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.

Host defense against infection and disease relies on the reciprocal communication between the immune and neuroendocrine systems where sex hormones exert negative and positive feedback actions on immune functions. Indeed, sex hormones have been implicated in gender dimorphic immune response and in the potentiation of immune-related disorders. The female hormone estrogen plays a role as an immunomodulator and may exert immunosuppressive and immunostimulatory effects. Though many studies focus on estrogen’s role in immunity within the female reproductive tract and autoimmunity, the modulatory effects of estrogen on vaccine responses are largely unexplored. The insufficient efficacy of some vaccines in certain target populations, as for example the elderly population, is well recognized. Hormones fluctuate throughout an individual’s life, and females in particular undergo several necessary reproductive (pregnancy and menopause) and lifestyle (oral contraceptive use) changes which involve sex hormones. Vaccine efficacy might be influenced by endogenous estrogen levels or by exogenous estrogen administration. Therefore, in the pursuit of improved vaccine efficacy, it is necessary to consider such hormonal factors and their contribution to immune status. We have studied estrogen’s role in modulation of vaccine responses using a mouse ovariectomy model where exogenous estrogen delivery can be controlled. Our studies included two different types of vaccines, a bacterial toxoid formulation and a bacterial secreted protein formulation. Results from these studies indicate that estrogen enhances vaccine-specific antibody production by likely supporting a general TH2 pathway and also modulates expression of genes encoding molecules critical in innate immune signaling and required for development of proper adaptive immune responses and antigen clearance through antibody-mediated mechanisms. The level at which estrogen modulates antibody responses appears to be dependent on the route of vaccine administration. The enhancement of specific humoral responses may involve mechanisms involving TLR2 and antibody Fc receptor expression on macrophages, cells that link innate and adaptive immune responses. Advances in our understanding of the relationship between sex hormones and the immune system may provide new insights into the mechanisms by which hormones act and thus may be exploited to guide the design of future vaccine strategies.

An effective vaccine to protect against HIV-1/AIDS remains elusive due to the extensive mechanisms employed by the HIV-1 virus to evade immune attack. Highly potent broadly neutralizing antibodies isolated from chronically infected individuals, however, show that such relevant antibodies can be naturally produced, implying that their elicitation through vaccination is a realistic possibility. These broadly neutralizing antibodies target different regions on the trimeric spikes formed by three protomers of the envelope (Env) protein. Each Env protein is comprised of the gp120 surface subunit in non-covalent association with the gp41 transmembrane subunit. Four regions have been identified: the CD4 binding site, the V1/V2 segment and the V3/glycan area all on the gp120 subunit as well as the MPER segment on the gp41 subunit. This dissertation focuses on the gp41 MPER segment given its highly conserved amino acid sequence among all HIV-1 clades and viral strain isolates and essential function in Env-mediated fusion and HIV entry. Of note, the MPER segment contains several adjacent epitopes targeted by broadly neutralizing antibodies, suggesting that the immune system is capable of producing neutralizing antibodies against this specific region. Analysis of both clade B and C MPER segments shows them to be L-shaped, consisting of two  helices separated by a hinge. We have found that the hinge region of the MPER segment provides the conformational flexibility necessary for the Env-mediated hemifusion and fusion processes. A significant reduction in virus infectivity is observed when the hinge region is disrupted by introduction of two amino acid mutations that eliminate -helical capping residues and the tandem hinge joints. The importance of the hinge region of the MPER segment is further supported by the action of four MPER-specific neutralizing antibodies 2F5, 4E10, 10E8 and Z13E1. These neutralizing antibodies block virus infection by disrupting MPER hinge-related function.

