WO1994009035A1

WO1994009035A1 - New influenza virus peptides, diagnostics and vaccines

Info

Publication number: WO1994009035A1
Application number: PCT/SE1993/000818
Authority: WO
Inventors: Jonas Blomberg; Rüdiger Pipkorn; Sergei Viazov; Elena Isaeva
Original assignee: Replico Medical Ab
Priority date: 1992-10-09
Filing date: 1993-10-11
Publication date: 1994-04-28
Also published as: SE9202968L; AU5289193A; SE9202968D0

Abstract

Provided are: Peptides, diagnostic antigens containing said peptides, antibodies against said peptides, a method of antibody purification, immunoassay methods, a vaccine composition containing said peptides and medicaments containing said peptides or antibodies against said peptides. The vaccine can protect humans and animals against infection caused by influenza A, B and C viruses. The immunoassay methods permit detection of influenza virus particles or viral antigens or antibodies to these antigens or disease caused by influenza viruses A, B and C. The medicaments can inhibit the reproduction of influenza viruses in infected humans and animals and modulate the course of influenza virus infection. The peptides are selected from the 7th segment of influenza A and B and the 6th segment of influenza C: (Influenza A virus) X-PHXLXXXFRRRPSFXTVFKXXXXXFEXKXIKNPQYXVQXXNDXXXXXXRXT-Z; (Influenza B virus) X-KKQIEAPISAFCISVRHLLFFIHSKAESRSNSFPPNQQCNFSASSALPSPSSV(23)ESR-Z; (Influenza C virus) X-PDIRGRCSFLISLAADLQADFPPVIAEIIAVLVSGATFFRNASVSAISISCAIV-Z (Influenza C virus) X-ISPHHLSHLPSTKPLTSSIMIFTNKYNIVIIPNPKEANVSMYPWVSLKQ-Z; and shorter variants thereof with at least 7 amino acids.

Description

NEW INFLUENZA VIRUS PEPTIDES, DIAGNOSTICS AND VACCINES.

The present invention relates to new peptides, to a dia¬ gnostic antigen, to the use of said diagnostic antigen, to vaccines, and to medicaments based on the peptides or anti¬ bodies to them. BACKGROUND ART 1.Influenza virus vaccines.

Currently available influenza virus vaccines, both live and inactivated, can significantly reduce the risk of influenza virus infection. The great handicap of these vaccines is that they do not provide complete protection, especially when the vaccine in use is a poor antigenic match with epidemic virus¬ es (Murphy B.R. and Webster R.G. in: Virology, Ed. by Fields B.N. et al.. Raven Press N.Y. ,1990,1091-1152; Glezen W.P. and Couch R.B. in: Viral infections of humans, Ed. by Evans A.S., Plenum Press, N.Y., 1991, 419-449). Thus new vaccines are needed that should stimulate a response that is both longer lasting and broader so as to provide protection against a spectrum of antigenic variants of the circulating influenza viruses.

2. Specific therapy of influenza.

Amantadine and rimantadine are partly effective in prophy¬ laxis and to a lesser extent in therapy against influenza A virus strains but not against influenza B and C viruses (Evans A.S. in:Viral infections of humans, Ed.by Evans A.S., Plenum Press, N.Y. ,1991.3-50; Zlydnikov D.M. et al., Rev.In-

SUBSTITUTE SHEET fect.Dis., 1981,3:408-421). Preliminary data have shown some therapeutic effect of ribavirin administered by aerosol (Gil¬ bert B.E., Knight V., Antimicrob. Agents Chemoter. , 1986, 30:201-205). The relatively low effectiveness of these drugs, the occurrence of severe side effects and the data on the appearance of drug-resistant viral strains (Murphy B.R. and Webster R.G. in: Virology, Ed. by Fields B.N. et all.,Raven Press, N.Y., 1990,10911152; Belshe R.B. et al. , J.Virol., 1988,62:1S08-1512) reflect the urgent need of further pro¬ gress in this field. One possible approach to solve this problem could be the use of analogs of conserved viral pro¬ teins, peptides derived from them or antibodies to them as the therapeutic medicament.

3. Diagnosis of influenza infection.

Virologic diagnosis of influenza virus infection is based on the demonstration of virus or its antigens in respiratory secretions or on the registration of a rise in influenza antibody activity between paired sera. Some of the existing serologic methods (including ELISA) are rather sensitive but neither of them is sensitive or informative enough to satisfy the practical demands. One reason for that could be the wrong choice of target antigens; antigen(s) of broader specificity would be desirable.

