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  • Vaccines against Avian Influenza — A Race against Time

    New England Journal of Medicine - NEJM

    Vaccines against Avian Influenza — A Race against Time
    Gregory A. Poland, M.D.
    <table border="0" cellpadding="0" cellspacing="0" height="49" width="640"><tbody><tr><td nowrap="nowrap" valign="top">
    </td> <td align="center" valign="top"><table border="0" cellpadding="0" cellspacing="0"> <tbody><tr><th align="center" nowrap="nowrap" valign="top">Volume 354:1411-1413</th> <td align="center" nowrap="nowrap"></td> <th align="center" nowrap="nowrap" valign="top">March 30, 2006</th> <td align="center" nowrap="nowrap"></td> <th align="center" nowrap="nowrap" valign="top">Number 13</th></tr></tbody></table>http://content.nejm.org/cgi/content/full/354/13/1411


    </td></tr></tbody></table>
    Avian influenza A (H5N1) virus poses an important pandemic threat.<sup> </sup>A study by the Congressional Budget Office estimates that the<sup> </sup>consequences of a severe pandemic could, in the United States,<sup> </sup>include 200 million people infected, 90 million clinically ill,<sup> </sup>and 2 million dead.<sup>1</sup> The study estimates that 30 percent of<sup> </sup>all workers would become ill and 2.5 percent would die, with<sup> </sup>30 percent of workers missing a mean of three weeks of work<sup> </sup>— resulting in a decrease in the gross domestic product<sup> </sup>of 5 percent. Furthermore, 18 million to 45 million people would<sup> </sup>require outpatient care, and economic costs would total approximately<sup> </sup>$675 billion. As of March 10, 2006, the World Health Organization<sup> </sup>(WHO) had reported 176 confirmed human cases of influenza A<sup> </sup>(H5N1) across seven countries, with 97 deaths (a 55 percent<sup> </sup>mortality rate for identified cases).<sup>2</sup>

    <sup> </sup> A major worry is that influenza A (H5N1) continues to evolve<sup> </sup>into antigenically distinct clades — infecting mammalian<sup> </sup>hosts other than humans, expanding its ecologic niche of domestic<sup> </sup>fowl to include wild migratory birds, and causing outbreaks<sup> </sup>among birds in more than 30 countries. The virus is close to<sup> </sup>meeting the criteria for a pandemic virus — one that is<sup> </sup>new, can cause human illness, and can be transmitted from human<sup> </sup>to human<sup>3</sup><sup>,</sup><sup>4</sup><sup>,</sup><sup>5</sup> — and the world is currently in phase three<sup> </sup>of the six WHO phases of alert for pandemic influenza (higher<sup> </sup>numbers represent greater seriousness). Influenza A (H5N1) is<sup> </sup>not yet pandemic because of a single factor: the inefficiency<sup> </sup>of human-to-human transmission. Once such transmission is efficient<sup> </sup>and sustained, even assuming that the current mortality rate<sup> </sup>of approximately 50 percent decreases, we will be in the midst<sup> </sup>of a serious pandemic.

    <sup> </sup>
    For this reason, maintaining the public health requires attempts<sup> </sup>to mitigate, avert, and treat infection with influenza A (H5N1)<sup> </sup>virus, and the key to meeting these goals is the development,<sup> </sup>testing, licensing, manufacturing, and stockpiling of vaccines.<sup> </sup>Safe and effective vaccines are likely to be the single most<sup> </sup>important public health tool for decreasing the morbidity, mortality,<sup> </sup>and economic effects of pandemic influenza — particularly<sup> </sup>in view of the reported resistance of influenza A (H5N1) to<sup> </sup>antiviral agents.<sup>6</sup>
    <sup>
    </sup>
    <sup> </sup>
    Thus, the data reported in this issue of the Journal by Treanor<sup> </sup>et al.<sup>7</sup> from their multicenter randomized, double-blind, placebo-controlled<sup> </sup>clinical trial of a subvirion influenza A (H5N1) vaccine are<sup> </sup>important and informative. Enrolled in the study were 451 healthy<sup> </sup>adults 18 to 64 years of age who received two doses of the vaccine<sup> </sup>without adjuvant, each of which contained 90, 45, 15, or 7.5<sup> </sup>µg of hemagglutinin antigen, or placebo. The vaccine was<sup> </sup>produced from a human isolate (A/Vietnam/1203/2004 [H5N1]) of<sup> </sup>a virulent clade 1 influenza A (H5N1) virus with the use of<sup> </sup>a plasmid rescue system, with only the hemagglutinin and neuraminidase<sup> </sup>genes expressed. The rest of the genes were derived from an<sup> </sup>avirulent egg-adapted influenza A/PR/8/34 strain. The hemagglutinin<sup> </sup>gene was further modified to replace six basic amino acids associated<sup> </sup>with high pathogenicity in birds at the cleavage site between<sup> </sup>hemagglutinin 1 and hemagglutinin 2. Immunogenicity was assessed<sup> </sup>by microneutralization and hemagglutination-inhibition assays<sup> </sup>with the use of the vaccine virus, although a subgroup of samples<sup> </sup>were tested with the use of the wild-type influenza A/Vietnam/1203/2004<sup> </sup>(H5N1) virus.

    <sup> </sup>
    The results of the vaccine in the study by Treanor et al. (referred<sup> </sup>to here as the "1203 vaccine") give pause. Although the 1203<sup> </sup>vaccine was safe, with an unremarkable adverse-event profile,<sup> </sup>its immunogenicity was poor to moderate at best. In fact, in<sup> </sup>only one group did more than 50 percent of the subjects reach<sup> </sup>the immunogenicity threshold (defined a priori) of an antibody<sup> </sup>titer of 1:40 or greater (typically thought of as seroprotective)<sup> </sup>— the subjects who received two doses of 90 µg each<sup> </sup>28 days apart — a total dose 12 times that of seasonal<sup> </sup>influenza vaccines. Notably, the current worldwide manufacturing<sup> </sup>capacity for influenza vaccine is estimated at only 900 million<sup> </sup>doses (at the dose level of 15 µg). The requirement of<sup> </sup>two doses of 90 µg per person means that only 75 million<sup> </sup>persons (1.25 percent of the world's population) could be fully<sup> </sup>immunized, and of those, only half would achieve seroprotection.<sup> </sup>Thus, vaccines must contain much less influenza hemagglutinin<sup> </sup>to be widely useful as a global public health measure.

