Written for the Faculty of Law, Biomedical Ethics Unit. Title from title page of PDF (viewed 2010/04/07). Includes bibliographical references.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: Gene therapy; immunology; vaccine; HIV; AIDS; codon optimization. Includes bibliographical references (p. 67-73).

Human immunodeficiency virus type 1 (HIV-1) has caused more than 70 million infections worldwide since its discovery, with half of the infected already died by the end of 2011 as a result of HIV-progression to acquired immunodeficiency syndrome (AIDS). Highly active antiretroviral therapy (HAART) is capable to extend the lifespan of HIV patients but economic burden and emergence of resistant HIV strains pose immediate problems in the care of HIV patients. Furthermore, HAART cannot clear virus. Therefore, tremendous efforts have tried to develop an effective HIV vaccine in the last thirty years but only partial efficacy (31%) was achieved in the recent Thai RV144 clinical trial. Hence, new vaccine and understanding the mechanism are required now and in the future. In this study, two novel DNA vaccine strategies that utilized programmed death-1 (PD-1) or its isoform to improve immunogenicity of DNA vaccine for HIV-1 Gag p24 by acting on dendritic cells are described. The molecule PD-1 delivers negative regulatory signals to T cells through interacting with its ligands PD-L1 and PD-L2, while blocking this signal could functionally rescue the “exhausted” T cells during chronic infection such as HIV-1. The first DNA vaccine involves the fusion between HIV-1 Gag p24 antigen and soluble PD1 for effective targeting to DCs while improving antigen uptake and DC maturation, which in turn elicited consistently high frequencies of HIV-1 Gag-specific, broadly reactive, polyfunctional, long-lived and cytotoxic CD8+ T cells, in addition to robust anti-Gag antibody titers in mouse. The mechanism behind the action of this vaccine (sPD1-p24fc) is based on engagement of cross-presentation to CD8+ T cells, and induction of Th1 cytokines. The second DNA vaccine utilized a novel isoform of human PD1 (Δ42PD1) that contains a 42-nucleotide in-frame deletion located at exon 2 domain discovered in healthy PBMC donors. Interestingly, Δ42PD1 does not engage PD-L1/PD-L2 but its recombinant form could induce pro-inflammatory cytokines. When Δ42PD1 was used as an intramolecular adjuvant to develop a fusion DNA vaccine with HIV-1 Gag p24 antigen (sΔ42PD1-p24fc), enhanced DC uptake was also observed. When mice was vaccinated with this vaccine, significantly enhanced anti-p24 IgG1/IgG2a antibody, p24-specific T cells responses with functionally improved proliferative and cytolytic capacities were also identified. Importantly, both of these vaccines enhanced antigen-specific immunity and provided protection against pathogenic viral challenge as well as tumor growth in mice. Overall, the induction of high frequency of durable and protective Gag-specific T cell immunity, especially CD8+ T cell immunity using these two vaccines have important implications for vaccine development and immunotherapy against HIV-1 and other pathogens. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Development of effective vaccines against mutable viruses (i.e HIV-1 and influenza) remains a big challenge. Antibody-dependent cell-mediated cytotoxicity (ADCC) has been found to be a key component of immune protection against viral infections in vivo. Therefore, vaccine immunogens that elicit broadly neutralizing antibodies with high ADCC are desired for vaccine development. This study is to identify primary immunogens that can initiate somatic maturation of germline antibodies of known broadly neutralizing HIV-1 antibodies (bnAbs) for HIV vaccine development and to localize dominant ADCC epitopes on hemagglutinin (HA) of pandemic H1N1 influenza virus for development of a flu vaccine. Based on the observations that known HIV-1 bnAbs have extensive somatic mutation compared to their germline versions and that HIV-1 envelope (Env) glycoprotein lacks measurable binding to putative germline antibodies of known bnAbs, we hypothesized that non-HIV-derived (poly)peptides may serve as primary immunogens to trigger somatic maturation of germline antibodies of bnAbs, leading to elicitation of intermediate antibodies (iAbs) that can further mature to HIV-1 bnAbs upon Env vaccination or HIV-1 infection. Using b12 as a model bnAb, we identified five non-HIV-derived (poly)peptides that bound to putative b12 germline and iAbs, and immunized rabbits with the (poly)peptide priming followed by Env boosting. Rabbit immunization with (poly)peptides alone induced high titers of antibodies that were cross-reactive with gp140SF162 trimer and resurfaced Env RSC3, and the serum IgGs neutralized SF162 and JRFL. These results suggest that the (poly)peptides might structurally mimic CD4bs of Env. Priming rabbits with (poly)peptides followed by boosts with gp140SF162 and RSC3 resulted in antibodies capable of competing with b12 for binding to gp140SF162 trimer and neutralizing cross-clade isolates, while control rabbits without priming produced antibodies that were unable to compete with b12 for gp140SF162 trimer binding, and the serum IgGs neutralized only 3 clade B isolates. Our results provide proof of concept that non-HIV-derived (poly)peptides may serve as primary vaccine immunogens to initiate guided immune responses towards bnAbs. HA protein has high level of immunogenicity and considered the most important target for immune protection against influenza virus infection. Several potent HA-specific bnAbs have been reported with their conserved neutralizing epitopes revealed, but there has been no report so far about ADCC epitopes on HA. Using yeast display and flow cytometry assisted cell sorting, we mapped the epitope of convalescent plasma IgGs with different ADCC activity, we identified two dominant ADCC epitopes, designated HA-E1 [AA92-117] and HA-E2 (AA 124-159), on HA of 2009 pandemic H1N1 influenza virus. E1 and E2 overlapped with the immunodominant epitopes of HA. Depletion of purified patient plasma IgGs with yeast cells expressing E1 or E2 peptides decreased ADCC activity of the IgGs. E1 and E2 sequences are highly conserved in H1N1 strains, but less so in other subtypes of influenza A viruses. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent bnAbs and ADCC epitopes may confer a comprehensive immune protection against influenza virus infection. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: DNA; Vaccine; Immunology; Virology; Influenza; Antibody; VIrus. Includes bibliographical references (leaves 51-54).

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2006. / Includes bibliographical references.

published_or_final_version / Community Medicine / Master / Master of Public Health

No description available.

No description available.

No description available.