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) or matrix protein (M) epitopes that are restricted to the most prevalent HLA molecules in different human populations.

SUMMARY OF THE INVENTION

According to the present invention, influenza peptide epitopes reactive with human cells were expressed in Salmonella flagellin and tested for efficacy in a human/mouse radiation chimera in which human PBMC were functionally engrafted. Clearance of the virus after challenge and resistance to lethal infection was found only in the vaccinated mice and production of virus specific human antibodies was also higher in this group. FACS analysis showed that most 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 human synthetic peptide-based influenza vaccine for intranasal administration comprising a mixture of flagella containing at least four epitopes of influenza virus each expressed individually in Salmonella flagellin, said influenza virus epitopes being reactive with human cells and being selected from the group consisting of: (i) one B-cell hemagglutinin (HA) epitope; (ii) one T-helper hemagglutinin (HA) or nucleoprotein (NP) epitope that can bind to many HLA molecules; and (iii) at least two cytotoxic lymphocyte (CTL) nucleoprotein (NP) or matrix protein (M) epitopes that are restricted to the most prevalent HLA molecules in different human populations.

The preferred B-cell HA epitope is the influenza virus hemagglutinin epitope 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 (SEQ ID NO:1) The preferred T-helper epitopes are the influenza virus hemagglutinin epitope 307 319 [HA 307 319] of the sequence:

Pro-Lys-Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr (SEQ ID NO:2) and the HA epitope 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-Va- l (SEQ ID NO:3)

The cytotoxic T-lymphocyte (CTL) epitopes used in the vaccine of the invention will change according to the population type, namely Caucasian or non-Caucasian (of Asian or African origin). For Caucasian populations, the preferred CTL epitopes are the influenza virus nucleoprotein (NP) epitope 335 350 [NP 335 3501] of the sequence:

Ser-Ala-Ala-Phe-Glu-Asp-Leu-Arg-Val-Leu-Ser-Phe-Ile-Arg-Gly-Tyr (SEQ ID NO:4) and the NP epitope 380 393 [NP 380 393] of the sequence:

Glu-Leu-Arg-Ser-Arg-Tyr-Trp-Ala-Ile-Arg-Thr-Arg-Ser-Gly (SEQ ID NO:5)

In a preferred embodiment of the invention, the intranasal influenza vaccine consists of a mixture of the four influenza virus epitopes: hemagglutinin epitopes HA91 108 and HA307 319, and nucleoprotein epitopes NP335 350 and NP380 393, expressed individually in Salmonella flagellin. 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 influenza virus each expressed individually in Salmonella flagellin, as described above, for the preparation of a human synthetic influenza vaccine for intranasal administration.

The present invention further relates to a method for inducing a human immune response and conferring protection against influenza virus in humans, which comprises administering intranasally to human individuals a synthetic peptide-based influenza vaccine comprising a mixture of flagella, as described above.

DETAILED DESCRIPTION OF THE INVENTION

The concept of peptide-based vaccine holds several advantages over traditional vaccines, including safety considerations, the relatively long shelf-life, the ability to target the immune response towards specific epitopes that are not suppressive nor hazardous for the host and the possibility of preparing multi-pathogen vaccine. 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 B and T cells epitopes was previously tested in a mouse model, and it has been shown that such a "vaccine" could induce specific local response in the lungs that led to protection of the immunized mice from viral challenge (Arnon and Levi, 1996). In the mice model used there, it was shown that the B cell epitope indeed induced high Ab production, while the T helper epitope elicited specific lymphocyte proliferation and the CTL epitope was important for cytotoxic activity against infected cells. However, efficient protection was achieved only when the mice were immunized with a mixture of all three epitopes (Levi and Arnon, 1996).

According to the present invention, for the purpose of human use, appropriate epitopes had to be selected because the T-cell epitopes are MHC-restricted. First, we have identified that at least four influenza epitopes are necessary for human use: one B-cell HA epitope, one T-helper HA or NP epitope that can bind to many HLA molecules, and at least two CTL NP or matrix epitopes that are restricted to the most prevalent HLA molecules in the different populations.

According to the invention, a preferred B-cell influenza epitope is HA 91 108. Preferred T-helper influenza epitopes are HA 307 319 and HA 306 324 (Rothbard, 1988), but also NP 206 229 (SEQ ID NO:11; Brett, 1991) may be used.

