An open‐label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults
Identifieur interne : 000977 ( Pmc/Corpus ); précédent : 000976; suivant : 000978An open‐label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults
Auteurs : Kawsar R. Talaat ; Ruth A. Karron ; Philana H. Liang ; Bridget A. Mcmahon ; Catherine J. Luke ; Bhagvanji Thumar ; Grace L. Chen ; Ji-Young Min ; Elaine W. Lamirande ; Hong Jin ; Kathy L. Coelingh ; George W. Kemble ; Kanta SubbaraoSource :
- Influenza and Other Respiratory Viruses [ 1750-2640 ] ; 2012.
Abstract
Url:
DOI: 10.1111/j.1750-2659.2012.00350.x
PubMed: 22417012
PubMed Central: 3527634
Links to Exploration step
PMC:3527634Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">An open‐label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults</title><author><name sortKey="Talaat, Kawsar R" sort="Talaat, Kawsar R" uniqKey="Talaat K" first="Kawsar R." last="Talaat">Kawsar R. Talaat</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Karron, Ruth A" sort="Karron, Ruth A" uniqKey="Karron R" first="Ruth A." last="Karron">Ruth A. Karron</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Liang, Philana H" sort="Liang, Philana H" uniqKey="Liang P" first="Philana H." last="Liang">Philana H. Liang</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Mcmahon, Bridget A" sort="Mcmahon, Bridget A" uniqKey="Mcmahon B" first="Bridget A." last="Mcmahon">Bridget A. Mcmahon</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Luke, Catherine J" sort="Luke, Catherine J" uniqKey="Luke C" first="Catherine J." last="Luke">Catherine J. Luke</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Thumar, Bhagvanji" sort="Thumar, Bhagvanji" uniqKey="Thumar B" first="Bhagvanji" last="Thumar">Bhagvanji Thumar</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Chen, Grace L" sort="Chen, Grace L" uniqKey="Chen G" first="Grace L." last="Chen">Grace L. Chen</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Min, Ji Oung" sort="Min, Ji Oung" uniqKey="Min J" first="Ji-Young" last="Min">Ji-Young Min</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Lamirande, Elaine W" sort="Lamirande, Elaine W" uniqKey="Lamirande E" first="Elaine W." last="Lamirande">Elaine W. Lamirande</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Jin, Hong" sort="Jin, Hong" uniqKey="Jin H" first="Hong" last="Jin">Hong Jin</name><affiliation><nlm:aff id="a3"></nlm:aff></affiliation></author><author><name sortKey="Coelingh, Kathy L" sort="Coelingh, Kathy L" uniqKey="Coelingh K" first="Kathy L." last="Coelingh">Kathy L. Coelingh</name><affiliation><nlm:aff id="a3"></nlm:aff></affiliation></author><author><name sortKey="Kemble, George W" sort="Kemble, George W" uniqKey="Kemble G" first="George W." last="Kemble">George W. Kemble</name><affiliation><nlm:aff id="a3"></nlm:aff></affiliation></author><author><name sortKey="Subbarao, Kanta" sort="Subbarao, Kanta" uniqKey="Subbarao K" first="Kanta" last="Subbarao">Kanta Subbarao</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22417012</idno><idno type="pmc">3527634</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527634</idno><idno type="RBID">PMC:3527634</idno><idno type="doi">10.1111/j.1750-2659.2012.00350.x</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">000977</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000977</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">An open‐label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults</title><author><name sortKey="Talaat, Kawsar R" sort="Talaat, Kawsar R" uniqKey="Talaat K" first="Kawsar R." last="Talaat">Kawsar R. Talaat</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Karron, Ruth A" sort="Karron, Ruth A" uniqKey="Karron R" first="Ruth A." last="Karron">Ruth A. Karron</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Liang, Philana H" sort="Liang, Philana H" uniqKey="Liang P" first="Philana H." last="Liang">Philana H. Liang</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Mcmahon, Bridget A" sort="Mcmahon, Bridget A" uniqKey="Mcmahon B" first="Bridget A." last="Mcmahon">Bridget A. Mcmahon</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Luke, Catherine J" sort="Luke, Catherine J" uniqKey="Luke C" first="Catherine J." last="Luke">Catherine J. Luke</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Thumar, Bhagvanji" sort="Thumar, Bhagvanji" uniqKey="Thumar B" first="Bhagvanji" last="Thumar">Bhagvanji Thumar</name><affiliation><nlm:aff id="a1"></nlm:aff></affiliation></author><author><name sortKey="Chen, Grace L" sort="Chen, Grace L" uniqKey="Chen G" first="Grace L." last="Chen">Grace L. Chen</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Min, Ji Oung" sort="Min, Ji Oung" uniqKey="Min J" first="Ji-Young" last="Min">Ji-Young Min</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Lamirande, Elaine W" sort="Lamirande, Elaine W" uniqKey="Lamirande E" first="Elaine W." last="Lamirande">Elaine W. Lamirande</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author><author><name sortKey="Jin, Hong" sort="Jin, Hong" uniqKey="Jin H" first="Hong" last="Jin">Hong Jin</name><affiliation><nlm:aff id="a3"></nlm:aff></affiliation></author><author><name sortKey="Coelingh, Kathy L" sort="Coelingh, Kathy L" uniqKey="Coelingh K" first="Kathy L." last="Coelingh">Kathy L. Coelingh</name><affiliation><nlm:aff id="a3"></nlm:aff></affiliation></author><author><name sortKey="Kemble, George W" sort="Kemble, George W" uniqKey="Kemble G" first="George W." last="Kemble">George W. Kemble</name><affiliation><nlm:aff id="a3"></nlm:aff></affiliation></author><author><name sortKey="Subbarao, Kanta" sort="Subbarao, Kanta" uniqKey="Subbarao K" first="Kanta" last="Subbarao">Kanta Subbarao</name><affiliation><nlm:aff id="a2"></nlm:aff></affiliation></author></analytic><series><title level="j">Influenza and Other Respiratory Viruses</title><idno type="ISSN">1750-2640</idno><idno type="eISSN">1750-2659</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Please cite this paper as:</italic> Talaat <italic>et al.</italic> (2012) An open‐label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750‐2659.2012.00350.x.</p><p><bold>Background </bold> Live attenuated influenza vaccines (LAIV) against a variety of strains of pandemic potential are being developed and tested. We describe the results of an open‐label phase I trial of a live attenuated H2N2 virus vaccine.</p><p><bold>Objectives </bold> To evaluate the safety, infectivity, and immunogenicity of a live attenuated H2N2 influenza virus vaccine.</p><p><bold>Participants/methods </bold> The A/Ann Arbor/6/60 (H2N2) virus used in this study is the attenuated, cold‐adapted, temperature‐sensitive strain that provides the genetic backbone of seasonal LAIV (MedImmune). We evaluated the safety, infectivity, and immunogenicity of two doses of 10<sup>7</sup> TCID<sub>50</sub> of this vaccine administered by nasal spray 4 weeks apart to normal healthy seronegative adults.</p><p><bold>Results </bold> Twenty‐one participants received a first dose of the vaccine; 18 participants received a second dose. No serious adverse events occurred during the trial. The most common adverse events after vaccination were headache and musculoskeletal pain. The vaccine was restricted in replication: 24% and 17% had virus detectable by culture or rRT‐PCR after the first and second dose, respectively. Antibody responses to the vaccine were also restricted: 24% of participants developed an antibody response as measured by either hemagglutination‐inhibition assay (10%), or ELISA for H2 HA‐specific serum IgG (24%) or IgA (16%) after either one or two doses. None of the participants had a neutralizing antibody response. Vaccine‐specific IgG‐secreting cells as measured by enzyme‐linked immunospot increased from a mean of 0·5 to 2·0/10<sup>6</sup> peripheral blood mononuclear cells (PBMCs); vaccine‐specific IgA‐secreting cells increased from 0·1 to 0·5/10<sup>6</sup> PBMCs.</p><p><bold>Conclusions </bold> The live attenuated H2N2 1960 AA <italic>ca</italic> vaccine demonstrated a safety profile consistent with seasonal trivalent LAIV but was restricted in replication and minimally immunogenic in healthy seronegative adults.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type="iso-abbrev">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type="doi">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type="publisher-id">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type="ppub">1750-2640</issn><issn pub-type="epub">1750-2659</issn><publisher><publisher-name>Blackwell Publishing Ltd</publisher-name><publisher-loc>Oxford, UK</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22417012</article-id><article-id pub-id-type="pmc">3527634</article-id><article-id pub-id-type="doi">10.1111/j.1750-2659.2012.00350.x</article-id><article-id pub-id-type="publisher-id">IRV350</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Articles</subject></subj-group><subj-group subj-group-type="overline"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>An open‐label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults</article-title><alt-title alt-title-type="left-running-head">Talaat <italic>et al.</italic></alt-title><alt-title alt-title-type="right-running-head">H2N2 live attenuated influenza vaccine trial</alt-title></title-group><contrib-group><contrib id="cr1" contrib-type="author"><name><surname>Talaat</surname><given-names>Kawsar R.</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr2" contrib-type="author"><name><surname>Karron</surname><given-names>Ruth A.</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr3" contrib-type="author"><name><surname>Liang</surname><given-names>Philana H.</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr4" contrib-type="author"><name><surname>McMahon</surname><given-names>Bridget A.</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr5" contrib-type="author"><name><surname>Luke</surname><given-names>Catherine J.</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr6" contrib-type="author"><name><surname>Thumar</surname><given-names>Bhagvanji</given-names></name><xref ref-type="aff" rid="a1"><sup>1</sup></xref></contrib><contrib id="cr7" contrib-type="author"><name><surname>Chen</surname><given-names>Grace L.</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr8" contrib-type="author"><name><surname>Min</surname><given-names>Ji‐Young</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr9" contrib-type="author"><name><surname>Lamirande</surname><given-names>Elaine W.</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib><contrib id="cr10" contrib-type="author"><name><surname>Jin</surname><given-names>Hong</given-names></name><xref ref-type="aff" rid="a3"><sup>3</sup></xref></contrib><contrib id="cr11" contrib-type="author"><name><surname>Coelingh</surname><given-names>Kathy L.</given-names></name><xref ref-type="aff" rid="a3"><sup>3</sup></xref></contrib><contrib id="cr12" contrib-type="author"><name><surname>Kemble</surname><given-names>George W.