Feline leukemia viruses (FeLVs) are pathogenic retroviruses of the domestic cat. FeLV transmission and emergence of pathogenic variants show striking similarity to HIV pathogenesis. The emergence of pathogenic subgroup-C FeLV from the transmitted subgroup-A FeLV coincides with a switch in host receptor used for infection as a result of mutations in the viral envelope protein (Env). I have characterized a novel FeLV Env that may represent an evolutionary intermediate between FeLV-A and FeLV-C. I have also reported evidence suggesting that FeLVs may use co-factors/co-receptors for infection. I have found that FeLVs inefficiently infect murine NIH3T3 cells overexpressing FeLV receptors (NIH3T3/Receptor). I have provided evidence that the low infection is caused by a block at a post-binding but pre-entry stage of FeLV infection. Furthermore, fusion of NIH3T3/Receptor cells with highly susceptible cells rescues inhibition to infection suggesting that FeLVs, like HIV, may also use co-receptors for infection.

Human immunodeficiency virus type 1 (HIV-1), the pathogen responsible for the onset of acquired immuno-deficiency syndrome (AIDS) in humans has reached pandemic proportions. To this day, no cure is available for infection with this virus and the only treatment option for this chronic infection is the life-long adherence to anti-retroviral therapy. Efforts in the quest to control the worldwide AIDS pandemic include the search for an effective anti-HIV-1 vaccine. Providing hope in this endeavor are a few monoclonal antibodies possessing broad neutralizing characteristics (bnmAbs) that have been isolated from the sera of rare patients that have a delayed progression to AIDS. In this thesis, one of these bnmAbs, 2F5 is extensively characterized at the atomic level to better understand its binding and neutralization mechanism. In total, 27 crystal structures of the 2F5 Fab’ in complex with various peptides representing its linear gp41 membrane proximal external region (MPER) epitope are presented. Furthermore, expression of the 2F5 Fab in a bacterial system allowed to design mutants of the 2F5 Fab and therefore investigate the implication of specific domains of 2F5 in mediating binding and neutralization. Atomic level characterization of this immune complex revealed a somewhat promiscuous recognition of 2F5 for its 664DKW666 epitope as long as the following characteristics were conserved: the aspartate’s negative charge, the hydrophobic alkyl-pi stacking arrangement between the beta-turn lysine and tryptophan, and the positive charge of the former. Moreover, it was demonstrated that 2F5 has an elongated and flexible complementary determining region 3 loop of the heavy chain (CDR H3), which is required for neutralization and is involved in secondary binding interactions other than to its core linear epitope. These contributions will significantly help in guiding the structure-based design of an HIV-1 vaccine looking to elicit 2F5-like antibody responses.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Stephen Higgs / The genetic composition of arboviruses is a critical determinant of viral infectivity and the capacity for virus dissemination in arthropod vectors. Due to concerns related to a hypothetical potential for loss of attenuation, the supression of vector infection and dissemination is a critical component for the rationale-based design of live-attenuated flavivirus vaccine candidates. The yellow fever virus (YFV) 17D vaccine virus is not only attenuated in vertebrates, but also has low infectivity for Aedes agypti mosquitoes and since it does not disseminate, it is not transmissible. Using a reverse genetics system, the mutations present in the envelope protein YFV 17D virus were characterized in Ae. aegypti to determine the role of mutations in limiting the viral infectivity and dissemination capacity. This knowledge would contribute to the rational design of live attenuated vaccines with the desirable phenotype of being nontransmissible by arthropod vectors. The upper lateral portion of the YFV 17D envelope (E) protein domain III (EDIII) habors the T380R mutation in the FG loop. Experiments demonstrated that the T380R mutation was associated with the viral infectivity phenotype for mosquitoes, but did not influence dissemination into the secondary tissues. The G52R mutation in the molecular hinge region that is located between E protein domains I (EDI) and II, significantly reduced viral infectivity for mosquitoes. In contrast, when cloned into the Asibi wildtype virus genetic backbone, the T173I mutation in the loop structure between the G0 and H0 β- strands did not attenuate viral infection and dissemination. The double mutant virus containing both the G52R and T173I mutations in the E protein, showed a similar attenuated reduced infectivity to the single G52R mutant. The M299I mutation in the linker region between EDI and EDIII resulted in a significantly lower viral infectivity at the initial phase of viral infection at 7 days post-infection in Ae. aegypti. In conclusion, the characterization on four mutations in the YFV 17D vaccine E protein have demonstrated three genetic loci, that can influence the process of YFV infection in Ae. aegypti. These results provide new knowledge and understanding which may have broad applications for the rationale design of safe flavivirus vaccines, via targeting genetic loci and introducing specific mutations that preclude infection of, and transmission by arthropod vectors.

