A human multi-epitope recombinant vaccinia virus as a universal T cell vaccine candidate against influenza virus.

There is a need to develop a universal vaccine against influenza virus infection to avoid developing new formulations of a seasonal vaccine each year. Many of the vaccine strategies for a universal vaccine target strain-conserved influenza virus proteins, such as the matrix, polymerase, and nucleoproteins, rather than the surface hemagglutinin and neuraminidase proteins. In addition, non-disease-causing viral vectors are a popular choice as a delivery system for the influenza virus antigens. As a proof-of-concept, we have designed a novel influenza virus immunogen based on the NP backbone containing human T cell epitopes for M1, NS1, NP, PB1 and PA proteins (referred as NPmix) as well as a construct containing the conserved regions of influenza virus neuraminidase (N-terminal) and hemagglutinin (C-terminal) (referred as NA-HA). DNA vectors and vaccinia virus recombinants expressing NPmix (WR-NP) or both NPmix plus NA-HA (WR-flu) in the cytosol were tested in a heterologous DNA-prime/vaccinia virus-boost vaccine regimen in mice. We observed an increase in the number of influenza virus-specific IFNγ-secreting splenocytes, composed of populations marked by CD4(+) and CD8(+) T cells producing IFNγ or TNFα. Upon challenge with influenza virus, the vaccinated mice exhibited decreased viral load in the lungs and a delay in mortality. These findings suggest that DNA prime/poxvirus boost with human multi-epitope recombinant influenza virus proteins is a valid approach for a general T-cell vaccine to protect against influenza virus infection.

Improved innate and adaptive immunostimulation by genetically modified HIV-1 protein expressing NYVAC vectors.

Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

Subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein.

The coronavirus nucleocapsid (N) protein is a viral RNA-binding protein with multiple functions in terms of virus replication and modulating cell signalling pathways. N protein is composed of three distinct regions containing RNA-binding motif(s), and appropriate signals for modulating cell signalling. The subcellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) N protein was studied. In infected cells, SARS-CoV N protein localized exclusively to the cytoplasm. In contrast to the avian coronavirus N protein, overexpressed SARS-CoV N protein remained principally localized to the cytoplasm, with very few cells exhibiting nucleolar localization. Bioinformatic analysis and deletion mutagenesis coupled to confocal microscopy and live-cell imaging, revealed that SARS-CoV N protein regions I and III contained nuclear localization signals and region II contained a nucleolar retention signal. However, cytoplasmic localization was directed by region III and was the dominant localization signal in the protein.

Cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein.

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) spike (S) protein is the major surface antigen of the virus and is responsible for receptor binding and the generation of neutralizing antibody. To investigate SCoV S protein, full-length and individual domains of S protein were expressed on the surface of insect cells and were characterized for cleavability and reactivity with serum samples obtained from patients during the convalescent phase of SARS. S protein could be cleaved by exogenous trypsin but not by coexpressed furin, suggesting that the protein is not normally processed during infection. Reactivity was evident by both flow cytometry and Western blot assays, but the pattern of reactivity varied according to assay and sequence of the antigen. The antibody response to SCoV S protein involves antibodies to both linear and conformational epitopes, with linear epitopes associated with the carboxyl domain and conformational epitopes associated with the amino terminal domain. Recombinant SCoV S protein appears to be a suitable antigen for the development of an efficient and sensitive diagnostic test for SARS, but our data suggest that assay format and choice of S antigen are important considerations.

Vaccination of pigs with a DNA construct expressing an influenza virus M2-nucleoprotein fusion protein exacerbates disease after challenge with influenza A virus.

In mice, vaccines inducing antibodies to the extracellular domain of the M2 protein (M2e) can confer protection to influenza A virus infection. Unlike the surface glycoproteins, haemagglutinin and neuraminidase, this domain of M2 is highly conserved and is therefore a potential broad-spectrum immunogen. In this study, the protection conferred by vaccines inducing antibodies to M2e was evaluated in a challenge model for swine influenza in pigs. A protein resulting from the fusion between M2e and the hepatitis B virus core protein (M2eHBc), with or without adjuvant, was evaluated. In addition, a DNA construct expressing a fusion protein between M2e and influenza virus nucleoprotein (M2eNP) was evaluated to see if the broad-spectrum protection conferred by antibodies could be further enhanced by T helper cells and cytotoxic T cells. All vaccines induced an antibody response against M2e, and the M2eNP DNA vaccine additionally induced an influenza virus-specific lymphoproliferation response. However, after challenge with a swine influenza virus (H1N1), no protection was observed in the vaccinated groups compared with the non-vaccinated control group. On the contrary, vaccinated pigs showed more severe clinical signs than the control pigs. The M2eNP DNA-vaccinated pigs showed the most severe clinical signs and three out of six pigs died on days 1 and 2 post-challenge. These results indicate that antibodies to M2e, especially in combination with cell-mediated immune responses, exacerbate disease. Thus, clinical signs after infection should be observed closely in further studies using M2e as an immunogen and caution should be exercised in using M2e in humans.

Respiratory and systemic humoral and cellular immune responses of pigs to a heterosubtypic influenza A virus infection.

The level of heterosubtypic immunity (Het-I) and the immune mechanisms stimulated by a heterosubtypic influenza virus infection were investigated in pigs. Pigs are natural hosts for influenza virus and, like humans, they host both subtypes H1N1 and H3N2. Marked Het-I was observed when pigs were infected with H1N1 and subsequently challenged with H3N2. After challenge with H3N2, pigs infected earlier with H1N1 did not develop fever and showed reduced virus excretion compared with non-immune control pigs. In addition, virus transmission to unchallenged group-mates could be shown by virus isolation in the non-immune control group but not in the group infected previously with H1N1. Pigs infected previously with homologous H3N2 virus were protected completely. After challenge with H3N2, pigs infected previously with H1N1 showed a considerable increase in serum IgG titre to the conserved extracellular domain of M2 but not to the conserved nucleoprotein. These results suggest that antibodies against external conserved epitopes can have an important role in broad-spectrum immunity. After primary infection with both H1N1 and H3N2, a long-lived increase was observed in the percentage of CD8(+) T cells in the lungs and in the lymphoproliferation response in the blood. Upon challenge with H3N2, pigs infected previously with H1N1 again showed an increase in the percentage of CD8(+) T cells in the lungs, whereas pigs infected previously with H3N2 did not, suggesting that CD8(+) T cells also have a role in Het-I. To confer broad-spectrum immunity, future vaccines should induce antibodies and CD8(+) T cells against conserved antigens.