Assessment of Human Immune Responses to H7 Avian Influenza Virus of Pandemic Potential: Results from a Placebo–Controlled, Randomized Double–Blind Phase I Study of Live Attenuated H7N3 Influenza Vaccine

<div><p>Introduction</p><p>Live attenuated influenza vaccines (LAIVs) are being developed to protect humans against future epidemics and pandemics. This study describes the results of a double–blinded randomized placebo–controlled phase I clinical trial of cold–adapted and temperature sensitive H7N3 live attenuated influenza vaccine candidate in healthy seronegative adults.</p><p>Objective</p><p>The goal of the study was to evaluate the safety, tolerability, immunogenicity and potential shedding and transmission of H7N3 LAIV against H7 avian influenza virus of pandemic potential.</p><p>Methods and Findings</p><p>Two doses of H7N3 LAIV or placebo were administered to 40 randomly divided subjects (30 received vaccine and 10 placebo). The presence of influenza A virus RNA in nasal swabs was detected in 60.0% and 51.7% of subjects after the first and second vaccination, respectively. In addition, vaccine virus was not detected among placebo recipients demonstrating the absence of person–to–person transmission. The H7N3 live attenuated influenza vaccine demonstrated a good safety profile and was well tolerated. The two–dose immunization resulted in measurable serum and local antibody production and in generation of antigen–specific CD4⁺ and CD8⁺ memory T cells. Composite analysis of the immune response which included hemagglutinin inhibition assay, microneutralization tests, and measures of IgG and IgA and virus–specific T cells showed that the majority (86.2%) of vaccine recipients developed serum and/or local antibodies responses and generated CD4⁺ and CD8⁺ memory T cells.</p><p>Conclusions</p><p>The H7N3 LAIV was safe and well tolerated, immunogenic in healthy seronegative adults and elicited production of antibodies broadly reactive against the newly emerged H7N9 avian influenza virus.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="http://clinicaltrials.gov/show/NCT01511419" target="_blank">NCT01511419</a></p></div

Trial profile.

<p>CONSORT 2010 Flow Diagram. The schema graphically outlines the design and conduct of the clinical study. One subject dropped out from the study prior to receiving the second dose of vaccine because of an adverse event not related to the vaccination (adenovirus infection on Day 28 confirmed by PCR).</p

Detection of influenza A virus in nasal swabs.

1<p>All subjects were negative before vaccination and revaccination, respectively.</p>2<p>Total number of positive subjects.</p

Genetic stability of attenuating mutations of LAIV vaccine reassortant A/17/mallard/Netherlands/00/95 (H7N3) isolates derived from vaccinated subjects.

1<p>wild–type strain A/Leningrad/134/57 (H2N2);</p>2<p>cold–adapted MDV A/Leningrad/134/17/57 (H2N2);</p>3<p>LAIV A/17/mallard/Netherlands/00/95 (H7N3) strain.</p

Restriction of growth of H7N3 vaccine isolates at different temperatures.

1<p>From the titer at permissive temperature (32°C);</p>2<p>A/Leningrad/134/17/57 (H2N2) master donor virus was used as a positive control of <i>ts</i> and <i>ca</i> markers.</p

Antibody responses to A/17/mallard/Netherlands/00/95 (H7N3) LAIV.

