Evolution and neutralization escape of the SARS-CoV-2 BA.2.86 subvariant (preprint)

Omicron BA.2.86 subvariant differs from Omicron BA.2 as well as recently circulating variants by over 30 mutations in the spike protein alone. Here we report on the first isolation of the live BA.2.86 subvariant from a diagnostic swab collected in South Africa which we tested for escape from neutralizing antibodies and viral replication properties in cell culture. BA.2.86 did not have significantly more escape than Omicron XBB.1.5 from neutralizing immunity elicited by infection of Omicron subvariants ranging from BA.1 to XBB, either by infection alone or as breakthrough infection in vaccinated individuals. Neutralization escape was present relative to earlier strains: BA.2.86 showed extensive escape both relative to ancestral virus in sera from pre-Omicron vaccinated individuals and relative to Omicron BA.1 in sera from Omicron BA.1 infected individuals. We did not observe substantial differences in viral properties in cell culture relative to XBB.1.5. Both BA.2.86 and XBB.1.5 produced infection foci of similar size, had similar cytopathic effect (both lower than ancestral SARS-CoV-2), and had similar replication dynamics. We also investigated the relationship of BA.2.86 to BA.2 sequences and found that the closest were BA.2 samples from Southern Africa circulating in early 2022. These observations suggest that BA.2.86 is more closely related to sequences from Southern Africa than other regions and so may have evolved there, and that evolution led to escape from neutralizing antibodies similar in scale to recently circulating strains of SARS-CoV-2.

Emergence of neutralizing antibodies associates with clearance of SARS-CoV-2 during HIV-mediated immunosuppression (preprint)

To design effective vaccines and other immune interventions against a pathogen, it is necessary to know which aspect of immunity associates with protection. We investigated whether neutralizing antibodies associate with infection clearance in long-term SARS-CoV-2 infection during HIV-mediated immunosuppression. We monitored neutralizing antibody activity against SARS-CoV-2 in five participants with advanced HIV disease and delayed control of HIV viremia. These participants had persistent SARS-CoV-2 infection ranging from 110 to 289 days which was associated with low or undetectable neutralizing antibody responses. SARS-CoV-2 clearance was associated with the emergence of neutralizing antibodies and occurred in two participants before suppression of HIV viremia, but after some CD4 T cell reconstitution. Vaccination only further increased neutralizing antibody levels in the advanced HIV disease participants who achieved HIV suppression pre-vaccination. During the prolonged SARS-CoV-2 infection we observed widespread evolution which was particularly pronounced in one Delta variant infection. This resulted in high-level escape from Delta-elicited neutralizing antibodies and a virus antigenically distinct from both ancestral SARS-CoV-2 and Omicron XBB in hamster experimental infections. The results offer new evidence that neutralizing antibodies associate with SARS-CoV-2 protection and argue that successful management of HIV may be necessary to curtail long-term infection and evolution of co-infecting pathogens.

SARS-CoV-2 evolves increased infection elicited cell death and fusion in an immunosuppressed individual (preprint)

The milder clinical manifestations of Omicron infection relative to pre-Omicron SARS-CoV-2 raises the possibility that extensive evolution results in reduced pathogenicity. To test this hypothesis, we quantified induction of cell fusion and cell death in SARS-CoV-2 evolved from ancestral virus during long-term infection. Both cell fusion and death were reduced in Omicron BA.1 infection relative to ancestral virus. Evolved virus was isolated at different times during a 6-month infection in an immunosuppressed individual with advanced HIV disease. The virus isolated 16 days post-reported symptom onset induced fusogenicity and cell death at levels similar to BA.1. However, fusogenicity was increased in virus isolated at 6 months post-symptoms to levels intermediate between BA.1 and ancestral SARS-CoV-2. Similarly, infected cell death showed a graded increase from earlier to later isolates. These results may indicate that, at least by the cellular measures used here, evolution in long-term infection does not necessarily attenuate the virus.

