The impact of neutralizing antibody and ADCC responses on HIV-1 envelope evolution in early infection
The development of an effective HIV-1 vaccine remains a global priority. Neutralizing antibodies (nAbs), which block infection by cell-free virus, are likely to be an important response for vaccines to elicit. However, evidence from the RV144 vaccine trial and non-human primate vaccine studies suggest antibody-dependent cellular cytotoxicity (ADCC) responses, which target virus-infected cells, may also be protective. This thesis uses deep sequencing, together with immune assays, to characterise HIV-1 Envelope evolution associated with both nAb and ADCC responses in early infection, and investigates broadly neutralizing and non-neutralizing monoclonal antibody ADCC activity against subtype C viruses. Recent advances in deep sequencing approaches, coupled with the primer ID method which barcodes each viral genome, enabled us to generate thousands of viral sequences to accurately track viral population dynamics in early infection. In all participants investigated, there was a significant drop in the relative frequency of wildtype (WT) virus following nAb responses. However, in three of the seven participants, when controlling for changes in viral load (VL) over time, we observed that the WT load (frequency of the WT residue x total VL) remained relatively stable despite an effective nAb response. Instead, there was an outgrowth of the escaped virus with a concomitant increase in viral loads. We found that nAbs were inefficient at blocking cell-cell transmission of early WT and escape viruses, identifying this as one mechanism by which viruses may persist despite the presence of nAbs. These results suggest that other antibody effector functions such as ADCC, which target infected cells, may be important to elicit in a protective HIV-1 vaccines. If ADCC responses are important in controlling viral populations, one would expect to find evidence of viral escape from these responses. In all nine participants investigated, we found ADCC responses emerged prior to nAb responses, and in three individuals we observed sequence changes prior to detectable nAbs. To evaluate if these changes were due to ADCC pressure on the virus, we introduced select mutations into infectious molecular clones encoding the cognate early/acute envelope (Env-IMCs). In one participant, the mutation introduced conferred resistance to both nAb and ADCC responses, while in two participants, mutations were identified which resulted in resistance to ADCC but had no effect on neutralization, suggesting escape from ADCC. Longitudinal analysis in one of these participants, which targeted the CD4- binding site, revealed three distinct escape pathways, of which two conferred resistance to ADCC, and confirmed that ADCC responses can directly drive viral evolution in vivo. Finally, we investigated the ADCC activity of eleven anti-HIV-1 monoclonal antibodies (mAbs), including seven broadly neutralizing antibodies (bnAbs) and four non-neutralizing antibodies (nnAbs), against a panel of nine acute subtype C Env-IMCs. We found bnAbs had low to moderate ADCC breadth (11-66%). In contrast, while the two V2 nnAbs we tested were narrow and weak, the two nnAbs targeting CD4-induced epitopes (A32 and C11) mediated the broadest (78-100%) and most potent (0.06-0.81 μg/mL) ADCC against this panel. In addition, a nonlinear relationship was found between ADCC activity and strength of mAb binding to the infected cell surface (rs = -0.5309, p=0.0001). In conclusion, in contrast to studies which evaluated limited number of sequences, utilizing deep sequencing approaches, we found that the WT load remained relatively stable following early nAb pressure, albeit at lower relative frequency to the escape variant. Evasion of antibody responses through cell-cell transmission may contribute to the persistence of WT virus, providing further motivation for the importance of antibody effector functions that target infected cells in a protective HIV-1 vaccine. For the first time, we provide evidence of ADCC-mediated immune pressure in early infection, showing that these responses can exert selective pressure on HIV-1. However, the limited number of sequence changes relative to those observed following nAb pressure suggests that this response does not put as much selective pressure on the virus as nAbs. Lastly, the moderate breadth of bnAb ADCC activity provides evidence that there are common epitopes on free virions and on the surface of infected cells. This indicates bnAbs with potent and broad ADCC should be identified to include in antibody-based treatment and cure strategies, which aim to eliminate infected cells. Altogether, these data suggest that while eliciting nAbs should be the primary goal of HIV-1 vaccine design, ADCC-mediating antibodies may also play an important role.
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