Antibodies can enhance or suppress the immune responses against their specific antigens. This phenomenon is known as antibody-mediated feedback regulation. We have studied the mechanisms underlying IgE-, IgM-, and IgG3-mediated enhancement of immune responses in mouse models using intravenous immunization. We attempted to answer the following questions: 1) Which cell type presents IgE-complexed antigens to CD4+ T cells? 2) Is complement activation required for specific IgM to enhance antibody responses? 3) Does IgM enhance CD4+ T-cell responses? 4) How are IgG3-antigen complexes transported into B-cell follicles? We found that CD23+ B cells transporting IgE-antigen complexes into B-cell follicles were not required to prime the antigen-specific CD4+ T cells in vivo, whereas CD11c+ cells were indispensable. After examining the three most common subpopulations of CD11c+ cells in the spleen, we determined that it was CD8α- conventional dendritic cells migrating into the T-cell zone following immunization that presented IgE-complexed antigens to CD4+ T cells. Next, we showed that specific IgM from Cµ13 mice, which is unable to activate complement, failed to enhance either antibody or germinal center responses whereas wild-type IgM enhanced both responses. Therefore, specific IgM must activate complement to enhance humoral responses. In addition, wild-type IgM did not up-regulate CD4+ T-cell responses. Finally, we showed that IgG3-antigen complexes were transported by marginal zone B cells into B-cell follicles via binding to complement receptors 1 and 2 (CR1/2) on those cells. The immune complexes were captured by follicular dendritic cells as early as 2 h after immunization. Germinal center responses were also enhanced by IgG3. Using bone marrow chimeric mice, we found that CR1/2 expression was required on both marginal zone B cells and follicular dendritic cells to provide an optimal enhancement of antibody responses.

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: In South Africa alone, 30% of women of child-bearing age are infected with HIV. With the increasing focus and success of prevention of mother-to-child transmission (PMTCT) programmes, an estimated 300 000 infants are born exposed to HIV every year. The underlying impact of in utero HIV exposure on infant immune health has not been extensively characterised. Clinical follow-up of these HIV-exposed uninfected (HEU) infants reveals increased infectious morbidity and mortality compared to their unexposed (UE) counterparts. Objectives: (i) To evaluate and characterise adaptive immune properties by measuring vaccine-specific antibody levels in children from 2 weeks to 2 years of age in the presence and absence of maternal HIV infection. (ii) To investigate specific cellular markers of immune activation, immune regulation, apoptosis and B cell memory on T and B cell populations in HEU and UE children measured at 18 and 24 months of age. Methods: This sub-investigation formed part of a collaborative pilot study between the universities of British Columbia (Vancouver, Canada) and Stellenbosch. A total of 95 HIV-positive and HIV-negative mothers were recruited after delivery at Tygerberg Hospital, and signed informed consent for their infants to be included in the study. Of these infants, only 27 HEU and 30 UE infants were eventually enrolled and followed up at various time points, starting at two weeks of age. Four of these infants were confirmed to be HIV-positive at 2 weeks and clinically followed up according to the protocol, but were excluded from statistical data analyses. Blood was collected at 2, 6 and 12 weeks and again at 6, 12, 18 and 24 months of age. Quantitative IgG-specific antibodies to Haemophilus influenzae B (Hib), Bordetella pertussis, tetanus and pneumococcus were measured at each time point, using commercially available ELISA (Enzyme-Linked ImmunoSorbent) kits. Cellular markers of immune activation, immune regulation, apoptosis and memory were measured in various populations of T and B cells at 18 and 24 months only, by using four-colour