4. Evidence for new influenza proteins.

In the 7th segment of virion RNA (i.e. of negative polarity) of all influenza A and B viruses sequenced so far and in the 6th segment of virion RNA of influenza C virus there is a

SUBSTITUTE SHEET small open reading frame (ORF). In influenza A virus RNA this ORF encodes a protein consisting of 68 amino acids. In in¬ fluenza B virus=RNA such an ORF encodes a protein consisting of 69 amino acids. In influenza C virus RNA there are two open reading frames coding for proteins of 83 and 95 amino acids. The sequences encoded by these ORF in influenza A,B and C viruses seem to be very conserved and include few amino acid changes. The proteins encoded by these open reading frames are the subject of the present invention.

Benefits of the present invention.

A vaccine based on a peptide or a combination of peptides derived from open reading frames found, respectively, in the 7th segments of virion RNA (i.e. of negative polarity) of influenza A and B viruses and in the 6th segment of virion RNA of influenza C viruses, comprises a system of well con¬ served amino acid sequences present in a majority, if not all, of influenza virus strains. This combination of "universal" sequences represents a novel type of influenza vaccine. This vaccine may be capable , in contrast to current vaccines, of inducing a broad immune response in immunized subjects thus providing protection against a spectrum of influenza viruses. Such an effective vaccine can be widely used, first of all for immunization of high risk groups including adults and children with chronic disorders of the cardiovascular or pulmonary systems and persons confined to nursing homes and other chronic-care facilities; such a vac¬ cine can also be used for immunisation of other vulnerable

SUBSTITUTE SHEET segments of population such as school children, children in day care, college students, military personnel. It is quite possible that to achieve control of epidemic influenza this vaccine may have to be administered to a larger number of persons of general population. Thus, practically everyone could benefit from the vaccine. The peptides can also be used to augment already existing influenza virus vaccines. Antibodies to whole proteins or to parts of the proteins encoded by open reading frames present, respectively in the 7th segments of virion RNA of influenza A and B viruses and in the 6th segment of virion RNA of influenza C viruses, may selectively attack infected cells which express corresponding influenza viral proteins; these antibodies may also block viral replication. Such an antibody then may be an efficient therapeutic or prophylactic antiinfluenza medicament. One or a combination of several peptides, or analogs thereof, encoded by open reading frames present respectively in the 7th or 6th segments of virion RNA of influenza A, B and C viruses, may inhibit the influenza virus reproduction by blocking specific receptors; such peptides or their analogs then may be efficient antiinfluenza medicaments. The peptides or their analogs can be used as diagnostic antigens to detect specific antibodies in human or animal sera, which is of diagnostic and prognostic value in influ¬ enza infection and disease. Of diagnostic value are also antibodies to such amino acid sequences; such antibodies can be used in immunoassays (ELISA, immunofluorescence, etc) to detect viral antigens in respiratory secretions.

SUBSTITUTE SHEET DESCRIPTION OF THE INVENTION

Throughout this document, the internationally accepted single letter code for amino acids is used.

In one aspect of the invention, there are provided peptides derived from proteins encoded by open reading frames present in the 7th segment of virion RNA of influenza A and B viruses and in the 6th segment of virion RNA of influenza C viruses characterised by the following amino acid sequences, where at least 90% of the amino acids are those indicated below:

( Influenza A virus)

X-PH(1)L(2) (3)(4)FRRRPSF(5)TVFK(6)(7)(8) (9) (10)FE(11)K(12)I

KNPQY(13)VQ(14) (15)ND(16) (17) (18) (19) (20) (21)R(22)T-Z

( Influenza B virus)

X-KKQIEAPISAFCISVRHLLFFIHSKAESRSNSFPPNQQCNFSASSALPSPSSV(23) ESR-Z;

( Influenza C virus)

X-PDIRGRCSFLISLAADLQADFPPVIAEIIAVLVSGATFFRNASVSAISISCAIV-Z;

( Influenza C virus)

