Cross-Reactive Immunity to Clade 2 Strains of Influenza Virus ASubtype H5N1 Induced in Adults and Elderly Patients by Fluval, a
Prototype Pandemic Influenza Virus Vaccine Derived byReverse Genetics, Formulated with a Phosphate
Adjuvant, and Directed to Clade 1 Strains�
Gyorgy Fazekas,1 Rita Martosne-Mendi,1 Istvan Jankovics,2 Istvan Szilvasy,3 and Zoltan Vajo3*Omninvest Ltd., Pilisborosjeno,1 and National Institute of Epidemiology2 and State Health Center,3 Budapest, Hungary
Received 9 September 2008/Returned for modification 3 November 2008/Accepted 10 November 2008
High fatality rates and multiple cases of transmission of avian H5N1 influenza viruses to humans illustratethe urgent need for an efficacious, cross-protective vaccine against H5N1 strains. Extensive genetic charac-terization of H5N1 strains has elucidated the natural evolutionary relationship of these strains, linking groupsknown as clades to a common ancestor. Although the clades and subclades probably differ sufficiently in theirantigenic structure to warrant the preparation of different vaccines, there is some evidence that cross-reactiveimmunity can be afforded. We aimed to assess the immunogenicity of a clade 1 H5N1 (NIBRG-14) whole-virusvaccine with an aluminum phosphate adjuvant and to determine whether it can induce cross-reactive immunityagainst 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 �g) of the vaccine, were studied. Asjudged by U.S. and European licensing criteria based on hemagglutination inhibition, the subjects developedcross-reactive immunity against all studied H5N1 strains belonging to a clade different from that of the strainutilized to produce the vaccine. Our findings highlight the importance of stockpiling, since cross-immunereactions induced by prepandemic vaccines will likely reduce morbidity and mortality in case of a pandemic.
Influenza continues to have a major worldwide impact, re-sulting in considerable human suffering and economic burden.Influenza pandemics occurring over the past centuries havecost the lives of tens of millions of people. The regular recur-rence of influenza epidemics and pandemics is thought to becaused by antigenic drift. To meet the challenge of antigenicdrift, vaccines that confer broad protection against heterovari-ant strains that circulate in influenza epidemics and pandemicsare needed (1). Also, because of the time required to identifyand produce an antigenically well matched pandemic vaccine,vaccines that offer broader cross-reactive immunity and pro-tection are desirable (15).
High fatality rates and multiple cases of transmission ofhighly pathogenic avian influenza (HPAI) H5N1 viruses tohumans illustrate the urgent need for an efficacious, cross-protective vaccine against H5N1 strains. Ideally, inactivatedvaccines will induce substantial intrasubtypic cross-protectionin humans so as to warrant the option of use either prior to orjust after the start of a pandemic outbreak.
The HPAI H5N1 viruses that have circulated in Asia since1997 have undergone genetic evolution in domestic poultry.Extensive genetic characterization of H5N1 strains has eluci-dated 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 hemagglu-tinins (HAs) within the same genetic clade and distinguishedrepresentatives of different clades. Although the clades andsubclades probably differ sufficiently in their antigenic struc-ture to warrant the preparation of different vaccines, there issome evidence that cross-reactive immunity can be afforded(14, 24).
We aimed to assess the immunogenicity of a clade 1 H5N1whole-virus vaccine formulated with an aluminum phosphateadjuvant system and to determine whether it can induce cross-reactive immunity to antigenically drifted clade 2 H5N1strains, both strains derived by reverse genetics and wild-typeisolates, in adult and elderly patients.
(This study was orally presented in part at the FDA/NIH/WHO Public Workshop on Immune Correlates of ProtectionAgainst Influenza A Viruses in Support of Pandemic VaccineDevelopment, 10 to 11 December 2007 [http://www.fda.gov/Cber/pandemic/panflu121007lp.pdf], and at the Third Meetingon 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].)