Master of Science / Department of Biology / A. Lorena Passarelli / Cydia pomonella granulovirus (CpGV) is a member of the Baculoviridae family of viruses. The CpGV open reading frame 46 (CpGV-ORF46) predicts a 545 amino acid protein that shares homology with matrix metalloproteases (MMPs), a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. In silico analyses revealed the presence of putative mmp genes in all species from the Betabaculovirus genus, while no mmps were identified in members of the Alphabaculovirus, Gammabaculovirus or Deltabaculovirus genera. Unlike most cellular MMPs, baculovirus MMPs do not have a propeptide domain, a domain involved in regulating MMP activation, or a hemopexin-like domain, which is necessary for substrate binding and specificity in many MMPs. However, Betabaculovirus MMPs do contain a predicted conserved zinc-binding motif (HEXGHXXGXXHS/T) within their catalytic domain. The function of CpGV-MMP and its effects on baculovirus replication in cultured cells and insect larvae were investigated. CpGV-MMP was expressed in and purified from Escherichia coli, and activity was measured using a generic MMP substrate in vitro. CpGV-MMP had in vitro activity and its activity was specifically inhibited by MMP inhibitors. To study the effects of CpGV-MMP on virus replication and dissemination, CpGV-MMP was expressed from Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of a strong and constitutive promoter, the Drosophila heat shock 70 protein promoter. Expression of CpGV-MMP did not affect virus replication in cultured cells. The effects of expressing CpGV-MMP from AcMNPV during larval infection were evaluated in the presence or absence of the AcMNPV chitinase and cathepsin genes. Insect bioassays showed that the absence of cathepsin resulted in a significant delay in larval time of death; however, this delay was compensated by expression of CpGV-MMP. In addition, larval time of death was accelerated when cathepsin, chitinase, and CpGV-MMP were all expressed. Finally, we determined the effects of CpGV-MMP on larvae melanization and liquefaction. CpGV-MMP was able to promote larvae melanization in the absence of cathepsin. CpGV-MMP, in the absence of cathepsin, was not able to promote larvae liquefaction. When chitinase was engineered to be secreted from cells, CpGV-MMP rescued liquefaction in the absence of cathepsin. In conclusion, CpGV-MMP is a functional MMP which can enhance larvae mortality with the presence of cathepsin. In addition, CpGV-MMP can promote larvae melanization; however, it can only promote liquefaction when chitinase is engineered to be secreted from cells.

The Epstein-Barr virus (EBV) EBNA1 protein mediates the replication and mitotic segregation of the EBV genomes via interactions with the viral oriP sequences. C666-1 is the only known nasopharyngeal carcinoma (NPC) cell line that stably maintains EBV in culture and I investigated whether this is due to differences in oriP-mediated functions in replication and segregation. I found that both C666-1 and EBV-negative NPC cell lines can replicate and maintain oriP plasmids for extended periods but that high EBNA1 levels interfered with plasmid segregation. The segregation element within oriP was recently shown to contain 29 repeated sequences instead of the 20 repeats in initial oriP isolates. I compared the functions of oriP with 20 or 29 repeats and found that the higher number of repeats decreased plasmid replication but increased plasmid maintenance, consistent with a segregation effect. Finally, I identified a potential role for promyelocytic leukemia nuclear bodies in oriP plasmid replication.

The Epstein-Barr virus (EBV) EBNA1 protein mediates the replication and mitotic segregation of the EBV genomes via interactions with the viral oriP sequences. C666-1 is the only known nasopharyngeal carcinoma (NPC) cell line that stably maintains EBV in culture and I investigated whether this is due to differences in oriP-mediated functions in replication and segregation. I found that both C666-1 and EBV-negative NPC cell lines can replicate and maintain oriP plasmids for extended periods but that high EBNA1 levels interfered with plasmid segregation. The segregation element within oriP was recently shown to contain 29 repeated sequences instead of the 20 repeats in initial oriP isolates. I compared the functions of oriP with 20 or 29 repeats and found that the higher number of repeats decreased plasmid replication but increased plasmid maintenance, consistent with a segregation effect. Finally, I identified a potential role for promyelocytic leukemia nuclear bodies in oriP plasmid replication.

