WO2009040343A1

WO2009040343A1 - Inactivated influenza vaccine

Info

Publication number: WO2009040343A1
Application number: PCT/EP2008/062694
Authority: WO
Inventors: Henricus Lodewijk Glansbeek; Jacobus Gerardus Maria Heldens
Original assignee: Nobilon International B.V.
Priority date: 2007-09-24
Filing date: 2008-09-23
Publication date: 2009-04-02
Also published as: EP2195020A1; CN101808659A; BRPI0817230A2; AR068536A1; TW200927928A

Abstract

The present invention is concerned with inactivated adjuvanted influenza vaccines. The present invention provides a vaccine that overcomes many of the drawbacks of existing inactivated influenza vaccines. The present invention provides an inactivated influenza vaccine, comprising beta propiolactone (BPL) inactivated whole influenza virus and comprising, as adjuvant, one or more mono-or disaccharide derivatives having at least one butnot more than N-1 fatty acid ester groups and, optionally, one but not more than N-1 sulphate ester groups, wherein N is the number of hydroxyl groups of the mono-or disaccharide from which the derivative is derived. The influenza virus in a vaccine according to the invention is preferably cell culture derived. Methods for producing influenza virus in cell culture are known in the art. The virus may be grown on cells of mammalian, avian, or human origen, such as Madin Darby Canine Kidney (MDCK), Vero, MDBK, CLDK, EBx or PerC6 cells.

Description

INACTIVATED INFLUENZA VACCINE

The present invention is concerned with inactivated adjuvanted influenza vaccines.

Influenza virus is a RNA virus of the family Orthomyxoviridae (the influenza viruses) capable of infecting birds and mammals. Influenza viruses have a segmented genome of eight negative sense, single strands (segments) of RNA, abbreviated as PB2, PB1 , PA, HA, NP, NA, M and NS. These segments encode 10 genes. The HA segment encodes the haemagglutinin protein, which is an antigenic protein found in the protein coat (viral envelope) of the viral particle. The protein is involved in cellular entry of the virus. The NA segment encodes the neuraminidase, which is an antigenic glycosylated enzyme also found on the surface of the influenza viral particle. It facilitates the release of progeny virus from infected cell.

There are three types of influenza viruses: A, B, and C.

Humans can be infected with influenza types A, B, and C viruses. Influenza A viruses are further classified by subtype on the basis of the two main surface glycoproteins haemagglutinin (HA) and neuraminidase (NA). No different subtypes of H and N have been identified for influenza B and C. There are 16 known HA subtypes and 9 known NA subtypes for influenza A viruses. For example, an "H5N1 " virus has an HA protein belonging to subtype 5 and an NA protein belonging to subtype 1. Subtypes of influenza A that are currently circulating among people worldwide include H1 N 1 , H1 N2, and H3N2 viruses. However, infections of humans with other subtypes, such as H9N2, H7N7 or H2N2, causing morbidity and mortality have been reported.

Influenza B viruses are not further classified although two distinct genetic and antigenic lineages (Victoria and Yamagata) are described.

There are two types of antigenic variation in influenza viruses, referred to as "antigenic drift" and 'antigenic shift".

Antigenic drift is part of the continuing occurrence of new influenza strains that differ form their ancestors by mutations (point mutations) in the HA and NA genes. The amount of change can be subtle or dramatic. The second type of antigenic variation is "antigenic shift". A genetic shift can occur when two different influenza viruses, co-infecting the same host, exchange a whole genomic segment. This could result in a "reassortant" virus with a novel gene constellation and consequently with new properties. A genetic shift can also occur when a virus subtype crosses the species barrier directly without reassortment in an intermediate host.

The occurrence antigenic shift may give rise genetic changes enabling new influenza viruses able to replicate in humans and more importantly to spread among humans efficiently. When such a virus has a subtype to which the human population is immunological naϊve, a pandemic might occur.

In the past century 3 influenza pandemics took place of which the Spanish flu in 1918 was the most severe. This pandemic killed approximately 50 million people worldwide.

