Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus that causes a contagious lung adenocarcinoma in sheep. One unique feature of JSRV is that its envelope (Env) glycoprotein functions as an active oncogene that induces oncogenic transformation in vitro and in vivo. While oncogenesis by JSRV Env protein has been extensively studied, the entry mechanism of JSRV has not been investigated. In this study, we showed that JSRV entry was specifically inhibited by lysosomotropic agents and bafilomycin Al (BafAl), indicating that JSRV is pH-dependent. Interestingly, oncoretroviral pseudotypes bearing JSRV Env protein were not inactivated by an acidic pH treatment, suggesting that additional factors besides low pH are involved in JSRV entry. Indeed, we found that JSRV entry was also blocked by dominant-negative mutants of dynamin and caveolin, raising the possibility that JSRV may use a dynamin-dependent, caveolae-associated pathway for entry. To determine a possible role of JSRV receptor, hyaluronidase 2 (Hyal2), in JSRV entry, we replaced the glycosylphosphatidyl-inositol (GPI) anchor of Hyal2 with the membrane-spanning domain and cytoplasmic tail of vesicular stomatitis virus G protein (VSV-G). We showed that although the transmembrane version of Hyal2 functioned efficiently as a JSRV receptor, JSRV entry mediated by the mutated Hyal2 was no longer inhibited by lysosomotropic agents and BafAl. Taken together, we conclude that JSRV entry is pH-dependent, but appears to use a non-classical pathway for entry. JSRV may provide an exciting novel model for a better understanding of retrovirus entry.

No description available.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 132-154).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 155-209).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 115-136).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 124-147).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 98-107).

Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function. First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens. Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells. Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies. Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.