HIV skews the SARS-CoV-2 B cell response toward an extrafollicular maturation pathway (preprint)

Background: HIV infection dysregulates the B cell compartment, affecting memory B cell formation and the antibody response to infection and vaccination. Understanding the B cell response to SARS-CoV-2 in people living with HIV (PLWH) may explain the increased morbidity, reduced vaccine efficacy, reduced clearance, and intra-host evolution of SARS-CoV-2 observed in some HIV-1 coinfections. Methods: We compared B cell responses to COVID-19 in PLWH and HIV negative (HIV-ve) patients in a cohort recruited in Durban, South Africa, during the first pandemic wave in July 2020 using detailed flow cytometry phenotyping of longitudinal samples with markers of B cell maturation, homing and regulatory features. Results: This revealed a coordinated B cell response to COVID-19 that differed significantly between HIV-ve and PLWH. Memory B cells in PLWH displayed evidence of reduced germinal center (GC) activity, homing capacity and class-switching responses, with increased PD-L1 expression, and decreased Tfh frequency. This was mirrored by increased extrafollicular (EF) activity, with dynamic changes in activated double negative (DN2) and activated naive B cells, which correlated with anti-RBD-titres in these individuals. An elevated SARS-CoV-2 specific EF response in PLWH was confirmed using viral spike and RBD bait proteins. Conclusions: Despite similar disease severity, these trends were highest in participants with uncontrolled HIV, implicating HIV in driving these changes. EF B cell responses are rapid but give rise to lower affinity antibodies, less durable long-term memory, and reduced capacity to adapt to new variants. Further work is needed to determine the long-term effects of HIV on SARS-CoV-2 immunity, particularly as new variants emerge.

Omicron sub-lineages BA.4/BA.5 escape BA.1 infection elicited neutralizing immunity (preprint)

The SARS-CoV-2 Omicron (B.1.1.529) variant first emerged as the BA.1 sub-lineage, with extensive escape from neutralizing immunity elicited by previous infection with other variants, vaccines, or combinations of both. Two new sub-lineages, BA.4 and BA.5, are now emerging in South Africa with changes relative to BA.1, including L452R and F486V mutations in the spike receptor binding domain. We isolated live BA.4 and BA.5 viruses and tested them against neutralizing immunity elicited to BA.1 infection in participants who were Omicron/BA.1 infected but unvaccinated (n=24) and participants vaccinated with Pfizer BNT162b2 or Johnson and Johnson Ad26.CoV.2S with breakthrough Omicron/BA.1 infection (n=15). In unvaccinated individuals, FRNT50, the inverse of the dilution for 50% neutralization, declined from 275 for BA.1 to 36 for BA.4 and 37 for BA.5, a 7.6 and 7.5-fold drop, respectively. In vaccinated BA.1 breakthroughs, FRNT50 declined from 507 for BA.1 to 158 for BA.4 (3.2-fold) and 198 for BA.5 (2.6-fold). Absolute BA.4 and BA.5 neutralization levels were about 5-fold higher in this group versus unvaccinated BA.1 infected participants. The observed escape of BA.4 and BA.5 from BA.1 elicited immunity is more moderate than of BA.1 against previous immunity. However, the low absolute neutralization levels for BA.4 and BA.5, particularly in the unvaccinated group, are unlikely to protect well against symptomatic infection. This may indicate that, based on neutralization escape, BA.4 and BA.5 have potential to result in a new infection wave.

Beta infection combined with Pfizer BNT162b2 vaccination leads to broadened neutralizing immunity against Omicron (preprint)

Omicron (B.1.1.529) shows extensive escape from vaccine immunity, although vaccination reduces severe disease and death. Boosting with vaccines incorporating variant spike sequences could possibly broaden immunity. One approach to choose the variant may be to measure immunity elicited by vaccination combined with variant infection. Here we investigated Omicron neutralization in people infected with the Beta (B.1.351) variant and subsequently vaccinated with Pfizer BNT162b2. We observed that Beta infection alone elicited poor Omicron cross-neutralization, similar to what we previously found with BNT162b2 vaccination alone or in combination with ancestral or Delta virus infection. In contrast, Beta infection combined with BNT162b2 vaccination elicited neutralization with substantially lower Omicron escape.

