

MICROBIOLOGY
Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5N1 influenza virus vaccines
<nobr>Erich Hoffmann<sup> *</sup></nobr>, <nobr>Aleksandr S. Lipatov<sup> *</sup></nobr>, <nobr>Richard J. Webby</nobr>, <nobr>Elena A. Govorkova</nobr>, and <nobr>Robert G. Webster<sup>

Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105-2794
Contributed by Robert G. Webster, July 27, 2005
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If H5N1 influenza viruses become transmissible among humans,<sup> </sup>vaccination will offer the most effective option to limit their<sup> </sup>spread. Two human vaccine candidates recently generated by reverse<sup> </sup>genetics are based on antigenically different hemagglutinin<sup> </sup>(HA) glycoproteins derived from the A/HK/213/03 (H5N1) and A/Vietnam/1203/04<sup> </sup>(H5N1) viruses. Their HA1 amino acid sequences differ at 10<sup> </sup>positions, one of which (N<sub>154</sub>) introduces a potential glycosylation<sup> </sup>site in A/Vietnam/1203/04 (H5N1). To assess the impact of five<sup> </sup>amino acids in the putative antigenic sites on immunogenicity<sup> </sup>and immune protection, we generated a series of whole-virus<sup> </sup>vaccines that differed only in one or two HA amino acids. Sera<sup> </sup>from ferrets vaccinated with these inactivated preparations<sup> </sup>had high virus neutralization titers, but their hemagglutination<sup> </sup>inhibition (HI) titers were usually low. Interestingly, a recombinant<sup> </sup>virus in which the HA amino acid S<sub>223</sub> (characteristic of 2004<sup> </sup>viruses) was converted to N<sub>223</sub> (as in A/HK/213/03) resulted<sup> </sup>in higher HI titers. This observation indicates that specific<sup> </sup>HA residues, such as N<sub>223</sub>, increase the sensitivity of the HI<sup> </sup>assay by altering receptor specificity and/or antibody-antigen<sup> </sup>binding. Ferrets vaccinated with mutant vaccine viruses were<sup> </sup>protected against lethal challenge with wild-type A/Vietnam/1203/04<sup> </sup>virus. Our results suggest that inclusion of the N<sub>223</sub> residue<sup> </sup>in the HA glycoproteins of diagnostic reference viruses may<sup> </sup>facilitate the evaluation of vaccine efficacy in humans.<sup> </sup>
reverse genetics | receptor specificity
<hr align="center" noshade="noshade" size="1" width="50%"> The recent outbreaks of highly pathogenic H5N1 influenza A viruses<sup> </sup>in poultry and humans across nine countries in Asia, from Japan<sup> </sup>in the north to Indonesia in the south, are unprecedented. In<sup> </sup>2003, the H5N1 viruses in Southeast Asia comprised different<sup> </sup>cocirculating genotypes, but in 2004, a single genotype (the<sup> </sup>"Z-genotype") became dominant (1). The current evidence suggests<sup> </sup>that the fatal human cases resulted from direct transmission<sup> </sup>of virus from birds to humans. The virus also caused disease<sup> </sup>in cats and was transmitted from cat to cat in experiments (2).<sup> </sup>This and other evidence of the changing host range and widespread<sup> </sup>distribution of this virus raised concern that H5N1 viruses<sup> </sup>may acquire the characteristics that allow transmission from<sup> </sup>human to human. Humans would have no immunity to such new H5N1<sup> </sup>viruses, which could cause catastrophic pandemic influenza (3).<sup> </sup> Readily available vaccines would provide the most effective<sup> </sup>tool against pandemic influenza. After the 1997 H5N1 outbreak<sup> </sup>in Hong Kong, vaccines produced by two different approaches<sup> </sup>were tested in humans. Conventional subunit H5 vaccine produced<sup> </sup>from A/duck/Singapore/3/97 (4) was poorly immunogenic in humans,<sup> </sup>even against antigenically closely related strains and after<sup> </sup>multiple vaccination (5). The use of the adjuvant MF59 increased<sup> </sup>the antibody titer of this H5 vaccine (6). Vaccination with<sup> </sup>inactivated "split" vaccine derived from nonpathogenic A/duck/HK/836/80<sup> </sup>(H3N1) virus and the modified H5 hemagglutinin from A/HK/156/97<sup> </sup>(H5N1) virus induced barely detectable titers of neutralizing<sup> </sup>antibodies in mice (7). Thus, although these H5N1 vaccines were<sup> </sup>well tolerated, they appeared to be poorly immunogenic.<sup> </sup>
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</nobr> </td><td align="left" valign="top"> Fig. 1. HI antibody titers in ferrets inoculated with H5N1 influenza viruses isolated in 2003 and 2004 (A) and HI and virus neutralizing titers in ferrets immunized with




