Dissertations / Theses on the topic 'Antibody response to HIV'

Since being identified as the cause of AIDS in 1983, HIV-1 infection has reached pandemic proportions. Despite public awareness about prevention, the growing incidence of HIV-1 infection and the limitations of current antiretroviral therapy underscore the imperative need for a vaccine. Understanding the basis of an immune response that controls infection or provides sterilizing immunity remains a major goal in the search for effective vaccines or immunotherapies. Research into correlates of immunity to HIV-1 have largely focused on CD8⁺ T cells or neutralising antibodies (NAbs) but to date these responses have not proved effective in containing viral replication in vaccinees who become infected. Natural killer cells (NKs), monocytes (MCs), and neutrophils (PMNs) are cells of the innate immune system with intrinsic cytotoxic function that can be enhanced by antibodies (Abs) in what is termed antibody-dependent cellular cytotoxicity (ADCC). In my studies I investigated the production of PMNs from human stem cells, the elimination of HIV-1 infected cells by these effector cells, the modulation of cellular cytotoxicity by Ab, and characterized how Abs facilitate a potent ADCC response. I developed a novel flow cytometry assay to measure cytotoxic activity against HIV-1 infected CD4⁺ T cells. Using this, effector cells were shown to have different cytotoxic capacities which were enhanced by Ab. Comparing ADCC mediated by patient serum revealed that higher levels correlated with IgG binding to infected cells. I observed no correlation between serum-mediated ADCC and markers of disease progression including patient status, viral RNA load, CD4⁺ T cell count, or NAb titers. The data presented here have implications for acquisition and control of early HIV-1 infection by NKs, MCs, and PMNs prior to activation of an adaptive immune response, at later stages in the presence of HIV-1-specific Abs, and are relevant to vaccine-induced anti- HIV-1 Ab-based effector mechanisms.

Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function. First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens. Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells. Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies. Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.

Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function. First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens. Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells. Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies. Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.

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.

This thesis examines the effects of psychosocial factors and physical activity on antibody response to vaccination in healthy young, older, and HIV+ populations. Chapter Two found that a brisk walk prior to vaccination did not improve antibody response to pneumococcal or influenza vaccinations in young (18-30yrs) or older (50-64yrs) adults. Chapter Three examined whether a lifestyle physical activity intervention affected antibody response to pneumococcal vaccination in sedentary middle-aged women. There was no effect on antibody response, body composition or fitness measures, although there was an improvement in quality of life for the intervention group. Finally, Chapter Four investigated the relationship between psychosocial and physical activity status and antibody response to vaccination in HIV+ patients. Antibody response to some strains of the pneumococcal vaccine were predicted by higher physical activity levels (pn1, pn6b, pn18c), greater social support (pn3) and lower life events stress (pn1). However, the majority of analyses found that antibody response to vaccination was not affected by these measures. In conclusion, neither acute nor chronic walking interventions improve antibody response to vaccination, and only limited relationships are seen between psychosocial factors, physical activity status and antibody response to a variety of vaccinations.

Despite 30 years of research, a prophylactic vaccine against HIV-1 is still lacking and is urgently needed in order to control the global AIDS pandemic. The discovery of broadly neutralising antibodies (BNAbs) was an important step for HIV-1 research but no vaccine candidate tested so far has been able to reproduce responses containing such antibodies, and it remains unclear how this could be achieved via immunisation. In this thesis, I attempted to explore this gap of knowledge in two ways. First, certain features (‘signatures’) of the Env protein that were associated with a broadly neutralising response were identified through machine learning. Further characterisation of these signatures revealed several ways by which these naturally-occurring mutations might alter the immunogenicity of the Env protein that could result in the elicitation of a broadly neutralising response. The incorporation of such signatures in future vaccine design could be useful as the Env protein might adopt a conformation that encourages the elicitation of a broadly neutralising response. Second, 3 novel vaccination approaches were proposed aiming to induce a BNAb antibody response. The development of 2 approaches proved to be difficult and was not continued. For the third approach, non-neutralising immunogen-derived antibodies were used to mask immunodominant epitopes on the Env protein (i.e. ‘antibody-shielding’), thus allowing the antibody response to be focused to the highly conserved CD4 binding site (CD4bs). Subsequent immunisation of the antibody-shielded gp120 proteins in mice and rabbits demonstrated that antibody-shielding was able to significantly dampen the V3-specific antibody response while retaining the CD4bs-specific response. However, the antibody response to the V1/V2 loop was enhanced upon V3-dampening which indicates that further optimisation of the antibody-shield is needed in order to eliminate any antibody response towards the immunodominant regions. In conclusion, these results are the first description of a number of novel vaccination ideas and provide valuable insights into how these approaches could be optimised to become effective HIV-1 vaccines that can lead to the elicitation of a broadly neutralising antibody response.

Includes bibliographical references.
Inducing broadly neutralizing antibodies targeting the HIV-1 envelope is thought to be crucial for developing an effective vaccine. The Membrane Proximal External Region (MPER) within the HIV- 1 gp41 envelope is a promising vaccine target. The MPER is highly conserved, functionally constrained, facilitates virus fusion and is targeted by broadly neutralizing monoclonal antibodies. The objectives of this research were 1) To evaluate the neutralization breadth of antibodies induced by epitopes within the MPER compared to the PG9/16-site in chronically HIV-1-infected individuals, 2) to identify neutralization resistant HIV-1 isolates (using plasma samples infected with the same subtype) and to characterize their sensitivity to anti-MPER antibodies and 3) to determine the accessibility of the MPER to HIV-1 induced polyclonal anti-MPER antibodies in a highly neutralization resistant virus (253-11; CRF02_AG subtype).