X-ISPHHLSHLPSTKPLTSSIMIFTNKYNIVIIPNPKEANVSMYPWVSLKQ-Z;

and shorter variants thereof which can be down to 7 amino acids long, and functional or structural molecular mimetics thereof some of which may block binding of said proteins or peptides to their receptors, where 1 denotes R or G, 2 W or R, 3 H,R or Y, 4 S or P, 5 Q or H, 6 A or I, 7 A,T or S, 8 I or V, 9 D or

SUBSTITUTE SHEET N, 10 A or T, 11 K or I, 12 M or T, 13 K or Q, 14 D or N, 15 P or S, 16 A, I or T, 17 C or R, 18 C, G or S, 19 D, N or S, 20 D or N, 21 :£ or K, 22 I or V, 23 N or S, and X can be hydrogen or binding-facilitating and spacing amino acids or other atoms, and Z is NH2 or OH or binding-facilitating and spacing amino acids or other atoms with this function. In a further aspect of the invention there is provided a method for diagnosis of influenza virus infection , with the aid of a sample of body fluid, wherein said sample is sub¬ jected to an immunoassay using as diagnostic antigen at least one peptide chosen from said peptides or structural molecular mimetics thereof, and in case said sample of body fluid contains antibodies which bind to said diagnostic antigen(s), the patient is likely to have or have had influenza virus infection. The sample of body fluid can be any hody fluid having a detectable amount of antibodies which bind to the above defined antigen(s).

In a preferred embodiment of this aspect of the invention the immunoassay is enzyme linked immunoassay (ELISA). In another aspect of the invention there is provided an antibody reacting with at least one peptide chosen from said peptide or structural molecular mimetics thereof. In an embodiment of this aspect of the invention said anti¬ body is a monoclonal antibody. The antibodies according to the invention are optionally coupled to a carrier. Examples of suitable carriers are lysine octa ers, human or bovine serum albumin, plastic or glass surfaces, latex- or gelatin particles.

6 SUBSTITUTE SHEET In another embodiment of this aspect of the invention said antibody is a genetically engineered antibody-like molecule having binding affinity to said peptides or their mimetics. These molecules are optionally coupled to a carrier. Examples of suitable carriers are lysine octamers, human or bovine serum albumin, plastic or glass surfaces, latex- or gelatin particles.

In still another aspect of the invention there is provided a method of isolating antibodies reacting with at least one peptide chosen from a combination of said peptides or structural molecular mimetics thereof, from blood samples of humans or animals, wherein said samples are subjected to affinity adsorption and desorption or affinity chromatogra- phy, in a per se known manner. The blood samples can be whole blood, serum or plasma.

In a further aspect of invention there is provided a method of for diagnosis of influenza virus infection, with the aid of a sample of body fluid or tissue, wherein said sample is subjected to an immunoassay using antibodies to said pep- tide(s) or structural mimetics thereof, and in case said sample contains antigens which bind to said antibodies, the patient is likely to have influenza virus infection. In yet another aspect of the invention there is provided a medicament for prophylactic or therapeutic use in the preven¬ tion or treatment of an influenza virus infection, in which the active principle of said medicament comprises antibodies or other affinity molecules reacting with at least one pep¬ tide chosen from a combination of said peptides or structural

7 SUBSTITUTE SHEET molecular mimetics thereof.

In one embodiment of this aspect of the invention the anti¬ bodies are polyclonal antibodies derived from blood samples of humans or apes, and in another embodiment said antibodies are monoclonal antibodies.

In a further embodiment of this aspect of the invention said antibodies are genetically engineered antibody-like molecules having binding affinity to said peptides or their mimetics. These affinity molecules are optionally coupled to a carrier. Examples of suitable carriers are lyεine octamers, human or bovine serum albumin, plastic or glass surfaces, latex or gelatin particles.

In a further aspect of the invention there is provided a vaccine composition which comprises as an immunizing com¬ ponent one or several peptides chosen from said peptides or structural molecular mimetics thereof.

In one embodiment of this aspect of the invention the immuni¬ zing component is present in an amount effective to protect a subject from influenza virus infection.

In another embodiment of this aspect of the invention the immunizing component may be used alone or in combination with other known immunogenic molecules, e.g. current influenza virus preparations based on whole virus particles or split vaccines.

8 SUBSTITUTE SHEET In yet another embodiment of this aspect of the invention an adjuvant is included in an amount, which together with an amount of said immunizing component, is effective to protect a subject from influenza virus infection.

