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H5N1 Vaccine-Specific B Cell Responses in Ferrets Primed with Live Attenuated Seasonal Influenza Vaccines

Live attenuated influenza H5N1 vaccines have been produced and evaluated in mice and ferrets that were never exposed to influenza A virus infection (Suguitan et al., Plos Medicine, e360:1541, 2006). However, the preexisting influenza heterosubtypic immunity on live attenuated H5N1 vaccine induced immune response has not been evaluated.Primary and recall B cell responses to live attenuated H5N1 vaccine viruses were examined using a sensitive antigen-specific B cell ELISpot assay to investigate the effect of preexisting heterosubtypic influenza immunity on the development of H5N1-specific B cell immune responses in ferrets. Live attenuated H5N1 A/Hong Kong/213/03 and A/Vietnam/1203/04 vaccine viruses induced measurable H5-specific IgM and IgG secreting B cells after intranasal vaccination. However, H5-specific IgG secreting cells were detected significantly earlier and at a greater frequency after H5N1 inoculation in ferrets previously primed with trivalent live attenuated influenza (H1N1, H3N2 and B) vaccine. Priming studies further revealed that the more rapid B cell responses to H5 resulted from cross-reactive B cell immunity to the hemagglutinin H1 protein. Moreover, vaccination with the H1N1 vaccine virus was able to induce protective responses capable of limiting replication of the H5N1 vaccine virus to a level comparable with prior vaccination with the H5N1 vaccine virus without affecting H5N1 vaccine virus induced antibody response. vaccine and the heterosubtypic immunity may be beneficial for pandemic preparedness

GM-CSF Increases Mucosal and Systemic Immunogenicity of an H1N1 Influenza DNA Vaccine Administered into the Epidermis of Non-Human Primates

Background: The recent H5N1 avian and H1N1 swine-origin influenza virus outbreaks reaffirm that the threat of a worldwide influenza pandemic is both real and ever-present. Vaccination is still considered the best strategy for protection against influenza virus infection but a significant challenge is to identify new vaccine approaches that offer accelerated production, broader protection against drifted and shifted strains, and the capacity to elicit anti-viral immune responses in the respiratory tract at the site of viral entry. As a safe alternative to live attenuated vaccines, the mucosal and systemic immunogenicity of an H1N1 influenza (A/New Caledonia/20/99) HA DNA vaccine administered by particle-mediated epidermal delivery (PMED or gene gun) was analyzed in rhesus macaques. Methodology/Principal Findings: Macaques were immunized at weeks 0, 8, and 16 using a disposable single-shot particlemediated delivery device designed for clinical use that delivers plasmid DNA directly into cells of the epidermis. Significant levels of hemagglutination inhibiting (HI) antibodies and cytokine-secreting HA-specific T cells were observed in the periphery of macaques following 1-3 doses of the PMED HA DNA vaccine. In addition, HA DNA vaccination induced detectable levels of HA-specific mucosal antibodies and T cells in the lung and gut-associated lymphoid tissues of vaccinated macaques. Importantly, co-delivery of a DNA encoding the rhesus macaque GM-CSF gene was found to significantly enhance both the systemic and mucosal immunogenicity of the HA DNA vaccine. Conclusions/Significance: These results provide strong support for the development of a particle-mediated epidermal DNA vaccine for protection against respiratory pathogens such as influenza and demonstrate, for the first time, the ability of skindelivered GM-CSF to serve as an effective mucosal adjuvant for vaccine induction of immune responses in the gut and respiratory tract. © 2010 Loudon et al

P -glycoprotein expression in murine T lymphocytes: Implications for development, function, and immunosenescence.

The P-glycoprotein (P-gp) transporter is normally expressed by subsets of both CD4 and CD8 T cells in mice, and the proportion in each subset that expresses P-gp goes up considerably with age. In addition, CD4 memory T cells that express P-gp are hyporesponsive to T cell receptor (TCR) stimulation in many in vitro assays. The physiological significance of P-gp expression in T cells, however, has not been well established. To elucidate whether certain T cell functions may require P-gp, we compared a variety of responses using T cells from normal and P-gp-deficient mice. These comparisons showed that P-gp is not required for proliferation, cytokine secretion, or allospecific cytotoxic responses. We also monitored the development of T cells in different lymphoid compartments to see whether P-gp expression affects T cell development or survival. Although circulating T cells were found to develop normally in P-gp-deficient mice, the relative proportions of T cell subsets in the intestinal lymphoid compartment became dramatically altered. The molecular basis for the hyporesponsive nature of P-gp-expressing CD4 memory T cells was also studied by analyzing TCR activation-dependent events in P-gphigh and P-gplow subsets of CD4 memory cells. Recruitment of key signaling proteins, including LAT and PKC-theta, to the stimulator cell interface (immunological synapse) was greatly impaired in P-gphigh CD4 memory T cells. In contrast, c-Cbl, a negative regulator of signaling, was clustered at the synapse in a high proportion of CD4 memory T cells regardless of P-gp subset. Moreover, while both c-Cbl and LAT were frequently recruited together to the synapse in individual P-gplow cells, P-gphigh cells often showed clustering of c-Cbl without LAT. Alterations in the ratios of positive and negative signaling regulators that assemble at the synapse during activation thus appear to contribute to the hyporesponsiveness of P-gp high CD4 memory T cells. Based on these data, P-gp has a negligible role in peripheral T cell effector functions, but its expression is necessary for normal intestinal lymphocyte development and is apparently linked to other cellular changes that lead to a state of anergy in an age-dependent CD4 memory T cell population.Ph.D.Biological SciencesCellular biologyHealth and Environmental SciencesImmunologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132340/2/9963774.pd