One potential approach to vaccine development against HIV involves generating an immunogen that can elicit the production of broadly neutralizing monoclonal antibodies (bnmAbs), which target specific sites on the HIV-1 envelope. Using site-directed mutagenesis and ELISA assays, this thesis investigates the idea of a secondary binding site of one of the bnmAbs, 2F5, as suggested by previous studies that identified residues Asp64, Thr65, and Arg82B on 2F5 that are recognized by its anti-idiotypic antibody 3H6. Results show that 2F5 binds only very weakly to the gp41 ectodomain in its post-fusion conformation. However, a small but significant difference was observed between the binding of the mutants and the T-20 peptide, a fusion inhibiting drug. Due to the limited effect, the results need to be confirmed using more quantitative techniques and more optimal conformations of the antigen, but raise the prospect that design of immunogens to elicit HIV-specific antibodies might have to incorporate this novel interaction site.

One potential approach to vaccine development against HIV involves generating an immunogen that can elicit the production of broadly neutralizing monoclonal antibodies (bnmAbs), which target specific sites on the HIV-1 envelope. Using site-directed mutagenesis and ELISA assays, this thesis investigates the idea of a secondary binding site of one of the bnmAbs, 2F5, as suggested by previous studies that identified residues Asp64, Thr65, and Arg82B on 2F5 that are recognized by its anti-idiotypic antibody 3H6. Results show that 2F5 binds only very weakly to the gp41 ectodomain in its post-fusion conformation. However, a small but significant difference was observed between the binding of the mutants and the T-20 peptide, a fusion inhibiting drug. Due to the limited effect, the results need to be confirmed using more quantitative techniques and more optimal conformations of the antigen, but raise the prospect that design of immunogens to elicit HIV-specific antibodies might have to incorporate this novel interaction site.

T follicular helper cells (T-FH) are a specialized subset of CD4 T cells that reside in B cell follicles and promote B cell maturation into plasma cells and long-lived memory B cells. During chronic infection prior to the development of AIDS, HIV-1 (HIV) replication is largely concentrated in T-FH. Paradoxically, T-FH numbers are increased in early and midstages of disease, thereby promoting HIV replication and disease progression. Despite increased T-FH numbers, numerous defects in humoral immunity are detected in HIV-infected individuals, including dysregulation of B cell maturation, impaired somatic hypermutation, and low quality of antibody production despite hypergammaglobulinemia. Clinically, these defects are manifested by increased vulnerability to bacterial infections and impaired vaccine responses, neither of which is fully reversed by antiretroviral therapy (ART). Deficits in T-FH function, including reduced HIV-specific IL-21 production and low levels of co-stimulatory receptor expression, have been linked to these immune impairments. Impairments in T-FH likely contribute as well to the ability of HIV to persist and evade humoral immunity, particularly the inability to develop broadly neutralizing antibodies. In addition to direct infection of T-FH, other mechanisms that have been linked to T-FH deficits in HIV infection include upregulation of PD-L1 on germinal center B cells and augmented follicular regulatory T cell responses. Challenges to development of strategies to enhance T-FH function in HIV infection include lack of an established phenotype for memory T-FH as well as limited understanding of the relationship between peripheral T-FH and lymphoid tissue T-FH. Interventions to augment T-FH function in HIV-infected individuals could enhance immune reconstitution during ART and potentially augment cure strategies.

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

The HIV-1 envelope glycoprotein, Env, facilitates the translocation of the viral capsid across the cellular membrane. Env is a trimer of hetero-dimers composed of a gp120 subunit and gp41 transmembrane protein. The gp120 subunit binds the primary receptor, CD4, leading to conformational changes of Env that then promote binding to the coreceptor, principally CCR5 or CXCR4. As the sole protein on the surface of the virion, Env is under continuous pressure from the host's antibody response. Two classes of antibodies target the highly conserved receptor-binding sites of gp120: CD4-binding site (CD4bs) and CD4-induced (CD4i) antibodies.