The best known correlate of protection provided by vaccines is the presence of pathogen specific antibodies after immunization. However, against the Human Immunodeficiency Virus-1 (HIV-1) the mere presence of antibodies specific for the viral Envelope (Env) protein is not sufficient to provide protection. This necessitates in depth study of the humoral responses elicited during infection and by vaccination. While a significant amount of effort has been invested in studying the evolution of antibody responses to viral infection, only limited progress in understanding antibody responses elicited through vaccination has been made. In the studies described here, I attempt to rectify this deficiency by investigating how the quality of a humoral response is altered with the use of different immunization regimens, in particular a DNA prime-protein boost regimen, or with the use of different model HIV-1 Env gp120 immunogens. In a New Zealand White (NZW) rabbit model, we demonstrate that the broader neutralizing activity elicited with the DNA prime-protein boost regimen may be the result of the elicitation of a higher avidity antibody response and a unique profile of antibody specificities. Specifically, use of a DNA prime-protein boost regimen elicits antibodies targeted to the CD4 binding domain of the HIV-1 Env, a specificity that was not frequently observed when only protein based immunizations were administered. We extended this analysis to sera from healthy human volunteers who participated in early phase HIV vaccine trials utilizing either a protein alone immunization regimen, a canarypox prime-protein boost immunization regimen, or a DNA prime-protein boost immunization regimen. Evaluation of sera from these trials demonstrated that the use of a DNA prime-protein boost regimen results in an antibody response with greater neutralization breadth characterized by an increased frequency and titer of antibodies targeted toward the CD4 binding site (CD4bs). In addition to this, the antibody response elicited by the DNA prime-protein boost regimen also exhibited the capability to mediate antibody dependent cell-mediated cytotoxicity (ADCC) activity as well as activation of the complement system. Additionally, in an attempt to better understand the capabilities of antibodies elicited by a DNA prime-protein boost regimen, we generated gp120 specific monoclonal antibodies (mAbs) from a single DNA primed-protein boosted NZW rabbit. Analysis of mAbs produced from this animal revealed that use of this immunization regimen elicits an antibody repertoire with diverse epitope specificity and cross reactivity. Furthermore, these select mAbs are capable of neutralizing heterologous HIV isolates. Further application of mAb generation in rabbits may provide a valuable tool to study immunogenicity of different vaccines and immunization regimens. Concurrently, while demonstrating that a DNA prime-protein boost regimen elicits a higher quality antibody response than that observed with other leading techniques, we also demonstrated that immunogen selection can play a vital role in the quality of the resulting antibody response. By immunizing with two closely related but phenotypically distinct model gp120 immunogens, known as B33 and LN40, we demonstrated that disparate gp120s have different intrinsic abilities to raise a heterologous neutralizing antibody response. Additionally, we showed that residues found within and flanking the b12 and CD4 binding sites play critical roles in modulating neutralizing activity of sera from animals immunized with LN40 gp120, indicating that the broader neutralizing activity seen with this immunogen may be due to differential elicitation of antibodies to this domain.

Our research laboratory has recently reported that mucosal priming with a replicating modified vaccinia Tiantan virus (MVTTgpe)-based vaccine elicits durable protection against pathogenic SIVmac239 infection in rhesus monkeys. However, the protective role of vaccine-elicited antibody responses remains poorly understood. Here, a novel yeast surface displayed (YSD) antigen library was established to quantitatively map the antigenic determinants presented by MVTTgpe-based and control vaccines as well as by SIVmac239 infection. The YSD-library allows the mapping of linear and some conformational epitopes as a major technical innovation, as validated by testing SIV-specific mAbs KK65, KK8 and VM-18S. While eight antigenic domains are characterized covering the entire SIVmac239 gp160, the MVTTgpe/Ad5gpe regimen uniquely induces antibody responses against a distinct major antigenic determinant (MAD) in V2 region as compared with the Ad5gpe/Ad5gpe vaccination and SIV infection. This MAD is associated with a higher titer of anti-V2 antibody responses, which inversely correlates with peak viral load. Unexpectedly, the MVTTgpe/Ad5gpe vaccine- challenge. The results showed that instead of recalling B cell memory response to V2, viral infection presents a distinct set of antigenic determinants with anti-V1V2 antibodies primarily directed to V1 region. Moreover, the anti-V1V2 antibody responses disappear in two infected macaques after they enter the stage of simian AIDS. SIVmac239 infection, therefore, can modulate vaccine-elicited B cell immunity by diminishing anti-V2 antibody memory responses in rhesus monkeys. These findings implicated that vaccine efforts with focus on V2 region would require periodic vaccinations to maintain a long-lasting high level of antibody responses for protection. In the absence of an effective vaccine for eliciting HIV-1-specific broadly neutralizing antibodies (bNAbs), passive immunization with bNAbs or Ab-like agents (e.g. immunoadhesin) becomes an attractive alternative for HIV-1 prevention. In this study, we aimed to design, optimize and produce secretory immunoadhesins (IAs) based on gene engineering of existing HIV-1 specific bNAbs for potency and production improvements. IAs are chimeric, antibody-like molecules that combine the functional domain of bNAb with immunoglobulin constant domains, including the hinge and Fc regions. We found that the modified secretory IAs not only preserved the neutralization activity of the parental bNAbs, but also had enhanced expression and smaller molecular size that is suitable for antibody gene-based in vivo delivery. Furthermore, we defined the synergistic effects of five IAs against HIV-1 infection and subsequently engineered two types of bi-specific IAs by combining the functional domains of Hu5A8, a humanized anti-CD4 antibody, and the bNAb PGT128. Significantly, one of the bi-specific IA, namely Bi-IA-Mono, neutralized 100% of the 33 viruses tested, including the transmitted/founder viruses and viruses resistant to both parental IAs. The remarkably enhanced neutralization activity of Bi-IA-Mono, either in potency and breadth, indicated the great potential of modified bi-specific IA to provide complete or nearly complete protection against major HIV-1 subtypes. Overall, our results demonstrated that the engineering of IA and bi-specific IA is an attractive way to improve anti-HIV-1 properties of existing bNAbs, which have significant implications for antibody-based prophylactics in blocking diverse HIV-1 transmissions and infections.
published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy

The purpose of this descriptive investigation was to study the reactions of women, specifically women who were seeking pregnancy counseling, to the psychological and psychosocial impact of HIV antibody testing. A questionnaire was developed that surveyed women on HIV knowledge, behaviors associated with HIV infection, and locus of control issues related to the disease. Additionally, questions regarding the stigma and discrimination associated with the disease were posed that may have acted as barriers to taking the HIV antibody test. Finally, supplemental questions related to HIV and the issue of abortion were also presented.;The sample for this research consisted entirely of women who were new or returning clients of Planned Parenthood. Four sites from Planned Parenthood in the Hampton Roads area of Virginia were utilized. One hundred and four women completed the survey. The ages of women sampled ranged from 12 to above 50. Median age range for this sample was 21-25 years of age. Other demographic information presented a composite of mostly white, lower socioeconomic status, high school to college educated women.;Eighty four women indicated that they would take the HIV antibody test; twelve indicated that they would decline taking it. However, many of the women who said that they would take the HIV antibody test had not taken the test at the time the survey was conducted. It was concluded that the majority of women had sufficient knowledge regarding the disease, although over half of the women surveyed did not know that HIV could be spread by an HIV infected mother who breast feed her newborn. A majority of women also demonstrated an internal locus of control with regards to HIV infection. Using a Likert Scale, ninety-six women answered the question posed about HIV infection as it pertained to the option of abortion. Twenty-one women (21.8%) strongly agreed to aborting their fetus if they were HIV infected, while seventeen participants (17.7%) agreed. However, forty-three women (44.7%) were uncertain as to their decision to abort their fetus; seven women (7.2%) disagreed and eight (8.3%) strongly disagreed to having an abortion as a result of being infected with HIV.;Further study is needed in this area which would demonstrate a more diversified sample of women in order to increase the external validity of the findings of this study. as research into the disease continues to divulge new methodologies for using established drugs to combat HIV, such as the preliminary revelation that AZT significantly cuts the risk of HIV transmission from mother to fetus, questions arise that deal with new psychological consequences and conflicts for the female population.