</td></tr></tbody></table> </td></tr></tbody></table></center> Serum antibody titers, mainly those determined by hemagglutination<sup> </sup>inhibition (HI) and virus neutralization assays, are the accepted<sup> </sup>surrogate measures of immune protection. We used reverse genetics<sup> </sup>to dissect the antigenic features of the HAs of the H5N1 viruses<sup> </sup>isolated in 2003 and 2004. Ferrets are considered the best model<sup> </sup>for evaluating the potential human immune response to influenza<sup> </sup>virus infection and vaccination. We vaccinated ferrets with<sup> </sup>inactivated H5 vaccines derived from virus isolated from a fatally<sup> </sup>infected human patient (A/Vietnam/1203/04) and investigated<sup> </sup>the contribution of single H5 amino acids to the induction,<sup> </sup>degree of protection, and detectability of the immune response<sup> </sup>in the ferret model.<sup> </sup> <!-- null -->
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Virus Strains and Generation of Recombinant Viruses. Highly<sup> </sup>pathogenic H5N1 viruses were obtained from World Health Organization-collaborating<sup> </sup>laboratories in Asia. All work with these viruses was performed<sup> </sup>in BL3+ facilities at St. Jude Children's Research Hospital.<sup> </sup>Recombinant viruses rendered nonpathogenic by modification of<sup> </sup>H5 HA at the cleavage site were generated by DNA transfection<sup> </sup>as described in ref. 8. Point mutations were inserted into the<sup> </sup>HA during PCR by using the QuikChange Site-Directed Mutagenesis<sup> </sup>Kit (Stratagene) and a set of H5 HA-specific primers. Reassortant<sup> </sup>viruses contained the HA gene or the HA and neuraminidase genes<sup> </sup>from H5N1 viruses in the genetic background of A/PR/8/34 (H1N1)<sup> </sup>virus (see Table 1 for viruses generated for this study and<sup> </sup>their abbreviated names). Allantoic fluid harvested after a<sup> </sup>single passage in embryonated chicken eggs was frozen at -80?C<sup> </sup>and used in experiments. The HA genes of the recombinant viruses<sup> </sup>were amplified by RT-PCR and sequenced to verify that only the<sup> </sup>designated mutations were present.<sup> </sup>
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</nobr> </td><td align="left" valign="top"> Table 1. Recombinant H5-PR/8/34 viruses generated for use in this study
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Preparation of Inactivated Virus for Immunization. Viruses were<sup> </sup>propagated in the allantoic cavities of 10-day-old embryonated<sup> </sup>chicken eggs at 35?C for 48 h. Vaccine viruses were inactivated,<sup> </sup>concentrated, and purified as described in refs. 9 and 10.<sup> </sup> Standardization of HA Protein Content of Vaccines. The single<sup> </sup>radial immunodiffusion technique was used to standardize

