Browsing by Author "Moyo, Thandeka"

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    Chinks in the armor of the HIV-1 Envelope glycan shield: implications for immune escape from anti-glycan broadly neutralizing antibodies
    (Elsevier, 2017-01-15) Moyo, Thandeka; Ferreira, Roux-cil; Davids, Reyaaz; Sonday, Zarinah; Moore, Penny L; Travers, Simon A; Wood, Natasha T; Dorfman, Jeffrey Robert
    Glycans on HIV-1 Envelope serve multiple functions including blocking epitopes from antibodies. We show that removal of glycan 301, a major target of anti-V3/glycan antibodies, has substantially different effects in two viruses. While glycan 301 on Du156.12 blocks epitopes commonly recognized by sera from chronically HIV-1-infected individuals, it does not do so on CAP45.G3, suggesting that removing the 301 glycan has a smaller effect on the integrity of the glycan shield in CAP45.G3. Changes in sensitivity to broadly neutralizing monoclonal antibodies suggest that the interaction between glycan 301 and the CD4 binding site differ substantially between these 2 viruses. Molecular modeling suggests that removal of glycan 301 likely exposes a greater surface area of the V3 and C4 regions in Du156.12. Our data indicate that the contribution of the 301 glycan to resistance to common neutralizing antibodies varies between viruses, allowing for easier selection for its loss in some viruses.
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    Role of envelope compactness and glycosylation in HIV-1 resistance to neutralising antibody responses
    (2017) Moyo, Thandeka; Dorfman, Jeffrey R
    Understanding the mechanisms used by HIV-1 to evade antibody neutralisation may contribute to the design of a high-coverage vaccine. This thesis explores the mechanism used by a Tier 3 virus leading to its high antibody neutralisation resistance phenotype. This thesis also describes how the glycans at the base of the V3 loop contribute to (i) breadth and potency in a cohort of unselected HIV-1-infected individuals and (ii) the selective pressures resulting from the V3/glycans shielding the virus from neutralisation and the glycans themselves being targets of broad antibody responses. HIV-1 isolates that are highly resistant to broadly neutralising antibodies could limit the efficacy of an antibody-based vaccine. For this reason, it is important to understand the mechanisms behind high HIV-1 resistance to neutralising antibodies. Chapter 2 and Chapter 3 of this thesis describe virus 253-11, a highly neutralisation resistant virus, which is particularly resistant to commonly-elicited, anti-membrane proximal external region (MPER) antibodies in sera. To further understand its resistance, mutations in the MPER were introduced that are known to delay fusion following CD4-binding and thus increase the time the virus spends in the open conformation. Interestingly, we found that these mutations affect the 253-11 Envelope (Env) spike before CD4-binding by destabilising the closed trimer structure. From these data, we hypothesized that the neutralisation resistance of 253-11 was due to an unusually tight, compact pre-fusion Env trimer that resists transient changes to the open conformation. The open conformation frequently exposes narrowly-neutralising antibody epitopes. Because the unliganded 253-11 Env presumably transitions infrequently into the open conformation, it would be able to evade these responses. 253-11 was sensitive to most but not all of the most potent broadly neutralising antibodies (bnAbs) tested, most likely because those broadly neutralising antibodies can access their epitopes in the pre-fusion Env conformation. To gain further information about the structure of the 253-11 Env, we designed a recombinant 253-11 SOSIP trimer and found it to be stable and predominantly adopt a closed conformation. The crystal structure of the SOSIP trimer revealed structural elements likely responsible for 253-11 Env compactness including the inward disposition of the heptad repeat helices and gp120 protomers towards the trimer axis. Taken together, the data from Chapter 2 and Chapter 3 highlight an underappreciated Env compactness mechanism of HIV-1 resistance to neutralising antibodies and these data may be useful in HIV-1 immunogen design research. Previous candidate HIV vaccines have failed to induce wide-coverage neutralising antibodies capable of substantially protecting vaccinees. A key approach in HIV immunogen development has been to define and model epitopes recognised by anti-HIV bnAbs. Candidate immunogen models identified by bnAbs include the V3/glycans, the V2/apex and the MPER epitopes. Autoreactivity and polyreactivity of anti-V3/glycan and anti-MPER antibodies are thought to pose both direct and indirect barriers to achieving neutralisation breadth. Chapter 4 of this thesis explored which of these bnAb epitopes were associated with breadth and potency in a South African cohort of chronically HIV-infected individuals. The study found that antibodies targeting the V3/glycans were associated with breadth and potency. In contrast, antibodies to the V2/apex were not associated with neutralisation breadth/potency. This suggests that auto/polyreactivity are not critical factors in the development of breadth and potency and that the V3/glycans should remain a high-priority vaccine candidate. Since targeting the V3/glycans was associated with breadth and potency in this cohort, the study continued to look at this epitope to investigate the role of these glycans in neutralisation resistance of Tier 2 viruses. The HIV-1 Env is surrounded by glycans that often prevent antibody neutralisation, leading to the term the "glycan shield", however some bnAbs have evolved to recognise these carbohydrates. Chapter 4 of this thesis describes how the N-linked glycan at position N301 is critical for maintaining neutralisation resistance of one subtype C virus (Du156.12), but not for another subtype-matched virus (CAP45.2.00.G3). Thus, the loss of the N301 glycan may have a substantial antibody-related fitness cost for some viruses but not others. The N301 glycan, as well as glycans at positions 332 and 334, are the primary targets of the anti-V3/glycan class of neutralising antibodies, which may select for loss of the targeted glycan. The evidence presented in Chapter 4 suggests that in some viruses, loss of the N301 glycan may result in evasion of anti-V3/glycan antibody responses while maintaining overall neutralisation resistance. This phenomenon may impair efficacy of passively-infused anti-V3/glycan bnAbs or a therapeutic vaccine.
