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PhD, Virology, Faculty of Health Sciences, University of the Witwatersrand / HIV-associated cardiomyopathy is a multifactorial disease with a broad spectrum of aetiologies that arise due to chronic immunosupression during HIV infection. The intricate relationship between HIV infection and cardiac co-morbidity was investigated with the aid of HIV-neutralizing aptamers. These synthetic nucleic acid ligands with antibody-like properties are molecular tools with multifunctional applications ranging from drug discovery to diagnostics and therapeutics. The advent of the HIV/AIDS pandemic has naturally married the field of HIV therapy and diagnostics with that of aptamer technology. By employing a HIV-1 neutralizing aptamer, named UCLA1, raised against the viral surface envelope glycoprotein 120, I dissected some of the pathways leading to cardiomyocyte apoptosis in a cell culture system. In chapter one I investigated the potential cytotoxic effects of UCLA1 by comparing it against a panel of 17 antiretrovirals (ARVs) in clinical use with the goal of establishing a safety portfolio geared towards its use as a therapeutic agent. Using cultured human cardiomyocytes and primary peripheral blood mononuclear cells (PBMCs), I selected some of the major biological markers of ARV-induced cytotoxicity and found no measurable deleterious effect, especially when compared to other ARVs used in the same study. In chapter two, the permissiveness of cardiomyocytes to HIV infection as well as the relationship between virus-host interaction and caspase-mediated apoptosis were investigated. Non-productive, receptor and tropism-independent infection was observed, which was arrested after the reverse transcription stage. However, interaction between the virus gp120 and the host’s CXCR-4 chemokine receptor preferentially activated caspase-9 triggering robust mitochondria-mediated apoptosis. A shift from mitochondrial-initiated, caspase-9 mediated to Fas-ligand initiated, caspase-8 mediated was observed when CM were co-cultured with HIV-infected MDM. UCLA1 protected against caspase-9 mediated vii apoptosis but not caspase-8 mediated. Finally in chapter three I provided answers for the shift in caspase activation by showing that supraphysiological levels of IL-1β and IL-6 during HIV infection of MDM augment the effects of tumor necrosis factor (TNF). These observation provide new insight into the complex pathophysiology of HIVCM and highlight the potential of UCLA1 as a novel therapeutic agent to fight HIV and some of its associated diseases.

Recombinant antibodies have increased in prominence as therapeutics and diagnostic tools since their introduction to the market in the mid-1980s. They are used to treat diverse conditions from Crohn's disease to cancer. Since the Anthrax letter attacks of 2001, a great deal of work has been carried out to develop therapeutics to this disease, and antibodies that neutralize the toxic action of Bacillus anthracis are prominent among them. This dissertation describes the elucidation of the structure of the 14B7 family of neutralizing antibodies directed at protective antigen (PA) of B. anthracis and the complex of PA domain 4 (PAD4) with an ultra-high affinity neutralizing antibody (M18), and then utilizes this information to aid in the engineering of the antibody to various ends. Chapter 2 presents the structure of the M18-PAD4 complex and of the 14B7 family of antibodies, which aids in the understanding of the affinity maturation process for this antibody family. Chapter 3 describes the affinity maturation of M18 to a PA variant by applying the knowledge gained from the complex structure. This previously intractable challenge was met by employing saturation mutagenesis in highly focused libraries to M18 directed by the complex structure to the area of variation on PA. These results indicate that this could be a generalizable method for the engineering of M18 to natural and deliberate variation of PA. Chapter 4 reports work toward the development of a reversible, photoresponsive antibody using small molecule and polymer-protein conjugates. The results indicate that a probable site on M18 was located for placement of the polymer appendage, although further work is necessary to empirically refine the properties of the photoresponsive polymer. Chapter 5 presents an unrelated project, which was to confirm the existence of a proposed RNA thermosensor in the 5' untranslated region of LcrF from the pathogenic bacterium Yersinia pestis, the causative agent of plague. Overall, these studies reveal the power of structure-based engineering in this antibody-antigen system. In addition, the structural elucidation of the M18-PAD4 complex and the 14B7 family of antibodies furthers our basic understanding of protein-protein interactions and the process of affinity maturation of antibodies. / text

