WO2011138032A2

WO2011138032A2 - Universal influenza vaccines and methods for their generation

Info

Publication number: WO2011138032A2
Application number: PCT/EP2011/002237
Authority: WO
Inventors: Amir Maksyutov; Denis Antonets; Anastasia Bakulina; Rinat Maksyutov
Original assignee: Artemev, Timur
Priority date: 2010-05-05
Filing date: 2011-05-05
Publication date: 2011-11-10
Also published as: WO2011138032A3

Abstract

The invention relates to novel methods for creating universal broad-spectrum influenza vaccines based on influenza virus hemagglutinin protein (HA). Also disclosed herein are several specific examples of seasonal and pandemic influenza vaccines based on peptide libraries or representative peptide panels (RPPs) created using the methods of the invention.

Description

UNIVERSAL INFLUENZA VACCINES AND METHODS FOR THEIR

GENERATION

TECHNICAL FIELD OF THE INVENTION

The invention relates to novel methods for creating universal broad-spectrum influenza vaccines based on influenza virus hemagglutinin protein (HA). Also disclosed herein are several specific examples of seasonal and pandemic influenza vaccines based on peptide libraries or representative peptide panels (RPP) created using the methods of the invention.

BACKGROUND OF THE INVENTION

Influenza virus infection in humans is a respiratory disease that ranges in severity from subclinical infection to primary viral pneumonia that can result in death. Influenza- associated complications include, among others, Reye's syndrome, myocarditis, pericarditis, myositis, encephalopathy and transverse myelitis. The persistence and unfettered nature of influenza virus leads to yearly epidemics as well as sporadic pandemics with potential to cause catastrophic loss of life. Palese et al., Nature Medicine 8(9):927 (2002). Seasonal influenza is the seventh leading cause of death in the United States and the leading cause of death in children ages 1 to 4 years. Every year in the United States, approximately 36,000 people die, 1 14,000 are hospitalized, and the country incurs more than $1 billion in direct economic costs.

An influenza pandemic occurs when a new influenza virus emerges and starts spreading as easily as normal influenza. The 1918 "Spanish flu" (H1N1) pandemic is the catastrophe against which all modern pandemics are measured. More than 20 million people were killed worldwide; 500,000 died in the United States alone. So far, the world has not seen a virus that severe again. "Asian influenza" (H2N2) in 1957, and "Hong Kong influenza"(H3N2) in 1968 were much milder, with an estimated 2 million deaths in 1957 and 1 million deaths in 1968. Influenza pandemics are recurring events. World Health Organization (WHO) estimates that next pandemic would cause from 2 million to 7.4 million deaths.

"Avian influenza" (H5N1) is one of the main candidates for the next calamitous pandemic virus. Since 2003, H5N1 virus has caused the largest and most severe outbreaks in poultry. According to the WHO report of March 12, 2010, 488 human cases were confirmed, and 289 persons have died. The spread of H5N1 virus from person to person has been limited and has not continued beyond one person. Nonetheless, because all influenza viruses have the ability to change, scientists are concerned that H5N1 virus one day would be able to infect humans and spread easily from one human to another. Because these viruses do not commonly infect humans, there is little or no immune protection against them in the human population. If H5N1 virus gains the capacity to spread easily from human to human, an worldwide outbreak of disease could begin.

Three types of influenza viruses (A, B, and C) are distinguishable by antigenic reactivities of their internal antigens. Influenza A, B and C belong to the family Orthomyxoviridae and have a segmented negative strand RNA genome that is replicated in the nucleus of the infected cell and consists of eight negative-sense RNA (nsRNA) gene segments that encode 10 polypeptides, including RNA-directed RNA polymerase proteins (PB2, PB1 and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (HA, which after enzymatic cleavage is made up of the association of subunits HA1 and HA2), the matrix proteins (Ml and M2) and the non-structural proteins (NS1 and NS2). Krug et al., In The Influenza Viruses, R.M. Krug, ed., Plenum Press, New York, 1989, pp. 89-152.

The HA and NA proteins embedded in the viral envelope are the primary antigenic determinants of the influenza virus (Air et al., Structure, Function, and Genetics, 1989, 6:341- 356; Wharton et al., In The Influenza Viruses, R. M. Krug, ed., Plenum Press, New York, 1989, pp. 153-174). Due to reassortment of influenza segmented genome, new HA and NA variants are constantly created for which a newly infected organism has no anamnestic immune response. Such constant generation of new antigenic variants from a vast number of circulating strains (genetic shift) creates enhanced danger of emergence of new highly pathogenic strains and creates the need for annual vaccination and development of antiviral agents that are effective against many or all strains. Palese, Nature Medicine 10(12 Suppl):S82 (2004); Garcia-Sastre and Biron, Science 312(5775):879 (2006); Li et al., Nature 2004, 430:209; Kuiken et al., Science 2004, 306:241. This has forced the World Health Organization to monitor current strains and constantly update the composition of the annual vaccine. For the production of a safe and effective vaccine it is believed to be important that the selected vaccine strains are closely related to the circulating strains, thereby ensuring that the antibodies in the vaccinated population are able to neutralize the antigenically similar virus.

HA glycoprotein is the major antigen for neutralizing antibodies and is involved in the binding of virus particles to receptors on host cells. HA molecules from different virus strains show significant sequence similarity at both the nucleic acid and amino acid levels. This level of similarity varies when strains of different subtypes are compared, with some strains clearly displaying higher levels of similarity than others (Air, Proc. Natl. Acad. Sci. USA, 1981, 78:7643). The levels of amino acid similarity vary between virus strains of one subtype and virus strains of other subtypes (Air, Proc. Natl. Acad. Sci. USA, 1981, 78:7643). This variation is sufficient to establish discrete subtypes and the evolutionary lineage of the different strains, but the DNA and amino acid sequences of different strains are still readily aligned using conventional bioinformatics techniques (Air, Proc. Natl. Acad. Sci. USA, 1981, 78:7643; Suzuki and Nei, Mol. Biol. Evol. 2002, 19:501).

Among the three types of influenza viruses, influenza A and B viruses cause significant morbidity and mortality in humans. Fields et al., Lippincott Williams & Wilkins, Philadelphia, PA, 2007. Thus, annual vaccines used to combat influenza virus infection include a combination of two influenza A strains with a single influenza B strain. Palese, Nature Medicine 10(12 Suppl):S82 (2004).

Propagation of these viral strains is usually performed in embryonated chicken eggs, where the virus can grow to very high titers. The virus particles generated in eggs are subsequently purified and used as stocks for vaccine preparations.

The influenza vaccines currently licensed by public health authorities for use in the United States and Europe are inactivated influenza vaccines as well as the live attenuated FLUMIST vaccine in the United States.

Current vaccine strategies focus on live attenuated influenza virus (LAIV) strains through the development of temperature-sensitive mutants or the removal of pathogenic factors such as the NS1 protein. Talon, J. et al., Proc. Natl. Acad. Sci. USA, 97:4309-4314 (2000); Nichol, Vaccine, 19:4373-4377 (2001); Palese et al., J. Infect. Dis., 1997, 176 Suppl l :S45-9. For example, FLUMIST (Influenza Virus Vaccine Live, Intranasal) contains influenza virus strains which are (a) cold-adapted (i.e., they replicate efficiently at 25°C, a temperature that is restrictive for replication of many wild-type influenza viruses); (b) temperature-sensitive (i.e., they are restricted in replication at 37°C (Type B strains) or 39°C (Type A strains), temperatures at which many wild-type influenza viruses grow efficiently); and (c) attenuated (they do not produce classic influenza-like illness in the ferret model of human influenza infection).

Still, current LAIV vaccines are too attenuated to stimulate a strong immune response in elderly people, the major group of the 20,000-40,000 individuals in the US dying each year as a result of influenza infection. Most importantly, present LAIV vaccines are subject to replicative impairment in embryonated chicken eggs because they have been adapted to growth at suboptimal temperatures required for proper egg development, thereby limiting the subsequent scale of vaccine production. Li et al., Nature 430(6996):209 (2004) and Krug, Science 31 1(5767):1562 (2006).

After the 1997 H5N1 outbreak in Hong Kong, vaccines produced by two different approaches were tested in humans. Conventional subunit H5 vaccine produced from A/duck/Singapore/3/97 was poorly immunogenic in humans, even against antigenically closely related strains and after multiple vaccinations (Nicholson et al., Lancet 2001 , 357: 1937; Stephenson et al., Journal of Infectious Disease 2005, 191 : 1210). The use of the adjuvant MF59 increased the antibody titer of this H5 vaccine (Stephenson et al., Vaccine 2003, 21 :1687). Vaccination with inactivated "split" vaccine derived from nonpathogenic A/duck/HK/836/80 (H3N1) virus and the modified H5 hemagglutinin from A/HK/156/97 (H5N1) virus induced barely detectable titers of neutralizing antibodies (Takada et al., Journal of Virology 1999, 73:8303). Thus, although these H5N1 vaccines were well tolerated, they appeared to be poorly immunogenic. The current lack of effective vaccines against H5N1 virus strains increases the threat of these viruses to cause pandemic disease.

A different "seasonal flu" vaccine is made every year based on the strains of influenza viruses that are in circulation at the time. It also includes viruses expected to circulate the following winter. The main challenge during the development of influenza vaccines arises from the extremely high genetic variability of the virus. To be efficient against a broad range of influenza variants, potential vaccines should elicit broadly neutralizing antibodies which, taken together, would^" be specific to multiple viral variants, both currently existing and potentially emerging. However, because current vaccines are composed of small numbers of currently circulating viral strains, none of the current vaccines represent a sufficiently broad range of immunogens of major isolates for representing the entire range of circulating influenza variants.

Sometimes, an unpredicted new influenza strain may appear after the vaccine has been made and distributed to doctor's offices and clinics. Because of this, even if a person has received the seasonal flu vaccine, he/she still may get infected. For example, the 2003 seasonal vaccines provided little protection against the variants of the virus spreading throughout the world during 2003-2004 seasons. The reason is that spreading viruses had antigenic drift away because of the mutations in the hemagglutinin (HA) gene, and this led to "escaping" the host organism's immunity to the 2003 year's vaccines.

Another limitation of currently available influenza vaccines is a short period for development and production of new seasonal vaccines.

Thus, there is a great need in the art for new universal influenza vaccines that are safe, efficient for generating protective immunity and are amenable to rapid large-scale production.

SUMMARY OF THE INVENTION

The present invention addresses these and other needs in the art by providing novel methods for creating universal broad-spectrum influenza vaccines based on peptide libraries and representative peptide panels (RPPs) containing a plurality of influenza virus hemagglutinin (HA) antigenic sites. The present invention further addresses these and other needs by providing several specific seasonal and pandemic influenza vaccines comprising peptide libraries or RPPs.

In one embodiment, the invention provides an immunogenic composition comprising one or more peptides selected from the group consisting of: rA_g51

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKS SWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVL GIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 1) ; rA 52

PLILRDCSVAGWLLGNPMCDEFI VPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQI I PKS SWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 2) ; rAg53

PLILRDCSVAGWLLGNPMCDEFI VPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQI IPRS SWSNHDASSGVSSACPYNGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 3 ) ; rAg54

PLILRDCSVAGWLLGNPMCDEFI VPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQI IPKS SWSNHEASSGVSSACPYQGTPSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNNEAEQTRLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 4), and rAg55

PLILRDCSVAG LLGNPMCDEFINVPE SYIVEKDNPVNGLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS SWSDHEASLGVSSACPYLGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIKLYQ NPNTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPENAYKIVKKGDSTI MKSEL (SEQ ID NO: 5) .

In a preferred embodiment, such immunogenic composition comprising peptides rA_g51

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKS SWSDHEASSGVSSACPYLGSPSFFRNVV LIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 1 ) ; rA 52

PLILRDCSVAG LLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS SWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 2) ; rAg53 PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPRS SWSNHDASSGVSSACPYNGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 3 ) ; rAg54

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKS SWSNHEASSGVSSACPYQGTPSFFRNVV LIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNNEAEQTRLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 4), and rAg55

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKDNPVNGLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS S SDHEASLGVSSACPYLGRSSFFRNVV LIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIKLYQ NPNTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTI MKSEL (SEQ ID NO: 5) .

In another embodiment, the invention provides an immunogenic composition comprising a peptide library L5 having the following amino acid composition:

Amino acids composition, %

P=100

K=85;R=15

S=95;N=5

S=100

W=100

S=95;P=5

D=55;N=25;S=20

H=100;

E=80;D=20

A=100

S=100

S=70;L=30 Amino acids composition, %

G=100

V=100

S=100

A=100

C=100

P=100

Y=100

Q=60;L=25;N=15

G=100

R=40;K=30;S=20;T=10

S=70;P=30

S=100

F=100

G=100

C=100

G=100

In yet another embodiment, the invention provides an immunogenic composition comprising a peptide library L5_noss having the following amino acid composition:

Amino acids composition, %

P=100

K=85;R=15

S=95;N=5

S=100

W=100 Amino acids composition, %

S=95;P=5

D=55;N=25;S=20

H==100;

E=80;D=20

A=100

S=100

S=70;L=30

G==100

V=100

S=100

s= 100

A= =100

P=100

Y==100

Q=60;L=25;N=15

G=100

R=40;K=30;S=20;T=10

S=70;P=30

S=100

F=100

G=100

In a separate embodiment, the invention provides an immunogenic composition comprising one or more peptides selected from the group consisting of:

A51 : PKSSWSDHEASSGVSSACPYLGSPSFGCG (SEQ ID NO: 6), A52: PKSSWSSHEASLGVSSACPYQGKSSFGCG (SEQ ID NO: 7),

A53: PRSSWSNHDASSGVSSACPYNGRSSFGCG (SEQ ID NO: 8),

A54: PKSSWSNHEASSGVSSACPYQGTPSFGCG (SEQ ID NO: 9), and

A55: PKSSWSDHEASLGVSSACPYLGRSSFGCG (SEQ ID NO: 10).

In a further embodiment, the invention provides an immunogenic composition comprising the peptides:

A51_noss: PKSSWSDHEASSGVSSAAPYLGSPSFG (SEQ ID NO: 1 1),

A52_noss: PKSSWSSHEASLGVSSAAPYQGKSSFG (SEQ ID NO: 12),

A53_noss: PRSSWSNHDASSGVSSAAPYNGRSSFG (SEQ ID NO: 13),

A54_noss: PKSSWSNHEASSGVSSAAPYQGTPSFG (SEQ ID NO: 14), and

A55_noss: PKSSWSDHEASLGVSSAAPYLGRSSFG (SEQ ID NO: 15).

Any of the above immunogenic compositions can further comprise one or more peptides selected from the group consisting of:

B51 : PNDAAEQTKLYQNPTT (SEQ ID NO: 16),

B52: PNNEAEQTRLYQNPTT (SEQ ID NO: 17),

B53: PNDAAEQIKLYQNPNT (SEQ ID NO: 18), and

C5: D ESTQKAIDGVTNKVNSIIDK (SEQ ID NO: 19).

rA_g31

ILDGENCTLIDALLGDPQCDGFQNKK DLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFN TG VTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIP IGSRPRVRNIPSRISIYWTIVKPGDILLI STGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 20) ; rA 32

ILDGENCTLIDALLGDPQCDGFQNKK DLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRGSNNSFFSRLNWLTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPSTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 21) ; rAg33

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQISLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

( SEQ ID NO: 22) ; rAg34

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNKSFFSRLNWLTQLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIY TIVKPGDILLINSTGNLIAPRGYFKIRSGKSSI RSDA

(SEQ ID NO: 23) ; rAg35

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNKSFFSRLNWLTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQTFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 2 ) , and rAg36

ILDGENCTLIDALLGDPQCDGFQNKN DLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSYACIRRSNNSFFSRLNWLTHLEFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYARASGR ITVSTKRSQQTVIP IGSRPRVRNI PSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 25) .

In a preferred embodiment, such immunogenic composition comprises peptides rAg31

ILDGENCTLIDALLGDPQCDGFQNKK DLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEQFDKLYI GVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 20) ; rA_g32

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRGSNNSFFSRLNWLTHSKFKYPALNVTMPNNEQFDKLYI GVHHPSTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 21) ; rAg33 ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNNSFFSRLN LTHLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQISLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 22) ; rAg34

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNKSFFSRLNWLTQLKFKYPALNVTMPNNEQFDKLYI GVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVI PNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 23) ; rAg35

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNKSFFSRLN LTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQTFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 24 ) , and rAg36

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSYACIRRSNNSFFSRLNWLTHLEFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYARASGR ITVSTKRSQQTVI PNIGSRPRVRNIPSRISIY TIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 25) .

In another embodiment, the invention provides an immunogenic composition comprising a peptide library L3A having the following amino acid composition:

Amino acids composition, %

N=100

G=100

T=100

S=100

S=45; Y=45; F=10;

A=90; T=10

C=100

KRG=20

KRR=30

IRR=50

S=100

N=80; D=10; 1=10; Amino acids composition, %

N=35; K=35; S=10; 1=10; H=10

S=100

F=100

G=100

C=100

G=100

In yet another embodiment, the invention provides an immunogenic composition comprising a peptide library L3 A_noss having the following amino acid composition:

Amino acids composition, %

N=100

G=100

T=100

S=100

S=45; Y=45; F=10;

A=90; T=10

A=100

KRG=20

KRR=30

IRR=50

S=100

N=80; D=10; 1=10;

N=35; K=35; S=10; 1=10; H=10

S=100

F=100

G=100

A31 : NGTSSACIRRSN SFGCG (SEQ ID NO: 26), A32: NGTSSACKRGSNNSFGCG (SEQ ID NO: 27),

A33: NGTSSACKRRSNNSFGCG (SEQ ID NO: 28),

A34: NGTSSACKRRSNKSFGCG (SEQ ID NO: 29),

A35: NGTSSACIRRSNKSFGCG (SEQ ID NO: 30), and

A36: NGTSYACIRRSNNSFGCG (SEQ ID NO: 31).

In a further embodiment, the invention provides an immunogenic composition comprising one or more peptides selected from the group consisting of:

A31_noss: NGTSSAAIRRSNNSFG (SEQ ID NO: 32),

A31_noss: NGTSSAAKRGSNNSFG (SEQ ID NO: 33),

A31_noss: NGTSSAAKRRSNNSFG (SEQ ID NO: 34),

A31_noss: NGTSSAAKRRSNKSFG (SEQ ID NO: 35),

A31_noss: NGTSSAAIRRSNKSFG (SEQ ID NO: 36), and

A31_noss: NGTSYAAIRRSNNSFG (SEQ ID NO: 37).