It is widely held that for an HIV-1 vaccine to provide sterilizing immunity, it would need to elicit broadly neutralizing antibodies (bnAbs). However, factors underlying the development of these antibodies are not clear. There is evidence to suggest that in some individuals who develop bnAbs, the development of breadth is influenced by the co-evolution of the transmitted/founder (t/f) virus and earlier strain-specific neutralizing antibody (ssnAb) responses. Here we characterized the viral evolution, ssnAb and bnAbs responses in CAP292, an HIV-1 infected woman who developed bnAb responses from one year post infection. We used single genome amplification (SGA) to characterize viral evolution at four time points: at acute infection; after the development of strain-specific neutralizing responses; at the first detection of the broadly neutralizing antibody response; and lastly, at the peak of the broad response. We identified the t/f virus, and generated chimeric viruses from this to determine the targets of the ssnAb responses. A panel of site-directed mutant viruses were used to map the specificity of the bnAb responses. Our data indicated that infection was most likely founded by a single virus and that the first wave of ssnAbs emerged at 14 weeks post infection (w.p.i), targeting the V1V2 loop of Envelope (Env). A second wave of ssnAbs, possibly targeting the C3V4 region, emerged by 30 w.p.i. Two distinct viral clusters were detected by the time the bnAb response peaked, suggesting the presence of distinct escape pathways. Mapping of the bnAb specificities indicated that CAP292 produced PGT128-like bnAb responses targeted toward the 332 glycan.

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.

Despite huge strides being made towards decreasing the number of individuals getting newly infected with HIV-1, and in reducing AIDS-related deaths, unfortunately current predictions are that the 2020 UNAIDS goals (90-90-90 targets, where 90% of people living which HIV-1 are diagnosed as such, from which 90% will will receive sustained antiretroviral therapy, resulting in viral suppression in 90% of these individuals by 2020) are out of reach. This of course means that the numbers of newly infected indivuals and AIDS-related deaths will be above the target derived from the 2020 UNAIDS goals. The development of an effective HIV vaccine could therefore be an important step towards realising these objectives. In work done for this thesis, a heterologous HIV-1 vaccine platform regimen was developed using antigen sequences from the predominant circulating HIV-1 subtype (subtype C) in South Africa. Specifically, this involved use of the envelope glycoprotein sequence of the CAP256 superinfecting virus (CAP256_SU) from the CAPRISA 002 cohort, and a mosaic Gag sequence which resulted in robust autologous Tier 2 neutralisation of CAP256_SU pseudovirions. The envelope glycoprotein sequence was modified so as to replace the native leader sequence with the tissue plasminogen activator leader, the furin cleavage site with a glycine rich flexible linker, and to introduce an I559P mutation. DNA and modified vaccinia virus Ankara (MVA) vaccines were generated where Env was truncated to gp150, thereby retaining the transmembrane domain and a partial cytoplasmic tail (Env). The Env sequence for the protein vaccine was further trimmed by removal of the transmembrane domain to give gp140, leading to a soluble, secreted protein (soluble Env). This allowed for the latter vaccine to be affinity purified using lectin (soluble Env (GNL)), and after generating stable cell lines, soluble Env yields were high enough to enable size exclusion chromatography which allowed isolation of the trimeric fraction of Env as determined by molecular weight (soluble trimeric Env). A Cterminal His-tagged version of soluble Env was generated as well. Surprisingly, the folding of Env-His was inferior to soluble Env, with a switch in profile from mainly trimeric Env to mainly monomeric Env. Nevertheless, soluble Env-His (GNL) and soluble trimeric Env-His were assessed for the presence of Env broadly neutralising antibody (bnAb) epitopes in an ELISA assay. The V3-glycan supersite (binding of bnAbs PGT128 and PGT135), the CD4-binding site (VRC01) and the V2-glycan site (PG9) were detected for both Env-His (GNL) and soluble trimeric proteins, whereas low signals for PG16, PGT145 and CAP256-VRC26.08, bnAbs which specifically recognise Env trimers in a native-like conformation, were only detectable for soluble trimeric Env-His. Soluble Env (GNL) was subsequently used as a protein vaccine in rabbits to test the immunomodulatory effects of the two adjuvants AlhydroGel (similar to alum) or the MF59-like squalene-based oil-in-water nano-emulsion AddaVax. Soluble Env (GNL) adjuvanted in AlhydroGel resulted in improved immune response in rabbits, with significantly higher serum binding antibodies to soluble Env (GNL) and scaffolded CAP256 V1V2-loop in comparison to AddaVax and unadjuvanted protein. Furthermore, significantly higher neutralisation titres to Tier 1A subtype C virus (MW965.26), in combination with an improved breadth to subtype C Tier 1A and 1B viruses, were observed in the AlhydroGel group. However, no neutralisation of Tier 2 viruses was detected. Nonetheless, AlhydroGel was selected as the best protein adjuvant for all further rabbit immunogenicity studies. Furthermore, in all subsequent experiments, soluble trimeric Env was used as a protein vaccine. DNA and recombinant MVA vaccines were generated using a membrane anchored gp150 (Env) with the aim that co-expression with mosaic Gag (GagM) would lead to the incorporation of Env into Gag virus-like particles (VLPs). Electron microscopy of cells expressing Env+GagM from DNA and recombinant MVA vaccines verified VLP formation from these constructs, and the presence of Env was observed in VLPs purified using a two-step OptiPrep gradient centrifugation protocol. The presence of Env bnAb epitopes in cellular membrane-bound Env was verified by qualitative immunofluorescent microscopy of live-cell stainings and a quantitative FACS assay. The same bnAb epitopes as for the Env protein vaccine were detectable, including bnAbs recognising only native-like Env trimers (PG16, PGT145 and CAP256-VRC26_08). However, expression levels of native-like Env trimers were lower, at approximately 20% when normalised to VRC01. These HIV-1 DNA, rMVA and soluble trimeric Env protein vaccines were tested in different heterologous vaccine platform immunogenicity studies in rabbits. These consisted of either priming with two recombinant MVA vaccines and boosting with three protein vaccines (MMPPP), or priming with DNA vaccines followed by two MVA vaccines, followed by two protein vaccines (DDMMPP). Furthermore, the inclusion of GagM into the DNA and MVA vaccines was compared to use of Env alone. Both vaccine regimens resulted in binding antibodies to soluble trimeric Env and a scaffolded CAP256 V1V2-loop; however, these were induced by MVA and protein vaccines, but not by DNA vaccines. Despite the lack of Env binding antibodies after DNA vaccination, better neutralisation was observed for the DDMMPP regimen compared to MMPPP, resulting in higher sera neutralisation titres towards vaccinematched, autologous Tier 2 CAP256_SU virus. Most encouragingly, when compared to Env alone, the inclusion of Gag (Env+GagM) into DNA and MVA vaccines improved the immunogenicity of the DDMMPP regimen even further. For Env+GagM DDMMPP, more animals developed Tier 2 neutralising antibodies, and improved titres, whereas Tier 2 neutralisation in general started to develop after fewer vaccinations, as for most rabbits this was observed after the second MVA inoculation. In an attempt to improve the spike density of Env on VLPs and the plasma membrane, two Env chimaeras were made replacing parts of gp41 with the corresponding elements of influenza A H5 haemagglutinin (HA2) (Env:HA2 chimaeras). Increased Env spike density was observed in a previous study using this strategy for the gp41 transmembrane domain and cytoplasmic tail (gp140HA2tr). A similar construct was generated here for CAP256_SU and a second chimaera was included replacing the whole of gp41 with HA2 (gp120HA2). Surprisingly, in experiments where VLPs were purified from OptiPrep gradients or the whole-cell bnAb FACS assay conducted with these Env:HA2 chimaeras, there was no evidence of increased spike density on VLPs or the plasma membrane as compared to Env. Furthermore, the folding of Env was severely impacted, especially regarding gp120HA2 where no binding of PG16, PGT145 and CAP256 VRC26.08 - bnAbs recognising native-like Env trimers - was observed. Although results for gp140HA2tr was improved over gp120HA2, in general the data for gp150 (Env) was superior in both the bnAb live-cell staining and FACS assay. Consequently, when both Env:HA2 chimaeras in combination with GagM were tested in the DDMMPP regimen, no improvement was observed with regard to autologous Tier 2 neutralisation. For rabbits receiving gp120HA2, no animals developed Tier 2 nAbs, whereas for gp140HA2tr, Tier 2 neutralisation in general developed later and to lower titres compared to Env+GagM. In conclusion, different HIV-1 DNA, recombinant MVA and protein vaccines were generated and characterised both in vitro and in vivo, leading to a vaccination regimen that induced both high titre Env binding and vaccine-matched Tier 2 neutralising antibodies in rabbits. Furthermore, a new Env sequence, the first from the South African CAPRISA cohort, has been added to the small list of Env sequences that can induce Tier 2 neutralisation.

