A Candidate H1N1 Pandemic Influenza Vaccine Elicits Protective Immunity in Mice

BackgroundIn 2009 a new pandemic disease appeared and spread globally. The recent emergence of the pandemic influenza virus H1N1 first isolated in Mexico and USA raised concerns about vaccine availability. We here report our development of an adenovirus-based influenza H1N1 vaccine tested for immunogenicity and efficacy to confer protection in animal model.MethodsWe generated two adenovirus(Ad5)-based influenza vaccine candidates encoding the wildtype or a codon-optimized hemagglutinin antigen (HA) from the recently emerged swine influenza isolate A/California/04/2009 (H1N1)pdm. After verification of antigen expression, immunogenicity of the vaccine candidates were tested in a mouse model using dose escalations for subcutaneous immunization. Sera of immunized animals were tested in microneutalization and hemagglutination inhibition assays for the presence of HA-specific antibodies. HA-specific T-cells were measured in IFN? Elispot assays. The efficiency of the influenza vaccine candidates were evaluated in a challenge model by measuring viral titer in lung and nasal turbinate 3 days after inoculation of a homologous H1N1 virus.Conclusions/SignificanceA single immunization resulted in robust cellular and humoral immune response. Remarkably, the intensity of the immune response was substantially enhanced with codon-optimized antigen, indicating the benefit of manipulating the genetic code of HA antigens in the context of recombinant influenza vaccine design. These results highlight the value of advanced technologies in vaccine development and deployment in response to infections with pandemic potential. Our study emphasizes the potential of an adenoviral-based influenza vaccine platform with the benefits of speed of manufacture and efficacy of a single dose immunizatio

Verification of gene expression by the H1N1 vaccine candidates.

<p>HA expression in A549 cells transduced with AdHA.wt, AdHA.cod or Ad5. <b>A,</b> HA detection in cell lysates by western blot analysis (60 ug of total protein loaded in each lane) using ferret antiserum. <b>B</b>, Flow cytometric analysis of HA expression at the cell surface using ferret or mouse antiserum. Shown are the percentages of HA positive cells.</p

Induced protection against H1N1 by the H1N1 vaccine candidates.

<p>Protection of immunized animals (5 or 6 weeks after immunization with AdHA.wt vs Ad5 control or AdHA.cod vs Ad5 control) against an intranasal challenge with 1000 pfu of A/Ohio/7/09 (H1N1)pdm was measured as viral titers in lung and nasal turbinate determined 3 days post-inoculation in a plaque formation assay using MDCK-L cells. Shown are log10 values of mean titer for each group ± SEM. The horizontal dashed line represents the lower limit of detection of the assay.</p

Protection against influenza A/Ohio/7/09 (H1N1)pdm.

(1)<p>number of animals from which virus was cultured/number of animals inoculated.</p

Induction of humoral and cellular immune responses by the H1N1 vaccine candidates.

<p>Induction of influenza H1N1-specific immune responses after subcutaneous immunization of Balb/c mice with adenovirus encoding wildtype (AdHA.wt) or codon-optimized (AdHA.cod) HA antigen or control adenovirus (Ad5). <b>A,</b> Antibody to influenza A/Texas/05/09 (H1N1)pdm in sera of mice after receiving dose escalations of the vaccine candidates (AdHA.wt or AdHA.cod). Shown are log2 values of HI-titer. Horizontal lines represent geometric means in each group. <b>B</b>, Induction of H1N1-specific neutralizing antibodies in sera 5 weeks after immunization with AdHA.wt, AdHA.cod or Ad5 control. Shown are neutralizing antibody titer to influenza A/Texas/05/09 (H1N1)pdm measured by microneutralization assay. Horizontal lines represent geometric means in each group. <b>C,</b> HA peptide specific T cell responses in splenocytes detected in IFNγ Elispot assays. A representative result is shown as means of SFC ± SEM of triplicate determinations in each group.</p