Currently avian influenza (H5N1 ) is of growing concern worldwide concern because of the ongoing outbreaks in Asia, Europe and Africa in the poultry population, and the considerable amount of human cases (317, dd 29 June 2007) of which 60 % were fatal. The virus is highly contagious and already over 200 million domestic birds have either been culled or died following infection. Until now human-to-human transmission is highly inefficient but the virus may acquire this ability upon adaptation. Such an event would increase the risk on a pandemic outbreak dramatically.

In the control of a pandemic, vaccines may play an important role. If the causing infectious agent can not be combated by chemical or pharmaceutical products (e.g. antibiotics or antivirals), due to non-susceptibility or resistance, or by sanitary measures, control might depend even fully on vaccination.

It is well known that antibodies that are induced after vaccination play a crucial role in protection against influenza. In addition to humoral immunity, cell-mediated immunity plays an important role to kill the infection.

The induction of cell-mediated immunity is important because T-cells recognize conserved epitopes which results in a broad protection against different strains.

Current inactivated epidemic influenza vaccines are being produced with three influenza virus strains that are recommended annually by the World Health Organization on the basis of information obtained from global influenza surveillance. Antigens for these vaccines are commonly produced in embryonated chicken eggs. The seasonal vaccines contain 15 μg haemagglutinin of each strain. Because the population is immunological naϊve for the pandemic influenza strain a much higher dose of a pandemic influenza vaccine is required for the induction of protective immunity. It has been shown that for induction of protective immunity against an H5N1 strain two doses of 90 μg haemagglutinin were required. In view of the limited amount of antigen that will be available such a high dose is inconvenient.

The use of an adjuvant might overcome the poor response against the pandemic influenza vaccine strain.

The composition of the vaccine, especially nature of the adjuvant, plays an important role in the kinetics of the immune response. Not only the level, but also the onset and duration of the immune reaction are affected by the vaccine composition. It is well known that repository (oily) adjuvants such as water-in-oil emulsions are strong adjuvants, induce a steadily increasing immune response, which reach high maximal levels and last for long periods of time. At the other hand, aqueous adjuvants induces rapid onset of immunity but in general, reach much lower maximal levels which last for much shorter periods of time.

In order to circumvent insufficient level of immunity, a booster immunization is often given three or more weeks after the first injection which extends significantly the total time required to establish immunity ('time to immunity').

In case of a pandemic rather than an epidemic, time and production capacity are crucial factors. A vaccine that establishes protective immunity short (e.g. one week) after a single injection ('one-shot'), with minimal concentrations of antigen, would open opportunities for controlling a pandemic which are beyond the possibilities of products that need several weeks or even a second administration with preparations containing high concentrations of antigen. A vaccine inducing high levels of antibodies in combination with the induction of cell mediated immunity may protect the population not only against the homologous strain (exact pandemic strain), but also against heterologous, or not complete matching influenza strains of the same subtype. This would enable an even more timely response to a potential pandemic outbreak as exemplified by the control of H5N1 outbreaks in poultry using an adjuvanted H5N2 vaccine. Such a vaccine that could be produced rapidly and at large scale (number of doses) after the first alarming signals of an emerging pandemic are manifest, would favour control. In fact, each and any measure that reduces significantly the time between the first alarming signal ('departure time') and the situation that the human population is protected at sufficient level ('arrival time') has a positive impact.

So, the ideal pandemic vaccine is available as early as possible during the course of an emerging pandemic to as large as possible population and establishes, as early as possible, sufficient levels of protective immunity (humoral and cellular) in as many as possible subjects.

The present invention provides a vaccine that overcomes many of the drawbacks of existing inactivated influenza vaccines.

The present invention provides an inactivated influenza vaccine, comprising beta propiolactone (BPL) inactivated whole influenza virus and comprising, as adjuvant, one or more mono-or disaccharide derivatives having at least one but not more than N-1 fatty acid ester groups and, optionally, one but not more than N-1 sulphate ester groups, wherein N is the number of hydroxyl groups of the mono-or disaccharide from which the derivative is derived.

