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Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

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  • Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

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    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> </sup></nobr>
    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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    <table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> Abstract </th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left"> Top
    Abstract
    Materials and Methods
    Results
    Discussion
    References
    </th></tr></tbody></table>

    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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    <center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top">
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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 H5N1/03 and H5N1/04 viruses (B). (A) Sera were collected on day 28 after inoculation with 10<sup>6</sup> eID<sub>50</sub> of H5N1 viruses and titrated against 4 HAUs of homologous virus. Data are representative values from two or four sera. (B) Sera were collected from ferrets vaccinated twice with 7 ?g of HA (see Materials and Methods) of H5N1/03 and H5N1/04 viruses and titrated against 4 HAUs and 100 tissue culture 50% infective dose of homologous virus, respectively.
    </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 -->
    <table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> Materials and Methods </th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left"> Top
    Abstract
    Materials and Methods
    Results
    Discussion
    References
    </th></tr></tbody></table>
    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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    <center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top"> View this table:
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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 H5N1/03<sup> </sup>(10). The remaining recombinant viruses were separated by 12%<sup> </sup>SDS/PAGE, the stained gels were analyzed by densitometry on<sup> </sup>the FUJIFILM Luminescent Image Analyzer LAS-1000plus, and HA<sup> </sup>was quantified by comparison with a reference protein preparation.<sup> </sup>
    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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    <table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> Results </th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left"> Top
    Abstract
    Materials and Methods
    Results
    Discussion
    References
    </th></tr></tbody></table>
    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 H5N1-A/PR/8/34 (6 + 2) vaccines that included<sup> </sup>the HA and neuraminidase of A/HK/213/03 and A/Vietnam/1203/04<sup> </sup>indicated that differences in the H5 could be a major contributor<sup> </sup>to this effect (Fig. 1B). Vaccination with two separate 7-?g<sup> </sup>doses of H5N1/03 vaccine induced high levels of serum antibodies<sup> </sup>detectable in both HI and virus neutralization tests (Fig. 1B).<sup> </sup>After identical vaccination with H5N1/04, very low (1:20) titers<sup> </sup>were detected in the HI test, whereas neutralizing titers were<sup> </sup>much higher (about half that induced by H5N1/03). Previous studies<sup> </sup>found that inactivated vaccine derived from A/duck/Singapore/3/97<sup> </sup>(H5N3) induced little or no detectable serum antibody (4, 6).<sup> </sup>Taken together, these results indicate that some H5 isolates<sup> </sup>may have unusual immunogenic and/or antigenic properties. Alignment<sup> </sup>of the H5 amino acid sequences revealed that the HAs of A/HK/213/03<sup> </sup>and A/Vietnam/1203/04 viruses differ in 10 amino acids in the<sup> </sup>HA1 region (Table 2). A/Vietnam/1203/04 virus has a potential<sup> </sup>glycosylation site at asparagine N<sub>154</sub> (N<sub><sup>*</sup>154</sub>-S<sub>155</sub>-T<sub>156</sub>, N-X-S/T,<sup> </sup>X P). Sequence comparison revealed three amino acids (S<sub>120</sub>,<sup> </sup>K<sub>189</sub>, and S<sub>223</sub>) that were present in all of the 2004 viruses<sup> </sup>but were not present in A/HK/213/03. K<sub>212</sub> was characteristic<sup> </sup>for A/Vietnam/1203/04 virus.<sup> </sup>
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    <center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top"> View this table:
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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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    <center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top">
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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 H5N1/04 or H5/04 recombinant viruses were inoculated intranasally with 10<sup>6</sup> eID<sub>50</sub> of A/Vietnam/1203/04 virus. Titers are the mean values (log<sub>10</sub> eID<sub>50</sub>/0.1 ml) ? SD determined in the nasal washes of three ferrets.
    </td></tr></tbody></table> </td></tr></tbody></table></center> Generation and Antigenic Characterization of Recombinant H5-A/PR/8/34<sup> </sup>Viruses. To test the impact of the identified amino acids on<sup> </sup>immunogenicity and protection against virus challenge, we used<sup> </sup>the 8-plasmid reverse genetics system to generate recombinant<sup> </sup>viruses with seven gene segments of A/PR/8/34 and the HA gene<sup> </sup>segment of A/Vietnam/1203/04, containing single point mutations<sup> </sup>(8). The amino acid change was verified by sequencing the HA<sup> </sup>segment of the recombinant viruses (Table 1). To evaluate the<sup> </sup>antigenic properties and diversity of the recombinant HAs, we<sup> </sup>performed HI assays with a panel of six anti-HA monoclonal antibodies<sup> </sup>(Table 3). Five mAbs reacted at relatively high titers with<sup> </sup>the H5N1/03 virus, but only three reacted with H5/04 HA. The<sup> </sup>reactivity patterns of H5<sub>S155N</sub>, <sub>T156A</sub>/04, H5<sub>S120N</sub>/04, and H5<sub>R212K</sub>/04<sup> </sup>viruses were similar, in general, to that of H5/04 virus. The<sup> </sup>reactions of H5<sub>S120N</sub>, <sub>S155N</sub>, <sub>T156A</sub>/04 virus were similar to<sup> </sup>those of H5N1/03 virus. Four mAbs recognized H5/04 HA with mutation<sup> </sup>S<sub>223</sub>N(H5<sub>S223N</sub>/04). The reverse mutation N<sub>223</sub>S in the HA of the<sup> </sup>2003 virus (H5<sub>N223S</sub>/03) resulted in significantly decreased<sup> </sup>HI titers or the loss of recognition by mAbs.<sup> </sup> <sup> </sup>