MATERIALS AND METHODS
Vaccine. The vaccine was produced as described previously (22). Briefly, withthe exception of the virus strain, the vaccine was made by essentially the samemethod as the yearly interpandemic influenza vaccine “Fluval AB,” which hasbeen used in Hungary for the past 11 years (19; license OGYI-T-8998/01, Na-tional Institute of Pharmacy, Budapest, Hungary, 1995). The method has beenvalidated by meeting the requirements of the European Agency for the Evalu-ation of Medicinal Products with regard to interpandemic influenza vaccines
* Corresponding author. Mailing address: State Health Center, Var-osmajor u. 49, Budapest 1122, Hungary. Phone: 36 70 948 9731. Fax: 3623 360 566. E-mail: [email protected].
each year since 1995 and by having been safely administered to humans inHungary in a total of more than 16 million cases since 1995 (3).
The virus strain (NIBRG-14), a reverse-genetics-derived 2:6 reassortant be-tween A/Vietnam/1194/2004 (H5N1) and A/Puerto Rico (PR)/8/34, was obtainedfrom the National Institute for Biological Standards and Control (NIBSC),London, United Kingdom, in May 2005. It is one of the reference virusesindicated as suitable for use in a mock-up vaccine by the Committee for Medic-inal Products for Human Use (2). Briefly, the vaccine strain was produced froma 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 reassortantvaccine virus and the avirulent laboratory reference strain A/PR/8/34, which wasused as the donor of the polymerase, nucleoprotein, matrix, and nonstructuralprotein genes.
A hen’s egg-grown, formaldehyde-inactivated, whole-virus vaccine, developedand produced by Omninvest Ltd. (Hungary), containing 6 �g of HA/dose, wasused. The HA content was determined before the addition of the aluminumphosphate adjuvant by a single radial immunodiffusion test using reagents sup-plied by NIBSC (London, United Kingdom), as described previously (25).
Purity was evaluated by endotoxin content (determined by a chromogenicassay utilizing a modified Limulus amebocyte lysate and a synthetic color-pro-ducing substrate to detect the presence of endotoxin), which was determined tobe less than 0.05 IU/dose, and the amount of ovalbumin, determined by anenzyme-linked immunosorbent assay (ELISA), which was less than 5 ng/dose.
Both values are much lower than the concentrations considered acceptable bythe European Pharmacopoeia, which are 100 IU/human dose and 1,000 ng/human dose, respectively (5). Aluminum phosphate (AlPO4) was used as theadjuvant, in the amount of 0.31 mg Al/ampoule, and Merthiolate was added asa preservative at 0.1 mg/ml, meeting the requirements of the European Phar-macopoeia (5).
Subjects. Sera of a total of 88 volunteers, including 44 adult (age, 18 to 60years) and 44 elderly (age, �60 years) individuals, were studied. The subjectswhose sera were studied were participants in a clinical trial registered underEUDRA CT 2006-003448-40 by the European Union Drug Regulatory Author-ities, European Medicines Agency (http://eudract.emea.europa.eu/). Detailedsafety 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, andI. Jankovics, submitted for publication). This study examined only immunoge-nicity and cross-immunity data.
Briefly, a negative urine or serum pregnancy test was required for women ofchildbearing potential. Also, for women of childbearing potential, use of anacceptable contraception method was required, and these women were not tobecome pregnant for the duration of the study. Acceptable contraception in-cluded implants, injectables, combined oral contraceptives, intrauterine devices,sexual abstinence, or a vasectomized partner. Exclusion criteria included diag-nosed immunodeficiency, history of Guillain-Barre syndrome, severe concomi-tant disease states (e.g., uncontrolled diabetes, autoimmune disease, malignancy)
FIG. 1. Evolution of the H5N1 hemagglutinin gene. The arrows indicate the vaccine strain (Vietnam/1194/2004) and the strains against whichcross-reactive immunity was tested.
that could affect the immune reactivity of the individual, use of immunosuppres-sive medication (corticosteroid nasal sprays were permitted), medical or psychi-atric conditions that precluded subject compliance with the study protocol, re-ceipt of an inactivated vaccine 14 days prior to the study, use of live attenuatedvaccines within 60 days of the study, use of investigational agents within 30 daysprior to the study, receipt of blood products or immunoglobulins in the past 6months, acute febrile illness 1 week before vaccination, pregnancy or nursing,known allergies to any component of the vaccine, including thiomerosal, and ahistory of allergy to eggs or egg products.
