Thèses sur le sujet « Broadly neutralizing antibodies (bNAbs) »

L'infection par le virus de l'immunodéficience humaine de type 1 (VIH-1) reste un problème majeur de santé publique à l'échelle mondiale, avec environ 37,7 millions de personnes vivant avec le virus et de nouvelles contaminations dépassant le million de cas par an. Des antirétroviraux efficaces permettent maintenant de traiter durablement les personnes infectées. Ces thérapies contribuent également à améliorer la prévention et à ralentir la progression de l'épidémie. Cependant, un vaccin reste nécessaire, en particulier pour contrôler l'épidémie dans les régions à faible revenu et les environnements précaires.Le rôle protecteur des anticorps neutralisants (AcN) a été démontré sans équivoque dans les modèles animaux d'infection par le VIH et chez l'homme. Par conséquent, le développement d'un vaccin visant à la production, par les cellules B, d'anticorps (Ac) capables de neutraliser la majorité des virus en circulation, à savoir des AcN à large spectre (AcNLS), pourrait être envisagé comme une réponse à la pandémie de VIH.L'étude du développement des AcNLS chez certains individus, dénommés neutraliseurs d’élite du VIH-1, fournit des informations précieuses pour la conception de tels vaccins. Jusqu'à présent, la plupart des études entreprises se sont appuyées sur le tri conventionnel de cellules B uniques par cytométrie en flux (FACS) pour isoler les AcNLS. Dans la présente étude, nous avons utilisé l'approche "Chromium Single Cell Immune Profiling" à haut débit sur cellules uniques (scRNA-seq) pour réaliser une exploration longitudinale du répertoire des cellules B chez un neutraliseur d'élite du VIH-1. Cette méthode permet d'utiliser comme appâts pour l'identification des cellules B spécifiques un nombre beaucoup plus important de glycoprotéines d’enveloppe (Env) du VIH par rapport aux approches d'isolement d'Ac basées sur le FACS, ce qui permet d'obtenir une analyse plus complète du répertoire en Ac anti-Env. En outre, cette approche fournit une multitude d'informations sur la nature des Ac spécifiques identifiés et sur les cellules B correspondantes.Notre étude a permis d'identifier la séquence de 12 130 anticorps spécifiques de la protéine Env du VIH. Des Ac de 39 lignées ont été produits et testés pour leurs capacités de neutralisation, révélant 21 lignées neutralisantes. Ces résultats démontrent la capacité de la méthode à explorer de vastes répertoires spécifiques d'antigènes à partir d'échantillons longitudinaux. L'activité neutralisante des Ac de quatre lignées récapitulait l'activité sérique du donneur, permettant de neutraliser 62,4 % d'un large panel prédictif de 126 pseudovirus. Une de ces lignées neutralisantes ciblait la région riche en mannose de la gp120. Par ailleurs, les Ac de cette lignée étaient sensibles à la présence d'un glycane en position N332. Un seul de ces Ac était responsable de la plus grande partie de cette neutralisation (51,1 %) avec une activité à faible concentration (IC50 moyenne de 91,1 ng.mL-1). Cet Ac possède un CDRH3 de 23 AA de long et 20 % d'hypermutation somatique (SMH). La lignée a montré une maturation continue sur 6,5 ans, avec des taux de SMH observés de 2,0 % à 30,6 % pour la chaîne lourde, sans insertion ou délétion.Un tri conventionnel basé sur la méthode FACS avait été utilisé précédemment pour isoler des AcNLS du même donneur. En comparaison, l'approche scRNA-seq a permis d'isoler des Ac en nombre bien supérieur. En outre, les AcN nouvellement isolés étaient globalement plus neutralisants et de plus large spectre que ceux isolés précédemment, ce qui indique la supériorité de la nouvelle méthode pour l'identification de lignées neutralisantes. Les études structurales en cours permettront d'élucider les épitopes responsables de la neutralisation observée chez ce donneur. L'ensemble de ces résultats pourrait contribuer à la conception d'approches de "vaccinologie inverse", qui représentent à l'heure actuelle un espoir pour la mise au point d'un vaccin contre le VIH