    <sup> </sup>
    And there are some additional provisos. An antibody titer of<sup> </sup>1:40 does not guarantee protection from infection. People with<sup> </sup>lower titers show protection against influenza, and people with<sup> </sup>higher titers can have symptomatic infection. Moreover, the<sup> </sup>assumption that a titer of 1:40 is seroprotective is based on<sup> </sup>circulating strains of seasonal influenza.
    Whether the same<sup> </sup>will prove to be true for new influenza viruses in people whose<sup> </sup>immune systems have not been primed is unknown. However, even<sup> </sup>moderate levels of seroprotection could be useful for the public<sup> </sup>health by preventing or decreasing transmissibility, severe<sup> </sup>symptoms, complications, or death.

    <sup> </sup>
    An important issue is whether the 1203 vaccine offers cross-protection<sup> </sup>against other H5N1 strains of influenza A. A lethal human infection<sup> </sup>with an antigenically distinct influenza A (H5N1) strain is<sup> </sup>discussed elsewhere in this issue of the Journal.<sup>8</sup> From an immunologic<sup> </sup>standpoint, it is probable that more than one H5N1 vaccine will<sup> </sup>be needed. We know that the Indonesian clade 2 influenza A (H5N1)<sup> </sup>viruses are antigenically distinct from the clade 1 viruses<sup> </sup>from which the 1203 vaccine was developed. Preliminary evidence<sup> </sup>from serologic studies of laboratory-confirmed cases of influenza<sup> </sup>A (H5N1) infection also suggests that cross-protection between<sup> </sup>these two influenza A (H5N1) clades may be limited (Katz J:<sup> </sup>personal communication).
    Therefore, further studies are warranted<sup> </sup>to establish the level of cross-neutralizing antibody against<sup> </sup>heterologous influenza A (H5N1) viruses, such as those in clade<sup> </sup>2, that is generated by vaccination with the 1203 vaccine. Such<sup> </sup>cross-neutralization is of great importance, because at the<sup> </sup>current time, the 1203 vaccine is being stockpiled for use in<sup> </sup>the event of an influenza A (H5N1) pandemic. In any case, one<sup> </sup>candidate for a clade 2 vaccine is now available, and others<sup> </sup>are being developed by the WHO Influenza Network.

    <sup> </sup>
    Additional factors for which data are needed include differences<sup> </sup>in vaccine-induced immunity according to age, sex, immune status,<sup> </sup>and ethnic group. Some of these data could be derived from the<sup> </sup>results of Treanor et al. on further analysis. Age may be particularly<sup> </sup>important; those who have died in past pandemics and from influenza<sup> </sup>A (H5N1) infection are disproportionately children, adolescents,<sup> </sup>and young adults.

    <sup> </sup>
    Studies of different dose levels of vaccines administered with<sup> </sup>MF59 (a licensed adjuvant in Europe), aluminum hydroxide, or<sup> </sup>other adjuvants are urgently needed. We know from previous work<sup> </sup>that new hemagglutinin proteins (including H5) in people who<sup> </sup>have not been primed are poorly immunogenic.<sup>9</sup><sup>,</sup><sup>10</sup> In recognition<sup> </sup>of this fact, the Department of Health and Human Services and<sup> </sup>the National Institutes of Health have funded studies of more<sup> </sup>than 30 candidate vaccines. Early results from some of these<sup> </sup>trials should be available in the next 6 to 12 months. Previous<sup> </sup>studies of a new influenza A (H5N3) vaccine administered with<sup> </sup>MF59 adjuvant showed that vaccine administered without adjuvant<sup> </sup>was poorly immunogenic but that vaccine administered with MF59<sup> </sup>adjuvant in two doses, each as low as 7.5 µg, was highly<sup> </sup>immunogenic and resulted in cross-neutralizing antibodies against<sup> </sup>influenza A (H5N1).<sup>11</sup><sup>,</sup><sup>12</sup> Studies of an influenza A (H2N2) vaccine<sup> </sup>administered with alum adjuvant had similar results:
    hemagglutination-inhibition<sup> </sup>titers increased significantly at doses as low as 1.9 µg.<sup>9</sup><sup> </sup>


    The immediate development and testing of such antigen-sparing<sup> </sup>vaccines administered with adjuvant are imperative both to improve<sup> </sup>immunogenicity and to increase the number of doses available<sup> </sup>(if lower doses are effective). In addition, live attenuated<sup> </sup>cold-adapted influenza vaccines are safe, are immunogenic, and<sup> </sup>have the relevant advantage of cross-protection against heterologous<sup> </sup>influenza strains — suggesting a promising avenue to the<sup> </sup>development of pandemic vaccines. A contract for the development<sup> </sup>of such vaccines has been awarded to MedImmune. Other approaches<sup> </sup>to vaccine development involve DNA, adenovirus vectors,<sup>13</sup> and<sup> </sup>cell-culture manufacturing techniques to increase the speed<sup> </sup>and capacity of vaccine production. These approaches are promising,<sup> </sup>particularly since reverse-genetics reassortant vaccine candidates<sup> </sup>can be generated within weeks.<sup>14</sup>
    <sup>
    </sup>
    <sup> </sup>
    Thirty years ago, the United States attempted to respond to<sup> </sup>the threat of pandemic influenza with a vaccine approach. Now,<sup> </sup>armed with a greater understanding of the science, we have the<sup> </sup>capacity and the responsibility to embark on multiple, parallel<sup> </sup>avenues of vaccine development. In addition, we need efficient,<sup> </sup>rapid, high-yield, low-cost manufacturing innovations; the rapid<sup> </sup>generation of candidate vaccines for other, potentially pandemic<sup> </sup>influenza viruses (including emerging clade-2 influenza A [H5N1]<sup> </sup>viruses); and the rapid movement of those vaccines into clinical<sup> </sup>trials. In turn, this effort will require creativity along the<sup> </sup>entire pipeline: in the development and manufacture of candidate<sup> </sup>vaccines; the synchronization among countries of regulatory<sup> </sup>approaches; the resolution of issues concerning liability and<sup> </sup>intellectual property; ensuring the efficiency of clinical trials;<sup> </sup>and the use of methods to stockpile and rapidly deploy these<sup> </sup>vaccines. To do otherwise, with the pandemic clock ticking,<sup> </sup>could prove to be too little, too late.

    <sup> </sup>
    Dr. Poland reports serving as the chair of a data monitoring<sup> </sup>and safety board for an investigational trial of an influenza<sup> </sup>peptide vaccine being conducted by Merck Research Laboratories.<sup> </sup>No other potential conflict of interest relevant to this article<sup> </sup>was reported.<sup> </sup>

    Source Information
    From the Mayo Vaccine Research Group, the Program in Translational Immunovirology and Biodefense, and the Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, Minn.


