated for haemagglutination and presented as Log EID5o (Thompson, 1947). 13. Statistical analysis The statistical analysis was carried out using the Stat View II program (Abacus Concepts Inc., Berkeley, CA, USA) on Macintosh llCi. The F test was used to calculate the probability values (p). The results are presented as mean and standard error of at least two independent repeated experiments, including 5-10 animals per group.

EXAMPLE 1 Response of chimeric mice to completely inactivated influenza virus In order to establish the desirability of the irradiated human / mouse chimeras for the evaluation of the synthetic peptide-based vaccine, the inventors first evaluated their immune response towards the purified inactive influenza virus which is known to be protective. The mice were immunized i.p. with 50μp of the virus on the day of the PBMC transplant, followed by a sublethal viral test with influenza strain A / Texas / 1/77 14 days after the immunization. Vaccination of the human / mouse chimeras radiated with the killed whole virus vaccine, without any adjuvant, induced the production of specific antibodies - the antibody titer in serum was significantly higher (2.4 times) in the immunized chimeras compared to the group control. In addition, this vaccination markedly reduced the subsequent viral infection. The titer of lung virus after the test was significantly lower (by 2.7 orders of magnitude) in the immunized chimeras compared to the control group. After thus demonstrating the desirability of the irradiated human / mouse chimeras for the evaluation of the anti-influenza response followed by immunization with inactivated influenza virus, the inventors proceeded with the evaluation of the peptide-based synthetic recombinant vaccine designed for humans in This humanized mouse model.

EXAMPLE 2 FACS analysis of the mice immunized for the evaluation of the human PBMC graft in human / BALB chimeras Successful grafting of human cells into the human / mouse chimera was demonstrated in a preliminary experiment that showed that most of the lymphocytes in the peritoneum (50-80%) and in the lungs of the mice (30-60%) were of human origin. For the evaluation of the human cells grafted on the human / mouse chimeras, the presence of human cells in the mice grafted by FACS was analyzed. Figure 1 is a FACS histogram that describes the pattern of lymphocytes in the human lung after immunization with tetraconstruction without additional test infection. The cells were stained with anti-CD45 antibodies or together with anti-CD3 or together with anti-CD19. As shown, most human cells (stained with anti-CD45) are CD3 +, called T cells (80% -90%) and only a minor population is CD19 + (3% -10%). Similar data obtained from human lymphocytes in the peritoneum. It is of interest that the CD8 + / CD4 + ratio in the immunized mice had a range between 1 and 2 as compared to the ratio of 0.3-0.5 in the untreated chimeras. This disproportionate expression of CD8 cells may suggest that they play a role in the observed protection.

EXAMPLE 3 Virus purification from the lungs following the sublevel test Influenza infection is a respiratory disease, therefore, a local immune response induced by an intranasal administration vaccine could be more effective than parenteral administration. The immunization scheme was modified in order to adapt it for intranasal immunization. Mice (6-8 per group in 7 repeat experiments) were immunized intranasally (i.n.) 10-12 days after transplantation of PBMC, as described in the methods. Ten days later, i.n. with 10"4 HAU in 50μl of the allantoic fluid of live strain A / Texas / 1/77 of the influenza virus, five days later they were sacrificed and their lungs were removed for viral titration as shown in figure 2, which exhibits the cumulative results, the vaccination with the tetraconstruction enabled the chimaera to purify the virus from its lungs significantly more efficiently than the group vaccinated with native flagella, or the group that was not transplanted with PMBC but that was immunized with the tetraconstruction. Although the same percentage of human T lymphocytes were detected in both transplanted groups (figure 1), only mice vaccinated with hybrid flagellin showed the ability to reduce viral load, indicating a specific and efficient local response in the lungs. The production of human antibodies in these mice was evaluated both in the serum (before the test) and in the lungs (after the test). Immunization with the tetraconstruction resulted in a significantly higher titer of human antibodies specific for the virus in both the serum and lung samples (figure 3). It seems that although the proportion of CD19 + lymphocytes as detected by the FACS analysis was similarly low in the immunized mice and in the control transplants, the production of the anti-influenza specific antibody response differed significantly between the two groups.

EXAMPLE 4 Survival and weight loss pattern after the lethal dose of viral infection Beyond the sublethal infection test experiment, the ability of the tetraconstruction preparation to protect the human / mouse chimeras from the lethal dose of influenza virus was examined. Figure 4 describes the results of two repeated experiments and demonstrates the survival of vaccinated and unvaccinated mice (both transplanted with human PBMC), as well as another control group that was not transplanted but was vaccinated with the tetraconstruction. As can be seen, while all the control mice that were immunized with the tetraconstruction but were not transplanted with the human lymphocytes died within 19 days after the injection, 100% survival was observed in the mice that received the PBMC before of immunization. This indicates that survival is due to the response of transplanted human immunocompetent cells. The PBMC by itself provides a limited beneficial effect, as 50% of the survival was observed in the control group that was vaccinated with the native flagellin, which by itself does not induce any anti-influenza protective response. In Figure 5, the body weight pattern of the tested mice is evidenced: the transplanted group that was immunized with the tetraconstruction of flagellin, shows only a slight reduction in their body weight following infection with lethal dose and a rapid return to normal, whereas the control group that was transplanted with human PBMC but was immunized with native flagellin, lost less weight (the body weight is significantly different between the experimental group and the control groups at days 22-33 after transplantation ) and mice that survived began weight recovery only in the last days of the experiment. Control groups not transplanted or vaccinated lost weight quickly and did not recover. The survival of the transplanted groups that were immunized with native flagella is better than that of the groups not transplanted, probably due to some anti-influenza memory response of the donor cells.