flow cytometry and validated whole-blood staining methods. In addition, a functional assay was developed to evaluate cell susceptibility to apoptosis (spontaneously) by measuring the expression of Annexin V on both CD4+ T and CD20+ B cells after 16 and 24-hour incubation periods. The statistical analysis of the antibody data was conducted by repeated-measures ANOVA (i.e. analysis of variance), using a mixed-model approach. Differences in the expression of the two groups’ cellular markers were compared by employing one-way ANOVA. An F test p value (which assumes normality) was reported, while the non-parametric Mann-Whitney U test served as confirmatory tool. Repeated-measures ANOVA was used for the evaluation of the functional spontaneous apoptosis assay at three time points (ex vivo, 16 and 24 hours) on the 18-month samples, while one-way ANOVA was used for the 24-month samples. Results: The HEU group (n = 23) displayed significantly lower levels of antibodies to pertussis (20.80 vs 28.01 Food and Drug Administration [FDA] U/ml; p = 0.0237), tetanus (0.08 vs 0.53 IU/ml; p < 0.001) and pneumococcus (31.67 vs 80.77 mg/l; p = 0.003) than the UE group (n = 23) at 2 weeks of age. No statistical differences were noted for Hib antibody levels between the two groups at this time point. At 6 weeks of age, HEU infants displayed lower mean levels of all antibodies measured; however, these differences did not reach statistical significance. Following vaccination, compared to UE controls, the HEU group presented with statistically significantly higher antibody levels to pertussis at 6 months (155.49 vs 63.729 FDA U/ml; p = 0.0013), 12 months (26.54 vs 8.50 FDA U/ml; p < 0.001) and 18 months of age (1658.94 vs 793.03 FDA U/ml; p = 0.0362). A significant difference in tetanus antibody levels between the two groups was only evident at 24 months, with the HEU group displaying higher levels (3.28 vs 1.70 IU/ml; p = 0.018) than the UE group. No differences were observed between the two groups following vaccination for Hib. At 18 and 24 months, the HEU group showed increased expression of cellular markers of immune activation (CD69 and CD40L) on CD4+ T cells compared to UE controls. The two groups showed similar expression of the cellular marker of activation CD38 on CD8+ T cells. The HEU group displayed significantly higher levels of CD127, the interleukin (IL) 7 receptor, on CD4+ T cells compared to UE controls at 18 months of age. The HEU group also showed increased expression of cellular markers of apoptosis on both CD4+ T and CD8+ T cells. No statistical significance was noted for the expression of Fas on CD4+ T cells at 18 and 24 months of age. However, at 24 months, the HEU group showed significantly increased expression of FasL on both CD4+ T and CD8+ T cells. During cell culture experiments, the HEU group displayed increased susceptibility to spontaneous apoptosis shown by increased Annexin V expression on CD4+ T cells after a 16-hour incubation period at both 18 and 24 months. At 18 and 24 months, no difference was noted in the two groups’ immune regulation as measured by the expression of CTLA-4. The HEU group displayed increased levels of the cellular markers of immune activation CD80 on CD20+ B cells at 18 and 24 months of age. The HEU group also showed significantly increased levels of CD69 on CD19+ B cells at 24 months. No statistical significance was reached for the expression of CD62L and CD10 at either 18 or 24 months. Although the HEU group displayed increased levels of apoptosis (Fas) on CD20+ B cells, no statistical significance was reached at 18 or 24 months of age. In addition, the HEU group showed no difference in the expression of programmed death 1 (PD-1) at 18 and 24 months. HEU and UE groups showed similar expression of Annexin V after 16 hours of incubation in the 18 and 24-month samples. The expression of the biomarker of B cell memory CD27 on CD20+ B and CD19+ B cells was comparable between the two groups at both time points. Conclusion: At 2 and 6 weeks, lower mean antibody responses in HEU infants