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    <center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top"> View this table:
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    </nobr> </td><td align="left" valign="top"> Table 3. H1 analysis of H5 recombinant viruses with anti-H5 monoclonal antibodies
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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>H5<sub>S223N</sub>/04 virus was less able to agglutinate 1% horse RBCs<sup> </sup>but it agglutinated chicken RBCs to a high titer (1:1,024).<sup> </sup>None of the remaining recombinant viruses differed in their<sup> </sup>reaction to chicken and horse RBCs to the same extent (data<sup> </sup>not shown).<sup> </sup> Vaccination of Ferrets with H5-Mutant Recombinant Viruses. We<sup> </sup>assessed the immunogenicity and protective efficacy of the inactivated<sup> </sup>vaccines by vaccinating groups of three ferrets by intramuscular<sup> </sup>injection with preparations of H5N1/04, H5/04, H5<sub>S155N, T156A</sub>/04,<sup> </sup>H5<sub>S120N</sub>/04, and H5<sub>S223N</sub>/04 virus standardized for HA content.<sup> </sup>After receiving two injections of 7 ?g of HA, each animal<sup> </sup>was inoculated with A/Vietnam/1203/04 (H5N1) as described in<sup> </sup>Materials and Methods.<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 H5<sub>S120N</sub>/04 virus, showed severe<sup> </sup>neurological signs and was euthanized on day 7 after inoculation.<sup> </sup>This ferret had shown severe viral conjunctivitis on day 4 after<sup> </sup>inoculation, and subsequent virus spread to the brain. It is<sup> </sup>likely that the virus was transferred to the eyes during the<sup> </sup>nasal lavage on day 3 and that rapid neuronal spreading to the<sup> </sup>brain caused encephalitis. The remaining vaccinated ferrets<sup> </sup>demonstrated decreased activity, loss of body weight, and increased<sup> </sup>body temperature during the first 3 days after virus challenge.<sup> </sup>These signs disappeared by day 5, and all animals recovered<sup> </sup>rapidly. Thus, all vaccine viruses tested protected ferrets<sup> </sup>from lethal challenge with A/Vietnam/1203/04. Vaccination decreased<sup> </sup>viral titers in the upper respiratory tract and decreased the<sup> </sup>duration of virus shedding.<sup> </sup>
    HI and Neutralization Tests of the Immunogenicity of Recombinant<sup> </sup>H5-A/PR/8/34 Viruses. Serum from vaccinated ferrets was tested<sup> </sup>against the recombinant viruses in HI and virus neutralization<sup> </sup>assays (Tables 4 and 5, respectively). Sera from ferrets vaccinated<sup> </sup>with the wild-type single-gene reassortant virus (H5/04, reference<sup> </sup>virus) produced HI titers of 1:20. The construct in which the<sup> </sup>glycosylation site was removed (H5<sub>S155N; T156A</sub>/04) induced HI<sup> </sup>titers of 1:10-1:20. Mutant H5 HA <sub>S120N</sub>/04 resulted in HI titers<sup> </sup>of 1:20-1:80. In contrast, vaccination with H5<sub>S223N</sub>/04 resulted<sup> </sup>in an HI titer of 1:640, and the other immune sera tested reacted<sup> </sup>with H5<sub>S223N</sub>/04 virus at high HI titers (1:160 to 1:320). Thus,<sup> </sup>although the vaccination induced protective immunity, the levels<sup> </sup>of detectable antibody were different.<sup> </sup>
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    </nobr> </td><td align="left" valign="top"> Table 4. Immunogenicity of A/Vietnam/1203/04 H5 HA recombinant viruses in ferrets
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    <center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top"> View this table:
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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 H5/04 viruses produced high titers of virus-neutralizing<sup> </sup>antibodies after vaccination (1:320-1:1,280) (Table 5). No substantial<sup> </sup>differences were observed between homologous and heterologous<sup> </sup>neutralizing titers. Therefore, the differences observed between<sup> </sup>the antisera in recognition of the HA did not reflect the ability<sup> </sup>of the antibodies to neutralize virus.<sup> </sup> To further evaluate the reactivity of the recombinant viruses,<sup> </sup>we used HI assays to test hyperimmune mouse and chicken serum<sup> </sup>obtained after vaccination with the H5N1/03 and A/HK/213/03<sup> </sup>viruses against recombinant viruses with altered HAs (Table 6).<sup> </sup>The mean HI titers to homologous H5N1/03 virus were 1:2,560.<sup> </sup>HI titers to H5/04 were 1:160. HI titers against recombinant<sup> </sup>H5<sub>S223N</sub>/04 virus were at least twice the titers against the<sup> </sup>other mutants.<sup> </sup>
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    </nobr> </td><td align="left" valign="top"> Table 6. HI testing of antisera to 2003 H5N1 against mutant viruses
    </td></tr></tbody></table> </td></tr></tbody></table></center>
    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>S point mutation (Table 1). This recombinant<sup> </sup>H5<sub>N223S</sub>/03 virus had lower HI titers in chicken and horse RBCs<sup> </sup>than did the H5N1/03 virus. To further characterize the impact<sup> </sup>of amino acid 223 on antigen-antibody recognition, we generated<sup> </sup>recombinant viruses that contained wild-type HA and mutated<sup> </sup>S<sub>223</sub>NHAfromA/duck/Singapore/3/97 (see Table 1). These viruses<sup> </sup>were tested by HI assay against a panel of anti-H5 antisera<sup> </sup>and mAbs (Table 7). The S<sub>223</sub>N substitution in the HA dramatically<sup> </sup>increased the HI titers (by a factor of 4 or more). However,<sup> </sup>this mutation did not significantly alter the reactivity pattern<sup> </sup>of A/duck/Singapore/3/97 HA, especially in the reactions with<sup> </sup>mAbs: neither the original nor the mutant HA reacted with mAbs<sup> </sup>HK03-3 and CP46, and both reacted at a low titer with CP46 (Table 7).<sup> </sup>These results demonstrate that the S<sub>223</sub>N substitution in<sup> </sup>HA increases the sensitivity of the HI assay.<sup> </sup> <sup> </sup>
    <!-- null -->