</given-names></name><xref ref-type="aff" rid="a3"><sup>3</sup></xref></contrib><contrib id="cr13" contrib-type="author"><name><surname>Subbarao</surname><given-names>Kanta</given-names></name><xref ref-type="aff" rid="a2"><sup>2</sup></xref></contrib></contrib-group><aff id="a1"><label><sup>1</sup></label>Center for Immunization Research, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.</aff><aff id="a2"><label><sup>2</sup></label>Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.</aff><aff id="a3"><label><sup>3</sup></label>MedImmune, Mountain View, CA, USA.</aff><author-notes><corresp id="correspondenceTo">Kawsar R. Talaat, Department of International Health, Hampton House Room 249, 624 N Broadway, Baltimore, MD 21205, USA. E‐mail: <email>ktalaat@jhsph.edu</email></corresp></author-notes><pub-date pub-type="epub"><day>14</day><month>3</month><year>2012</year></pub-date><pub-date pub-type="ppub"><month>1</month><year>2013</year></pub-date><volume>7</volume><issue>1</issue><issue-id pub-id-type="doi">10.1111/irv.2012.7.issue-1</issue-id><fpage>66</fpage><lpage>73</lpage><history>Accepted 14 January 2012. Published Online 14 March 2012.</history><permissions><copyright-statement content-type="article-copyright">Published 2012. This article is a US Government work and is in the public domain in the USA.</copyright-statement></permissions><self-uri content-type="pdf" xlink:type="simple" xlink:href="file:IRV-7-66.pdf"></self-uri><abstract><p><italic>Please cite this paper as:</italic> Talaat <italic>et al.</italic> (2012) An open‐label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750‐2659.2012.00350.x.</p><p><bold>Background </bold> Live attenuated influenza vaccines (LAIV) against a variety of strains of pandemic potential are being developed and tested. We describe the results of an open‐label phase I trial of a live attenuated H2N2 virus vaccine.</p><p><bold>Objectives </bold> To evaluate the safety, infectivity, and immunogenicity of a live attenuated H2N2 influenza virus vaccine.</p><p><bold>Participants/methods </bold> The A/Ann Arbor/6/60 (H2N2) virus used in this study is the attenuated, cold‐adapted, temperature‐sensitive strain that provides the genetic backbone of seasonal LAIV (MedImmune). We evaluated the safety, infectivity, and immunogenicity of two doses of 10<sup>7</sup> TCID<sub>50</sub> of this vaccine administered by nasal spray 4 weeks apart to normal healthy seronegative adults.</p><p><bold>Results </bold> Twenty‐one participants received a first dose of the vaccine; 18 participants received a second dose. No serious adverse events occurred during the trial. The most common adverse events after vaccination were headache and musculoskeletal pain. The vaccine was restricted in replication: 24% and 17% had virus detectable by culture or rRT‐PCR after the first and second dose, respectively. Antibody responses to the vaccine were also restricted: 24% of participants developed an antibody response as measured by either hemagglutination‐inhibition assay (10%), or ELISA for H2 HA‐specific serum IgG (24%) or IgA (16%) after either one or two doses. None of the participants had a neutralizing antibody response. Vaccine‐specific IgG‐secreting cells as measured by enzyme‐linked immunospot increased from a mean of 0·5 to 2·0/10<sup>6</sup> peripheral blood mononuclear cells (PBMCs); vaccine‐specific IgA‐secreting cells increased from 0·1 to 0·5/10<sup>6</sup> PBMCs.</p><p><bold>Conclusions </bold> The live attenuated H2N2 1960 AA <italic>ca</italic> vaccine demonstrated a safety profile consistent with seasonal trivalent LAIV but was restricted in replication and minimally immunogenic in healthy seronegative adults.</p></abstract><kwd-group><kwd id="k1">H2N2</kwd><kwd id="k2">human</kwd><kwd id="k3">influenza</kwd><kwd id="k4">influenza vaccine</kwd><kwd id="k5">live attenuated influenza vaccines</kwd><kwd id="k6">neuraminidase inhibition</kwd></kwd-group><counts><fig-count count="0"></fig-count><table-count count="3"></table-count><page-count count="8"></page-count></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>January 2013</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_NLMPMC version:4.9.1 mode:remove_FC converted:12.07.2016</meta-value></custom-meta></custom-meta-group></article-meta><notes><fn-group><fn id="fn1"><p>This work was carried out at the Center for Immunization Research, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205 (ClinicalTrials.gov Identifier: NCT00722774).</p></fn></fn-group></notes></front><body><sec id="ss1"><title>Introduction</title><p>Influenza A viruses cause periodic pandemics in addition to the yearly outbreaks and epidemics. H1N1, H2N2, and H3N2 influenza viruses entered the human population and led to pandemics in the 20th century; the novel 2009 H1N1 influenza virus is the first pandemic influenza virus of the 21st century.<xref rid="b1" ref-type="ref"><sup>1</sup></xref>, <xref rid="b2" ref-type="ref"><sup>2</sup></xref> The H1N1 2009 pandemic, as well as repeated introductions of avian influenza strains into human populations, underscore the importance of developing vaccines for influenza viruses of pandemic potential.</p><p>H2N2 influenza viruses emerged in the 1950s through reassortment of the circulating H1N1 human influenza virus with an avian H2N2 virus.<xref rid="b3" ref-type="ref"><sup>3</sup></xref>, <xref rid="b4" ref-type="ref"><sup>4</sup></xref> The first H2N2 influenza cases occurred in China in 1956, and the disease became widespread in 1957–1958, resulting in the ‘Asian Influenza’ pandemic that was responsible for more than one million deaths worldwide.<xref rid="b5" ref-type="ref"><sup>5</sup></xref>, <xref rid="b6" ref-type="ref"><sup>6</sup></xref> H2N2 viruses have not circulated in humans since 1968, when they were replaced by H3N2 influenza viruses, and consequently, a large proportion of the population is now susceptible to infection with H2 influenza viruses.</p><p>H2N2 viruses continue to circulate in birds, leading to the potential reintroduction of these viruses into susceptible human populations. There was a small, unsustained outbreak of H2N2 in Leningrad in 1980.<xref rid="b7" ref-type="ref"><sup>7</sup></xref> In 2004–2005, influenza A H2N2 virus (A/Japan/305/57) was inadvertently included in an influenza proficiency testing panel sent to 3748 laboratories in 18 countries.<xref rid="b8" ref-type="ref"><sup>8</sup></xref>, <xref rid="b9" ref-type="ref"><sup>9</sup></xref> There were no cases of infection reported in persons who handled the proficiency panel. Given the waning immunity to H2N2 in the human population as the proportion of individuals born after 1968 increases, the development of a vaccine against this subtype that is a proven cause of illness and death in humans is an important priority in the development of a library of vaccines against potential pandemic strains of influenza.</p><p>In 1967, Maassab<xref rid="b10" ref-type="ref"><sup>10</sup></xref> attenuated an A/Ann Arbor/6/60 H2N2 influenza virus strain by serial passage at successively lower temperatures in specific pathogen‐free primary chick kidney cells (23 passages followed by seven plaque purifications) followed by amplification in embryonated eggs (three passages). The resulting virus was shown to be attenuated (<italic>att</italic>) in ferrets, cold adapted (<italic>ca</italic>), and temperature sensitive (<italic>ts</italic>). The virus was evaluated in 13 human subjects who had low to moderate levels of pre‐existing antibody to A/AA/6/60 influenza.<xref rid="b11" ref-type="ref"><sup>11</sup></xref> A dose of 3·0 × 10<sup>5</sup> TCID<sub>50</sub> of the virus was immunogenic in seven of eight individuals with low levels of pre‐existing antibody.</p><p>The A/Ann Arbor/6/60 Master Donor Virus (MDV‐A) (H2N2 1960 AA <italic>ca</italic>) provides the genetic backbone [i.e. genes encoding proteins other than the hemagglutinin (HA) and neuraminidase (NA) proteins] for seasonal live attenuated influenza vaccines (LAIV). As such, its genetic backbone has been combined with many different human H1 and H3 HA genes and human N1 and N2 NA genes to produce reassortant LAIV strains that have been extensively evaluated in support of licensure in several countries for protection against seasonal and pandemic influenza.<xref rid="b12" ref-type="ref"><sup>12</sup></xref>, <xref rid="b13" ref-type="ref"><sup>13</sup></xref>, <xref rid="b14" ref-type="ref"><sup>14</sup></xref> In addition, we have prepared and evaluated several reassortant vaccine viruses bearing avian HA and NA genes and the internal protein genes of the H2N2 1960 AA <italic>ca</italic> virus, including candidate vaccines for H9, H5, and H7 influenza. In clinical trials, these vaccines were similar to seasonal LAIV in safety and were attenuated but varied in their capacity to induce antibody responses.<xref rid="b15" ref-type="ref"><sup>15</sup></xref>, <xref rid="b16" ref-type="ref"><sup>16</sup></xref>, <xref rid="b17" ref-type="ref"><sup>17</sup></xref>, <xref rid="b18" ref-type="ref"><sup>18</sup></xref> Although there is extensive clinical experience with reassortant vaccine strains containing the A/Ann Arbor/6/60 backbone, the H2N2 1960 AA <italic>ca</italic> virus itself has been subjected to limited clinical testing. Here, we report the phase I evaluation of the safety, infectivity, and immunogenicity of the H2N2 1960 AA <italic>ca</italic> virus in H2‐naïve adults.</p></sec><sec id="ss2"><title>Participants, materials and methods</title><sec id="ss3"><title>Vaccine virus</title><p>H2N2 1960 AA <italic>ca</italic> is a live attenuated, cold‐adapted, temperature‐sensitive influenza virus derived from the A/Ann Arbor/6/60 <italic>ca</italic> (H2N2) Master Donor Virus (MedImmune, Mountain View, CA, USA) that contains all eight gene segments from the MDV‐A virus. The HA and NA from this vaccine share 97·5% and 98·1% amino acid homology with the HA and NA of the 1957 pandemic virus, A/Japan/57 (H2N2).</p><p>The H2N2 1960 AA <italic>ca</italic> vaccine virus was manufactured at MedImmune using plasmid‐based reverse genetics as previously described.<xref rid="b13" ref-type="ref"><sup>13</sup></xref>, <xref rid="b14" ref-type="ref"><sup>14</sup></xref>, <xref rid="b15" ref-type="ref"><sup>15</sup></xref> The bulk virus was subjected to characterization, and lot release testing, including virus titration, was formulated with sucrose phosphate buffer plus the stabilizing agent gelatin‐arginine‐glutamate (SP + GAG) and was filled into Accuspray™ devices (Becton‐Dickinson, Franklin Lakes, NJ, USA) at a volume of 0·2 ml per sprayer. Filled sprayers were stored frozen at −60°C or below.</p></sec><sec id="ss4"><title>Study population</title><p>This phase 1 clinical trial was conducted during the summer of 2008 at the Center for Immunization Research (CIR) outpatient clinic and at the CIR isolation unit at the Johns Hopkins Bayview Medical Center as previously described.