B31 : PGTDNDQIFLYAQASG (SEQ ID NO: 38),

B32: PGTDNDQIFLYARASG (SEQ ID NO: 39),

B33: PGTDNDQISLYAQASG (SEQ ID NO: 40),

B34: PGTDNDQTFLYAQASG (SEQ ID NO: 41), and

B35: PSTDNDQIFLYAQASG (SEQ ID NO: 42). Any of the above immunogenic compositions can further comprise the peptide library L3B having the following amino acid composition:

Amino acids composition, %

P=100

S=45; G=45; V=10

T=100

D=90; Y=10

N=33; S=33; K=33

D=100

Q=100

l=50; T=50

F=50; S=50

L=100

Y=100

A=50; V=50

Q=50; R=50

A=100

S=90; P=10

G=100

Any of the above immunogenic compositions can further comprise one or more peptides selected from the group consisting of

C3 HA1 : VPNGTIV TITNDQIEVTNAT (SEQ ID NO: 43),

C31_HA2: DLKSTQAAINQINGKLNRLIGK (SEQ ID NO: 44),

C32 HA2: DLKSTQAAINQINGKLNRLEGK (SEQ ID NO: 45),

C33_HA2: DLKSTQAADNQINGKLNRLIGK (SEQ ID NO: 46), and

C34 HA2: DLKSTQAADNQINGKLNRLEGK (SEQ ID NO: 47). Any of the above immunogenic compositions can further comprise one or more peptides selected from the group consisting of

C3 HA1 : VPNGTIVKTITNDQIEVTNAT (SEQ ID NO: 43),

C31 HA2: DLKSTQAAINQING LNRLIG (SEQ ID NO: 44),

C32 HA2: DLKSTQAAINQINGKLNRLEGK (SEQ ID NO: 45),

C33 HA2: DLKSTQAADNQINGKLNRLIGK (SEQ ID NO: 46), and

C34 HA2: DLKSTQAADNQINGKLNRLEGK (SEQ ID NO: 47).

rAgll:

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKS SWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHT ENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRI YYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGI I NSNA (SEQ ID NO: 48) ; rAgl2:

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKE SS PNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQMTLYH KENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYY TLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGI INSNA (SEQ ID NO: 49) ; rAgl3:

PLQLGNCSVAGWILGNPECELLISKES SYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKE SSWPNHTVTKGVTASCSHNGKSSFYRNLLWLTGKNGLYPNLSMSYVNNKEKEVLVL GVHHPPNIGDQRALY HTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTI IFEANGNLIAPRYAFALSRGFGSG IINSNA (SEQ ID NO: 50), and rAgl4:

PLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKT SSWPNHDSNKGVTAACPHAGAKSFYKNLI LVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLY QNADAYVFVGSSRYSKKFKPEIAIRP VRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSG

IIISDT (SEQ ID NO: 51) .

In a preferred embodiment, such immunogenic composition comprises peptides rAgll:

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKS SWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHT ENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTI IFEANGNLIAPRYAFALSRGFGSGI I NSNA (SEQ ID NO: 48) ; rAgl2:

PLQLGNCSVAG ILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKE SS PNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQMTLYH KENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTI IFEANGNLIAPRYAFALSRGFGSGI INSNA (SEQ ID NO: 49) ;

rAgl3:

PLQLGNCSVAGWILGNPECELLISKES SYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKE SS PNHTVTKGVTASCSHNGKSSFYRNLLWLTGKNGLYPNLSMSYVNNKEKEVLVLWGVHHPPNIGDQRALY HTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSG IINSNA (SEQ ID NO: 50), and

rAgl4:

PLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKT SSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLY QNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSG IIISDT (SEQ ID NO: 51) .

Al 1 : ESSWPNHTVTGVSASCSH GKSSFGCG (SEQ ID NO: 52),

A12: ESSWPNHTVTGVSASCSHNGESSFGCG (SEQ ID NO: 53),

A13: ESSWPNHTVTKGVTASCSHNGKSSFGCG (SEQ ID NO: 54), and

A14: TS S WPNHDSNKG VT AACPH AGAKSFGCG (SEQ ID NO: 55).

Al l noss: ESSWPNHTVTGVSASASHNGKSSFG (SEQ ID NO: 56),

A12_noss: ESSWPNHTVTGVSASASHNGESSFG (SEQ ID NO: 57), A13_noss: ESSWPNHTVTKGVTASASHNGKSSFG (SEQ ID NO: 58), and

A14_noss: TSSWPNHDSNKGVTAAAPHAGAKSFG (SEQ ID NO: 59).

Any of the above immunogenic compositions can further comprise the peptide DQKSTQNAIDGITNKVNSVIEK (CI) (SEQ ID NO: 60).

In any of the above immunogenic compositions, one or more (or all, or none) of the peptides can contain disulfide bonds.

Any of the above immunogenic compositions can be vaccine compositions. In a preferred embodiment, the present invention provides the following peptide vaccines:

1. rAg51+rAg52+rAg53+rAg54+rAg55 (vaccine for H5N1 viruses);

2. rAg31+rAg32+rAg33+rAg34+rAg35+rAg36 (vaccine for H3N2 viruses);

3. r Ag 1 1 +r Ag 12+r Ag 13 +rAg 14 (vaccine for H 1 N 1 viruses) .

The vaccine compositions of the invention can further comprise an adjuvant.

In conjunction with the vaccine compositions, the invention provides methods of inducing a protective immune response to an influenza infection in a mammal, said method comprising administering to said mammal a vaccine composition of the invention. In a preferred embodiment, the mammal is human. The vaccine compositions of the invention can be administered by any route, including, e.g., mucosally, subcutaneously (s.c), intramuscularly (i.m.), intradermally (i.d.), and orally. Preferably, the vaccine compositions of the invention are administered mucosally, most preferably, intranasally.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing alignment entropy for HlNl HA, averaged with the window 8, for two periods of time (1934-1944 and 1934-2007). DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the development of new methods for generating immunogenic libraries of peptides corresponding to a broad range of influenza HA molecules. The present invention further provides effective broad-spectrum influenza vaccines generated based on such immunogenic peptide libraries and/or representative peptide panels (RPPs) derived from such libraries.

The use of peptide libraries and RPPs of the present invention as immunogens (e.g., as part of vaccine compositions) allows to generate protective immune response against a broad range of influenza strains.

The present invention is based on the realization that, to be efficient against a broad range of influenza variants, a vaccine should elicit broadly neutralizing antibodies which would recognize multiple viral variants, currently existing and newly emerging.

The present invention is further based on the observation that simultaneous exposure to multiple antigenic determinants often induces immune responses to each of them (Thomson, S. A. et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92:5845-5849; Thomson, S. A. et al. J. Virol. 1998, 72: 2246-2252; and Woodberry, T. et al. J. Virol. 1999, 73:5320-5325). However, efficient methods for selection of suitable antigenic determinants are not currently available.

Antibodies directed to influenza HA which are formed during a normal immune response, primarily interact with a limited number of HA regions. These regions are termed "antigenic sites" or "antigenic determinants". In order for a viral variant to successfully spread in a population, it has to be antigenically different from variants which have already caused an immune response. Thus, there should be amino acid changes in antigenic sites. The boundaries of antigenic sites in different naturally occurring and laboratory HA variants are determined by the accumulation of amino acid changes. For example, the antigenic sites of H3 and HI HA molecules are described in Wiley et al, 1981 , Nature 289, 373-378 and Caton et al, 1982, Cell 31, 417^127. For HI , the antigenic sites are designated as Ca, Cb, Sa and Sb; for H3, the antigenic sites are designated as A, B, C, D, E. For H5, the determination of antigenic sites is highly preliminary due to the fact that the virus circulating in birds remains rather antigenically stable (Kaverin et al, 2002, J Gen Virol. 83, 2497-2505).

All HA molecules are structurally similar to each other and can be classified into four structural groups: (1) including H8, H12, H9; (2) including HI , H2, H5, H6, Hl l , H13; (3) including H3, H4, H14, and (4) including H10, H7, H15 (Ha et al, 2002, EMBO J. 21 , 865- 875). Position of antigenic sites within the HA structure is determined by various factors, including glycosylation which is different between various HA subtypes.

The present invention proposes a new method for determination of antigenic sites based on calculation of alignment entropy of amino acid sequence variants dominating in the course of evolution. The term "dominating amino acid" (or "dominating residue") refers to an amino acid which is found more frequently than others in a given position within a given sequence (e.g., HA) in a given year. Alignment entropy is used as a measure of variability and is calculated using the following formula: sum[p(i)*log(p(i))], where p(i) is a frequency of occurrence of i dominating amino acid. p(i) is calculated as a ratio of the number of years during which the i amino acid was dominating to the total number of years during which a given subtype was observed. For determination of potential antigenic sites, values of alignment entropy are averaged with the window equals 8, which approximately corresponds to the minimal size of an epitope recognized by an antibody. Maximal peak values on the graph of relationship of average alignment entropy to the amino acid position within the sequence correspond to the regions with the highest variability of dominating amino acids and thus have a high probability to correspond to antigenic sites (see Figure 1). Interestingly, in the course of influenza evolution, positions of such regions with the highest variability of dominating amino acids are generally preserved. For example, as demonstrated in Figure 1 , for HA of HlNl influenza, position of the four regions with the highest variability of dominating amino acids determined based on alignment entropy for the 1934-1944 period is the same as for the 1934-2007 period.

Visual analysis of HA structure with superimposed positions of glycosylation sites and/or known escape mutations and/or alignment entropy values may be also useful for determination of antigenic sites. If the structure of a specific HA molecule is not available, a structure of a closely homologous HA molecule can be used (e.g., an HA molecule belonging to the same structural group as defined above).

Antigenic sites can be also mapped using algorithms for predicting B-cell epitopes, such as, e.g., ADEPT (Maksyutov et al, 1993, Comput Appl Biosci. 9(3), 291-297).

The novel immunogenic strategy disclosed in the present application relies on the use of peptide libraries which adequately represent the diversity of currently existing and potentially emerging variants of antigenic sites. As used herein, the term "peptide library" refers to a combination of different peptides. Such peptide library can be described by amino acid composition in % for each position within the sequence. The size of the library is determined by the number of peptides of different sequence contained in such library. Preferably, such library includes between 100 and 10000 different peptides. Specific libraries used in the Examples section, below, contained 270, 576 and 2304 peptides, respectively. Peptides within the library can have any useful length, but are preferably 6 to 60 amino acids long, more preferably 6 to 40 amino acids long, and most preferably 16 to 28 amino acids long.

As disclosed herein, the peptide libraries of the invention can be used as a polyvalent immunogens to induce efficient cellular and/or humoral immune responses to both existing viral strains and strains which will emerge in the future. Indeed, it has been shown in animal models that simultaneous exposure to multiple antigenic determinants often induces immune responses to each of them (Thomson, S. A. et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92:5845-5849; Thomson, S. A. et al. J. Virol. 1998, 72: 2246-2252; and Woodberry, T. et al. J. Virol. 1999, 73:5320-5325). This indicates that multiple antigenic determinants are suitable as components of a single vaccine construct.

Peptide libraries can be generated using various methods known in the art. These standard methods include exclusive solid phase synthesis, automated solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis, and recombinant DNA technology (See, e.g., Merrifield J. Am. Chem. Soc. 1963 85:2149; Merrifield et al., 1982, Biochemistry, 21 :502; Lebl M. and Krchnak V. (1997) Solid-Phase Peptide Synthesis, Methods in Enzymology, 289, Academic Press, Minneapolis, Minn.). A preferred method for peptide synthesis is solid phase synthesis. Solid phase peptide synthesis procedures are well-known in the art (see, e.g., Stewart, Solid Phase Peptide Syntheses, Freeman and Co.: San Francisco, 1969; 2002/2003 General Catalog from Novabiochem Corp, San Diego, USA; Goodman, Synthesis of Peptides and Peptidomimetics, Houben- Weyl, Stuttgart 2002). The synthesis can be a combinatorial synthesis, in which mixtures of amino acid are coupled to the growing peptide, and a mixture of peptide sequences is formed. In a combinatorial synthesis, amino acids are added to a coupling reaction in proportions dictated by the desired ratio of residues at each position in the peptide, adjusted for the relative coupling rate for each amino acid. Alternatively, the library can be produced by parallel synthesis, in which each sequence is synthesized separately, and the library can be a group of individually pure peptide sequences. Such a library can be converted to a mixture by mixing the pure peptides. Automated combinatorial synthesis can be preferable when a large library is to be produced. Split synthesis can be preferable when a small library of individual sequences is to be produced.

Peptide libraries also can be synthesized using split&mix technology (Furka and Bennett, Comb Chem High Throughput Screen. 1999 2(2): 105-22). In one embodiment of such method, for introduction of amino acid residues into a variable position, peptidyl- polymer is washed with dimethylchloride and dimethyl ether, and dried to constant weight in vacuo. Then it is separated by weighing on parts that corresponded to quota of each peptide containing replacement in this position. Each part of peptidyl-polymer is transferred into separate reaction vessel, deprotected and condensed with derivative of required amino acid. If it is required, additional couplings can be performed in order to get a negative ninhydrin test. After attachment of all residues in the variable position the polymers are combined, and washed with dimethylchloride during 5-10 minutes. If the next position is variable, then the peptidyl polymer is separated in parts in required ratio (by weight), and all stages of synthesis are repeated. If the next position is constant, then synthesis is continued without separation of the matrix in parts.

In addition to chemical synthesis, the peptides of the present invention can be synthesized by employing recombinant DNA technology by expressing one or more polynucleotide comprising a peptide coding region. Thus, provided herein are isolated polynucleotides that encode the peptides of the present invention as well as recombinant vectors and host cells (both eukaryotic and prokaryotic) that have been genetically modified to express or overexpress the peptides of the present invention.

The peptide libraries of the present invention can be synthesized in the form of a multiple antigenic peptide (MAP). As used herein, the term "Multiple Antigenic Peptide" (MAP) refers to peptide multimer formed from a core (e.g., polylysine) and containing a branched scaffolding onto which peptides are conjugated (see, e.g., Tarn, J. Immunol. Meth. 196:17, 1996; Nardin et al., Adv. Immunol. 60: 105, 1995). Methods for MAP synthesis are well known and are disclosed in, e.g., Tarn, Proc. Natl. Acad. Sci. USA 85:5409, 1988; Tarn, Meth. Enzymol. 168:7, 1989. The MAPs useful in the peptide libraries of the present invention can include, for example, from 2 to 16 peptide chains, preferably 4 peptide chains. The MAP can include a hydrophobic group, such as an alkyl chain derived from palmitic acid. The hydrophobic group can facilitate assembly of the MAP with liposomes or viruslike particles.

The immunogenic peptides of the invention can include the 20 naturally occurring L- amino acids, corresponding D-amino acids, or mixtures of D and L-amino acids. Non-natural amino acids can likewise be included in the peptides. Common examples of conventional amino acids include stereoisomers (e.g., D-amino acids) and unnatural amino acids such as, for example, L-ornithine, L-homocysteine, L-homoserine, L-citrulline, 3-sulfino-L-alanine, N-(L-arginino)succinate, 3,4-dihydroxy-L-phenylalanine, 3 -iodo-L- tyrosine, 3,5-diiodo-L- tyrosine, triiodothyronine, L-thyroxine, . L-selenocysteine, N-(L-arginino)taurine, 4- aminobutylate, (R,S)-3-amino-2-methylpropanoate, a,a-disubstituted amino acids, N-alkyl amino acids, lactic acid, β-alanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine, N- methylglycine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, nor- leucine, and other similar amino acids and imino acids. A general method for site-specific incorporation of unnatural amino acids into proteins and peptides is described, e.g., in Noren et al., Science, 244: 182-188 (April 1989).

In addition to immunogenic applications, the peptide libraries of the invention can be used in diagnostic applications, for example to determine the presence of antibodies to any one of a range of influenza strains. For example, the libraries may be used an antigens for an enzyme-linked immunosorbent assay (ELISA) to determine the presence of antibodies in a subject sample. In a solid-phase assay such as an ELISA, the peptides of the library are immobilized on a substrate (for example, on a polystyrene well). The substrate is then exposed to a test sample (e.g., serum from a subject) under conditions that allow specific peptide-antibody complex formation. Any unbound antibodies are then washed away. Antibodies associated with the substrate by virtue of a specific peptide-antibody interaction are then detected. Detection can include exposing the antibodies to a secondary antibody specific for immunoglobulins from the subject. The secondary antibody can be radiolabeled, or coupled to an enzyme such as horseradish peroxidase or alkaline phosphatase, that catalyzes the formation of a colored product.

There are a number of important structural and functional constraints affecting amino acid sequences of existing and emerging influenza HA molecules. In order for influenza virus to enter the target cell, HA needs to bind to sialic acid on the cell surface with a certain affinity. A weaker interaction will prevent binding and a stronger interaction will prevent conformational change of HA which is essential for viral entry inside the cell. These structural and functional constraints affect the composition and location of antigenic sites and should be taken into consideration when constructing peptide libraries.

Amino acids found in each position of the peptide library should be present in different proportions. The amino acids which are more frequently found in a given position in currently circulating variants and variants which are likely to emerge in the near future should be present more frequently within the library. A frequency for the presence of a given amino acid in a given position in the strains which will emerge in the future can be predicted based on their frequency in the strains which had emerged in the past. These predicted frequencies are termed "effective frequencies of occurrence." Effective frequencies of occurrence are represented the same way as the peptide library composition, i.e., by amino acid composition in % for each position within the peptide sequence. A collection of effective frequencies of occurrence for each amino acid position within the antigenic site of interest represents a preliminary version of the library.

The terms "filter" or "filtration" are used herein to refer to an algorithm which excludes certain peptide variants from a library following certain criteria. The term "renormalization" is used herein to refer to a procedure, wherein the composition in % for a given position changes following a certain algorithm depending on the % value.

An immunogenic peptide library of the present invention can be constructed using the following algorithm:

1. Calculation of effective frequencies of occurrence of amino acids in each position of the peptide;

2. Filtration;

3. Renormalization.

Ways for determining effective frequencies of occurrence and calculating filtration and renormalization can be chosen depending on available information on HA evolution pattern of a particular viral subtype.

To determine effective frequencies of occurrence, a database containing HA sequences for a given influenza subtype should be used. For each HA sequence used, the host, year of isolation and place of isolation should be known. For example, NCBI database can be used (www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). Effective frequencies of occurrence of amino acids can be calculated using various methods depending on available information on HA evolution pattern of a particular viral subtype.

Frequently found HA variants of a given subtype can belong to vastly different groups. For example, swine HI HA variant belongs to two distinct lineages (Kanegae et al., 1994, Arch Virol.134, 17-28). If this is the case, it is useful to cluster HA sequences using some algorithm, for example, using Bayesian clustering or bootstrap HMM-based clustering (see, e.g., Sequence Learning - Paradigms, Algorithms, and Applications, 2001 , Lecture Notes In Computer Science; Vol. 1828, ISBN:3-540-41597-1 Editors: Ron Sun, C. Lee Giles), and then determine effective frequencies of occurrence independently for each cluster.

Database for determination of effective frequencies of occurrence should preferably contain only sequences from hosts for which the vaccine is being constructed, although in some cases sequences from other hosts may be also included. For example, currently H5 subtype of influenza is transmitted from birds to humans but not from humans to humans. Since the determinants of cross-species transmission are not clearly understood (Peiris et al, 2007 Clin Microbiol Rev. 20, 243-267), immune protection from all avian viral H5 HA variants is desired, and all of these variants should be taken into consideration for determination of effective frequencies of occurrence.

To account for differences in the numbers of HA sequences obtained in each given year and at each given location, effective frequencies of occurrence should be normalized to year and/or geographic location. Normalization to years can be performed as follows: for each amino acid residue in each position, effective frequency of occurrence is p_eff = sum(pi)/n (formula 1), where p; is a frequency of occurrence of a given amino acid residue in a given position in year i; n is the total number of years of observation, and sum() is the sum for all years of observation.

Different years can have different impact on effective frequencies of occurrence. For example, in some cases, earlier years are less important. In such cases, the frequency of occurrence in formula 1 can be multiplied by empirical coefficient xj, so that sum(xj) = 1. Then peff = sum(pj*Xj)/n (formula 2), where pi is a frequency of occurrence of a given amino acid residue in a given position in year i; n is the total number of years of observation; x_¾ coefficient for each year, and sum() is the sum for all years of observation. For example, for the last 10 years of observation Xj = 0.1 , while for other years x; = 0. Or a linear function xi = a + b*i can be used, where a and b are coefficients which correspond the following condition: xi for the earliest year of observation is 0, sum(xi) = 1. In some cases, earlier years should be considered as equally important. Such cases include sequences where no significant antigenic drift occurred or where viral sequences are known for only a short range of years.

