Microbiology & Immunology - Theses
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ItemCharacterisation of Neutralising and Functional Antibody Responses to Different HIV-1 Env Vaccines in Bovines( 2020)Two main challenges have impeded the development of an effective HIV-1 envelope (Env) vaccine, with antibodies eliciting neutralisation of virions as well as Fc-effector functions, such as antibody-dependent cytotoxicity (ADCC), phagocytosis (ADP) or complement deposition (ADCD). On one hand, designing the right Env vaccine to elicit humoral or cellular protection has been challenging and, to date, SOSIP-Env trimers which are covalently constrained in the closed, pre-fusion conformation are the best vaccine candidate over uncleaved (Unc), open-structured trimers. On the other hand, eliciting heterologous neutralising antibodies in several animal models (including humans) has been difficult. Cows nevertheless produce unique antibodies with long CDRH3 regions, capable of accessing neutralising epitopes beneath the glycan shield, inaccessible for other animals. We tested how differences in clade and/or structure of HIV-1 Env vaccines affect the neutralising activity and Fc-effector functions of antibodies elicited, using recombinant trimers of clades A (KNH1,BG505), B (AD8, PSC89) and C (MW), which exposed either an open structure (Unc gp140) or a closed structure (SOSIP gp140). KNH1/BG505 SOSIP gp140 vaccine elicited the best neutralising IgGs against heterologous tier-2 pseudoviruses with high potency and breadth. While AD8 Unc gp140 also induced neutralisation, it was against only tier-1 pseudoviruses. Nevertheless, it was the only vaccine able to elicit IgGs that engaged CD32 (FcgRIIa), induced phagocytosis and complement-activation. The different antibody profile observed with both vaccines was explained by the Env immunogen structure, as KNH1/BG505 SOSIP gp140 induced mostly IgGs targeting the V1/V2 loop, whereas AD8 Unc gp140 induced antibodies targeting CD4-binding site and CD4-induced epitopes. In addition, analysis of IgG repertoires from animals of KNH1/BG505 SOSIP 100 and AD8 Unc 500 groups showed that KNH1/BG505 SOSIP gp140 induced higher rates of somatic hypermutation in germline genes compared to AD8 Unc gp140, with each animal presenting a unique antibody profile, and with germline antibodies already presenting high affinity towards HIV-1 Env trimers, as high levels of affinity maturation were not required to obtain antibodies with high neutralising activity. Overall, the results in this work show that open structured trimers elicit antibodies which highly activate antibody-effector functions, while SOSIP trimers focus antibody responses to concealed neutralising epitopes. The high neutralising responses observed in bovines against HIV-1 Env are due to antibodies which do not need high levels of somatic hypermutations and, in particular for KNH1/BG505 SOSIP, this antigen induced high levels of affinity maturation, probably favouring the improvement of both binding and neutralisation. Our study suggests that an effective vaccine regimen may include both uncleaved gp140 and SOSIP gp140, in order to target epitopes required for antibody-dependent effector functions as well as neutralisation, or a new trimeric structure with flexibility in the gp120-gp41 interface, exposing both epitopes involved in Fc-effector functions as well as neutralising ones.
