Leveraging South African HIV research to define SARS‐CoV‐2 immunity triggered by sequential variants of concern

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the virus that causes coronavirus disease 2019 (COVID‐19), has shifted our paradigms about B cell immunity and the goals of vaccination for respiratory viruses. The development of population immunity, through responses directed to highly immunogenic regions of this virus, has been a strong driving force in the emergence of progressively mutated variants. This review highlights how the strength of the existing global virology and immunology networks built for HIV vaccine research enabled rapid adaptation of techniques, assays, and skill sets, to expeditiously respond to the SARS‐CoV‐2 pandemic. Allying real‐time genomic surveillance to immunological platforms enabled the characterization of immune responses elicited by infection with distinct variants, in sequential epidemic waves, as well as studies of vaccination and hybrid immunity (combination of infection‐ and vaccination‐induced immunity). These studies have shown that consecutive variants of concern have steadily diminished the ability of vaccines to prevent infection, but that increasing levels of hybrid immunity result in higher frequencies of cross‐reactive responses. Ultimately, this rapid pivot from HIV to SARS‐CoV‐2 enabled a depth of understanding of the SARS‐CoV‐2 antigenic vulnerabilities as population immunity expanded and diversified, providing key insights for future responses to the SARS‐CoV‐2 pandemic.


| PANDEMIC PREPAREDNE SS THROUG H 3 0 YE AR S OF HIV VACCINE RE S E ARCH
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in a global pandemic causing more than 6 million global deaths and resulting in significant social and economic challenges. This pandemic was also coupled with massively accelerated scientific research, the speed and impact of which has never been seen before. This rapid response to SARS-CoV-2 has in large part been facilitated by more than 30 years of HIV vaccine research, which has long since benefited research on other pathogens of medical significance, but has been most pronounced during the SARS-CoV-2 pandemic.
The HIV vaccine research field has pioneered immunological and virological research through in-depth studies of virus-host interplay during chronic infection. This has included technical advances in single B cell isolation and characterization, and massively deep nextgeneration sequencing of both antibody and viral genes. The field has also driven increased reliance on structural biology and rigorous immunization studies, both in preclinical settings and in accelerated designs for human clinical trials, including experimental medicine.
Initial HIV vaccine research efforts were directed to eliciting T cell responses; however, it soon became apparent that while T cells mediated control of viral loads in some individuals, infection had to be completely blocked given the ability of HIV to integrate into human DNA and remain latent for months to years following initial infection.
Protection from infection could only be mediated through the presence of high titer antibodies at mucosal sites. Therefore, the characterization of the antibody response to HIV has been a focus for many years, resulting in strong data regarding the role of antibodies in protection from infection in passive immunization studies in animal models. [1][2][3][4] More recently, the phase 2b AMP trial (HVTN703 and HVTN704) provided further proof that antibodies could prevent infection in humans. 5 This emphasis on antibody research and the realization of the need to accurately compare results across many studies in different laboratories resulted in the development and use of standardized pseudovirus neutralization assays, 6 using engineered cell lines rather than primary cells, the latter which resulted in a high level of variability. Apart from the reproducibility of the pseudovirus neutralization assay for HIV studies, the single cycle infectious nature of pseudoviruses ensured a built-in safety feature, which allowed for these assays to be performed in BSL2 rather than BSL3 environments, making such assays more accessible in under-resourced areas of the world. In addition, the use of the same HIV backbone with different envelopes allowed for relatively rapid characterization of multiple different forms of HIV, with advances in sequencing (and reduced cost) enhancing our understanding of viral envelope quasispecies.
These assays have been fundamental to HIV vaccine research and are now a routine technique that has been implemented in laboratories across the world. Along with the development of more standardized assays came the implementation of proficiency panels using well-characterized serum panels and sets of viruses, with a further emphasis on ensuring comparability of data. Following the results of the RV144 Thai vaccine trial, which implicated non-neutralizing antibody functions in protection, 7 assays to investigate the role of antibody functions such as phagocytosis and antibody-dependent cellular cytotoxicity were established. [8][9][10][11] In addition to this, several HIV laboratories have also refined technologies to isolate antigenspecific monoclonal antibodies with broad B cell function, including binding, neutralization, and Fc effector function.
