Host factors and broadly neutralizing antibodies in South African women infected with HIV-1 subtype C

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 2015Broadly 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

Dependence on a variable residue limits the breadth of an HIV MPER neutralizing antibody, despite convergent evolution with broadly neutralizing antibodies

Broadly neutralizing antibodies (bNAbs) that target the membrane-proximal external region (MPER) of HIV gp41 envelope, such as 4E10, VRC42.01 and PGZL1, can neutralize \u3e 80 % of viruses. These three MPER-directed monoclonal antibodies share germline antibody genes (IGHV1 - 69 and IGKV3 - 20) and form a bNAb epitope class. Furthermore, convergent evolution within these two lineages towards a 111.2GW111.3 motif in the CDRH3 is known to enhance neutralization potency. We have previously isolated an MPER neutralizing antibody, CAP206 - CH12, that uses these same germline heavy and light chain genes but lacks breadth (neutralizing only 6 % of heterologous viruses). Longitudinal sequencing of the CAP206-CH12 lineage over three years revealed similar convergent evolution towards 111.2GW111.3 among some lineage members. Mutagenesis of CAP206-CH12 from 111.2GL111.3 to 111.2GW111.3 and the introduction of the double GWGW motif into CAP206-CH12 modestly improved neutralization potency (2.5 3 -fold) but did not reach the levels of potency of VRC42.01, 4E10 or PGZL1. To explore the lack of potency/breadth, viral mutagenesis was performed to map the CAP206-CH12 epitope. This indicated that CAP206-CH12 is dependent on D674, a highly variable residue at the solvent-exposed elbow of MPER. In contrast, VRC42.01, PGZL1 and 4E10 were dependent on highly conserved residues (W672, F673, T676, andW680) facing the hydrophobic patch of the MPER. Therefore, while CAP206-CH12, VRC42.01, PGZL1 and 4E10 share germline genes and show some evidence of convergent evolution, their dependence on different amino acids, which impacts orientation of binding to the MPER, result in differences in breadth and potency. These data have implications for the design of HIV vaccines directed at the MPER epitope

Serum glycan-binding IgG antibodies in HIV-1 infection and during the development of broadly neutralizing responses.

CAPRISA, 2017.Abstract available in pdf

Inferred Allelic Variants of Immunoglobulin Receptor Genes: a system for their evaluation, documentation, and naming

Immunoglobulins or antibodies are the main effector molecules of the B-cell lineage and are encoded by hundreds of variable (V), diversity (D), and joining (J) germline genes, which recombine to generate enormous IG diversity. Recently, high-throughput adaptive immune receptor repertoire sequencing (AIRR-seq) of recombined V-(D)-J genes has offered unprecedented insights into the dynamics of IG repertoires in health and disease. Faithful biological interpretation of AIRR-seq studies depends upon the annotation of raw AIRR-seq data, using reference germline gene databases to identify the germline genes within each rearrangement. Existing reference databases are incomplete, as shown by recent AIRR-seq studies that have inferred the existence of many previously unreported polymorphisms. Completing the documentation of genetic variation in germline gene databases is therefore of crucial importance. Lymphocyte receptor genes and alleles are currently assigned by the Immunoglobulins, T cell Receptors and Major Histocompatibility Nomenclature Subcommittee of the International Union of Immunological Societies (IUIS) and managed in IMGT®, the international ImMunoGeneTics information system® (IMGT). In 2017, the IMGT Group reached agreement with a group of AIRR-seq researchers on the principles of a streamlined process for identifying and naming inferred allelic sequences, for their incorporation into IMGT®. These researchers represented the AIRR Community, a network of over 300 researchers whose objective is to promote all aspects of immunoglobulin and T-cell receptor repertoire studies, including the standardization of experimental and computational aspects of AIRR-seq data generation and analysis. The Inferred Allele Review Committee (IARC) was established by the AIRR Community to devise policies, criteria, and procedures to perform this function. Formalized evaluations of novel inferred sequences have now begun and submissions are invited via a new dedicated portal (https://ogrdb.airr-community.org). Here, we summarize recommendations developed by the IARC—focusing, to begin with, on human IGHV genes—with the goal of facilitating the acceptance of inferred allelic variants of germline IGHV genes. We believe that this initiative will improve the quality of AIRR-seq studies by facilitating the description of human IG germline gene variation, and that in time, it will expand to the documentation of TR and IG genes in many vertebrate species

Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization

The emergence of Omicron (Pango lineage B.1.1.529), first identified in Botswana and South Africa, may compromise vaccine effectiveness and lead to re-infections1. We investigated whether Omicron escapes antibody neutralization in South Africans vaccinated with Pfizer BNT162b2. We also investigated if Omicron requires the ACE2 receptor to infect cells. We isolated and sequence confirmed live Omicron virus from an infected person in South Africa and compared plasma neutralization of Omicron relative to an ancestral SARS-CoV-2 strain, observing that Omicron still required ACE2 to infect. For neutralization, blood samples were taken soon after vaccination from participants who were vaccinated and previously infected or vaccinated with no evidence of previous infection. Neutralization of ancestral virus was much higher in infected and vaccinated versus vaccinated only participants but both groups showed a 22-fold escape from vaccine elicited neutralization by the Omicron variant. However, in the previously infected and vaccinated group, the level of residual neutralization of Omicron was similar to the level of neutralization of ancestral virus observed in the vaccination only group. These data support the notion that, provided high neutralization capacity is elicited by vaccination/boosting approaches, reasonable effectiveness against Omicron may be maintained

Emergence of SARS-CoV-2 Omicron lineages BA.4 and BA.5 in South Africa

Three lineages (BA.1, BA.2 and BA.3) of the SARS-CoV-2 Omicron variant of concern predominantly drove South Africa's fourth COVID-19 wave. We have now identified two new lineages, BA.4 and BA.5, responsible for a fifth wave of infections. The spike proteins of BA.4 and BA.5 are identical, and comparable to BA.2 except for the addition of 69-70del (present in the Alpha variant and the BA.1 lineage), L452R (present in the Delta variant), F486V and the wild type amino acid at Q493.The two lineages only differ outside of the spike region. The 69-70 deletion in spike allows these lineages to be identified by the proxy marker of S-gene target failure, on the background of variants not possessing this feature . BA.4 and BA.5 have rapidly replaced BA.2, reaching more than 50% of sequenced cases in South Africa by the first week of April 2022. Using a multinomial logistic regression model, we estimate growth advantages for BA.4 and BA.5 of 0.08 (95% CI: 0.08 - 0.09) and 0.10 (95% CI: 0.09 - 0.11) per day respectively over BA.2 in South Africa. The continued discovery of genetically diverse Omicron lineages points to the hypothesis that a discrete reservoir, such as human chronic infections and/or animal hosts, is potentially contributing to further evolution and dispersal of the virus

Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic in southern Africa has been characterised by three distinct waves. The first was associated with a mix of SARS-CoV-2 lineages, whilst the second and third waves were driven by the Beta and Delta variants, respectively1-3. In November 2021, genomic surveillance teams in South Africa and Botswana detected a new SARS-CoV-2 variant associated with a rapid resurgence of infections in Gauteng Province, South Africa. Within three days of the first genome being uploaded, it was designated a variant of concern (Omicron) by the World Health Organization and, within three weeks, had been identified in 87 countries. The Omicron variant is exceptional for carrying over 30 mutations in the spike glycoprotein, predicted to influence antibody neutralization and spike function4. Here, we describe the genomic profile and early transmission dynamics of Omicron, highlighting the rapid spread in regions with high levels of population immunity

A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa.

The progression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in Africa has so far been heterogeneous, and the full impact is not yet well understood. In this study, we describe the genomic epidemiology using a dataset of 8746 genomes from 33 African countries and two overseas territories. We show that the epidemics in most countries were initiated by importations predominantly from Europe, which diminished after the early introduction of international travel restrictions. As the pandemic progressed, ongoing transmission in many countries and increasing mobility led to the emergence and spread within the continent of many variants of concern and interest, such as B.1.351, B.1.525, A.23.1, and C.1.1. Although distorted by low sampling numbers and blind spots, the findings highlight that Africa must not be left behind in the global pandemic response, otherwise it could become a source for new variants

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century