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.

Elicitation of neutralizing antibodies (NAbs) remains a major goal in the generation of a successful vaccine against human immunodeficiency virus type 1 (HIV-1). One of the major obstacles in generating such NAbs is the vast sequence diversity of their target, the HIV-1 envelope (Env) protein. This dissertation will describe multiple vaccination strategies utilizing a soluble form of Env (gp140) to address the sequence diversity of HIV-1 Env and to improve the NAb responses elicited through vaccination in the guinea pig model. A bioinformatically optimized mosaic gp140, designed as a single sequence to cover global HIV-1 sequence diversity, was utilized in combination with a clade C gp140, resulting in a complementary repertoire of NAbs when compared to vaccination with each single protein. Additionally, a quadrivalent mixture of four novel clade C gp140s with distinct antigenic properties was found to elicit a greater magnitude of NAbs when compared to any single protein in the mixture. Furthermore, longitudinal, heterologous prime/boost vaccination regimens resulted in binding antibodies to a greater number of distinct sequences within a single epitope of variable loop 2, and NAb of a greater magnitude, than a homologous prime/boost regimen. Finally, a panel of rationally designed variable loop 2 and 3 sequence modified trimers was designed. When utilized in a mixture, the variable loop 2 modified trimers elicited a greater magnitude and breadth of NAbs than the single, unmodified wild type protein alone. These data show that multivalent vaccination strategies with HIV-1 Env trimers result in improved NAb responses compared to single Env protein vaccinations and may help to guide the development of future vaccination regimens.
Medical Sciences