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine in the specialty of Virology, June 2018 / The discovery of broad and potent neutralizing HIV antibodies (bNAbs) has opened up new opportunities of passive immunization for HIV-1 prevention. In this study, we have engineered CAP256-VRC26.25, a V1V2 bNAb that neutralizes 70% of clade C viruses, as a single domain antibody (sdAb). These small antigen binding entities are derived from naturally occurring heavy chain only antibodies present in members of the dromedary families, and are characterized by the absence of a light chain, long complementarity-determining regions (CDR) heavy (H) chain 3 and high stability. Since CAP256.25 contains a highly charged and protruding CDR-H3 that binds mainly through its heavy chain, we hypothesized that it may function well as an sdAb. Multiple camelization approaches to engineer CAP256.25 as a sdAb were tested in silico utilising structural modelling software. Parameters such as germline sequence homology, hydrophobicity and solubility, folding energy, torsion angles and native conformation of CAP256.25 in complex with its binding epitope were major factors considered during the modelling process. Four CAP256.25 sdAb derivatives were generated from parental antibody, the mut_0 or a wild type (WT), which was used as a base line for downstream optimization. CAP256.25 mut_4 in which residues involved in LC interactions were replaced with residues strongly conserved in camel sdAbs, which minimize hydrophobic interface of the sdAb. Mut_8 variant, which included four additional substitutions to increase solubility and mut_9 contained a single additional mutation at the base of CDR-H3 to improve the energetic landscape of sdAb. All genes were synthesized and sub-cloned into a mammalian expression vector and recombinant proteins expressed in HEK293T cell line, and purified by Immobilized Metal Ion Affinity Chromatography (IMAC) and Fast Protein Liquid Chromatography (FPLC). CAP256.25_mut0 expression was below the detectable level and whilst mut_4 expressed at low levels, it showed no neutralization activity. CAP256.25 sdAb mut_8 and mut_9 expressed at significantly lower levels compared to m36, a previously described sdAb used a positive control. Nevertheless CAP256.25mut_8 sdAb showed neutralization capability although it lost significant potency in comparison to the parental antibody, yet still within the therapeutic window of the VRC01 bNAb. Importantly, CAP256.25 sdAb was unable to neutralize the K169E mutant confirming that it retained specificity for the V2 epitope. These data suggest that camelization of human antibodies is possible although further engineering is required to increase expression and improve stability. As such, sdAb engineering could be an encouraging step for the generation of small antigen binding fragments for future therapeutic purposes including topical delivery at mucosal surfaces, to interrupt or block sexual transmission of HIV. / XL2018

Foot-and-mouth disease (FMD) is one of the most contagious infectious diseases of domestic and wild cloven-hoofed animals, particular in cattle, sheep, pigs, goats and domestic buffalo, as well as wild ruminants such as deer. In Taiwan, there was a severe outbreak of FMD after more than 60 years freedom from the disease. The virus strain, O Taiwan 97 from the March 1997 outbreak of FMD in Taiwan, however, has been shown to have a species-specific adaptation to pigs. Although there are 7 distinct serotypes of FMD found in different regions of the world, this study focuses on the pig-adapted type O strain of FMD. After the FMD outbreak commenced in