To ensure that the antigenic peptide library reflects antigenic properties of the currently circulating variants as well as variants that are likely to emerge in the future, the size of the initial library sometimes should be reduced. The term "initial library" refers to the effective frequencies of amino acids, and the term "final library" refers to the initial library after filtering and renormalization. As specified above, for the purposes of the present invention, a preferred size of the library is between 100 and 10000 peptides. However, the size of the initial library for a 15 -residues peptide with 4 variants of amino acids in each position is more than 1 billion. To reduce the size of the initial library, the following filters can be used.

Correlation filter. Using methods of statistical analysis, for example, mutual information calculation (Guiasu, Silviu (1977), Information Theory with Applications, McGraw-Hill, New York), and analysis of structure- function properties of influenza HA molecules, correlations between amino acid changes in various positions can be identified. Sequence variants which do not correspond to determined correlations can be excluded from the library.

Evolutionary trend filter. Size of the library can be decreased based on properties of HA molecules which are known to be retained throughout evolution. For example, H3 HA is known to accumulate N-glycosylation sites. Accordingly, sequence variants containing fewer N-glycosylation sites than currently circulating strains can be excluded from the library.

Single nucleotide change filter. Library can be also limited to currently circulating variants and those that are likely to emerge in the near future. For antigenically important sites, the maximal probability of amino acid change is for changes associated with a single nucleotide change in a codon. Thus, amino acid changes which cannot result from a single nucleotide change can be excluded from the library. For example, for GCT codon encoding A, single nucleotide change can result only in replacement of A with G, D, P, T, S, and V. Thus, the remaining amino acids, C, E, F, H, I, K, L, M, N, R, Q, Y and W can be excluded from the given position in the library..

Rare amino acid filter. HA variants present in databases have different abilities to cause an epidemic. When looking at the frequencies of occurrence of amino acids in each given position of antigenically important sites for each year, amino acids found with very low frequency point to the variants with low ability to cause epidemics or even to non-existing variants (e.g., sequencing mistakes). A threshold can be introduced to exclude such rarely occurring amino acids for a given position. Antigenic similarity can be expressed in Antigenic Similarity Matrix (ASM) (see, e.g., Maksyutov et al., Mol. Biol. 1987, 21 :30-47) or any other distance matrix reflecting physico-chemical or antigenic properties of amino acids, for example, a distance matrix derived from BLOSUM substitution matrix (Henikoff, S.; Henikoff, J.G. (1992) PNAS 89: 10915-10919). For example, if the initial amino acid composition is G=l%, 1=4%, N=57%, L=38%, after using the filter with the threshold of 5%, G and I are removed leaving N=60%, L=40%.

In some instances (as discussed below), the library can incorporate some amino acids which have not been found in a given position or have been found at a frequency below the threshold.

Filter for close replacements. When compiling an initial library, some positions may contain antigenically similar amino acids. The following procedure can be used for filtering out such antigenically similar amino acids. For each position, antigenic distances between all pairs of amino acids are examined. If a pair of amino acids has a distance below a given threshold on ASM, an amino acid with the lower value of effective frequency of occurrence is excluded, and the other amino acid is used instead of the excluded one. For example, if for some position an initial library contains =30%, R=45%, S=25%, and K and R are considered antigenically similar to each other, but antigenically distinct from S, the application of the filter for close replacements will result in R=75%, S=25%. A typical threshold value is 30. However, other values from 20 to 50 also can be used (e.g., if a certain amount of peptides is needed in the library after the filter is applied).

Renormalization. After application of one or more filters, peptides comprising the resulting library can be renormalized. Renormalization is an averaging of a library. It is needed since there is no actual difference between, for example, 26% and 32%, or between 3% and 5%. An amino acid in a given position of a library can be considered either as a major or as a minor one; it can be more or less important for a protective immune response. Renormalization makes all major amino acids equally important in the library, and the same is for minor amino acids. A threshold between major and minor amino acids is usually 10%. For example, for a given position, all amino acids can be taken at the same %. Alternatively, all amino acids found in frequencies below a certain level can be included at minimal levels (for example, at 5% or 10%), while amino acids present in frequencies above a certain level can be taken at the same %. The resulting ratios can be also averaged to 5% or 10%. In addition to immunogenic libraries, the present invention provides representative peptide panels (RPP) which reflect a diversity of antigenic sites. As used herein, the term "representative peptide panel" or "RPP" refers to a collection of peptides compiled based on known currently circulating sequences and sequences which are likely to emerge in a given antigenic site. RPP is much smaller than a library and preferably contains less than 10 peptides. Depending on evolutionary properties of a given region of an HA molecule, different methods of generating RPPs can be used. Non-limiting examples of such methods are provided below.

RPP design based on clustering of known sequences. Known variants of sequences of antigenic sites of HA can be grouped and the centers of such clusters or the most frequently found variant within each cluster can be used as a representative peptide (RP). Clustering algorithm can vary depending on the evolutionary properties of a given HA molecule. Clustering can be performed using a distance between peptides on ASM or another distance matrix, such as a distance matrix derived from BLOSUM substitution matrix (Henikoff, S.; Henikoff, J.G. (1992) PNAS 89: 10915-10919). See also McBurney SP, Ross TM (2007) Curr Pharm Des. 13(19): 1957-64.

RPP design based on a library. RPP can be designed to encompass the most important variants of antigenic sites. The importance of a given amino acid in a given position is reflected in its content (%) in this position in the library. To design RPP, peptides which are present in a given library are sorted based on % amino acid content. The first peptide in RPP has the highest content (%) in the library. When each new peptide is considered, its distance on ASM from the other peptides in RPP is taken into consideration. If such next peptide is sufficiently distant from the other peptides in RPP (for example, has a distance more than about 90 on ASM), it becomes included. The process is repeated until RPP of a desired size (e.g., from 3 to 10) is generated.

Peptides representing selected HA antigenic sites in a form of a peptide library or a RPP can be used as immunogens to induce a protective immune responses to a wide range of influenza viruses. In a specific embodiment, such peptides can be used as MAPs (e.g., a MAP with lysine core and 4-8 branches) or other branched constructs, for example, a synthetic construction where core polypeptide branch has several peptidic scions (http://www.aurorafinechemicals.com/peptide-s The peptide libraries and RPPs of the present invention can be formulated in pharmaceutical and vaccine compositions.

To stabilize peptide structures, peptides within such compositions may be stabilized using disulfide bonds or other chemical modifications, for example, using thiol-reactive sulfonated alkyne-based cross-linker (see, e.g., Fuzhong Zhang, Oleg Sadovski, Steven J. Xin, and G. Andrew Woolley. J. Am. Chem. Soc, 2007, 129 (46), pp 14154-14155) or a hydrazone link (Edelmira Cabezas and Arnold C. Satterthwait. J. Am. Chem. Soc, 1999, 121 (16), pp 3862-3875).

To further enhance immunogenicity, peptides from RPP can be incorporated in or conjugated to a carrier such as a recombinant HA protein or its fragments, or to another protein, for example, BSA. Any incorporation and conjugation method known in the art can be used. For example, the peptide from RPP and the carrier protein can be expressed as a single polypeptide chain.

According to the present invention, different vaccination strategies can be used to achieve a protective immune response. For example, a library can be used for the first vaccination, while boosting can be done using RPP or recombinant HA fragments, and vice versa. A DNA vaccine which corresponds to a RPP-based immunogen also can be used for vaccination or for priming. Non- variable fragments of HA also can be used as an additional component of a vaccine.

Taken together, the novel vaccine strategy of the present invention provides the versatility, safety, and efficacy required for rapid generation of large quantities of vaccines for newly emerging influenza strains.

Definitions

The term "influenza virus" is used herein to define a viral species of which pathogenic strains cause the disease known as influenza or flu. The term influenza is meant to include any strain or serotype of the influenza virus, including any combination of HA, e.g., HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16; and NA, e.g., Nl, N2, N3, N4, N5, N6, N7, N8 or N9 genes. In one embodiment, influenza refers to H5N1 influenza (bird flu or pandemic influenza). In one embodiment, influenza refers to other strains or subtypes of the influenza virus, including but not limited to H1N1 , H2N2, and H3N2.

As used herein, the term "influenza virus variant" encompasses known influenza isolates as well as their modifications which are still unknown.

In the context of influenza virus biology, "coding region" refers to areas of viral RNA which encode amino acids that are represented in the mature viral proteins.

The term "peptide", as used herein, refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, polypeptides, oligopeptides, and proteins are included within the definition of peptide. This term also includes peptides having post- translational modifications such as, for example, glycosylations, acetylations, phosphorylations, and the like.

As used herein, the term "Multiple Antigenic Peptide" (MAP) refers to peptide multimer formed from a core (e.g., polylysine) and containing a branched scaffolding onto which peptides are conjugated.

As used herein, the term "peptide library" refers to a combination of different peptides.

The term "alignment entropy" is used herein to refer to a measure of the variability at each alignment position (see, e.g., Korber et al., J Virol., 1994, , 68(1 1):7467-7481).

The terms "dominating amino acid" or "dominating residue" are used interchangeably to refer to an amino acid which is found more frequently than others in a given position within a given sequence (e.g., HA) in a given year.

In the context of influenza virus biology, the term "escape mutation" is used to refer to a mutation within one of the viral proteins which significantly reduces its interaction with antisera against the virus without the mutation. An "individual" or "subject" or "animal", as used herein, refers to vertebrates that support a negative strand RNA virus infection, specifically influenza virus infection, including, but not limited to, birds (such as water fowl and chickens) and members of the mammalian species, such as canine, feline, lupine, mustela, rodent (racine, murine, etc.), equine, bovine, ovine, caprine, porcine species, and primates, the latter including humans. In a specific embodiment, the subject is a ferret, which is a good animal model for studying influenza. In another embodiment, the subject is a human.

As used herein, the term "immunogenic" means that an agent is capable of eliciting a humoral or cellular immune response, and preferably both. An immunogenic entity is also antigenic. An immunogenic composition is a composition that elicits a humoral or cellular immune response, or both, when administered to an animal having an immune system.

A molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. An antigenic polypeptide contains an "epitope" of at least about five, and preferably at least about 10, amino acids. An antigenic portion of a polypeptide, also called herein the "epitope", can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier polypeptide for immunization. A molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier.

Serum antibody titer methods are the accepted surrogate measures of immune protection after vaccination or viral infection. The predominantly used serum antibody titer methods are virus neutralization titer assays and hemagglutinin inhibition (HI) titer assays. These assays are based on the ability of influenza antibodies from human serum to cross react with antigens under in vitro conditions. Briefly stated, the virus neutralization assay examines the ability of antibodies from a serum sample to block the infection of cultured cells by influenza virus. The assay is carried out by creating serial dilutions (titers) of a serum sample and combining each of these dilutions with a standard amount of infectious virus. Each dilution mixture is then presented to a defined cell culture and the resulting infection rates assayed. The hemagglutinin inhibition (HI) assay similarly examines the ability of antibodies from a serum sample to bind with a standardized reference virus. The basis for this assay is the fact that influenza viruses will bind to and agglutinate erythrocytes. In the HI assay, serial dilutions of serum sample are mixed with standard amounts of reference virus and after a set incubation period added to erythrocytes. The association between reference viruses and erythrocytes into complexes is then detected visually. The highest dilution of serum that inhibits hemagglutinin is read as the hemagglutinin inhibition titer.

The term "protective immunity" refers to an immune response in a host animal (either active/acquired or passive/innate, or both) which leads to inactivation and/or reduction in the load of said antigen and to generation of long-lasting immunity (that is acquired, e.g., through production of antibodies), which prevents or delays the development of a disease upon repeated exposure to the same or a related antigen. A "protective immune response" comprises a humoral (antibody) immunity or cellular immunity, or both, effective to, e.g., eliminate or reduce the load of a pathogen or infected cell (or produce any other measurable alleviation of the infection) in an immunized (vaccinated) subject.

The term "vaccine" refers to a composition (e.g., one or more recombinant influenza virus proteins or peptides with or without an adjuvant) that can be used to elicit protective immunity in a recipient. It should be noted that to be effective, a vaccine of the invention can elicit immunity in a portion of the immunized population, as some individuals may fail to mount a robust or protective immune response, or, in some cases, any immune response. This inability may stem from the individual's genetic background or because of an immunodeficiency condition (either acquired or congenital) or immunosuppression (e.g., due to treatment with chemotherapy or use of immunosuppressive drugs). Vaccine efficacy can be established in animal models.

The term "adjuvant" refers to a compound or composition that augments the host's immune response to another antigen (e.g., live attenuated influenza virus) when administered conjointly with that antigen. Adjuvants useful in the vaccine compositions of the present invention include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (Γ- 2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine, Bacille Calmette- Guerin (BCG), and Corynebacterium parvum. Preferably, the adjuvant is pharmaceutically acceptable.

Within the meaning of the present invention, the term "conjoint administration" is used to refer to administration of an immune adjuvant and an antigen simultaneously in one composition, or simultaneously in different compositions, or sequentially within a specified time period (e.g., 24 hours).

The term "protect" is used herein to mean prevent or treat, or both, as appropriate, development or continuance of a disease (e.g., flu) in a subject.

The terms "protective immune response" or "protective immunity" comprise a humoral (antibody) immunity or cellular immunity, or both, effective to, e.g., eliminate or reduce the load of a pathogen (e.g., influenza virus) or infected cell or produce any other measurable alleviation of the infection in an immunized (vaccinated) subject.

The term "therapeutically effective amount/dose" is used herein interchangeably with the term "immunogenically effective amount/dose" and refers to that quantity of a live attenuated influenza virus or a pharmaceutical composition or vaccine comprising such virus that is sufficient to produce a protective immune response upon administration to a mammal.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "carrier" applied to pharmaceutical or vaccine compositions of the invention refers to a diluent, excipient, or vehicle with which a compound (e.g., a live attenuated influenza virus) is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution, saline solutions, and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th Edition.

As used herein, the term "isolated" means that the referenced material (e.g., a cell or virus) is removed from its native environment. Thus, an isolated biological material can be free of some or all cellular components, i.e., components of the cells in which the native material occurs naturally (e.g., cytoplasmic or membrane component). A material shall be deemed isolated if it is present in a cell extract or supernatant. In the case of nucleic acid molecules, an isolated nucleic acid includes, without limitation, a PCR product, an isolated RNA (e.g., mRNA or miRNA), a DNA (e.g., cDNA), or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the cellular or viral genome in which it may be found, and, e.g., is no longer joined or proximal to other genes or regulatory sequences located upstream or downstream of this nucleic acid. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like, i.e., when it forms part of a chimeric recombinant nucleic acid construct. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism. An isolated material may be, but need not be, purified.

The term "about" or "approximately" means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term "about" or "approximately" depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds.

(1985) ]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press,

(1986) ]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, F.M. et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include site directed mutagenesis as described in Kunkel, Proc. Natl. Acad. Sci. USA 82: 488- 492 (1985), U. S. Patent No. 5,071 , 743, Fukuoka et al. , Biochem. Biophys. Res. Commun. 263: 357-360 (1999); Kim and Maas, BioTech. 28: 196-198 (2000); Parikh and Guengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech. 13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang and Malcolm, BioTech. 26: 680- 682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Patents Nos. 5,789, 166 and 5,932, 419, Hogrefe, Strategies 14. 3: 74-75 (2001), U. S. Patents Nos. 5,702,931, 5,780,270, and 6,242,222, Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson, Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996), Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch and Joly, Nuc. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28: 197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22: 541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al., Meth. Molec. Biol. 67: 209- 218.

Nucleotide changes can be introduced using any of the methods of site directed mutagenesis known in the art. See, e.g., Kunkel, Proc. Natl. Acad. Sci. USA 82: 488- 492 (1985), U. S. Patent No. 5,071, 743, Fukuoka et al. , Biochem. Biophys. Res. Commun. 263: 357-360 (1999); Kim and Maas, BioTech. 28: 196-198 (2000); Parikh and Guengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech. 13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang and Malcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Patents Nos. 5,789, 166 and 5,932, 419, Hogrefe, Strategies 14. 3: 74-75 (2001), U. S. Patents Nos. 5,702,931, 5,780,270, and 6,242,222, Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson, Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996), Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch and Joly, Nuc. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28: 197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22: 541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al., Meth. Molec. Biol. 67: 209-218.

Vaccine Compositions of the Invention

The present invention provides novel improved vaccine compositions comprising one or more immunogenic peptides of the invention and a pharmaceutically acceptable carrier or diluent. The vaccine may be used in a method of prophylaxis of a disease condition caused by the influenza virus by administering to a subject in need thereof a therapeutically effective amount of the vaccine.

In a specific embodiment, the immunogenic peptides of the invention may be administered as a nucleic acid vaccine, e.g., as DNA vectors expressing said peptides. Examples of suitable expression vectors include a pDNAVACCultra vector family in which expression is driven from optimized versions of either the CMV or the mouse VL30 NVL-3 enhancer-promoter leading to dramatically augmented expression (compared to unaltered CMV and VL30 NVL-3 enhancer-promoter) in a wide variety of cell lines (Li et. al.., Gene Ther., 1999, 6(12):2005-1 1).

Preferably, in the disclosed compositions, the immunogenic peptides or vectors encoding them are present in immunogenically effective amounts. For each specific composition, the optimal immunogenically effective amount should be determined experimentally (taking into consideration specific characteristics of a given subject and/or type of treatment). Generally, this amount is in the range of 2-100 μg of an antigen per capita.

Strategies to further enhance influenza vaccine effectiveness include, e.g., the conjoint administration of adjuvants (see above) or immunostimulatory molecules such as cytokines, lymphokines, or chemokines (e.g., interleukins IL-1 , IL-2, IL-3, IL-4, IL-12, IL-13, granulocyte-macrophage colony stimulating factor (GM-CSF) and other colony stimulating factors, macrophage inflammatory factor, Flt3 ligand, B7.1 , B7.2, etc.). Salgaller and Lodge, J. Surg. Oncol. 1998, 68: 122; Lyman, Curr. Opin. Hematol., 5: 192, 1998. Adjuvants or immunostimulatory molecules can be delivered systemically or locally (e.g., directly as proteins or by expression from a vector). See Wood and Williams, In: Nicholson, Webster and May (eds.), Textbook of Influenza, Chapter 23, pp. 317-323; Salgaller and Lodge, J. Surg. Oncol. 1998, 68: 122.

A therapeutically effective protective amount of the vaccine of the invention can be administered by various administration routes known in the art. Preferably, the immunogenic formulations of the invention are delivered by mucosal (e.g., intranasal), subcutaneous (s.c), intramuscular (i.m.), intradermal (i.d.), or oral administration. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as excipients, suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The immunogenic compositions of the invention can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Mucosal administration is particularly preferred, since influenza infection occurs via the mucosa and the mucosa harbors dendritic cells, which are important targets for immunotherapy. Examples of useful mucosal vaccination strategies include, among others, encapsulating the immunogen in microcapsules (U.S. Patents Nos. 5,075,109; 5,820,883, 5,853,763) and using an immunopotentiating membranous carrier (PCT Publication No. WO 98/0558). In a specific embodiment, the vaccines of the invention can be administered mucosally in an admixture with, or as a conjugate or chimeric fusion protein with, cholera toxin (CT), such as CT B or a CT A/B chimera (Hajishengallis, J Immunol., 154: 4322-32, 1995; Jobling and Holmes, Infect Immun., 60: 4915-24, 1992). Mucosal vaccines based on the use of the CT B subunit have been described (Lebens and Holmgren, Dev Biol Stand 82: 215-27, 1994). In another embodiment, an admixture with heat labile enterotoxin (LT) can be prepared for mucosal vaccination. The immunogenicity of inhalation-based administered vaccine can be also enhanced by using red blood cells (rbc) or rbc ghosts (U.S. Patent No. 5,643,577), or by using blue tongue antigen (U.S. Patent No. 5,690,938).