Human Immunodeficiency Virus type 1 (HIV-1) infection remains a major global health concern, with an estimated 37.7 million people living with the virus worldwide and new contaminations above a million cases yearly. Efficient anti-retroviral therapies are available, allowing a sustained relief for infected individuals. These therapeutics have also contributed to a better prevention and helped curb the epidemic, notably in high-income countries. However, a vaccine is still highly awaited for controlling this epidemic, especially in lower-income regions and precarious settings.The protective role of neutralizing antibodies (NAbs) has been unequivocally demonstrated in both animal models of HIV infection and in human settings. Consequently, the development of a B-cell-based vaccine capable of eliciting antibodies (Abs) with the ability to neutralize the majority of circulating viruses, namely broadly NAbs (bNAbs), could be foreseen as an answer to the HIV pandemic.The investigation of bNAb development in HIV-1 elite neutralizers provides valuable insights to inform the design of such vaccines. To date, most of the undertaken studies have relied on conventional single B-cell FACS sorting to isolate bNAbs. In the present study, we have used the Chromium Single Cell Immune Profiling approach to conduct a high-throughput longitudinal single-cell exploration of the B-cell repertoire in an HIV-1 elite neutralizer. Importantly, this novel method enables the use of a much greater number of HIV envelope glycoprotein (Env) baits compared to regular FACS-based Ab isolation studies, providing a more comprehensive view of the anti-Env Ab repertoire. In addition, this approach yields a wealth of information on the nature of the specific Abs identified and the corresponding B-cells.The study enabled the uncovering of the sequence of 12,130 putative HIV Env specific Abs. Antibodies from 39 lineages were produced and tested for neutralization, revealing 21 distinct neutralizing lineages. The results thus demonstrated the ability of the method to explore large antigen-specific Ab repertoires from longitudinal samples. The neutralizing activity of Abs from four neutralizing lineages together recapitulated the serum activity of the donor, achieving neutralization against 62.4 % of a large predictive panel of 126 pseudoviruses. One of these neutralizing Ab lineages was shown to target the gp120 high-mannose patch supersite with great breadth and potency; Abs from this lineage were sensitive to the presence of a glycan in position N332. A single of those Abs achieved most of the neutralization breadth (51.1 %) with a high potency (mean IC50 of 91.1 ng.mL-1). This Ab exhibited a 23 AA-long CDRH3 and 20 % somatic hypermutation (SMH). The lineage showed continuous evolution over 6.5 years of maturation, with observed SHM rates ranging from 2.0 % to 30.6 % for the heavy chain, without any insertions or deletions.Conventional FACS-based sorting was previously used to isolate bNAbs from the same donor. In comparison, the single cell high-throughput approach made possible the isolation of orders of magnitude more Abs. Furthermore, the newly isolated NAbs were overall more potent and broader than those isolated previously, indicating the superiority of the novel method in recovering neutralizing lineages. Ongoing structural studies will elucidate the epitopes responsible for the broad neutralization observed in this donor. Together, the findings may help the design of reverse vaccine approaches, which show promise in the development of an effective AIDS vaccine

