Thesis (M.S.)--University of Wisconsin--Madison. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 222-236).

The lack of an effective HIV vaccine calls for efforts to investigate the mechanism of protective immunity against AIDS viruses. It has been previously demonstrated that the live replication-competent modified vaccinia virus Tiantan (MVTT) is superior to non-replication vaccinia MVA in inducing high levels of neutralizing antibodies against SARS-CoV infection via mucosal vaccination. Therefore, the hypothesis was that MVTT could be a better HIV vaccine vector given its highly attenuated phenotypes such as no neurovirulence and safe in severe combined immunodeficiency disease (SCID) mice. Here, a recombinant MVTT expressing SIVmac239 Gag-Pol and Env (rMVTTSIVgpe) was constructed and its immunogenicity was assesed when administered via different routes using homologous prime-boost strategies or in heterologous regimens boosted with a recombinant adenovirus-based vaccine inserted matched SIVmac239 genes (rAd5SIVgpe). Results show that the heterologous prime-boost immunization with rMVTTSIVgpe and rAd5SIVgpe induces significantly greater humoral and T cell responses specific to SIV Gag, Pol and Env than homologous inoculations in mice with remarkable improvements in quality and quantity. The further study comparing different combinations of rMVTTSIVgpe and rAd5SIVgpe demonstrates that the rMVTTSIVgpe prime-rAd5SIVgpe boost regimens elicit systemic CD8+ T cell responses with augmented magnitude and polyfunctionality, as compared with rAd5SIVgpe-rMVTTSIVgpe and rAd5SIVgpe-rAd5SIVgpe regimens. Priming with rMVTTSIVgpe also increases frequencies of gut-homing Gag-specific CD8+ T cells (CCR9+47+ and CCR6+47+) and levels of CD8+ T cell ELISPOT responses against Gag, Pol and Env in mesenteric lymph nodes (MLNs) post-boost. The mucosal route of immunization is essential for rMVTTSIVgpe to induce rectal IgG with detectable neutralizing activity against SIVmac1A11. Furthermore, the regimen involving mucosal prime with rMVTTSIVgpe followed by systemic boost with rAd5SIVgpe proves to be efficient in protecting monkeys from mucosal challenge of a high dose of SIVmac239, a CCR5-tropic strain with high pathogenicity and neutralization-resistance. SIV-specific T cell ELISPOT responses specific to Gag and Pol but not Env and the frequency of Gag-specific IFN-+TNF-+CD8+ effector memory T cells (TEM) are likely associated with virological control after challenge. Mucosal immunity induced by this vaccination strategy also has important implications to the effectiveness of protection against disease progression. A hypothesis was generated that removal of non-protective but immune dominant determinant of SIVmac239 Env may drive antibody responses to protective domains. It was found that the neutralization-resistance of SIVmac239 could be partially explained by its high immunogenicity in eliciting CD4-induced neutralizing antibodies, which are unable to protect the CCR5-binding site due to the conformational masking and steric restriction. It was discovered that the immunodominance of CD4-induced neutralizing antibodies on SIV envelope is determined by a single highly conserved N-linked glycosylation site (N277) in the C2 domain. Substitution of this N-linked site abolishes viral entry and the immunogenicity of the CD4i domain while promotes V2-specific antibody responses, which have recently been identified as an important immunological correlate to HIV-1/SIV protection. Our findings demonstrate the concept that B cell immunodominance is relative and eliminating the dominant antigenic region can result in redirection of B cell recognition, which have critical implications for immunogen design and the development of protective antibody-based HIV vaccine. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Retroviruses are obligate intracellular parasites that carry the information necessary for replication within their genomes. The three polyproteins, Gag, Pol, and Env, encoded by all retroviruses, function to generate progeny virions inside the host cell. Formation of new viral particles requires detailed instructions contained within the Gag polyprotein. Herein we describe our investigation into assembly of the Mason-Pfizer monkey virus (M-PMV). During retrovirus assembly, the transition from immature to a fully infectious mature particle is associated with the operation of molecular switches that trigger dramatic conformational changes of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid lattice is located in the C-terminus of the capsid (CA) protein in many retroviral Gags. The HIV-1 Gag contains a stretch of five amino acid residues termed the 'clasp motif', important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of the HIV-1 and M-PMV highlighted a spacer-like domain in M-PMV that may provide comparable function. In the present study we report an examination of the role of the clasp motif in the M-PMV life cycle. Our results demonstrate that claps motif mutants display major defects in virion assembly and release, and even larger defects in infectivity. Our data identifies the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient viral infection. The retroviral life cycle, unlike that of any other viral family, leads to the obligate integration of a proviral DNA into the host genome of somatic cells and in some cases even into the germ line. This remarkable feature of the Retroviridae family of viruses accounts for their extraordinary persistence through time and widespread abundance among vertebrate hosts. Because retroviral infection can have serious consequences to the host, there is great selective pressure to evolve strong networks that act to control incoming viruses. In the second study presented here, we report a novel cofactor of an antiviral system, Riplet, which operates to augment HIV-1 restriction by ZAP. The zinc-finger antiviral protein (ZAP) is an interferon-stimulated gene (ISG) with potent intrinsic antiviral activity. ZAP inhibits replication of retroviruses including MLV and HIV-1, as well as alphaviruses, filoviruses, hepatitis B virus, etc. ZAP operates at the post-transcriptional stage, reducing the number of viral transcripts available for translation in the cytoplasm, although additional pathways might be at play. The exact mechanisms by which ZAP restricts viral replication are not fully understood. ZAP lacks enzymatic activity and utilizes other cellular proteins to suppress viral replication. TRIM25 and the nuclease KHNYN have been identified as ZAP cofactors, but its activity may well involve other cellular proteins. Here we identify Riplet, a protein known to play a central role in the activation of the retinoic acid-inducible gene I (RIG-I), as a novel ZAP cofactor that acts to augment ZAP’s antiviral activity. Our data demonstrates that Riplet significantly augments ZAP-mediated restriction of HIV-1. Additionally, we show that Riplet interacts with ZAP via its PRY/SPRY domain and that the ubiquitin ligase activity of Riplet is not required to stimulate ZAP-mediated inhibition. Moreover, we show that Riplet interacts with TRIM25 suggesting that both Riplet and TRIM25 may operate synergistically to augment ZAP-mediated inhibition of HIV-1. The intracellular tropism of viruses is determined by a diverse combination of host proteins that allow infection to proceed efficiently. To achieve successful infection the virus needs the contribution of numerous cellular factors that assist at various steps of the life cycle. Conversely, replication requires resistance to species-specific restriction factors that act to suppress virus infection. The replication of M-PMV has been found to be highly restricted in mouse cell lines. The mechanism underlying the restriction of M-PMV replication in mouse cells has not been characterized. In the third study presented here, we examined this potent post-entry block and performed an unbiased genome-wide CRISPR-Cas9 screen, selecting for knock-out of host factors that relieved the block. Our data identified several candidate genes that encode proteins involved in virus trafficking and innate immune activation.

Thesis (Ph.D.)--Mississippi State University. Department of Wildlife and Fisheries. / Title from title screen. Includes bibliographical references.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).