With the emergence of SARS-CoV-2 and the declaration of a global pandemic, rapid, and reproducible assays were urgently needed to measure antibody responses, initially after infection and later after vaccination. The urgency for these assays, and the need for them to be rapidly adapted, became particularly evident after the first detection of mutated variants with suspected immune escape potential, with potential implications for reinfection and vaccine efficacy. Multiple HIV laboratories, including our own, quickly converted the HIV pseudovirus neutralization assay and several Fc effector function assays to investigate SARS-CoV-2 antibody resistance, in an effort to link genotype to phenotype. [12][13][14] As the scientific field scrambled to pivot to SARS-CoV-2 research, more than 30 different neutralization assays were adapted for SARS-CoV-2, using a wide array of platforms including lentiviral backbones, VSV-based and live virus neutralization assays in many different cell lines. [15][16][17][18][19] As had previously been seen in the HIV vaccine field, this many assays produced quantitatively different results and required local standardization to enable cross-comparisons of data. These standardization approaches included inter-laboratory comparisons, external quality assessments, and the development of an international standard for use in assay calibration. 20,21 Through these endeavors, the SARS-CoV-2 antibody assays, many adapted from the HIV field, have successfully been employed to map antibody responses following infection and to confirm the immunogenicity of numerous SARS-CoV-2 candidate and licensed vaccines. 13,22,23 Lastly, the characterization of these SARS-CoV-2 responses led to the isolation of monoclonal antibody therapeutics to prevent severe SARS-CoV-2 illness and death, [24][25][26][27] in large part due to the contribution of foundational HIV vaccine research.

| LE VER AG ING HIV VACCINE RE S E ARCH IN SOUTH AFRIC A TO CONTRIBUTE TO SAR S -COV-RE S E ARCH
In South Africa, as the pandemic emerged, we too leveraged many years of investment in immunology and virology to pivot to SARS-CoV-2. South Africa has the largest antiretroviral treatment program in the world, with more than 4.8 million people accessing drugs. This necessitated the development of a large national program to monitor the emergence of antiretroviral drug resistance mutations, both at the population level and in persons failing treatment. As a consequence, HIV researchers in South Africa had built up their next-generation sequencing expertise and infrastructure through routine HIV antiretroviral drug resistance surveillance 28,29 and basic virological studies of HIV [30][31][32][33][34] to inform vaccine design. 35,36 In June 2020, several South African scientists established a network of sequencing and diagnostic laboratories, led by eight individuals, seven of whom had previously been involved in HIV research. The newly formed Network for Genomic Surveillance in South Africa (NGS-SA) 37 centralized four sequencing hubs in Durban, Cape Town, Bloemfontein, and Johannesburg, to receive specimens from SARS-CoV-2 testing laboratories, in both the public and private sectors, to monitor the evolution of SARS-CoV-2. The expertise of this network was quickly capitalized upon and strengthened by the Africa Centres for Disease Control and Prevention (Africa CDC)'s Pathogen Genomics Initiative (PGI), which was launched in November 2019, fortuitously timed to contribute to the SARS-CoV-2 pandemic. The Africa CDC PGI used two hubs within NGS-SA to initially provide SARS-CoV-2 genomic surveillance to numerous countries in southern Africa. Later, this partnership systematically expanded the reach of next-generation sequencing across Africa through virtual and in-person training conducted by NGS-SA.
