Evaluation of Measles Vaccine Virus as a Vector to Deliver Respiratory Syncytial Virus Fusion Protein or Epstein-Barr Virus Glycoprotein gp350.

Live attenuated recombinant measles vaccine virus (MV) Edmonston-Zagreb (EZ) strain was evaluated as a viral vector to express the ectodomains of fusion protein of respiratory syncytial virus (RSV F) or glycoprotein 350 of Epstein-Barr virus (EBV gp350) as candidate vaccines for prophylaxis of RSV and EBV. The glycoprotein gene was inserted at the 1(st) or the 3(rd) position of the measles virus genome and the recombinant viruses were generated. Insertion of the foreign gene at the 3(rd) position had a minimal impact on viral replication in vitro. RSV F or EBV gp350 protein was secreted from infected cells. In cotton rats, EZ-RSV F and EZ-EBV gp350 induced MV- and insert-specific antibody responses. In addition, both vaccines also induced insert specific interferon gamma (IFN-γ) secreting T cell response. EZ-RSV F protected cotton rats from pulmonary replication of RSV A2 challenge infection. In rhesus macaques, although both EZ-RSV F and EZ-EBV gp350 induced MV specific neutralizing antibody responses, only RSV F specific antibody response was detected. Thus, the immunogenicity of the foreign antigens delivered by measles vaccine virus is dependent on the nature of the insert and the animal models used for vaccine evaluation.

Molecular and cellular response profiles induced by the TLR4 agonist-based adjuvant Glucopyranosyl Lipid A.

BACKGROUND: Toll-like receptor (TLR)4 agonists are known potent immunostimulatory compounds. These compounds can be formulated as part of novel adjuvants to enhance vaccine medicated immune responses. However, the contribution of the formulation to the innate in vivo activity of TLR4 agonist compounds is not well understood. METHODOLOGY AND PRINCIPAL FINDINGS: We evaluated synthetic TLR4 agonist Glucopyranosyl Lipid A (GLA) for its effects on molecular and cellular innate immune responses in the murine model. Microarray techniques were used to compare the responses to GLA in an aqueous formulation or in an oil-in-water Stable Emulsion formulation (GLA-SE) versus either SE alone or the mineral salt aluminum hydroxide (alum) at the muscle injection site over multiple timepoints. In contrast to the minimal gene upregulation induced by SE and alum, both GLA and GLA-SE triggered MyD88- and TRIF-dependent gene expression. Genes for chemokines, cytokine receptors, signaling molecules, complement, and antigen presentation were also strongly upregulated by GLA and GLA-SE. These included chemokines for T(H)1-type T cells (CXCL9 and CXCL10) and mononuclear leukocytes (CCL2, CCL3) among others. GLA-SE induced stronger and more sustained gene upregulation than GLA in the muscle; GLA-SE induced genes were also detected in local draining lymph nodes and at lower levels in peripheral blood. Both GLA and GLA-SE resulted in increased cellular trafficking to the draining lymph nodes and upregulated MHC molecules and ICAM1 on local dendritic cells. GLA and GLA-SE transiently upregulated circulating MCP-1, TNFα, IFNγ and IP-10 in blood. CONCLUSIONS/SIGNIFICANCE: While GLA and GLA-SE activate a large number of shared innate genes and proteins, GLA-SE induces a quantitatively and qualitatively stronger response than GLA, SE or alum. The genes and proteins upregulated could be used to facilitate selection of appropriate adjuvant doses in vaccine formulations.

Generation of live attenuated novel influenza virus A/California/7/09 (H1N1) vaccines with high yield in embryonated chicken eggs.

Several live attenuated influenza virus A/California/7/09 (H1N1) (CA09) candidate vaccine variants that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the CA09 virus and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (H2N2) virus were generated by reverse genetics. The reassortant viruses replicated relatively poorly in embryonated chicken eggs. To improve virus growth in eggs, reassortants expressing the HA and NA of CA09 were passaged in MDCK cells and variants exhibiting large-plaque morphology were isolated. These variants replicated at levels approximately 10-fold higher than the rate of replication of the parental strains in embryonated chicken eggs. Sequence analysis indicated that single amino acid changes at positions 119, 153, 154, and 186 were responsible for the improved growth properties in MDCK cells and eggs. In addition, the introduction of a mutation at residue 155 that was previously shown to enhance the replication of a 1976 swine influenza virus also significantly improved the replication of the CA09 virus in eggs. Each variant was further evaluated for receptor binding preference, antigenicity, attenuation phenotype, and immunogenicity. Mutations at residues 153, 154, and 155 drastically reduced viral antigenicity, which made these mutants unsuitable as vaccine candidates. However, changes at residues 119 and 186 did not affect virus antigenicity or immunogenicity, justifying their inclusion in live attenuated vaccine candidates to protect against the currently circulating 2009 swine origin H1N1 viruses.

Multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (FluMist) derived from cold-adapted A/Ann Arbor/6/60.

FluMist influenza A vaccine strains contain the PB1, PB2, PA, NP, M, and NS gene segments of ca A/AA/6/60, the master donor virus-A strain. These gene segments impart the characteristic cold-adapted (ca), attenuated (att), and temperature-sensitive (ts) phenotypes to the vaccine strains. A plasmid-based reverse genetics system was used to create a series of recombinant hybrids between the isogenic non-ts wt A/Ann Arbor/6/60 and MDV-A strains to characterize the genetic basis of the ts phenotype, a critical, genetically stable, biological trait that contributes to the attenuation and safety of FluMist vaccines. PB1, PB2, and NP derived from MDV-A each expressed determinants of temperature sensitivity and the combination of all three gene segments was synergistic, resulting in expression of the characteristic MDV-A ts phenotype. Site-directed mutagenesis analysis mapped the MDV-A ts phenotype to the following four major loci: PB1(1195) (K391E), PB1(1766) (E581G), PB2(821) (N265S), and NP(146) (D34G). In addition, PB1(2005) (A661T) also contributed to the ts phenotype. The identification of multiple genetic loci that control the MDV-A ts phenotype provides a molecular basis for the observed genetic stability of FluMist vaccines.