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

Cryptococcus sp é um fungo saprófita, cosmopolita, que causa micose sistêmica, geralmente, subaguda ou crônica, conhecida, sobretudo, por sua localização meníngea, após aquisição da infecção por via respiratória Embora seja ubíquo, a criptococose ocorre predominantemente em indivíduos imunodeficientes e podendo ocorrer, também, em indivíduos imunocompetentes. Os estudos experimentais e em humanos avaliando a ativação do sistema complemento e a produção de anticorpos específicos mostram que a resposta inata e de anticorpos são importantes para a delimitação do processo infeccioso por Cryptococcus sp, como também, a administração de anticorpos monoclonais podem induzir uma resposta eficaz na disseminação da doença. O sistema complemento contribui para a defesa do organismo contra o Cryptococcus sp de diferentes maneiras: secretando opsoninas e fatores quimiotáticos e colaborando com a ação dos anticorpos específicos, aumentando a interação entre a imunidade inata e adquirida. Os anticorpos antiglicuroxilomanana (GXM) possuem numerosas atividades biológicas: a) opsonização para fagocitose, b) ativação da via clássica do complemento resultando na deposição precoce de fragmentos de C3 no fungo, c) supressão do excesso de acúmulo de C3 pela via alternativa; d) facilitação do clareamento do GXM do soro in vivo, resultando no maior acúmulo de GXM nos tecidos ricos em células do sistema fagocítico mononuclear; e) proteção em modelos murinos da criptococose e f) facilitação de vários aspectos da imunidade celular ao Cryptococcus sp. O objetivo desse estudo foi avaliar a resposta humoral ao GXM e às proteínas da parede celular (Ag S) avaliando a atividade do sistema complemento como também a produção de anticorpos específicos em amostras séricas de adultos com e sem neurocriptococose. Foram coletadas 106 amostras de soro e divididas em 3 grupos: grupo 1- 21 indivíduos com neurocriptococose e baixa exposição a levedura, grupo 2- foi composto por 23 indivíduos saudáveis com alta exposição ao fungo e HIV negativos, granjeiros da cidade de Jumirim localizada a 164 km de São Paulo, na região de Sorocaba e, o grupo 3- 60 indivíduos saudáveis, HIV negativos e com baixa exposição ao Cryptococcus sp. Dois pacientes foram excluídos do estudo por apresentarem tumores (timona e câncer de pulmão). O sistema complemento foi avaliado por ensaio hemolítico (CH 50 e AP 50) e, a dosagem da proteína ligadora de manose (MBL) foi feita por ELISA. Os valores de CH 50 estiveram dentro da normalidade em 17/21, 13/23, 59/60 indivíduos dos grupos 1, 2 e 3 respectivamente. A média dos valores de CH 50 foi diferente significativamente entre o três grupos (P < 0,0001). O grupo 2 mostrou níveis reduzidos significantes em comparação aos dois outros grupos. Os valores de AP 50 estiveram dentro da normalidade em 11/21; 21/23 e 60/60 indivíduos dos grupos 1, 2 e 3 respectivamente. Houve diferença nos valores de AP 50 (P = 0,0005) e apenas um paciente do grupo 1 apresentou valores indetectáveis desta via. Houve diferença significante na dosagem de MBL entre os três grupos (P = 0,0277). Anticorpos IgG anti-GXM foram quantificados por ELISA e expressos por densidade óptica (DO). IgG anti GXM foi detectado em todos os grupos com diferença significante entre eles (P= 0,0127). As médias de IgG anti- GXM (DO) foram: 1.191 (0,49 a 1.217) no grupo 1, 1.572 (0,815 a 2.479) no grupo 2 e 0,965 (0,321 a 1.295) no grupo 3. Dois indivíduos assintomáticos do grupo 2 tiveram títulos de GXM detectáveis (1/256 e 1/32). Quatro pacientes com neurocriptococose faleceram (19%) e seus resultados mostravam: CH 50 normal, 2/4 tinham valores de AP 50 baixo (12 UI/mL) e indetectável; 3/4 tinham altos níveis de MBL e apenas um tinha baixa DO de IgG anti-GXM. Baseado em nosso estudo, podemos concluir que a resposta humoral (sistema complemento e anticorpos) não é suficiente para explicar a susceptibilidade a neurocriptococose, porém a alta e constante exposição ao Cryptococcus sp pode prevenir o desenvolvimento de doença, ou seja, a constante e intensa exposição ao fungo induz a produção de anticorpos que previnem a doença clínica mas não a infecção. Por outro lado fatores genéticos que determinam as concentrações de MBL podem influenciar na susceptibilidade a neurocriptococose. Os anticorpos contribuem para o clearence de GXM, entretanto as concentrações séricas não se correlacionam com resistência à doença
Cryptococcus sp is a fungal pathogen with a worldwide distribution. Although it is ubiquitous in the environment, cryptococcal disease occurs predominantly in immunocompromised hosts and can also occur in apparently immunocompetent individuals. The innate immunity is of special relevance for the antifungal reaction, as it allows an immediate reaction and recognizes a broad variety of fungal pathogens. The host immune response is a major determinant of the outcome of cryptococcal infection; however, the antibodies response is poorly understood. In addition, most of the studies are experimental and there is restricted knowledge concerning the human immune response. Complement system has soluble factors, restrictive regulator proteins and cellular receptors involved in defense mechanism. Glucuroxylomannan (GXM) monoclonal antibodies (MAbs) have numerous biological activities: a) opsonization for phagocytosis, b) activation of the classical complement pathway leading to early deposition of C3 fragments on the yeast, c) suppression overall accumulation of C3 via the alternative pathway; d) clearance facilitation of GXM from serum in vivo, leading to increased accumulation of GXM in tissues rich in mononuclear phagocyte system; e) protection in murine models of cryptococcosis and f) facilitation of various aspects of cellular immunity to Cryptococcus sp. The goal of our study was to evaluate if the antibody response to GXM and cell wall proteins regarding specific antibodies as well as complement system in sera of immunocompetent adults with and without neurocryptococcosis. The aim of our research was to evaluate classical and alternative complement system pathway, to quantify mannose-binding lectin (MBL) as well antibody response to GXM and cell wall proteins (AgS) regarding specific antibodies in sera of immunocompetent adults with and without neurocryptococcosis. One hundred and six samples were collected and classified in 3 groups: group 1- 21 individuals with neurocryptococcosis and low exposure to the yeast; group 2- was composed by 23 healthy individuals, chicken farmings from Jurumirim, a town 164 km to São Paulo, and with high exposure to Cryptoccocus spp and HIV negative. The third group included 60 healthy HIV negative individuals with presumed low exposure to Cryptococcus. Two patients were excluded by report of previous malignancies (timoma and pulmonary cancer). The complement system was evaluated by hemolytic assay and ELISA to MBL. CH 50 and AP 50 values were within the normal range in 17/21; 13/23; 59/60 patients in groups 1, 2 and 3 respectivelly. Mean CH 50 values were significantly different among the three groups (P < 0,0001). Group 2 showed significantly reduced levels in comparison with groups 1 and 3. AP 50 values were within the normal range in 11/21; 21/23; 60/60 patients in groups 1, 2 and 3 respectivelly. There was difference in the AP 50 values (P=0,0005) and one no activation of this pathway in group 1. There was significant difference in MBL among the groups (P = 0,0277). GXM antibodies IgG was measured by ELISA and expressed as optical density (OD). GXM- IgG was detected in all the groups with significant difference among them (P = 0,0127). The means of IgG anti-GXM (OD) were: 1.191 (range 0,49 to 1.217) in group 1, 1.572 (range 0,815 to 2.479) in group 2 and 0,965 (range 0,321 to 1.295) in the group 3. Two of the group 2 individuals had low GXM titers (1/256 and 1/32) and no symptoms. Four patients (4/21; 19%) with neurocryptococcosis died and the results showed: normal classical pathway activation, 2/4 had low (12 UI/mL) or undetectable alternative pathway values ; 3/4 had high MBL concentrations and only one had low OD for IgG anti-GXM. In conclusion, our results suggest that constant and high exposure to Cryptococcus sp can prevent the development of cryptococcosis, i.e. constant and intensive fungal exposition induces protective antibodies to clinical disease but not to the infection. In the other side, genetic factors which determine MBL concentrations could influence the susceptibility to neurocryptococcosis. The antibodies contribute to GXM clearance, however, the concentrations did not correlate with the resistance to the disease