In the space of 2 years and under the extremely heavy constraints of the pandemic, the Africa CDC PGI 38 has expanded next-generation sequencing capacity in Africa from just 7 countries in November 2019 to 31 countries in January 2022. 39 South African laboratories have also been heavily involved in HIV vaccine and microbicide research since the 1990s, with significant global investment in South African laboratories and clinical trials infrastructure. As a result, we had substantial experience in the pseudovirus neutralization assays and the single B cell isolation

| THE SOUTH AFRI C AN EPIDEMI C AND THE EMERG EN CE OF T WO VO C S WITH G LOBAL R AMIFIC ATIONS
The South African SARS-CoV-2 epidemic has been characterized by four epidemiologic waves between March 2020 and February 2022 40 (Figure 1). The first wave of infection was protracted due to F I G U R E 1 The SARS-CoV-2 epidemic in South Africa from 2020 to 2022. Number of diagnosed SARS-CoV-2 cases and deaths (7- Wave periods are staggered for the deaths compared to cases. Dominant variants for each wave are represented in colored text boxes with D614G in grey, Beta in purple, Delta in green, and Omicron in red. SARS-CoV-2 case data obtained from the National Institute for Communicable Diseases National COVID-19 Daily Report. SARS-CoV-2 genomics surveillance data obtained from the Network of Genomics Surveillance Weekly Report a "hard lockdown" with extremely restricted interprovincial movement, school closures, and the requirement that non-essential workers cease work or function remotely. The first wave was characterized by multiple introductions of ancestral D614G lineages, which subsequently diversified into at least 16 novel lineages including C.1 and several B.1.1 lineages. 41,42 Despite several lineage-defining mutations distinguishing each of these novel lineages, none of these mutations coded for amino acid substitutions in the spike protein. 42 This first wave resulted in >650 000 infections and >18 000 deaths  46 The clustering of mutations in these two regions, both of which were known to be targets of the neutralizing antibody response, suggested more than just adaptation toward enhanced engagement with human angiotensin-converting enzyme 2 (hACE2), the cellular receptor for this virus.
We sought to investigate the impact of these spike mutations by testing convalescent plasma from individuals who had been infected with ancestral D164G variants, against the Beta variant. Within 6 weeks of identification of the variant, we showed that these mutations mediated significant escape from neutralizing antibodies, with convalescent plasma from 48% of individuals completely unable to neutralize the Beta variant and a 13-fold reduction in geometric mean titer. 13 However, an RBD chimera, which possessed only the Beta RBD mutations within a D614G background, was resistant to only 27% of plasma, showing that a substantial fraction of the neutralization escape was mediated by the NTD Beta mutations. 13 In addition, although therapeutic monoclonal antibodies were not (and are still not) available in South Africa, we confirmed that the Beta variant showed resistance to three classes of therapeutically relevant antibodies. 13 These data, which were the first to show that the Beta VOC represented a public health threat, were a harbinger of the threat of viral evolution for the global response to SARS-CoV-2.
In addition, in individuals who had received the AstraZeneca ChAdOx1 nCoV-19 COVID-19 vaccine, we showed that more than 75% did not have neutralizing antibodies against the Beta variant. 47 This lack of neutralization corroborated the lack of protection from infection provided by this vaccine in a clinical trial in South Africa, where only 10.4% vaccine efficacy was observed. 47 At this time, the primary goal of vaccination was to prevent SARS-CoV-2 infection. Therefore, the neutralization resistant profile of the Beta variant resulted in the roll-out of the AstraZeneca ChAdOx1 nCoV-19 COVID-19 vaccine in South Africa being halted, only days after the arrival of the vaccine in South Africa. The ability of this vaccine to provide protection from severe illness and hospitalization could not be accurately assessed in this trial, which was conducted in a relatively young population. Given that we now know that the ChAdOx1 nCoV-19 COVID-19 vaccine performs well in preventing illness and hospitalization, this decision, which has yet to be reversed in South Africa, was an unfortunate set-back for South Africa's vaccine program.
Interestingly, although neutralization was significantly diminished against Beta, binding antibody 13  of the CD4 T cell response-targeted peptides where mutations in the Beta variant had arisen. 50 While CD8 T cell responses were rarely detected, those that were detected were often directed to regions that were conserved between the ancestral D614G and Beta variants and were therefore not significantly affected by the Beta mutations. 50 Despite this conservation of T cell epitopes which is associated with protection from severe disease, 51-53 the second wave of infections in South Africa which were caused almost exclusively by the Beta variant, accounted for 34% (>33 000) of the total number of documented SARS-CoV-2 deaths in South Africa. 54 This was likely a result of relatively low population immunity through infection, and a very slow vaccine roll-out following wave 1 which was further delayed by the emergence of the Beta variant and concerns of the lack of efficacy of vaccines against this variant ( Figure 1). Prior to the emergence of Omicron, the Beta variant was the most neutralization resistant variant to be described. However, despite this immune evasive phenotype, it failed to expand globally, perhaps due to competition from the highly transmissible Alpha variant, 55,56 which emerged and came to dominate globally at the concurrent time. It is difficult to disentangle the differences in transmissibility, infectivity or founder effects between the Alpha and Beta variants, given the severe travel restrictions and bans placed on South Africa during the dominance of the Beta variant.
