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Clinical and Vaccine Immunology, April 2009, p. 437-443, Vol. 16, No. 4
1071-412X/09/$08.00+0     doi:10.1128/CVI.00327-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cross-Reactive Immunity to Clade 2 Strains of Influenza Virus A Subtype H5N1 Induced in Adults and Elderly Patients by Fluval, a Prototype Pandemic Influenza Virus Vaccine Derived by Reverse Genetics, Formulated with a Phosphate Adjuvant, and Directed to Clade 1 Strains

György Fazekas,¹ Rita Martosne-Mendi,¹ Istvan Jankovics,² Istvan Szilvasy,³ and Zoltan Vajo^3*

Omninvest Ltd., Pilisborosjeno,¹ National Institute of Epidemiology,² State Health Center, Budapest, Hungary³

Received 9 September 2008/ Returned for modification 3 November 2008/ Accepted 10 November 2008

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High fatality rates and multiple cases of transmission of avian H5N1 influenza viruses to humans illustrate the urgent need for an efficacious, cross-protective vaccine against H5N1 strains. Extensive genetic characterization of H5N1 strains has elucidated the natural evolutionary relationship of these strains, linking groups known as clades to a common ancestor. Although the clades and subclades probably differ sufficiently in their antigenic structure to warrant the preparation of different vaccines, there is some evidence that cross-reactive immunity can be afforded. We aimed to assess the immunogenicity of a clade 1 H5N1 (NIBRG-14) whole-virus vaccine with an aluminum phosphate adjuvant and to determine whether it can induce cross-reactive immunity against antigenically drifted clade 2 H5N1 strains, both those derived by reverse genetics and wild-type isolates. A total of 88 (44 adult and 44 elderly) subjects, who received one dose (6 µg) of the vaccine, were studied. As judged by U.S. and European licensing criteria based on hemagglutination inhibition, the subjects developed cross-reactive immunity against all studied H5N1 strains belonging to a clade different from that of the strain utilized to produce the vaccine. Our findings highlight the importance of stockpiling, since cross-immune reactions induced by prepandemic vaccines will likely reduce morbidity and mortality in case of a pandemic.

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Influenza continues to have a major worldwide impact, resulting in considerable human suffering and economic burden. Influenza pandemics occurring over the past centuries have cost the lives of tens of millions of people. The regular recurrence of influenza epidemics and pandemics is thought to be caused by antigenic drift. To meet the challenge of antigenic drift, vaccines that confer broad protection against heterovariant strains that circulate in influenza epidemics and pandemics are needed (1). Also, because of the time required to identify and produce an antigenically well matched pandemic vaccine, vaccines that offer broader cross-reactive immunity and protection are desirable (15).

High fatality rates and multiple cases of transmission of highly pathogenic avian influenza (HPAI) H5N1 viruses to humans illustrate the urgent need for an efficacious, cross-protective vaccine against H5N1 strains. Ideally, inactivated vaccines will induce substantial intrasubtypic cross-protection in humans so as to warrant the option of use either prior to or just after the start of a pandemic outbreak.

The HPAI H5N1 viruses that have circulated in Asia since 1997 have undergone genetic evolution in domestic poultry. Extensive genetic characterization of H5N1 strains has elucidated the natural evolutionary relationship of these strains, linking groups known as clades to a common ancestor (11). Reciprocal cross-reactions in hemagglutination inhibition (HI) tests have demonstrated the antigenic similarity of hemagglutinins (HAs) within the same genetic clade and distinguished representatives of different clades. Although the clades and subclades probably differ sufficiently in their antigenic structure to warrant the preparation of different vaccines, there is some evidence that cross-reactive immunity can be afforded (14, 24).

We aimed to assess the immunogenicity of a clade 1 H5N1 whole-virus vaccine formulated with an aluminum phosphate adjuvant system and to determine whether it can induce cross-reactive immunity to antigenically drifted clade 2 H5N1 strains, both strains derived by reverse genetics and wild-type isolates, in adult and elderly patients.

(This study was orally presented in part at the FDA/NIH/WHO Public Workshop on Immune Correlates of Protection Against Influenza A Viruses in Support of Pandemic Vaccine Development, 10 to 11 December 2007 [http://www.fda.gov/Cber/pandemic/panflu121007lp.pdf], and at the Third Meeting on Influenza Vaccines That Induce Broad Spectrum and Long-Lasting Immune Responses, 3 to 4 December 2007, Geneva, Switzerland [http://www.who.int/vaccine_research/diseases/influenza/Fazekas_Omninvest_3rdBroadspectrum.pdf].)