Such adjuvants are disclosed in WO 0140240, Hilgers LA., and Blom A.G. Sucrose fatty acid sulphate esters as novel vaccine adjuvant. Vaccine 24: S2-81 (2006), and Blom A.G., and Hilgers LA. Sucrose fatty acid sulphate esters as novel vaccine adjuvants: effect of the chemical composition. Vaccine 23: 743-54 (2004).

This type of adjuvant does not form a depot of antigen, as oily adjuvants do, which results in immediate availability of antigen to the host immune system. Especially in a pandemic situation rapid onset of immunity is important. The adjuvant preferably is CoVaccine HT™ . CoVaccine HT™ contains a sucrose fatty acid sulphate ester incorporated in a submicron squalane-in-water emulsion. The dose of sucrose fatty acid sulphate ester is between 0.1 and 40 mg. Preferably, the dose of sucrose fatty acid sulphate ester is between 0.25 and 10 mg. Most preferably, the dose of sucrose fatty acid sulphate ester is between 0.5 and 4 mg. The dose of squalane is between 0.4 and 160 mg. Preferably, the dose of squalane is between 1 and 40 mg. Most preferably, the dose of squalane is between 2 and 16 mg. The dose of haemagglutinin is between 0.1 and 60 μg. Preferably, the dose of haemagglutinin is between 0.25 and 15 μg. Most preferably, the dose is between 1 and 3 μg.

CoVaccine HT stimulates both Th1 and Th2 response (important for induction of cell mediated immunity), while, for example, Aluminium hydroxide gives only a Th2 response. CoVaccine HT does not induce enhanced pathology after challenge infection.

The influenza virus in a vaccine according to the invention is preferably cell culture derived. Methods for producing influenza virus in cell culture are known in the art. The virus may be grown on cells of mammalian, avian, or human origen, such as Madin Darby Canine Kidney (MDCK), Vero, MDBK, CLDK, EBx or PerC6 cells.

MDCK cells are cells known in the art. The MDCK cell line was derived from a kidney of an apparently normal adult female cocker spaniel, September, 1958, by S. H. Madin and N. B. Darby. The original MDCK cell line (NBL-2) was deposited at the ATCC (catalogue number ATCC CCL 34).

MDCK cells may be grown adherent, for example in roller bottles or on microcarriers, preferably in serum free medium (Merten, O.W., et al. Production of influenza virus in cell cultures for vaccine preparation. Adv Exp Med Biol.; 397:141-51 (1996); Kalbfuss, B., et al. Harvesting and concentration of human influenza A virus produced in serum-free mammalian cell culture for the production of vaccines. Biotechnology and Bioengeneering, 97 (2007).

MDCK cells can also be grown in suspension culture (Nakamura, K., et al. Method of suspension culture for MDCK cells and isolation of influenza virus in MDCK suspension cultured cells. Kansenshogaku Zasshi; 54:306-12 (1980).

Vaccines for pandemic use aim to protect humans against infection with a highly pathogenic (avian) influenza virus with a pandemic potential, such as the H5N1 strain. A vaccine according to the invention is preferably based on an inactivated influenza virus of the H5 type, especially the H5N1 type.

A vaccine based on the influenza virus strain NIBRG-14, cultured on MDCK cells, inactivated with BPL and adjuvanted with the CoVaccine HT™ was tested. Surprisingly, a single injection of this vaccine in ferrets, the animal model for influenza vaccines, conferred high virus-neutralizing antibody titres indicating high degree of protection.

NIBRG-14 virus was engineered by the National Institute for Biological Standards and Control (Potters Bar, England) with a view to its use as a human influenza vaccine. NIBRG-14 is an attenuated reassortant virus containing 2 surface genes

(modified HA & NA) from AΛ/ietnam/1 194/2004 (H5N1 ) and 6 internal genes from the egg-high growth A/PR/8/34 (H 1 N1 ). To improve the safety of the strain the polybasic cleavage site in the haemagglutinin gene was removed. The non-pathogenicity of this vaccine strain was confirmed in embryonated eggs, chickens, and ferrets. (Wood,

J. M., et al. From lethal virus to life saving vaccine: developing inactivated vaccines for pandemic influenza. Nature Reviews in Microbiology 2, 842-847 (2004).