Antibody-dependent enhancement (ADE) is the process by which virus-specific antibodies enhance entry or replication of a virus, leading to increased infection and the potential to exacerbate disease progression or severity. In terms of HIV infection, ADE could make the difference between an ineffective vaccine and a dangerous one. There is also the possibility that ADE affects disease progression in natural infection. Here, one aspect of ADE, complement-mediated ADE (C'-ADE), was investigated in detail. An assay was developed to study C'-ADE in both R5- and X4-tropic HIV-1 strains in the context of early, primary infection and vaccination. A CD4+ T cell line was used for these studies which expressed the complement receptor 2 (CR2) and HIV-1 co-receptors CXCR4 and CCR5. C'-ADE in primary infection was investigated using serum samples collected longitudinally from infected individuals from as early as 12 days post onset of primary symptoms. When tested against autologous viruses isolated early after infection, C'-ADE was detected in 9 out of 10 patients. In some cases this was potent enough to produce increases in infection greater than 100-fold. Later virus isolates that evolved to escape antibody neutralisation were enhanced by sera that neutralised early virus. Competition studies carried out with neutralising and enhancing IgG showed neutralisation to be the dominant activity. Enhancing, but not neutralising, activity of patient sera was detected against heterologous primary isolates. Post-vaccination C'-ADE was investigated using sera from volunteers vaccinated with a monomeric gpl20 vaccine from a dual-tropic HIV-1 strain. Sera from vaccinees enhanced infection of the vaccine virus strain but did not neutralise it. Neutralisation was seen for an X4 virus strain, MN, and individuals that produced the most potent neutralisation against MN also produced the most potent enhancement of the vaccine strain. CR2-mediated increased attachment of opsonised viruses to the target cell was shown to be the principle mechanism of C'-ADE. The use of a CR2 cytoplasmic tail mutant showed that receptor signalling was not necessary for C'-ADE. Results from these studies show that antibodies that appear to be non-neutralising in neutralisation assay systems can actually have dramatic effects on virus infection in a different context. These new findings are considered in relation to existing knowledge on neutralisation and ADE of HIV.

Germinal centres (GCs) are the sites where V-gene hypermutation and B cell selection are taking place. Testing specificity and affinity of GC B cell receptor by interaction with antigen on follicular dendritic cells (FDCs) may be an important selection process to select high affinity B cell clone. As antigen on FDC is present in the form of antigen-antibody immune complex, GC B cells are expected to have to compete with antibody to get access antigen. Initially this antibody will be of low affinity. However, during the course of an immune response, this affinity may increase. We have tested this competitive selection model by following the replacement of antibodies in the GC over the course of an immune response. The speed of this replacement is dependent on affinity. Antibody added during an ongoing GC reaction can replace antibody in the GC, but only, if it is of high enough affinity. Presence of high or low affinity antibodies on FDC influences centrocyte selection, leading to variations in apoptosis within the GC, serum affinity, and plasma cell output. Parallel in silico experiments support the idea that a dynamic GC selection threshold, dependent on the affinity of GC output cells increases affinity maturation, because it enhances selection efficiency over a longer period during the course of a GC reaction. A dynamic selection threshold may explain the termination of the GC reaction, when affinity of new B cell variants is not sufficient to overcome the affinity of antibodies produced outside the GC. IRF4 is essential for the plasma cell differentiation and Ig class switch. IRF4 mRNA and protein rapidly upregulate within one hour after naive B cells get stimulation with NP-Ficoll in QM×C57BL/6 mice, and then activated B blasts expressing intermediate level of IRF4 either go into the red pulp to form the early extrafollicular response by upregulating high level of IRF4, or travel into the follicle to differentiate into GC founding cells. IRF4 completely shuts down when cells becomes proliferating centrocytes. But IRF4 expresses again in centorcytes, which have been committed to differentiate into the plasmablasts. Its high level expression shows the GC emigrants. Here IRF4 is selected as the marker for the early plasmablats appearance on the GC-T zone interface at the beginning of the GC reaction. And further experiments by using cytokines such as IL-6, IL-10, IL-21, costimulatory signals OX40, CD30 deficient mice show that these signals can affect the development of these early IRF4\(^+\) plasmablasts on the GC-T zone interface in the TD antigen response.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2019
Cataloged from PDF version of thesis. "The pagination in this thesis reflects how it was delivered to the Institute Archives and Special Collections. The Table of Contents does not accurately represent the page numbering"--Disclaimer Notice page.
Includes bibliographical references (pages 95-102).
One of the barriers to rational vaccine design against evolving pathogens is our lack of mechanistic understanding of how innate and adaptive immune response systematically emerge and evolve. Immune response is comprised of dynamic events that require many components to cooperate collectively in a manner that spans a range of scales. These characteristics make it hard to predict mechanisms for immune response based solely on experimental observations. This thesis investigates various aspects of affinity maturation that are relevant to vaccination and therapeutic strategies but are not yet fully understood mechanistically, ranging from the evolution of the heterogeneity of the antibody population with respect to affinity to optimal design parameters for temporal dosing of vaccines. Our approach is to apply computational techniques to mathematically model the immune system, and being synergistic with complementary experiments. 1.
As affinity maturation ensues, average affinity of antibodies increase with time while resulting affinity distribution becomes increasingly heterogeneous. To shed light on how the extent of this heterogeneity evolves with time during affinity maturation, we have taken advantage of previously published data of antibodies isolated from individual serum samples. Using the ratio of the strongest to the weakest binding subsets as a metric of heterogeneity (or affinity inequality), we find that after a single injection of small antigen doses, the ratio decreases progressively over time. This is consistent with Darwinian evolution in the strong selection limit. By contrast, neither the average affinity nor the heterogeneity evolves much with time for high doses of antigen, as competition between clones of the same affinity is minimal. 2.
What are the aspects of affinity maturation being altered by various temporal patterns of antigen dosing? Certain extended-duration dosing profiles increase the strength of the humoral response, with exponentially-increasing(EI) dosage providing the greatest enhancement. While this is an exciting result, it is necessary to establish a mechanistic understanding of how immune response be enhanced to further engineer and optimize the temporal patterns. From our computational model, the effect is driven by enhanced capture of antigen in lymph nodes by evolving higher-affinity antibodies early in the GC response. We validate the prediction from independent experimental data, where EI dosage result in promoted capture and retention of the antigen in lymph nodes. To our knowledge, this work is the first to demonstrate a key mechanism for vaccine kinetics in the response of B cells to immunization, and may prove to be an effective method for increasing the efficacy of subunit vaccines. 3.
Are there optimal dosing profiles that maximize total protection? That is, lead to the evolution of the most antibodies of high affinity? In extension of mechanistic studies in 2, we propose a stochastic simulation method that can be used as a tool for optimizing dosage protocols for vaccine delivery. Using this tool, we analyze experimental conditions for EI dosage induce suboptimal immune response and investigate two approaches for the optimization. Specifically, reducing the total dosage optimizes affinity of resulting antibodies, while total protection is optimal neither at constant or EI dosage but that corresponding to a "linear-like" dosing profile. Our approach can be extended to broader applications in vaccine design.
by Myungsun (Sunny) Kang.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering

Background: HIV rapid antibody assays are important for screening children aged <18 months for HIV exposure and children ≥ 18 months for HIV infection. Limited available data indicate variable performance of different HIV rapid tests in comparison to laboratory HIV antibody assays. The aim of this study was to evaluate the diagnostic accuracy of 6 HIV rapid tests currently used in South Africa for screening children using whole blood. Methods: A prospective descriptive cross-sectional laboratory study was conducted at two paediatric healthcare facilities in South Africa. Sensitivity and specificity analyses and positive and negative likelihood ratios were performed. The reference standard was the laboratory HIV enzyme-linked immunosorbent assay (ELISA) test and HIV polymerase chain reaction (PCR) test. Results: Blood samples from 1159 children (896 <18 months of age) with valid HIV ELISA test results were included in the analysis. A total of 5768 HIV rapid tests (4446 in children <18 months of age) were performed. Sensitivity of HIV rapid tests for detecting HIV exposure among children <18 months of age ranged from 38.7% to 94.7%. Four HIV rapid tests attained specificity in excluding HIV exposure among children <18 months of age of >98%. Seroreversion rates were lowest with the Determine rapid test. Three HIV rapid tests (Abon, Advanced Quality, Determine) detected 100% of HIV-infected children <18 months of age, the Reveal, SD Bioline and Insti rapid tests missed 27 (41.5%), 1 (4.5%) and 1 (1.5%) of the HIV-infected children respectively. In children ≥ 18 months of age, sensitivity of rapid tests for detecting HIV infection ranged from 69.2% to 100% and specificity of all rapid tests was 100%. Conclusions: None of the 6 HIV rapid tests evaluated achieved both the World Health Organisation recommended sensitivity and specificity standards for any antibody assay used in screening for HIV exposure in children <18 months. The Determine test showed the best overall diagnostic accuracy and is therefore recommended as the preferred screening test for children. Recommendations: on the use of specific HIV rapid tests in infants and young children should be based on evaluation of their performance in the population to be tested.

In an attempt to more fully understand the generation of antibody diversity to carbohydrate antigens, we produced and characterized a panel of hybridoma cell lines specific for group A streptococcal carbohydrate from mice injected with the intact bacteria (minus the hyaluronic acid capsule and cell wall protein antigens). We have analyzed the use of heavy and light chain variable region genes in the early (day 7) and late response (hyperimmune) and have determined the nucleotide sequence of the dominant VH gene used in several of our hybridomas. Our data allowed us to assess the extent to which the recombination of various V, D, and J gene segments and somatic mutation contribute to antibody diversification in this system. In this report we confirm that a minimum of two VH and four VK gene segments are used to encode this response. We extend this analysis to show that multiple D and J gene segments are used and that a significant amount of junctional variability is tolerated in CDR 3. Our results also suggest that there is a positive selection for somatic mutation in CDR 1 during the hyperimmune response to group A streptococcal carbohydrate.

The purpose of this study was to assess which particular circumstances of HIV antibody testing are most important to Ball State University students when making the decision whether or not to be tested. This study also looked at psychological variables within the individual that may influence one's decision whether or not to be tested. A descriptive correlational study design was used. Subjects were recruited from the psychological science subject pool. These students were enrolled in the Psychology 100 class at Ball State University during the Spring, 1992 Semester. Subjects were also recruited from sororities, fraternities, and business fraternities. A total of 397 subjects (210 males and 187 females) were recruited for the study.Subjects filled out four surveys: an HIV Antibody Testing Inventory, an AIDS Knowledge Survey, the MultiDimensional Health Locus of Control Scale, and the Social Desirability Scale. Results indicated students in this sample preferred going off campus for HIV testing versus on campus. They preferred a medical setting with a medical counselor doing the testing. These subjects did not want peers doing the HIV testing or counseling. The level of AIDS Knowledge subject had did not correlate with their stated likelihood of being tested for HIV. Subjects preferred anonymous testing, but appeared to recognize the benefits of recording basic demographic information.
Institute for Wellness

Generation of neutralising antibodies with broad specificity would be one of the effective approaches to control HIV-1 spread. It is clear that a method that allows rapid generation of neutralising antibodies is needed. This project aims at developing a novel approach to rapidly access human anti-HIV-1 antibodies in vitro by using ribosome display and selection from DNA libraries of HIV-1 patients. Two single-chain antibody libraries (M325 and K530) were constructed from two HIV-1 long-term non-progressors, whose sera showed cross-neutralising activities against various HIV-1 strains across a range of clades. In each library, total RNA was extracted from blood of each donor and used to synthesise cDNA. Families of 4 κ light chains, 9 λ light chains and 8 heavy chains were generated by using RT-PCR amplification. These fragments were then assembled with all possible combinatorial pairs to form diversified repertories in the form of VL-link-VH-partial CH. Both libraries were subjected to ribosome display for in vitro selection of functional antibodies. Ribosome display is a cell-free technique used to generate proteins that can bind to an immobilised antigen. During this process, the translated proteins are associated with their mRNAs, enabling a simultaneous selection of functional proteins and their gene. The employment of ribosome display facilitated rapid screening of two large libraries against recombinant gp120 (generated from patient K530).

Introduction. The overarching goal of this study is to determine whether a long-lived, transgenic helminth parasite might express a potentially therapeutic antibody, using HIV-1 as the model target of a broadly neutralizing antibody expressed and secreted by schistosomes.

Methods. Cultured schistosomes were transfected by square wave electroporation with a plasmid encoding a human immunoglobulin G1 that is broadly neutralizing for HIV-1.

Results. Following introduction of an expression plasmid into cultured schistosomes by square wave electroporation, and extraction of total RNA and soluble lysates of the parasites, transcripts encoding both the light and heavy chains of the anti-HIV-1 broadly neutralizing antibody b12 and fragments of the human IgG1 antibody b12 were detected by reverse transcription PCR and western blot analysis, respectively.