Procedures. Baseline evaluations included demographic data, medical history,and a physical examination, with recording of preexisting conditions, concomi-tant medications, and vital signs (blood pressure and pulse rate). For femalesubjects of childbearing age, pregnancy tests were performed. Blood sampleswere taken from the cubital vein to test for specific antibodies against the H5N1virus by HI and microneutralization (MN). The purpose of the day 0 serologicalexamination 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, con-taining 6 �g of HA, was administered on one side into the deltoid muscle by deepintramuscular injection. The injection was not repeated. On day 21, a medicalhistory and a list of any medications used since the last visit were taken, a physicalexamination was performed, and blood samples were taken from the cubital veinto test for specific antibodies against the H5N1 virus by HI and MN. With theexception of the blood draw, the procedures listed for day 21 were repeated ondays 90 and 180.
Virus strains used for testing cross-reactions. The following four strains wereused to test for immune cross-reactions: first and second, two different clade 2H5N1 candidate vaccine viruses recommended for pilot lot vaccine production,rgA/Anhui/01/2005 (H5N1)-PR8-IBCDC-RG5 (kindly provided by Ruben Do-nis, Centers for Disease Control and Prevention, Atlanta, GA) and rgA/Barheaded goose/Qinghai/1A/2005 (kindly provided by Richard Webby, St. JudeChildren’s Research Hospital, Memphis, TN); third, a clade 2 H5N1 strainA/Swan/Nagybaracska/01/2006 (H5N1)-like A/PR/8/34 reassortant (producedfrom wild-type virus by classical reassortant technology in the biosafety level 3facility of Omninvest Ltd. [Fig. 1]); and last, a non-H5N1 influenza virus strainA/Solomon Island/13/2006(H1N1)-like IVR-145 reassortant (kindly provided byJohn Wood, NIBSC, Potters Bar, United Kingdom).
Laboratory tests. Serum antibody titers were measured by HI using chickenred 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 deter-mined by titration in MDCK cells by using high-binding 96-well styrene immu-noassay 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 dilutionswere made in a 50-�l volume of viral diluent in immunoassay plates with aninitial dilution of 1:2. The diluted sera were mixed with an equal volume of viraldiluent containing influenza virus at 2 � 103 TCID50/ml (100 TCID50/50 �l).Four control wells of virus plus viral diluent or viral diluent alone were includedon each plate. The plates were gently shaken. After a 2-h incubation at 37°Cunder a 5% CO2 humidified atmosphere, 100 �l of MDCK cells at 1.5 � 105/mlwas 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 wasdetected by ELISA as described elsewhere (23).
ELISA for measuring total antibody titers. Ninety-six-well MaxiSorp microti-ter plates (Nunc, Rochester, NY) were coated with a 1:100 dilution of viruscontaining monovalent bulks in 60 �l phosphate buffer containing 0.01% NaN3.Plates were incubated at 37°C for 2 h or at 4°C overnight and were washed threetimes with PBS-Tween 20. A blocking step was performed by incubation with 200�l of blocking solution (1% bovine serum albumin in PBS [PBS-BSA]) at 37°Cfor 1 h. Human serum samples were diluted in PBS-BSA. After the plates werewashed three times with PBS-Tween 20, 60 �l of serum dilutions (1:102, 1:103,1:104, and 1:105) was added in two parallel wells, and the mixture was incubatedfor 2 h at 37°C, washed four times with PBS-Tween 20, and further incubatedwith 60 �l of horseradish peroxidase-conjugated goat anti-human immunoglob-ulin (Southern Biotechnology, Birmingham, AL) diluted in PBS-BSA (1:5,000dilution). After three washing steps with PBS-Tween 20, color development wasperformed by the addition of 60 �l of a freshly prepared substrate solution(tetramethylbenzidine). The reaction was stopped by the addition of 60 �l of 1M 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 thereagent control.
ELISA was performed on days 0 and 21. The results for day 0 were subtractedfrom the results for day 21 in order to increase specificity by eliminating back-ground values.