    References
    1. <!-- null -->
    2. Congressional Budget Office. A potential influenza pandemic: possible macroeconomic effects and policy issues. December 8, 2005. (Accessed March 10, 2006, at http://www.dhhs.state.nh.us/DHHS/CDC...bo-economy.htm.)<!-- HIGHWIRE ID="354:13:1411:1" --> <!-- /HIGHWIRE --><!-- null -->
    3. World Health Organization. Avian influenza. (Accessed March 10, 2006, at http://www.who.int/csr/disease/avian_influenza/en/.)<!-- HIGHWIRE ID="354:13:1411:2" --><!-- /HIGHWIRE --><!-- null -->
    4. Buxton Bridges C, Katz JM, Seto WH, et al. Risk of influenza A (H5N1) infection among health care workers exposed to patients with influenza A (H5N1), Hong Kong. J Infect Dis 2000;181:344-348.<!-- HIGHWIRE ID="354:13:1411:3" --> [CrossRef][ISI][Medline]<!-- /HIGHWIRE --><!-- null -->
    5. Katz JM, Lim W, Bridges CB, et al. Antibody response in individuals infected with avian influenza A (H5N1) viruses and detection of anti-H5 antibody among household and social contacts. J Infect Dis 1999;180:1763-1770.<!-- HIGHWIRE ID="354:13:1411:4" --> [CrossRef][ISI][Medline]<!-- /HIGHWIRE --><!-- null -->
    6. Ungchusak K, Auewarakul P, Dowell SF, et al. Probable person-to-person transmission of avian influenza A (H5N1). N Engl J Med 2005;352:333-340.<!-- HIGHWIRE ID="354:13:1411:5" --> <nobr>[Abstract/Full Text]</nobr><!-- /HIGHWIRE --><!-- null -->
    7. de Jong MD, Thanh TT, Khanh TH, et al. Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med 2005;353:2667-2672.<!-- HIGHWIRE ID="354:13:1411:6" --> <nobr>[Abstract/Full Text]</nobr><!-- /HIGHWIRE --><!-- null -->
    8. Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M. Safety and immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. N Engl J Med 2006;354:1343-1351.<!-- HIGHWIRE ID="354:13:1411:7" --> <nobr>[Abstract/Full Text]</nobr><!-- /HIGHWIRE --><!-- null -->
    9. Shu Y, Yu H, Li D. Lethal avian influenza A (H5N1) infection in a pregnant woman in Anhui Province, China. N Engl J Med 2006;354:1421-1422.<!-- HIGHWIRE ID="354:13:1411:8" --> <nobr>[Full Text]</nobr><!-- /HIGHWIRE --><!-- null -->
    10. Hehme N, Engelmann H, Kuenzel W, Neumeier E, Saenger R. Immunogenicity of a monovalent, aluminum-adjuvanted influenza whole virus vaccine for pandemic use. Virus Res 2004;103:163-171.<!-- HIGHWIRE ID="354:13:1411:9" --> [CrossRef][ISI][Medline]<!-- /HIGHWIRE --><!-- null -->
    11. Hehme N, Engelmann H, Kuenzel W, Neumeier E, Saenger R. Pandemic preparedness: lessons learnt from H2N2 and H9N2 candidate vaccines. Med Microbiol Immunol (Berl) 2002;191:203-208.<!-- HIGHWIRE ID="354:13:1411:10" --> [CrossRef][ISI][Medline]<!-- /HIGHWIRE --><!-- null -->
    12. Nicholson KG, Colegate AE, Podda A, et al. Safety and antigenicity of non-adjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against H5N1 influenza. Lancet 2001;357:1937-1943.<!-- HIGHWIRE ID="354:13:1411:11" --> [CrossRef][ISI][Medline]<!-- /HIGHWIRE --><!-- null -->
    13. Stephenson I, Nicholson KG, Colegate A, et al. Boosting immunity to influenza H5N1 with MF59-adjuvanted H5N3 A/Duck/Singapore/97 vaccine in a primed human population. Vaccine 2003;21:1687-1693.<!-- HIGHWIRE ID="354:13:1411:12" --> [CrossRef][ISI][Medline]<!-- /HIGHWIRE --><!-- null -->
    14. Hoelscher MA, Garg S, Bangari DS, et al. Development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human H5N1 strains in mice. Lancet 2006;367:475-481.<!-- HIGHWIRE ID="354:13:1411:13" --> [CrossRef][ISI][Medline]<!-- /HIGHWIRE --><!-- null -->
    15. Wood JM, Robertson JS. From lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza. Nat Rev Microbiol 2004;2:842-847.<!-- HIGHWIRE ID="354:13:1411:14" --> [CrossRef][ISI][Medline]<!-- /HIGHWIRE -->

  • #2
    Re: New England Journal of Medicine

    New England Journal of Medicine - NEJM
    <table border="0" cellpadding="0" cellspacing="0" width="640"><tbody><tr><td nowrap="nowrap" valign="top">
    </td> <td align="center" valign="top"><table border="0" cellpadding="0" cellspacing="0"> <tbody><tr><th align="center" nowrap="nowrap" valign="top">Volume 354:1343-1351</th> <td nowrap="nowrap"></td> <th align="center" nowrap="nowrap" valign="top">March 30, 2006</th> <td nowrap="nowrap"></td> <th align="left" nowrap="nowrap" valign="top">Number 13</th></tr></tbody></table></td></tr></tbody></table>
    Safety and Immunogenicity of an Inactivated Subvirion Influenza A (H5N1) Vaccine
    <center> John J. Treanor, M.D., James D. Campbell, M.D., Kenneth M. Zangwill, M.D., Thomas Rowe, M.S., and Mark Wolff, Ph.D.

    ABSTRACT Background Influenza A (H5N1) viruses could cause a severe worldwide<sup> </sup>epidemic, with high attack rates, large numbers of deaths and<sup> </sup>hospitalizations, and wide disruption. Effective vaccines against<sup> </sup>these viruses in humans are urgently needed.

    <sup> </sup>

    Methods We conducted a multicenter, double-blind two-stage study<sup> </sup>involving 451 healthy adults 18 to 64 years of age who were<sup> </sup>randomly assigned in a 2:2:2:2:1 ratio to receive two intramuscular<sup> </sup>doses of a subvirion influenza A (H5N1) vaccine of 90, 45, 15,<sup> </sup>or 7.5 µg of hemagglutinin antigen or placebo. The subjects<sup> </sup>were followed for the safety analysis for 56 days. Serum samples<sup> </sup>obtained before each vaccination and again 28 days after the<sup> </sup>second vaccination were tested for H5 antibody by microneutralization<sup> </sup>and hemagglutination inhibition.