HIV-1 primarily utilizes the CCR5 receptor as a co-receptor, but over time, viruses can evolve to use the CXCR4 protein. Changes in the viral envelope V3 loop mediate this switch. The emergence of CXCR4-utilizing viruses has been presumed to occur as a consequence of decreased humoral immunity. We show that exclusively CXCR4-using (X4) viruses contain a 2 to 3 amino acid insertion in the V3 loop. Structural modeling revealed that this insertion caused a protrusion in the V3 loop, which impacts CCR5 receptor interaction. These genotypic and structural motifs affected neutralization susceptibility because X4, as compared to co-circulating CCR5-utilizing (R5) viruses, were less neutralization sensitive to autologous contemporaneous and heterologous plasma. Individuals with co-circulating X4 and R5, as compared to those with only R5, viruses had similar neutralization breadth and potency indicating that the emergence of X4 viruses is not associated with decreased humoral immunity. These results suggest that X4 viruses are neutralization escape variants and arise due to humoral selective pressure. This work has implications for future antibody-based therapeutics. Along with providing a framework for developing an HIV-1 vaccine, broadly neutralizing antibodies (bnAbs) are also being investigated as a potential therapeutic. BnAbs target a limited number of conserved HIV-1 envelope structures, including glycans in and around the V1/V2 and V3 domains. Along with the V3 loop, changes in V1/V2 are also known to impact co-receptor usage. We show that viruses that exclusively use the CXCR4 co-receptor, as compared to variants that only utilize CCR5, were less neutralization sensitive to V1/V2 and V3 directed bnAbs. In contrast, R5 and X4 viruses did not demonstrate neutralization differences to bnAbs that target non-V1/V2 and V3 envelope regions, such as the CD4 binding site and the membrane proximal external region. Structural modeling revealed that the predicted orientation of the V1/V2 loop among diverse HIV-1 variants predicts susceptibility to V3 loop directed bnAbs. In aggregate, our results suggest that viruses with different co-receptor usage have differing bnAb susceptibility. Furthermore, structural modeling may be used as a tool to predict neutralization susceptibility to bnAbs against regions associated with co-receptor usage. / 2020-06-12T00:00:00Z

All challenges associated with influenza A viruses including antigenic variation in hemagglutinin (HA) and neuraminidase (NA), the evolving drug resistance and the drawbacks of current vaccines hinder our ability to control this constant threat. Furthermore, gene reassortment as well as the direct transmission of highly pathogenic avian viruses to humans can result in an occasional emergence of novel influenza strains with devastating pandemic potential. Therefore, it is crucial to investigate alternative approaches to better control these viruses and to develop new prophylactic and treatment options. Targeting highly conserved epitopes or antigens among the different subtypes of influenza A virus could offer protection against broad range of influenza viruses, including emerging strains. In my research, I have investigated the potential of broadly neutralizing antibodies against HA and conducted mechanistic study of a prototype vaccine based on the highly conserved nucleoprotein (NP). We recently found that the 14 amino acids of the amino-terminus of the fusion peptide of influenza HA2 subunit is the only universally conserved sequence in all HA subtypes of influenza A and the two lineages of influenza B viruses. Here, I show that universal antibodies targeting this linear sequence in the viral HA (Uni-1 antibodies) can cross-neutralize multiple subtypes of influenza A virus by inhibiting the pH-dependant fusion of viral and cellular membranes. It is noted that the influenza NP is a highly conserved antigen and has the potential to induce heterosubtypic immunity against divergent subtypes of influenza A virus. However, NP-based vaccination only affords weak protective immunity compared to HA. This is mostly due to the non-sterilizing immunity induced by NP. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, I show that single immunization with recombinant adenovirus carrying a fused gene encoding the secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced both B-cell and T-cell responses and augmented the role of both NP-specific antibodies and CTLs in protection. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent. The rapid evolution of the influenza A viruses necessitates the development of new alternatives to contain this medically important pathogen. The results of these studies could significantly contribute to future vaccine development and avert the necessity of yearly vaccine updates.