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ItemFc-functional antibody immunity to HIV: the role of neutrophils and IgA( 2020)A safe and effective prophylactic vaccine against HIV-1 is an essential component to limit the HIV-1 epidemic. The RV144 HIV vaccine efficacy trial has highlighted the importance of generating Fc functional antibodies to prevent the further spread of HIV infection. Fc functional antibody responses have also been shown to correlate with delayed HIV disease progression. Despite the intensification of interest in Fc-mediated responses to HIV infection, there has been limited research focused on the Fc functional capacity of neutrophils, which are a key innate immune cell at mucosal surfaces and in the blood. The majority of Fc-effector studies in HIV focus upon examining NK cells and/or monocytes responses, while other effector cells such as neutrophils remain understudied. NK cells lack the FcalphaR and cannot mediate any IgA-dependent Fc-mediated effector responses therefore, other immune cells like neutrophil are necessary for IgA to be studied. Neutrophils are highly functional innate effector cells with the potential to induce both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis. In chapter 2, methods were optimized to evaluate antibody-dependent neutrophil phagocytosis (ADNP) and neutrophil-mediated rapid fluorometric antibody-dependent cellular cytotoxicity (RFADCC) effector responses, using freshly isolated primary human neutrophils from blood. In vitro, neutrophil-mediated RFADCC responses peaked at 4 hours, which was faster than primary NK cells or monocyte mediated responses. There was a large spectrum of responses of both ADNP and neutrophil-mediated RFADCC responses across a cohort of 41 viremic antiretroviral-therapy naive HIV positive subjects. ADNP and RFADCC responses correlated well with each other, suggesting that they measure overlapping functions. The viral load of the patients inversely correlated with the ADNP responses, suggesting that these antibody-mediated neutrophil-based assays could prove useful in dissecting HIV-specific immunity. The role that IgA plays in active HIV infection remains controversial, with some reports of HIV-specific IgA being able to inhibit HIV infection and potentially being protective. Chapter 3 investigated if HIV progression was influenced by HIV-specific ADNP and neutrophil-mediated RFADCC responses and the effects of IgA on these responses. It was shown that, although neutrophil-mediated RFADCC responses were higher in the plasma of subjects who controlled their viremia levels (viremic controllers), IgA from both viremic controllers and viremic non-controllers inhibited both ADNP and neutrophil-mediated RFADCC responses similarly. The IgG mediated ADNP responses from both viremic controller and viremic non-controllers were broadly inhibited by both autologous HIV positive IgA and HIV negative pooled purified IgA. The IgA inhibition was able to be blocked by pretreating neutrophils with an Fc alpha receptor (FcalphaR) blocking antibody. This suggests that IgA inhibition of ADNP responses can be mediated by 2 mechanisms; 1) antigen dependent, FcalphaR independent and 2) antigen independent, FcalphaR dependent. The RV144 vaccine trial has generated interest in Fc functional antibodies and in the role that HIV-specific IgA can play during HIV vaccination strategies and in HIV infection. The RV144 vaccine induced IgG antibodies that were able to mediate ADCC responses. However, the vaccine efficacy was reduced in the presence of high concentrations of HIV-specific IgA. Monoclonal IgA that was isolated from the plasma of the RV144 vaccinees was able to block the potentially protective IgG antibodies from binding similar epitopes, thus preventing ADCC responses with NK cells. This indicates there was epitope competition between IgA and IgG antibodies in the RV144 vaccine trial. NK cells lack the FcalphaR and cannot mediate any IgA-dependent Fc-mediated effector responses. Chapter 4 assessed plasma samples from the RV144 vaccine trial for their ability to induce neutrophil-mediated responses and if IgA was able to inhibit these responses. IgG from the RV144 vaccinees was able to induce modest HIV-specific ADNP and neutrophil-mediated RFADCC responses. Plasma IgA from the vaccinees was able to inhibit ADNP responses but not neutrophil-mediated RFADCC responses. Using pooled IgG from the vaccinees, it was shown that pooled purified IgA from vaccinees, pooled purified IgA from HIV positive donors and pooled purified HIV negative IgA were able to inhibit the IgG mediated ADNP responses. Overall, this thesis shows that neutrophils can mediate HIV-specific antibody-dependent phagocytosis and neutrophil-mediated RFADCC responses. HIV-specific IgG mediated neutrophil responses, induced by either infection or vaccination, can be inhibited by plasma IgA in an antigen dependent mechanism and an antigen independent mechanism that is a FcalphaR dependent mechanism. The inhibitory effects of IgA may assist in understanding HIV pathogenesis and improving future HIV vaccine designs.