In early 2021, the Delta variant of SARS-CoV-2 emerged in India 57 and was characterized by mutations associated with increased spike cleavage and messenger RNA expression, which resulted in increased replicative fitness 58 and particle assembly. 59 As a consequence, even infected but asymptomatic individuals had 1000 times more virus than D614G variant infections, 60  While individuals who received mRNA rather than Ad-vectored vaccines had higher neutralization titers against Delta, breakthrough infections were occurring at a higher rate following the emergence of Delta than the D614G or Alpha variants. 66,67 Despite this, SARS-CoV-2 hospitalizations and deaths were drastically skewed toward unvaccinated individuals globally and in South Africa during the dominance of the Delta variant, regardless of the vaccine platform used. [67][68][69][70][71] Following the third wave of infections in South Africa, the 7day rolling average for SARS-CoV-2 case numbers were at an "all time low" with less than 100 cases per day between the months of were generated by NGS-SA within 7 days from diagnostic specimens sampled in 3 other provinces, including two coastal provinces between 600-1500 km away from Gauteng. 73 In addition, using the SGTF as a proxy for the detection of this new lineage, we saw rapid dissemination throughout South Africa, coupled with an increase in the hazards ratio for reinfection risk, 74 the first time this signal had been seen in South Africa. This resulted in this lineage, designated B.1.1.529, 43 being classified by the WHO as the Omicron VOC within a week of its detection. 45 The Omicron lineage has since been further classified into multiple sub-lineages, 43  South Africa, 76 India, 61,62 Denmark, 77 and the United Kingdom. 78 As in many other parts of the word, Omicron was associated with lower levels of hospitalization and deaths, and despite causing >750 000 infections in South Africa, the country recorded only 10 478 deaths in the fourth wave. 70,72,79 This disconnect between hospitalization and deaths was widely attributed both to potentially lower pathogenicity, but also to the fact that as many as 70% of the population were estimated to have been infected in South Africa, at the end of the third wave, 79 described in more detail below.
In addition to Omicron, two other highly mutated lineages, namely C.1.2 64  BNT162b vaccine plasma, 81 given the containment of this lineage, it was never evaluated for sensitivity to convalescent plasma. It is highly likely that many more such mutated variants emerge but do not cause substantial numbers of infections and therefore go undetected. While the structure of the NGS-SA has resulted in an efficient genomics surveillance system across South Africa, less than 1% of the diagnosed cases in South Africa are sequenced. 40 Additionally, the majority of cases are asymptomatic, 82 not diagnosed and therefore do not filter into the national genomics surveillance program.
As a striking example of the risk of what remains relatively low-level genomic surveillance, the Omicron parental lineage (B.1.1) was not detected in South Africa for months prior to the emergence of the VOC. 41 These examples suggest that multiple lineages are likely continually circulating at low frequency, each with the potential to evolve given the appropriate driving selection pressures.
A further factor which may contribute to the risk that highly mu- this, the spike protein was suspected to be a major target for antibodies, given its surface exposure and its functional importance for cell entry. The SARS-CoV-2 spike is a metastable heterotrimer (Figure 2A and B) consisting of an S1 subunit, which mediates hACE2 binding, and an S2 subunit, which facilitates fusion of the host and viral membranes. 95 The RBD is located at the trimer apex (Figure 2A and B), but is only accessible to hACE2 once a hinge-like conformation occurs, shifting RBD from the down to the up conformation. 95 Following hACE2 engagement by the RBD, the S1 subunit is shed, and the S2 subunit is able to transition into a fusion-compatible conformation. 95 Two highly antigenic regions of the spike protein were identified through studies of the immune response in convalescent individuals infected with ancestral variants. The most immunodominant of these regions, the RBD and accounted for 65%-90% of the binding activity of convalescent plasma and contains the receptor binding motif (RBM), which engages with the hACE2 receptor. 49,96 The second highly antigenic region, the NTD (Figure 2A and B), is thought to act as a co-receptor by engaging with DC-SIGN/L-SIGN on cells that do not express hACE2 and is located at the trimer face that is proximal to the RBD. NTD binding antibodies were present at lower frequency (4%-20%). 96 Finally, the smallest fraction of the binding antibody response was targeted to the S2 subunit or other undefined regions. These regions were the targets of a large proportion of binding (non-neutralizing) antibodies, while the neutralizing fraction consisted of a small minority of the overall antibody response. 49,96,97 In addition, the T cell response, like non-neutralizing antibodies, targets epitopes much more dispersed throughout the spike and are less dependent on the quaternary structures of RBD and NTD. 50,65 The T cells response is heavily CD4-driven, with CD8 responses rarely detected. The relative immunodominance of the RBD and NTD regions in the neutralizing response resulted in a global population immunity profile that was relatively similar in multiple geographic locations as described below and resulted in convergent evolution of similar escape mutations in multiple populations. The targets of these neutralizing responses is described in more detail below.