Immunogenicity assessment. HI and MN antibody titers were determined atbaseline and on day 21 after the vaccination. HI titers were used to calculateseroconversion rates, seroprotection rates, and increases in geometric meantiters (GMTs). Immunogenicity was assessed according to the criteria of theEuropean Union Committee for Human Medicinal Products and the EuropeanCentre for Disease Prevention and Control with regard to interpandemic andprepandemic influenza vaccines (3, 4). In order to confirm compliance withEuropean licensing criteria for adult patients, one of the following three require-ments must be met: (i) seroprotection, i.e., achievement of an HI titer of �1:40in �70% of subjects; (ii) seroconversion, i.e., a �4-fold increase in the HIantibody 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 criteriawere used: (i) seroconversion, i.e., a �4-fold increase in the HI antibody titer, orreaching a titer of �1:40, in �30% of subjects; (ii); seroprotection, i.e., achieve-ment of an HI titer of �1:40 in 60% of subjects; and (iii) a �2-fold increase inGMTs (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 crite-rion (21). Since there are no guidelines for MN, we applied the same conven-tional criteria as those described above for HI.
RESULTS
In the adult group, the age range was 19 to 60 years (mean �standard deviation, 38.5 � 12.9 years), and there were 27 malesand 17 females. The elderly group included 26 males and 18females (age range, 60 to 83 years; mean age � standard
FIG. 2. Seroconversion rates (percentages of subjects) for adult and elderly subjects by hemagglutination inhibition.
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deviation, 67.9 � 6.8 years). All study subjects were Cauca-sians.
The results of immunogenicity assessments are shown in Fig.2 to 7. By HI, the vaccine induced seroconversion at ratesexceeding the licensing criteria (40% in adult and 30% inelderly patients), not only against the NIBRG-14 strain, whichwas used to create the vaccine, but also against H5N1 strainsbelonging to different phylogenetic clades (Fig. 2). Interest-ingly, in elderly patients, the rate of seroconversion exceededlicensing criteria even for a non-H5N1 strain: it was found thatthe seroconversion rate was 56.3% against the influenza Avirus H1N1 (A/Solomon Island/13/2006 [H1N1]-like IVR-145reassortant) strain, exceeding the 30% criterion for elderlysubjects (Fig. 2).
Ratios of postvaccination GMTs to prevaccination GMTs,determined by HI, exceeded the licensing criteria for bothadult and elderly patients (2.5- and 2-fold increases, respec-tively) for all virus strains studied, including the different H5N1clades and the non-H5N1 strain (Fig. 3).
The licensing criterion for the seropositivity rate was met forstrain NIBRG-14 in both adult and elderly subjects. However,for the other strains studied, seropositivity remained below therequired 70% and 60% rates (Fig. 4).
Because there are no guidelines for MN, we applied thesame 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 require-ments for rates determined by HI) for most H5N1 strains
studied (Fig. 5). Similarly, GMT ratios measured by MN metthe established criteria of 2.5- and 2.0-fold increases in adultsand elderly individuals, respectively, for all H5N1 strains stud-ied except for the rgA/Bar headed goose/Qinghai/1A/05 strainin adults (Fig. 6).
Total antibody titers measured by ELISA showed substantialincreases after vaccination for all H5N1 strains studied (Fig. 7).Interestingly, in elderly individuals, antibody titers were higheragainst all clade 2 H5N1 strains studied than against the clade1 NIBRG-14 strain used for vaccination (Fig. 7).
DISCUSSION
The results discussed above confirm our earlier findings,indicating that the present vaccine triggered immune responsesagainst the influenza A virus (H5N1) strain NIBRG-14, meet-ing all applicable licensing criteria for 146 adult subjects afteronly one injection (22). By the present study, those findings areconfirmed for adults and extended to elderly individuals.Moreover, the vaccine studied induced cross-reactive HI andneutralizing antibody responses against all three H5N1 strainsexamined belonging to different phylogenetic clades. One ofthe strains tested, the A/Swan/Nagybaracska strain, is a classi-cal reassortant from wild-type avian influenza virus. Further-more, the present H5N1 vaccine induced marked increases inthe postvaccination/prevaccination GMT ratio against a non-H5N1 strain, an influenza virus A/Solomon Island 13/2006(H1N1)-like IVR-145 reassortant.
FIG. 3. Postvaccination/prevaccination GMT ratios for adult and elderly subjects by hemagglutination inhibition.
FIG. 4. Seropositivity rates (percentages of subjects) for adult and elderly subjects by hemagglutination inhibition.