1<p>The HAI antibody GMT after second vaccination was statistically significantly higher than pre–vaccination GMT (Wilcoxon Matched Pairs Test with Bonferroni adjustment: p = 0,0024);</p>2<p>There was no statistically significant difference between serum HAI antibody GMTs at three time points in placebo group (Friedman ANOVA: ANOVA Chi Sqr. (N = 10, df = 2) = 0,6667, p = 0,7165);</p>3<p>The HAI antibody GMT after first vaccination was statistically significantly higher than pre–vaccination GMT (Wilcoxon Matched Pairs Test with Bonferroni adjustment: p = 0,0012);</p>4<p>The HAI antibody GMT after second vaccination was statistically significantly higher than pre–vaccination GMT (Wilcoxon Matched Pairs Test with Bonferroni adjustment: p = 0,0004);</p>5<p>There was no statistically significant difference between serum HAI antibody GMTs at three time points in placebo group (Friedman ANOVA: ANOVA Chi Sqr. (N = 10, df = 2) = 2,6667; p = 0,2636);</p>6<p>The serum neutralizing antibody GMT after first vaccination was statistically significantly higher than pre–vaccination GMT (Wilcoxon Matched Pairs Test with Bonferroni adjustment: p = 0,0166);</p>7<p>The serum neutralizing antibody GMT after second vaccination was statistically significantly higher than pre–vaccination GMT (Wilcoxon Matched Pairs Test with Bonferroni adjustment: p = 0,0001);</p>8<p>The serum neutralizing antibody GMT after second vaccination was statistically significantly higher than GMT after first vaccination (Wilcoxon Matched Pairs Test with Bonferroni adjustment: p = 0,0025);</p>9<p>There was no statistically significant difference between serum neutralizing antibody GMTs at three time points in placebo group (Friedman ANOVA: ANOVA Chi Sqr. (N = 10, df = 2) = 2,000, p = 0,3679);</p>10<p>There was no statistically significant difference between serum IgG GMTs at three time points in vaccinated group (Friedman ANOVA: ANOVA Chi Sqr. N = 29, df = 2) = 0,2222, p = 0,8948);</p>11<p>There was no statistically significant difference between serum IgG GMTs at three time points in placebo group (Friedman ANOVA: ANOVA Chi Sqr. (N = 10, df = 2) = 3,000, p = 0,2231);</p>12<p>The serum IgA GMT after second vaccination was statistically significantly higher than pre–vaccination GMT (Wilcoxon Matched Pairs Test with Bonferroni adjustment: p = 0,0157);</p>13<p>There was no statistically significant difference between serum IgA GMTs at three time points in placebo group (Friedman ANOVA: ANOVA Chi Sqr. (N = 10, df = 2) = 2,6667, p = 0,2636);</p>14<p>The mucosal IgA GMT after first vaccination was statistically significantly higher than pre–vaccination GMT (Wilcoxon Matched Pairs Test with Bonferroni adjustment: p = 0,0031);</p>15<p>There was no statistically significant difference between mucosal IgA GMTs at three time points in placebo group (Friedman ANOVA: ANOVA Chi Sqr. (N = 10, df = 2) = 0,3077, p = 0,8574);</p>16<p>Percent with ≥fourfold HAI antibody titer rise after two doses of LAIV A(H7N3) was statistically significantly higher than in placebo group (Fisher exact (two–tailed) p = 0,0161).</p>17<p>Percent with ≥four fold serum neutralizing antibody titer rise after two doses of LAIV A(H7N3) was statistically significantly higher than in placebo group (Fisher exact (two–tailed) p = 0,0172).</p

Cumulative data on immune responses to A/17/Mallard/Netherlands/00/95 (H7N3) LAIV in vaccinated subjects after the first and/or the second doses.

1<p>Occurrences of antibody conversions or cellular responses after the first and/or the second doses of A (H7N3) LAIV.</p

T cell induction after <i>in vitro</i> stimulation of PBMC from subjects vaccinated with LAIV A/17/Mallard/Netherlands/00/95 (H7N3).

1<p>Data exceeding 3 SD of placebo mean value.</p>2<p>LAIV (n = 29), placebo (n = 9). When calculating placebo mean value the data from one of the subjects were not available because of the sample damage.</p

Cross–reactivity provided by H7N3 LAIV to H7N9 avian influenza virus.

1<p>A/17/mallard/Netherlands/00/95 (H7N3) LAIV;</p>2<p>A/Anhui/1/2013 (H7N9).</p

Percentage of adult subjects vaccinated with H7N3 LAIV with solicited local and systemic reactions within 7 days of vaccination.

1<p>All reactions observed were mild and included sore throat, fever, nasal congestion and catarrhial nasopharinx, sneeze and headache.</p>2<p>All reactions observed were mild and included sore throat, fever, cough and nasal congestion.</p>3<p>95% confidence interval.</p