Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine

The target of neutralizing antibodies that protect against influenza virus infection is the viral protein HA. Genetic and antigenic variation in HA has been used to classify influenza viruses into subtypes (H1–H16). The neutralizing antibody response to influenza virus is thought to be specific for a few antigenically related isolates within a given subtype. However, while heterosubtypic antibodies capable of neutralizing multiple influenza virus subtypes have been recently isolated from phage display libraries, it is not known whether such antibodies are produced in the course of an immune response to influenza virus infection or vaccine. Here we report that, following vaccination with seasonal influenza vaccine containing H1 and H3 influenza virus subtypes, some individuals produce antibodies that cross-react with H5 HA. By immortalizing IgG-expressing B cells from 4 individuals, we isolated 20 heterosubtypic mAbs that bound and neutralized viruses belonging to several HA subtypes (H1, H2, H5, H6, and H9), including the pandemic A/California/07/09 H1N1 isolate. The mAbs used different VH genes and carried a high frequency of somatic mutations. With the exception of a mAb that bound to the HA globular head, all heterosubtypic mAbs bound to acid- sensitive epitopes in the HA stem region. Four mAbs were evaluated in vivo and protected mice from challenge with influenza viruses representative of different subtypes. These findings reveal that seasonal influenza vaccination can induce polyclonal heterosubtypic neutralizing antibodies that cross-react with the swine-origin pandemic H1N1 influenza virus and with the highly pathogenic H5N1 virus

Influenza H5 Hemagglutinin DNA Primes the Antibody Response Elicited by the Live Attenuated Influenza A/Vietnam/1203/2004 Vaccine in Ferrets

Priming immunization plays a key role in protecting individuals or populations to influenza viruses that are novel to humans. To identify the most promising vaccine priming strategy, we have evaluated different prime-boost regimens using inactivated, DNA and live attenuated vaccines in ferrets. Live attenuated influenza A/Vietnam/1203/2004 (H5N1) candidate vaccine (LAIV, VN04 ca) primed ferrets efficiently while inactivated H5N1 vaccine could not prime the immune response in seronegative ferrets unless an adjuvant was used. However, the H5 HA DNA vaccine alone was as successful as an adjuvanted inactivated VN04 vaccine in priming the immune response to VN04 ca virus. The serum antibody titers of ferrets primed with H5 HA DNA followed by intranasal vaccination of VN04 ca virus were comparable to that induced by two doses of VN04 ca virus. Both LAIV-LAIV and DNA-LAIV vaccine regimens could induce antibody responses that cross-neutralized antigenically distinct H5N1 virus isolates including A/HongKong/213/2003 (HK03) and prevented nasal infection of HK03 vaccine virus. Thus, H5 HA DNA vaccination may offer an alternative option for pandemic preparedness

Eurasian-Origin Gene Segments Contribute to the Transmissibility, Aerosol Release, and Morphology of the 2009 Pandemic H1N1 Influenza Virus

The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus

Kinetics of the development of H5 HA-specific serum IgG (A), IgA (B), and neutralizing antibodies (C) elicited by various H5N1 prime-boost regimens.

<p>Ferrets were immunized with either 10⁷ PFU of VN04 <i>ca</i> virus (weeks 0 and 4) or 200 µg of H5 HA DNA (weeks 0, 2 and/or 4) and serum samples were collected regularly at 2-week intervals. H5 HA-specific IgG and IgA in ferret serum samples (N = 4/group) were determined by ELISA using recombinant VN04 H5 HA as antigen. Neutralizing antibody titers against 100 TCID₅₀ of the VN04 <i>ca</i> virus are depicted. Geometric mean titers from each group are shown.</p

Protection conferred by different H5 vaccine prime-boost regimens in ferrets.

<p>Four weeks after boost immunization, ferrets were intranasally infected with 10⁷ PFU of HK03 <i>ca</i> virus. Nasal turbinates were collected on day 3 post-infection and viral titers in tissue homogenates were determined by EID₅₀. Horizontal lines depict the mean virus titers for each group. An asterisk above a horizontal bar denotes statistical significance compared to the vector DNA group (<i>p</i><0.05, Mann-Whitney U Test).</p

H5 HA DNA vaccine construct.