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Vaccine. The vaccine was produced as described previously (22). Briefly, with the exception of the virus strain, the vaccine was made by essentially the same method as the yearly interpandemic influenza vaccine "Fluval AB," which has been used in Hungary for the past 11 years (19; license OGYI-T-8998/01, National Institute of Pharmacy, Budapest, Hungary, 1995). The method has been validated by meeting the requirements of the European Agency for the Evaluation of Medicinal Products with regard to interpandemic influenza vaccines each year since 1995 and by having been safely administered to humans in Hungary in a total of more than 16 million cases since 1995 (3).

The virus strain (NIBRG-14), a reverse-genetics-derived 2:6 reassortant between A/Vietnam/1194/2004 (H5N1) and A/Puerto Rico (PR)/8/34, was obtained from the National Institute for Biological Standards and Control (NIBSC), London, United Kingdom, in May 2005. It is one of the reference viruses indicated as suitable for use in a mock-up vaccine by the Committee for Medicinal Products for Human Use (2). Briefly, the vaccine strain was produced from a human isolate (A/Vietnam/1194/2004 [H5N1]) of a virulent clade 1 influenza A (H5N1) virus used for the preparation of a reverse-genetics-modified reassortant vaccine virus and the avirulent laboratory reference strain A/PR/8/34, which was used as the donor of the polymerase, nucleoprotein, matrix, and nonstructural protein genes.

A hen's egg-grown, formaldehyde-inactivated, whole-virus vaccine, developed and produced by Omninvest Ltd. (Hungary), containing 6 µg of HA/dose, was used. The HA content was determined before the addition of the aluminum phosphate adjuvant by a single radial immunodiffusion test using reagents supplied by NIBSC (London, United Kingdom), as described previously (25).

Purity was evaluated by endotoxin content (determined by a chromogenic assay utilizing a modified Limulus amebocyte lysate and a synthetic color-producing substrate to detect the presence of endotoxin), which was determined to be less than 0.05 IU/dose, and the amount of ovalbumin, determined by an enzyme-linked immunosorbent assay (ELISA), which was less than 5 ng/dose. Both values are much lower than the concentrations considered acceptable by the European Pharmacopoeia, which are 100 IU/human dose and 1,000 ng/human dose, respectively (5). Aluminum phosphate (AlPO₄) was used as the adjuvant, in the amount of 0.31 mg Al/ampoule, and Merthiolate was added as a preservative at 0.1 mg/ml, meeting the requirements of the European Pharmacopoeia (5).

Subjects. Sera of a total of 88 volunteers, including 44 adult (age, 18 to 60 years) and 44 elderly (age, >60 years) individuals, were studied. The subjects whose sera were studied were participants in a clinical trial registered under EUDRA CT 2006-003448-40 by the European Union Drug Regulatory Authorities, European Medicines Agency (http://eudract.emea.europa.eu/). Detailed safety data are under publication with that trial (Z. Vajo, J. Wood, L. Kosa, I. Silvasy, M. Gondos, Z. Pauliny, K. Bartha, I. Visontay, M. Jankovics, A. Kis, and I. Jankovics, submitted for publication). This study examined only immunogenicity and cross-immunity data.

Briefly, a negative urine or serum pregnancy test was required for women of childbearing potential. Also, for women of childbearing potential, use of an acceptable contraception method was required, and these women were not to become pregnant for the duration of the study. Acceptable contraception included implants, injectables, combined oral contraceptives, intrauterine devices, sexual abstinence, or a vasectomized partner. Exclusion criteria included diagnosed immunodeficiency, history of Guillain-Barré syndrome, severe concomitant disease states (e.g., uncontrolled diabetes, autoimmune disease, malignancy) that could affect the immune reactivity of the individual, use of immunosuppressive medication (corticosteroid nasal sprays were permitted), medical or psychiatric conditions that precluded subject compliance with the study protocol, receipt of an inactivated vaccine 14 days prior to the study, use of live attenuated vaccines within 60 days of the study, use of investigational agents within 30 days prior to the study, receipt of blood products or immunoglobulins in the past 6 months, acute febrile illness 1 week before vaccination, pregnancy or nursing, known allergies to any component of the vaccine, including thiomerosal, and a history of allergy to eggs or egg products.

Procedures. Baseline evaluations included demographic data, medical history, and a physical examination, with recording of preexisting conditions, concomitant medications, and vital signs (blood pressure and pulse rate). For female subjects of childbearing age, pregnancy tests were performed. Blood samples were taken from the cubital vein to test for specific antibodies against the H5N1 virus by HI and microneutralization (MN). The purpose of the day 0 serological examination was to test for the absence of such antibodies prior to vaccination.