Although the above approaches are promising for improved future vaccination strategies, their use in specific situations requires validation and surveillance to ensure vaccine effectiveness.

Immunogenic compositions of the present invention can be formulated in any conventional manner using one or more pharmaceutically acceptable carriers. Suitable carriers are, for example, water, saline, buffered saline, dextrose, glycerol, ethanol, sterile isotonic aqueous buffer or the like and combinations thereof. In addition, if desired, the preparations may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or immune stimulators (e.g., adjuvants) that enhance the effectiveness of the pharmaceutical composition or vaccine.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the immunogenic formulations of the invention. The kit may also optionally include one or more physiologically acceptable carriers and/or auxiliary substances. Associated with the kit can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Effective Dose and Safety Evaluations

According to the methods of the present invention, the pharmaceutical and immunogenic compositions described herein are administered to a patient at immunogenically effective doses, preferably, with minimal toxicity. Following methodologies which are well-established in the art (see, e.g., reports on evaluation of several vaccine formulations containing novel adjuvants in a collaborative effort between the Center for Biological Evaluation and Food and Drug Administration and the National Institute of Allergy and Infectious Diseases [Goldenthal et al., National Cooperative Vaccine Development Working Group. AIDS Res. Hum. Retroviruses 1993, 9:545-9]), effective doses and toxicity of the compounds and compositions of the instant invention are first determined in preclinical studies using small animal models (e.g., mice) in which the antiens of the invention have been found to be immunogenic and that can be reproducibly immunized by the same route proposed for the human clinical trials. Specifically, for any pharmaceutical composition or vaccine used in the methods of the invention, the therapeutically effective dose can be estimated initially from animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms). Dose-response curves derived from animal systems are then used to determine testing doses for the initial clinical studies in humans. In safety determinations for each composition, the dose and frequency of immunization should meet or exceed those anticipated for use in the clinical trial.

As disclosed herein, the dose of immunogenic peptides in the compositions of the present invention is determined to ensure that the dose administered continuously or intermittently will not exceed a certain amount in consideration of the results in test animals and the individual conditions of a patient. A specific dose naturally varies depending on the dosage procedure, the conditions of a patient or a subject animal such as age, body weight, sex, sensitivity, feed, dosage period, drugs used in combination, seriousness of the disease. The appropriate dose and dosage times under certain conditions can be determined by the test based on the above-described indices and should be decided according to the judgment of the practitioner and each patient's circumstances according to standard clinical techniques. In this connection, the dose of an immunogenic HA peptide is generally in the range of 2-100 μ&

Toxicity and therapeutic efficacy of the immunogenic peptides in immunogenic compositions of the invention can be determined by standard pharmaceutical procedures in experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions that exhibit large therapeutic indices are preferred. As disclosed herein, the immunogenic peptides of the invention are not only highly immunostimulating at relatively low doses but also possess low toxicity and do not produce significant side effects.

As specified above, the data obtained from the animal studies can be used in formulating a range of dosage for use in humans. The therapeutically effective dosage for use in humans lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. Ideally, a single dose should be used.

Methods for Assessing Immunogenicity

Serum antibody titer methods are the accepted surrogate measures of immune protection after vaccination or viral infection. The predominantly used serum antibody titer methods are virus neutralization titer assays and hemagglutinin inhibition (HI) titer assays. These assays are based on the ability of influenza antibodies from human serum to cross react with antigens under in vitro conditions. Assays are selected for a given situation based not only on their ability to provide consistent and applicable results but also based on their ease of use and the facility requirements for each type of assay.

Briefly stated, the virus neutralization assay examines the ability of antibodies from a serum sample to block the infection of cultured cells by influenza virus. The assay is carried out by creating serial dilutions (titers) of a serum sample and combining each of these dilutions with a standard amount of infectious virus. Each dilution mixture is then presented to a defined cell culture and the resulting infection rates assayed. The virus neutralization titer assay is useful and reliable test to examine the level of immunoprotective antibodies present in a given individual.

The hemagglutinin inhibition (HI) assay similarly examines the ability of antibodies from a serum sample to bind with a standardized reference virus. The basis for this assay is the fact that influenza viruses will bind to and agglutinate erythrocytes. In the HI assay, serial dilutions of serum sample are mixed with standard amounts of reference virus and after a set incubation period added to erythrocytes. The association between reference viruses and erythrocytes into complexes is then detected visually. The highest dilution of serum that inhibits hemagglutinin is read as the hemagglutinin inhibition titer.

EXAMPLES

The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

Example 1

In the library for H5 HA region 125-147 (here and below aa numbering for H5 HA corresponds to structure 2FK0.PDB (Stevens, J., Blixt, O., Tumpey, T.M., Taubenberger, J.K., Paulson, J.C., Wilson, LA. (2006) Science 312: 404-410), the following amino acids can be present (see Table 1.1). These are the amino acids which were present in more than two H5 HA isolates existing in NCBI database in 2008. For positions which had more than one type of amino acid, amino acid content varied from 0% to 100%. In position 12, there is a deletion to account for which split and mix technology can be used.

Table 1.1

Number Composition

1 P

2 K R Number Composition

3 DGNS

4 S Y

5 W

6 AFP ST

7 DNS

8 H Y

9 DENY

10 AST

11 ST

12 -LS V

13 G

14 G V

15 S

16 AS

17 AS V

18 C

19 LPS

20 FH S Y

21 DGHILNQS

22 EGR

23 A E G K M N Q R S T V

24 P S

25 S

26 FL

Example la

A library variant from Example 1 , which used for designing an initial library frequencies of occurrence of amino acids for all sequenced H5 HA variants for all hosts as available in the NCBI database. A filter for rare amino acids was used with the threshold of 3% and rounding to 5%. The library composition is provided in Table 1.2. Table 1.2.

Number Amino acid composition, %

24 S=70;P=30

25 S=100

26 F=100

All values in the above tables are approximate values. For example, values S=70; L=30 (line 12) should be considered as S being in the range 20-90 and L being in the range 5-50.

Example lb

The construct from example la, where GC was added to the C-termini of peptides and a disulfide bond is formed between the C-terminal cysteine and CI 8 (for stabilization of the peptide structure).

Example lc

The construct from example lb, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G) can be added between the C-terminus of each peptide and MAP core.

Example Id

The construct from example 1 c, where linker is G. Example le

The construct from example la, where CI 8 is replaced with A. Example If

The construct from example le, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G) can be added between the C-terminus of each peptide and MAP core.

Example lg

The construct from example If, where linker is GS. Example 2

For H5 HA region 125-138 RPP was constructed using clustering of known variants of the sequence of this region and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. PKSSWSDHEASSGVSSA (SEQ ID NO: 97)

2. PKSS SSHEASLGVSSA (SEQ ID NO: 98)

3. PRSSWSNHDASSGVSSA (SEQ ID NO: 99)

4. PKSSWSNHEASSGVSSA (SEQ ID NO: 100)

5. PKSSWSDHEASLGVSSA (SEQ ID NO: 101)

Example 3

For H5 HA region 134-147 RPP was constructed using clustering of known variants of the sequence of this region and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. GVSSACPYQGKSSF (SEQ ID NO: 108)

2. GVSSACPYLGRSSF (SEQ ID NO: 109)

3. GVSSACPYNGRSSF (SEQ ID NO: 110)

4. GVSSACPYQGTPSF (SEQ ID NO: 111)

5. GVSSACPYLGSPSF (SEQ ID NO: 112)

Example 4 The following peptide constructs corresponding to H5 HA region 125-147 were created based on the sequences of examples 2 and 3:

1. PKSSWSDHEASSGVSSACPYLGSPSF (SEQ ID NO: 77)

2. PKSSWSSHEASLGVSSACPYQGKSSF (SEQ ID NO: 78)

3. PRSSWSNHDASSGVSSACPYNGRSSF (SEQ ID NO: 79)

4. PKSSWSNHEASSGVSSACPYQGTPSF (SEQ ID NO: 80)

5. PKSSWSDHEASLGVSSACPYLGRSSF (SEQ ID NO: 81)

When combining constructs from examples 2 and 3, the frequencies of their combination in naturally occurring HA sequences were taken into account. Preference was given to combinations closest to the naturally occurring.

Example 4a

The construct from example 4, where GC was added to the C-termini of peptides and a disulfide bond is formed between the C-terminal cysteine and CI 8 (for stabilization of the structure).

Example 4b

The construct from example 4a, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G) can be added between the C-terminus of each peptide and MAP core.

Example 4c

The construct from example 4b, where linker is G. Example 4d

The construct from example 4, where CI 8 is replaced with A. Example 4e The construct from example 4d, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G) can be added between the C-terminus of each peptide and MAP core.

Example 4f

The construct from example 4e, where linker is GS.

Example 5

For H5 HA region 185-200 RPP was constructed using clustering of known variants of the sequence of this region and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. PNDAAEQTKLYQNPTT (SEQ ID NO: 16)

2. PNNEAEQTRLYQNPTT (SEQ ID NO: 17)

3. PNDAAEQIKLYQNPNT (SEQ ID NO: 18)

Example 6

For H5 HA region 90-106 RPP was constructed using clustering of known variants of the sequence of this region and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. KANPANDLCYPGNFNDYE

2. KANPVNDLCYPGDFNDYE

3. KASPANDLCYPGDFNDYE

. KANPTNDLCYPGSFNDYE

5. KDNPVNGLCYPGDFNDYE Example 7

The constructs from examples 4, 5 and 6 inserted into H5 HA fragment, for example H5 HA fragment 59-272.

Example 7a

The constructs from example 7 containing the following flanking and spacer sequences.

For N-terminal flanking sequence, H5 HA region 60-89 which was prevalent in 2008 was chosen:

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVE (SEQ ID NO: 76)

For spacer sequences between the peptides from example 4 and 5, H5 HA region 107-124 prevalent in 2008 was chosen:

ELKHLLSRINHFEKIQII (SEQ ID NO: 96)

For spacer between peptides of examples 5 and 6, RPP was constructed using clustering of known variants of the sequence of the H5 HA region 148-184 and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. FRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHH (SEQ ID NO: 105)

2. FRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHH (SEQ ID NO: 106)

3. FRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHH (SEQ ID NO: 107)

For C-terminal flanking sequence, RPP was constructed using clustering of known variants of the sequence of the H5 HA region 201-272 and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. YISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSE L (SEQ ID NO: 117) 2. YISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTIMKSE L (SEQ ID NO: 118)

Final constructs based on H5 HA fragment 59-272 have the following sequence:

1. PLILRDCSVAGWLLGNPMCDEFINVPE SYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIP KSS SDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKL YQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDS TIMKSEL (SEQ ID NO: 1)

2. PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIP KSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKL YQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDS TIMKSEL (SEQ ID NO: 2)

3. PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIP RSSWSNHDASSGVSSACPYNGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVL GIHHPNDAAEQTKL YQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDS TIMKSEL (SEQ ID NO: 3)

4. PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIP KSSWSNHEASSGVSSACPYQGTPSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVL GIHHPNNEAEQTRL YQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDS TIMKSEL (SEQ ID NO: 4)

5. PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKDNPVNGLCYPGDFNDYEELKHLLSRINHFEKIQI IP KSSWSDHEASLGVSSACPYLGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIKL YQNPNTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPENAYKIVKKGDS TIMKSEL (SEQ ID NO: 5)

Example 7b

The construct from example 7a limited to a fragment corresponding to H5 HA fragment 90- 262:

1. KANPANDLCYPGNFNDYEELKHLLSRINHFEKIQI IPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLI KKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQS GRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVK (SEQ ID NO: 61) 2. KANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQI IPKSSWSSHEASLGVSSACPYQGKSSFFRNVV LI KKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQS GRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVK (SEQ ID NO: 62)

3. KASPANDLCYPGDFNDYEELKHLLSRI HFEKIQIIPRSSWSNHDASSGVSSACPYNGRSSFFRNVVWLI KKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQS GRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVK (SEQ ID NO: 63)

4. KANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSNHEASSGVSSACPYQGTPSFFRNVV LI KKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNNEAEQTRLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQS GRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVK (SEQ ID NO: 64)

5. KDNPVNGLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASLGVSSACPYLGRSSFFRNVV LI KKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIKLYQNPNTYISVGTSTLNQRLVPRIATRSKVNGQS GRMEFFWTILKPNDAINFESNGNFIAPENAYKIVK (SEQ ID NO: 65)

Example 7c

The construct from example 7a in the form of DNA vaccine. Example 7d

The construct from example 7b in the form of DNA vaccine.

Example 8

A conservative H5 HA fragment such as, e.g., HA2 subunit region 37-58, can be used as an additional vaccine component.

DKESTQKAIDGVTNKVNSIIDK (SEQ ID NO: 19)

Example 8a

The construct from example 8 in the form of MAP (e.g., 4-branched MAP). Example 9

Any combination of constructs from examples 1-8.

Example 10

In the library for H3 HA region 133-147 (here and below aa numbering for H3 HA corresponds to structure 1HGF.PDB (Sauter, N.K., Hanson, J.E., Glick, G.D., Brown, J.H., Crowther, R.L., Park, S.J., Skehel, J.J., Wiley, D.C. (1992) Biochemistry 31 : 9609- 9621), the following amino acids can be present (see Table 10.1). These are the amino acids which were present in more than two human H3 HA isolates existing in NCBI database in 2008. For positions which had more than one type of amino acid, amino acid content varied from 0%to 100%.

Table 10.1

Number Amino acid composition

1 DNS

2 G

3 ADEGKT

4 S

5 ACFNS Y

6 AST

7 C

8 EIKQR

9 KR

10 EGKRS

11 PST

12 DGIKNSTV

13 IKNQRS Number Amino acid composition

14 G S

15 F

Example 10a

A library variant from Example 10, which used for designing an initial library frequencies of occurrence of amino acids for all sequenced human H3 HA variants available in the NCBI database and normalized to years. The following filters were applied; correlation filter (region 140-143); evolutionary trend filter (preservation of predicted glycosylation sites in positions 133 and 144 imposes limitations on amino acids in positions 133, 144, 135, and 136; since until recently position 144 was highly variable, this filter was not applied to it); filter for residues which are close (i.e., contain antigenically similar amino acids) and have a distance below threshold 30 on an antigenic similarity matrix (ASM); filter for rare amino acids with the threshold of 7%. This was followed by renormalization: for each position, the amino acids which were contained within the library but were not prevalent in any year after 1980, were taken at 10%, and the remaining residues were taken in equal amounts. For position 144, which has a likelihood of preserving a glycosylation site, all amino acids except N, were taken at 10%. The library composition is provided in Table 10.2.

Table 10.2

Number Library composition

1 N=100

2 G=100

3 T=100

4 S=100

5 S=45; Y=45; F=10;

6 A=90; T=10

7 C=100

8-10 KRG=20

KRR=30

IRR=50 Number Library composition

1 1 S=100

12 N=80; D=10; 1=10;

13 N=35; K=35; S=10; 1=10;

H=10

14 S=100

15 F=100

All values in the above table are approximate values. For example, values S=45; Y=45; F=10 (line 5) should be considered as S and Y are in a range 20-90 and F is in a range 5-50.

Example 10b

The construct from example 10a, where GC was added to the C-termini of peptides and a disulfide bond is formed between the C-terminal cysteine and C7 (for stabilization of the structure).

Example 10c

The construct from example 10b, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G), can be added between the C-terminus of each peptide and the MAP core.

Example lOd

The construct from example 10c, where the linker is G. Example lOe

The construct from example 10a, where C7 is replaced with A. Example lOf

The construct from example lOe, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G) can be added between the C-terminus of each peptide and the MAP core.

Example lOg The construct from example lOf, where the linker is GS.

Example 11

For H3 HA region 133-147, RPP was constructed using the library from example 10a. The resulting RPP has the following composition:

1. GTSSACIRRSNNSF (SEQ ID NO: 84)

2. GTSSACKRGSNNSF (SEQ ID NO: 85)

3. GTSSACKRRSNNSF (SEQ ID NO: 86)

4. GTSSACKRRSNKSF (SEQ ID NO: 87)

5. GTSSACIRRSNKSF (SEQ ID NO: 88)

6. GTSYACIRRSNNSF (SEQ ID NO: 89)

Example 11a

The construct from example 1 1 , where GC was added to the C-termini of peptides and a disulfide bond is formed between the C-terminal cysteine and C7 (for stabilization of the structure).

Example lib

The construct from example 1 la, which in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G) can be added between the C-terminus of each peptide and the MAP core.

Example 11c

The construct from example 1 lb, where the linker is G. Example lid

The construct from example 1 1 , where C7 is replaced with A. Example lie

The construct from example l id, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G), can be added between the C-terminus of each peptide and the MAP core.

Example llf

The construct from example l ie, where the linker is GS.

Example 12

In the library for H3 HA region 185-200, the following amino acids can be present (see Table 12.1). These are the amino acids which were present in more than two human H3 HA isolates existing in NCBI database in 2008. For positions which had more than one type of amino acid, amino acid content varied from 0% to 100%.

Table 12.1

Number Amino acid composition

1 P

2 D G I S V

3 T

4 D E G N Y

5 K N Q R S Y

6 D E G N V

7 Q

8 I T V

9 D F K N R S

10 I L P V

1 1 F H Y

12 A I T V

13 H Q R

14 A E S T Number Amino acid composition

15 A L P S

16 G

Example 12a

A library variant from Example 12, which used for designing an initial library frequencies of occurrence of amino acids for all sequenced human H3 HA variants available in the NCBI database and normalized to years. The following filters were applied; filter for residues which are close (i.e., contain antigenically similar amino acids) and have a distance below 30 on an antigenic similarity matrix (ASM), and filter for rare amino acids with the threshold of 10%. This was followed by renormalization: for each position, the amino acids which were contained within the library but were not prevalent in any year after 1980, were taken at 10%, and the remaining residues were taken in equal amounts. The library composition is provided in Table 12.2.

Number Library composition

1 P=100

2 S=45; G=45; V=10

3 T=100

4 D=90; Y=10

5 N=33; S=33; K=33

6 D=100

7 Q=100

8 l=50; T=50

9 F=50; S=50

10 L=100

1 1 Y=100

12 A=50; V=50

13 Q=50; R=50

14 A=100 Number Library composition

15 S=90; P=10

16 G=100

All values in the above table are approximate values. For example, values S=45; G=45; V=10 (line 2) should be considered as S and G are in a range 20-90 and V is in a range 5-50.

Example 13

For H3 HA region 155-165, RPP design is based on library, which was built for human sequenced variants available in the NCBI database. The resulting RPP has the following composition:

1. THLEFKYPALN (SEQ ID NO: 124)

2. THLKFKYPALN (SEQ ID NO: 125)

3. THSKFKYPALN (SEQ ID NO: 126)

4. TQLKFKYPALN (SEQ ID NO: 127)

Example 14

For H3 HA region 185-200, RPP was constructed based on the library of example 12a. The resulting RPP has the following composition:

1. PGTDNDQIFLYAQASG (SEQ ID NO: 38)

2. PGTDNDQIFLYARASG (SEQ ID NO: 39)

3. PGTDNDQISLYAQASG (SEQ ID NO: 40)

4. PGTDNDQTFLYAQASG (SEQ ID NO: 41)

5. PSTDNDQIFLYAQASG (SEQ ID NO: 42)

Example 15 The constructs from examples 1 1, 13 and 14 inserted into a carrier, which carrier is H3 HA fragment (e.g., H3 HA fragment 58-272).

Example 15a

The constructs from example 15 containing the following flanking and spacer sequences.