The CTL influenza epitopes are different in the Caucasian, the Asia- or the Africa-originated population. For the Caucasian population, the preferred influenza CTL epitopes are NP335 350 and NP380 393 (Dyer and Middleton, 1993; Gulukota and DeLisi, 1996), that are restricted to the most prevalent HLA molecules in the Caucasian population. Other influenza epitopes that can be used according to the invention for the Caucasian population are the nucleoprotein epitopes: NP305 313 (SEQ ID NO:13; 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 (SEQ ID NO:12; DiBrino, 1994); and NP365 380 (Townsend, 1986); and the matrix protein (M) epitopes M2 22, M2 12 (SEQ ID NO:10), 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 influenza CTL epitopes that can be used are HA458 467 of the sequence Asn-Val-Lys-Asn-Leu-Tyr-Glu-Lys-Val-Lys (NVKNLYEKVK; SEQ ID NO:6), a CTL epitope for allele All with high frequency in Japanese, Chinese, Thais and Indian populations (J. Immunol. 1997, 159(10): 4753-61); M59 68 and M60 68 of the sequences Ile-Leu-Gly-Phe-Val-Phe-Thr-Leu-Thr-Val (ILGFVFTLTV; SEQ ID NO:7) and Leu-Gly-Phe-Val-Phe-Thr-Leu-Thr-Val (LGFVFTLTV; SEQ ID NO:8), respectively, two CTL epitopes for HLA-B51 with high frequency in Thais population (Eur. J. Immunol. 1994, 24(3): 777 80); and M128 135 of the sequence Ala-Cys-Ser-Met-Gly-Leu-Ile-Tyr (ACSMGLIY; SEQ ID NO:9), a CTL epitope for allele B35 with high frequency in negroid West African population (Eur. J. Immunol. 1996, 26(2) 335 39)

Since peptides are usually poor immunogens, the efficacy of peptide-based vaccine depends on the adequate presentation of the epitopes to the immune system. The influenza epitopes were expressed in the flagellin gene of Salmonella vaccine strain, which provides both carrier and adjuvant function. After cleavage of the flagella from the bacteria and the purification steps, the fine suspension of the flagella was used for vaccination. All immunizations were performed with a mixture of the four epitopes: HA91 108, HA307 319, NP335 350 and NP380 393, expressed in Salmonella flagellin, in the absence of any adjuvant. The mixture of said four epitopes is referred to as "tetra construct" throughout the specification.

The three T-cell epitopes used in the vaccine of the present invention were selected due to their specific recognition by the prevalent HLA's in the Caucasian population, and were included in the vaccine together with the HA 91 108 B cell epitope. In order to overcome the problem of antigenic variation of the virus, all these epitopes are derived from conserved regions in the virus proteins and hence, can induce cross-strain protection. The two CTL epitopes from the inner nucleoprotein are recognized by the prevalent HLAs of the Caucasian population: the NP 335 350 epitope is restricted to A2, A3, Aw68.1 and B37 HLA haplotypes, and the NP 380 393 epitope is restricted to B8 and B27 HLA haplotypes. The T-helper epitope from the hemagglutinin, HA 307 319, is a "universal" epitope restricted to most of MHC class II molecules, including DR1, DR2, DR4, DR5, DR7, DR9, DR52A, and others. These T-cell epitopes, together with the B-cell epitope HA 91 108, were expressed individually in flagellin and the: mixture of resultant flagella was used without any adjuvant for intranasal vaccination of human/mouse radiation chimera, thus inducing a human immune response and conferring protection. The vaccinated mice were also protected from a lethal infection and their recovery was quicker.

To evaluate the capacity of such tetra construct to act as a vaccine and stimulate a response of the human immune system, a humanized mouse model was employed. The observation that human PBMC can be adoptively transferred i.p. into the SCID mouse and that the engrafted cells survive for an extended period of time producing high levels of human Ig, has offered many new possibilities in clinical immunology research (reviewed in Mosier, 1991). In particular, many researchers have been utilizing this model for studying the capacity of engrafted lymphocytes to generate primary and secondary human humoral responses, and for viral research studies.

Recently, Lubin et al, 1994, described a new approach enabling engraftment of human PBMC in normal strains of mice following split-dose lethal irradiation which allows an effective and rapid engraftment of human cells. As previously reported, in such human/mouse radiation chimera, a marked human humoral as well cellular (CTL) responses could be generated by immunization with either foreign antigens or with allogeneic cells (Marcus et al, 1995; Segal et al, 1996), rendering advantages to this model in comparison to the previously used Mosier's SCID mouse model. Further advantages of this model is that the dissemination of engrafted lymphocytes is very rapid and both B and T lymphocytes were found by FACS analysis in significant numbers in the lymphoid tissues within a few days post transplantation (Burakova et al, 1997).