EXAMPLE 5 Protection of infection with different strains of influenza One of the main problems with currently available Influenza vaccines is that they are effective only against the strains that are included in the vaccine. Therefore, it was of interest to examine the ability of hybrids to flagellin expressing influenza epitopes to protect mice from different strains of influenza carrying several hemagglutinins and neuraminidase glycoproteins. The epitope of B cell that is expressed in flagellin is conserved in all influenza subtypes, H3, while the T cell epitopes are from highly conserved regions of hemagglutinin and nucleoprotein in other subtypes as well. In the first step, it is shown that rabbit antibodies to these epitopes can in fact recognize and react in ELISA with different strains of influenza including A / Texas / 1/77, A / Aichi / 68, A / PR / 8 / 34 and A / Japanese / 57 (table 1). To further test the potential of these epitopes to confer cross-protection in humans, the human / mouse radiation chimera (8 mice per group) was immunized i.n. with the tetraconstruction. Its resistance to different test influenza strains was detected 7 days later and compared with non-transplanted mice that were immunized with the same mixture of flagella. The influenza strains that were used for the infections were: A / Texas / 1/77 (H3N2), A Japanese / 57 (H2N2) and A / PR / 8/34 (H1 N1). Protective immunity was observed against all three strains, as presented in figure 6. The specific human Ig for each influenza strain was detected in the serum of all transplanted and vaccinated mice, but not in the control group, as It is shown in figure 7.

TABLE 1 Rabbits immunized with four influenza epitopes (NP 335-350, NP 380-393, HA 91-108 and HA 307-319) were conjugated to BSA, producing antibodies whose specificity was determined by ELISA. These antibodies recognized different strains of influenza virus that were covering the ELISA microplates. The recognition between antibodies raised against whole viruses (A / Texas / 1/77) serves as a positive control. Serum samples were tested in 1: 150 dilutions, and antibody recognition was scaled according to the maximum D.O .: +++ = D.O > 2; ++ = D.O. 1-2; + = D.O. 0.5-1; + - = D.O.O.5.

References 1. Arnon, R, and Levi, R. Synthetic recombinant vaccine induces anti-influenza long-term immunity and cross strain protection, In: Novel Strategies in Design and Production of Vaccines (Ed .: Cohen, S. and Shafferman, A.) Plenum Press, NY, 1996, p.23. 2. Barret, T and Inglis, S.C. Growth purification and titration influenza viruses, In: Virology. A practical approach (Ed. Mahy, W.J.) IRL Press, Wash. D.C., 1985, pp. 119-151. 3. Brett et al., J. Immunol. 1991. 147: 984-991. 4. Bullas.I.R. and Ryu, J. J. of Bacterio !. 1983. 156: 471-74. 5. Burakova, T., Marcus, H., Canaan, A., Dekel, B., Shezen, E., David, M., Lubin, [., Segal, H. and Reisner, Y. Engrafted human T and B lymphocytes form mixed follicles in lymphoid organs of human / mouse and human / rat radiation chimera. Transplantation 1997. 63: 1166-1171. 6. Carreno, B.M., Koenig, S., Coligan, J.E. and Biddison, W.E. The peptide binding specificity of HLA class I molecules is largely allele-specific and non-overlapping. Mol Immunol 1992. 29: 1131: 1140. 7. Cerundoio et al. Proc. R. Soc. Lon. 1991. 244: 169.7. 8. DiBrino et al. PNAS 90. 1993. (4): 1508-12. 9. Dyer, P. and Middleton, D. In: Histocompatibility testing, a practical approach (Ed .: Rickwood, D. and Hames, B.D.) IRL Press, Oxford, 1993, p. 292. 10. Gulukota, K. and DeLisi, C. HLA aliéis selection for designing peptide vaccines. Genetic Analysis: Biomolecular Engineering 1996.13: 81. 11. Ibrahim, G.F. et al. J. Clin. Microbiol. 1985. 22: 1040-1044. 12. Kvist et al. Nature 1991. 348: 446-448. 13. Laver, W.G., Air, G.M., Dopheide, T.A. and Ward, C.W. Amino acids sequence changes in the Hemagglutinin of A / Hong Kong (H3N2) influenza virus during the period 1968-77. Nature 1980. 283: 454-457. 14. Laver, W., Air., G., Webster, R., Gerhard, W., Ward, C and Dopheid, T. Antigenic drift in type A influenza virus: sequence differences in the Hemagglutinin of Hong Kong (H3N2 ) variants selected with monoclonal hybridoma antibody. Virology 1980a. 98: 226-237. 15. Levi, R. and Arnon, R. Synthetic recombinant vaccine induces efficient long-term immunity and cross strain protection. Vaccine 1996. 14: 85-92. 16. Levite, M., Meshorer, A. and Reisner, Y. A rapid method for obtaining murine bone marrow cells in high yield. Bone Marrow Transpl. 1991. 8: 1-3. 17. Lubin, I., Segall, H., Marcus, H., David, M., Kulova, L., Steinitz, M., Eriich, P., Gan, J. and Reisner, Y. Engraftment of human blood lymphocytes in normal strains of mice. Blood 1994. 83: 2368-2381. 18. Marcus, H., David, M., Cnaan, A., Kulova, I. Lubin, I. Segal, H., Denis, L., Eriich, P., Galun, E., Gan., J. , Laster., M and Reisner, Y. Human / mouse radiation chimera are capable of mounting a human primary humoral response. Blood 1995. 86: 398-406. 19. Mosier, D.E. Adoptive transfer of human lymphoid cells to severely immunodeficient mice: modeis for normal human immune function, autoimmunity, lymphomagenesis, and AIDS. Adv. Immunol. 1991. 50: 303-325.