    <center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top"> View this table:
    <nobr>[in this window]
    [in a new window]
    </nobr> </td><td align="left" valign="top"> Table 7. Antigenic analysis of H5/97 and H5<sub>S223N</sub>/97 viruses with polyclonal and monoclonal antibodies
    </td></tr></tbody></table> </td></tr></tbody></table></center>
    <sup> </sup> <!-- null -->

    <center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top">
    View larger version (42K):
    <nobr>[in this window]
    [in a new window]
    </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.
    </td></tr></tbody></table> </td></tr></tbody></table></center> <!-- null -->
    <table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> Discussion </th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left"> Top
    Abstract
    Materials and Methods
    Results
    Discussion
    References
    </th></tr></tbody></table>
    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>N substitution resulted in higher HI titers. The epitopes<sup> </sup>reported to be important for recognition of H5 HA by mAbs (12)<sup> </sup>did not include position 223. Alignment of all published H5N1<sup> </sup>virus HA sequences shows that all recent isolates in Asia have<sup> </sup>S<sub>223</sub>. Some avian H5N1 viruses isolated in Central and South<sup> </sup>America have a basic amino acid, R<sub>223</sub>. The basic amino acid<sup> </sup>N<sub>223</sub> is found only in the HA of the human isolate A/HK/213/03<sup> </sup>and is located in the 220-loop of the receptor binding domain<sup> </sup>between Q<sub>222</sub> and G<sub>224</sub> (Fig. 3). The experimental evidence suggests<sup> </sup>that the higher HI titers reflect a change in receptor specificity.<sup> </sup>Indeed, Q<sub>222</sub> and G<sub>224</sub> bind directly to the sialic acid receptor.<sup> </sup>Amino acids in the 220 loop or adjacent are important for the<sup> </sup>conformation of the receptor binding pocket (18). Possibly,<sup> </sup>substitution of S<sub>223</sub> to N<sub>223</sub> results in conformational changes<sup> </sup>and altered receptor specificity. This finding is consistent<sup> </sup>with the fact that H5<sub>S223N</sub>/04 virus did not agglutinate horse<sup> </sup>RBCs, whose sialic acid receptors have the N-glycosyl sialic<sup> </sup>acid 2,3 linkage (20). The sialic acid receptors on chicken<sup> </sup>RBCs have 2,3 and 2,6 linkages. Thus, if H5<sub>S223N</sub>/04 virus<sup> </sup>binds only to sialic acid receptors with 2,6 linkage, the resultant<sup> </sup>lower binding on chicken RBCs would require a smaller quantity<sup> </sup>of antibody to inhibit hemagglutination.<sup> </sup>
    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 H5-N<sub>223</sub>. A 2-fold or 4-fold increase in sensitivity<sup> </sup>could be especially significant in situations in which the endpoints<sup> </sup>of conventional titration methods are below the limit of detection.<sup> </sup>The strategy applied here to H5 may be useful in increasing<sup> </sup>HI titers against other avian HA subtypes, such as H9 and H7.<sup> </sup>In addition to the use of reference viruses with increased sensitivity<sup> </sup>in human vaccine clinical trials, those viruses can be used<sup> </sup>in seroepidimiology studies. The availability of data showing<sup> </sup>how many humans were infected with H5N1 viruses by rapid and<sup> </sup>simple detection methods like HI assays would provide important<sup> </sup>information on the prevalence of H5N1 viruses in humans. This<sup> </sup>data could be used to asses the probability of H5N1 viruses<sup> </sup>to spread from human to human.<sup> </sup>
