A Whole Virus Pandemic Influenza H1N1 Vaccine Is Highly Immunogenic and Protective in Active Immunization and Passive Protection Mouse Models

The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine

Comprehensive Size-Determination of Whole Virus Vaccine Particles Using Gas-Phase Electrophoretic Mobility Macromolecular Analyzer, Atomic Force Microscopy, and Transmission Electron Microscopy

Biophysical properties including particle size distribution, integrity, and shape of whole virus vaccine particles at different stages in tick-borne encephalitis (TBE) vaccines formulation were analyzed by a new set of methods. Size-exclusion chromatography (SEC) was used as a conservative sample preparation for vaccine particle fractionation and gas-phase electrophoretic mobility macromolecular analyzer (GEMMA) for analyzing electrophoretic mobility diameters of isolated TBE virions. The derived particle diameter was then correlated with molecular weight. The diameter of the TBE virions determined after SEC by GEMMA instrumentation was 46.8 ± 1.1 nm. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) were implemented for comparison purposes and to gain morphological information on the virion particle. Western blotting (Dot Blot) as an immunological method confirmed biological activity of the particles at various stages of the developed analytical strategy. AFM and TEM measurements revealed higher diameters with much higher SD for a limited number of virions, 60.4 ± 8.5 and 53.5 ± 5.3 nm, respectively. GEMMA instrumentation was also used for fractionation of virions with specifically selected diameters in the gas-phase, which were finally collected by means of an electrostatic sampler. At that point (i.e., after particle collection), AFM and TEM showed that the sampled virions were still intact, exhibiting a narrow size distribution (i.e., 59.8 ± 7.8 nm for AFM and 47.5 ± 5.2 nm for TEM images), and most importantly, dot blotting confirmed immunological activity of the collected samples. Furthermore dimers and virion artifacts were detected, too

Th-1 and Th-2 cytokine responses in mice immunized with seasonal and pandemic H1N1, and pandemic H5N1 vaccines.

<p>Balb/c mice were immunized with pandemic H1N1 (H1N1 A/California/7/2009), seasonal H1N1 (H1N1 A/Brisbane/59/2007), and pandemic H5N1 (H5N1 A/Vietnam/1203/2004) vaccines. Spleen cells were collected 7 days after the first, or 21 days after the booster immunization (i.e. 42 days after the first), and stimulated with various seasonal or pandemic influenza virus antigens, before determination of cells responding by secretion of either IFN-g or IL-4 by an ELISPOT assay. Anti-HA IgG subclass responses were analyzed by ELISA using sera collected on day 42.</p

Protection of mice from lung viremia.

<p>Groups of CD1 mice were immunized twice with five-fold serial dilutions of pandemic H1N1 (H1N1 A/California/7/2009) whole virus vaccine, before being challenged intranasally with 10⁵ TCID₅₀. Lungs were harvested at day three after challenge, and virus titers determined as described (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009349#s2" target="_blank">Methods</a>). Lack of detection of virus in lungs was considered indicative of protection.</p

Dose-dependent immunogenicity of H1N1 A/California/7/2009 candidate vaccine in mice.

<p>CD1 mice were immunized twice with different doses of the candidate vaccine, and HI titers were determined 21 days after the first (d21) and 21 days after the booster immunization (d42) to calculate the percentage of seroconversion (%SC), geometric mean titers (GMT), and effective dose 50 (ED₅₀) based on an HI titer of ≥40.</p

H1N1 challenge and passive protection of SCID mice.

<p>SCID mice were challenged with 10⁵ TCID₅₀ pandemic H1N1 (H1N1 A/California/7/2009) by intranasal instillation, and survival monitored for 30 days. For passive protection, 200 µl immune mouse or guinea pig (GP) sera, or naïve mouse serum, were intraperitoneally administered to mice both at days one and two prior to virus challenge.</p