For N-terminal flanking sequence, RPP was constructed using clustering of known 2008 variants of the sequence of the H3 HA region 58-132 and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGV TQ (SEQ ID NO: 82)

2. ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGV TQ (SEQ ID NO: 83)

For spacer sequences between the peptides from examples 11 and 13, H3 HA region 148-154 prevalent in 2008 was chosen:

FSRLNWL (SEQ ID NO: 102)

For spacer between peptides of examples 13 and 14, RPP was constructed using clustering of known 2008 variants of the sequence of the H3 HA region 166-184 and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. TMPNNEQFDKLYIWGVHH (SEQ ID NO: 113)

2. TMPNNEKFDKLYIWGVHH (SEQ ID NO: 114)

For C-terminal flanking sequence, the prevalent 2008 sequence for the region 201 -272 was chosen:

RITVSTKRSQQTVIPNIGSRPRVR IPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 119) Various peptides from examples 1 1 , 13, and 14 and flanking and spacer sequences were combined in such a way as to reflect naturally occurring correlations. Final constructs based on H3 HA fragment 58-272 have the following sequences:

1. ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNW TGVTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQAS GRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLI STGNLIAPRGYFKIRSGKSSIMRSD

A (SEQ ID NO: 20)

2. ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNW TGVTQNGTSSACKRGSNNSFFSRLNWLTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPSTDNDQIFLYAQAS GRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSD

A (SEQ ID NO: 21)

3. ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNW TGVTQNGTSSACKRRSNNSFFSRLN LTHLKF YPALNVTMPNNEQFDKLYIWGVHHPGTDNDQISLYAQAS GRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSI RSD

A (SEQ ID NO: 22)

4. ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFN TGVTQNGTSSACKRRSNKSFFSRLNWLTQLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQAS GRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSD

A (SEQ ID NO: 23)

5. ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFN TGVTQNGTSSACIRRSNKSFFSRLNWLTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQTFLYAQAS GRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIY TIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSD

A (SEQ ID NO: 24)

6. ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNW TGVTQNGTSYACIRRSNNSFFSRLNWLTHLEFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYARAS GRI VSTKRSQQTVIP IGSRPRVR IPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSD

A (SEQ ID NO: 25)

Example 15b

The construct from example 15a limited to region 90-262: 1. RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLKF KYPALNVTMPNNEQFDKLYI GVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRIS IYWTIVKPGDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 66)

2. RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRGSNNSFFSRLNWLTHSKF KYPALNVTMPNNEQFDKLYIWGVHHPSTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRIS IYWTIVKPGDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 67)

3. RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKF KYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQISLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRIS IYWTIVKPGDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 68)

4. RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRRSNKSFFSRLNWLTQLKF KYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRIS IYWTIVKPGDILLI STGNLIAPRGYFKIRS (SEQ ID NO: 69)

5. RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNKSFFSRLNWLTHSKF KYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQTFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRIS IYWTIVKPGDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 70)

6. RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSYACIRRSNNSFFSRLNWLTHLEF KYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYARASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRIS IYWTIVKPGDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 71)

Example 15c

The construct from example 15a in the form of a DNA vaccine. Example 15d

The construct from example 17b in the form of a DNA vaccine. Example 16

A conservative fragment of H3 HA can be used as an additional component of a vaccine. For example, one or several prevalent sequences for H3 HA2 subunit region 37-58 can be used:

1. DLKSTQAAINQINGKLNRLIGK (SEQ ID NO: 44)

2. DLKSTQAAINQINGKLNRLEGK (SEQ ID NO: 45) 3. DLKSTQAADNQINGKLNRLIGK (SEQ ID NO: 46)

4. DLKSTQAADNQINGKLNRLEGK (SEQ ID NO: 47)

Example 16a

H3 HA region 20-40 can be used as an additional conserved component of the vaccine:

VPNGTIVKTITNDQIEVTNAT (SEQ ID NO: 43)

Example 17

Any combinations of constructs from examples 10-16 can be used, including combinations with conserved H3 HA fragments.

Example 18

In the library for HI HA region 124-147 (here and below aa numbering for HI HA corresponds to structure 1RU7.PDB (Gamblin, S.J., Haire, L.F., Russell, R.J., Stevens, D.J., Xiao, B., Ha, Y., Vasisht, N., Steinhauer, D.A., Daniels, R.S., Elliot, A., Wiley, D.C., Skehel, J.J. (2004) Science 303 : 1838-1842), the following amino acids can be present (see Table 18.1). These are the amino acids which were present in more than two human HI HA isolates existing in NCBI databsae in 2008. For positions which had more than one type of amino acid, amino acid content varied from 0% to 100%. In position 1 1 , there is a deletion to account for which split and mix technology can be used.

Table 18.1

Number Amino acid composition

1 A D E G K T

2 N R S

3 A S

4 W Number Amino acid composition

5 L P

6 D H K N S Y

7 H

8 D E I N T

9 A F I T V

10 I N S T

1 1 - I K R

12 G

13 G V

14 S T

15 A T V

16 A s

17 C

18 P s

19 H Y

20 A E K N R S

21 G K R

22 A E K R

23 C N S

24 S

25 F

Example 18a

The construct from example 18, where GC was added to the C-temini of peptides and a disulfide bond is formed between the C-terminal cysteine and CI 7 (for stabilization of the structure).

Example 18b

The construct from example 18a, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G) can be added between the C-terminus of each peptide and MAP core. Example 18c

The construct from example 18b, where linker is G. Example 18d

The construct from example la, where CI 7 is replaced with A. Example 18e

The construct from example 18d, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G) can be added between the C-terminus of each peptide and MAP core.

Example 18f

The construct from example 18e, where linker is GS.

Example 19

For HI HA region 124-147 RPP was constructed using clustering of known variants of the sequence of this region and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. ESSWPNHTVTGVSASCSHNGKSSF (SEQ ID NO: 92)

2. ESSWPNHTVTGVSASCSHNGESSF (SEQ ID NO: 93)

3. ESSWPNHTVTKGVTASCSHNGKSSF (SEQ ID NO: 94)

. TSS PNHDSNKGVTAACPHAGAKSF (SEQ ID NO: 95)

Example 19a

The construct from example 19, where GC was added to the C-termini of peptides and a disulfide bond is formed between the C-terminal cysteine and CI 7 (for stabilization of the structure). Example 19b

The construct from example 19a, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G) can be added between the C-terminus of each peptide and MAP core.

Example 19c

The construct from example 19b, where linker is G. Example 19d

The construct from example 19, where CI 7 is replaced with A. Example 19e

The construct from example 19d, which is in the form of MAP (e.g., 4-branched MAP). A linker (e.g., G) can be added between the C-terminus of each peptide and MAP core.

Example 19f

The construct from example 19e, where linker is GS.

Example 20

For HI HA region 156-199 RPP was constructed using clustering of known variants of the sequence of this region and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. GKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTEN (SEQ ID NO: 120)

2. GKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQMTLYHKEN (SEQ ID NO: 121)

3. GKNGLYPNLSMSYVNNKEKEVLVLWGVHHPPNIGDQRALYHTEN (SEQ ID NO: 122)

4. KKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNAD ( SEQ ID NO: 123) Example 21

The constructs from examples 19 and 20 inserted into HI HA fragment (e.g., HI HA fragment 52-273).

Example 21a

The constructs from example 21 containing the following flanking and spacer sequences.

For N-terminal flanking sequence, RPP was constructed using clustering of known variants of the sequence of the HI HA region 53-123 and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFP K (SEQ ID NO: 90)

2. PLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFP K (SEQ ID NO: 91)

For spacer between peptides from examples 19 and 20, RPP was constructed using clustering of known variants of the sequence of HI HA region 148-155 and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition:

1. YRNLLWLT (SEQ ID NO: 103)

2. YKNLIWLV (SEQ ID NO: 104)

For C-terminal flanking sequence, RPP was constructed using clustering of known variants of the sequence of HI HA region 200-273 and selection of the most frequently found sequences of each cluster. The resulting RPP has the following composition: l .

AYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGI INSNA

(SEQ ID NO: 115)

2.

AYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGI IISDT

(SEQ ID NO: 116) Different peptide variants from examples 19 and 20 and flanking and spacer sequences were combined in a way so that they correspond to the naturally occurring correlations. Final constructs based on HI HA fragment 53-273 have the following sequences:

1. PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIF PKSS PNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQ KALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFAL SRGFGSGIINSNA (SEQ ID NO: 48)

2. PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIF PKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGD QMTLYHKENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRI YYWTLLEPGDTI IFEANGNLIAPRYAFA LSRGFGSGIINSNA (SEQ ID NO: 49)

3. PLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIF PKESSWPNHTVTKGVTASCSHNGKSSFYRNLLWLTGKNGLYPNLSMSYVNNKEKEVLVL GVHHPPNIG DQRALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAF ALSRGFGSGIINSNA (SEQ ID NO: 50)

4. PLHLGKCNIAG ILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIF PKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVK GNSYPKLSKSYINDKGKEVLVLWGIHHPSTSA DQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAF AMERNAGSGIIISDT (SEQ ID NO: 51)

Example 21b

The construct from example 21a truncated to the fragment 86-262:

1. KPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKSSWPNHTVTGVSASCSHNGESSFYRNLLWLT GKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAYVSVVSSHYSRKFTPEIAKRPKVR DQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSR (SEQ ID NO: 72)

2. KPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWL TGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQMTLYHKENAYVSVVSSHYSRKFTPEIAKRPKV RDQEGRINYYWTLLEPGDTI IFEANGNLIAPRYAFALSR (SEQ ID NO: 73)

3. TPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTKGVTASCSHNGKSSFYRNLLW LTGKNGLYPNLSMSYVNNKEKEVLVLWGVHHPPNIGDQRALYHTENAYVSVVSSHYSRKFTPEIAKRPK VRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSR (SEQ ID NO: 74) 4. TSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSS PNHDSNKGVTAACPHAGAKSFYKNLIW LVKKGNSYPKLSKSYINDKGKEVLVL GIHHPSTSADQQSLYQNADAYVFVGSSRYS KFKPEIAIRPK VRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMER (SEQ ID NO: 75)

Example 21c

The construct from example 21 a in the form of DNA vaccine. Example 2 Id

The construct from example 21b in the form of DNA vaccine. Example 22

A conserved HI HA fragment ca be used as an additional vaccine component. For example, HI HA2 subunit region 37-58 can be used:

DQKSTQNAIDGITNKVNSVIEK (SEQ ID NO: 60)

Example 22a

The construct of example 22 in the form of a 4-branched MAP. Example 23

Any combinations of the constructs from examples 18-22. Peptide Synthesis

Solid-phase peptide synthesis methods are used.

Peptides were synthesized in linear form or in the form of a 4-branched MAP or 8- branched MAP.

Peptide libraries were synthesized using split&mix technology. Specifically, for introduction of amino acid residues into a variable position, peptidyl-polymer was washed with dimethylchloride and dimethyl ether, and dried to constant weight in vacuo. Then it was separated by weighing on parts that corresponded to quota of each peptide containing replacement in this position. Each part of peptidyl-polymer was transferred into separate reaction vessel, deprotected and condensed with derivative of required amino acid. If it was required, additional couplings were performed in order to get a negative ninhydrin test. After attachment of all residues in the variable position the polymers were combined, and washed with dimethyl chloride during 5-10 minutes. If the next position was variable, then the peptidyl polymer was separated on parts in required ratio (by weight), and all stages of synthesis were repeated. If the next position was constant, then synthesis was continued without separation of the matrix on parts. Additional information on the split and mix technology can be found, e.g., in Furka and Bennett, Comb Chem High Throughput Screen. 1999 2(2): 105-22. See also www.combichemistry.com/synthesis_combinatorial_library.html

Nucleic acid design and synthesis

Standard plasmid vectors, for example, a pDNAVACCultra (see above), were used for expression of DNA vaccines in mammalian cells. Design of nucleic acids encoding vaccine components (e.g., rAg51, rAg52, rAg53, rAg54, rAg55, rAg31, rAg32, rAg33, rAg34, rAg35, rAg36, rAgl l, rAgl2, rAgl3, and rAgl4) involved codon optimization for GC content, mRNA secondary structure, premature polyadenylation sites, inhibition sites of DNA replication, internal ribosome entry sites, etc. to allow optimal expression in the following expression systems:

•E. coli,

•baculovirus,

•mammalian cells.

After nucleic acid synthesis by standard solid-phase synthesis method, their sequence was confirmed by sequencing.

Expression of recombinant antigens

Recombinant antigens were produced via expression in E.coli or in mammalian cells (e.g., Vero or CHO) or using baculovirus expression system following standard protocols.

Immunization of mice Groups of 6 female Balb/c mice, weighing from 13 to 15 g (Laboratory Animal Farm, Siberian Branch of Russian Academy of Medical Sciences, Novosibirsk, Russia), were inoculated intraperitoneally on days 0, 14, and 28 with 100 of a suspension containing 20 μg of antigens emulsified in either CFA (Sigma) (day 0) or IFA (Sigma) (other days). Animals were bled 10 days after the third immunization.

List of constructs injected: for H5N1 :

A=A51+A52+A53+A54+A55, A51 , A51_noss, A55, B=B51+B52+B53, C5, A+B, A+B+C5, L5, L5+B, L5→rAg51 ("→" mean that first injection was with L5 and second with rAg51), L5→rAg51→rAg51 (three injections, first with L5, second and third with rAg51), rAg51→L5, rAg51, rAg52, rAg53, rAg54, rAg55, rAg5=∑rAg5j, rAg51bac, ("bac" is mean that protein was manufactured in baculoviral expression system), rAg52bac, rAg53bac, rAg54bac, rAg55bac, rAg5bac=∑rAg5jbac. for H3N2:

L3A, L3B, L3A+L3B, C3_HA1, C3_HA2, rAg31 , rAg32, rAg33, rAg34, rAg35, rAg36, rAg3=∑rAg3j. for HlNl : rAgl l, rAgl2, rA_g13, rA_g14, rAgl=∑rAglj.

For immunization with nucleic acid vaccines, groups of 6 female Balb/c mice, weighting from 13 to 15 g (Laboratory Animal Farm, Siberian Branch of Russian Academy of Medical Sciences, Novosibirsk, Russia), were inoculated intramuscularly on days 0, 21, and 42 with 100 of a suspension containing 100 μg of antigens. Animals were bled 14 days after the 3rd immunization. List of constructs injected (in the form of DNA vaccines): pAg51 , pAg52, pAg53, pAg54, pAg55, pAg5=∑pAg5j - Example 7c, pAgl4 - Example 21c. Letter "p" in the name (pAgij) is indicated the plasmid (DNA vaccine) encoding the corresponding peptide (rAgij).

Results

Immunogenicity was tested using ELISA in a routine manner. Briefly, 96-well polystyrene plates (GOSNII Polymer, Moscow, Russia) were coated with 100 μΐ of 0.05 M bicarbonate-carbonate (BCC) buffer (pH 9.6) containing 5 μg/ml of CPL or RPP peptide tested and incubated for 20 hours at 20-25°C. After two washings with phosphate buffered saline with the addition of Tween (PBST) (0.015 M NaCl, 10 mM phosphate buffer [pH 7.3], 0.05% Tween-20), plates were blocked with a solution of 0.2% casein in phosphate buffered saline (PBS) for 2 hours at 20-25°C. Serum samples were diluted in a diluting solution of 0.2% casein in PBST. 100 μΐ of the sample was loaded to each well and incubated in a wet chamber for 30 min at 37°C. After five washings in PBST, an anti-mouse IgG peroxidase conjugate was added and the mixture was incubated for 30 min at 37°C. After five washings in PBST, the color reaction with a substrate mixture (0.1 mg/ml TMB, 0.0044% hydrogen peroxide in 0.1 M sodium-acetate buffer, pH 6.0) was allowed to develop for 20 min at 20- 25°C and then stopped with 50 μΐ of 2M H₂S0₄. Optical density (OD) was recorded with a spectrophotometer at 450 nm. Critical value of OD (OD_cutofr) was calculated as the mean OD of wells assayed without serum plus 0.2 on the same plate. Titers of antibodies in the sample were calculated by the formula: T = (OD/OD_cutoff)* , where K is reciprocal of the sample dilution in the range of linear function OD = f(K) of titration curve.

Hemagglutination inhibition (HAI) reaction of mice antisera against vaccine constructs were done in a routine manner according to WHO_Manual on Animal Influenza Diagnosis and Surveillance (WHO/CDS/CSR/NCS/2002.5).

Result ELISA H5N1

ELISA* Antigens coated on the plate

Sera A51 A52 A53 A54 A55 A51noss L5 B B51 B52 B53 C5 A+B+C5 A+B

A55 219 146 219 219 73 375 13 8 - - - - - 219 219

B - - - - - - - - 44 33 33 24 - 24 33

C5 - - - - - - - - - - - - 5 1 -

A+B+C5 219 73 219 146 33 219 - 5 37 16 24 24 1 146 146

A+B 300 73 328 162 49 219 - 5 37 16 24 40 - 111 135

L5 73 33 49 33 24 49 13 146 - 73 73

L5+B 49 33 49 24 24 49 - 111 49 24 33 24 - 111 111

L5→rAg51 13 8 13 8 8 24 - 5 13 8 8 13 - 13 24

L5→rAg51→rAg51 49 49 33 24 24 73 - 8 49 24 33 49 - 73 73 rAg51→L5 5 3 5 3 3 8 - 8 8 8 13 13 - 5 13 rAg51 219 219 73 125 125 299 33 13 49 33 49 49 - 73 111 rAg52 146 73 219 73 125 219 24 24 33 24 33 33 - 33 73 rAg53 125 44 73 146 73 146 13 8 49 24 33 33 - 49 73 rAg54 146 73 146 73 219 125 8 24 49 33 13 49 - 33 49 rAg55 219 219 146 125 146 429 13 13 73 73 24 49 - 73 49 rAg5 219 219 146 146 146 299 13 24 49 33 33 49 - 73 111 rAg51bac - - - - - - - - - - - - - - - rAg52bac - - - - - - - - - - - - - - - rAg53bac - - - - - - - - - - - - - - - rAg54bac - - - - - - - - - - - - - - - rAg55bac - - - - - - - - - - - - - - - rAg5bac - - - - - - - - - - - - - - -

RPP23 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PBS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Flu51 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

Result ELISA_H5N1 (continuation

ELISA* Antigens coated on the plate Controls

Sera rAg51 rAg52 rAgS3 rAgM rAgS5 rAgS rAgSibac rAg52bac rAg53ba< rAg54bac rAg55bac rAgSbac RPP23 Flu51

A51 33 13 24 8 13 24 33 13 24 13 13 33 0 13

A51 noss 8 5 8 5 8 13 13 8 8 5 8 13 0 5

A55 13 24 13 13 33 24 13 24 13 13 33 24 0 13

B 5 3 3 8 5 5 3 3 5 5 3 8 0 5

C5 - - - - - - - - - - - - 0 0

A+B+C5 13 13 8 5 13 24 13 13 8 8 13 24 0 13

A+B 13 24 13 8 24 24 24 13 13 8 24 24 0 24

L5 8 8 8 13 8 8 13 8 13 8 8 13 0 5

L5+6 5 13 8 8 5 8 13 8 13 8 8 24 0 8

L5→rAg51 13 8 8 8 5 13 13 8 8 5 5 13 0 5

L5→rAg51→rAg51 73 24 24 24 13 49 73 33 24 24 13 49 0 8 rAg51— >L5 8 5 5 3 3 8 5 5 3 3 5 5 0 3 rAg51 350 219 116 219 146 219 316 130 146 219 146 219 - 49 rAg52 219 219 219 120 146 219 219 316 219 125 316 330 - 33 rAg53 219 219 105 219 146 146 219 316 146 219 219 219 - 33 rAg54 350 429 316 429 219 429 520 520 320 429 219 429 - 49 rAg55 219 219 146 146 219 219 219 219 146 125 219 429 - 33 rAg5 330 219 133 219 146 219 330 310 219 219 146 429 - 49 rAg51bac 350 310 219 219 146 219 429 219 316 310 219 310 - 73 rAg52bac 219 316 219 146 146 219 316 429 219 219 219 330 - 73 rAg53bac 219 219 146 219 219 310 316 316 219 310 219 316 - 49 rAg54bac 429 429 219 316 219 429 590 580 316 429 219 429 - 111 rAg55bac 219 219 146 146 219 219 219 310 146 219 219 429 - 73 rAg5bac 350 219 219 330 146 219 350 219 330 316 219 429 - 111

RPP23 - - - - - - - - - - - - 219 0

PBS 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Flu51 13 24 13 13 13 24 24 24 33 49 24 49 0 437

Titers are measured as thousands. All numbers in the table should be multiplied by Note. RPP23 - Control peptide TRKGIHIGPGQAWYTTGDITG (SEQ ID NO: 128) was in the form of a four-branched MAP.