| Receptor binding domain
The RBD of SARS-CoV-2 only possess two glycan sites, which have between 80-100% low density complex glycan occupancy. 98 This region elicits multiple antibody responses, 49,99,100,101 but is less able to induce CD4 T cell responses. 65 RBD-targeting antibodies can be broadly divided into four main classes 100 with class 1 antibodies targeting the receptor binding motif, therefore competing for hACE2 engagement and only able to bind the RBD up conformation of spike.
Class 1 antibodies were most frequently elicited following infection and commonly use the VH3-53/66 germline antibody genes. Class 2 and 3 antibodies target the outer face of RBD, which is exposed in both the RDB up and down conformations. Class 2 antibodies, which also bind to the RBM, typically use a more diverse VH-gene pool, although multi-donor VH1-2 public antibodies belonging to this class have also been identified. Class 3 antibodies bind an epitope that is relatively conserved within sarbecoviruses and in line with this, these antibodies have been less affected by escape mutations. 102 Finally, the class 4 antibodies target the inner RBD face, or the cryptic epitope, which is only exposed in the RBD up conformation and contains antibodies that neutralize less potently compared to class 1 and 2 antibodies.
As with most low-resolution classification strategies, antibodies do not always fit neatly into one of these four broad classes. Within class 1 antibodies, for example, neutralization can be achieved either through trimer destabilization and/or blocking hACE2 engagement.
In addition, like the HIV envelope protein, which is heavily shrouded in 26-30 host-derived glycans, the SARS-CoV-2 spike protein has 22 glycan sites. 98 However, while approximately 60% of the HIV glycan shield consists of large oligomannose type glycans, these high mannose glycans are only present in less than 30% of the SARS-CoV-2 spike glycan sites, 98,103 leaving a substantial proportion of the protein surface beneath exposed to immune attack. For this reason, antibodies can bind to the SARS-CoV-2 spike and especially the RBD, which only possess two N-linked glycan sites, without much constraint by glycosylation. This has led to the further sub-categorization of antibodies with distinct binding footprints and functional characteristics.
This higher resolution classification therefore includes seven RBD binding groups and 14 sub-groups, which align in terms of antibody angle of approach and the stoichiometry of antibody binding. 104 Antibodies within class 1 can be further categorized into three overlapping groups, namely RBD 1 to 3, which have decreasing degrees of overlap with the RBM and can bind 2-3 fabs per spike. 104

| N-terminal domain
In addition to antibodies targeting the RBD, several potent monoclonal neutralizing antibodies isolated from infected donors bind to the spike NTD. The NTD ioverall elicits fewer B cell responses 96 than RBD, likely due to the presence of 8 glycans with five having 80-100% complex glycan occupancy, and 3 possessing 30-100% oligomannose glycan occupancy. 98 In contrast to RBD, however NTDtargeted antibodies are derived from diverse VH-genes, potentially reflective of the inherent flexibility of this sub-domain, and therefore, these antibodies bind the spike through multiple angles of approach for convergent recognition of this region. Six antigenically distinct sites have been identified within the NTD (sites i-vi), with the immunodominant site i preferentially eliciting VH3-21 antibodies. Antibodies elicited to sites ii-vi can bind trimer, but are unable to neutralize. Despite the overall lower frequency of NTD-directed antibodies, these potent responses exhibit significant pressure on the virus, given that NTD is the most diverse region of the spike trimer, with not only amino acid substitutions but also indels used to re-structure this domain. Some NTD-directed antibodies may also block entry via the endosomal or TMPRSS-independent route, therefore changes within this region may have impacts on the variant preference for cell surface or endosomal entry, as has been observed with Omicron, which has substantial NTD rearrangement. 96

| S2 subunit