    <!-- null -->
    <table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> Acknowledgements </th></tr></tbody></table>
    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>


    <table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> Footnotes </th></tr></tbody></table>
    <!-- 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>
    <!-- null --> <sup></sup> To whom correspondence should be addressed. E-mail: robert.webster@stjude.org<script type="text/javascript"><!-- var u = "robert.webster", d = "stjude.org"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></script>.
    ? 2005 by The National Academy of Sciences of the USA
    <!-- null -->
    <table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> References </th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left"> Top
    Abstract
    Materials and Methods
    Results
    Discussion
    References
    </th></tr></tbody></table>
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    4. Fouchier, R., Kuiken, T., Rimmelzwaan, G. & Osterhaus, A. (2005) Nature 435, 419-420.<!-- HIGHWIRE ID="102:36:12915:3" -->[CrossRef][ISI][Medline] <!-- /HIGHWIRE --><!-- null -->
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    10. Liu, M., Wood, J. M., Ellis, T., Krauss, S., Seiler, P., Johnson, C., Hoffmann, E., Humberd, J., Hulse, D., Zhang, Y., et al. (2003) Virology 314, 580-590.<!-- HIGHWIRE ID="102:36:12915:9" -->[CrossRef][ISI][Medline] <!-- /HIGHWIRE --><!-- null -->
    11. Webby, R. J., Perez, D. R., Coleman, J. S., Guan, Y., Knight, J. H., Govorkova, E. A., McClain-Moss, L. R., Peiris, J. S., Rehg, J. E., Tuomanen, E. I. & Webster, R. G. (2004) Lancet 363, 1099-1103.<!-- HIGHWIRE ID="102:36:12915:10" -->[CrossRef][ISI][Medline] <!-- /HIGHWIRE --><!-- null -->
    12. Govorkova, E. A., Rehg, J. E., Krauss, S., Yen, H. L., Guan, Y., Peiris, M., Nguyen, T. D., Hanh, T. H., Puthavathana, P., Long, H. T., et al. (2005) J. Virol. 79, 2191-2198.<!-- HIGHWIRE ID="102:36:12915:11" --><nobr>[Abstract/Free Full Text]</nobr><!-- /HIGHWIRE --><!-- null -->
    13. Kaverin, N. V., Rudneva, I. A., Ilyushina, N. A., Lipatov, A. S., Krauss, S. & Webster, R. G. (2004) J. Virol. 78, 240-249.<!-- HIGHWIRE ID="102:36:12915:12" --><nobr>[Abstract/Free Full Text]</nobr><!-- /HIGHWIRE --><!-- null -->
    14. Kohler, G. & Milstein, C. (1976) Eur. J. Immunol. 6, 511-519.<!-- HIGHWIRE ID="102:36:12915:13" -->[ISI][Medline] <!-- /HIGHWIRE --><!-- null -->
    15. Palmer, D. F., Dowdle, M. T. Coleman, M. T. & Schild, G. C. (1975) Advanced Laboratory Techniques for Influenza Diagnosis. U.S. Department of Health, Education and Welfare Immunology Series 6 (U.S. Dept. of Health, Education, and Welfare, Washington, DC).<!-- HIGHWIRE ID="102:36:12915:14" --><!-- /HIGHWIRE --><!-- null -->
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    18. Lipatov, A. S., Webby, R. J., Govorkova, E. A., Krauss, S. & Webster, R. G. (2005) J. Infect. Dis. 191, 1216-1220.<!-- HIGHWIRE ID="102:36:12915:17" -->[CrossRef][ISI][Medline] <!-- /HIGHWIRE --><!-- null -->
    19. Ha, Y., Stevens, D. J., Skehel, J. J. & Wiley, D. C. (2001) Proc. Natl. Acad. Sci. USA 98, 11181-11186.<!-- HIGHWIRE ID="102:36:12915:18" --><nobr>[Abstract/Free Full Text]</nobr><!-- /HIGHWIRE --><!-- null -->
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  • #2
    Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