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    The screening of neutralising antibodies against a resistant HIV-1 strain to identify novel epitopes
    (2014) Moyo, Thandeka; Dorfman, Jeffrey
    Since the start of the HIV/AIDS pandemic in the 1980s, over 75 million individuals have been infected with the virus and it has been the cause of approximately 36 million deaths worldwide. With such a high morbidity and mortality in HIV-1 infected individuals, there is a need to find ways of controlling the disease. Development of an HIV-1 vaccine would help in the fight against HIV/AIDS. It is clear that other prevention strategies poorly reach vulnerable groups such as intravenous drug users and people living in war zones. More importantly, they generally provide very transient protection and do not provide the durable and affordable protection that could be expected from a vaccine. Antiretroviral therapy (ART) may be effective in reducing death and morbidity; but, treatment is life-long and ART is not a cure. However, producing immunogens that elicit neutralising antibodies that are protective against HIV-1 acquisition has proven difficult. This is not only because of the genetic diversity of the viruses circulating in the human population but is also as a result of an incomplete understanding of how to design effective immunogens based on the known targets for broadly neutralising antibodies (BnAbs). The availability of more potential targets for BnAbs is also an important goal. In this project, we designed a system to help identify novel targets of BnAbs if one or more does exist. We selected the QH343.A10 virus as the basis for this system. We found this virus to be moderately resistant to sera and resistant to all the BnAbs we initially tested against it (which target various epitopes on the HIV-1 envelope). QH343.A10 is resistant to monoclonal antibodies (mAbs) b12 (anti-CD4-binding site), 2G12 (anti-V3/glycan), 2F5 and 4E10 (both anti-membrane proximal external region). Of note, the virus is resistant to the extremely broad and potent mAb VRC01 (anti-CD4-bs). QH343.A10 was also found to be resistant to neutralisation by soluble CD4 (sCD4). This made the virus attractive to use in our system as antibodies that recognise QH343.A10 in the same manner as these mAbs are also unlikely to neutralise the virus. Therefore, we tested the ability of 474 serum samples, from ART-naïve chronically HIV-1-infected individuals from a Cape Town cohort, to neutralise QH343.A10. Sixty-six sera (14%) were able to neutralise the virus by an ID50 value of 150 or higher and were retained for further analysis. The sera which recognise the MPER, CD4-bs and V3/glycan regions in a similar way to the mAbs that are unable to neutralise QH343.A10 would presumably be similarly unable to neutralise the virus. Thus, just by identifying sera able to neutralise QH343.A10, we propose that we are already partially enriched against sera that recognise these three targets. Because we expected this enrichment to be only partially effective, we then systematically tested for and removed QH343.A10-recoginising sera that recognised the MPER, the V2/glycan-site and V3/glycan region. For technical reasons, we have not yet attempted to remove sera that recognise QH343.A10 through the CD4-binding site and CD4-inducible site (3BC176 mAb site), which are both targets for BnAbs. After exclusion of sera recognising the MPER, V2/glycan-site and V3/glycan region, we were left with 19 samples. We analysed neutralisation breadth and potency of these remaining 19 serum samples as we wanted to retain sera containing potent BnAbs. We remained with 12 sera samples which were broad and potent and did not detectably neutralise QH343.A10 through the MPER, V2/glycan -site or V3/glycan region. In this manner, we believe we have selected heavily for sera that could plausibly neutralise QH343.A10 through the recognition of a novel target of BnAbs. We propose that further study of this very select set of sera taken from a large serum cohort may allow identification of a novel target of broadly neutralising anti-HIV-1 antibodies, if such a target does exist. Our unique system can be used to screen a large panel of serum samples and allows the scientist to focus on those few samples that are broadly neutralising but do not detectably neutralise most of the already identified targets of broadly neutralising anti-HIV-1 antibodies.