The (combined) use of cell culture (instead of eggs) for virus production, whole-virus (instead of split or subunits), BPL (instead of cross-linking agents), aqueous (instead of oily), non-repository (instead of repository) adjuvant and/or simply adding (instead of emulsifying) antigen and adjuvant offers important advantages.

First of all, the vaccine results in a rapid onset of immunity, which is crucial in the face of a pandemic threat.

Moreover, a vaccine according to the invention is easier to produce with higher yields

(number of doses).

Due to the fact that with a vaccine according to the invention only a single shot is required, the amount of antigen required is reduced. Due to the fact that the vaccine induces high antibody and cell mediated responses, not only protection against homologous, but also against heterologous strains may be obtained.

The invention is further exemplified by the Examples given below. The experiments presented show that a vaccine according to the present invention reached unexpectedly high HI antibody titres after a single immunization. These titres could not be reached with comparable vaccines that differed only in the choice of adjuvant (aluminium hydroxide instead of CoVaccine HT™). EXAMPLES

Example 1 : Generation of vaccines

Influenza virus NIBRG14 (H5N1 ) was grown on MDCK cells. After 3-5 days of fermentation, the virus supernatant was harvested and clarified prior to inactivation with BPL (0.025 % w/v). After inactivation the inactivated virus was concentrated by ultrafiltration and further purified. The antigen concentration was determined by single radial immunodiffusion (SRID) analysis. Vaccines were formulated by mixing virus antigen with the required amount of adjuvant and/or phosphate buffered saline (PBS) (Table 1 ). The adjuvant CoVaccine HT™ was kindly provided by CoVaccine BV (Utrecht, The Netherlands).

Example 2: Vaccination/challenge experiment with CoVaccine HT™ adjuvanted cell culture-derived, inactivated whole-virus vaccine in mice

Experimental design

Female Swiss mice, 6-8 weeks of age were randomly divided over five groups (n=5). Vaccine was administered by intramuscular (IM) injection in the hind legs as 0.1 mL. The vaccine formulations are indicated in Table 1.

Challenge virus (A/Puerto Rico/8/34) (H1 N1 ) was produced by inoculation of 0.2 mL virus into 9-11 days old embryonated SPF eggs. After an incubation period of three days at 34^CC-37°C the allantoic fluid was harvested and titrated on MDCK cells.

To evaluate the induction of antibodies blood samples were taken 24 days after vaccination. Antibody titres were determined by haemagglutination inhibition assay. Antigen specific IgGI and lgG2a antibody titres were measured by enzyme linked immunoassay (ELISA).

Four weeks after the vaccination all animals where challenged with mouse-adapted (A/Puerto Rico/8/34; H1 N1 ). Body weight was measured daily to evaluate protection against clinical symptoms. Twelve days after the challenge all animals were sacrificed. Table 1 : Vaccine formulations

Results:

To evaluate the ability of adjuvant CoVaccine HT to improve the immunogenicity of a cell culture derived inactivated whole virus influenza vaccine mice were vaccinated with different vaccine formulations.

Three weeks after the vaccination HI antibody titres were determined in the sera taken from vaccinated and control animals from groups. As shown in Table 2

CoVaccine HT™ did not strongly enhance the HI titres against A/Pr/8/34. The HI titres were even lower than the titres induced by the vaccine that contained aluminium hydroxide.

Table 2: HI titres against A/Pr/8/34 in sera taken 24 days after vaccination

The IgG isotypes were analysed to determine whether CoVaccine HT has an effect on the type of immunity that is induced. As shown in Table 3 co-delivery of aluminium hydroxide induced high titres of IgGI and lgG2a. Co-delivery of CoVaccine HT clearly induced a shift in IgG isotypes as the lgG1/lgG2a ratio decreased strongly. This shift to a decreased lgG1/lgG2a ratio might be important as a low lgG1/lgG2a ratio is in mice associated with a good induction of cell-mediated immunity (Th1 response) while a high ratio is associated with a poor induction of cell-mediated immunity (Th2 response).