Immunization of Ferrets. Male and female outbred ferrets were<sup> </sup>obtained through a special breeding program of the Animal Resources<sup> </sup>Center at St. Jude Children's Research Hospital. Animals were<sup> </sup>3-5 months old and were seronegative by HI tests for exposure<sup> </sup>to currently circulating influenza A H1N1, H3N2, and H5N1 viruses<sup> </sup>and influenza B viruses. Groups of three ferrets were vaccinated<sup> </sup>by intramuscular injection of 250 ?l of sterile PBS containing<sup> </sup>7 ?g of HA from inactivated purified viruses. Three control<sup> </sup>animals were injected with 250 ?l of sterile PBS alone.<sup> </sup>On day 21 after vaccination, serum was collected and a second<sup> </sup>intramuscular injection of 7 ?g of HA was given. Two weeks<sup> </sup>later, serum was again collected and animals were inoculated<sup> </sup>with challenge virus.<sup> </sup>
Challenge Infection of Ferrets. Vaccinated and control animals<sup> </sup>were inoculated intranasally as previously described with 10<sup>6</sup><sup> </sup>50% egg infective doses (eID<sub>50</sub>) of A/Vietnam/1203/04 virus (11).<sup> </sup>Clinical signs of infection, body weight, and temperature were<sup> </sup>monitored daily for 2 weeks. Ferrets that showed signs of severe<sup> </sup>disease were killed. To estimate the postinfective immune response,<sup> </sup>additional groups of ferrets were inoculated with 10<sup>6</sup> eID<sub>50</sub><sup> </sup>of the human and avian H5N1 isolates A/HK/213/03, A/Vietnam/3046/04,<sup> </sup>A/Vietnam/3062/04, A/chicken/Vietnam/39/04, and A/falcon/HK/D0028/04.<sup> </sup>Sera were collected from the animals on day 28 after inoculation.<sup> </sup>
Determination of Virus Titers in Upper Respiratory Tract. Specimens<sup> </sup>were obtained by nasal lavage on days 3, 5, and 7 by methods<sup> </sup>described in ref. 11. Virus in the samples was titrated in 10-day-old<sup> </sup>embryonated chicken eggs and expressed as log<sub>10</sub> eID<sub>50</sub> per 0.1<sup> </sup>ml.<sup> </sup>
mAbs to H5 Hemagglutinin. mAbs CP24, CP46, CP58, and 406/7 to<sup> </sup>the HA of A/chicken/Pennsylvania/1370/83 (H5N3) virus were produced<sup> </sup>in the Infectious Diseases Department of St. Jude Children's<sup> </sup>Research Hospital. mAb VN04-6 to the HA of A/Vietnam/1203/04<sup> </sup>virus and mAb HK03-3 to the HA of A/HK/213/03 virus were prepared<sup> </sup>by a modification of the method described by Kohler and Milstein<sup> </sup>(12, 13).<sup> </sup>
Serologic Tests. Sera collected from ferrets were treated overnight<sup> </sup>with Vibrio cholerae receptor-destroying enzyme (Denka-Seiken,<sup> </sup>Tokyo), heat inactivated at 56?C for 30 min, and adsorbed<sup> </sup>with a 0.5% suspension of chicken erythrocytes. Standard HI<sup> </sup>and virus neutralization tests in Madin Darby canine kidney<sup> </sup>cells were performed as described in refs. 14 and 15. Four hemagglutinating<sup> </sup>units (HAUs) of virus were used in each HI assay and 100 50%<sup> </sup>tissue culture infective doses were used in each neutralization<sup> </sup>assay.<sup> </sup>
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Serum Antibody Titers of Inoculated Ferrets. To compare the<sup> </sup>immunogenicity of the 2003 influenza viruses of Z genotype,<sup> </sup>which became dominant in 2004, with that of the 2004 viruses,<sup> </sup>we inoculated ferrets with the H5N1 virus isolated from a fatal<sup> </sup>human case (A/HK/213/03) (16) and with four H5N1 viruses isolated<sup> </sup>from humans, chickens, and falcons in 2004 (Fig. 1A). Serum<sup> </sup>antibodies were titrated by HI assay with challenge viruses<sup> </sup>28 days after inoculation. A/HK/213/03 virus induced high antibody<sup> </sup>titers (1:640-1:1,280), whereas the four 2004 strains induced<sup> </sup>very low HI titers (1:20-1:40).<sup> </sup>
Virus Neutralization and Sequence Analysis of HA. The relatively<sup> </sup>low HI titers to the 2004 H5N1 viruses could have been the result<sup> </sup>of virus-induced general immune suppression. However, the results<sup> </sup>of vaccination with