<xref rid="b15" ref-type="ref"><sup>15</sup></xref>, <xref rid="b16" ref-type="ref"><sup>16</sup></xref> The clinical protocol was reviewed and approved by the Western Institutional Review Board (WIRB). Informed written consent was obtained from each participant. Healthy adult men and non‐pregnant women between 18 and 39 years of age who were seronegative to H2N2 viruses were enrolled in the clinical trial if they met eligibility criteria and were willing to remain on the isolation unit for the duration of the inpatient portion of the trial. Key exclusion criteria included immunosuppression, a history of anaphylaxis, Guillain‐Barre Syndrome, recent asthma, significant systemic disease, previous receipt of a LAIV or an H2N2 vaccine, a positive test for HIV, Hepatitis B or C, current narcotic use, allergy to egg, and recent receipt of another vaccine.</p></sec><sec id="ss5"><title>Study design</title><p>This study was conducted as an open‐label phase 1 inpatient trial with all participants receiving vaccine. Participants were screened to establish health status with a thorough medical history, physical examination, and laboratory work including hematology, chemistries, urine, and serological tests for HIV, Hepatitis B, and C. If eligible, participants were given vaccine as a nasal spray using the Accuspray™ (Becton‐Dickinson) device and examined daily while on the isolation unit by a health care provider (physician or physician’s assistant). Four weeks after the first admission, participants were re‐admitted to the isolation unit and received a second dose of vaccine. Participants were isolated for 12 days (from days −2 to 9 after vaccination) for each dose of the vaccine.</p><p>The isolation unit, study design, and study procedures have been previously described.<xref rid="b15" ref-type="ref"><sup>15</sup></xref>, <xref rid="b16" ref-type="ref"><sup>16</sup></xref>, <xref rid="b17" ref-type="ref"><sup>17</sup></xref> Participants were discharged from the isolation unit on study day 9 if rRT‐PCR assays for influenza virus were negative on study days 7 and 8.</p></sec><sec id="ss6"><title>Isolation, quantitation, and identification of the H2N2 virus</title><p>Nasal washes were obtained prior to vaccination and then daily from the day of vaccination until the day of discharge. Specimens were tested for the presence of vaccine virus by quantitative viral culture and by rRT‐PCR amplification of a portion of the influenza A M2 gene.<xref rid="b15" ref-type="ref"><sup>15</sup></xref>, <xref rid="b16" ref-type="ref"><sup>16</sup></xref>, <xref rid="b17" ref-type="ref"><sup>17</sup></xref> The limit of detection of vaccine virus was 50 copies/ml.</p></sec><sec id="ss7"><title>Immunologic assays</title><sec id="ss8"><title>Hemagglutination inhibition and microneutralization</title><p>Sera were tested for hemagglutination‐inhibiting (HI) antibodies to H2N2 as previously described.<xref rid="b19" ref-type="ref"><sup>19</sup></xref> The sera were tested for neutralizing antibodies using a modified version of a previously described microneutralization assay.<xref rid="b20" ref-type="ref"><sup>20</sup></xref> Our assay differed from the previously published microneutralization assay as previously described.<xref rid="b16" ref-type="ref"><sup>16</sup></xref> In addition, our test virus was not the wild‐type A/AA/6/60 H2N2 influenza virus, but rather the vaccine virus, H2N2 1960 AA <italic>ca</italic>. HI and MN were performed at baseline and day 28 after each vaccination.</p></sec><sec id="ss9"><title>ELISA</title><p>Sera were also tested for IgA and IgG antibody to the H2 HA by ELISA. Immulon 2 plates were coated with 30 ng/well of recombinant baculovirus‐expressed H2 HA (from the H2N2 1960 AA <italic>ca</italic> vaccine virus) produced in insect cells (Protein Sciences, Meriden, CT, USA), and the ELISA was performed using endpoint titration.<xref rid="b19" ref-type="ref"><sup>19</sup></xref> Nasal wash specimens were concentrated<xref rid="b21" ref-type="ref"><sup>21</sup></xref> and were tested using the same antigen to measure vaccine‐specific IgA, expressed as a percent of total IgA, as previously described.<xref rid="b21" ref-type="ref"><sup>21</sup></xref> ELISA for IgG and IgA was performed at baseline and day 28 after each vaccination.</p></sec><sec id="ss10"><title>Antibody‐secreting cells</title><p>Ten milliliters of heparinized whole blood was diluted with 30 ml of Hank’s Balanced Salt Solution (Invitrogen, Grand Island, NY, USA), and peripheral blood mononuclear cell (PBMCs) were isolated using Ficoll gradient centrifugation followed by re‐suspension in RPMI 1640 (containing 10% fetal bovine serum, <sc>l</sc>‐glutamine and 1× penicillin/streptomycin). Total and influenza vaccine‐specific IgG‐ and IgA antibody‐secreting cells (ASCs) were measured using an enzyme‐linked immunospot (ELISPOT) assay based on an assay by Sasaki,<xref rid="b22" ref-type="ref"><sup>22</sup></xref> modified as previously described.<xref rid="b16" ref-type="ref"><sup>16</sup></xref> For enumeration of total and influenza vaccine‐specific ASCs, 3·33 × 10<sup>5</sup> cells and 1 × 10<sup>5</sup> PBMC, respectively, were added to each well. Briefly, our assay differed from the published assay in which the wells were coated with beta‐propiolactone (BPL)‐treated H2N2 1960 AA <italic>ca</italic> virus stock diluted to 5000 HA/ml in Dulbecco’s phosphate‐buffered saline (D‐PBS; Invitrogen) or purified goat anti‐human IgA plus IgG plus IgM (Kierkegaard & Perry Laboratories, Gaithersburg, MD, USA) at a concentration of 5 μg/ml in D‐PBS. PBS alone and human CCRF‐CEM cells (ATCC, Manassas, VA, USA) were used as negative controls; human IM9 cells (ATCC) were used as a positive control. Plate images were recorded and counted using I<sc>mmun</sc>S<sc>pot</sc> 4 software (Cellular Technologies, Ltd., Shaker Heights, OH, USA). Human IgA ASCs were visualized as red spots, and IgG ASC were visualized as blue spots. The number of vaccine‐specific IgG and IgA ASC was expressed as the percentage of total IgG and IgA ASC. ELISPOT for ASCs was performed at baseline and day 7 after each vaccination.</p></sec><sec id="ss11"><title>Neuraminidase inhibition assay</title><p>NA‐specific antibodies were measured by a previously described miniaturized neuraminidase inhibition (NAI) assay.<xref rid="b23" ref-type="ref"><sup>23</sup></xref> Measurement of NAI titers requires the use of a virus with an irrelevant hemagglutinin so that anti‐hemagglutinin antibodies do not interfere with the detection of anti‐NA antibodies. Therefore, two H6N2 reassortant viruses were generated, containing the HA from an A/Teal/W312/HK/97 (H6N1) virus and the N2 NA of the A/Uruguay/716/2007 (H3N2) virus that was representative of viruses circulating at the time of the clinical trial or the N2 NA of the H2N2 1960 AA <italic>ca</italic> virus. The A/Uruguay/716/2007 (H3N2) strain was the A/Brisbane 10/2007‐like H3N2 component of the seasonal LAIV in 2008–2009. Briefly, the NA activity of each virus was standardized by colorimetric analysis of sialic acid released from the substrate fetuin. NAI activity in the sera was determined by comparing the NA activity of the virus alone with the activity measured following incubation of the virus with serially diluted sera. The dilution of serum that resulted in a 50% reduction in NA activity of the virus without serum was recorded as the NAI titer.</p></sec><sec id="ss12"><title>Rhinovirus and enterovirus rRT‐PCR</title><p>RNA was extracted from nasal washes using the Nuclisens MiniMAG system (bioMerieux, Hazelwood, MO, USA). rRT‐PCR was used to detect rhinovirus,<xref rid="b16" ref-type="ref"><sup>16</sup></xref> and a modified version of a previously published rRT‐PCR assay<xref rid="b24" ref-type="ref"><sup>24</sup></xref> was used to detect enterovirus.</p></sec></sec><sec id="ss13"><title>Data analysis</title><p>Infection after immunization with the H2N2 1960 AA <italic>ca</italic> vaccine virus was defined as: (i) shedding of vaccine virus detected by culture and/or (ii) shedding of vaccine virus detected by rRT‐PCR any time after study day 1 and/or (iii) a ≥fourfold rise in serum HI antibody, neutralizing antibody, or H2‐specific IgG or IgA serum antibodies as measured by ELISA. Participants whose nasal washes were rRT‐PCR positive on study day 1 but had no other evidence of infection were not considered infected because we could not exclude the possibility that input virus, rather than replicating virus, was being detected. To calculate mean titers, HI antibody, neutralizing antibody, and ELISA reciprocal titers were log<sub>2</sub> transformed.</p></sec></sec><sec id="ss14"><title>Results</title><sec id="ss15"><title>Study participants</title><p>Fifty‐nine potential participants were screened for the H2N2 1960 AA <italic>ca</italic> virus vaccine trial, and 21 eligible individuals were enrolled and vaccinated in August 2008. The age of the participants ranged from 20 to 39 years (mean age 28·8 years, SD 4·6); 18 were Black and 11 were men. Eighteen of the 21 participants received a second dose of vaccine 4 weeks later. Three participants chose not to return for the second dose for personal reasons unrelated to the vaccine, but they returned for their days 28 and 61 follow‐up visits. One subject who received both doses left the country prior to her day 28 follow‐up visit after the second dose. No participant withdrew because of vaccine‐related events.</p></sec><sec id="ss16"><title>Reactogenicity</title><p>The vaccine was generally well tolerated with the exception of a few reported minor illnesses. After the first dose, five participants complained of headache, the most commonly reported symptom. One of these subject also complained of sore throat and nasal congestion on day 2 (<xref rid="t1" ref-type="table-wrap">Table 1</xref>). Vaccine virus was detected by rRT‐PCR in two of the participants who reported headache, including the subject with complaints of sore throat and nasal congestion. A middle ear effusion (without evidence of otitis media) was detected in a subject who had reported decreased hearing in the left ear, but this finding was not associated with the detection of vaccine virus by culture or rRT‐PCR in nasal wash specimens. Other illnesses reported following the first dose of vaccine included nausea and vomiting (two individuals); and localized musculoskeletal complaints including backache (three individuals), shoulder pain (two individuals), and muscle soreness (two individuals). The detection of vaccine virus by culture or rRT‐PCR in the remaining symptomatic participants did not coincide with symptoms. Adventitious viruses were not detected by culture or rRT‐PCR in nasal washes obtained from any participants after the first dose.