All challenges associated with influenza A viruses including antigenic variation in hemagglutinin (HA) and neuraminidase (NA), the evolving drug resistance and the drawbacks of current vaccines hinder our ability to control this constant threat. Furthermore, gene reassortment as well as the direct transmission of highly pathogenic avian viruses to humans can result in an occasional emergence of novel influenza strains with devastating pandemic potential. Therefore, it is crucial to investigate alternative approaches to better control these viruses and to develop new prophylactic and treatment options. Targeting highly conserved epitopes or antigens among the different subtypes of influenza A virus could offer protection against broad range of influenza viruses, including emerging strains. In my research, I have investigated the potential of broadly neutralizing antibodies against HA and conducted mechanistic study of a prototype vaccine based on the highly conserved nucleoprotein (NP). We recently found that the 14 amino acids of the amino-terminus of the fusion peptide of influenza HA2 subunit is the only universally conserved sequence in all HA subtypes of influenza A and the two lineages of influenza B viruses. Here, I show that universal antibodies targeting this linear sequence in the viral HA (Uni-1 antibodies) can cross-neutralize multiple subtypes of influenza A virus by inhibiting the pH-dependant fusion of viral and cellular membranes. It is noted that the influenza NP is a highly conserved antigen and has the potential to induce heterosubtypic immunity against divergent subtypes of influenza A virus. However, NP-based vaccination only affords weak protective immunity compared to HA. This is mostly due to the non-sterilizing immunity induced by NP. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, I show that single immunization with recombinant adenovirus carrying a fused gene encoding the secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced both B-cell and T-cell responses and augmented the role of both NP-specific antibodies and CTLs in protection. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent. The rapid evolution of the influenza A viruses necessitates the development of new alternatives to contain this medically important pathogen. The results of these studies could significantly contribute to future vaccine development and avert the necessity of yearly vaccine updates.

Le VIH est la cause de la pandémie de SIDA depuis les années 1980. Avec plus d’un million de nouvelles infections chaque année, un vaccin prophylactique est indispensable pour bloquer de façon définitive la propagation du virus. Parmi les stratégies vaccinales, l’induction d’anticorps neutralisants à large spectre est une des plus prometteuses, car ceux-ci pourraient protéger contre l’infection par la grande diversité génétique des souches de VIH circulantes dans le monde. A ce jour, aucun immunogène n’a permis l’induction de tels anticorps, mais ils ont été isolés à partir de personnes infectées par le VIH. En effet, une faible fraction d’individus infectés développe des anticorps neutralisants à large spectre qui ciblent des régions vulnérables et conservées de la glycoprotéine d’enveloppe. La région du patch riche en mannose, centrée autour du glycane en position N332 de la gp120, est la plus fréquemment ciblée, et est à cet égard attractive d’un point de vue vaccinal.Afin de mieux comprendre comment se développent les anticorps ciblant le patch riche en mannose, nous avons étudié un donneur sélectionné de la cohorte du Protocole C de l’International AIDS Vaccine Initiative, et ayant une activité neutralisante sérique exceptionnelle. Nous avons isolé, à partir des cellules sanguines de cet individu, deux lignées d’anticorps ciblant la région N332, que nous avons caractérisées pour leur activité neutralisante et dont nous avons cartographié l’épitope. Nous avons également cartographié le paratope d’une lignée d’anticorps issue d’un autre donneur du Protocole C ciblant également la région N332. Nos résultats font apparaître la diversité de solutions adoptées pour atteindre une neutralisation à large spectre contre cette région. Les études de lignées, telles que nous l’avons entrepris, permettent d’appréhender comment la coévolution anticorps-virus conduit à la sélection d’anticorps neutralisants à large spectre. Le but ultime est d’utiliser les connaissances ainsi générées, pour mettre au point des immunogènes et des protocoles d’immunisations, visant à induire des lignées d’anticorps spécifiques et à conduire leur évolution vers la neutralisation à large spectre. / HIV has been the cause of the AIDS pandemic since the 1980s. With over a million new infections each year, a prophylactic vaccine is needed to stop the virus spread. Among vaccine strategies, the induction of broadly neutralizing antibodies is one of the most promising, as they could protect against infection by the huge genetic diversity of circulating HIV strains. To date, no immunogen has induced such antibodies, but they have been isolated from HIV infected people. Indeed, a small fraction of infected individuals eventually develops broadly neutralizing antibodies that target vulnerable and conserved sites of the envelope glycoprotein. The region of the high-mannose patch, centred around a glycan at position N332 of gp120, is the most frequently targeted, and is therefore attractive from a vaccination standpoint.In order to better understand how antibodies targeting the high-mannose patch develop, we studied a donor selected from the International AIDS Vaccine Initiative Protocol C cohort with exceptional serum neutralizing activity. We isolated two antibody lineages targeting the N332 region from this individual's blood cells, which we characterized for their neutralizing activity and mapped their epitope. We also mapped the paratope of an antibody lineage from another Protocol C donor, also targeting the N332 region. Our results show the great diversity of solutions to achieve broad neutralization against this region. Lineage studies, as we have undertaken, provide an understanding of how antibody-virus coevolution leads to the selection of broadly neutralizing antibodies. The ultimate goal is to use this knowledge to develop immunogens and immunization protocols, to induce specific antibody lineage and drive their evolution towards broad neutralization.