La transmission sexuelle du VIH-1 se fait principalement via du sperme infecté contaminé, contenant à la fois des virions libres et des leucocytes infectés. Les facteurs présents dans le liquide séminal peuvent aussi moduler l’infectivité du sperme et la réponse immunitaire de l’hôte. Ainsi, le macaque infecté par le virus de l’immunodéficience simienne (VIS) sera utilisé comme modèle expérimental afin d’étudier l’infectivité des cellules séminales, les réponses immunitaires anti-virales et évaluer l’effet inhibiteur d’anticorps neutralisant à large spectre (bNAbs) sur la transmission du virus intercellulaire (TVI).Chez les macaques infectés avec le SIVmac251, les réponses immunitaires innées et adaptatives spécifiques du VIS ont été étudiées dans le sperme, en se focalisant sur les réponses T CD8, humorale et sur l’expression des cytokines, chimiokines et facteurs de croissance. L’infection VIS induit l’expression de cytokines pro-inflammatoires et immuno-régulatrices dans le sperme concordant avec une augmentation des cellules activées T CD8+ CD69+ et T CD8+ CXCR3+ CCR5+. Ni les cellules T CD8+ spécifiques du VIS, ni la réponse humorale ne permettent le contrôle de la dissémination du virus dans le sperme. L’absence de contrôle de la réplication virale dans le sperme infecté VIS est associée à une inflammation générale et à une activation immunitaire, pouvant refléter ce qui se produit dans le tractus reproducteur masculin, et qui pourrait mener à l’augmentation de la transmission VIH-1/VIS.De plus, nous avons développé un modèle d’étude in vitro de TVI en utilisant soit la lignée cellulaire TZM-bl, soit des PBMC humains comme cellules cibles, et des splénocytes ou des leucocytes CD45+ du spermes infectés avec le SHIV163P3 comme cellules infectieuses. Ce modèle nous a permis d’évaluer l’inhibition de TVI par des bNAbs. Nous avons testé 4 bNabs de 1ère génération et 8 bNabs de 2nde génération. La combinaison de bNabs de 1ère génération (2F5 + 2G12 + 4E10) permet d’inhiber TVI, alors qu’en combinaison double ou seule, aucune inhibition de la transmission n’est observée. Cependant, les bNabs de 2nde génération peuvent, seules, induire une inhibition de TVI aussi efficacement qu’en combinaison. Ainsi, un bNabs de 2nde génération anti-V3 a été sélectionné afin de tester son effet inhibiteur de TVI dans un modèle in vivo
HIV-1 sexual transmission occurs mostly through contaminated semen, which contains both free virions and infected leukocytes. Moreover, factors in seminal plasma (SP) can influence both semen infectivity and host’s response. Therefore, we used the experimental model of Simian Immunodeficiency Virus (SIV) infection of macaques, to investigate semen cells infectivity and the antiviral immune responses and to evaluate the potency of broadly neutralizing antibodies (bNAbs) to block cell-to-cell virus transmission.In SIVmac251 infected cynomolgus macaques, we investigated SIV-specific innate and adaptive responses in semen, including CD8+ T cell response, humoral response and levels of cytokines, chemokines and growth factors. SIV infection induced pro-inflammatory and immunoregulatory cytokines in semen and a concomitant upregulation of activated CD69+ CD8+ T cells and CCR5+ CXCR3+ CD8+ T cells. Neither SIV-specific CD8+ T-cell responses nor humoral responses controlled seminal viral shedding. Failure to control viral replication in SIV-infected semen is related to a general inflammation and immune activation, which possibly mirrors what happen in the male genital tract and which could lead to enhanced HIV/SIV transmission.Moreover, we developed cell-to-cell transmission assays, using either TZM-bl or human PBMC as target cells and SHIV162P3-infected splenocytes and CD45+ semen leukocytes as donor cells, and evaluated bNAbs-mediated inhibition. The bNAb panel included four 1st generation bNAbs and eight 2nd generation bNAbs. A combination of 1st generation bNAbs (2F5+2G12+4E10) was able to efficiently inhibit CAV transmission, while double combination or single bNAbs showed reduced potency. Of note, individual 2nd generation bNAbs inhibited transmission as efficiently as bNAbs combinations. An anti-V3 bNAb has been selected to evaluate its potential to block cell-to-cell transmission in vivo