In yet another aspect of the invention, a medicament con¬ taining as active principle at least one peptide chosen from a combination of said peptides or structural molecular mime¬ tics thereof some of which may block binding of said proteins or peptides to their receptors, is provided, said medicament having the property of modulation of influenza virus in¬ fection in human beings or animals, in order to weaken or abolish the symptoms of the influenza virus infection.

SUBSTITUTESHEET EXPERIMENTAL

Summary

Peptides corresponding to part of the amino acid sequences encoded by the open reading frame present in the 7th segment of virion RNA of two influenza A viruses - A/FW/1/50 (H1N1) and A/Singapore/1/57 (H2N2) - were synthesized. These pep¬ tides when used as an antigen in solid-phase ELISA, reacted with: (1) rabbit antisera to purified influenza A viruses; (2) convalescent sera of influenza patients; (3) sera of healthy blood donors who have antibodies to hemagglutinins of influenza A viruses. These synthetic peptides were also used for the immunization of rabbits. In solid-phase ELISA such rabbit anti-peptide sera demonstrated positive reactions with purified particles of influenza A viruses. These anti-peptide sera were able to inhibit the reproduction of influenza A viruses in Madin Darby canine kidney (MDCK) cells. Mice immunized with the synthetic peptides were protected against challenge with virulent mice-adapted influenza viruses.

Description

Two peptides corresponding to fragments of proteins encoded by an open reading frame present in the 7th segment of virion RNA of two influenza A viruses - A/FW/1/50 (H1N1) and A/Singapore/1/57 (H2N2) were synthesized in the octameric form (Tam J.P.,Methods Enzymol. ,1989,168:7-15) . These pep¬ tides have been designated P1359 and P1360 (Table 1). Three other synthetic peptides - P1358, P3 and P527- were used in this study as the control ones (Table 1). P1358 and P3 cor-

10 SUBSTITUTE SHEET respond respectively to fragments of HA2 proteins of influ¬ enza A viruses belonging to H3 and H2 subtypes. The third control peptide (P527) corresponds to part of the sequence of the GP41 protein of HIV1 (Ratner L. et al.,Nature, 1985,313:277-284).

All synthesized peptides were used as antigens in solid phase ELISA. Table 2 demonstrates the reaction of these peptides with the rabbit antisera to purified preparations of differ¬ ent influenza A viruses. The majority of such rabbit antisera reacted with P1359 and P1360, but not with P527. The peptides P1358 and P3 reacted only with some antisera to homologous influenza viruses.

In parallel all peptides were sorbed on solid-phase and then tested in ELISA with paired sera of influenza patients (Table 3). Antibodies to P1359 and P1360 were present in all tested human sera; moreover, in many cases a rise in antibody titers to P1359 and P1360 was registered. Reactions with control peptides were negative (P527) or sometimes positive (P1358 and P3) .

In a subsequent series of experiments all peptides were adsorbed on solid phase and tested in ELISA against sera from healthy blood donors (Table 4). Peptides P1359 and P1360 reacted with most of donor's sera, which contained antibodies to influenza A viruses (HlNl and H3N2 ) . Control peptides did not react with these sera at all (P527) or only with a few of them (P1358 and P3) .

Peptides P1358, P1359 and P1360 were used to immunize rab¬ bits; because these peptides were synthesized in octameric

11

SUBSTITUTESHEET form no adjuvant was used in the immunization protocol. These anti-peptide sera were tested for the ability to react with purified preparations of some influenza A viruses (Table 5). Antisera to P1359 and to P1360 reacted not only with most of homologous viruses but also with some of heterologous virus strains.

The rabbit anti-peptide sera were used in a series of neutra¬ lization experiments in vitro. Test-viruses belonging to HlNl and H3N2 subtypes were incubated with different dilutions of anti-peptide or control sera and then used for the inocu¬ lation of Madin Darby kidney (MDCK) cells. Two methods were used for the virus titration: (1) quantitative antigen ana¬ lysis of viral antigens by ELISA and (2) plaque forming assay.

Table 6 demonstrates the ELISA data. Antisera to P1359 caused a significant titer reduction of HlNl influenza viruses and a less marked inhibition of H3N2 viruses. On the other hand, antibodies to P1360 only slightly interfered with the re¬ production of HlNl influenza viruses but caused a distinct reduction of titers of H3N2 viruses, especially of A/Bang- kok/1/79 virus. Neither normal rabbit serum nor antiserum to P1358 inhibited the reproduction of any tested viruses.On the other hand, homologous but not heterologous antisera to puri¬ fied influenza viruses caused a significant reduction of virus titers.