After the physical examination and blood draw, 0.5 ml of the vaccine, containing 6 µg of HA, was administered on one side into the deltoid muscle by deep intramuscular injection. The injection was not repeated. On day 21, a medical history and a list of any medications used since the last visit were taken, a physical examination was performed, and blood samples were taken from the cubital vein to test for specific antibodies against the H5N1 virus by HI and MN. With the exception of the blood draw, the procedures listed for day 21 were repeated on days 90 and 180.

Virus strains used for testing cross-reactions. The following four strains were used to test for immune cross-reactions: first and second, two different clade 2 H5N1 candidate vaccine viruses recommended for pilot lot vaccine production, rgA/Anhui/01/2005 (H5N1)-PR8-IBCDC-RG5 (kindly provided by Ruben Donis, Centers for Disease Control and Prevention, Atlanta, GA) and rgA/Bar headed goose/Qinghai/1A/2005 (kindly provided by Richard Webby, St. Jude Children's Research Hospital, Memphis, TN); third, a clade 2 H5N1 strain A/Swan/Nagybaracska/01/2006 (H5N1)-like A/PR/8/34 reassortant (produced from wild-type virus by classical reassortant technology in the biosafety level 3 facility of Omninvest Ltd. [Fig. 1]); and last, a non-H5N1 influenza virus strain A/Solomon Island/13/2006(H1N1)-like IVR-145 reassortant (kindly provided by John Wood, NIBSC, Potters Bar, United Kingdom).

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FIG. 1. Evolution of the H5N1 hemagglutinin gene. The arrows indicate the vaccine strain (Vietnam/1194/2004) and the strains against which cross-reactive immunity was tested.

Laboratory tests. Serum antibody titers were measured by HI using chicken red blood cells by following standard procedures (12, 13).

The MN assay was modified from a previously described procedure (18). Briefly, all assays were performed with Madin-Darby canine kidney (MDCK) cells. The 50% tissue culture infectious dose (TCID₅₀) of the virus was determined by titration in MDCK cells by using high-binding 96-well styrene immunoassay plates and was calculated by the method of Reed and Muench (17). Human sera were heat inactivated for 30 min at 56°C, and twofold serial dilutions were made in a 50-µl volume of viral diluent in immunoassay plates with an initial dilution of 1:2. The diluted sera were mixed with an equal volume of viral diluent containing influenza virus at 2 x 10³ TCID₅₀/ml (100 TCID₅₀/50 µl). Four control wells of virus plus viral diluent or viral diluent alone were included on each plate. The plates were gently shaken. After a 2-h incubation at 37°C under a 5% CO₂ humidified atmosphere, 100 µl of MDCK cells at 1.5 x 10⁵/ml was added to each well. The plates were incubated for 20 h at 37°C under 5% CO₂. The monolayers were washed with phosphate-buffered saline (PBS) and fixed in ice-cold 80% acetone for 10 min. The presence of viral protein was detected by ELISA as described elsewhere (23).

ELISA for measuring total antibody titers. Ninety-six-well MaxiSorp microtiter plates (Nunc, Rochester, NY) were coated with a 1:100 dilution of virus containing monovalent bulks in 60 µl phosphate buffer containing 0.01% NaN₃. Plates were incubated at 37°C for 2 h or at 4°C overnight and were washed three times with PBS-Tween 20. A blocking step was performed by incubation with 200 µl of blocking solution (1% bovine serum albumin in PBS [PBS-BSA]) at 37°C for 1 h. Human serum samples were diluted in PBS-BSA. After the plates were washed three times with PBS-Tween 20, 60 µl of serum dilutions (1:10², 1:10³, 1:10⁴, and 1:10⁵) was added in two parallel wells, and the mixture was incubated for 2 h at 37°C, washed four times with PBS-Tween 20, and further incubated with 60 µl of horseradish peroxidase-conjugated goat anti-human immunoglobulin (Southern Biotechnology, Birmingham, AL) diluted in PBS-BSA (1:5,000 dilution). After three washing steps with PBS-Tween 20, color development was performed by the addition of 60 µl of a freshly prepared substrate solution (tetramethylbenzidine). The reaction was stopped by the addition of 60 µl of 1 M phosphoric acid. The absorbance at 450 nm was read with an ELISA reader (EL 800 Biotech). Titers were calculated according to the optical densities of the reagent control.

ELISA was performed on days 0 and 21. The results for day 0 were subtracted from the results for day 21 in order to increase specificity by eliminating background values.