Conclusions. These findings revealed that a human antibody, which is known to be broadly neutralizing for HIV-1, was expressed in schistosomes. Whereas this investigation is a work in progress, the findings thus far provide impetus to explore whether schistosomes transgenic with the gene encoding b12 might be harnessed to inhibit HIV-1 infection in vivo.

HIV-1 has evolved a number of strategies in response to current anti-retroviral drugs and the selection pressure of humoral and cellular immunity. In particular, R5 viral strains that are essential for AIDS pathogenesis are very resistant to neutralization by antibodies. Therefore, the aim of this thesis was to develop synthetic nucleic acid ligands, aptamers, against gp120 of an R5 strain of HIV-1, with a view of using aptamers as novel neutralization molecules and analytical tools to study HIV-1 entry into target cells. The central hypothesis of this thesis was that aptamers by virtue of their small size and slow dissociation rates, compared to antibodies, would easily access and bind occluded gp120 neutralization sites. Using the SELEX protocol and SPR technology, I isolated 2'-Fluoro-pyrimidine-RNA aptamers against HIV-l_Ba-L monomeric gp120. Most of these aptamers not only bound gp120 with high affinities but also neutralized R5 primary isolates in human PBMC by 1,000 to 100,000-fold, truly unprecedented when compared with natural ligands such as antibodies. Some aptamers, like B4, defined a conserved site of gp120 that could not mutate to escape neutralization following stringent selection, in vitro, for breakthrough virus. This was consistent with subsequent findings that B4 aptatope (binding site) overlaps a poorly immunogenic but highly conserved CD4-induced epitope as determined by competition with 17b and 48d mAbs that map to this neutralization epitope on the gp120. This study was thus the first of its kind to describe neutralization of HIV-1 primary isolates by a ligand against the CD4-induced epitope. Most intriguing, although B4 potently neutralized HIV-1_Ba-L infection in PBMC, which is a mixed T cell and macrophage population, it modestly neutralized infection of the same virus in a purified culture of macrophages. These findings are intriguing in that they suggest that aptamers could be used to dissect unique sites on the virus that interact with target cell surface in ways that have not been revealed heretofore, and would help understand better HIV-1 entry pathways, especially in macrophages. Thus neutralizing aptamers such as these could be exploited to provide leads in developing alternative anti-HIV-1 drugs and a deeper understanding of the molecular interactions between the virus and its host cell.

Protection against the highly contagious foot and mouth disease virus (FMDV) coincides with neutralising antibody titres. Infection in cattle is characterised by 100% morbidity of an acute vesicular disease whilst infection of their closest relative, the African buffalo, is sub-clinical despite having diverged only 5.7 – 9.3 million years ago (Glanzmann et al., 2016; 1). The germline and antibody repertoire in African buffalo has not previously been characterised and so the cause of their differential disease response may be the production of a more specific and / or avid antibody response to FMDV than cattle. The cattle and African buffalo antibody germline was sought to characterise the recombinatorial potential of the antibody loci and their subsequent primary antibody repertoire. Expression of the antibody heavy chain (IGH) and antibody lambda light chain (IGL) was investigated with qPCR and RNA-seq. The antibody repertoire in response to FMDV infection was interrogated in African buffalo infected with SAT1 FMDV and compared to the cattle IGH repertoire inoculated with highly purified SAT1 FMDV antigen. The recombinatorial potential of the cattle and African buffalo IGH and IGL is severely limited compared to other species such as mice and human. The characterisation of the cattle IGH and IGL is the most accurate to date and reveals internal duplications of the IGH, disrupting the expected IGHV-IGHD-IGHJ-IGHC ordering seen in mammalian immune loci and resulting in four IGHD regions, containing long and ultra-long IGHD. These IGHD provide a novel diversification mechanism that can compensate for limited germline diversity by forming long and ultra-long CDR3H loops that are highly diverse in their length and amino acid composition. The African buffalo antibody repertoire also forms highly diverse long and ultra-long CDR3H, despite lack of evidence for the existence of the duplications in their IGH. Limited variability is seen in the length and amino acid composition of the IGL in both species, suggesting they are playing a structural role to support these unusual long and ultra-long CDR3H. In response to FMDV infection in African buffalo, a dramatic increase in specific long and ultra-long CDR3H sequence abundance occurs but this change in frequency of specific transcripts is absent in cattle. The differential antibody response may account for the protection of African buffalo against FMD.

Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the functional envelope spike, which consists of trimers of gp120 bound to gp41. Most variants of HIV-1 enter cells through attachment of the envelope spike to the main cellular receptor CD4, allowing interaction with a co-receptor and eventually fusion of viral and cellular membranes. Neutralising antibodies inhibit HIV-1 entry by targeting epitopes on the functional spike. HIV-1 has, however, evolved several ways to evade recognition by antibodies, including variable regions, carbohydrates, and conformational masking. As a result, the neutralising antibody response in HIV-1 infection and post-immunisation is generally narrow, and only a handful of broadly neutralising monoclonal antibodies have been reported. In this thesis, the isolation and characterisation of novel, broadly neutralising antibody fragments derived from llamas is described. Llamas produce antibodies devoid of light chains, which have their antigen-binding properties confined to a single fragment, the VHH, and a preference for cleftrecognition. VHH were isolated from llamas immunised with recombinant gp120 using phage display-based methods. In order increase the chances of isolating neutralising VHH, a functional selection strategy was employed, involving a competitive elution with soluble CD4. Three VHH able to neutralise HIV-1 primary isolates of subtype B and C were characterised. These VHH bound to gp120 with high affinities and competed with soluble CD4 and antibodies to the CD4-binding site for this binding, indicating that their mechanism of neutralisation involves interacting with the functional envelope spike prior to binding to CD4. These results indicate that llama VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for HIV-1 microbicide development. Anti-gp120 VHH might also prove useful in defining neutralising and non-neutralising epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design.

The prevalence of HIV-1 is highest in Sub-Saharan Africa. Protective immune responses directed against HIV are complex and involve both cellular and humoral immunity. Based on the recent finding that the best correlate of protection against the first protective prophylactic RV144 vaccine were HIV-specific antibody responses, including those mediating natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC), there has been considerable interest in measuring alternative roles for HIV-specific binding antibodies. The aim of this MSc dissertation was to optimise and compare two high-throughput flow cytometry based approaches - the GranToxilux and PanToxilux assays - to measure HIV-specific ADCC responses. To do this, NK cells from a panel of healthy HIV-negative individuals were screened for their ability to directly kill the tumour cell line K562, as a measure of direct NK cell cytotoxicity. The individual with the highest granzyme B and caspase activity against K562 cells was chosen as the universal NK cell donor for this study.