Seule une minorité des individus infectés par le virus de l’immunodéficience humaine (VIH) développe une réponse immunitaire capable de contrôler le virus. Chez la plupart des individus, on observe un échappement virologique et un épuisement des lymphocytes T CD8+ spécifiques du VIH. L’infection chronique non-traitée altère également les lymphocytes T CD4+ spécifiques du VIH caractérisé par l’expression accrue des récepteurs co-inhibiteurs et une signature des cellules auxiliaires T folliculaires (Tfh). La thérapie antirétrovirale (TAR) est très efficace pour supprimer durablement la charge virale dans le plasma. Néanmoins, elle ne permet pas une éradication complète du VIH car le virus persiste, intégré dans le génome des cellules réservoirs, desquelles le virus réapparaît lors de l’interruption de la thérapie. Cela démontre que l'immunité adaptive spécifiques du VIH n'est pas restaurée. Les anticorps neutralisants à large spectre (bNAbs) représentent une alternative potentielle à la TAR. En plus de la neutralisation du virus – et contrairement à la TAR – les bNAbs ne limitent pas la disponibilité de l'antigène et peuvent engager le système immunitaire. L'administration de bNAbs à des macaques rhésus induit des réponses immunitaires adaptatives associées à un contrôle prolongé de la virémie, mais cela n’a pas été établi chez l’Homme. Dans cette thèse, nous avons donc exploré la modulation des réponses des lymphocytes T spécifiques du VIH lors d'une TAR standard et d’une immunothérapie utilisant des bNAbs. Dans un premier objectif nous avons analysé la modulation persistante des réponses des lymphocytes T CD4+ spécifiques du VIH chez les individus sous TAR. Nous avons pu démontrer l'expansion persistante des Tfh spécifiques au VIH avec des caractéristiques phénotypiques et fonctionnelles les distinguant des Tfh spécifiques d’antigènes viraux comparatifs (cytomégalovirus, virus de l’hépatite B). Ces caractéristiques ont été induites au cours de l’infection chronique non-traitée, persistaient pendant la TAR et étaient associées au réservoir du VIH compétent pour la traduction. Ces données suggèrent qu’une stimulation antigénique persistante, malgré une TAR efficace, maintient des modifications immunologiques notamment au niveau des Tfh. Dans un second objectif, nous avons caractérisé les réponses T spécifiques du VIH à la suite d’un traitement utilisant des bNAbs et une interruption structurée de la TAR (IST). Des individus inclus dans une étude clinique de phase Ib ont reçu une perfusion d’une combinaison des bNAbs 10-1074 et 3BNC117 et ont démontré une suppression virale prolongée après l’IST. Chez ces participants, nous avons observé une augmentation des réponses immunitaires des lymphocytes T CD8+ et CD4+ spécifiques du VIH due à l'expansion des réponses immunitaires préexistantes et au développement de réponses ciblant de nouveaux épitopes. Cela suggère que la combinaison d’un traitement par bNAbs avec l’IST est associée au maintien de la charge virale plasmatique indétectable et à une intensification de la réponse immunitaire des lymphocytes T spécifiques du VIH. Nos travaux permettent une meilleure compréhension des réponses des lymphocytes T spécifiques du VIH au cours de la TAR et lors d’une immunothérapie. Ils peuvent contribuer au développement de stratégies thérapeutiques plus efficaces visant à contrôler la réplication virale sans la TAR. / Only a small fraction