The influence of anti-peptide sera on the reproduction of the same test-viruses was also measured by plaque assay (Table 7). The results obtained went in parallel with those presen-

12 SUBSTITUTE SHEET ted in Table 6, namely antisera to P1359 and P1360 caused a significant titer reduction of homologous viruses and a less marked inhibition of reproduction of heterologous virus strains. Thus, antisera to peptides P1359 and P1360 caused a significant inhibition of reproduction of influenza A viruses in vitro.

The next set of experiments was conducted in order to estab¬ lish the protective potential of peptides P1359 and P1360 against infectious influenza viruses in vivo. Groups of out- bred mice were immunized with different peptides, according to the following protocol of immunization: The first inocu¬ lation was with 30 ug of peptide per animal, intraperi- toneally, with complete Freund's adjuvant; the second in¬ oculation took place after 3 weeks, when 30 ug of peptide per animal were introduced intravenously. 10 days after the se¬ cond inoculation some of the mice were bleeded and tested by ELISA for the presence of anti-peptide antibodies (Table 8). The other animals were infected intranasally with mice-adapt¬ ed virulent variants of A/PR8/34 and A/Aichi/2/68 viruses. In preliminary experiments we established a dose of the virus which caused the death of approximately 50-70% of infected mice. 3 days after challenge 5 mice from the each group were anaesthetized with ether and lung extracts were prepared by homogenization. Table 9 demonstrates the virus titers in the lung extracts of these animals. The results showed that in comparison with control peptides, P1359 and P1360 caused a significant reduction of virus titers in lungs of all chal-

13 SUBSTITUTE SHEET lenged animals. The peptide P1359 inhibited the reproduction of A/PR8/34 virus more than the A/Aichi/2/68 strain. On the contrary, P1360 was able to cause significant reduction of titers of both test-viruses.

The other challenged animals (10 mice in each group) were observed for 30 days in order to estimate the possible in¬ fluence of the peptides on the mortality. The results of this experiment are presented in Table 10. In comparison with control peptides, P1359 and P1360 were able to dramatically increase the survival rates of challenged animals. P1359 caused a 75% protection (i.e. 8/10 survived compared to 2/10 in the control group) against challenge with a highly viru¬ lent variant of A/PR8/34 virus and P1360 provide protection of 87.5% and 71.4% of mice against infection caused by A/PR8/34 and A/Aichi/2/68 viruses, respectively. Thus, the infection of mice with highly virulent influenza viruses could be significantly influenced by immunization with pep¬ tides bearing the sequences encoded by an open reading frame present in the 7th segment of virion RNA of influenza virus¬ es.

14

SUBSTITUTE SHEET TABLE 1. AMINO ACID SEQUENCES OF THE INVESTIGATED PEPTIDES

PEPTIDE SEQUENCE

P1359 FEKKTIKNPQYQVQDPNDTCGNNKRIT

P1360 FEKKTIKNPQYQVQDPNDTRGNNKRVT

P1358 VEGRIQDLEKYVEDT

P3 CYMENERTLDLH

P527 LQARILAVERYLKDQQL

15

SUBSTITUTE SHEET TABLE 2. REACTION OF SYNTHETIC PEPTIDES WITH RABBIT ANTISERA TO PURIFIED INFLUENZA VIRUSES.

Antiserum to Antibodies* to

*) Sera were considered positive if their titer was higher than 1:2 by ELISA (S/N>= 2.5); **) Reciprocal titer

16 SUBSTITUTE SHEET TABLE 3. REACTION OF SYNTHETIC PEPTIDES WITH PAIRED SERA OF INFLUENZA PATIENTS

*) Sera were considered positive if their titer was higher than 1:200 by ELISA (S/N>=2.5);

**) 1 - First of paired serum (day 2-3 of the disease): ***) 2 - Second of paired serum (day 10-16 of the disease); ****) Reciprocal titer

17

SUBSTITUTE SHEET TABLE 4. REACTION OF SYNTHETIC PEPTIDES WITH SERA OF BLOOD DONORS

Donor Antibodies* to

HlNl H3N2 influenza influenza virus virus P1359 P1360 P1358 P3 P527

* ) Sera were considered positive if their titer was higher than 1: 200 by ELISA (S/N >= 2.5); ** ) Reciprocal titer

18

SUBSTITUTE SHEET TABLE 5. REACTION OF RABBIT ANTISERA TO SYNTHETIC PEPTIDES WITH INFLUENZA A VIRUSES.