    <sup> </sup>

    Results Mild pain at the injection site was the most common<sup> </sup>adverse event for all doses of vaccine. The frequency of a serum<sup> </sup>antibody response was highest among subjects receiving doses<sup> </sup>of 45 µg or 90 µg. Among those who received two<sup> </sup>doses of 90 µg, neutralization antibody titers reached<sup> </sup>1:40 or greater in 54 percent, and hemagglutination-inhibition<sup> </sup>titers reached 1:40 or greater in 58 percent. Neutralization<sup> </sup>titers of 1:40 or greater were seen in 43 percent, 22 percent,<sup> </sup>and 9 percent of the subjects receiving two doses of 45, 15,<sup> </sup>and 7.5 µg, respectively. No responses were seen in placebo<sup> </sup>recipients.

    <sup> </sup>

    Conclusions A two-dose regimen of 90 µg of subvirion influenza<sup> </sup>A (H5N1) vaccine does not cause severe side effects and, in<sup> </sup>the majority of recipients, generates neutralizing antibody<sup> </sup>responses typically associated with protection against influenza.<sup> </sup>A conventional subvirion H5 influenza vaccine may be effective<sup> </sup>in preventing influenza A (H5N1) disease in humans. (ClinicalTrials.gov<sup> </sup>number, NCT00115986<!-- HIGHWIRE EXLINK_ID="354:13:1343:1" VALUE="NCT00115986" TYPEGUESS="CLINTRIALGOV" --> [ClinicalTrials.gov] <!-- /HIGHWIRE -->.)<sup> </sup>
    <sup> </sup>

    <hr>Avian influenza A viruses of the H5N1 subtype are currently<sup> </sup>causing widespread infections in bird populations throughout<sup> </sup>Southeast Asia, with spread into Central Asia, Africa, and Europe.<sup>1</sup><sup> </sup>There have been numerous instances of transmission of these<sup> </sup>viruses to humans, resulting in severe disease or death.<sup>2</sup>

    <sup> </sup> These viruses possess a new H5 subtype of hemagglutinin, against<sup> </sup>which at present there is little immunity in human populations.<sup> </sup>The viruses have the potential to cause extremely severe respiratory<sup> </sup>illness in humans, and of the 169 cases reported to the World<sup> </sup>Health Organization as of February 13, 2006, 91 (54 percent)<sup> </sup>have been fatal.<sup>3</sup> Many of the viruses isolated from humans have<sup> </sup>been found to be genotypically resistant to the adamantanes,<sup>4</sup><sup> </sup>and resistance to oseltamivir has also been described.<sup>5</sup> Although<sup> </sup>human-to-human transmission appears at present to be rare,<sup>6</sup><sup> </sup>the development of an effective vaccine against influenza A<sup> </sup>(H5N1) virus is a matter of considerable urgency.

    <sup> </sup>
    Inactivated influenza vaccines used annually for the control<sup> </sup>of human influenza are made from purified virions grown in embryonated<sup> </sup>hens' eggs and are formulated to contain not less than 15 µg<sup> </sup>of the hemagglutinin of the represented strains per 0.5-ml dose;<sup> </sup>the vaccine is administered intramuscularly without adjuvant.<sup> </sup>Efficacy in adults for the prevention of laboratory-confirmed<sup> </sup>influenza is typically in the range of 70 to 90 percent.<sup>7</sup><sup>,</sup><sup>8</sup><sup> </sup>An influenza A (H5N1) vaccine that contained a new antigen but<sup> </sup>was otherwise similar to licensed vaccine could be considered<sup> </sup>by regulatory authorities to represent a change in strain, rather<sup> </sup>than a completely new product, thereby facilitating the rapid<sup> </sup>licensure of the vaccine. Therefore, we evaluated an egg-grown,<sup> </sup>inactivated subvirion H5 vaccine administered without adjuvant.<sup> </sup>Because an immunologically naive population would probably require<sup> </sup>a two-dose schedule of administration, as is used in children,<sup> </sup>and might require higher doses than are used in annual influenza<sup> </sup>vaccinations, we evaluated a two-dose regimen, with doses ranging<sup> </sup>from 7.5 to 90 µg.

    <sup> </sup>
    Methods
    Vaccine
    The seed virus for the production of the experimental influenza<sup> </sup>A (H5N1) vaccine was generated from the human isolate influenza<sup> </sup>A/Vietnam/1203/2004 (H5N1) virus with the use of a plasmid rescue<sup> </sup>system.<sup>9</sup> The gene segments encoding the hemagglutinin and neuraminidase<sup> </sup>were derived from the A/Vietnam/2004 virus, and all other genes<sup> </sup>were derived from the A/PR/8/34 virus, a laboratory strain commonly<sup> </sup>used as a platform for influenza vaccines. The hemagglutinin<sup> </sup>gene was further modified to replace the stretch of six basic<sup> </sup>amino acids at the cleavage site between hemagglutinin 1 and<sup> </sup>hemagglutinin 2, associated with high pathogenicity in birds<sup> </sup>with an avirulent avian sequence. The resulting influenza rgA/Vietnam/1203/2004xA/PR/8/34<sup> </sup>influenza (H5N1) virus was antigenically identical to the wild-type<sup> </sup>A/Vietnam/1203/2004 virus, reached high titers in eggs, and<sup> </sup>was apathogenic in chickens, allowing the virus to be handled<sup> </sup>under the containment conditions of a biosafety level 2 laboratory,<sup> </sup>with additional safety precautions.

    <sup> </sup>
    The vaccine product was generated according to standard techniques.<sup> </sup>The seed virus was grown to a high titer in eggs, the virions<sup> </sup>were purified by means of centrifugation, inactivated with the<sup> </sup>use of formalin, disrupted with the use of Triton X-100, and<sup> </sup>filtered to remove bacteria. The vaccine underwent further purification<sup> </sup>and was formulated at concentrations of 90 µg of hemagglutinin<sup> </sup>protein per milliliter (lot U10915C) and 30 µg of hemagglutinin<sup> </sup>protein per milliliter (lot U10914C) in vials containing 0.7<sup> </sup>ml without preservative. The content of immunologically active<sup> </sup>hemagglutinin in the final formulation was determined with the<sup> </sup>use of single-radial-immunodiffusion. Sheep antiserum to bromelain-cleaved<sup> </sup>native H5 hemagglutinin was used in the agar. Placebo consisted<sup> </sup>of normal saline. Both vaccine and placebo were stored at 4°C<sup> </sup>until use.