Finally, the S2 region of the spike, which overall bears between 34-43% similarity with non-beta coronaviruses, possess 6 glycans of which 5 have 90-100% complex glycan occupancy. 98 Antibodies elicited by common cold coronaviruses, such as hCoV-HKU1 and hCoV-OC43, and those elicited by prior SARS-CoV-1 infection, frequently target the S2 subunit, but are not neutralizing. 105 Figure 2A) and, moreover, that these polyclonal responses were neutralizing. 106 Since then two other neutralizing anti-S2 epitopes distal from the fusion peptide and targeting the S2 stem-helix region (Figure 2A and B), which is functionally conserved to maintains the fusion machinery, have been identified from convalescent plasma. [107][108][109] The S2 stem antibodies also appear to target a linear epitopes and inhibit fusion; however, these epitopes are distinct from each other with one spanning the stem helix N-terminus (1140-1157) 108,109 and the other over the stem helix C-terminus and hinge region (1153-1165). 107

| Population immunity as a selector of variants
The shared population immunity profile described above resulted in convergent evolution of SARS-CoV-2 by mid-2020, 110

| DECIPHERING THE EFFEC T OF E XP OSURE TO MULTIPLE S PIK E VARIANTS ON P OPUL ATION IMMUNIT Y
The infection of a naive host with a new pathogen that has immunodominant epitopes can result in a population immune response that is very predictable and similar across multiple individuals from various geographic locations, as we have seen with SARS-CoV-2 in humans. 49,96,97,100,115 As the pandemic progressed and SARS-CoV As with the Beta variant, Delta also exhibited a different profile of breadth but likely due to a different mechanism. Even with 6 changes in the Delta spike, the increased replicative fitness, in large part due to the spike L452R, 120 P681R 58 and nucleocapsid R203M 59 substitutions allowed this variant to reach viral loads 1000 times that of ancestral strains even during mild or asymptomatic SARS-CoV-2 infection. 60 As has been shown in both HIV and SARS-CoV-2 infection studies, in general the higher the viral load, the higher the virus-specific antibody levels. 32,121 In the case of Delta infections, this very likely contributed to the higher overall neutralizing titers and therefore cross-neutralizing titers that were observed following Delta infections. 48,64 The strain-specific antigenicity of the currently globally dominant variant, Omicron, is likely linked to its significant evolution away from ancestral and other variants. This evolution resulted in 16 mutations in the RBD alone and two alternate strategies for restructuring the NTD, involving multiple substitutions, deletions, and insertions, the latter of which was not seen in previous variants. 73 These changes in the immunodominant regions of the spike are likely what, in the case of primary infection with or exposure by Omicron, results in elicitation of strain-specific responses that can only weakly cross-neutralize other variants. 119 In conclusion, individuals infected with either the Beta and Delta variants had antibodies with greater capacity to neutralize other variants. 23,48,64 However, this was likely caused by the different mechanisms discussed above, following infection with each. In In LMICs, the maintenance of protracted lockdown periods and enforcing of restriction to curb SARS-CoV-2 transmission, has been economically disastrous. The result of more relaxed restrictions or the limited periods with which they were implemented, has contributed to a more rapid transmission of SARS-CoV-2, and is reflected in by high sero-prevalence study performed in Gauteng Province, 79 which was the initial epicenter of Omicron, in November 2021, between 22 October and December 9, 2021. In this province, an overall sero-prevalence of 73% despite only 36% vaccine coverage in individuals 12 years of age or older was observed. At this time when Omicron emerged, case numbers rapidly increased in Gauteng province to peak within a month, in comparison to the third wave driven by Delta, where increase to peak occurred over 2 months. 40 Despite this, a decoupling of cases, hospitalizations and deaths was seen during this fourth wave of infections, likely in part due to the high sero-prevalence seen in this province and by extension, South Africa and Africa as a whole. 72,79 This high seroprevalence of SARS-CoV-2, despite relatively low vaccine coverage in Africa, resulted in an immunological landscape that is different to other parts of the globe where primary exposure to SARS-CoV-2 is through vaccination.