All tested peptide constructs except C5 and all tested recombinant proteins showed high immunogenicity. High level of cross-reacting antibodies correlated with the presence of disulfide bond in antigenic region A. Antibodies directed against rAg51, rAg52, rAg53, rAg54, and rAg55 recombinant proteins showed a much higher level of cross-reactivity with each other and with the virus (Flu51 recombinant vaccine strain ADuck/86/92 H5N2). This high level of cross-reactivity indicates that rAg51 , rAg52, rAg53, rAg54, and rAg55 can elicit broadly reactive immune response and are therefore suitable constructs for the generation of a universal influenza vaccine. Since rAg51, rAg52, rAg53, rAg54, and rAg55 are chimeric HA molecules of H5N1 influenza virus containing directed modifications of a number of regions, they represent the most suitable constructs for the generation of a universal H5N1 influenza vaccine. Among those, the most promising are the recombinant proteins generated via baculovirus expression as they produce higher titers.

Result ELISA H5N1 DNA vaccines

Note. * - Titers are measured as thousands. All numbers in the table should be multiplied by recombinant vaccine strain ADuck/86/92 H5N2. Immunization with DNA-vaccine constructs demonstrated that such constructs can be successfully used for generating immune response to H5N1 influenza virus. Further titer increase can be achieved by using more efficient immunization protocols. See, e.g., DNA vaccines: methods and protocols / edited by W. Mark Saltzman, Hong Shen, Janet L. Brandsma. - 2nd ed. 2006.

Result_HAI_H5N1 : Hemagglutination-lnhibition (HAI) Reactions of Mice

Antisera against Vaccine Constructs

Hemagglutination inhibition (HAI) reaction of mice antisera against tested vaccine constructs have shown that protein constructs rAg51, rAg52, rAg53, rAg54, and rAg55 as well as DNA vaccines pAg51 , pAg52, pAg53, pAg54, and pAg55 elicit immune response with high HAI titers. The highest HAI titers were observed when animals were immunized with baculovirus-expressed rAg51bac-rAg55bac. This observation confirms usefulness of rAg51 , rAg52, rAg53, rAg54, and rAg55 for a universal vaccine against H5N1 influenza virus.

Result ELISA H3N2

ELISA* Antigens coated on the plate Controls

·¾ L3B L3A+L3B C3_HA1 C3_HA; rAg31 rAg32 rAg33 rAg34 rAg35 rAg36 rAg3 RPP23 Flu32

L3A 125 - 73 - - 3 5 5 8 5 5 8 0 3

L3B - 194 278 - - 8 8 8 13 8 8 5 0 5

L3A+

125 380 437 - - 8 8 8 13 8 8 13 0 5 L3B

C3_H

- - - 219 - - - - - - - - 0 0

A1

C3 H

- - - - 73 - - - - - - - 0 0

A2

rAg31 5 24 24 - 316 219 219 316 146 219 219 - 39 rAg32 8 8 8 - 219 219 316 219 146 219 219 - 24 rAg33 8 24 24 - 316 219 430 316 219 316 316 - 49 rAg34 13 13 24 - 219 219 316 350 219 219 316 - 24 rAg35 8 13 24 - 219 146 219 219 219 146 146 - 49 rAg36 13 8 13 - 146 146 219 146 146 219 146 - 39 rAg3 13 13 24 - 316 219 316 219 146 219 219 - 39

RPP2

0 0 0 0 0 - - - - - - - 146 0

3

PBS 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Flu32 5 13 13 2 1 37 24 39 13 49 24 37 0 330

Note. * - Titers are measured as thousands. All numbers in the table should be multip ied by 1000.

Flu32 - vaccine strain A/Brisbane/10/2007.

All constructs, including peptide libraries and recombinant proteins, demonstrated high immunogenicicty. Antibodies directed against rAg31, rAg32, rAg33, rAg34, rAg35, and rAg36 recombinant proteins showed a high level of cross-reactivity with each other and with the virus (Flu32 - vaccine strain A/Brisbane/10/2007). This high level of cross-reactivity indicates that rAg31, rAg32, rAg33, rAg34, rAg35, and rAg36 can elicit broadly reactive immune response and are therefore suitable constructs for the generation of a universal influenza vaccine. Since rAg31, rAg32, rAg33, rAg34, rAg35, and rAg36 are chimeric HA molecules of H3N2 influenza virus containing directed modifications of a number of regions, they represent the most suitable constructs for the generation of a universal H3N2 influenza vaccine. Result_HAI_H3N2: Hemagglutination-lnhibition (HAI) Reactions of Mice

Antisera against Vaccine Constructs

Hemagglutination inhibition (HAI) reaction of mice antisera against tested vaccine constructs have shown that protein constructs rAg31 , rAg32, rAg33, rAg34, rAg35, and rAg36 elicit immune response with high HAI titers. This observation confirms usefulness of rAg31, rAg32, rAg33, rAg34, rAg35, and rAg36 for a universal vaccine against H3N2 influenza virus.

Result ELISA H1N1

Note. * - Titers are measured as thousands. All numbers in the table should be multiplied by 1000.

Flul 1 - vaccine strain A/New Caledonia/20/99.

All recombinant proteins demonstrated high immunogenicicty. Antibodies directed against rAgl 1, rAgl2, rAgl3, and rAgl4 recombinant proteins showed a high level of cross- reactivity with each other and with the virus (Flul l - vaccine strain A/New Caledonia/20/99). This high level of cross-reactivity indicates that rAgl 1, rAgl 2, rAgl 3, and rAgl 4 can elicit broadly reactive immune response and are therefore suitable constructs for the generation of a universal influenza vaccine. Since rAgl 1, rAgl2, rAgl3, and rAgl4 are chimeric HA molecules of H1N1 influenza virus containing directed modifications of a number of regions, they represent the most suitable constructs for the generation of a universal H1N1 influenza vaccine.

Result_HAI_H1N1 : Hemagglutination-lnhibition (HAI) Reactions of Mice

Antisera against Vaccine Constructs

Hemagglutination inhibition (HAI) reaction of mice antisera against tested vaccine constructs have shown that protein constructs rAgl l, rAgl2, rAgl3, and rAgl4 elicit immune response with high HAI titers. This observation confirms usefulness of rAgl l, rAgl2, rAgl3, and rAgl4 for a universal vaccine against H1N1 influenza virus.

* * *

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification. SEQUENCE DISCLOSURES H5N1 :

Peptide library based on site A (L5):

Amino acids composition, %

P=100

K=85;R=15

S=95;N=5

S=100

W=100

S=95;P=5

D=55;N=25;S=20

H=100;

E=80;D=20

A=100

S=100

S=70;L=30

G=100

V=100

S=100

A=100

C=100

P=100

Y=100

Q=60;L=25;N=15 Amino acids composition, %

G=100

R=40;K=30;S=20;T=10

S=70;P=30

S=100

F=100

G=100

C=100

G=100

Peptide library based on site A without disulfide bonds (L5_noss):

Amino acids composition, %

P=100

K=85;R=15

S=95;N=5

S=100

W=100

S=95;P=5

D=55;N=25;S=20

H=100;

E=80;D=20

A=100

S=100

S=70;L=30

G=100

V=100 Amino acids composition, %

S=100

A=100

P=100

Y=100

Q=60;L=25;N=15

G=100

R=40;K=30;S=20;T=10

S=70;P=30

S=100

F=100

G=100

Set of site A representative peptides with disulfide bonds:

A51: PKSSWSDHEASSGVSSACPYLGSPSFGCG (SEQ ID NO 6)

A52: PKSSWSSHEASLGVSSACPYQGKSSFGCG (SEQ ID NO 7)

A53: PRSSWSNHDASSGVSSACPYNGRSSFGCG (SEQ ID NO 8)

A54: PKSSWSNHEASSGVSSACPYQGTPSFGCG (SEQ ID NO 9)

A55: PKSSWSDHEASLGVSSACPYLGRSSFGCG (SEQ ID NO 10)

A51 is a fragment of natural H5N1 flu HA sequences from Indonesia, years 2006-2007. A52 is a fragment of natural human and avian H5N1 flu HA sequences from Thailand and Vietnam, year 2004.

A53 is a fragment of natural avian only H5N1 flu HA sequences from USA, Europe and Japane, various years.

A54 is a chimeric sequence, the closest natural fragment is PKSSWSNHEASSGVSSACPYQGNPSF from A/duck/Shantou/4610/2003 and A/Dk/ST/4003/2003 isolates.

A54: PKSSWSNHEASSGVSSACPYQGTPSFGCG (SEQ ID NO: 9) A/duck/Shantou/4610/2003: N...

A55 is a fragment of natural human H5N1 flu HA sequences from Indonesia, year 2006.

Set of site A representative peptides without disulfide bonds:

A51_noss: PKSSWSDHEASSGVSSAAPYLGSPSFG (SEQ ID NO: 11)

A52 noss: PKSSWSSHEASLGVSSAAPYQGKSSFG (SEQ ID NO: 12) A53_noss: PRSSWSNHDASSGVSSAAPYNGRSSFG (SEQ ID NO: 13)

A54_noss: PKSSWSNHEASSGVSSAAPYQGTPSFG (SEQ ID NO: 14)

A55_noss: PKSS SDHEASLGVSSAAPYLGRSSFG (SEQ ID NO: 15).

Set of site B2 representative peptides:

B51: PNDAAEQTKLYQNPTT (SEQ ID NO: 16)

B52: PNNEAEQTRLYQNPTT (SEQ ID NO: 17)

B53: PNDAAEQIKLYQNPNT (SEQ ID NO: 18)

B51 is a fragment of natural poultry H5N1 flu HA sequences from Thailand, year 2004. B52 is a fragment of natural human H5N1 flu HA sequences from Indonesia, years 2006- 2007.

B53 is a fragment of natural poultry H5Nlflu HA sequences from Mexico, years 1994-1995. rAg51:

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKS SWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPEYAY IVKKGDSTI MKSEL (SEQ ID NO: 1) rAg52:

PLILRDCSVAGWLLGNPMCDEFI VPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQI IPKS SWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVL GIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 2) rAg53:

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPRS S SNHDASSGVSSACPYNGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 3) rAg54:

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKS SWSNHEASSGVSSACPYQGTPSFFRNVV LIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNNEAEQTRLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 4) rAg55:

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKD PVNGLCYPGDFNDYEELKHLLSRINHFEKIQI IPKS SWSDHEASLGVSSACPYLGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIKLYQ NPNTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTI MKSEL (SEQ ID NO: 5) rAg51 is chimeric, the closest natural sequence is

PLILRDCSVAGWLLGNPMCDEFI VPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQI IPKS SWSDHEASSGVSSACPYLGTPSFFRNVV LIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQ NPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAI MKSEL which is contained in A/Indonesia/6/2005 and A/QUAIL/JOMBANG/P2KH/2005 isolates.

rAg51 :

PLILRDCSVAGWLLGNPMCDEFI VPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQI I PKS SWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 1)

A/Indonesia/6/2005:

T.

K. .A. rAg52 is a fragment of natural human and avian H5N1 flu HA sequences from Thailand and Vietnam, year 2004.

rAg53 is a chimeric sequence, the closest natural is

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS S SNHEASSGVSSACPYNGKSSFFRNVV LIKKDNAYPTIKRSYNNTNQEDLLILWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPEYAYKIVKKGDSAI MKSEL from A/SCk/Hong Kong/YU 100/2002 isolate.

rAg53:

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPRS SWSNHDASSGVSSACPYNGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVL GIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI

MKSEL A/SCk/Hong Kong/YU 100/2002:

N K. E K I K A.

rAg54 is chimeric, the closest natural is

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS SWSNHEASSGVSSACPYQGKPSFFRNVVWLIKKNSAYPTIKRSYNNTNQEDLLVLWGIHHPNNEAEQTKLYQ NPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAI

MKSEL from A/goose/Viet Nam/324/2001 isolate.

rAg54 :

PLILRDCSVAG LLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKS SWSNHEASSGVSSACPYQGTPSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNNEAEQTRLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 4) A/goose/Viet Nam/324/2001:

S.A D

K S K...

K A.

rAg55 is chimeric, the closest natural is

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKDNPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWS SHEASLGVSSACPYQGKSSFFRNVV LIKKNSTYPTIKRSYNNTNQEDLLVMWGIHHPNDAAEQAKLYQNPTTYI SVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSEL

from A/chicken/Viet Nam/1/2004 and A/duck/Vietnam/ 17/2003 isolates.

rAg55 :

PLILRDCSVAGWLLGNPMCDEFINVPEWSYI EKDNPVNGLCYPGDFNDYEELKHLLSRINHFEKIQI IPKSSWS DHEASLGVSSACPYLGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIKLYQNPNTYI SVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTIMKSEL

(SEQ ID NO: 5)

A/chicken/Viet Nam/1/2004:

D

S Q.K M A T...

Y

Conservative 37-58 HA2 peptide (C5):

DKESTQKAIDGVTNKVNSIIDK (SEQ ID NO: 19)

Multiple nature H5N1 flu isolate contain this fragment.

The possible vaccine compositions for H5N1:

1. L5

2. L5_noss

3. A51, A52, A53, A54, A55

4. A51_noss, A52_noss, A53_noss, A54_noss, A55_noss

5. any of above and B51 , B52, B53

6. any of above and C5

7. rAg51 , rAg52, rAg53, rAg54, rAg55

H3N2:

Peptide library based on site A (L3A):

Amino acids composition, % Amino acids composition, %

N=100

G=100

T=100

S=100

S=45; Y= 45; F=10;

A=90; T= 10

C=100

KRG=20

KRR=30

IRR=50

S=100

N=80; D=10; 1 = 10;

N=35; K=35; S=10; 1=10; H=10

S=100

F=100

G=100

C=100

G=100

Peptide library based on site A without disulfide bonds(L3A_noss):

Amino acids composition, %

N=100

G=100

T=100

S=100

S=45; Y=45; F=10;

A=90; T=10

A=100

KRG=20

KRR=30

IRR=50

S = 100 Amino acids composition, %

N=80; D=10; 1=10;

N=35; K=35; S=10; 1=10; H=10

S=100

F=100

G=100

Peptide library based on site B (L3B):

Amino acids composition, %

P=100

S=45; G=45; V=10

T=100

D=90; Y=10

N=33; S=33; K=33

D=100

Q=100

l=50; T=50

F=50; S=50

L=100

Y=100

A=50; V=50

Q=50; R=50

A=100

S=90; P=10

G=100

Set of site A representative peptides with disulfide bonds:

A31 NGTSSACIRRSNNSFGCG (SEQ ID NO 26)

A32 NGTSSACKRGSNNSFGCG (SEQ ID NO 27)

A33 NGTSSACKRRSNNSFGCG (SEQ ID NO 28)

A34 NGTSSACKRRSNKSFGCG (SEQ ID NO 29)

A35 NGTSSACIRRSNKSFGCG (SEQ ID NO 30)

A36 NGTSYACIRRSNNSFGCG (SEQ ID NO 3D A31 is a fragment of natural human H3N2 flu HA sequences, isolated on 2007-2008, such as the former WHO vaccine strain A/Brisbane/10/2007.

A32 is a fragment of natural human H3N2 flu HA sequences, isolated on 2006-2007, for example A/Pennsylvania/05/2007.

A33 is a fragment of natural human H3N2 flu HA sequences, isolated on 2004-2007, such as former WHO vaccine strains A/Wisconsin/67/2005 and A/California/7/2004.

A34 is a fragment of natural human H3N2 flu HA sequences, isolated on 1999-2005, such as former WHO vaccine strains A/Panama/2007/99, A/Fujian/41 1/2002, A/Wellington 1/2004. A35 is a fragment of natural human H3N2 flu HA sequences, A/Minnesota/08/2007, A Managua 2/2007 and A Finland/305/2003 isolates.

A36 is not a fragment of any natural flu sequence, the closest natural sequence is A35.

A36: NGTSSACIRRSNKSF

A35: .... Y N..

Set of site A representative peptides without disulfide bonds:

A31 noss : NGTSSAAIRRSNNSFG (SEQ ID NO 32

A32^~ noss : NGTSSAAKRGSNNSFG (SEQ ID NO 33

A33^~ noss : NGTSSAAKRRSNNSFG (SEQ ID NO 34

A34^~ noss : NGTSSAAKRRSNKSFG (SEQ ID NO 35

A35^~ noss : NGTSSAAIRRSNKSFG (SEQ ID NO 36

A36^~ noss : NGTSYAAIRRSNNSFG (SEQ ID NO 37

Set of site B2 representative peptides:

B31 PGTDNDQI FLYAQASG (SEQ ID NO 38)

B32 PGTDNDQI FLYARASG (SEQ ID NO 39)

B33 PGTDNDQI SLYAQASG (SEQ ID NO 40)

B34 PGTDNDQTFLYAQASG (SEQ ID NO 41)

B35 PSTDNDQI FLYAQASG (SEQ ID NO 42)

B31 is a fragment of the former WHO vaccine strains A/Brisbane/10/2007.

B32 is not a part of a natural sequence, the closest natural is PGTDNDQIFLYAKASG,

A/FUKUSHIMA/20/2006 isolate.

B32: PGTDNDQI FLYARASG (SEQ ID NO: 39)

A/FUKUSHIMA/20/2006: K...

B33 is a fragment of the former WHO vaccine strains A/Wellington/ 1/2004

B34 is a fragment of A/Georgia 05/2007 and several other isolates.

B35 is not a part of a natural sequence, the closest natural sequence is PGTDNDQIFLYAQASG, which is contained in multiple isolates (for example A/Florida/02/2007), years 2005-2008.

B35: PSTDNDQI FLYAQASG (SEQ ID NO: 42)

A/Florida/02/2007 : .G

Conservative 20-40 HA1 peptide (C3_HA1):

VPNGTIVKTITNDQIEVTNAT (SEQ ID NO: 43)

C3_HA1 is a fragment of multiple H3N2 flu sequences. Set of 37-58 HA2 peptides (C3_HA2):

C31 _HA2 : DLKSTQAAINQINGKLNRLIGK (SEQ ID NO: 44 )

C32 _HA2 : DLKSTQAAINQINGKLNRLEGK (SEQ ID NO: 45)

C33 _HA2 : DLKSTQAADNQINGKLNRLIGK (SEQ ID NO: 46)

C34 HA2 : DLKSTQAADNQINGKLNRLEGK (SEQ ID NO: 47)

C31_HA2 is a fragment of former WHO vaccine strains A/California/7/2004,

A/Wellington/ 1/2004 and A/Wisconsin/67/2005.

C32_HA2 is not natural, the closest natural is C31_HA2.

C32_HA2: DLKSTQAAINQINGKLNRLEGK (SEQ ID NO: 45)

C31_HA2 : I ..

C33_HA2 is not natural, the closest natural is C31_HA2.