Taiwan, the spread of disease was very rapid and the whole of the western parts of Taiwan was affected within a few days after the diagnosis of FMD was confirmed. In some situations airborne transmission of FMD virus was suspected and it was speculated that this was the explanation for such rapid spread in Taiwan. Therefore, studies were conducted to investigate the transmissibility of O Taiwan/97 FMDV to susceptible pigs by direct and indirect spread including airborne spread in an enclosed animal house. This study showed that pigs in direct contact with challenged pigs became infected but none of the close-contact pigs became infected. These experiments clearly demonstrated that the pig adapted strain O Taiwan/97 was only efficiently transmitted by direct contact. This indicates that effective control against future outbreaks of pig adapted FMDV strains could be achieved by restriction of pig movement and stamping out if the outbreak has been detected in the early stages and prior to the movements of pigs from the infected premises. The measures used to control the Taiwanese FMD outbreak in 1997 were initially the slaughter of whole herds in the infected premises. However, with the rapid spread and large numbers of cases, the decision was taken to use universal compulsory vaccination of pig herds to control the outbreak when sufficient supply of vaccines was organized. Type O FMD vaccines were imported from a number of major FMD vaccine manufactures from around the world. Initially, vaccine efficacy for the imported vaccines was tested by measurement of neutralizing antibody titers in vaccinated pigs. To establish the relationship between serum neutralizing titers and protection from foot and mouth disease in pigs after vaccination, challenge studies were conducted with O/Taiwan/97 FMD in vaccinated pigs. Additionally, antibody responses to structural (neutralizing antibody) and non-structural proteins (NSP) were evaluated in vaccinated pig herds after primary and secondary vaccination in herds infected before and after vaccination. In order to be able to monitor the circulation of virus in vaccinated pig populations, valid diagnostic kits based on the detection of antibody against NSP were required. These tests needed to be evaluated against pig sera derived from challenge studies and natural FMD outbreaks. Three commercially available ELISAs (Cedi, UBI and Checkit), which were available to differentiate infected from vaccinated pigs, were tested and results showed that the Cedi test had the optimal sensitivity and specificity for pig adapted type O FMD testing. This test was used to retrospectively evaluate the sera collected from infected and non-infected pig herds collected sequentially in the year after the 1997 FMD outbreak in Taiwan. These studies also showed that the early vaccines used, stimulated NSP antibody production in swine herds that were vaccinated but not infected. This resulted in the requirement for purified FMD vaccines to be used when monitoring programs for FMD infection by NSP testing were in place. In these studies, it was also demonstrated that the purified FMD vaccines used later in the control program did not induce NSP antibody after multiple double dosage to pigs. Although clinical FMD appeared to be successfully controlled with vaccination program in Taiwan it was essential for the eradication plan to maintain active surveillance for NSP reactors in the pig population. The UBI and Cedi NSP kits were applied as screening and confirmatory tests, respectively, to pig sera collected in auction markets distributed around Taiwan to monitor for evidence of the circulation of FMD virus. Herds with positive reactors were followed-up by clinical inspection and 15 sera from suspected herds were further sampled. Negative results were obtained from all these investigation. With the absence of clinical outbreaks and the lack of evidence of FMDV circulation in the field from the NSP reactor surveillance, the Taiwanese government has progressed the eradication plan to a progressive cessation of vaccination, commencing with banning of vaccination on one isolated island in December 2006. The absence of outbreaks on that island, paved the way for further cessation of FMD vaccination in Taiwan from July 2008.