^* NC= not calculated as the mean IgGI titre was less that two times the background. Sera were prediluted 200-fold and serially diluted 2-fold in 96-well ELISA plates coated with antigen.

Twentyseven days after vaccination all mice were challenged with homologous mouse adapted A/Pr/8/34. Body weights were measured daily. All animals in the PBS control group decreased in body weight and died within 8 days. Animals in group 2 (antigen only) demonstrated clear group mean body weight loss until day 6 after challenge, after which body weights increased to normal levels. Animals in groups 3- 5 were better protected as these animals did not lose weight after the challenge.

Although CoVaccine HT did not induce higher HI titres than aluminium hydroxide, it had a beneficial effect on the Th1/Th2 ratio of the response.

Example 3: lmmunogenicity of CoVaccine HT adjuvanted cell culture derived inactivated whole virus vaccine (strain A/Pr/8/34) in ferrets (trial I), (vaccination/challenge):

The effects of CoVaccine HT on the immunogenicity of a cell culture derived inactivated whole virus influenza vaccine was evaluated also in ferrets.

Experimental design Four groups of castrated male ferrets (n=7) were used for the experiment. One week prior to vaccination the ferrets received a transponder (Biomedic Data Systems IPTT-

200) subcutaneously to measure body temperature and to enable identification. All animals were vaccinated once with different formulations by intramuscular injection of

0.5 ml_. HI titres were determined in blood samples were taken 21 days and 49 days after the vaccination. Eight weeks after vaccination the ferrets were challenged with infectious

A/Puerto Rico/8/34 (H1 N 1 ). Challenge virus (A/Puerto Rico/8/34)(H1 N1 ) was produced by inoculation of 0.2 ml_ virus into 9-1 1 days old embryonated SPF eggs. After an incubation period of three days at 34°C-37°C the allantoic fluid was harvested and titrated on MDCK cells. Prior challenge, body weight and body temperature were measured frequently to establish normal baseline values. Following challenge, body weights were measured and body temperature was monitored twice a day. On Day 4 after the challenge infection the animals were exsanguinated by heart puncture where after gross pathology was performed. The formulations are shown in Table 4.

Table 4: Vaccine formulations in ferret trial I

Group Antigen (inactivated whole Adjuvant virus)

1 PBS -

2 A/Pr/8/34 10-15 μg - HA/dose

3 A/Pr/8/34 10-15 μg Aluminium hydroxide (0.2%)(Brenntag HA/dose Biosector, Denmark)

4 A/Pr/8/34 10-15 μg CoVaccine HT (4 mg/dose)(CoVaccine BV) HA/dose

Results: As shown in Table 5, immunization with CoVaccine HT resulted in 6- to 7-fold higher HI titres than immunization with aluminium hydroxide.

Table 5: HI titres against A/Pr/8/34 in sera taken 21 and 48 days after vaccination

The ferrets were challenged with homologous infectious virus 8 weeks post vaccination. On day 4 after the challenge infection ferrets were sacrificed and lung tissue was taken for histology. Unvaccinated animals of Group 1 showed the most severe pathological lesions in the lungs. The animals of the vaccinated groups (Group 2 to 4) showed only minor inflammation of bronchioli/bronchi and alveoli. After challenge, lymphoid stimulation was present with perivascular lymfocytic infiltration of small vessels (highest score in Group 3) and diffuse interstitial mononuclear cell infiltration (highest score in Group 2 and 3, but most severe in control group). The differences between the different vaccinated groups were not enormously and not clearly cut.

It was concluded that in ferrets but not in mice, CoVaccine HT induced significantly higher HI antibody titres than aluminium hydroxide.

Example 4: lmmunogenicity of CoVaccine HT adjuvanted cell culture derived inactivated whole virus vaccine (strain NIBRG-14) in ferrets (trial II).

To evaluate whether CoVaccine HT also improves the immunogenicity of a H5N1 strain, a new vaccination experiment was performed. For production of the vaccines inactivated whole virus antigen produced under GMP was used.

Experimental design: Seven groups of male ferrets (n=7) were used for the experiment.