Antigens Antisera* to

*) Sera were considered positive if their titer was higher than 1:200 by ELISA (S/N>= 2.5); **) reciprocal titer

19

SUBSTITUTE SHEET TABLE 6. NEUTRALIZATION OF INFLUENZA A VIRUSES BY RABBIT ANTI¬ SERA TO SYNTHETIC PEPTIDES IN MDCK CELL CULTURE (ELISA)

*) Two observations per dilution were made. **) m.o.i.-lOO TCID50/cell

20

SUBSTIT'ITE SHEET TABLE 7. NEUTRALIZATION OF INFLUENZA A VIRUSES BY RABBIT ANTISERA TO SYNTHETIC PEPTIDES IN MDCK CELL CULTURES (PFU METHOD) .

Rabbit Dilution Index of neutralization (in loglO/ml)* anti of serum anti- to serum

HlNl H3N2 A/PR8/34 A/USSR/90/77 A/AICHI/2/68 A/BANGKOK/ 1/79

100 3.5/4.5** 2.5/3.5 200 2.0/3.0 2.0/2.0

P1359 400 1.5/2.0 1.0/1.0 800 < <

100 2.5/3.0 2.0/2.5 200 1.5/2.5 1.0/2.0

P1360 400 < < 800 < <

100 < < 200 < <

P1378 400 < < 800 < <

100 6.0/6.0 <

200 6.0/6.0 <

A/PR8/ 400 6.0/6.0 <

34 800 5.0/5.0 <

100 2.0/3.0 <

200 1.0/2.0 <

A/USSR/ 400 < 1.0

90/77 800 <

100 < <

200 < <

A/AICHI 400 < <

/2/68 800 < <

100 < <

200 < <

A/BANG- 400 < <

KOK/1/ 800 < <

79

*) Indexes : 1.0 loglO/ml are shown;

**) Two virus doses were used: Numerator m.o.i. =0.01 pfu/cell; denominator n =0.001 pfu/cell; in the experiments with antibodies to whole virus only one dose (m.o.i.=0.001 pfu/ml) was used.

21

SUBSTITUTE SHEET .

Peptide No of Antibody titer Mean animals towards the geometric immunizing titer peptide

278 + 6.1

308 + 5.1

172 + 5.6

82 + 3.9

284 + 5.2

22

SUBSTITUTE SHEET . SYNTHETIC PEPTIDES.

Antigen Animal Virus titer

A/PR8/34 A/AICHI/2/68

1 2

P1359 3

4 5

1 2

P1360 3 4 5

1 2

P1358 3 4 5

1 2

P3 3 4 5

1 2

P527 3 4 5

1 2

Un- 3 immu 4 nized 5

mice

23

SUBSTITUTE SHEET TABLE 10. PROTECTION OF MICE FROM VIRUS CHALLENGE FOLLOWING IMMUNIZATION WITH SYNTHETIC PEPTIDES

Immuno Challenge with Challenge with gen A/PR8/34 A/Aichi/2/68

9 87.5 <0.01

8 75.0 <0.01

5 37.5 >

3 12.5 >

3 12.5 >

*) Fisher exact test

24

SUBSTITUTE SHEE I

Claims

1. Peptides derived from proteins encoded by open readin frames present in the 7th segment of virion RNA of influenz A and B viruses and in the 6th segment of virion RNA o influenza C viruses characterised by the following amino aci sequences, where at le st 90% of the amino acids are thos indicated below:

( Influenza A virus)

X-PH(1)L(2)(3)(4)FRRRPSF(5)TVFK(6)(7)(8)(9)(10)FE(11)K(12)I

KNPQY(13)VQ(14)(15)ND(16)(17)(18)(19)(20)(21)R(22)T-Z

( Influenza B virus)

X-KKQIEAPISAFCISVRHLLFFIHSKAESRSNSFPPNQQCNFSASSALPSPSSV(23) ESR-Z;

( Influenza C virus) X-PDIRGRCSFLISLAADLQADFPPVIAEIIAVLVSGATFFRNASVSAISISCAIV-Z;

( Influenza C virus)