Immunogenicity assessment. HI and MN antibody titers were determined at baseline and on day 21 after the vaccination. HI titers were used to calculate seroconversion rates, seroprotection rates, and increases in geometric mean titers (GMTs). Immunogenicity was assessed according to the criteria of the European Union Committee for Human Medicinal Products and the European Centre for Disease Prevention and Control with regard to interpandemic and prepandemic influenza vaccines (3, 4). In order to confirm compliance with European licensing criteria for adult patients, one of the following three requirements must be met: (i) seroprotection, i.e., achievement of an HI titer of 1:40 in >70% of subjects; (ii) seroconversion, i.e., a >4-fold increase in the HI antibody titer, or reaching a titer of >1:40, in >40% of subjects; and (iii) a >2.5-fold increase in GMTs. For patients >60 years old, the following criteria were used: (i) seroconversion, i.e., a >4-fold increase in the HI antibody titer, or reaching a titer of >1:40, in >30% of subjects; (ii); seroprotection, i.e., achievement of an HI titer of 1:40 in 60% of subjects; and (iii) a >2-fold increase in GMTs (3, 4). These criteria have also been proposed in a guideline by the U.S. Food and Drug Administration, with the exception of the GMT increase criterion (21). Since there are no guidelines for MN, we applied the same conventional criteria as those described above for HI.

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In the adult group, the age range was 19 to 60 years (mean ± standard deviation, 38.5 ± 12.9 years), and there were 27 males and 17 females. The elderly group included 26 males and 18 females (age range, 60 to 83 years; mean age ± standard deviation, 67.9 ± 6.8 years). All study subjects were Caucasians.

The results of immunogenicity assessments are shown in Fig. 2 to 7. By HI, the vaccine induced seroconversion at rates exceeding the licensing criteria (40% in adult and 30% in elderly patients), not only against the NIBRG-14 strain, which was used to create the vaccine, but also against H5N1 strains belonging to different phylogenetic clades (Fig. 2). Interestingly, in elderly patients, the rate of seroconversion exceeded licensing criteria even for a non-H5N1 strain: it was found that the seroconversion rate was 56.3% against the influenza A virus H1N1 (A/Solomon Island/13/2006 [H1N1]-like IVR-145 reassortant) strain, exceeding the 30% criterion for elderly subjects (Fig. 2).

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FIG. 2. Seroconversion rates (percentages of subjects) for adult and elderly subjects by hemagglutination inhibition.

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FIG. 7. Total antibody levels measured by ELISA. The difference between the titers for NIBRG-14 on day 21 and day 0 was taken as 100% for purposes of comparison.

Ratios of postvaccination GMTs to prevaccination GMTs, determined by HI, exceeded the licensing criteria for both adult and elderly patients (2.5- and 2-fold increases, respectively) for all virus strains studied, including the different H5N1 clades and the non-H5N1 strain (Fig. 3).

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FIG. 3. Postvaccination/prevaccination GMT ratios for adult and elderly subjects by hemagglutination inhibition.

The licensing criterion for the seropositivity rate was met for strain NIBRG-14 in both adult and elderly subjects. However, for the other strains studied, seropositivity remained below the required 70% and 60% rates (Fig. 4).

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FIG. 4. Seropositivity rates (percentages of subjects) for adult and elderly subjects by hemagglutination inhibition.

Because there are no guidelines for MN, we applied the same conventional criteria as those described above for HI. When examined by MN, seroconversion rates surpassed 40% in adults and 30% in elderly individuals (the licensing requirements for rates determined by HI) for most H5N1 strains studied (Fig. 5). Similarly, GMT ratios measured by MN met the established criteria of 2.5- and 2.0-fold increases in adults and elderly individuals, respectively, for all H5N1 strains studied except for the rgA/Bar headed goose/Qinghai/1A/05 strain in adults (Fig. 6).

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FIG. 5. Seroconversion rates (percentages of subjects) for adult and elderly subjects by microneutralization.

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FIG. 6. Postvaccination/prevaccination GMT ratios for adult and elderly subjects by microneutralization.

Total antibody titers measured by ELISA showed substantial increases after vaccination for all H5N1 strains studied (Fig. 7). Interestingly, in elderly individuals, antibody titers were higher against all clade 2 H5N1 strains studied than against the clade 1 NIBRG-14 strain used for vaccination (Fig. 7).

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The results discussed above confirm our earlier findings, indicating that the present vaccine triggered immune responses against the influenza A virus (H5N1) strain NIBRG-14, meeting all applicable licensing criteria for 146 adult subjects after only one injection (22). By the present study, those findings are confirmed for adults and extended to elderly individuals. Moreover, the vaccine studied induced cross-reactive HI and neutralizing antibody responses against all three H5N1 strains examined belonging to different phylogenetic clades. One of the strains tested, the A/Swan/Nagybaracska strain, is a classical reassortant from wild-type avian influenza virus. Furthermore, the present H5N1 vaccine induced marked increases in the postvaccination/prevaccination GMT ratio against a non-H5N1 strain, an influenza virus A/Solomon Island 13/2006 (H1N1)-like IVR-145 reassortant.