The success of adenoviral mediated airway gene therapy is hindered by host immune responses against adenoviral vectors. Helper-dependent adenoviral vectors (HD-Ad) are devoid of viral coding sequences and have an improved safety profile compared to earlier generation adenoviral vectors. However, intranasal delivery of HD-Ad vectors potentiates a pulmonary adaptive immune response, described in chapter 2, which is a barrier to gene therapy. One of the ways to reduce the immunogenicity of HD-Ad vectors is to increase the efficiency of HD-Ad mediated gene transfer to the airways, which would lessen the immunogen availability, limiting immune response against HD-Ad vectors. In chapter 3, a viral formulation strategy using Nacystelyn and DEAE-Dextran to substantially increase the efficacy of adenoviral mediated gene transfer to the airways is described. To further reduce the immune response to HD-Ad vectors, I have developed two novel strategies to induce vector-specific tolerance. The first strategy, described in chapter 4, involves the use of dendritic cells (DCs) differentiated in presence of IL-10, which are refractory to HD-Ad induced maturation and instead prime generation of regulatory T cells which suppress HD-Ad induced T cell proliferation. Delivery of these DCs pulsed with HD-Ad vectors to mice results in induction of immunological tolerance along with sustained gene expression following multiple rounds of HD-Ad readministrations. The second strategy, described in chapter 5, involves delivery of apoptotic DCs followed by delivery of antigen towards which tolerance needs to be generated. Apoptotic DCs are readily taken up by viable DCs, which suppresses DC maturation and induces TGF-β1 secretion, driving generation of regulatory T cells towards the delivered antigen. This strategy has shown remarkable success in achieving tolerance towards ovalbumin. Therefore, these strategies can be used to induce immunological tolerance towards gene therapy vectors which will likely allow for sustained and long term therapeutic transgene expression.

The success of adenoviral mediated airway gene therapy is hindered by host immune responses against adenoviral vectors. Helper-dependent adenoviral vectors (HD-Ad) are devoid of viral coding sequences and have an improved safety profile compared to earlier generation adenoviral vectors. However, intranasal delivery of HD-Ad vectors potentiates a pulmonary adaptive immune response, described in chapter 2, which is a barrier to gene therapy. One of the ways to reduce the immunogenicity of HD-Ad vectors is to increase the efficiency of HD-Ad mediated gene transfer to the airways, which would lessen the immunogen availability, limiting immune response against HD-Ad vectors. In chapter 3, a viral formulation strategy using Nacystelyn and DEAE-Dextran to substantially increase the efficacy of adenoviral mediated gene transfer to the airways is described. To further reduce the immune response to HD-Ad vectors, I have developed two novel strategies to induce vector-specific tolerance. The first strategy, described in chapter 4, involves the use of dendritic cells (DCs) differentiated in presence of IL-10, which are refractory to HD-Ad induced maturation and instead prime generation of regulatory T cells which suppress HD-Ad induced T cell proliferation. Delivery of these DCs pulsed with HD-Ad vectors to mice results in induction of immunological tolerance along with sustained gene expression following multiple rounds of HD-Ad readministrations. The second strategy, described in chapter 5, involves delivery of apoptotic DCs followed by delivery of antigen towards which tolerance needs to be generated. Apoptotic DCs are readily taken up by viable DCs, which suppresses DC maturation and induces TGF-β1 secretion, driving generation of regulatory T cells towards the delivered antigen. This strategy has shown remarkable success in achieving tolerance towards ovalbumin. Therefore, these strategies can be used to induce immunological tolerance towards gene therapy vectors which will likely allow for sustained and long term therapeutic transgene expression.

Chronic viral infection is a significant burden on healthcare systems worldwide. Robust anti-viral immune responses are essential for viral clearance. Persistent viruses use a variety of mechanisms to evade immune surveillance including the upregulation of host immunesuppressive factors. Secreted fibrinogen-like protein 2 (FGL2) has been identified as an inhibitory effector molecule in suppressing immune responses in patients with chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) disease. In a murine model of chronic infection caused by Lymphocytic choriomeningitis virus (LCMV) clone 13, we demonstrate that mice deficient in Fgl2 have increased numbers of mature antigen-presenting cells (APC), improved virus-specific cytotoxic T cell immunity and enhanced viral clearance when compared to wild-type mice. These results highlight the importance of the FGL2 inhibitory pathway in immune evasion and provide a rationale to investigate the effects of blocking FGL2 as a novel immune therapeutic in patients suffering from persistent infections.

Chronic viral infection is a significant burden on healthcare systems worldwide. Robust anti-viral immune responses are essential for viral clearance. Persistent viruses use a variety of mechanisms to evade immune surveillance including the upregulation of host immunesuppressive factors. Secreted fibrinogen-like protein 2 (FGL2) has been identified as an inhibitory effector molecule in suppressing immune responses in patients with chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) disease. In a murine model of chronic infection caused by Lymphocytic choriomeningitis virus (LCMV) clone 13, we demonstrate that mice deficient in Fgl2 have increased numbers of mature antigen-presenting cells (APC), improved virus-specific cytotoxic T cell immunity and enhanced viral clearance when compared to wild-type mice. These results highlight the importance of the FGL2 inhibitory pathway in immune evasion and provide a rationale to investigate the effects of blocking FGL2 as a novel immune therapeutic in patients suffering from persistent infections.