The properties of human immunodeficiency virus type 1 (HIV-1) and its simian counterpart SIV that enable persistent replication in the face of robust cellular, antibody, and innate immune responses have complicated efforts to develop a safe and effective vaccine. Vaccine protection against HIV-1 infection may require a combination of immune mechanisms. However, the types of immune responses that can be induced by vaccination to prevent HIV-1 infection remain unclear. The features of the viral envelope glycoprotein (Env) that confer inherent resistance to neutralization by antibodies also interfere with the development of antibody responses. We therefore vaccinated rhesus macaques with single-cycle SIV (scSIV) strains expressing Env proteins mutated to remove features that interfere with the induction of antibody responses. Antibodies capable of neutralizing Env-modified but not wild-type SIV were selectively enhanced. Identifying the immune responses underlying complete protection by live-attenuated SIV against pathogenic SIV challenge may provide guidance for HIV-1 vaccine design. To test the hypothesis that antibodies not measurable by assays for virus neutralization correlate with protection by live-attenuated SIV, we developed a novel assay for antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity increased progressively over time after inoculation, and was measurable against viruses expressing heterologous Env proteins from independent SIV isolates when neutralization was undetectable. Two separate pathogenic \(SIV_{mac}251\) challenge experiments took advantage of either the strain specificity or the time-dependent development of immunity to overcome complete protection by live-attenuated SIV. In both experiments, macaques inoculated with live-attenuated SIV that remained uninfected by \(SIV_{mac}251\) had significantly higher ADCC activity than those that became infected. We also measured ADCC for the primary immune correlates analysis of a recent HIV-1 vaccine clinical trial in Thailand (RV144) that reported modest vaccine protection (31%). There was a nonsignificant trend towards lower risk of infection among vaccinees with high versus low relative ADCC activity. However, Env-specific IgA correlated with risk, prompting an analysis stratified by IgA levels. Among vaccinees with low Env-specific IgA, there was lower risk of infection among those with higher ADCC activity. These observations suggest that antibodies that direct ADCC may contribute to vaccine protection against SIV and HIV-1 infection.

One potential approach to vaccine development against HIV involves generating an immunogen that can elicit the production of broadly neutralizing monoclonal antibodies (bnmAbs), which target specific sites on the HIV-1 envelope. Using site-directed mutagenesis and ELISA assays, this thesis investigates the idea of a secondary binding site of one of the bnmAbs, 2F5, as suggested by previous studies that identified residues Asp64, Thr65, and Arg82B on 2F5 that are recognized by its anti-idiotypic antibody 3H6. Results show that 2F5 binds only very weakly to the gp41 ectodomain in its post-fusion conformation. However, a small but significant difference was observed between the binding of the mutants and the T-20 peptide, a fusion inhibiting drug. Due to the limited effect, the results need to be confirmed using more quantitative techniques and more optimal conformations of the antigen, but raise the prospect that design of immunogens to elicit HIV-specific antibodies might have to incorporate this novel interaction site.

One potential approach to vaccine development against HIV involves generating an immunogen that can elicit the production of broadly neutralizing monoclonal antibodies (bnmAbs), which target specific sites on the HIV-1 envelope. Using site-directed mutagenesis and ELISA assays, this thesis investigates the idea of a secondary binding site of one of the bnmAbs, 2F5, as suggested by previous studies that identified residues Asp64, Thr65, and Arg82B on 2F5 that are recognized by its anti-idiotypic antibody 3H6. Results show that 2F5 binds only very weakly to the gp41 ectodomain in its post-fusion conformation. However, a small but significant difference was observed between the binding of the mutants and the T-20 peptide, a fusion inhibiting drug. Due to the limited effect, the results need to be confirmed using more quantitative techniques and more optimal conformations of the antigen, but raise the prospect that design of immunogens to elicit HIV-specific antibodies might have to incorporate this novel interaction site.

No description available.