</p><table-wrap id="t1" xml:lang="en" orientation="portrait" position="float"><label>Table 1</label><caption><p> Clinical response and vaccine virus shedding following two 10<sup>7</sup> TCID<sub>50</sub> doses of H2N2 1960 AA <italic>ca</italic></p></caption><table frame="hsides" rules="groups"><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><thead valign="bottom"><tr style="border-bottom:solid 1px #000000"><th rowspan="3" valign="bottom" align="left" colspan="1">Subjects</th><th rowspan="3" valign="bottom" align="left" colspan="1">No. of subjects</th><th rowspan="3" valign="bottom" align="left" colspan="1">% Infected*</th><th rowspan="2" colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom">Virus detection (culture) (nasal wash)</th><th rowspan="2" colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom">Virus detection (rRT‐PCR) (nasal wash)</th><th colspan="5" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Symptoms reported</th></tr><tr style="border-bottom:solid 1px #000000"><th colspan="5" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">No. with indicated illness</th></tr><tr style="border-bottom:solid 1px #000000"><th align="left" valign="bottom" rowspan="1" colspan="1">No. shedding virus**</th><th align="left" valign="bottom" rowspan="1" colspan="1">Peak titer mean log<sub>10</sub>***</th><th align="left" valign="bottom" rowspan="1" colspan="1">Duration of shedding mean days<sup>†</sup></th><th align="left" valign="bottom" rowspan="1" colspan="1">No. pos. on day 1</th><th align="left" valign="bottom" rowspan="1" colspan="1">No. pos. after day 1</th><th align="left" valign="bottom" rowspan="1" colspan="1">Duration of shedding mean days</th><th align="left" valign="bottom" rowspan="1" colspan="1">Fever</th><th align="left" valign="bottom" rowspan="1" colspan="1">URI</th><th align="left" valign="bottom" rowspan="1" colspan="1">LRI</th><th align="left" valign="bottom" rowspan="1" colspan="1">Headache</th><th align="left" valign="bottom" rowspan="1" colspan="1">Reacto‐genicity event<sup>††</sup></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1">First dose</td><td align="left" valign="top" rowspan="1" colspan="1">21</td><td align="left" valign="top" rowspan="1" colspan="1">29</td><td align="left" valign="top" rowspan="1" colspan="1">4</td><td align="left" valign="top" rowspan="1" colspan="1">1·4</td><td align="left" valign="top" rowspan="1" colspan="1">2</td><td align="left" valign="top" rowspan="1" colspan="1">8</td><td align="left" valign="top" rowspan="1" colspan="1">5</td><td align="left" valign="top" rowspan="1" colspan="1">2·3</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">1</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">5</td><td align="left" valign="top" rowspan="1" colspan="1">5</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Second dose</td><td align="left" valign="top" rowspan="1" colspan="1">18</td><td align="left" valign="top" rowspan="1" colspan="1">33</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">≤0·6</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">4</td><td align="left" valign="top" rowspan="1" colspan="1">4</td><td align="left" valign="top" rowspan="1" colspan="1">1·3</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">1</td><td align="left" valign="top" rowspan="1" colspan="1">1</td></tr></tbody></table><table-wrap-foot><fn id="t1_note46"><p>No., number; pos., positive; URI, upper respiratory tract symptoms include rhinorrhea, nasal congestion, cough, pharyngitis; LRI, lower respiratory tract symptoms include pneumonia, wheezing, rhonchi</p></fn><fn id="t1_note47"><p>*Infection is defined as shedding of vaccine virus and/or a fourfold or greater rise in HI antibody titer or in serum microneutralization or ELISA response.</p></fn><fn id="t1_note48"><p>**Number shedding virus are those volunteers who had virus recoverable by culture.</p></fn><fn id="t1_note49"><p>***Viral titers are expressed as log<sub>10</sub> TCID<sub>50</sub>/ml. Geometric Mean peak titers were calculated for all infected subjects for the first dose. Peak titers of 0·6 TCID<sub>50</sub>/ml were assigned to culture‐negative samples.</p></fn><fn id="t1_note50"><p><sup>†</sup>Calculated for all infected subjects. One subject had virus recovered days 1 and 2, two subjects had virus cultured day 2 only, and one subject had culturable virus days 2–6.</p></fn><fn id="t1_note51"><p><sup>††</sup>Reactogenicity events are defined as fever, nasal congestion, rhinorrhea, pharyngitis, cough, otitis media, pneumonia, headache, myalgia, chills, conjunctivitis, wheezing, rhonchi and epistaxis. Transient abnormalities in blood pressure, pulse and respiratory rate are not included here,nor are adverse events that were thought to be unrelated or not likely to be related.</p></fn></table-wrap-foot></table-wrap><p>Following the second dose of vaccine, one subject reported headache, and one additional subject described having an ‘itchy’ throat that did not meet criteria for a diagnosis of pharyngitis (<xref rid="t1" ref-type="table-wrap">Table 1</xref>). Vaccine virus was not detected in either subject. Two participants reported gastrointestinal symptoms, one of whom had moderate (grade 2) nausea, vomiting, and diarrhea for several days starting on day 2. Of note, a staff member had similar gastrointestinal symptoms preceding the participants’ symptoms. The subject with gastroenteritis had vaccine virus recovered by rRT‐PCR from the nasal washes on days 1 and 2. The other subject complained of abdominal bloating and loose stools and did not have vaccine virus recovered from the respiratory tract. Again, three participants had musculoskeletal complaints, only one of whom had virus detected by rRT‐PCR on days 1 and 2 (the subject reported back pain from days 2 through 12). One subject had transient tachycardia detected during routine physical assessment on day 9, which was not associated with virus recovery. Adventitious viral cultures were negative for each of these participants.</p></sec><sec id="ss17"><title>Vaccine virus replication</title><p>The H2N2 1960 AA <italic>ca</italic> vaccine virus was restricted in replication. After the first dose, 4/21 (19%) participants had vaccine virus recovered by culture from the nasal washes: one on days 3 through 6, two on day 2 only, and one on days 1 and 2 (<xref rid="t1" ref-type="table-wrap">Table 1</xref>). In these four participants, peak titers of vaccine virus shed ranged from 10<sup>0·75</sup> to 10<sup>4·5</sup> TCID<sub>50</sub>/ml; the geometric mean peak titer was 10<sup>1·8</sup> TCID<sub>50</sub>/ml. Two additional subjects met the criteria for infection (see <xref rid="ss2" ref-type="sec">Participants, materials and methods</xref> for the definitions of infection): one subject was positive by rRT‐PCR and one by serology. For the six infected participants, the geometric mean peak titer was 10<sup>1·4</sup> TCID<sub>50</sub>/ml (<xref rid="t1" ref-type="table-wrap">Table 1</xref>). When the nasal wash specimens were tested by rRT‐PCR, vaccine virus was detected on day 1 only (six individuals), on day 2 only (two individuals), on days 1 and 2 (two individuals), and on days 2 through 6 (one individual). Vaccine virus was not recovered by culture from any nasal wash specimen following the second dose of vaccine but was detected by rRT‐PCR in five participants: one on day 1 only, one on day 2 only, and three on days 1 and 2 (<xref rid="t1" ref-type="table-wrap">Table 1</xref>). All five participants who had vaccine virus recovered after the second dose also had detectable virus after the first dose.</p></sec><sec id="ss18"><title>Antibody responses</title><p>None of the participants had a detectable HI response following the first dose of vaccine. Two of the participants (12%) who received two doses of vaccine had ≥fourfold rises in HI titer (<xref rid="t2" ref-type="table-wrap">Table 2</xref>). Neutralizing antibody responses were not detected in any of the participants (<xref rid="t2" ref-type="table-wrap">Table 2</xref>).</p><table-wrap id="t2" xml:lang="en" orientation="portrait" position="float"><label>Table 2</label><caption><p> Antibody responses following two 10<sup>7</sup> TCID<sub>50</sub> doses of H2N2 1960 AA <italic>ca</italic></p></caption><table frame="hsides" rules="groups"><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><thead valign="bottom"><tr style="border-bottom:solid 1px #000000"><th rowspan="3" valign="bottom" align="left" colspan="1">Subjects</th><th rowspan="3" valign="bottom" align="left" colspan="1">No. of subjects</th><th rowspan="3" valign="bottom" align="left" colspan="1">% Infected*</th><th colspan="15" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Reciprocal mean (SD) log<sub>2</sub> antibody</th></tr><tr style="border-bottom:solid 1px #000000"><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">HI</th><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Serum microneut Ab response</th><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Serum HA‐IgG ELISA</th><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Serum HA‐IgA ELISA</th><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Nasal wash HA‐IgA ELISA</th></tr><tr style="border-bottom:solid 1px #000000"><th align="left" valign="bottom" rowspan="1" colspan="1">Pre</th><th align="left" valign="bottom" rowspan="1" colspan="1">Post**</th><th align="left" valign="bottom" rowspan="1" colspan="1">% With ≥fourfold rise</th><th align="left" valign="bottom" rowspan="1" colspan="1">Pre</th><th align="left" valign="bottom" rowspan="1" colspan="1">Post**</th><th align="left" valign="bottom" rowspan="1" colspan="1">% With ≥fourfold rise</th><th align="left" valign="bottom" rowspan="1" colspan="1">Pre</th><th align="left" valign="bottom" rowspan="1" colspan="1">Post**</th><th align="left" valign="bottom" rowspan="1" colspan="1">% With ≥fourfold rise</th><th align="left" valign="bottom" rowspan="1" colspan="1">Pre</th><th align="left" valign="bottom" rowspan="1" colspan="1">Post**</th><th align="left" valign="bottom" rowspan="1" colspan="1">% With ≥fourfold rise</th><th align="left" valign="bottom" rowspan="1" colspan="1">Pre</th><th align="left" valign="bottom" rowspan="1" colspan="1">Post**</th><th align="left" valign="bottom" rowspan="1" colspan="1">% With ≥fourfold rise</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1">First dose</td><td align="left" valign="top" rowspan="1" colspan="1">21</td><td align="left" valign="top" rowspan="1" colspan="1">29</td><td align="left" valign="top" rowspan="1" colspan="1">1·1 (0·4)</td><td align="left" valign="top" rowspan="1" colspan="1">1·2 (0·4)</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">2·3 (0)</td><td align="left" valign="top" rowspan="1" colspan="1">2·3 (0)</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">9·4 (2·0)</td><td align="left" valign="top" rowspan="1" colspan="1">9·5 (1·5)</td><td align="left" valign="top" rowspan="1" colspan="1">14***</td><td align="left" valign="top" rowspan="1" colspan="1">5·5 (1·0)</td><td align="left" valign="top" rowspan="1" colspan="1">5·7 (1·2)</td><td align="left" valign="top" rowspan="1" colspan="1">10</td><td align="left" valign="top" rowspan="1" colspan="1">2·5 (0·8)</td><td align="left" valign="top" rowspan="1" colspan="1">2·6 (0·9)</td><td align="left" valign="top" rowspan="1" colspan="1">0</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Second