Our results may support the importance of regular vaccina-tion with seasonal influenza vaccines in developing cross-reac-tive immunity against pandemic viruses, since higher titers ofvirus-neutralizing antibodies and cross-reactive antibodieswere detected in elderly patients by HI, MN, and ELISAs.Elderly individuals receive annual vaccinations against influ-enza, and there is evidence that vaccination with seasonal in-fluenza vaccines may provide at least some cross-immunityagainst H5N1 strains (8, 10, 20). Thus, priming with H1N1strains may have been a factor in the H5N1 and H1N1 re-sponses we have seen in elderly patients.
Our findings also highlight the importance of stockpiling,since immune cross-reactions induced by prepandemic vac-cines will likely reduce morbidity and mortality in the case of apandemic. In June of 2007, the World Health Organization(WHO) announced that it is working with vaccine manufac-turers to create a global stockpile of vaccine for the H5N1avian influenza virus. The announcement followed a request bythe World Health Assembly in May 2007 for WHO to establishan international stockpile of H5N1 vaccine. The vaccine de-scribed in the present study has been officially offered forinclusion in the WHO stockpile (www.who.int/mediacentre/news/statements/2007/s14/en/index.html).
A recent animal study suggested that H5N1 vaccines maystimulate an immune response that is more cross-protectivethan might be predicted by in vitro assays and thus that theyhave potential for being stockpiled as “initial” pandemic vac-
cines (9). In animal studies, subtype cross-reactive anti-HAantibody responses were associated with heterosubtypic pro-tection against lethal infection with an HPAI H5N1 virus strain(10, 20). Thus, although challenge studies with humans are notpossible for obvious ethical reasons, our results detectingcross-reactive anti-HA antibody responses are encouraging.One desirable feature of a pandemic vaccine is the ability toinduce cross-reactive immune responses sufficient to protectagainst variants that have undergone antigenic drift. Thepresent vaccine was tested against H5N1 viruses with substan-tial antigenic differences. Phylogenetic analysis of the H5 HAgenes showed that all three H5N1 viruses used in the presentstudy belonged to different subclades (Fig. 1) (26).
Most of the H5N1 strains in circulation in the past 3 years canbe separated into two distinct phylogenetic clades on the basis oftheir HA sequences. The A/Vietnam/1194/2004 strain, used toproduce the vaccine, belongs to clade 1, but the viruses that arecurrently circulating and have caused most of the human deathsin the past year belong to clade 2 (http://www.who.int/csr/disease/avian_influenza/guidelines/recommendationvaccine.pdf). Thestrains tested for cross-immunity in the present study are allexamples of clade 2 viruses (26). Our data show that the H5N1vaccine formulated with the adjuvant can induce cross-reactiveHI and neutralizing antibody responses both to strains derivedby reverse genetics and to wild-type isolates. The cross-cladeneutralizing antibody responses recorded imply that such avaccine could be deployed before a pandemic outbreak, which
FIG. 5. Seroconversion rates (percentages of subjects) for adult and elderly subjects by microneutralization.
FIG. 6. Postvaccination/prevaccination GMT ratios for adult and elderly subjects by microneutralization.
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is an important mitigation strategy proposed for pandemicinfluenza (6, 7).
These findings are important because the identification of acandidate H5N1 pandemic influenza vaccine that can be man-ufactured commercially on a large scale, is immunogenic at lowantigen doses, and confers cross-clade immunity againstdrifted H5N1 strains is an important global health objective.As we expected, all of our participants were immunologicallynaïve for H5N1 viruses before vaccination.
There is no doubt that, as with the annual human influenzavaccines, it would be optimal to select a vaccine strain from thepandemic strains. However, at the beginning of a pandemic,vaccines antigenically matched to the circulating viruses cannotbe supplied in a timely manner, and an undersupply of vaccineis expected (16). Theoretically, if vaccines derived from anti-genically distinct viruses can induce protective immunityagainst coming pandemic viruses and if that efficacy lasts longenough, as a possible preventive measure, we can immunizenaïve populations with the present vaccine, containing A/Viet-nam/1194/2004, as a first-priming vaccine prior to a pandemicand then boost with a vaccine produced from the pandemicstrain.
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