Mosquito-borne viruses, including dengue virus (DENV), Usutu virus (USUV), West Nile virus (WNV), and Japanese encephalitis virus (JEV), are rapidly emerging, global pathogens. Though the number of people impacted by each virus varies, there have been thousands to millions of people infected. The focus of this thesis work centers around USUV and WNV; both have RNA genomes and belong to the Flaviviridae virus family. Both WNV and USUV were initially isolated in Africa and have since spread to Europe; interestingly, WNV has also spread globally and is considered endemic in the Americas. Similar to other flaviviruses, USUV and WNV are maintained in a mosquito vector-avian host transmission cycle, with spillover infection into humans. Human infections of WNV and USUV are usually asymptomatic, but in severe cases can cause neuroinvasive disease. WNV and USUV belong to the JEV serocomplex group, which indicates that antibodies produced against these viruses share a common antigen; the common antigen is hypothesized to be the envelope (E) protein on the outside of the virion. Neutralizing antibodies against both WNV and USUV have been found in birds and humans across Europe. In vitro cross-neutralization of WNV and USUV has been modeled experimentally and been observed in clinical settings. The neutralizing antibody response generated against WNV has been studied extensively in mouse models; however, there are few studies which examine the neutralizing antibody response generated against USUV. Whether prior WNV exposure protects against USUV disease is also unknown. The main goal of this thesis was to characterize how a primary flavivirus exposure would influence a secondary flavivirus exposure; specifically, we wanted to observe if WNV exposure would protect against USUV disease in vivo and generate a cross-neutralizing antibody response in vitro. For the WNV exposure, we used an attenuated vaccine strain of WNV that contains the WNV E gene (D2/WN-V3) developed by our collaborators. We hypothesized that treatment with D2/WN-V3 would protect against USUV infection. Two in vivo models were used: CD-1 mice and interferon alpha-beta receptor 1 deficient (Ifnar1-/-) mice. We discovered that sera from mice vaccinated with D2/WN-V3 neutralized both WNV and USUV in vitro. In the Ifnar1-/- model, we observed that vaccinated mice had higher survival rates and lower USUV viremia levels after USUV challenge. This work helps characterize the consequences of flavivirus antibody cross-neutralization in vitro and cross-protection in vivo. As the flavivirus field moves toward the goal of creating a pan-flavivirus vaccine, both cross-reactive antibodies and cross-protection need to be considered. / Master of Science / West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses that were originally isolated in Africa during the 20th century. Both viruses are maintained through a transmission cycle between mosquito vectors and avian hosts. Mosquitos transfer the infectious agent (WNV or USUV) through feeding on a bird (usually a passerine species); once in the bird, the virus can replicate to high levels. Human infections of WNV and USUV from mosquitos can also occur, with symptoms ranging from mild febrile illness to severe encephalitis or meningitis. Over the past few decades, WNV and USUV have spread to Europe, most likely through infected migratory birds. Interestingly, mosquito surveillance studies in mainland Europe have found mosquitos that tested positive for both USUV and WNV. In Europe, antibodies for both viruses have been found in humans and birds, indicating a previous exposure to WNV, USUV, or both. The neutralizing antibody response is a critical immune defense against viral infections. Neutralizing antibodies bind strongly to the outside of the virion (virus particle), preventing the virion from interacting with and infecting the host cell. For WNV and USUV, one of the targets that neutralizing antibodies bind to is the outer envelope (E) protein of the virion. In clinical settings and experimental studies, cross-neutralization of WNV and USUV has been documented. During cross-neutralization, a serum sample containing neutralizing antibodies against WNV can also neutralize USUV, and vice versa. Although the neutralizing response against WNV has been characterized in humans and lab animal models such as mice, there is little research regarding the neutralizing response against USUV. Importantly, whether prior WNV exposure provides protection against USUV infection is currently unknown. The main goal of this thesis was to characterize the disease outcome and neutralizing response against USUV after a WNV exposure. For the WNV exposure, we used a vaccine strain of WNV that contains the E gene (D2/WN-V3) developed by our collaborators. We predicted that vaccinated mice would avoid USUV clinical signs of disease and generate neutralizing responses to WNV and USUV. To do this work, we used two laboratory mouse models: mice with an intact immune response system (CD-1) and mice with a stunted immune response (Ifnar1-/-). We discovered that serum from vaccinated mice did cross-neutralize WNV and USUV. In the Ifnar1-/- model, vaccinated mice had higher survival rates and lower levels of virus in blood after USUV infection compared to unvaccinated mice. Ultimately, this work highlights the importance of characterizing the immune response against similar viruses and will inform the development of human vaccines for both viruses.