One week prior to vaccination the ferrets received a transponder (Biomedic Data Systems IPTT-200) subcutaneously to measure body temperature and to enable identification. All animals were vaccinated twice with different formulations by injecting 0.5 ml_ in the left musculus biceps femoris. For production of the vaccines inactivated whole virus antigen (strain NIBRG14 (H5N1 ), produced under GMP) was used. The different vaccine formulations are shown in Table 6. Blood samples were taken at different time points.

Table 6: Vaccine formulations in ferret trial Il

All animals were vaccinated twice with a 3-week interval. As shown in Table 7 co-delivery of 1 mg/dose CoVaccine HT™ resulted in a 4.9 fold increase in HI titre compared to the aluminium hydroxide adjuvanted vaccine. Co- delivery of 4 mg/dose CoVaccine HT™ even resulted in an 8.5 fold increase. The booster further increased the HI titres. The formulations with CoVaccine HT™ resulted in the highest HI titres on Day 35 but the differences with the aluminium hydroxide adjuvanted vaccine were smaller than on Day 21 .

Table 7: HI titres against NIBRG-14 in sera taken on Day 21 (21 days after first vaccination) and on Day 35 (14 days after booster vaccination)

Group Antigen Adjuvant HI titre (²Log) HI titre (²Log) (inactivated Day 21 Day 35 whole virus)

1 7.5 μg HA/dose - 3.5 ± 1.8 8.4 ± 1.6

2 7.5 μg HA/dose Aluminium hydroxide 5.1 ± 1.3 10.8 ± 0. 5

3 15 μg HA/dose - 4.7 ± 1.1 8.3 ± 0.7

4 15 μg HA/dose Aluminium hydroxide 6.2 ± 0.2 1 1.2 ± 1 . 0

5 7.5 μg HA/dose CoVaccine HT (4 8.2 ± 0.2 12.2 ± 0. 8 mg/dose)

6 7.5 μg HA/dose CoVaccine HT (1 7.4 ± 0.3 12.4 ± 1 . 1 mg/dose)

7 7.5 μg HA/dose CoVaccine HT (0.25 5.6 ± 0.2 10.8 ± 0. 5 mg/dose)

From the described experiments it can be concluded that protective HI titres could be obtained after a single vaccination with inactivated whole virus vaccines adjuvanted with CoVaccine HT (HI titres > 5.3 are considered to be protective in humans).

An H5N1 strain co-delivered with CoVaccine HT results in a 8.5 fold increase (4 mg/dose) or 4.9 fold increase (1 mg/dose) in HI titre compared to co-delivery of aluminium hydroxide.

After a single vaccination the HI titre induced by 7.5 μg HA/dose + CoVaccine HT is 4 fold higher that the titre induced by 15 μg HA/dose + Aluminium hydroxide.

Example 6: Comparison of CoVaccine HT and aluminium hydroxide adjuvanted influenza vaccines in nonhuman primates

The present invention was further illustrated by a vaccination study in nonhuman primates. For this purpose, CoVaccine HT™ and aluminium hydroxide adjuvanted H5N1 vaccines were injected into female Cynomolgus macaques {Macaca fascicularis) of about 3 years of age (Hartelust BV, Tilburg, The Netherlands) and antibody responses were measured after one and two injections. The animals were housed in groups of 6 animals in a normal cage using sawdust as bedding. The animal facility conditions were a day/night light cycle (12h/12h), a temperature of 21 degrees Celsius ± 2⁰C, and a relative humidity of 40-60%. The animals had ad libitum supply of tap water and food (pellets and fruit). They were checked daily for overt signs of disease.

The animal experiment was carried out in accordance with Dutch law for animal experimentation and in agreement with the "Guide for the care and use of laboratory animals", ILAR recommendations and AAALAC standards.