of individuals infected with the human immunodeficiency virus (HIV) develops effective immune responses able to control the virus. In most individuals, the virus escapes the antiviral immune response and HIV-specific CD8+ T cell responses become exhausted. Untreated progressive HIV infection also leads to alterations in HIV-specific CD4+ T cells. This includes increased expression of co-inhibitory receptors and skewing towards a T follicular helper cell (Tfh) signature. Antiretroviral therapy (ART) is highly effective in controlling the HIV viral load at undetectable levels in the plasma. However, ART does not represent a cure as the virus integrates into the genome of infected cells from where the virus rebounds once ART is stopped. This demonstrates that the HIV-specific T cell immunity is not restored. However, the changes that are introduced during progressive infection and that are maintained after viral suppression with ART are poorly known. Broadly neutralizing antibodies (bNAbs) represent a potential alternative to ART. In addition to virus neutralization and unlike ART, bNAbs to do not limit HIV antigen availability and can engage the immune system. bNAb administration elicited adaptive immune responses that were associated with long-lasting viral control in a simian animal model but this has not been established in HIV-infected individuals. In this thesis, we therefore proceeded to study the modulation of HIV-specific T cell responses during standard ART and after an immunotherapeutic intervention using bNAbs. The first objective was to better understand persistent modulation of HIV-specific CD4+ T cell responses in ART-treated individuals. Our results demonstrated the persistent expansion of HIV-specific Tfh cell responses with multiple phenotypic and functional features that differed from Tfh cells specific for comparative viral antigens (cytomegalovirus, hepatitis B virus). These features were induced during chronic untreated HIV infection, persisted during ART and correlated with the translation-competent HIV reservoir. This suggests that persistent HIV antigen expression, despite effective ART, maintains these altered immunological features specifically for Tfh responses. For the second objective, we characterized changes in the HIV-specific CD8+ and CD4+ T cell immunity after bNAb treatment and analytical treatment interruption (ATI). For this, we used samples obtained from participants enrolled in a clinical phase Ib study that received combined infusion of bNAbs 10-1074 and 3BNC117 and demonstrated prolonged viral suppression after ATI. In these individuals, we detected an increase of HIV-specific CD8+ and CD4+ T cell responses during ART interruption when compared to baseline. Increased T cell responses were due to both expansion of pre-existing responses and the emergence of responses to new epitopes. In contrast, HIV-specific T cell responses remained unchanged in ART-treated individuals who did not receive bNAb infusions. This suggests that bNAb treatment and ATI is associated with increased HIV-specific T cell immunity while viral suppression is maintained. Together our results contribute to a better understanding of HIV-specific T cell responses during ART and immunotherapy treatment. Our findings may help to develop more effective HIV treatment strategies to improve the host’s immune system so that HIV can be controlled without the need for ART.