<p>(A) Schematic diagram of H5-VN.tPA HA gene. The H5-VN.tPA DNA encodes the full length HA antigens with a human tissue plasminogen activator (tPA) leader substituting for the natural HA leader sequence. The cleavage site between HA1 and HA2 subunits are indicated. The numbers above the HA inserts denote the relevant amino acid positions in natural HA proteins. (B) Western-blot analysis of the expression of HA antigen by H5-VN.tPA DNA vaccine in transiently transfected 293T cell supernatant (S) and cell lysate (L).</p

Ferret serum antibody titers from vaccination with iVN04 and VN04 <i>ca</i>.

<p>Ferrets were inoculated with 10⁷ PFU of VN04 <i>ca</i> intranasally or 15 µg of iVN04 vaccine intramuscularly. Blood was collected 4 weeks after each dose and the H5N1-specific antibody levels (expressed as geometric mean titers) were determined by microneutralization assay.</p

Serum antibody titers against homologous and variant H5N1 <i>ca</i> viruses following different vaccine regimens in ferrets.

<p>Ferrets (N = 4/group) were inoculated with 10⁷ PFU of VN04 H5N1 <i>ca</i> intranasally or 200 µg of vector or H5 HA DNA vaccine intramuscularly as indicated. Blood was collected 4 weeks after each dose and H5N1-specific antibody titers in the serum were determined by microneutralization (left panel) and HAI (right panel) assays. Geometric mean titers of neutralizing and HAI Ab against (A) VN04 (A/Vietnam/1203/2004), (B) HK03 (A/Hong Kong/213/2003), (C) AH05 (A/Anhui/1/2005), and (D) IN05 (A/Indonesia/5/2005) <i>ca</i> viruses are depicted. Data points in black indicate titers of post-prime and data points in orange indicate titers of post-boost and the mean Ab titers are indicated in horizontal solid lines. The dashed line indicates the detection limit of the assays.</p

Survival of Mice Immunized with a Single Dose of H5N1 <i>ca</i> Vaccine Candidates Following Lethal Challenge with H5N1 1997 <i>wt</i> or H5N1 2004 <i>wt</i> Viruses

<p>Groups of eight mice that received a single dose of 10⁶ TCID₅₀ of the H5N1 1997 <i>ca</i> (▪), H5N1 2003 <i>ca</i> (▵), H5N1 2004 <i>ca</i> (̂), or AA <i>ca</i> (○) viruses, or were mock-immunized (L-15 medium; ♦) were challenged with the H5N1 1997 <i>wt</i> (A–C) or H5N1 2004 <i>wt</i> virus (D–F) at 50 (A and D), 500 (B and E), or 5,000 (C and F) LD₅₀. The mice were monitored daily for 21 d p.i. Immunization with the H5N1 1997 <i>ca,</i> H5N1 2003 <i>ca,</i> and H5N1 2004 <i>ca</i> viruses resulted in 100% survival of the mice following challenge, so the symbols for these groups are superimposed.</p

Efficacy in Ferrets of Two Doses of H5N1 <i>ca</i> Viruses against Challenge with Homologous and Heterologous H5N1 <i>wt</i> Viruses

<div><p>Ferrets were immunized with two doses of 10⁷ TCID₅₀ of H5N1 1997 <i>ca</i> or H5N1 2004 <i>ca</i> viruses 1 mo apart and challenged 1 mo later with 10⁷ TCID₅₀ of either H5N1 1997 <i>wt,</i> H5N1 2004 <i>wt,</i> or A/Indonesia/05/2005 (H5N1) <i>wt</i>. In order to determine the contribution of the internal protein genes of the AA <i>ca</i> virus to protection, an additional group of ferrets was immunized with H1N1 A/New Caledonia/99 <i>ca</i> and challenged with H5N1 1997 <i>wt</i>. These ferrets were not challenged with H5N1 2004 <i>wt</i> or A/Indonesia/05/2005 (H5N1) <i>wt</i>.</p> <p>Lungs, nasal turbinates, and brains were harvested 3 d after challenge and were homogenized and titered on MDCK cells. Three ferrets per group were evaluated. The lower limit of detection is indicated by the dashed line. *<i>p</i> < 0.05 compared to mock-immunized ferrets.</p></div