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</nobr> </td><td align="left" valign="top"> Table 2. Sequence differences in the HA1 protein of H5N1 influenza viruses
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</nobr> </td><td align="left" valign="top"> Fig. 2. Virus titers in nasal washes of vaccinated and control ferrets after challenge with A/Vietnam/1203/04 (H5N1). Ferrets vaccinated with


</td></tr></tbody></table> </td></tr></tbody></table></center> Generation and Antigenic Characterization of Recombinant























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</nobr> </td><td align="left" valign="top"> Table 3. H1 analysis of

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Another interesting observation was obtained in an HA test with<sup> </sup>chicken and horse red blood cells (RBCs). Interestingly, recombinant<sup> </sup>











Nasal washes of all vaccinated animals showed virus titers of<sup> </sup>2.5-4.5 log<sub>10</sub> eID<sub>50</sub> on day 3, 0.5-2.5 log<sub>10</sub> eID<sub>50</sub> on day 5,<sup> </sup>and 0.25 log<sub>10</sub> eID<sub>50</sub> or less on day 7 (Fig. 2). Unvaccinated<sup> </sup>ferrets had a mean titer of 4.0 log<sub>10</sub> eID<sub>50</sub> 1 week after infection.<sup> </sup>Two of the three control ferrets developed signs of severe disease<sup> </sup>(massive weight loss and paralysis) and were euthanized and<sup> </sup>one died of infection. Only one vaccinated ferret became seriously<sup> </sup>ill. This ferret, vaccinated with


HI and Neutralization Tests of the Immunogenicity of Recombinant<sup> </sup>











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</nobr> </td><td align="left" valign="top"> Table 4. Immunogenicity of A/Vietnam/1203/04

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</nobr> </td><td align="left" valign="top"> Table 5. Virus-neutralization titers of ferret sera after vaccination with viruses containing the modified HA of A/Vietnam/1203/04 virus
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All






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</nobr> </td><td align="left" valign="top"> Table 6. HI testing of antisera to 2003 H5N1 against mutant viruses
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To obtain additional information about the contribution of the<sup> </sup>amino acid at position 223 to serological reactivity, we generated<sup> </sup>a recombinant virus in which the H5 was derived from A/HK/213/03,<sup> </sup>with only the N<sub>223</sub>







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</nobr> </td><td align="left" valign="top"> Table 7. Antigenic analysis of H5/97 and H5<sub>S223