Animals were observed twice a day by the animal facility technicians. For handling, animals were sedated with ketamin (25 mg/kg; i.rm.), which provided deep sedation for approximately 20-40 minutes and which is a standard procedure. On Day 0, animals were immunized into the left hind leg femoral muscle (LH) where the skin was shaved. On study day 21 the vaccine was administered into the right hind (RH) leg femoral muscle where the skin was shaved. The injection site was inspected just before and 4 and 24 hrs after each immunization. The dose of antigen was 7.5 μg HA (inactivated NIBRG-14). Aluminium hydroxide was used at a concentration of 0.2 % (w/v). The dose of CoVaccine HT was 2 mg SFASE.

Results:

No local or systemic adverse events were noted except some local redness in a few cases.

For the haemaglutination inhibition (HI) assay a virus suspension was incubated with serial (2-fold) dilutions of serum sample pre-treated with cholerafiltrate (obtained from Vibrio cholerae cultures). Subsequently, erythrocytes were added to the dilutions and after incubation the maximum dilution of the agents showing complete inhibition of haemaglutination was defined as the HI antibody titre.

Table 8: HI antibody titres after one and two immunizations with aluminium hydroxide (Group 1 ) and CoVaccine HT-adjuvanted (Group 2), whole-virus H5N1 influenza vaccine in macaques.

GMT is geometric mean titre; SD is standard deviation of GMT; antilog is the 2^ΛGMT; factor of increase is the antilog at a certain day divided by the antilog at Day 0, i.e. 10.

The detection limit of the HI test system is 10. Three weeks after the first injection with cell-culture-derived, whole-virus, H5N1 influenza virus with aluminium hydroxide or CoVaccine HT as adjuvant HI titres were increased at least 1.6 and 17.2-fold, respectively as compared to Day 0 (before immunization) Three weeks after the second injection with cell-culture-derived, whole-virus, H5N1 influenza virus with aluminium hydroxide or CoVaccine HT as adjuvant HI titres were increased 12.3 and 175.1 -fold, respectively as compared to Day 0 (before immunization). The three EMEA criteria for assessment of influenza vaccine efficacy are: 1 ) the number of seroconversions or significant increase in HI titre should be > 40 %, 2) the increase in GMT should be > 2.5 and 3) the portion of subjects with a HI titre >_ 40 should be at least 70%.

Surprisingly, a single dose of the vaccine according to the present invention in unprimed animals resulted in an immune response that met easily these criteria while for the conventional aluminium hydroxide-adjuvanted vaccine, two immunizations were required. It was concluded that the time-to-immunity was reduced significantly by the present invention. This offers enormous advantages in combating and controlling an influenza pandemic.

Claims

CLAIMS:

1 . Inactivated influenza vaccine, comprising beta propiolactone (BPL) inactivated whole influenza virus and comprising as an adjuvant one or more mono- or disaccharide derivatives having at least one but not more than N-1 fatty acid ester groups wherein N is the number of hydroxyl groups of the mono- or disaccharide from which the derivative is derived.

2. Inactivated influenza vaccine according to claim 1 , characterized in that the adjuvant is sucrose fatty acid sulphate ester incorporated into a squalane-in- water emulsion.

3. Inactivated influenza vaccine according to claim 2, characterized in that the adjuvant is CoVaccine HT™.

4. Inactivated influenza vaccine according to any of claims 1 -3, characterised in that the influenza virus is cell-culture derived.

5. Inactivated influenza vaccine according to any of the preceding claims, characterised in that the cell culture is an MDCK cell culture.

6. Inactivated influenza vaccine according to any of the preceding claims, characterised in that the influenza is of the H5 type.

7. Inactivated influenza vaccine according to claim 6, characterised in that the influenza is of the H5N1 type.

8. Inactivated influenza vaccine according to claim 7, characterised in that the influenza is NIBRG-14.

9. Inactivated influenza vaccine according to any of the preceding claims, characterised in that one dose of the vaccine contains between 0.1 and 60 μg HA.

10. Inactivated influenza vaccine according to any of claims 1 -3, characterised in that one dose of the vaccine contains between 0.1 mg and 40 mg sucrose fatty acid (sulphate) ester.

1 1. Use of a vaccine according to any of claims 1 -9, in a method to protect a human or an animal against influenza.

12. Use according to claim 10, wherein the human or animal is vaccinated in a one-shot vaccine regimen.