dose</td><td align="left" valign="top" rowspan="1" colspan="1">17<sup>†</sup></td><td align="left" valign="top" rowspan="1" colspan="1">33</td><td align="left" valign="top" rowspan="1" colspan="1">1·2 (0·4)</td><td align="left" valign="top" rowspan="1" colspan="1">1·6 (0·9)</td><td align="left" valign="top" rowspan="1" colspan="1">12<sup>††</sup></td><td align="left" valign="top" rowspan="1" colspan="1">2·3 (0)</td><td align="left" valign="top" rowspan="1" colspan="1">2·4 (0·2)</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">9·6 (1·6)</td><td align="left" valign="top" rowspan="1" colspan="1">9·9 (1·7)</td><td align="left" valign="top" rowspan="1" colspan="1">11</td><td align="left" valign="top" rowspan="1" colspan="1">5·6 (1·3)</td><td align="left" valign="top" rowspan="1" colspan="1">5·6 (1·4)</td><td align="left" valign="top" rowspan="1" colspan="1">6</td><td align="left" valign="top" rowspan="1" colspan="1">2·6 (0·9)</td><td align="left" valign="top" rowspan="1" colspan="1">2·6 (1·1)</td><td align="left" valign="top" rowspan="1" colspan="1">6</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Any dose</td><td align="left" valign="top" rowspan="1" colspan="1">21</td><td align="left" valign="top" rowspan="1" colspan="1">38</td><td align="left" valign="top" rowspan="1" colspan="1">1·1 (0·4)</td><td align="left" valign="top" rowspan="1" colspan="1">1·6 (0·9)</td><td align="left" valign="top" rowspan="1" colspan="1">10</td><td align="left" valign="top" rowspan="1" colspan="1">2·3 (0)</td><td align="left" valign="top" rowspan="1" colspan="1">2·4 (0·2)</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">9·4 (2·0)</td><td align="left" valign="top" rowspan="1" colspan="1">9·7 (1·8)</td><td align="left" valign="top" rowspan="1" colspan="1">24</td><td align="left" valign="top" rowspan="1" colspan="1">5·5 (1·0)</td><td align="left" valign="top" rowspan="1" colspan="1">5·7 (1·4)</td><td align="left" valign="top" rowspan="1" colspan="1">16</td><td align="left" valign="top" rowspan="1" colspan="1">2·5 (0·8)</td><td align="left" valign="top" rowspan="1" colspan="1">2·5 (1·0)</td><td align="left" valign="top" rowspan="1" colspan="1">5</td></tr></tbody></table><table-wrap-foot><fn id="t2_note78"><p>No., number; SD, standard deviation; HI, hemagglutination‐inhibiting; Microneut, microneutralizing; Ab, antibody.</p></fn><fn id="t2_note79"><p>*Infection is defined as shedding of vaccine virus by culture on any day and/or by rRT‐PCR after day 1 and/or a fourfold or greater rise in HI antibody titer or in serum microneutralization or ELISA response. Those with a gradual rise of antibody responses over the two doses were designated as infected after dose 2.</p></fn><fn id="t2_note80"><p>**Post‐values drawn on day 26 for dose 1, day 29 for dose 2.</p></fn><fn id="t2_note81"><p>***One subject who received only one dose had a delayed response by ELISA with the rise in titer occurring during the day 56 visit.</p></fn><fn id="t2_note82"><p><sup>†</sup>Data is available on 17 subjects for the second dose. 1 subject did not return for the follow‐up visit.</p></fn><fn id="t2_note83"><p><sup>††</sup>One subject had a gradual rise of antibody response, with a twofold increase after each dose.</p></fn></table-wrap-foot></table-wrap><p>Serum IgG and IgA and nasal wash IgA antibodies to a recombinant baculovirus‐expressed H2 HA were measured by ELISA (<xref rid="t2" ref-type="table-wrap">Table 2</xref>). Five participants had fourfold rises in serum IgG titer: three (14%) following the first dose of vaccine and two (11%) following the second dose. Three participants had fourfold rises in serum IgA titer: two (10%) following the first dose of vaccine and one (6%) following the second dose. Nasal wash IgA was the least sensitive measure of antibody response to this vaccine: only one subject had a fourfold or greater rise in titer after the second dose of vaccine (<xref rid="t2" ref-type="table-wrap">Table 2</xref>).</p><p>The development of vaccine‐specific ASCs was also extremely limited. When measured prior to immunization, six participants had detectable vaccine‐specific IgG ASCs, all ≤3 cells/10<sup>6</sup> PBMCs. Seven days after the first vaccination, the mean (SD) number of vaccine‐specific IgG ASCs was 2·0 (3·2), and four of 21 participants had an increase of five or more specific IgG ASCs. By the time of the second dose, vaccine‐specific IgG ASCs were nearly undetectable: two participants each had one vaccine‐specific IgG‐secreting cell/10<sup>6</sup> PBMCs, and specific ASCs were not detected in any of the other participants. Seven days after the second vaccination, the mean (SD) number of vaccine‐specific IgG ASCs was 1·2 (2·6)/10<sup>6</sup> PBMCs, and only two of 18 participants (one of whom had also responded to the first dose) had an increase of five or more vaccine‐specific IgG ASCs/10<sup>6</sup> PBMCs. Vaccine‐specific IgA responses were of lower magnitude than vaccine‐specific IgG responses: none of the participants had an increase of ≥5 vaccine‐specific IgA ASCs either after the first or second dose of vaccine (<xref rid="t3" ref-type="table-wrap">Table 3</xref>).</p><table-wrap id="t3" xml:lang="en" orientation="portrait" position="float"><label>Table 3</label><caption><p> Antibody secreting cells and NAI responses following two 10<sup>7</sup> TCID<sub>50</sub> doses of H2N2 1960 AA <italic>ca</italic></p></caption><table frame="hsides" rules="groups"><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><thead valign="bottom"><tr style="border-bottom:solid 1px #000000"><th rowspan="3" valign="bottom" align="left" colspan="1">Subjects</th><th rowspan="3" valign="bottom" align="left" colspan="1">No. of subjects</th><th rowspan="3" valign="bottom" align="left" colspan="1">% Infected*</th><th colspan="6" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">Number of cells secreting antibody to vaccine virus (SD)</th><th colspan="6" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">NAI assay titer (SD)</th></tr><tr style="border-bottom:solid 1px #000000"><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">IgG ASC to vaccine virus</th><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">IgA ASC To vaccine virus</th><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">NAI against A/AA/6/60</th><th colspan="3" style="border-bottom:solid 1px #000000" align="left" valign="bottom" rowspan="1">NAI against A/Uruguay/716/2007</th></tr><tr style="border-bottom:solid 1px #000000"><th align="left" valign="bottom" rowspan="1" colspan="1">Pre</th><th align="left" valign="bottom" rowspan="1" colspan="1">Post**</th><th align="left" valign="bottom" rowspan="1" colspan="1">% With ≥5 cell rise</th><th align="left" valign="bottom" rowspan="1" colspan="1">Pre</th><th align="left" valign="bottom" rowspan="1" colspan="1">Post**</th><th align="left" valign="bottom" rowspan="1" colspan="1">% With ≥5 cell rise</th><th align="left" valign="bottom" rowspan="1" colspan="1">Pre</th><th align="left" valign="bottom" rowspan="1" colspan="1">Post***</th><th align="left" valign="bottom" rowspan="1" colspan="1">% With ≥fourfold rise</th><th align="left" valign="bottom" rowspan="1" colspan="1">Pre</th><th align="left" valign="bottom" rowspan="1" colspan="1">Post***</th><th align="left" valign="bottom" rowspan="1" colspan="1">% With ≥fourfold rise</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1">First dose</td><td align="left" valign="top" rowspan="1" colspan="1">21</td><td align="left" valign="top" rowspan="1" colspan="1">29</td><td align="left" valign="top" rowspan="1" colspan="1">0·5 (0·9)</td><td align="left" valign="top" rowspan="1" colspan="1">2·0 (3·2)</td><td align="left" valign="top" rowspan="1" colspan="1">14</td><td align="left" valign="top" rowspan="1" colspan="1">0·1 (0·5)</td><td align="left" valign="top" rowspan="1" colspan="1">0·5 (0·9)</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">13·2 (9·7)</td><td align="left" valign="top" rowspan="1" colspan="1">13·3 (5·1)</td><td align="left" valign="top" rowspan="1" colspan="1">5</td><td align="left" valign="top" rowspan="1" colspan="1">21·6 (36·6)</td><td align="left" valign="top" rowspan="1" colspan="1">26·8 (36·1)</td><td align="left" valign="top" rowspan="1" colspan="1">19</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Second dose</td><td align="left" valign="top" rowspan="1" colspan="1">17<sup>†</sup></td><td align="left" valign="top" rowspan="1" colspan="1">33</td><td align="left" valign="top" rowspan="1" colspan="1">0·1 (0·3)</td><td align="left" valign="top" rowspan="1" colspan="1">1·2 (2·6)</td><td align="left" valign="top" rowspan="1" colspan="1">12</td><td align="left" valign="top" rowspan="1" colspan="1">0·2 (0·4)</td><td align="left" valign="top" rowspan="1" colspan="1">0·3 (0·8)</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">13·3 (5·1)</td><td align="left" valign="top" rowspan="1" colspan="1">14·7 (5·3)</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">26·8 (36·1)</td><td align="left" valign="top" rowspan="1" colspan="1">24·9 (36·9)</td><td align="left" valign="top" rowspan="1" colspan="1">6</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Any dose</td><td align="left" valign="top" rowspan="1" colspan="1">21</td><td align="left" valign="top" rowspan="1" colspan="1">38</td><td align="left" valign="top" rowspan="1" colspan="1">0·5 (0·9)</td><td align="left" valign="top" rowspan="1" colspan="1">1·2 (2·6)</td><td align="left" valign="top" rowspan="1" colspan="1">19</td><td align="left" valign="top" rowspan="1" colspan="1">0·1 (0·5)</td><td align="left" valign="top" rowspan="1" colspan="1">0·3 (0·8)</td><td align="left" valign="top" rowspan="1" colspan="1">0</td><td align="left" valign="top" rowspan="1" colspan="1">13·2 (9·7)</td><td align="left" valign="top" rowspan="1" colspan="1">14·7 (5·3)</td><td align="left" valign="top" rowspan="1" colspan="1">5</td><td align="left" valign="top" rowspan="1" colspan="1">21·6 (36·6)</td><td align="left" valign="top" rowspan="1" colspan="1">24·9 (36·9)</td><td align="left" valign="top" rowspan="1" colspan="1">24</td></tr></tbody></table><table-wrap-foot><fn id="t3_note66"><p>No., number; SD, standard deviation; ASC, antibody secreting cells; NAI, neuraminidase Inhibition Assay.</p></fn><fn id="t3_note67"><p>*Infection is defined as shedding of vaccine virus by culture on any day and/or by rRT‐PCR after day 1 and/or a fourfold or greater rise in HI antibody titer or in serum microneutralization or ELISA response. Those with a gradual rise of antibody responses over the two doses were designated as infected after dose 2.</p></fn><fn id="t3_note68"><p>**Post‐values for ASC drawn on day 7 after each vaccination</p></fn><fn id="t3_note69"><p>***Post‐values drawn on day 26 for dose 1, day 29 for dose 2.</p></fn><fn id="t3_note70"><p><sup>†</sup>Data is available on 17 subjects for the second dose. 1 subject did not return for the follow‐up visit.