    Comment


    • #3
      Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

      Note that the Shantou isolates have S227R (S223R in the above paper) as well as K222R and V223I (K218R and V219I in the above paper).

      Comment


      • #4
        Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

        How does these H5N1 RBDs compare to the RBD configuration of circulating H1N1 and H3N2?

        .
        "The next major advancement in the health of American people will be determined by what the individual is willing to do for himself"-- John Knowles, Former President of the Rockefeller Foundation

        Comment


        • #5
          Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

          S227N has been shown in increase affinity for human receptors (2,6) and decrease affinity for avian (2,3). There are no structures or affinities available for the new combinations of changes.
          Last edited by AlaskaDenise; November 12, 2006, 03:49 AM.

          Comment


          • #6
            Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

            good news, isn't it ?

            Now, Switzerland,Singapore,France? might have ordered the
            wrong vaccine from Glaxo and they might start to change the
            vaccine now ?!
            Also, that prepandemic vaccines might become the preferred
            method now as compared with pandemic vaccines.

            I'm confused however, why the increase in HI-titers didn't
            correlate so well with ferret-protection ?!

            I also wonder, what these studies do cost and whether we
            couldn't/shouldn't afford more of this.
            I'm interested in expert panflu damage estimates
            my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

            Comment


            • #7
              Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

              Originally posted by gsgs
              good news, isn't it ?

              Now, Switzerland,Singapore,France? might have ordered the
              wrong vaccine from Glaxo and they might start to change the
              vaccine now ?!
              Also, that prepandemic vaccines might become the preferred
              method now as compared with pandemic vaccines.

              I'm confused however, why the increase in HI-titers didn't
              correlate so well with ferret-protection ?!

              I also wonder, what these studies do cost and whether we
              couldn't/shouldn't afford more of this.
              This article was published over a year ago. The effect of S227N on immunogenicity is well known.

              Comment


              • #8
                Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

                Mingus should tell us, when it's old !

                That make me curious, whether Glaxo does include that S227N in their current prepandemic vaccine ?

                (strain A/Vietnam/1203/2004)
                I'm interested in expert panflu damage estimates
                my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

                Comment


                • #9
                  Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

                  Originally posted by gsgs
                  Mingus should tell us, when it's old !

                  That make me curious, whether Glaxo does include that S227N in their current prepandemic vaccine ?

                  (strain A/Vietnam/1203/2004)
                  I believe that the only modification is the removal of the HA cleavage site.

                  Comment


                  • #10
                    Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection o

                    Originally posted by gsgs
                    Mingus should tell us, when it's old !
                    As you may have see, we reorganised the FluTrackers scientific library to make search & retreival of articles easier to those that may have need for.