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.

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.

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.

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

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine Johannesburg, 2018.
Southern Africa is currently the most affected region of the world in terms of the HIV/AIDS pandemic and despite decades of research into this virus, a vaccine that is adequately effective is yet to be developed. However, it is widely believed that broadly neutralizing antibodies (bNAbs) are likely to be required for protection. Eliciting bNAbs by vaccination has been challenging for several reasons, one of which is the failure of most viral proteins to bind the germline versions of bNAbs (bNAb precursors). For vaccine design, it will be necessary to either select or engineer suitable immunogens that bind the inferred bNAb precursors in vitro, which is the first step in driving such responses towards breadth. This study focused on an unusual set of six previously identified viral escape mutations (collectively referred to as CS-Mut) that enhance neutralization by bNAbs to the membrane proximal external region (MPER). These mutations were used to engineer and test a variety of viral constructs for their potential as vaccine candidates. We first tested the effect of each individual cleavage site mutation on viral entry and MPER exposure. We showed that individual mutations resulted in reduced viral entry potential, but not to the same extent as all six mutations combined in the CS-Mut. We next tested the effect of the individual mutations on neutralization by MPER bNAbs. We showed that most of the single mutations enhanced viral sensitivity to MPER bNAbs, with three of the six mutations most promising in terms of MPER exposure. However, the MPER enhancement was less marked than the combined set of six mutations, suggesting further improvement was needed. We therefore next combined the three most promising mutations into a single construct and showed that enhancement of MPER sensitivity was improved for this triple mutant compared to the individual mutants. Indeed, for some MPER bNAbs, the triple mutant was more sensitive to MPER bNAbs than the matched virus containing all six mutations. Moreover, the entry capacity of the triple mutant was significantly improved over the six mutant construct, making it more amenable to incorporation into virus-like particles for vaccine design. Lastly, we engineered dual germline targeting immunogens by simultaneously adding the MPER enhancing mutations to five viruses that were previously shown to have a high probability of binding V2 bNAb precursors (V2 “special strains”). The “special strain” CS-Mut viruses exhibited varying degrees of reduction in viral entry potential, with infectivity abrogated for two. For the remaining three “special strain” CS-Mut viruses, enhancement in MPER sensitivity was observed. Increased sensitivity was largely specific for bNAbs targeting the MPER epitope or interface, as bNAbs and plasma to other viral epitopes showed no enhanced neutralization. However, despite enhanced sensitivity to mature MPER bNAbs, no neutralization was observed using germline reverted MPER bNAbs, the best available approximate of MPER precursors. In conclusion, the cleavage site mutations resulted in favorable exposure of the MPER epitope, a trait that is promising for immunogen design. However, the fitness cost that results from the addition of the cleavage site mutations is problematic for use of these constructs in vaccines using virus-like particles. In future studies, a balance between viral entry potential and enhancement of MPER neutralization needs to be determined to optimize immunogen candidates. The CAP256 SU CS-Mut construct showed promise as a dual germline targeting immunogen, exhibiting limited reduction in entry potential while favorably exposing the MPER epitope, with minimal disruption to the native envelope trimer structure.
LG2018