</p></fn></table-wrap-foot></table-wrap><p>As immunological responses to the vaccine were limited, NAI assays were carried out to see whether pre‐existing NA antibodies blocked vaccine replication and immunogenicity (<xref rid="t3" ref-type="table-wrap">Table 3</xref>). Only 1/21 subjects had pre‐existing NAI antibodies to the vaccine NA prior to vaccination, and one subject developed a fourfold response to the vaccine NA. In contrast, 18/21 subjects had pre‐existing NAI titers to the seasonal N2 NA (Geometric mean titer 1:21·6; Standard error 36·6), and four subjects had modest increases in titer against this seasonal NA after vaccination; three of the four subjects went from having undetectable to detectable NAI antibodies to the seasonal N2 NA. None of the subjects with a response to the seasonal N2 NA showed a fourfold response to the vaccine N2 NA (<xref rid="t3" ref-type="table-wrap">Table 3</xref>).</p></sec></sec><sec id="ss19"><title>Discussion</title><p>We have reported results of an open‐label phase I study of a pandemic H2N2 influenza A virus vaccine using the H2N2 1960 AA <italic>ca</italic> virus strain. This vaccine demonstrated a safety profile consistent with current seasonal trivalent LAIV but was restricted in replication and minimally immunogenic in immunologically naïve healthy young adults who were born after H2N2 viruses stopped circulating in the human population.</p><p>One of the difficulties in evaluating vaccines for novel influenza strains is determining the correlates of vaccine‐induced immunity.<xref rid="b25" ref-type="ref"><sup>25</sup></xref> For seasonal inactivated influenza virus vaccines, the correlate of protection accepted by regulatory agencies is an HI titer of ≥1:40, measured in a standard assay using 4 U of HA.<xref rid="b26" ref-type="ref"><sup>26</sup></xref>, <xref rid="b27" ref-type="ref"><sup>27</sup></xref> However, seasonal LAIV have been shown to protect against natural infection or challenge with wild‐type influenza viruses even when HI titers ≥1:40 were not achieved, presumably through the induction of local mucosal antibody and cellular immunity.<xref rid="b28" ref-type="ref"><sup>28</sup></xref>, <xref rid="b29" ref-type="ref"><sup>29</sup></xref>, <xref rid="b30" ref-type="ref"><sup>30</sup></xref> Moreover, HI antibody may not be the best measure of protection against infection with H5 and H7 avian influenza viruses, as individuals infected with these viruses often fail to develop substantial HI antibody responses.<xref rid="b20" ref-type="ref"><sup>20</sup></xref>, <xref rid="b31" ref-type="ref"><sup>31</sup></xref> For this reason, alternative antibody assays, such as the microneutralization assay and ELISA<xref rid="b20" ref-type="ref"><sup>20</sup></xref> have been developed.</p><p>To assess the antibody response to the H2N2 1960 AA <italic>ca</italic> vaccine, we used several assays in addition to the standard HI and microneutralization assays. Using the HI assay, we found that only 12% of participants had a ≥fourfold rise in HI antibody titer after two doses of vaccine, with modest increases achieved (<xref rid="t1" ref-type="table-wrap">Table 1</xref>). We also measured neutralizing antibody, H2‐specific IgG and IgA antibody, and production of ASCs against the vaccine virus. Although the ability of each assay to detect a response differed slightly, minimal responses to the vaccine were detected by all assays (<xref rid="t2" ref-type="table-wrap">Tables 2, 3</xref>). Interestingly, unlike other pandemic live attenuated vaccines that we have assessed (H5N1, H7N3), serum IgA was not the most sensitive indicator of an immune response (<xref rid="t2" ref-type="table-wrap">Table 2</xref>). Additionally, measurement of vaccine‐specific ASCs detected minimal responses to either dose of the vaccine (<xref rid="t3" ref-type="table-wrap">Table 3</xref>), which is quite different from what was previously observed in children who received seasonal LAIV<xref rid="b22" ref-type="ref"><sup>22</sup></xref> or in adults administered a live attenuated H7N3 vaccine.<xref rid="b16" ref-type="ref"><sup>16</sup></xref></p><p>The substantial restriction in replication and poor antibody response to the H2N2 1960 AA <italic>ca</italic> vaccine were somewhat unexpected because the vaccine was shown to be infectious and immunogenic in mice and ferrets in preclinical studies.<xref rid="b32" ref-type="ref"><sup>32</sup></xref> Also, our study participants were born after 1968 and should have been susceptible to H2N2 influenza virus infection.<xref rid="b33" ref-type="ref"><sup>33</sup></xref> However, because of the extensive passage history of the seasonal LAIV MDV in chicken kidney cells and eggs, it is possible that this virus became less infectious for humans. In support of this concept, a previous study of a cold‐adapted, temperature‐sensitive, attenuated A/Ann Arbor/6/60 (H2N2) virus that had undergone less extensive passage in chicken cells was found to be immunogenic in seven of nine individuals when administered at a 30‐fold lower dose (3·0 × 10<sup>5</sup> TCID<sub>50</sub>) in 1973.<xref rid="b11" ref-type="ref"><sup>11</sup></xref> It should be noted that in both the 1973 study and the present study, virus replication could not be detected following nasal administration. There are several possible explanations for these observations. Anti‐NA antibodies can restrict the release and spread of influenza viruses. Although only one subject had anti‐NA antibody to the vaccine N2 NA, most subjects (18/21) had pre‐existing antibody to seasonal N2 NA (<xref rid="t3" ref-type="table-wrap">Table 3</xref>). Therefore, it is possible that pre‐existing antibodies against the seasonal N2 NA may have limited the replication and consequently the immunogenicity of the attenuated vaccine virus. However, the NA of the seasonal A/Uruguay/716/2007 H3N2 virus and the NA of the 1960 AA <italic>ca</italic> vaccine virus share only 84% homology at the amino acid level, and the NAI response was not cross‐reactive (<xref rid="t3" ref-type="table-wrap">Table 3</xref>). It appears that the MDV containing this particular H2 and N2 is inherently less infectious and immunogenic than the H1N1 and H3N2 LAIV that have been evaluated in multiple studies.<xref rid="b34" ref-type="ref"><sup>34</sup></xref>, <xref rid="b35" ref-type="ref"><sup>35</sup></xref> It is also possible that the H2N2 1960 AA <italic>ca</italic> virus might induce protective immunity in the absence of HI or neutralizing antibodies, as it protected mice against homologous viral challenge after one dose in the absence of a detectable antibodies to the homologous virus and protected mice and ferrets against heterologous viral challenge in the absence of detectable antibodies to heterologous H2N2 viruses.<xref rid="b32" ref-type="ref"><sup>32</sup></xref> It may be that the vaccine induces immune responses that were not measured in preclinical or clinical studies, including cellular immune responses as has been demonstrated with trivalent seasonal formulations of LAIV. Such responses should be evaluated in future studies.</p><p>In summary, we have shown that two doses of a live attenuated cold‐adapted H2N2 1960 AA <italic>ca</italic> vaccine resulted in a low incidence of side effects and were modestly immunogenic when given to healthy adults. Contemporary H2 and N2 genes obtained from low passage human virus isolates should be tested on the same backbone to study the human immune response to a pandemic H2N2 LAIV.</p></sec><sec id="ss21"><title>Addendum</title><p>K. R. Talaat contributed to protocol development, study conduct, data analysis, and manuscript writing; R. A. Karron involved in the study development and manuscript editing; P. H. Liang contributed to the study conduct, data collection, entry and analysis, and manuscript drafting; B. A. McMahon contributed to the source documents, study logistics, data collection, entry and analysis, and manuscript editing; C. J. Luke contributed to the study design, protocol and regulatory document development, and manuscript writing; B. Thumar oversaw laboratory assays, designed assays, and edited the manuscript; G. L. Chen, J.‐Y. Min, and E. W. Lamirande involved in assay design and performance and manuscript editing; H. Jin, K. L. Coelingh, G. W. Kemble contributed to the study design, protocol and regulatory document input, and manuscript editing; K. Subbarao contributed to the study design, protocol and regulatory document input, and manuscript writing.</p></sec></body><back><ack id="ss20"><title>Acknowledgements</title><p>We wish to thank Ruval Comendador, Jasmine Jennings, and Paula Williams‐Soro for their expert clinical assistance, Susan DiLorenzo, Nicole Yoder, and Celia Santos for their expert technical assistance. We would also like to thank Bin Lu, Weidong Cui, Silas Nwagwu, Alfred Pan, Lynne Fitch, Patricia Ryan, So‐Ching Brazer, David Prichett, Edith Matsuyama, and Zhongying Chen of MedImmune for vaccine manufacturing, testing, and making the reassortant H6 viruses for the NAI assay. This research was supported by the Intramural Research Program of the NIAID, NIH (N01‐AI‐15444). This research was performed as a Cooperative Research and Development Agreement (CRADA) (CRADA No: AI‐0155) between the Laboratory of Infectious Diseases, NIAID and MedImmune.</p></ack><ref-list id="ss22"><title>References</title><ref id="b1"><label>1</label><mixed-citation publication-type="journal" id="cit1"><string-name><surname>Nicholson</surname><given-names>KG</given-names></string-name>,
<string-name><surname>Wood</surname><given-names>JM</given-names></string-name>,
<string-name><surname>Zambon</surname><given-names>M</given-names></string-name>.
<article-title>Influenza</article-title>.
<source>Lancet</source><year>2003</year>; <volume>362</volume>:<fpage>1733</fpage>–<lpage>1745</lpage>.
<pub-id pub-id-type="pmid">14643124</pub-id></mixed-citation></ref><ref id="b2"><label>2</label><mixed-citation publication-type="book" id="cit2"><collab collab-type="authors">CDC</collab>.
<source>CDC Estimates of 2009 H1N1 Influenza Cases, Hospitalizations and Deaths in the United States, April–January 16, 2010</source>. Atlanta: <publisher-name>CDC</publisher-name>, <year>2010</year> Available at <ext-link ext-link-type="uri" xlink:href="http://www.cdc.gov/h1n1flu/estimates_2009_h1n1.htm">http://www.cdc.gov/h1n1flu/estimates_2009_h1n1.htm</ext-link> (cited 23 February 2010).
</mixed-citation></ref><ref id="b3"><label>3</label><mixed-citation publication-type="journal" id="cit3"><string-name><surname>Scholtissek</surname><given-names>C</given-names></string-name>,
<string-name><surname>Rohde</surname><given-names>W</given-names></string-name>,
<string-name><surname>Von Hoyningen</surname><given-names>V</given-names></string-name>,
<string-name><surname>Rott</surname><given-names>R</given-names></string-name>.
<article-title>On the origin of the human influenza virus subtypes H2N2 and H3N2</article-title>.
<source>Virology</source><year>1978</year>; <volume>87</volume>:<fpage>13</fpage>–<lpage>20</lpage>.
