The screening of neutralising antibodies against a resistant HIV-1 strain to identify novel epitopes

Master Thesis


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University of Cape Town

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.

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