Our results may support the importance of regular vaccination with seasonal influenza vaccines in developing cross-reactive immunity against pandemic viruses, since higher titers of virus-neutralizing antibodies and cross-reactive antibodies were detected in elderly patients by HI, MN, and ELISAs. Elderly individuals receive annual vaccinations against influenza, and there is evidence that vaccination with seasonal influenza vaccines may provide at least some cross-immunity against H5N1 strains (8, 10, 20). Thus, priming with H1N1 strains may have been a factor in the H5N1 and H1N1 responses we have seen in elderly patients.

Our findings also highlight the importance of stockpiling, since immune cross-reactions induced by prepandemic vaccines will likely reduce morbidity and mortality in the case of a pandemic. In June of 2007, the World Health Organization (WHO) announced that it is working with vaccine manufacturers to create a global stockpile of vaccine for the H5N1 avian influenza virus. The announcement followed a request by the World Health Assembly in May 2007 for WHO to establish an international stockpile of H5N1 vaccine. The vaccine described in the present study has been officially offered for inclusion in the WHO stockpile (www.who.int/mediacentre/news/statements/2007/s14/en/index.html).

A recent animal study suggested that H5N1 vaccines may stimulate an immune response that is more cross-protective than might be predicted by in vitro assays and thus that they have potential for being stockpiled as "initial" pandemic vaccines (9). In animal studies, subtype cross-reactive anti-HA antibody responses were associated with heterosubtypic protection against lethal infection with an HPAI H5N1 virus strain (10, 20). Thus, although challenge studies with humans are not possible for obvious ethical reasons, our results detecting cross-reactive anti-HA antibody responses are encouraging. One desirable feature of a pandemic vaccine is the ability to induce cross-reactive immune responses sufficient to protect against variants that have undergone antigenic drift. The present vaccine was tested against H5N1 viruses with substantial antigenic differences. Phylogenetic analysis of the H5 HA genes showed that all three H5N1 viruses used in the present study belonged to different subclades (Fig. 1) (26).

Most of the H5N1 strains in circulation in the past 3 years can be separated into two distinct phylogenetic clades on the basis of their HA sequences. The A/Vietnam/1194/2004 strain, used to produce the vaccine, belongs to clade 1, but the viruses that are currently circulating and have caused most of the human deaths in the past year belong to clade 2 (http://www.who.int/csr/disease/avian_influenza/guidelines/recommendationvaccine.pdf). The strains tested for cross-immunity in the present study are all examples of clade 2 viruses (26). Our data show that the H5N1 vaccine formulated with the adjuvant can induce cross-reactive HI and neutralizing antibody responses both to strains derived by reverse genetics and to wild-type isolates. The cross-clade neutralizing antibody responses recorded imply that such a vaccine could be deployed before a pandemic outbreak, which is an important mitigation strategy proposed for pandemic influenza (6, 7).

These findings are important because the identification of a candidate H5N1 pandemic influenza vaccine that can be manufactured commercially on a large scale, is immunogenic at low antigen doses, and confers cross-clade immunity against drifted H5N1 strains is an important global health objective. As we expected, all of our participants were immunologically naïve for H5N1 viruses before vaccination.

There is no doubt that, as with the annual human influenza vaccines, it would be optimal to select a vaccine strain from the pandemic strains. However, at the beginning of a pandemic, vaccines antigenically matched to the circulating viruses cannot be supplied in a timely manner, and an undersupply of vaccine is expected (16). Theoretically, if vaccines derived from antigenically distinct viruses can induce protective immunity against coming pandemic viruses and if that efficacy lasts long enough, as a possible preventive measure, we can immunize naïve populations with the present vaccine, containing A/Vietnam/1194/2004, as a first-priming vaccine prior to a pandemic and then boost with a vaccine produced from the pandemic strain.

 
* Corresponding author. Mailing address: State Health Center, Varosmajor u. 49, Budapest 1122, Hungary. Phone: 36 70 948 9731. Fax: 36 23 360 566. E-mail: zoltanvajo{at}gmail.com

Published ahead of print on 19 November 2008.

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Clinical and Vaccine Immunology, April 2009, p. 437-443, Vol. 16, No. 4
1071-412X/09/$08.00+0     doi:10.1128/CVI.00327-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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