<pub-id pub-id-type="pmid">664248</pub-id></mixed-citation></ref><ref id="b4"><label>4</label><mixed-citation publication-type="journal" id="cit4"><string-name><surname>Kawaoka</surname><given-names>Y</given-names></string-name>,
<string-name><surname>Krauss</surname><given-names>S</given-names></string-name>,
<string-name><surname>Webster</surname><given-names>RG</given-names></string-name>.
<article-title>Avian‐to‐human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics</article-title>.
<source>J Virol</source><year>1989</year>; <volume>63</volume>:<fpage>4603</fpage>–<lpage>4608</lpage>.
<pub-id pub-id-type="pmid">2795713</pub-id></mixed-citation></ref><ref id="b5"><label>5</label><mixed-citation publication-type="journal" id="cit5"><string-name><surname>Rajagopal</surname><given-names>S</given-names></string-name>,
<string-name><surname>Treanor</surname><given-names>J</given-names></string-name>.
<article-title>Pandemic (avian) influenza</article-title>.
<source>Semin Respir Crit Care Med</source><year>2007</year>; <volume>28</volume>:<fpage>159</fpage>–<lpage>170</lpage>.
<pub-id pub-id-type="pmid">17458770</pub-id></mixed-citation></ref><ref id="b6"><label>6</label><mixed-citation publication-type="journal" id="cit6"><string-name><surname>Dunn</surname><given-names>FL</given-names></string-name>.
<article-title>Pandemic Influenza in 1957: review of International Spread of New Asian Strain</article-title>.
<source>JAMA</source><year>1958</year>; <volume>166</volume>:<fpage>1140</fpage>–<lpage>1148</lpage>.
</mixed-citation></ref><ref id="b7"><label>7</label><mixed-citation publication-type="journal" id="cit7"><string-name><surname>Golubev</surname><given-names>DB</given-names></string-name>,
<string-name><surname>Karpukhin</surname><given-names>GI</given-names></string-name>,
<string-name><surname>Galitarov</surname><given-names>SS</given-names></string-name>,
<string-name><surname>Denisov</surname><given-names>GM</given-names></string-name>.
<article-title>Type A influenza (H2N2) viruses isolated in Leningrad in 1980</article-title>.
<source>J Hyg (Lond)</source><year>1985</year>; <volume>95</volume>:<fpage>493</fpage>–<lpage>504</lpage>.
<pub-id pub-id-type="pmid">4067301</pub-id></mixed-citation></ref><ref id="b8"><label>8</label><mixed-citation publication-type="journal" id="cit8"><string-name><surname>Nelson</surname><given-names>R</given-names></string-name>.
<article-title>Influenza A H2N2 saga remains unexplained</article-title>.
<source>Lancet Infect Dis</source><year>2005</year>; <volume>5</volume>:<fpage>332</fpage>.
</mixed-citation></ref><ref id="b9"><label>9</label><mixed-citation publication-type="journal" id="cit9"><string-name><surname>Schrauder</surname><given-names>A</given-names></string-name>,
<string-name><surname>Schweiger</surname><given-names>B</given-names></string-name>,
<string-name><surname>Buchholz</surname><given-names>U</given-names></string-name><italic>et al.
</italic><article-title>Laboratory exposure to influenza A H2N2, Germany, 2004–2005</article-title>.
<source>Emerg Infect Dis</source><year>2006</year>; <volume>12</volume>:<fpage>1997</fpage>–<lpage>1998</lpage>.
<pub-id pub-id-type="pmid">17354343</pub-id></mixed-citation></ref><ref id="b10"><label>10</label><mixed-citation publication-type="journal" id="cit10"><string-name><surname>Maassab</surname><given-names>HF</given-names></string-name>.
<article-title>Adaptation and growth characteristics of influenza virus at 25 degrees C</article-title>.
<source>Nature</source><year>1967</year>; <volume>213</volume>:<fpage>612</fpage>–<lpage>614</lpage>.
<pub-id pub-id-type="pmid">6040602</pub-id></mixed-citation></ref><ref id="b11"><label>11</label><mixed-citation publication-type="journal" id="cit11"><string-name><surname>Kitayama</surname><given-names>T</given-names></string-name>,
<string-name><surname>Togo</surname><given-names>Y</given-names></string-name>,
<string-name><surname>Hornick</surname><given-names>RB</given-names></string-name>,
<string-name><surname>Friedwald</surname><given-names>WT</given-names></string-name>.
<article-title>Low‐temperature‐adapted influenza A2/AA/6/60 virus vaccine in man</article-title>.
<source>Infect Immun</source><year>1973</year>; <volume>7</volume>:<fpage>119</fpage>–<lpage>122</lpage>.
<pub-id pub-id-type="pmid">16558072</pub-id></mixed-citation></ref><ref id="b12"><label>12</label><mixed-citation publication-type="journal" id="cit12"><string-name><surname>Nichol</surname><given-names>KL</given-names></string-name>,
<string-name><surname>Mendelman</surname><given-names>PM</given-names></string-name>,
<string-name><surname>Mallon</surname><given-names>KP</given-names></string-name><italic>et al.
</italic><article-title>Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial</article-title>.
<source>JAMA</source><year>1999</year>; <volume>282</volume>:<fpage>137</fpage>–<lpage>144</lpage>.
<pub-id pub-id-type="pmid">10411194</pub-id></mixed-citation></ref><ref id="b13"><label>13</label><mixed-citation publication-type="journal" id="cit13"><string-name><surname>Belshe</surname><given-names>RB</given-names></string-name>,
<string-name><surname>Edwards</surname><given-names>KM</given-names></string-name>,
<string-name><surname>Vesikari</surname><given-names>T</given-names></string-name><italic>et al.
</italic><article-title>Live attenuated versus inactivated influenza vaccine in infants and young children</article-title>.
<source>N Engl J Med</source><year>2007</year>; <volume>356</volume>:<fpage>685</fpage>–<lpage>696</lpage>.
<pub-id pub-id-type="pmid">17301299</pub-id></mixed-citation></ref><ref id="b14"><label>14</label><mixed-citation publication-type="miscellaneous" id="cit14"><collab collab-type="authors">US FDA</collab>.
<article-title>February 3, 2010 Approval Letter – Influenza A (H1N1) 2009 Monovalent Vaccine (MedImmune LLC)</article-title>. <year>2010</year> Available at <ext-link ext-link-type="uri" xlink:href="http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm201995.htm">http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm201995.htm</ext-link> (cited 20 December 2011).
</mixed-citation></ref><ref id="b15"><label>15</label><mixed-citation publication-type="journal" id="cit15"><string-name><surname>Karron</surname><given-names>RA</given-names></string-name>,
<string-name><surname>Callahan</surname><given-names>K</given-names></string-name>,
<string-name><surname>Luke</surname><given-names>C</given-names></string-name><italic>et al.
</italic><article-title>A Live Attenuated H9N2 Influenza Vaccine Is Well Tolerated and Immunogenic in Healthy Adults</article-title>.
<source>J Infect Dis</source><year>2009</year>; <volume>199</volume>:<fpage>711</fpage>–<lpage>716</lpage>.
<pub-id pub-id-type="pmid">19210163</pub-id></mixed-citation></ref><ref id="b16"><label>16</label><mixed-citation publication-type="journal" id="cit16"><string-name><surname>Talaat</surname><given-names>KR</given-names></string-name>,
<string-name><surname>Karron</surname><given-names>RA</given-names></string-name>,
<string-name><surname>Callahan</surname><given-names>KA</given-names></string-name><italic>et al.
</italic><article-title>A live attenuated H7N3 influenza virus vaccine is well tolerated and immunogenic in a Phase I trial in healthy adults</article-title>.
<source>Vaccine</source><year>2009</year>; <volume>27</volume>:<fpage>3744</fpage>–<lpage>3753</lpage>.
<pub-id pub-id-type="pmid">19464558</pub-id></mixed-citation></ref><ref id="b17"><label>17</label><mixed-citation publication-type="journal" id="cit17"><string-name><surname>Karron</surname><given-names>RA</given-names></string-name>,
<string-name><surname>Talaat</surname><given-names>K</given-names></string-name>,
<string-name><surname>Luke</surname><given-names>C</given-names></string-name><italic>et al.
</italic><article-title>Evaluation of two live attenuated cold‐adapted H5N1 influenza virus vaccines in healthy adults</article-title>.
<source>Vaccine</source><year>2009</year>; <volume>27</volume>:<fpage>4953</fpage>–<lpage>4960</lpage>.
<pub-id pub-id-type="pmid">19540952</pub-id></mixed-citation></ref><ref id="b18"><label>18</label><mixed-citation publication-type="miscellaneous" id="cit18"><collab collab-type="authors">WHO</collab>.
<article-title>Tables on the Clinical trials of pandemic influenza prototype vaccines</article-title>, <year>2009</year> Available at <ext-link ext-link-type="uri" xlink:href="http://www.who.int/vaccine_research/diseases/influenza/flu_trials_tables/en/index.html">http://www.who.int/vaccine_research/diseases/influenza/flu_trials_tables/en/index.html</ext-link> (accessed 25 August 2009, 23 February 2010).
</mixed-citation></ref><ref id="b19"><label>19</label><mixed-citation publication-type="journal" id="cit19"><string-name><surname>Murphy</surname><given-names>BR</given-names></string-name>,
<string-name><surname>Tierney</surname><given-names>EL</given-names></string-name>,
<string-name><surname>Barbour</surname><given-names>BA</given-names></string-name><italic>et al.
</italic><article-title>Use of the enzyme‐linked immunosorbent assay to detect serum antibody responses of volunteers who received attenuated influenza A virus vaccines</article-title>.
<source>Infect Immun</source><year>1980</year>; <volume>29</volume>:<fpage>342</fpage>–<lpage>347</lpage>.
<pub-id pub-id-type="pmid">7011972</pub-id></mixed-citation></ref><ref id="b20"><label>20</label><mixed-citation publication-type="journal" id="cit20"><string-name><surname>Rowe</surname><given-names>T</given-names></string-name>,
<string-name><surname>Abernathy</surname><given-names>RA</given-names></string-name>,
<string-name><surname>Hu‐Primmer</surname><given-names>J</given-names></string-name><italic>et al.
</italic><article-title>Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays</article-title>.
<source>J Clin Microbiol</source><year>1999</year>; <volume>37</volume>:<fpage>937</fpage>–<lpage>943</lpage>.
<pub-id pub-id-type="pmid">10074505</pub-id></mixed-citation></ref><ref id="b21"><label>21</label><mixed-citation publication-type="journal" id="cit21"><string-name><surname>Clements</surname><given-names>ML</given-names></string-name>,
<string-name><surname>O’Donnell</surname><given-names>S</given-names></string-name>,
<string-name><surname>Levine</surname><given-names>MM</given-names></string-name>,
<string-name><surname>Chanock</surname><given-names>RM</given-names></string-name>,
<string-name><surname>Murphy</surname><given-names>BR</given-names></string-name>.
<article-title>Dose response of A/Alaska/6/77 (H3N2) cold adapted reassortant vaccine virus in adult volunteers: role of local antibody in resistance to infection with vaccine virus</article-title>.