    <sup> </sup>
    Study Design
    We conducted a randomized, placebo-controlled, double-blind,<sup> </sup>multicenter trial. Written informed consent was obtained from<sup> </sup>potential subjects. Healthy adults 18 to 64 years of age were<sup> </sup>carefully screened for the absence of any chronic illnesses<sup> </sup>(enrollment criteria are described in detail in the Supplementary Appendix,<sup> </sup>available with the full text of this article at www.nejm.org).<sup> </sup>Eligible subjects were randomly assigned in a 2:2:2:2:1 ratio<sup> </sup>to receive two doses of vaccine at a dose of 90, 45, 15, or<sup> </sup>7.5 µg or placebo. Each dose was administered intramuscularly<sup> </sup>into the deltoid muscle, and the two doses were given 28 days<sup> </sup>apart. A permuted-block randomization was used, and subjects<sup> </sup>were stratified according to study site, age (18 to 39 years<sup> </sup>vs. 40 to 64 years), and history of receipt of the 2004 formulation<sup> </sup>of licensed influenza vaccine. All vaccinations were administered<sup> </sup>by a clinician who was not involved in the assessment of adverse<sup> </sup>events or the laboratory follow-up, and the contents of the<sup> </sup>syringe were shielded from the subject's view. The subjects<sup> </sup>were observed for 30 minutes after the receipt of each dose<sup> </sup>for adverse events, and for the next seven days, they recorded<sup> </sup>the presence and severity of local symptoms (pain and tenderness)<sup> </sup>and systemic symptoms (feverishness, malaise, myalgia, headache,<sup> </sup>and nausea) and oral temperature. Subjects used a standard scale<sup> </sup>to grade side effects during this seven-day period, in which<sup> </sup>symptoms were considered mild if they did not interfere with<sup> </sup>normal activities, moderate if they resulted in some interference<sup> </sup>with normal activities, and severe if they prevented subjects<sup> </sup>from engaging in normal daily activities.

    <sup> </sup>
    The subjects' observations were reviewed by members of the study<sup> </sup>staff on day 7 after each vaccination, and the medical history<sup> </sup>and record of adverse events occurring during the interval were<sup> </sup>also reviewed on days 28 and 56. Samples of serum for the assessment<sup> </sup>of antibody responses were obtained before each vaccination<sup> </sup>and again 28 days after the second vaccination.

    <sup> </sup>
    The study was conducted in two stages. In the first stage, 118<sup> </sup>subjects were enrolled and followed, as described. In addition,<sup> </sup>hemoglobin levels, total white-cell count, platelet count, creatinine<sup> </sup>levels, and serum alanine aminotransferase levels were determined<sup> </sup>in each subject before vaccination and on day 7 after each vaccination.<sup> </sup>After the safety data through the seven days after the first<sup> </sup>dose were reviewed by an independent data and safety monitoring<sup> </sup>board, the remaining 333 subjects were enrolled and treated,<sup> </sup>as described, but without the additional laboratory measurements.
    <sup> </sup>A second review of the initial group of 118 subjects was performed<sup> </sup>before the second dose was administered to the remaining subjects.<sup> </sup>

    Laboratory Assays
    Microneutralization assays and hemagglutination-inhibition assays<sup> </sup>were performed at a central laboratory (Southern Research Institute)<sup> </sup>with the use of the influenza rgA/Vietnam/1203/2004xA/PR/8/34<sup> </sup>influenza (H5N1) vaccine. In addition, a subgroup of samples<sup> </sup>were also tested with the use of the wild-type influenza A/Vietnam/1203/2004<sup> </sup>virus under conditions of enhanced biocontainment (biosafety<sup> </sup>level 3-plus laboratory).
    Microneutralization assays were performed<sup> </sup>as described previously.<sup>10</sup><sup>,</sup><sup>11</sup> Serum samples were tested at an<sup> </sup>initial dilution of 1:20, and those that were negative were<sup> </sup>assigned a titer of 10. Serum samples were tested separately<sup> </sup>and in duplicate; if the results showed a difference by a factor<sup> </sup>of 2, the samples were retested.

    <sup> </sup>
    Hemagglutination-inhibition assays were performed according<sup> </sup>to established procedures,<sup>12</sup><sup>,</sup><sup>13</sup> with the use of horse erythrocytes.<sup> </sup>After treatment with receptor-destroying enzyme to remove nonspecific<sup> </sup>inhibitors of agglutination, the serum samples were tested at<sup> </sup>an initial dilution of 1:20.

    <sup> </sup>
    Statistical Analysis
    The prespecified primary immunologic end point of the trial<sup> </sup>was the proportion of subjects in each group categorized according<sup> </sup>to dose of vaccine or receipt of placebo in whom a neutralizing<sup> </sup>titer of 1:40 or greater developed against the rgA/Vietnam/1203/2004<sup> </sup>virus at day 28 after the administration of the second dose<sup> </sup>of vaccine. The geometric mean of duplicate results for each<sup> </sup>specified time was used for the calculation. Exact (Clopper–Pearson)<sup> </sup>confidence intervals are reported for all proportional end points.<sup> </sup>Geometric mean titers of antibody and their confidence intervals<sup> </sup>were computed by transforming the results to a logarithmic scale,<sup> </sup>assuming asymptotic normality conditions were satisfied on the<sup> </sup>scale and converting back to the original scale.

    <sup> </sup>
    Rates of reactogenicity after each vaccination were based on<sup> </sup>the most severe response reported. The rates were compared by<sup> </sup>an exact linear-by-linear–association test (with the use<sup> </sup>of antigen dose levels and evenly spaced scores for reactogenicity).<sup> </sup>The overall comparison between vaccine and placebo for reactogenicity<sup> </sup>was based on an exact permutation test in which reactogenicity<sup> </sup>was dichotomized as none to mild or moderate to severe. The<sup> </sup>antibody dose–response relationship was assessed with<sup> </sup>the use of a general linear model. Comparisons of geometric<sup> </sup>mean titer between groups were performed with the use of the<sup> </sup>Wilcoxon rank-sum test, and response rates were compared with<sup> </sup>the use of the Mantel–Haenszel chi-square test. Spearman's<sup> </sup>correlation coefficient was used to assess the correlation in<sup> </sup>neutralization titers to different viruses. All reported P values<sup> </sup>are two-sided. StatXact software, version 6.1 (Statistical Solutions),<sup> </sup>was used to compute the exact tests. All other data manipulations<sup> </sup>and statistical computations were conducted with SAS software,<sup> </sup>version 8.2.<sup> </sup>
    The sample size for this study (100 subjects in each vaccine<sup> </sup>group and 50 subjects in the control [saline] group) was selected<sup> </sup>to provide a robust initial safety database as well as some<sup> </sup>information on the dose-related immune response in a timely<sup> </sup>fashion. Given the enrollment of 100 subjects in each vaccine<sup> </sup>group, the half-width of a 95 percent confidence interval for<sup> </sup>any observed event rate is no greater than 10 percent. In addition,<sup> </sup>the binomial probability of detecting three or more events is<sup> </sup>88.2 percent when the true event rates are 5 percent or higher.