A thesis submitted to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand in fulfilment of the degree of Doctor of Philosophy (PhD) Johannesburg, September 2015
Broadly neutralizing antibodies are capable of neutralizing a large number of HIV-1 strains and have shown to be protective against infection in non-human primate models. These antibodies are likely to play an important role in an effective vaccine against HIV. Eliciting them by vaccination has thus far been unsuccessful and their unusual features such as long CDHR3 lengths and high levels of somatic hypermutation make this a particularly challenging task. Approximately 15-30% of chronically HIV-1 infected individuals develop these types of antibodies but an understanding of the underlying mechanisms is limited. The aim of this study, therefore, was to investigate host factors associated with the development of broadly neutralizing antibodies in HIV-1 subtype C infected individuals. In particular we analysed genetic variation of the genes encoding the variable region of antibodies, the evolution of an HIV-1 specific antibody lineage and glycan-binding profiles of serum antibodies. The human heavy chain variable region genes (IGHV) are the largest and most variable of all human immunoglobulin genes and encode the major antigen-binding region. These genes are divided into seven subgroups, each subgroup contains numerous genes and alleles. Using genomic DNA from 28 HIV-1 subtype C infected individuals we performed next generation sequencing using both Illumina MiSeq and Roche 454 technologies. Included were 13 individuals who developed broadly neutralizing antibodies, 13 who did not despite chronic HIV-1 infection and two intermediate neutralizers. We found no genetic differences in the IGHV genes between these two groups. However, we identified 85 novel alleles and 38 alleles that had previously only been observed in rearranged antibody sequences. Of these alleles, eight were used by functional antibodies, two of which were HIV-1 specific. This study highlights the importance of a fully comprehensive database for inferring germline gene usage and the unmutated common ancestors of antibody lineages. In addition it showed that everyone has the same genetic potential of developing broadly neutralizing antibodies, which has positive implications for vaccine development. A number of studies have demonstrated the importance of strain-specific antibodies in the development of broadly neutralizing antibodies. In addition to being the forerunners of broadly neutralizing antibodies, strain-specific antibodies can help shape the viral populations that elicit different broadly neutralizing antibody lineages. We therefore studied the evolution of a strain-specific HIV antibody lineage (CAP88-CH06) in an individual who failed to develop neutralization breadth even after 5 years of infection, to understand why some strain-specific antibodies remain limited to autologous viruses. CAP88-CH06 was previously isolated as an IgA1 antibody using the IGHV4-39 gene with 8.8% divergence from donor CAP88 at 34 weeks post-infection, which mapped to the C3- V4 region of gp120. IgA and IgG antibodies using the same germline IGHV were sequenced on an Illumina MiSeq from 5 to 121 weeks post-infection. IgA sequences identical to that of the fully matured antibody with 8.8% divergence were detected from early infection and throughout until after 2 years, well after viral escape. This was consistent with plasma neutralization of the C3-V4 region within CAP88. However, very little evidence of evolution was seen within the IgA sequences. A group of related IgG sequences were also identified between 11 and 34 weeks but not at other time-points. Interestingly, within the 11 week transcripts we identified an identical sequence as both an IgA and IgG isotype, which likely gave rise to these transient IgG antibodies. The lack of neutralization breadth in this individual could therefore be the result of both limited evolution of the IgA isotype and well as the disappearance of the IgG isotype. The HIV envelope is surrounded by glycans, known as the glycan shield. These glycans contribute towards the structural integrity of the envelope and serve as protection against immune responses to conserved regions. However, glycans often form targets for broadly neutralizing antibodies. Thus we studied the glycan-binding profiles of HIVnegative and HIV-positive individuals (including 12 individuals who develop broadly neutralizing antibodies and 13 who did not despite chronic infection) to determine whether glycan-binding was specific to individuals who develop broadly neutralizing antibodies. Longitudinal samples were taken yearly for three years from all 47 individuals and their serum IgG levels were tested on glycan microarrays. We observed fluctuations in glycanbinding over time within the HIV-negative individuals and these were used to establish baseline values. The HIV-positive individuals were found to have elevated levels of antibodies targeting high mannose N-linked glycans, Tn-peptides and glycolipids during infection. Binding to Tn-peptides and glycolipids were elevated throughout infection, whereas high mannose N-linked glycans were elevated from 2-3 years post-infection. We observed no differences in these glycans between the individuals who developed broadly neutralizing antibodies compared to those who did not despite chronic HIV infection. This data suggests that the elevated levels of glycan-binding serum antibodies were a consequence of infection rather than specific to broadly neutralizing antibodies. Since glycan-binding antibodies against Tn-peptides and glycolipids were detected earlier than high mannose N-linked glycans and antibodies targeting these glycans were elevated during infection, they might warrant further investigation with respect to immunogen design. Collectively this study has contributed to a greater understanding of the role of various host factors in the development of broadly neutralizing antibodies to HIV. This includes showing that there were no differences in the IGHV genes between individuals who did and did not develop broadly neutralizing antibodies as well as providing a wealth of new data on human antibody genes that will have benefits beyond the field of HIV. Furthermore our study has reinforced the essential role of somatic hypermutation in developing neutralization breadth and the need for further co-evolution studies on strainspecific lineages to understand this roadblock. Finally our study using glycan arrays has highlighted that glycan-binding antibodies are induced in all HIV-infected individuals even though only a minority go on to develop broadly neutralizing antibodies. Overall these data suggest that all humans have the ability to develop broadly neutralizing antibodies but a vaccine capable of eliciting such protective responses remains a major hurdle.

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.

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

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.