                    Previously a similar organisation was set in the H5N1org site but it will be more convenient & accessible to record the articles directly here. It also prevent a confusing mess of article stockpile...

                    Since I have some access to some articles from science-direct & some others net-based library, I took the initiative to post some articles that we just haved the abstract previously.

                    So ... sorry but it should be very easy for you to check the publication date

                    -Mingus

                    Comment


                    • #11
                      Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

                      yes, now that I know it...
                      Usually I just press the red button "latest posts". How are
                      others following here ?

                      It was well worth a read, despite being old.
                      I'm interested in expert panflu damage estimates
                      my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

                      Comment


                      • #12
                        Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

                        Originally posted by niman
                        I believe that the only modification is the removal of the HA cleavage site.

                        maybe that's why Germany isn't yet buying that vaccine ?!?
                        I'm interested in expert panflu damage estimates
                        my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

                        Comment


                        • #13
                          Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

                          Originally posted by gsgs
                          maybe that's why Germany isn't yet buying that vaccine ?!?
                          The vaccine targeting a 2004 isolate from Vietnam has been obsolete for some time now. WHO has alreadu announced new targets (based on 2005 isolates, from Qingahi, China, and Indonesia), which will also soon be obsolete).

                          Comment


                          • #14
                            Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

                            gs the date of an article is usually right with the title somewhere; in this one, its right after the authors names. I understand its easy to miss, I got caught on that a few times myself when I first started following the flu boards. Now I've learned to look for it before I read the article.

                            One thing this and several similar articles Ive read about vaccine effectiveness or production has impressed on me- in most cases we judge common vaccine effectiveness by its ability to prevent disease or morbidity/mortality.

                            With H5, we may have to adjust our scale- if we are judging effectivenes of a prepandemic vaccine based on current technology and vaccine targets; we may have to judge vaccine effectiveness on its ability to diminish morbidity and mortality.

                            To me, thats a huge shift in thinking. I beleive it also has implications for spread- since to my knowledge theoretically an immunized, infected person can still shed infective virus, a vaccinated person can still spread disease.

                            So, even (in a perfect world where sufficient vaccine for this was available) if I as a healthcare worker and all my colleagues were vaccinated with a prepandemic vaccine, that might encourage otherwise reluctant HCW to stay on the job when a pandemic strikes.

                            But, will that mean they (we) can still get infected and spread the disease both to non-flu patients, visitors, other HCW and their families and communities?

                            I dont know how infective a person immunized with a prepandemic vaccine might be. I wish there were studies on this, at least in animal models-perhaps there are and I've missed them? But, I wonder if the infections that could result from an immunized person would be more likely to be resistant to the prepandemic vaccine, putting others at higher risk, ie, will shed virions that are able to subvert the prepandemic induced antibodies result in later waves with resistant strains? Will those strains have different virulence? will they be protected against by the interpandemic vaccine that is produced in response to the first wave?

                            Could mass vaccination with prepandemic vaccince actullly change the strain that eventually predominates, and will those changes be benefiical or deleterious?

                            Would this result in another later pandemic wave that the prepandemic vaccinated individuals will be susceptable to? Might this be similar to the reports in 1918 that many folks infected in wave 1 were reinfected in other waves? that the changes are great enough to create a situation where recovered individuals are still at risk, and perhaps greater risk of exhuberant immune response?
                            Upon this gifted age, in its dark hour,
                            Rains from the sky a meteoric shower
                            Of facts....They lie unquestioned, uncombined.
                            Wisdom enough to leech us of our ill
                            Is daily spun, but there exists no loom
                            To weave it into fabric..
                            Edna St. Vincent Millay "Huntsman, What Quarry"
                            All my posts to this forum are for fair use and educational purposes only.

                            Comment


                            • #15
                              Re: Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5

                              when you are vaccinated, you should be less likely to shed virus.
                              Looking at fig.2 in that paper I guess, about half as much as
                              nonvaccinated people in the first 3-5 days and then even less.
                              I don't expect people with H5N1 would go to work.
                              The idea is also, that when many are vaccinated, then
                              H5N1 would just not be able to spread like an epidemic.

                              There were some studies with graphics about the spread
                              with incomplete vaccination and/or other measures.
                              Maybe someone has a link
                              I'm interested in expert panflu damage estimates
                              my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

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