The differential exposure to distinct variants and to vaccines less commonly used in HICs has necessitated studies of "hybrid" immunity in the South African context. Studies of breakthrough infection in individuals who had received a single dose of the AD26.COV2.S vaccine, widely used in Africa, have been performed. 116,117 Neutralizing titers in uninfected individuals were low over the course of the first 6 months following vaccination, with GMT titers against multiple variants, including the ancestral D614G, not exceeding 1:200. 117 However, individuals who experienced a breakthrough infection between 4 and 5 months after vaccination displayed significant boosting of neutralizing titers between 62-and 185-fold with titers above 1:3000 for all variants except Omicron. While Omicron neutralization was lower than that for other variants, cross-neutralization of all SARS-CoV-2 variants has been confirmed for Delta breakthrough infections following vaccination with other regimens, including mRNA-based vaccines. 117,123,124 In addition, these cross neutralizing titers are apparent regardless of whether infection occurred before or after vaccination. 125,126 The cross-neutralizing capacity of antibodies elicited through hybrid immunity, extends to include other sarbecoviruses, like SARS-CoV-1. 117,127 SARS-CoV-1 and SARS-CoV-2 share an overall 76% amino acid similarity between their spikes with a similarity hierarchy of S2 region (88%), followed by the RDB (74%), and lastly NTD being the most variable (51%) ( Figure 2C). In addition, the electrostatic conservation of the spike surfaces between these two viruses is largely conserved, with the SARS-CoV-2 RBDs forming a more closed protective cap than the SARS-CoV-1 RBDs ( Figure 2B 117,127,128 In addition, a recent immunization study in non-human primates shows that ACE2-competing mAbs that are able to bind to multiple variants of SARS-CoV-2 are elicited after the priming dose of the mRNA-1237. These observations, together with the relatively limited diversity of SARS-CoV-2, even including the Omicron sub-lineages, suggest that primary vaccination with either single-or two-dose regimens may be enough to elicit antibodies, albeit at low titer, able to broadly recognize SARSrelated viruses. Given that it seems increasingly unlikely that we will be able to induce high neutralizing antibody titers in the respiratory mucosa for sustained periods, to be able to prevent infection with SARS-CoV-2, the lower titer presence of these anti-sarbecovirus antibodies as well as the induction of memory B cell responses, even after infection or vaccination only, may be sufficient to dampen viral load and therefore disease outcome in future infections with progressively mutated SARS-CoV-2 variants. This is supported by recent findings showing that vaccination after infection provided significant protection from re-infection. 129,130 Despite the public rhetoric in South Africa and elsewhere suggesting that this pandemic is over, these data provide evidence for why it remains critical to extend vaccine coverage in Africa and other LMICs, even if only single-dose vaccine regimens are feasible. While increased viral fitness, enhanced hACE2 engagement and increased transmissibility, likely caused the replacement of ancestral SARS-CoV-2 variants by the prior variants of concern, namely Alpha, Beta, Gamma, and Delta, the role in antibody immunity cannot be negated, given antibody durability of between 4 and 8 months following infection or vaccination. 101,131,132 Using the example of South Africa, where distinct waves of resurgence are observed every ±6 months, it does seem likely that the waning of antibody responses is a contributing factor to the timing of resurgence and therefore to the selection of new variants. Despite the fact that the current globally dominant variant, Omicron, appears to have decreased disease severity compared to previous VOCs, it is highly likely that we will continue to see the selection of new variants periodically. This is likely to occur, given what we know about SARS-CoV-2 evolution due to selection pressure induced by population immunity, which will increasingly become more hybrid-like and therefore more diverse. It is critical therefore that we continue genomic surveillance for SARS-CoV-2, through established sentinel surveillance systems like the Global Influenza Surveillance and Response System (GISRS), which has expanded to include routine monitoring of SARS-CoV-2 and respiratory syncytial virus (RSV), through influenza-like illness and severe acute respiratory illness detections and characterization.

ACK N OWLED G M ENTS
We would like to acknowledge Cathrine Scheepers for generating

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.