    <sup> </sup>
    The study was designed jointly by the investigators and the<sup> </sup>program staff at the National Institute of Allergy and Infectious<sup> </sup>Diseases (NIAID) (the NIAID influenza team), and Dr. Treanor<sup> </sup>served as the principal investigator. The study was approved<sup> </sup>by the institutional review boards of the University of Rochester,<sup> </sup>University of Maryland, and the Los Angeles Biomedical Research<sup> </sup>Institute at Harbor–UCLA Medical Center. The vaccine product<sup> </sup>was manufactured by Sanofi Pasteur under contract to NIAID,<sup> </sup>but Sanofi had no role in the conduct of the study or the preparation<sup> </sup>of this report. The article was written jointly by the investigators.<sup> </sup>Dr. Treanor assumed responsibility for the writing and preparation<sup> </sup>of the manuscript and vouches for its accuracy and completeness.

    <sup> </sup>
    Results
    In the first stage of the study, 118 subjects received the first<sup> </sup>dose of vaccine or placebo, and 117 of these subjects received<sup> </sup>the second dose 28 days later. Enrollment in the first stage<sup> </sup>occurred in April 2005, with 28 subjects randomly assigned to<sup> </sup>receive 90 µg, 25 subjects assigned to 45 µg, 25<sup> </sup>assigned to 15 µg, 28 assigned to 7.5 µg, and 12<sup> </sup>assigned to placebo. One subject was unable to complete the<sup> </sup>study because of military service. After a review of the safety<sup> </sup>data by the independent data and safety monitoring board, the<sup> </sup>remaining 333 subjects were enrolled and received the first<sup> </sup>of the two doses. Enrollment in the second stage occurred in<sup> </sup>May 2005, and 75 subjects were randomly assigned to receive<sup> </sup>90 µg, 73 were assigned to 45 µg, 76 were assigned<sup> </sup>to 15 µg, 73 were assigned to 7.5 µg, and 36 were<sup> </sup>assigned to placebo. Of these subjects, 13 did not receive the<sup> </sup>second dose, 11 because they were unable to make the follow-up<sup> </sup>visits, were unable to maintain compliance, or were ineligible<sup> </sup>and 2 because of adverse events. Two subjects were excluded<sup> </sup>from the immunogenicity analysis because they had participated<sup> </sup>in a previous study evaluating an H5 influenza vaccine. (A diagram<sup> </sup>of the disposition of the study subjects is available in the<sup> </sup>Supplementary Appendix.)

    <sup> </sup>
    The median age of the enrolled subjects was 39 years (range,<sup> </sup>18 to 64), and 54 percent of subjects were female; 79 percent<sup> </sup>of subjects were white, 11 percent were Asian, and 8 percent<sup> </sup>were black. Race was self-reported. Of the subjects, 42 percent<sup> </sup>had received conventional influenza vaccine in the previous<sup> </sup>year. The subjects' ages and demographic characteristics were<sup> </sup>similar in each of the study groups, but there were more women<sup> </sup>in the group assigned to 15 µg (as shown in Table 1 in<sup> </sup>the Supplementary Appendix).

    <sup> </sup>
    Safety Analysis
    The rates of symptoms reported during the first seven days after<sup> </sup>administration of each dose of vaccine are shown in Figure 1.<sup> </sup>Generally, the vaccine was well tolerated at all doses, and<sup> </sup>84 percent of all reported symptoms were graded as mild by the<sup> </sup>subjects. There was no indication that the frequency or severity<sup> </sup>of either local or systemic symptoms was greater after the second<sup> </sup>dose than after the first dose, and there were no instances<sup> </sup>of anaphylaxis, hives, or other serious allergic reactions.<sup> </sup>
    <!-- null --> <table cellpadding="0" cellspacing="0"><tbody><tr bgcolor="#e8e8d1"><td><table cellpadding="2" cellspacing="2"><tbody><tr bgcolor="#e8e8d1"><td align="center" bgcolor="#ffffff" valign="top">
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    </nobr> </td><td align="left" valign="top"> Figure 1. Rates of Local and Systemic Adverse Events during the Seven Days after Receipt of the First Dose (Panel A) or the Second Dose (Panel B) of Vaccine. Subjects used a subjective scale to grade adverse events. Symptoms were considered mild if they did not interfere with normal activities, moderate if they resulted in some interference with normal activities, and severe if they prevented subjects from carrying out normal daily activities.

    </td></tr></tbody></table></td></tr></tbody></table>
    The frequencies of pain and local tenderness at the injection<sup> </sup>site after each dose were greater among vaccine recipients than<sup> </sup>placebo recipients in a dose-dependent manner (P<0.001).<sup> </sup>In addition, moderate pain and tenderness were reported almost<sup> </sup>exclusively among recipients of the 90-µg dose. In general,<sup> </sup>reports of local pain were not accompanied by objective findings<sup> </sup>of erythema or swelling at the injection site. There were no<sup> </sup>severe local reactions.

    <sup> </sup>
    Systemic symptoms were relatively less common after either dose<sup> </sup>in all study groups and were not dependent on the dose; the<sup> </sup>frequencies of reports of feverishness, malaise, myalgia, headache,<sup> </sup>and nausea in all groups did not differ significantly from those<sup> </sup>in the placebo group (P>0.05). Eleven subjects reported fever<sup> </sup>(temperature, 37.8°C; maximum, 38.2°C) after vaccination:<sup> </sup>9 after the first dose (2 in the placebo group, 1 in the 7.5-µg<sup> </sup>group, 3 in the 15-µg group, and 3 in the 90-µg<sup> </sup>group) and 2 subjects after the second dose (1 in the placebo<sup> </sup>group and 1 in the 45-µg group). Clinical laboratory safety<sup> </sup>testing of subjects during the first stage did not reveal clinically<sup> </sup>significant abnormalities (described in the Supplementary Appendix).

    <sup> </sup>
    In one vaccine recipient, a rash developed after receipt of<sup> </sup>the first dose, and the subject did not receive the second dose.<sup> </sup>This subject noticed a nonpruritic, maculopapular rash over<sup> </sup>the abdomen and upper arms bilaterally on day 5 after the first<sup> </sup>dose of vaccine, without involvement of the face, hands or feet,<sup> </sup>or mucous membranes. The rash faded and resolved completely<sup> </sup>by day 42. Because the cause of the rash was unclear, the investigator<sup> </sup>elected not to administer the second dose of vaccine. This subject<sup> </sup>had no history of reaction to influenza vaccine, including rash.<sup> </sup>
    There was one serious adverse event in the study, but it was<sup> </sup>judged by investigators to be unrelated to vaccination. A 52-year-old<sup> </sup>man in the second stage of the study died 24 days after receipt<sup> </sup>of the first dose of 45 µg of vaccine. He had a history<sup> </sup>of alcohol abuse that had not been revealed on enrollment, and<sup> </sup>the subject was noted to be consuming alcohol heavily. Autopsy<sup> </sup>revealed marked steatosis of the liver, and the death was determined<sup> </sup>by the medical examiner to be due to chronic alcoholism. (Additional<sup> </sup>information on this event is included in the Supplementary Appendix.)