C33_HA2: DLKSTQAADNQINGKLNRLIGK _o( SEQ ID NO: 46)

C31_HA2: I

C34 HA2 is not natural, the closest natural is C31_HA2.

C34_HA2: DLKSTQAADNQINGKLNRLEGK (SEQ ID NO: 47)

C31 HA2 : I I .. rA 31

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLI STGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 20) rA_g32

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRGSNNSFFSRLNWLTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPSTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 21) rAg33

(SEQ ID NO: 22) rAg34

ILDGENCTLIDALLGDPQCDGFQNKK DLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNKSFFSRLN LTQLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 23) rAg35

(SEQ ID NO: 24) rAg36

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSYACIRRSNNSFFSRLNWLTHLEFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYARASGR ITVSTKRSQQTVIP IGSRPRVRNIPSRISIYWTI KPGDILLI STGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 25)

rAg31 is a fragment of natural human H3N2 flu HA sequences, isolated on 2007, for example A/Missouri/01/2008. rAg32 is not a natural sequence, the closest natural fragment is

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFN TG VTQNGTSSACKRGSNNSFFSRLNWLTHSKFKYPALNVTMPNNEEFDKLYIWGVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVI NIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA a fragment of natural human H3N2 flu HA sequences, isolated on 2006-2007, for example A/Thailand/CU228/2006.

rAg32 :

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRGSNNSFFSRLN LTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPSTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILL NSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 21)

A/Thailand/CU228/2006 :

rAg33 is not a natural sequence, the closest natural is a fragment of A/Hanoi/TN403/2005:

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYVSLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNNSFFSRLN LTHLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQISLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

rAg33:

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQISLYAQASGR ITVSTKRSQQTVIPNIGSRPRVR I PSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 22) A/Hanoi/TN403

rAg34 is not a natural sequence, the closest natural is a fragment of A/Denmark/50/2006 or A/Denmark/ 100/2006 isolates:

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNERFDKLYI GVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIY TIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

rAg34 :

ILDGENCTLIDALLGDPQCDGFQNKK DLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNKSFFSRLN LTQLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIY TIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 23)

A/Denmark/50/2006:

rAg35 is not a natural sequence, the closest natural is

ILDGENCTLIDALLGDPQCDGFQNKN DLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNNSFFSRLNWLTHSKFKYPALNVTMPNNENFDKLYIWGVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA which is a fragment of human H3N2 flu HA sequences, isolated on 2008 in USA, for example A/Washington/ AF 1696/2008

rAg35:

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNKSFFSRLNWLTHSKFKYPALNVTMPNNEQFDKLYI GVHHPGTDNDQTFLYAQASGR ITVSTKRSQQTVIP IGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 24)

A/ ashington/AF1696/2008 :

rAg36 is not a natural sequence, the closest natural is a fragment of A/Puno/FLU8262/2007:

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNNSFFSRLNWLTHLRFKYPALNVT PNNEKFDKLYIWGVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIY TIVKPGDILLINSTGNLIAPRGYFKIRSGKSSI RSDA rAg36:

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSYACIRRSNNSFFSRLNWLTHLEFKYPALNVTMPNNEKFDKLYI GVHHPGTDNDQIFLYARASGR ITVSTKRSQQTVIPNIGSRPRVRNI PSRISIYWTI KPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 25)

A/Puno/FLU8262/2007 :

K S R Q....

The possible vaccine compositions for H3N2:

1. L3A

2. L3A_noss

3. A31 , A32, A33, A34, A35, A36

4. A31_noss, A32_noss, A33_noss, A34_noss, A35_noss, A36_noss

5. any of above and L3B or B31, B32, B33, B34, B35.

6. any of above and C3 H A 1

7. any of above and C3 HA2

8. rAg31 , rAg32, rAg33, rAg34, rAg35, rAg36.

H1N1

Set of site A representative peptides with disulfide bonds:

All: ESSWPNHTVTGVSASCSHNGKSSFGCG (SEQ ID NO: 52)

A12: ESSWPNHTVTGVSASCSHNGESSFGCG (SEQ ID NO: 53)

A13: ESSWPNHTVTKGVTASCSHNGKSSFGCG (SEQ ID NO: 54)

A14: TSSWPNHDSNKGVTAACPHAGAKSFGCG (SEQ ID NO: 55)

Al l is a fragment of the former WHO vaccine strain A/New Caledonia/20/99.

A12 is a fragment of natural HlNl human flu HA sequences, isolated on 1998-2008.

A13 is a fragment of natural HlNl human flu HA sequences, isolated on 1985-2001.

A14 is a fragment of A/Wisconsin/ 10/98 isolate.

Set of site A representative peptides without disulfide bonds:

All_noss: ESSWPNHTVTGVSASASHNGKSSFG (SEQ ID NO: 56)

A12_noss: ESSWPNHTVTGVSASASHNGESSFG (SEQ ID NO: 57)

A13_noss: ESSWPNHTVTKGVTASASHNGKSSFG (SEQ ID NO: 58)

A14_noss: TSSWPNHDSNKGVTAAAPHAGAKSFG (SEQ ID NO: 59)

rAgll: PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKS S PNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHT ENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRI YYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGI I NSNA (SEQ ID NO: 48) rAgl2:

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKE SS PNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVL GVHHPPNIGDQMTLYH KENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGI INSNA (SEQ ID NO: 49) rAgl3:

PLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKE SSWPNHTVTKGVTASCSHNGKSSFYRNLLWLTGKNGLYPNLSMSYVNNKEKEVLVLWGVHHPPNIGDQRALY HTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSG IINSNA (SEQ ID NO: 50) rAgl4:

PLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKT SSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLY QNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSG IIISDT (SEQ ID NO: 51) rAgl 1 is not a natural sequence, the closest natural is

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKE SS PNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVL GVHHPPNIGBQKALYH TENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRI YYWTLLEPGDTI IFEANGNLIAPRYAFALSRGFGSGI INSNA which is a fragment of H1N1 sequences, isolated on 2007-2008, for example A/New York/01/2008.

rAgll :

PLQLGNCSVAG ILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPK- SSWPN

HTVTGVSASCSHNGESSFYRNLL LTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAY VSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTI IFEANGNLIAPRYAFALSRGFGSGIINSNA

A/New York/01/2008:

E

rAgl 2 is not a natural sequence, the closest natural is a fragment of A/Jiangxi/ 1140/2006 isolate: PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKE SSWPNHTVTGVSASCSHNGKSSFYKNLLWLTGKNGLYPNLSKSYANNKEKEVLVL GVHHPPNIGDQMTLYH KENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGI INSNA

rAgl2 :

PLQLGNCSVAG ILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKE SS PNHTVTGVSASCSHNGKSSFYRNLL LTGKNGLYPNLSKSYANNKEKEVLVL GVHHPPNIGDQMTLYH KENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGI INSNA (SEQ ID NO: 49)

A/Jiangxi/1140/2006:

K

rAgl3 is not a natural sequence, the closest natural is a fragment of A/RiodeJaneiro/21/00 isolate:

PIQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKE SSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSMSYVNNKEKEVLVL GVHHPPNIGDQRALYH TENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGI ITSNA

rAg!3:

PLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKE SSWPNHTVTKGVTASCSHNGKSSFYRNLL LTGKNGLYPNLSMSYVNNKEKEVLVLWGVHHPPNIGDQRALY HTENAYVS VSSHYSRKFTPEIAKRPKVRDQEGRINYY TLLEPGDTIIFEANGNLIAPRYAFALSRGFGSG IINSNA (SEQ ID NO: 50)

A/RiodeJaneiro/21/00 :

.W. rAgl4 is a fragment of pandemic H1N1 sequences, isolated on 2009.

Conservative 37-58 HA2 peptide (CI):

DQKSTQNAIDGITNKVNSVIEK (SEQ ID NO: 60)

The possible vaccine compositions for H1N1: 1. All, A12, A13, A14

2. Al l_noss, A12_noss, A13_noss, A14_noss

3. rAgll, rAgl2,rAgl3,rAgl4

LIST OF SEQUENCES (BY SEP ID NO):

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRI HFEKIQI IPKSSWS DHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSEL

(SEQ ID NO: 1)

PLILRDCSVAGWLLGNPMCDEFI VPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRI HFEKIQIIPKSSWS SHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSEL

(SEQ ID NO: 2)

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPRSSWS NHDASSGVSSACPYNGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSEL

(SEQ ID NO: 3)

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWS NHEASSGVSSACPYQGTPSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNNEAEQTRLYQNPTTYI SVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSEL

(SEQ ID NO: 4)

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKDNPVNGLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWS DHEASLGVSSACPYLGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIKLYQNPNTYI SVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTIMKSEL

(SEQ ID NO: 5)

A51: PKSSWSDHEASSGVSSACPYLGSPSFGCG (SEQ ID NO: 6)

A52: PKSSWSSHEASLGVSSACPYQGKSSFGCG (SEQ ID NO: 7)

A53: PRSSWSNHDASSGVSSACPYNGRSSFGCG (SEQ ID NO: 8)

A54: PKSSWSNHEASSGVSSACPYQGTPSFGCG (SEQ ID NO: 9)

A55: PKSSWSDHEASLGVSSACPYLGRSSFGCG (SEQ ID NO: 10).

A51_noss: PKSSWSDHEASSGVSSAAPYLGSPSFG (SEQ ID NO: 11)

A52_noss: PKSSWSSHEASLGVSSAAPYQGKSSFG (SEQ ID NO: 12)

A53_noss: PRSSWSNHDASSGVSSAAPYNGRSSFG (SEQ ID NO: 13)

A54_noss: PKSSWSNHEASSGVSSAAPYQGTPSFG (SEQ ID NO: 14)

A55_noss: PKSSWSDHEASLGVSSAAPYLGRSSFG (SEQ ID NO: 15)

B51: PNDAAEQTKLYQNPTT (SEQ ID NO: 16)

B52: PNNEAEQTRLYQNPTT (SEQ ID NO: 17)

B53: PNDAAEQIKLYQNPNT (SEQ ID NO: 18)

C5: DKESTQKAIDGVTNKVNSI IDK (SEQ ID NO: 19) I-.LDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQ NGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTK RSQQTVIPNIGSRPRVRNIPSRISIY TIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA (SEQ ID NO: 20)

I _^LDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQ NGTSSACKRGSNNSFFSRLN LTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPSTDNDQIFLYAQASGRITVSTK RSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA (SEQ ID NO: 21)

I _^LDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQ NGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVT PNNEQFDKLYIWGVHHPGTDNDQISLYAQASGRITVSTK RSQQTVIP IGSRPRVR IPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA (SEQ ID NO: 22)

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQ NGTSSACKRRSNKSFFSRLN LTQLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTK RSQQTVIPNIGSRPRVRNIPSRISIY TIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA (SEQ ID NO: 23)

ILDGENCTLIDALLGDPQCDGFQNKN DLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQ NGTSSACIRRSNKSFFSRLNWLTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQTFLYAQASGRITVSTK RSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA (SEQ ID NO: 24)

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQ NGTSYACIRRSNNSFFSRLNWLTHLEFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYARASGRITVSTK RSQQTVIP IGSRPRVR IPSRISIYW IVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA (SEQ ID NO: 25)

A31: NGTSSACIRRSNNSFGCG (SEQ ID NO: 26)

A32: NGTSSACKRGSNNSFGCG (SEQ ID NO: 27)

A33: NGTSSACKRRSNNSFGCG (SEQ ID NO: 28)

A34: NGTSSACKRRSNKSFGCG (SEQ ID NO: 29)

A35: NGTSSACIRRSNKSFGCG (SEQ ID NO: 30)

A36: NGTSYACIRRSNNSFGCG (SEQ ID NO: 31)

A31_noss: NGTSSAAIRRSNNSFG (SEQ ID NO: 32)

A31_noss: NGTSSAAKRGSNNSFG (SEQ ID NO: 33)

A31_noss: NGTSSAAKRRSNNSFG (SEQ ID NO: 34)

A31_noss: NGTSSAAKRRSNKSFG (SEQ ID NO: 35)

A31_noss: NGTSSAAIRRSNKSFG (SEQ ID NO: 36)

A31_noss: NGTSYAAIRRSNNSFG (SEQ ID NO: 37)

B31: PGTDNDQIFLYAQASG (SEQ ID NO: 38)

B32: PGTDNDQIFLYARASG (SEQ ID NO: 39) B33: PGTDNDQISLYAQASG (SEQ ID NO: 40)

B34: PGTDNDQTFLYAQASG ( SEQ ID NO: 41)

B35: PSTDNDQIFLYAQASG (SEQ ID NO: 42)

C3_HA1: VPNGTIVKTITNDQIEVTNAT (SEQ ID NO: 43)

C31_HA2: DLKSTQAAINQINGKLNRLIGK (SEQ ID NO: 44)

C32_HA2: DLKSTQAAINQINGKLNRLEGK (SEQ ID NO: 45)

C33_HA2: DLKSTQAADNQINGKLNRLIGK (SEQ ID NO: 46)

C34 HA2: DLKSTQAADNQINGKLNRLEGK (SEQ ID NO: 47)

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKSSWP NHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAYVS VVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNA (SEQ ID NO: 48)

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESS PNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQMTLYHKENAYV SVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTII FEANGNLIAPRYAFALSRGFGSGIINSNA

(SEQ ID NO: 49)

PLQLGNCSVAG ILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSW PNHTVTKGVTASCSHNGKSSFYRNLLWLTGKNGLYPNLSMSYVNNKEKEVLVLWGVHHPPNIGDQRALYHTENAY VSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTI IFEANGNLIAPRYAFALSRGFGSGIINSNA

(SEQ ID NO: 50)

PLHLGKCNIAGWILGNPECESLSTASS SYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSW PNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAY VFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFA ERNAGSGII ISDT

(SEQ ID NO: 51)

All: ESSWPNHTVTGVSASCSHNGKSSFGCG (SEQ ID NO: 52)

A12: ESSWPNHTVTGVSASCSHNGESSFGCG (SEQ ID NO: 53)

A13: ESS PNHTVTKGVTASCSHNGKSSFGCG (SEQ ID NO: 54)

A14: TSSWPNHDSNKGVTAACPHAGAKSFGCG (SEQ ID NO: 55)

All_noss: ESSWPNHTVTGVSASASHNGKSSFG (SEQ ID NO: 56)

A12_noss: ESSWPNHTVTGVSASASHNGESSFG (SEQ ID NO: 57)

A13_noss: ESSWPNHTVTKGVTASASHNGKSSFG (SEQ ID NO: 58)

A14_noss: TSSWPNHDSNKGVTAAAPHAGAKSFG (SEQ ID NO: 59)

CI: DQKSTQNAIDGITNKVNSVIEK (SEQ ID NO: 60) KANPANDLCYPGNFNDYEELKHLLSRINHFEKIQI I PKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNST YPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWT ILKPNDAINFESNGNFIAPEYAYKIVK (SEQ ID NO: 61)

KANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNST YPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWT ILKPNDAINFESNGNFIAPEYAYKIVK (SEQ ID NO: 62)

KASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPRSSWSNHDASSGVSSACPYNGRSSFFRNVVWLIKKDNA YPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWT ILKPNDAINFESNGNFIAPEYAYKIVK (SEQ ID NO: 63)

KANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQI IPKSSWSNHEASSGVSSACPYQGTPSFFRNVVWLIKKNNA YPTIKRSYNNTNQEDLLVLWGIHHPNNEAEQTRLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWT ILKPNDAINFESNGNFIAPEYAYKIVK (SEQ ID NO: 64)

KDNPVNGLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASLGVSSACPYLGRSSFFRNVVWLIKKNST YPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIKLYQNPNTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWT ILKPNDAINFESNGNFIAPENAYKIVK (SEQ ID NO: 65)

RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPAL NVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKP GDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 66)

RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRGSNNSFFSRLNWLTHSKFKYPAL NVT PNNEQFDKLYIWGVHHPSTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKP GDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 67)

RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPAL NVT PNNEQFDKLYIWGVHHPGTDNDQISLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKP GDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 68)

RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRRSNKSFFSRLNWLTQLKFKYPAL NVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKP GDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 69)

RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNKSFFSRLNWLTHSKFKYPAL NVTMPNNEQFDKLYIWGVHHPGTDNDQTFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKP GDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 70)

RSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSYACIRRSNNSFFSRLNWLTHLEFKYPAL NVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYARASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKP GDILLINSTGNLIAPRGYFKIRS (SEQ ID NO: 71)

KPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKSSWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLY PNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTL LEPGDTI IFEANGNLIAPRYAFALSR (SEQ ID NO: 72)

KPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGL YPNLSKSYANNKEKEVLVLWGVHHPPNIGDQMTLYHKENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWT LLEPGDTIIFEANGNLIAPRYAFALSR (SEQ ID NO: 73)

TPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTKGVTASCSHNGKSSFYRNLLWLTGKNG LYPNLSMSYVNNKEKEVLVLWGVHHPPNIGDQRALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYW TLLEPGDTI IFEANGNLIAPRYAFALSR (SEQ ID NO: 74) TSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSS PNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGN SYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYW TLVEPGDKITFEATGNLVVPRYAFAMER (SEQ ID NO: 75)

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVE (SEQ ID NO: 76)

PKSSWSDHEASSGVSSACPYLGSPSF (SEQ ID NO: 77)

PKSS SSHEASLGVSSACPYQGKSSF (SEQ ID NO: 78)

PRSSWSNHDASSGVSSACPYNGRSSF (SEQ ID NO: 79)

PKSSWSNHEASSGVSSACPYQGTPSF (SEQ ID NO: 80)

PKSS SDHEASLGVSSACPYLGRSSF (SEQ ID NO: 81)

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQ

(SEQ ID NO: 82)

ILDGENCTLIDALLGDPQCDGFQNKN DLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFN TGVTQ

(SEQ ID NO: 83)

NGTSSACIRRSNNSF (SEQ ID NO: 84)

NGTSSACKRGSNNSF (SEQ ID NO: 85)

NGTSSACKRRSNNSF (SEQ ID NO: 86)

NGTSSACKRRSNKSF (SEQ ID NO: 87)

NGTSSACIRRSNKSF (SEQ ID NO: 88)

NGTSYACIRRSNNSF (SEQ ID NO: 89)

PLQLGNCSVAGWILGNPECELLISKES SYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPK

(SEQ ID NO: 90)

PLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPK

(SEQ ID NO: 91)

ESSWPNHTVTGVSASCSHNGKSSF (SEQ ID NO: 92)

ESSWPNHTVTGVSASCSHNGESSF (SEQ ID NO: 93)

ESSWPNHTVTKGVTASCSHNGKSSF (SEQ ID NO: 94)

TSSWPNHDSNKGVTAACPHAGAKSF (SEQ ID NO: 95)

ELKHLLSRINHFEKIQII (SEQ ID NO: 96)

PKSSWSDHEASSGVSSA (SEQ ID NO: 97)

PKSSWSSHEASLGVSSA (SEQ ID NO: 98)

PRSSWSNHDASSGVSSA (SEQ ID NO: 99)

PKSSWSNHEASSGVSSA (SEQ ID NO: 100) PKSSWSDHEASLGVSSA (SEQ ID NO: 101)

FSRLNWL (SEQ ID NO: 102)

YRNLL LT (SEQ ID NO: 103)

YKNLIWLV (SEQ ID NO: 104)

FRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHH (SEQ ID NO: 105)

FRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHH (SEQ ID NO: 106)

FRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHH (SEQ ID NO: 107)

GVSSACPYQGKSSF (SEQ ID NO: 108)

GVSSACPYLGRSSF (SEQ ID NO: 109)

GVSSACPYNGRSSF (SEQ ID NO: 110)

GVSSACPYQGTPSF (SEQ ID NO: 111)

GVSSACPYLGSPSF (SEQ ID NO: 112)

VTMPNNEQFDKLYIWGVHH (SEQ ID NO: 113)

VTMPNNEKFDKLYIWGVHH (SEQ ID NO: 114)

AYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNA

(SEQ ID NO: 115)

AYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGI IISDT

(SEQ ID NO: 116)

YISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI KSEL

(SEQ ID NO: 117)

YISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTIMKSEL

(SEQ ID NO: 118)

RITVSTKRSQQTVIP IGSRPRVRNIPSRISIY TIVKPGDILLINSTGNLI PRGYFKIRSGKSSI RSDA

(SEQ ID NO: 119)

GKNGLYPNLS SYANNKEKEVLVLWGVHHPPNIGDQKALYHTEN (SEQ ID NO: 120)

GKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQMTLYHKEN (SEQ ID NO: 121)

GKNGLYPNLSMSYVNNKEKEVLVLWGVHHPPNIGDQRALYHTEN (SEQ ID NO: 122)

KKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNAD (SEQ ID NO: 123)

THLEFKYPALN (SEQ ID NO: 124)

THLKFKYPALN (SEQ ID NO: 125)

THSKFKYPALN (SEQ ID NO: 126) TQLKFKYPALN (SEQ ID NO: 127) TRKGIHIGPGQAWYTTGDITG (SEQ ID NO: 128)

Claims

WHAT IS CLAIMED IS:

1. An immunogenic composition comprising one or more peptides selected from the group consisting of:

rA_g51

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKS SWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 1 ) ; rA_g52

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS SWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 2) ; rAg53

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPRS SWSNHDASSGVSSACPYNGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 3) ; rAg54

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKS SWSNHEASSGVSSACPYQGTPSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNNEAEQTRLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 4), and rAg55

PLILRDCSVAGWLLGNPMCDEFINVPE SYIVEKDNPVNGLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS S SDHEASLGVSSACPYLGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIKLYQ NPNTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTI MKSEL (SEQ ID NO: 5) .