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</nobr> </td><td align="left" valign="top"> Fig. 3. Location of amino acid at positions 154 and 223 in the 3D structure of H5 HA. (A) The amino acids in the 3D structure of the HA of A/duck/Singapore/3/97 (H5N3) virus (18). The glycosylation site at position 154 is located at the top of the HA molecule. (B) The circle represents the interface between the monomer shown and two other monomers (data not shown) in the trimeric HA. Amino acid at position 223 is located at the surface of the trimer.
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The H5N1 viruses isolated in 2004 had antigenic properties different<sup> </sup>from those of the previous isolates and possessed 10 amino acids<sup> </sup>in the HA1 region that differed from those in A/HK/213/03 virus.<sup> </sup>Therefore, the World Health Organization-collaborating laboratories<sup> </sup>recommended that a new reassortant A/PR/8/34 virus with the<sup> </sup>HA from A/Vietnam/1203/04 (H5N1) be generated by reverse genetics<sup> </sup>for vaccine studies. In this study, most ferrets vaccinated<sup> </sup>with A/PR/8/34 single-gene reassortants that differed only in<sup> </sup>their H5s were protected against a lethal challenge with A/Vietnam/1203/04<sup> </sup>virus. These results on cross-protection extend those of previous<sup> </sup>studies that used a mouse model (17).<sup> </sup> A/Vietnam/1203/04 virus had a previously uncharacterized potential<sup> </sup>glycosylation site that might mask epitopes of the H5 HA from<sup> </sup>the previous vaccine candidate. The recombinant virus in which<sup> </sup>this glycosylation site at the top of the H5 (Fig. 3) was changed<sup> </sup>completely protected ferrets against virus challenge. These<sup> </sup>findings are consistent with those of a previous study in BALB/c<sup> </sup>mice vaccinated with an H5-DNA vaccine derived from the 1997<sup> </sup>H5N1 viruses (19). Mice vaccinated with HAs derived from A/HK/156/97<sup> </sup>containing single point mutations in the H5 were protected from<sup> </sup>lethal challenge. Thus, we conclude that the presence or absence<sup> </sup>of carbohydrate residues at position 154 of H5 is not crucial<sup> </sup>to the protective efficacy of H5 vaccines.<sup> </sup>
In several independent experiments, we found that vaccine viruses<sup> </sup>containing H5s from 2004 elicited low detectable HI antibody<sup> </sup>titers. This effect was observed after both intranasal inoculation<sup> </sup>and intramuscular vaccination. Interestingly, unlike the other<sup> </sup>three amino acid substitutions, assays using a virus with the<sup> </sup>S<sub>223</sub>









Within the last five years, plasmid-only systems have become<sup> </sup>powerful tools for generating high-yield influenza viruses of<sup> </sup>different serotypes (8, 10, 21, 22). To create a new vaccine<sup> </sup>representing the antigens of a new serotype, a combination of<sup> </sup>factors must be optimized, such as number of doses, formulation<sup> </sup>with or without adjuvants, dose range, antigenicity, and antibody<sup> </sup>testing. The first human vaccine generated by classical reassortment<sup> </sup>was derived from A/duck/Singapore/3/97 (H5N3) virus and was<sup> </sup>designed to provide protection against H5N1/97 viruses. This<sup> </sup>vaccine was reported to be poorly immunogenic in humans, but<sup> </sup>use of the adjuvant MF59 increased the antibody titer (6). The<sup> </sup>results of our study and of in vitro assays suggest that genetic<sup> </sup>engineering of specific residues in the H5 HA may provide an<sup> </sup>additional means of improving detection of a specific immune<sup> </sup>response to H5 antigens.<sup> </sup>
Our results in ferrets suggest that evaluation of postinfection<sup> </sup>human sera could be improved by genetic engineering of H5 antigens,<sup> </sup>such as

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We thank Scott Krauss, David Walker, Patrick Seiler, Jennifer<sup> </sup>Humberd, and Kelly Jones for excellent technical assistance<sup> </sup>and Sharon Naron for editorial assistance. These studies were<sup> </sup>supported by Grant AI95357 from the National Institute of Allergy<sup> </sup>and Infectious Diseases, Cancer Center Support Grant CA21765<sup> </sup>from the National Institutes of Health, and the American Lebanese<sup> </sup>Syrian Associated Charities.<sup> </sup>
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<!-- null --> Author contributions: E.H., A.S.L., R.J.W., E.A.G., and R.G.W.<sup> </sup>designed research; E.H., A.S.L., R.J.W., E.A.G., and R.G.W.<sup> </sup>performed research; E.H., A.S.L., R.J.W., E.A.G., and R.G.W.<sup> </sup>analyzed data; and E.H., A.S.L., R.J.W., E.A.G., and R.G.W.<sup> </sup>wrote the paper.<sup> </sup>
<!-- null --> Abbreviation: EID<sub>50</sub>, egg 50% infective dose; HI, hemagglutination<sup> </sup>inhibition; HAU, hemagglutinating unit; RBC, red blood cell.<sup> </sup>
<!-- null --> <sup>*</sup> E.H. and A.S.L. contributed equally to this work.<sup> </sup>

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? 2005 by The National Academy of Sciences of the USA
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