suggest poor placental transfer due to maternal HIV infection, while increased responses to specific antibodies may reflect an exaggerated immune response to immunisation. These robust responses may be due to the lack of competition with maternal antibodies, or may be ascribed to indirect stimulation of B cells via the activation of T cells. A hyper-inflammatory state is an imminent danger, with increased expression of cellular markers of immune activation and apoptosis that may be consistent with early HIV exposure that persists following infancy. These observations may serve as contributing factors to the extensively documented increased susceptibility to infections in the HEU population. Although these findings are consistent with a primed immune system, larger studies are required to confirm these observations in relation to clinical outcomes and to assess further whether these differences persist in later years. / AFRIKAANSE OPSMOMMING: Agtergrond: In Suid-Afrika alleen het 30% van vroue van ʼn vrugbare leeftyd MIV. Met die toenemende fokus en sukses van programme vir die voorkoming van moeder-na-kind-oordrag (sogenaamde PMTCT-programme) word ongeveer 300 000 babas jaarliks aan MIV blootgestel. Die onderliggende impak van intra-uteriene MIV-blootstelling op ʼn baba se immuunstelsel is nog nie omvattend beskryf nie. Kliniese opvolgondersoeke van hierdie MIV-blootgestelde dog onbesmette babas (sogenaamde HEU’s) dui op ʼn hoër siekte- en sterftesyfer weens infeksies as hul nieblootgestelde eweknieë (sogenaamde UE’s). Doelstellings: (i) Om kinders met MIV-positiewe en MIV-negatiewe moeders se aangepaste (verworwe) immuuneienskappe te beoordeel en te beskryf deur hulle vaksienspesifieke teenliggaamvlakke vanaf die ouderdom van twee weke tot twee jaar te meet. (ii) Om ondersoek in te stel na bepaalde sellulêre merkers van immuunaktivering, immuunregulering, apoptose en B-selgeheue by die T- en B-selgroepe van sowel HEU’s as UE’s op die ouderdom van 18 en 24 maande. Metodes: Hierdie subondersoek het deel uitgemaak van ʼn samewerkende loodsondersoek tussen die universiteite van Brits-Columbië (Vancouver, Kanada) en Stellenbosch. Altesaam 95 MIV-positiewe en MIV-negatiewe moeders is gewerf nadat hulle by Tygerberghospitaal geboorte geskenk het, en het ingeligte toestemming verleen dat hul babas by die studie ingesluit kon word. Van dié babas is slegs 27 HEU’s en 30 UE’s uiteindelik in die studie opgeneem en in verskillende stadia vanaf die ouderdom van twee weke opgevolg. Vier van die babas is op twee weke as MIV-positief bevestig en volgens die protokol klinies opgevolg, maar is van die statistiese dataontleding uitgesluit. Bloedmonsters is op twee, ses en 12 weke en weer op ses, 12, 18 en 24 maande geneem. Kwantitatiewe IgG-spesifieke teenliggame teen Haemophilus influenzae B (Hib), Bordetella pertussis, tetanus en pneumokokkus is telkens met behulp van kommersieel verkrygbare ELISA- (“Enzyme-Linked ImmunoSorbent”-)stelle bepaal. Sellulêre merkers van immuunaktivering, immuunregulering, apoptose en geheue is op slegs 18 en 24 maande by verskillende populasies T- en B-selle deur middel van ʼn vierkleurvloeisitometrie en geldig verklaarde volbloedkleuringsmetodes bepaal. Voorts is ʼn funksionele toets ontwikkel om selvatbaarheid vir apoptose te bepaal deur die ekspressie van Annexin V op sowel CD4+ T- as CD20+ B-selle ná 16 en 24 uur van inkubasie te meet. Die statistiese ontleding van die teenliggaamdata is met behulp van herhaaldemetings-ANOVA (d.w.s. afwykingsontleding) volgens ʼn gemengdemodel-benadering gedoen. Verskille in die twee groepe se sellulêre merkervlakke is deur middel van eenrigting-ANOVA vergelyk. ʼn F-toets-p-waarde (wat normaliteit veronderstel) is bereken, terwyl die nieparametriese Mann-Whitney-U-toets as bevestigende instrument gedien het. Vir die 18 maande-monsters is herhaaldemetings-ANOVA gebruik om die funksionele toets vir spontane apoptose in drie stadia (ex vivo, op 16 uur en op 24 uur) te beoordeel. Vir die 24 maande-monsters is eenrigting-ANOVA gebruik. Resultate: Op die ouderdom van