For evaluating the efficacy of a human influenza vaccine according to the invention, we used this human/mouse radiation chimera model. Although the number of human B cells after transplantation was low, the chimeric mice were able to produce specific antibodies in response to i.p. administration of antigens. This is in accord with previous findings, showing that towards the second week post-transplantation, the engrafted human B and T cells form follicles in the spleen and lymph nodes. Furthermore, their phenotype was that of memory cells, namely mostly CD45RO positive and CD45RA negative (Burakova et al, 1997).

According to the present invention, the human/mouse radiation chimera were immunized with the tetra construct administered by the intranasal route. This is the first report of induction of local immune response in the nasal cavity and lungs following 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 the lungs homogenates, by elevation of CD8.sup.+ lymphocytes proportion and by the viral clearance as a result of immunization with the tetra construct. The tetra flagellin construct could also protect the mice from a lethal dose challenge of the virus, which is the ultimate demonstration of the protective effect. Under these conditions, in which the challenge dose is orders of magnitude higher than that pertaining in natural infection, all the chimera were infected regardless of their immune state. However, whereas none of the immunized mice that had not been transplanted with the human lymphocytes survived the infection, and only 50% of the transplanted but not immunized mice survived, the transplanted and immunized group was completely protected and showed 100% survival.

The partial protection in the non-vaccinated mice is probably due to polyclonal stimulation and expansion of memory cells originating from the donor. This could be due to either previous exposure of the donor to the antigen or because it is cross-reactive to some extent with other recall antigens, a phenomena that was previously reported for other antigens (Marcus et al, 1995).

However, although such partial protection was indeed observed, a significant difference in the efficacy of the recovery process between the immunized and non-immunized groups was observed as evident both by survival rate and by their weight loss pattern (FIGS. 4, 5). Although the HLA phenotypes of the PMBC donors were not determined, all of the transplanted mice were protected as a result of the vaccination, indicating that the epitopes used in the present invention are indeed recognized by a wide range 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 strain-specific activity. The rapid variation in the viral surface glycoproteins leads to appearance of new strains with high variability in their serospecificity, and hence the vaccines containing the outer glycoproteins of some specific strains are limited in their efficacy to these strains. According to the present invention, we also established the cross-protection capacity of the tetra construct vaccine. All the epitopes that were included in the tetra construct are conserved regions in the respective proteins, and consequently, antibodies against the recombinant flagella could recognize various influenza strains. Consequently, immunization of the chimeric mice with the epitopes led to production of specific antibodies and to their protection from sub-lethal dose infection by three different influenza strains, of the H1, H2 or H3 specificity.

Thus, the results with the tetra construct according to the invention demonstrate the ability of a synthetic peptide-based vaccine to confer protection against influenza viral challenge. The recombinant flagellin construct indeed presents the influenza B and T-cell epitopes to the human immune cells in an efficient manner and induces both humoral and cellular responses. Since the employed T cell epitopes are recognized by a variety of HLA molecules, the vaccine was effective in all the experiments in which different donors with unknown HLA typing were utilized, indicating the applicability of this approach for a human vaccine in a heterologous population.
 

Claim 1 of 5 Claims

1. A human synthetic peptide-based influenza vaccine, comprising at least four epitopes of influenza virus, said influenza virus epitopes being reactive with human cells, wherein said epitopes comprise: (a) at least one B-cell hemagglutinin (HA) epitope consisting of the influenza virus hemagglutinin epitope 91 108 (HA 91 108) of SEQ ID NO:1; (b) at least one T-helper epitope selected from the group consisting of the influenza virus hemagglutinin epitope 307 319 (HA 307 319) of SEQ ID NO:2, influenza virus hemagglutinin epitope 306 324 (HA 306 324) of SEQ ID NO:3 and the nucleoprotein epitope 206 229 (NP 206 229) of SEQ ID NO:11, each epitope being capable of binding to many HLA molecules; and (c) at least two cytotoxic lymphocyte (CTL) epitopes selected from the group consisting of NP265 273 of SEQ ID NO:12, NP305 313 of SEQ ID NO:13 and influenza virus matrix protein epitope M2 12 of SEQ ID NO:10.
 

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