    <sup> </sup>
    Immunogenicity Analysis
    The results of immunogenicity testing with the use of both hemagglutination<sup> </sup>inhibition and microneutralization are shown in Table 1 and<sup> </sup>Figure 2. As expected, in the majority of subjects, antibody<sup> </sup>against the A/Vietnam/2004 virus was not detected by either<sup> </sup>method before immunization, although 15 subjects (3 percent)<sup> </sup>had a positive hemagglutination-inhibition test, and 12 (3 percent)<sup> </sup>had a positive microneutralization test. The reasons for these<sup> </sup>positive results are unknown, because none of the subjects reported<sup> </sup>exposures that would be likely to result in H5 virus infection,<sup> </sup>and preliminary analysis has not suggested any relationship<sup> </sup>between H5 antibody and antibody against conventional human<sup> </sup>influenza viruses.<sup> </sup>
    <!-- null --> <table cellpadding="0" cellspacing="0"><tbody><tr bgcolor="#e8e8d1"><td><table cellpadding="2" cellspacing="2"><tbody><tr bgcolor="#e8e8d1"><td align="center" bgcolor="#ffffff" valign="top"> View this table:
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    </nobr> </td><td align="left" valign="top"> Table 1. Geometric Mean Titers (GMT) of Antibody against the Influenza A/Vietnam/1203/2004 (H5N1) Virus in Subjects Receiving Two Doses of Vaccine, as Assessed by Hemagglutination Inhibition or Microneutralization.
    </td></tr></tbody></table></td></tr></tbody></table>
    <!-- null --> <table cellpadding="0" cellspacing="0"><tbody><tr bgcolor="#e8e8d1"><td><table cellpadding="2" cellspacing="2"><tbody><tr bgcolor="#e8e8d1"><td align="center" bgcolor="#ffffff" valign="top">
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    </nobr> </td><td align="left" valign="top"> Figure 2. Reverse Cumulative Distribution Curves for Serum Samples Collected 28 Days after the Second Vaccination. The proportions of subjects are based on the total number of subjects tested. Panel A shows the results of hemagglutination-inhibition testing with the use of horse erythrocytes. Panel B shows the results of microneutralization testing.

    </td></tr></tbody></table></td></tr></tbody></table>
    There was a very clear dose–response relationship with<sup> </sup>the use of either assay (P<0.001), with a large difference<sup> </sup>in response between the groups receiving vaccine at doses of<sup> </sup>45 µg or 90 µg and those receiving lower doses.<sup> </sup>Only the 90-µg dose was associated with antibody responses<sup> </sup>(increase in antibody titer by a factor of 4 or more) in either<sup> </sup>hemagglutination-inhibition or microneutralization assays in<sup> </sup>more than half the subjects (Table 1). Two doses of 45 µg<sup> </sup>also resulted in antibody responses in a substantial proportion<sup> </sup>of subjects, whereas lower doses of vaccine were much less immunogenic.<sup> </sup>Similarly, there were substantially higher geometric mean titers<sup> </sup>of both hemagglutination inhibition and microneutralization<sup> </sup>antibody after vaccination in the group receiving 90 µg,<sup> </sup>and there were significantly lower titers of both antibodies<sup> </sup>in the groups receiving lower doses of vaccine (P<0.001).

    <sup> </sup>
    The prespecified primary immunogenicity end point chosen for<sup> </sup>this study was the development of a microneutralization titer<sup> </sup>of 1:40 or greater after two doses of vaccine. Figure 2 shows<sup> </sup>the distribution of antibody titers according to the hemagglutination-inhibition<sup> </sup>assay (Figure 2A) and microneutralization assay (Figure 2B)<sup> </sup>after vaccination in each group categorized according to dose.<sup> </sup>Only in the group receiving the 90-µg dose was the primary<sup> </sup>end point reached by more than 50 percent of the recipients.<sup> </sup>In this group, 54 percent of the subjects (95 percent confidence<sup> </sup>interval, 43 to 64 percent) had microneutralization titers of<sup> </sup>1:40 or greater and 58 percent (95 percent confidence interval,<sup> </sup>47 to 67 percent) had hemagglutination-inhibition titers of<sup> </sup>1:40 or greater. The frequency of both these end points in the<sup> </sup>group receiving the 90-µg dose was significantly greater<sup> </sup>than in the other vaccine groups (P<0.001). Microneutralization<sup> </sup>titers of 1:20 or greater were seen in 70 percent of the group<sup> </sup>receiving the 90-µg dose and 57 percent of the group receiving<sup> </sup>the 45-µg dose, but in only 32 percent of the group receiving<sup> </sup>the 15-µg dose and 25 percent of the group receiving the<sup> </sup>7.5-µg dose.

    <sup> </sup>
    Because the highly pathogenic wild-type influenza A/Vietnam/1203/2004<sup> </sup>virus can be manipulated only under strict conditions of biocontainment,<sup> </sup>the majority of serologic tests used the antigenically identical<sup> </sup>but apathogenic influenza rgA/Vietnam/1203/2004xA/PR/8/34 vaccine<sup> </sup>virus. The ability of serum samples from this study to neutralize<sup> </sup>the wild-type virus was confirmed in a subgroup of 63 samples<sup> </sup>obtained on day 56 from randomly selected specimens representing<sup> </sup>a spectrum of antibody titers, which were weighted toward higher<sup> </sup>responses to the apathogenic vaccine virus and assayed against<sup> </sup>the wild-type virus. The agreement in the antibody titers assayed<sup> </sup>against the two viruses was good, with a Spearman's correlation<sup> </sup>coefficient of 0.74 (P<0.001). Of the 53 samples tested in<sup> </sup>which the titers of antibody against the vaccine virus were<sup> </sup>greater than 1:40, 51 also had titers of antibody against the<sup> </sup>wild-type virus of more than 1:40.

    <sup> </sup>
    Discussion
    This study demonstrates that is it possible to generate immunity<sup> </sup>against H5 influenza with the use of a purified, subvirion vaccine<sup> </sup>administered in two relatively high doses. Our results are similar<sup> </sup>to those observed in a study conducted with the use of a purified,<sup> </sup>recombinant H5 hemagglutinin in humans,<sup>14</sup> in which intramuscular<sup> </sup>administration of two doses of approximately 90 µg each<sup> </sup>of a baculovirus-expressed recombinant H5 hemagglutinin resulted<sup> </sup>in neutralizing antibody titers of 1:80 or greater in 56 percent<sup> </sup>of healthy adult recipients, whereas lower doses were considerably<sup> </sup>less immunogenic. Although that study used a different vaccine<sup> </sup>and slightly different assays to measure immune responses, both<sup> </sup>those results and ours show that high doses of relatively purified<sup> </sup>protein vaccines were required to induce immunity in most recipients.