X-ISPHHLSHLPSTKPLTSSIMIFTNKYNIVIIPNPKEANVSMYPWVSLKQ-Z;

and shorter variants thereof which can be down to 7 amin acids long, and functional or structural molecular mimetics thereof som of which may block binding of said proteins or peptides t their receptors, where 1 denotes or G, 2 W or R, 3 H,R or Y, 4 S or P, 5 or H, 6 A or I, 7 A,T or S, 8 I or V, 9 D or N, 10 A or T, 1

K or I , 12 M or T, 13 K or Q, 14 D or N, 15 P or S , 16 A,

25 SUBSTITUTE SHEET or T, 17 C or R, 18 C, G or S, 19 D, N or S, 20 D or N, 21 E or K, 22 I or V, 23 N or S, and X can be hydrogen or binding- facilitating and spacing amino acids or other atoms, and Z is NH2 or OH or binding-facilitating and spacing amino acids or other atoms with this function.

2. An antigen for use in the diagnosis of influenza virus infection characterised in that it contains one or a combi¬ nation of several peptides or their mimetics chosen from the group of peptides mentioned in claim 1.

3. A method for diagnosis of influenza virus infection char¬ acterised in that a sample of body fluid is subjected to an immunoassay using a diagnostic antigen according to claim 2, and in case said sample of body fluid contains antibodies which bind to said diagnostic antigen(s), the patient is likely to have or have had influenza virus infection. The sample of body fluid can be any body fluid having a detect¬ able amount of antibodies which bind to the above defined antigen(s) .

In a preferred embodiment of this aspect of the invention the immunoassay is enzyme linked immunoassay (ELISA).

4. An antibody characterised in that it reacts with at least one peptide or its mimetic according to claim 1. In an embodiment of this aspect of the invention said anti¬ body is a monoclonal antibody.

In another embodiment of this aspect of the invention said antibody is a genetically engineered antibody-like molecule having binding affinity to peptides or their mimetics ac¬ cording to claim 1.

26

SUBSTITUTE SHEET Said antibodies or affinity molecules are optionally couple to a carrier. Examples of suitable carriers are lysine bcta mers, human or bovine serum albumin, plastic or glas surfaces, latex- or gelatin particles.

5. A method of isolating antibodies reacting with at leas one peptide chosen from a combination of peptides or thei mimetics according to claim 1 , from blood samples of human or animals, characterised in that said samples are subjecte to affinity adsorption and desorption or affinity chromato graphy, in a per se known manner. The blood samples can b whole blood, serum or plasma.

6. A method for diagnosis of influenza virus infection char acterised in that a sample of body fluid or tissue is sub jected to an immunoassay using antibodies according to clai 4 and in case said sample contains antigens which bind t said antibodies the patient is likely to have influenza virus infection.

7. A medicament for prophylactic or therapeutic use in the prevention or treatment of an influenza virus infection, characterised in that the active principle of said medicament comprises antibodies or other affinity molecules reacting with peptides or their mimetics according to claim 1.

In one embodiment of this aspect of the invention the anti¬ bodies are polyclonal antibodies derived from blood samples of humans or apes.

In another embodiment said antibodies are monoclonal anti¬ bodies.

27

SUBSTITUTE SHEET In a further embodiment of this aspect of the invention said antibodies are genetically engineered antibody-like molecules having binding—affinity to peptides or their mimetics ac¬ cording to claim 1.

8. A vaccine composition characterised in that it comprises as an immunizing component one or several peptides chosen from peptides or their mimetics according to claim 1. In one embodiment of this aspect of the invention the immuni¬ zing component is present in an amount effective to protect a subject from influenza virus infection.

In another embodiment of this aspect of the invention the immunizing component may be used alone or in combination with other known immunogenic molecules, e.g. influenza virus pre¬ parations based on whole virus particles or split vaccines. In yet another embodiment of this aspect of the invention an adjuvant is included in an amount, which together with an amount of said immunizing component, is effective to protect a subject from influenza virus infection.

9. A medicament characterised by containing as active prin¬ ciple at least one peptide or a combination of peptides chosen from peptides or their mimetics according to claim 1, some of which may block binding of said proteins or peptides to their receptors, is provided, said medicament having the property of modulation of influenza virus infection in human beings or animals, in order to weaken or abolish the symptoms of the influenza virus infection.

28

SUBSTITUTE SHEET