twee weke het die groep HEU’s (n = 23) aansienlik laer teenliggaamvlakke teen kinkhoes (20.80 vs 28.01 Food and Drug Administration [FDA] U/ml; p = 0.0237), tetanus (0.08 vs 0.53 U/ml; p < 0.001) en pneumokokkus (31.67 vs 80.77 mg/l, p = 0.003) as die UE-groep (n = 23) getoon. In dié stadium is geen statistiese verskille in Hib-teenliggaamvlakke tussen die twee groepe opgemerk nie. Op ses weke het die groep HEU’s laer gemiddelde vlakke van ál die betrokke teenliggame getoon, hoewel hierdie verskille nie statisties beduidend was nie. In vergelyking met die UE-kontrolegroep het die groep HEU’s ná inenting statisties beduidend hoër teenliggaamvlakke teen kinkhoes getoon op ses maande (155.49 vs 63.729 FDA U/ml; p = 0.0013), 12 maande (26.54 vs 8.50 FDA U/ml; p < 0.001) én 18 maande (1658.94 vs 793.03 FDA U/ml; p = 0.0362). ʼn Beduidende verskil in die twee groepe se tetanus-teenliggaamvlakke het eers op 24 maande geblyk, met die groep HEU’s s’n hoër (3.28 vs 1.70 IE/ml; p = 0.018) as die UE’s s’n. Ná inenting teen Hib is geen verskille tussen die twee groepe waargeneem nie. Op 18 en 24 maande het die HEU’s verhoogde ekspressie van sellulêre merkers van immuunaktivering (CD69 en CD40L) op CD4+ T-selle getoon in vergelyking met die UE-kontrolegroep. Soortgelyke vlakke van die sellulêre merker van aktivering CD38 is ook op die CD8+ T-selle van die twee groepe opgemerk. Op 18 maande het die HEU-groep ʼn beduidend verhoogde ekspressie van CD127, die IL-7-reseptor, op CD4+ T-selle getoon in vergelyking met die UE-kontrolegroep. Die HEU groep het ook verhoogde ekspressie van sellulêre merkers van apoptose op sowel CD4+ T- as CD8+ T-selle getoon. FAS-ekspressie op CD4+ T-selle op 18 en 24 maande was nie statisties beduidend nie, hoewel die HEU-groep op 24 maande beduidend verhoogde ekspressie van FasL op CD4+ T- sowel as CD8+ T-selle getoon het. In selkwekingseksperimente het die HEU-groep ʼn verhoogde vatbaarheid vir apoptose getoon na aanleiding van die ekspressie van Annexin V op CD4+ T-selle ná 16 uur van inkubasie op sowel 18 as 24 maande. Op 18 en 24 maande was immuunregulering, aan die hand van die ekspressie van CTLA-4, bykans dieselfde by albei groepe. Op sowel 18 as 24 maande toon die HEU’s verhoogde ekspressie van die sellulêre merker van immuunaktivering CD80 op CD20+ B-selle. Op 24 maande het die HEU’s ook aansienlik hoër vlakke van CD69 by CD19+ B selle getoon. Op nóg 18 nóg 24 maande was die ekspressie van CD62L en CD10 statisties beduidend. Hoewel verhoogde vlakke van apoptose (Fas) by CD20+ B-selle by die HEU-groep opgemerk is, was dit nie statisties beduidend op 18 óf 24 maande nie. Daarbenewens was daar ook geen verskil in die ekspressie van geprogrammeerde seldood 1 (PD-1) op 18 en 24 maande nie. Op 18 en 24 maande het die HEU’s en UE’s ʼn soortgelyke ekspressie van Annexin V ná 16 uur van inkubasie getoon. Op sowel 18 as 24 maande was die twee groepe se ekspressie van die biomerker van B-selgeheue CD27 op CD20+ B- en CD19+ B-selle vergelykbaar. Gevolgtrekking: Op twee en ses weke dui laer gemiddelde teenliggaamreaksies by HEU’s op swak plasentale oordrag weens die moeder se MIV-infeksie, terwyl verhoogde reaksies op bepaalde teenliggame weer op oordrewe immuunreaksie op inenting dui. Hierdie robuuste reaksie kan toegeskryf word aan die gebrek aan mededinging met die moeder se teenliggame, of kan deur indirekte stimulasie van die B-selle via die aktivering van die T-selle veroorsaak word. ʼn Hiperinflammatoriese toestand is ʼn dreigende gevaar, met verhoogde ekspressie van sellulêre merkers van immuunaktivering en apoptose wat met vroeë MIV-blootstelling met ʼn latere nawerking verbind kan word. Hierdie waarnemings kan bydraende faktore wees tot HEU’s se goed gedokumenteerde verhoogde vatbaarheid vir infeksies. Hoewel hierdie bevindings met ʼn geaktiveerde immuunstelsel strook, moet groter studies dit aan die hand van kliniese uitkomste bevestig en ook bepaal of hierdie verskille in later jare voortduur. / The Harry Crossley Foundation, Poliomyelitis Research Foundation (PRF) / NHLS Research Grant Trust