<source>Infect Immun</source><year>1983</year>; <volume>40</volume>:<fpage>1044</fpage>.
<pub-id pub-id-type="pmid">6852910</pub-id></mixed-citation></ref><ref id="b22"><label>22</label><mixed-citation publication-type="journal" id="cit22"><string-name><surname>Sasaki</surname><given-names>S</given-names></string-name>,
<string-name><surname>Jaimes</surname><given-names>MC</given-names></string-name>,
<string-name><surname>Holmes</surname><given-names>TH</given-names></string-name><italic>et al.
</italic><article-title>Comparison of the influenza virus‐specific effector and memory B‐cell responses to immunization of children and adults with live attenuated or inactivated influenza virus vaccines</article-title>.
<source>J Virol</source><year>2007</year>; <volume>81</volume>:<fpage>215</fpage>–<lpage>228</lpage>.
<pub-id pub-id-type="pmid">17050593</pub-id></mixed-citation></ref><ref id="b23"><label>23</label><mixed-citation publication-type="journal" id="cit23"><string-name><surname>Sandbulte</surname><given-names>MR</given-names></string-name>,
<string-name><surname>Gao</surname><given-names>J</given-names></string-name>,
<string-name><surname>Straight</surname><given-names>TM</given-names></string-name>,
<string-name><surname>Eichelberger</surname><given-names>MC</given-names></string-name>.
<article-title>A miniaturized assay for influenza neuraminidase‐inhibiting antibodies utilizing reverse genetics‐derived antigens</article-title>.
<source>Influenza Other Respir Viruses</source><year>2009</year>; <volume>3</volume>:<fpage>233</fpage>–<lpage>240</lpage>.
<pub-id pub-id-type="pmid">21462400</pub-id></mixed-citation></ref><ref id="b24"><label>24</label><mixed-citation publication-type="journal" id="cit24"><string-name><surname>Lai</surname><given-names>KK</given-names></string-name>,
<string-name><surname>Cook</surname><given-names>L</given-names></string-name>,
<string-name><surname>Wendt</surname><given-names>S</given-names></string-name>,
<string-name><surname>Corey</surname><given-names>L</given-names></string-name>,
<string-name><surname>Jerome</surname><given-names>KR</given-names></string-name>.
<article-title>Evaluation of real‐time PCR versus PCR with liquid‐phase hybridization for detection of enterovirus RNA in cerebrospinal fluid</article-title>.
<source>J Clin Microbiol</source><year>2003</year>; <volume>41</volume>:<fpage>3133</fpage>–<lpage>3141</lpage>.
<pub-id pub-id-type="pmid">12843053</pub-id></mixed-citation></ref><ref id="b25"><label>25</label><mixed-citation publication-type="journal" id="cit25"><string-name><surname>Subbarao</surname><given-names>K</given-names></string-name>,
<string-name><surname>Joseph</surname><given-names>T</given-names></string-name>.
<article-title>Scientific barriers to developing vaccines against avian influenza viruses</article-title>.
<source>Nat Rev Immunol</source><year>2007</year>; <volume>7</volume>:<fpage>267</fpage>–<lpage>278</lpage>.
<pub-id pub-id-type="pmid">17363960</pub-id></mixed-citation></ref><ref id="b26"><label>26</label><mixed-citation publication-type="journal" id="cit26"><string-name><surname>Plotkin</surname><given-names>SA</given-names></string-name>.
<article-title>Vaccines: correlates of vaccine‐induced immunity</article-title>.
<source>Clin Infect Dis</source><year>2008</year>; <volume>47</volume>:<fpage>401</fpage>–<lpage>409</lpage>.
<pub-id pub-id-type="pmid">18558875</pub-id></mixed-citation></ref><ref id="b27"><label>27</label><mixed-citation publication-type="journal" id="cit27"><string-name><surname>Eichelberger</surname><given-names>M</given-names></string-name>,
<string-name><surname>Golding</surname><given-names>H</given-names></string-name>,
<string-name><surname>Hess</surname><given-names>M</given-names></string-name><italic>et al.
</italic><article-title>FDA/NIH/WHO public workshop on immune correlates of protection against influenza A viruses in support of pandemic vaccine development, Bethesda, Maryland, US, December 10–11, 2007</article-title>.
<source>Vaccine</source><year>2008</year>; <volume>26</volume>:<fpage>4299</fpage>–<lpage>4303</lpage>.
<pub-id pub-id-type="pmid">18582523</pub-id></mixed-citation></ref><ref id="b28"><label>28</label><mixed-citation publication-type="journal" id="cit28"><string-name><surname>Belshe</surname><given-names>RB</given-names></string-name>,
<string-name><surname>Gruber</surname><given-names>WC</given-names></string-name>,
<string-name><surname>Mendelman</surname><given-names>PM</given-names></string-name><italic>et al.
</italic><article-title>Correlates of immune protection induced by live, attenuated, cold‐adapted, trivalent, intranasal influenza virus vaccine</article-title>.
<source>J Infect Dis</source><year>2000</year>; <volume>181</volume>:<fpage>1133</fpage>–<lpage>1137</lpage>.
<pub-id pub-id-type="pmid">10720541</pub-id></mixed-citation></ref><ref id="b29"><label>29</label><mixed-citation publication-type="journal" id="cit29"><string-name><surname>Belshe</surname><given-names>R</given-names></string-name>,
<string-name><surname>Lee</surname><given-names>MS</given-names></string-name>,
<string-name><surname>Walker</surname><given-names>RE</given-names></string-name>,
<string-name><surname>Stoddard</surname><given-names>J</given-names></string-name>,
<string-name><surname>Mendelman</surname><given-names>PM</given-names></string-name>.
<article-title>Safety, immunogenicity and efficacy of intranasal, live attenuated influenza vaccine</article-title>.
<source>Expert Rev Vaccines</source><year>2004</year>; <volume>3</volume>:<fpage>643</fpage>–<lpage>654</lpage>.
<pub-id pub-id-type="pmid">15606348</pub-id></mixed-citation></ref><ref id="b30"><label>30</label><mixed-citation publication-type="journal" id="cit30"><string-name><surname>Basha</surname><given-names>S</given-names></string-name>,
<string-name><surname>Hazenfeld</surname><given-names>S</given-names></string-name>,
<string-name><surname>Brady</surname><given-names>RC</given-names></string-name>,
<string-name><surname>Subbramanian</surname><given-names>RA</given-names></string-name>.
<article-title>Comparison of antibody and T‐cell responses elicited by licensed inactivated‐ and live‐attenuated influenza vaccines against H3N2 hemagglutinin</article-title>.
<source>Hum Immunol</source><year>2011</year>; <volume>72</volume>:<fpage>463</fpage>–<lpage>469</lpage>.
<pub-id pub-id-type="pmid">21414368</pub-id></mixed-citation></ref><ref id="b31"><label>31</label><mixed-citation publication-type="journal" id="cit31"><string-name><surname>Meijer</surname><given-names>A</given-names></string-name>,
<string-name><surname>Bosman</surname><given-names>A</given-names></string-name>,
<string-name><surname>van de Kamp</surname><given-names>EE</given-names></string-name>,
<string-name><surname>Wilbrink</surname><given-names>B</given-names></string-name>,
<string-name><surname>van Beest Holle Mdu</surname><given-names>R</given-names></string-name>,
<string-name><surname>Koopmans</surname><given-names>M</given-names></string-name>.
<article-title>Measurement of antibodies to avian influenza virus A(H7N7) in humans by hemagglutination inhibition test</article-title>.
<source>J Virol Methods</source><year>2006</year>; <volume>132</volume>:<fpage>113</fpage>–<lpage>120</lpage>.
<pub-id pub-id-type="pmid">16271401</pub-id></mixed-citation></ref><ref id="b32"><label>32</label><mixed-citation publication-type="journal" id="cit32"><string-name><surname>Chen</surname><given-names>GL</given-names></string-name>,
<string-name><surname>Lamirande</surname><given-names>EW</given-names></string-name>,
<string-name><surname>Jin</surname><given-names>H</given-names></string-name>,
<string-name><surname>Kemble</surname><given-names>G</given-names></string-name>,
<string-name><surname>Subbarao</surname><given-names>K</given-names></string-name>.
<article-title>Safety, immunogencity, and efficacy of a cold‐adapted A/Ann Arbor/6/60 (H2N2) vaccine in mice and ferrets</article-title>.
<source>Virology</source><year>2010</year>; <volume>398</volume>:<fpage>109</fpage>–<lpage>114</lpage>.
<pub-id pub-id-type="pmid">20034647</pub-id></mixed-citation></ref><ref id="b33"><label>33</label><mixed-citation publication-type="journal" id="cit33"><string-name><surname>Beyer</surname><given-names>WE</given-names></string-name>,
<string-name><surname>Palache</surname><given-names>AM</given-names></string-name>,
<string-name><surname>de Jong</surname><given-names>JC</given-names></string-name>,
<string-name><surname>Osterhaus</surname><given-names>AD</given-names></string-name>.
<article-title>Cold‐adapted live influenza vaccine versus inactivated vaccine: systemic vaccine reactions, local and systemic antibody response, and vaccine efficacy. A meta‐analysis</article-title>.
<source>Vaccine</source><year>2002</year>; <volume>20</volume>:<fpage>1340</fpage>–<lpage>1353</lpage>.
<pub-id pub-id-type="pmid">11818152</pub-id></mixed-citation></ref><ref id="b34"><label>34</label><mixed-citation publication-type="journal" id="cit34"><string-name><surname>Ambrose</surname><given-names>CS</given-names></string-name>,
<string-name><surname>Levin</surname><given-names>MJ</given-names></string-name>,
<string-name><surname>Belshe</surname><given-names>RB</given-names></string-name>.
<article-title>The relative efficacy of trivalent live attenuated and inactivated influenza vaccines in children and adults</article-title>.
<source>Influenza Other Respi Viruses</source><year>2011</year>; <volume>5</volume>:<fpage>67</fpage>–<lpage>75</lpage>.
</mixed-citation></ref><ref id="b35"><label>35</label><mixed-citation publication-type="journal" id="cit35"><string-name><surname>Rhorer</surname><given-names>J</given-names></string-name>,
<string-name><surname>Ambrose</surname><given-names>CS</given-names></string-name>,
<string-name><surname>Dickinson</surname><given-names>S</given-names></string-name><italic>et al.
</italic><article-title>Efficacy of live attenuated influenza vaccine in children: a meta‐analysis of nine randomized clinical trials</article-title>.
<source>Vaccine</source><year>2009</year>; <volume>27</volume>:<fpage>1101</fpage>–<lpage>1110</lpage>.
<pub-id pub-id-type="pmid">19095024</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/H2N2V1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000977 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000977 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= H2N2V1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:3527634
|texte= An open‐label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:22417012" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a H2N2V1
| This area was generated with Dilib version V0.6.33. |  |