2. An immunogenic composition comprising peptides rA 51

PLILRDCSVAG LLGNPMCDEFINVPE SYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQII PKS S SDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 1 ) ; rA 52 PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS SWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 2 ) ; rAg53

PLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPRS SWSNHDASSGVSSACPYNGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVL GIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 3) ; rAg54

PLILRDCSVAG LLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRI HFEKIQI IPKS SWSNHEASSGVSSACPYQGTPSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNNEAEQTRLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEL (SEQ ID NO: 4), and rAg55

PLILRDCSVAGWLLGNPMCDEFINVPE SYIVEKDNPVNGLCYPGDFNDYEELKHLLSRINHFEKIQI IPKS SWSDHEASLGVSSACPYLGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIKLYQ NPNTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTI MKSEL (SEQ ID NO: 5) .

3. An immunogenic composition comprising a peptide library L5 having the amino acid composition:

Amino acids composition, %

P=100

K=85;R=15

S=95;N=5

S=100

W=100

S=95;P=5

D=55;N=25;S=20

H=100;

E=80;D=20 Amino acids composition, %

A=100

S=100

S=70;L=30

G=100

V=100

S=100

A=100

C=100

P=100

Y=100

Q=60;L=25;N=15

G=100

R=40;K=30;S=20;T=10

S=70;P=30

S=100

F=100

G=100

C=100

G=100

4. An immunogenic composition comprising a peptide library L5_noss having the amino acid composition:

Amino acids composition, %

P=100

K=85;R=15 Amino acids composition, %

S=95;N=5

S=100

W=100

S=95;P=5

D=55;N=25;S=20

H=100;

E=80;D=20

A=100

S=100

S=70;L=30

G=100

V=100

S=100

A=100

P=100

Y=100

Q=60;L=25;N=15

G=100

R=40;K=30;S=20;T=10

S=70;P=30

S=100

F=100

G=100

5. An immunogenic composition comprising one or more peptides selected from the group consisting of:

A51 : PKSSWSDHEASSGVSSACPYLGSPSFGCG (SEQ ID NO: 6),

A52: PKSSWSSHEASLGVSSACPYQGKSSFGCG (SEQ ID NO: 7),

A53: PRSSWSNHDASSGVSSACPYNGRSSFGCG (SEQ ID NO: 8),

A54: PKSSWSNHEASSGVSSACPYQGTPSFGCG (SEQ ID NO: 9), and

A55: PKSSWSDHEASLGVSSACPYLGRSSFGCG (SEQ ID NO: 10).

6. An immunogenic composition comprising the peptides:

A51_noss: PKSSWSDHEASSGVSSAAPYLGSPSFG (SEQ ID NO: 1 1), A52_noss: PKSSWSSHEASLGVSSAAPYQGKSSFG (SEQ ID NO: 12), A53_noss: PRSS WSNHD AS SG VS S A AP YNGRS SFG (SEQ ID NO: 13), A54_noss: PKSSWSNHEASSGVSSAAPYQGTPSFG (SEQ ID NO: 14), and A55_noss: PKSSWSDHEASLGVSSAAPYLGRSSFG (SEQ ID NO: 15).

7. The composition of any one of claims 3-6, further comprising one or more peptides selected from the group consisting of:

B51 : PNDAAEQTKLYQNPTT (SEQ ID NO: 16),

B52: PNNEAEQTRLYQNPTT (SEQ ID NO: 17),

B53: PNDAAEQIKLYQNPNT (SEQ ID NO: 18), and

C5: DKESTQKAIDGVTNKVNSIIDK (SEQ ID NO: 19).

8. An immunogenic composition comprising one or more peptides selected from the group consisting of:

rAg31

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEQFDKLYI GVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIP IGSRPRVR I PSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 20) ; rA 32

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRGSNNSFFSRLNWLTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPSTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINS GNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 21) ; rAg33

(SEQ ID NO: 22) ; rAg34

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNKSFFSRLNWLTQLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 23) ; rAg35

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNKSFFSRLNWLTHSKFKYPALNVTMPNNEQFDKLYI GVHHPGTDNDQTFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNI PSRISIY TIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 2 ) , and rAg36

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFN TG VTQNGTSYACIRRSNNSFFSRLNWLTHLEFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYARASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 25).

9. An immunogenic composition comprising peptides

rAg31

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQIFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSI RSDA

(SEQ ID NO: 20) ; rA_g32

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFN TG VTQNGTSSACKRGSNNSFFSRLN LTHSKFKYPALNVTMPNNEQFDKLYIWGVHHPSTDNDQIFLYAQASGR ITVSTKRSQQTVIP IGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 21) ; rAg33

ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEQFDKLYIWGVHHPGTDNDQISLYAQASGR ITVSTKRSQQTVIPNIGSRPRVR IPSRISIY TIVKPGDILLI STGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 22) ; rAg34

(SEQ ID NO: 23) ; rAg35

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSSACIRRSNKSFFSRLNWLTHSKFKYPALNVT PNNEQFDKLYIWGVHHPGTDNDQTFLYAQASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 24 ) , and rAg36

ILDGENCTLIDALLGDPQCDGFQNKNWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTG VTQNGTSYACIRRSNNSFFSRLNWLTHLEFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYARASGR ITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA

(SEQ ID NO: 25) .

10. An immunogenic composition comprising a peptide library L3A having the amino acid composition:

Amino acids composition, %

N=100

G=100

T=100

S=100

S=45; Y=45; F=10;

A=90; T=10 Amino acids composition, %

C=100

KRG=20

KRR=30

IRR=50

S=100

N=80; D=10; 1=10;

N=35; K=35; S=10; 1=10; H=10

S=100

F=100

G=100

C=100

G=100

1 1. An immunogenic composition comprising a peptide library L3 A noss having the amino acid composition:

I Amino acids composition, % I

N=100

G=100

T=100

S=100

S=45; Y=45; F=10;

A=90; T=10

A=100

KRG=20

KRR=30

IRR=50

S=100

N=80; D=10; 1=10;

N=35; K=35; S=10; 1=10; H=10

S=100 Amino acids composition, %

F=100

G=100

12. An immunogenic composition comprising one or more peptides selected from the group consisting of:

A31 : NGTSSACIRRSNNSFGCG (SEQ ID NO: 26),

A32: NGTSSACKRGSNNSFGCG (SEQ ID NO: 27),

A33: NGTSSACKRRSNNSFGCG (SEQ ID NO: 28),

A34: NGTSSACKRRSNKSFGCG (SEQ ID NO: 29),

A35: NGTSSACIRRSNKSFGCG (SEQ ID NO: 30), and

A36: NGTSYACIRRSN SFGCG (SEQ ID NO: 31).

13. An immunogenic composition comprising one or more peptides selected from the group consisting of:

A31_noss: NGTSSAAIRRSNNSFG (SEQ ID NO: 32),

A31_noss: NGTSSAAKRGSNNSFG (SEQ ID NO: 33),

A31_noss: NGTSSAAKRRSNNSFG (SEQ ID NO: 34),

A31_noss: NGTSSAAKRRSNKSFG (SEQ ID NO: 35),

A31_noss: NGTSSAAIRRSNKSFG (SEQ ID NO: 36), and

A31_noss: NGTSYAAIRRSNNSFG (SEQ ID NO: 37).

14. The composition of any one of claims 10-13, further comprising one or more peptides selected from the group consisting of:

B31 : PGTDNDQIFLYAQASG (SEQ ID NO: 38), B32: PGTDNDQIFLYARASG (SEQ ID NO: 39),

B33: PGTDNDQISLYAQASG (SEQ ID NO: 40),

B34: PGTDNDQTFLYAQASG (SEQ ID NO: 41), and

B35: PSTDNDQIFLYAQASG (SEQ ID NO: 42).

15. The composition of any one of claims 10-13, further comprising the peptide library L3B having the amino acid composition:

Amino acids composition, %

P=100

S=45; G=45; V=10

T=100

D=90; Y=10

N=33; S=33; K=33

D=100

Q=100

l=50; T=50

F=50; S=50

L=100

Y=100

A=50; V=50

Q=50; R=50

A=100

S=90; P=10

G=100

16. The composition of claim 14, further comprising one or more peptides selected from the group consisting of

C3_HA1 : VPNGTIVKTITNDQIEVTNAT (SEQ ID NO: 43), C31_HA2: DL STQAAINQINGKLNRLIGK (SEQ ID NO: 44),

C32 HA2: DLKSTQAAINQINGKLNRLEGK (SEQ ID NO: 45),

C33_HA2: DLKSTQAADNQINGKLNRLIGK (SEQ ID NO: 46), and

C34 HA2: DLKSTQAADNQINGKLNRLEGK (SEQ ID NO: 47).

17. The composition of claim 15, further comprising one or more peptides selected from the group consisting of

C3_HA1 : VPNGTIVKTITNDQIEVTNAT (SEQ ID NO: 43),

C31_HA2: DLKSTQAAINQINGKLNRLIGK (SEQ ID NO: 44),

C32 HA2: DLKSTQAAINQINGKLNRLEGK (SEQ ID NO: 45),

C33 HA2: DLKSTQAADNQINGKLNRLIGK (SEQ ID NO: 46), and

C34 HA2: DLKSTQAADNQINGKLNRLEGK (SEQ ID NO: 47).

18. An immunogenic composition comprising one or more peptides selected from the group consisting of:

rAgll:

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKS SWPNHTVTGVSASCSHNGESSFYRNLL LTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHT ENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTI IFEANGNLIAPRYAFALSRGFGSGII NSNA (SEQ ID NO: 48) ; rAgl2:

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKE SSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQMTLYH KENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYY TLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGI INSNA (SEQ ID NO: 49) ; rAgl3:

PLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKE SS PNHTVTKGVTASCSHNGKSSFYRNLLWLTGKNGLYPNLSMSYVNNKEKEVLVLWGVHHPPNIGDQRALY HTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSG IINSNA (SEQ ID NO: 50), and rAgl4:

19. An immunogenic composition comprising peptides

rAgll:

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKS SWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHT ENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTI IFEANGNLIAPRYAFALSRGFGSGII NSNA (SEQ ID NO: 48) ; rAgl2:

PLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKE SSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVL GVHHPPNIGDQMTLYH KENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYY TLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGI INSNA (SEQ ID NO: 49) ; rAgl3:

PLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKE SSWPNHTVTKGVTASCSHNGKSSFYRNLLWLTGKNGLYPNLSMSYVNNKEKEVLVLWGVHHPPNIGDQRALY HTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSG IINSNA (SEQ ID NO: 50), and rAgl4:

PLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKT SS PNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLY QNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSG IIISDT (SEQ ID NO: 51) .

20. An immunogenic composition comprising one or more peptides selected from the group consisting of:

Al 1 : ESSWPNHTVTGVSASCSH GKSSFGCG (SEQ ID NO: 52),

A12: ESSWPNHTVTGVSASCSHNGESSFGCG (SEQ ID NO: 53),

A13: ES S WPNHT VTKG VT ASCSHNGKS SFGCG (SEQ ID NO: 54), and

A14: TSSWPNHDSNKGVTAACPHAGAKSFGCG (SEQ ID NO: 55).

21. An immunogenic composition comprising one or more peptides selected from the group consisting of:

Al l noss: ESSWPNHTVTGVSASASHNGKSSFG (SEQ ID NO: 56),

A12_noss: ES S WPNHT VTG VS AS ASHNGES SFG (SEQ ID NO: 57),

A13_noss: ESSWPNHTVTKGVTASASHNGKSSFG (SEQ ID NO: 58), and

A14_noss: TSSWPNHDSNKGVTAAAPHAGAKSFG (SEQ ID NO: 59).

22. The composition of any one of claims 18-21, further comprising the peptide DQKSTQNAIDGITNKVNSVIEK (CI) (SEQ ID NO: 60).

23. The composition of any one of claims 1-6, 8-13, and 18-21, wherein one or more peptides contain disulfide bonds.

24. The composition of claim 23, wherein all peptides contain disulfide bonds.

25. The composition of any one of claims 1-6, 8-13, and 18-21, wherein none of the peptides contain disulfide bonds.

26. The composition of claim 7, wherein one or more peptides contain disulfide bonds.

27. The composition of claim 26, wherein all peptides contain disulfide bonds.

28. The composition of claim 7, wherein none of the peptides contain disulfide bonds.

29. The composition of claim 14, wherein one or more peptides contain disulfide bonds.

30. The composition of claim 29, wherein all peptides contain disulfide bonds.

31. The composition of claim 14, wherein none of the peptides contain disulfide bonds.

32. The composition of claim 15, wherein one or more peptides contain disulfide bonds.

33. The composition of claim 32, wherein all peptides contain disulfide bonds.

34. The composition of claim 15, wherein none of the peptides contain disulfide bonds.

35. The composition of claim 16, wherein one or more peptides contain disulfide bonds.

36. The composition of claim 35, wherein all peptides contain disulfide bonds.

37. The composition of claim 16, wherein none of the peptides contain disulfide bonds.

38. The composition of claim 17, wherein one or more peptides contain disulfide bonds.

39. The composition of claim 38, wherein all peptides contain disulfide bonds.

40. The composition of claim 17, wherein none of the peptides contain disulfide bonds.

41. The composition of claim 22, wherein one or more peptides contain disulfide bonds.

42. The composition of claim 41, wherein all peptides contain disulfide bonds.

43. The composition of claim 22, wherein none of the peptides contain disulfide bonds.

44. The composition of any one of claims 1-6, 8-13, and 18-21, which is a vaccine composition.

45. The vaccine composition of claim 44 further comprising an adjuvant.

46. A method of inducing a protective immune response to an influenza infection in a mammal, said method comprising administering to said mammal the vaccine composition of claim 44.

47. The method of claim 46, wherein said mammal is human.

48. The method of claim 46, wherein said vaccine composition is administered by a route selected from the group consisting of mucosal, subcutaneous (s.c), intramuscular (i.m.), intradermal (i.d.), and oral administration.

49. The method of claim 46, wherein said vaccine composition is administered mucosally.

50. The method of claim 46, wherein said vaccine composition is administered intranasally.

51. The composition of claim 7 which is a vaccine composition.

52. The vaccine composition of claim 51 further comprising an adjuvant.

53. A method of inducing a protective immune response to an influenza infection in a mammal, said method comprising administering to said mammal the vaccine composition of claim 51.

54. The method of claim 53, wherein said mammal is human.

55. The method of claim 53, wherein said vaccine composition is administered by a route selected from the group consisting of mucosal, subcutaneous (s.c), intramuscular (i.m.), intradermal (i.d.), and oral administration.

56. The method of claim 53, wherein said vaccine composition is administered mucosally.

57. The method of claim 53, wherein said vaccine composition is administered intranasally.

58. The composition of claim 14 which is a vaccine composition.

59. The vaccine composition of claim 58 further comprising an adjuvant.

60. A method of inducing a protective immune response to an influenza infection in a mammal, said method comprising administering to said mammal the vaccine composition of claim 58.

61. The method of claim 60, wherein said mammal is human.

62. The method of claim 60, wherein said vaccine composition is administered by a route selected from the group consisting of mucosal, subcutaneous (s.c), intramuscular (i.m.), intradermal (i.d.), and oral administration.

63. The method of claim 60, wherein said vaccine composition is administered mucosally.

64. The method of claim 60, wherein said vaccine composition is administered intranasally.

65. The composition of claim 15 which is a vaccine composition.

66. The vaccine composition of claim 65 further comprising an adjuvant.

67. A method of inducing a protective immune response to an influenza infection in a mammal, said method comprising administering to said mammal the vaccine composition of claim 65.

68. The method of claim 67, wherein said mammal is human.

69. The method of claim 67, wherein said vaccine composition is administered by a route selected from the group consisting of mucosal, subcutaneous (s.c), intramuscular (i.m.), intradermal (i.d.), and oral administration.

70. The method of claim 67, wherein said vaccine composition is administered mucosally.

71. The method of claim 67, wherein said vaccine composition is administered intranasally.

72. The composition of claim 16 which is a vaccine composition.

73. The vaccine composition of claim 72 further comprising an adjuvant.

74. A method of inducing a protective immune response to an influenza infection in a mammal, said method comprising administering to said mammal the vaccine composition of claim 72.

75. The method of claim 74, wherein said mammal is human.

76. The method of claim 74, wherein said vaccine composition is administered by a route selected from the group consisting of mucosal, subcutaneous (s.c), intramuscular (i.m.), intradermal (i.d.), and oral administration.

77. The method of claim 74, wherein said vaccine composition is administered mucosally.

78. The method of claim 74, wherein said vaccine composition is administered intranasally.

79. The composition of claim 17 which is a vaccine composition.

80. The vaccine composition of claim 79 further comprising an adjuvant.

81. A method of inducing a protective immune response to an influenza infection in a mammal, said method comprising administering to said mammal the vaccine composition of claim 79.

82. The method of claim 81, wherein said mammal is human.

83. The method of claim 81 , wherein said vaccine composition is administered by a route selected from the group consisting of mucosal, subcutaneous (s.c), intramuscular (i.m.), intradermal (i.d.), and oral administration.

84. The method of claim 81 , wherein said vaccine composition is administered mucosally.

85. The method of claim 81 , wherein said vaccine composition is administered intranasally.

86. The composition of claim 22 which is a vaccine composition.

87. The vaccine composition of claim 86 further comprising an adjuvant.

88. A method of inducing a protective immune response to an influenza infection in a mammal, said method comprising administering to said mammal the vaccine composition of claim 86.

89. The method of claim 88, wherein said mammal is human.

90. The method of claim 88, wherein said vaccine composition is administered by a route selected from the group consisting of mucosal, subcutaneous (s.c), intramuscular (i.m.), intradermal (i.d.), and oral administration.

91. The method of claim 88, wherein said vaccine composition is administered mucosally.

92. The method of claim 88, wherein said vaccine composition is administered intranasally.