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Claims (7)

NOVELTY OF THE INVENTION CLAIMS

1. - A synthetic intranasal influenza vaccine based on human peptide for intranasal administration comprising a mixture of flagella containing at least four epitopes of the influenza virus each expressed individually in the flageiin of Salmonella, said epitopes of influenza virus being reactive with human cells and being selected from the group consisting of: (i) an epitope (HA) of hemagglutinin from the B cell; (I) an epitope of hemagglutinin (HA) or nucleoprotein (NP) of the helper T cell that can bind to many HLA molecules; and (iii) at least two epitopes of cytotoxic lymphocyte (CTL) nucieoprotein (NP) or matrix protein (M) that are restricted to the most prevalent HLA molecules in different human populations.

2. The intranasal vaccine against human influenza according to claim 1, characterized in that the epitope HA of the B cell is the epitope of the hemagglutinin of the influenza virus 91-108 [HA 91-108] of the sequence : Ser-Lys-Ala-Phe-Ser-Asn-Cys-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ser-Leu.

3. The intranasal vaccine against human influenza according to claim 1 or 2, further characterized in that the epitope of the helper T cell is selected from the hemagglutinin epitope of influenza virus 307-319 [HA 307-319] of the sequence: Pro-Lys-Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr and the epitope HA 306-324 [HA 306-324] of the sequence: Cys-Pro-Lys -Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr-Giy-Met-Arg-Asn-Val.

4. The intranasal vaccine against human influenza according to any of claims 1-3, further characterized in that at least two epitopes of cytotoxic lymphocyte (CTL) are the epitope (NP) NP335-350 of the nucleoprotein of the human influenza virus. influenza of the sequence: Ser-Ala-Ala-Phe-Glu-Asp-Leu-Arg-Val-Leu-Ser-Phe-lle-Arg-GIy-Tyr and the epitope NP380-393 of the sequence: Glu-Leu Arg-Ser-Arg-Tyr-Trp-Ala-lle-Arg-Thr-Arg-Ser-Gly.

5. The intranasal vaccine against human influenza according to any of claims 1 to 4, further characterized in that it consists of a mixture of the four epitopes of the haemagglutinin epitope of influenza virus HA91-108, hemagglutinin epitope HA307- 319, nucleoprotein epitopes NP335-350 and NP380-393, expressed in flagellin from Salmonella.

6. The intranasal vaccine against human influenza according to any of claims 1 to 3, for non-Caucasian populations, wherein at least two lymphocytic cytotoxic epitopes (CTL) are selected from the influenza virus epitope HA458-467 of the sequence Asn-Val-Lys-Asn-Leu-Tyr-Glu-Lys-Val-Lys, and the epitopes of the matrix protein (M) M59-68, M60-68 and M128-135 of the sequences: Leu-Gly-Phe-Val-Phe-Leu-Thr-Val, lle-Leu-Gly-Phe-Val-Phe-Leu-Thr-Val, and Ala-Cys-Ser-Met-Gly-Leu-lle-Tyr , respectively.

7. The use of a mixture of flagella containing at least four epitopes of influenza viruses reactive with human cells each of which are individually expressed in flagellin of Salmonella, said epitopes of the influenza virus being selected from the group which consists of: (i) a hemagglutinin epitope of B cell (HA); (ii) a hemagglutinin epitope of the helper T cell (HA) or a nucleoprotein epitope (NP) that can bind to many HLA molecules; and (iii) at least two epitopes of cytotoxic lymphocyte (CTL) core protein (NP) or "matrix protein (M) that are restricted to the most prevalent HLA molecules in different human populations, for the preparation of a synthetic influenza vaccine. human for intranasal administration.