    <sup> </sup>
    The interpretation of the significance of these findings should<sup> </sup>be done in the context of our current understanding of immunity<sup> </sup>to H5 viruses. Our decision to use a neutralizing antibody titer<sup> </sup>of 1:40 as the primary immunogenicity end point was not based<sup> </sup>on observations of antibody-mediated protection in the field<sup> </sup>but, rather, on the development of criteria that appeared to<sup> </sup>distinguish between infected and uninfected persons during serologic<sup> </sup>surveys performed during the previous outbreak of H5 virus infection<sup> </sup>in 1997.<sup>15</sup> It is possible that lower titers of neutralizing<sup> </sup>antibody could be associated with protection, as has been observed<sup> </sup>in studies of conventional human influenza viruses.<sup>16</sup><sup>,</sup><sup>17</sup><sup>,</sup><sup>18</sup><sup>,</sup><sup>19</sup><sup> </sup>The development of a sensitive hemagglutination-inhibition assay<sup> </sup>for H5 viruses<sup>12</sup> should facilitate further studies, but the<sup> </sup>level of antibody associated with protection in this assay has<sup> </sup>yet to be determined.<sup> </sup>
    On the basis of these preliminary data, a two-dose schedule<sup> </sup>of 90 µg of subvirion H5 vaccine would probably have an<sup> </sup>acceptable tolerability profile and could be effective in preventing<sup> </sup>H5 influenza in healthy adult recipients. Elderly persons, persons<sup> </sup>with impaired immunity, or children may have a different response,<sup> </sup>and trials of the vaccine in these populations are in progress.<sup> </sup>Production of the vaccine and this clinical trial are important<sup> </sup>steps toward control of a pandemic, and the current vaccine<sup> </sup>would probably be acceptable for licensure, if needed. However,<sup> </sup>the need for a vaccine with a total dose of 180 µg would<sup> </sup>pose a considerable barrier to rapid production of a supply<sup> </sup>that would be adequate to meet the world's requirements should<sup> </sup>a pandemic occur. Therefore, dose-sparing approaches should<sup> </sup>be pursued aggressively. These approaches could include the<sup> </sup>use of adjuvants such as aluminum<sup>20</sup> or MF59<sup>21</sup> and the use of<sup> </sup>intradermal administration of vaccine,<sup>22</sup><sup>,</sup><sup>23</sup> both of which have<sup> </sup>been reported to be potentially dose sparing for influenza vaccines<sup> </sup>in small studies. In addition, the recent demonstration of a<sup> </sup>substantial increase in the immune response when a third dose<sup> </sup>of H5 vaccine was administered to subjects 16 months after a<sup> </sup>primary series<sup>24</sup> suggests that another strategy for improving<sup> </sup>the immune response would be prepriming, perhaps by including<sup> </sup>an H5 component in the annual vaccine. Combinations of these<sup> </sup>approaches may be needed. Finally, live attenuated vaccines<sup> </sup>are being developed. As the results of studies to evaluate each<sup> </sup>of these options become available, our results may be useful<sup> </sup>for comparison.

    <sup> </sup>
    <sup> </sup>
    <sup> </sup>
    Supported by contracts with the National Institute of Allergy<sup> </sup>and Infectious Diseases (N01 AI 25460 to Dr. Treanor, AI 25461<sup> </sup>to Dr. Campbell, AI 25463 to Dr. Zangwill, and AI 30068 to Dr.<sup> </sup>Rowe) and grants from the federal General Clinical Research<sup> </sup>Center (M01 RR00044 to Dr. Treanor, M01 RR165001 to Dr. Campbell,<sup> </sup>and M01-RR00425 to Dr. Zangwill). None of the authors report<sup> </sup>having received financial support from Sanofi Pasteur. Dr. Treanor<sup> </sup>reports having received or applying for grant support from Merck,<sup> </sup>Protein Sciences, VaxGen, AlphaVax, ID Biomedical, EpiVax, and<sup> </sup>PowderMed. No other potential conflict of interest relevant<sup> </sup>to this article was reported.

    <sup> </sup>
    We are indebted to the many subinvestigators, study nurses,<sup> </sup>and technicians who contributed to this study, including: C.<sup> </sup>Mhorag Hay, Diane O'Brien, and Carrie Nolan (University of Rochester);<sup> </sup>Wilbur Chen and Mary Lou Mullen (University of Maryland Center<sup> </sup>for Vaccine Development); Midi Mikasa, Pat Chatfield, Sacred<sup> </sup>Cartwright, Susan Partridge, Elizabeth Moore, Swei-ju Chang,<sup> </sup>Rita Manai, and Merlyn Dubria (Los Angeles Biomedical Research<sup> </sup>Institute); Michael McDowell, Lisa Slappey, Franziska Rosser,<sup> </sup>Diana Noah, Lucile White, and Mindy Sosa (Southern Research<sup> </sup>Institute); Fred Batzold (Clinical Trials Management, Division<sup> </sup>of Microbiology and Infectious Diseases, National Institutes<sup> </sup>of Health); John Coleman, Erich Hoffman, and Richard Webby (St.<sup> </sup>Jude Children's Research Hospital); Richard Hjorth, Desirée<sup> </sup>Taylor, and Charles Whittaker (Sanofi Pasteur); Heather Hill,<sup> </sup>Jill Kissel, Dewei She, Bernadette Jolles, and Ken Wilkins (EMMES);<sup> </sup>and Jean Hu-Primmer, Emily Kough, Linda Lambert, Pamela McInnes,<sup> </sup>Katherine Muth, and Shy Shorer (National Institute of Allergy<sup> </sup>and Infectious Diseases Influenza Team).<sup> </sup>

    Source Information
    From the Department of Medicine, University of Rochester, Rochester, N.Y.(J.J.T.); the Center for Vaccine Development, University of Maryland School of Medicine, Baltimore (J.D.C.); the Los Angeles Biomedical Research Institute and UCLA Center for Vaccine Research, Harbor–UCLA Medical Center, Los Angeles (K.M.Z.); Southern Research Institute, Birmingham, Ala. (T.R.); and EMMES, Rockville, Md. (M.W.).
    Address reprint requests to Dr. Treanor at the Department of Medicine, University of Rochester Medical Center, 601 Elmwood Ave., Rm. 3-6309, Rochester, NY 14642, or at john_treanor@urmc.rochester.edu<script type="text/javascript"><!-- var u = "john_treanor", d = "urmc.rochester.edu"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></script>.


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