Unsuppressed HIV infection impairs T cell responses to SARS-CoV-2 infection and abrogates T cell cross-recognition (preprint)

HIV infection has been identified as one of the major risk factors for severe COVID-19 disease, but the mechanisms underpinning this susceptability are still unclear. Here, we assessed the impact of HIV infection on the quality and epitope specificity of SARS-CoV-2 T cell responses in the first wave and second wave of the COVID-19 epidemic in South Africa. Flow cytometry was used to measure T cell responses following PBMC stimulation with SARS-CoV-2 peptide pools. Culture expansion was used to determine T cell immunodominance hierarchies and to assess potential SARS-CoV-2 escape from T cell recognition. HIV-seronegative individuals had significantly greater CD4+ and CD8+ T cell responses against the Spike protein compared to the viremic PLWH. Absolute CD4 count correlated positively with SARS-CoV-2 specific CD4+ and CD8+ T cell responses (CD4 r= 0.5, p=0.03; CD8 r=0.5, p=0.001), whereas T cell activation was negatively correlated with CD4+ T cell responses (CD4 r= 0.7, p=0.04). There was diminished T cell cross-recognition between the two waves, which was more pronounced in individuals with unsuppressed HIV infection. Importantly, we identify four mutations in the Beta variant that resulted in abrogation of T cell recognition. Together, we show that unsuppressed HIV infection markedly impairs T cell responses to SARS-Cov-2 infection and diminishes T cell cross-recognition. These findings may partly explain the increased susceptibility of PLWH to severe COVID-19 and also highlights their vulnerability to emerging SARS-CoV-2 variants of concern.

SARS-CoV-2 spike T cell responses induced upon vaccination or infection remain robust against Omicron (preprint)

The SARS-CoV-2 Omicron variant has multiple Spike (S) protein mutations that contribute to escape from the neutralizing antibody responses, and reducing vaccine protection from infection. The extent to which other components of the adaptive response such as T cells may still target Omicron and contribute to protection from severe outcomes is unknown. We assessed the ability of T cells to react with Omicron spike in participants who were vaccinated with Ad26.CoV2.S or BNT162b2, and in unvaccinated convalescent COVID-19 patients (n = 70). We found that 70-80% of the CD4 and CD8 T cell response to spike was maintained across study groups. Moreover, the magnitude of Omicron cross-reactive T cells was similar to that of the Beta and Delta variants, despite Omicron harbouring considerably more mutations. Additionally, in Omicron-infected hospitalized patients (n = 19), there were comparable T cell responses to ancestral spike, nucleocapsid and membrane proteins to those found in patients hospitalized in previous waves dominated by the ancestral, Beta or Delta variants (n = 49). These results demonstrate that despite Omicron's extensive mutations and reduced susceptibility to neutralizing antibodies, the majority of T cell response, induced by vaccination or natural infection, cross-recognises the variant. Well-preserved T cell immunity to Omicron is likely to contribute to protection from severe COVID-19, supporting early clinical observations from South Africa.

SARS-CoV-2 Omicron has extensive but incomplete escape of Pfizer BNT162b2 elicited neutralization and requires ACE2 for infection (preprint)

The emergence of the SARS-CoV-2 Omicron variant, first identified in South Africa, may compromise the ability of vaccine and previous infection (1) elicited immunity to protect against new infection. Here we investigated whether Omicron escapes antibody neutralization elicited by the Pfizer BNT162b2 mRNA vaccine in people who were vaccinated only or vaccinated and previously infected. We also investigated whether the virus still requires binding to the ACE2 receptor to infect cells. We isolated and sequence confirmed live Omicron virus from an infected person in South Africa. We then compared neutralization of this virus relative to an ancestral SARS-CoV-2 strain with the D614G mutation. Neutralization was by blood plasma from South African BNT162b2 vaccinated individuals. We observed that Omicron still required the ACE2 receptor to infect but had extensive escape of Pfizer elicited neutralization. However, 5 out of 6 of the previously infected, Pfizer vaccinated individuals, all of them with high neutralization of D614G virus, showed residual neutralization at levels expected to confer protection from infection and severe disease (2). While vaccine effectiveness against Omicron is still to be determined, these data support the notion that high neutralization capacity elicited by a combination of infection and vaccination, and possibly by boosting, could maintain reasonable effectiveness against Omicron. If neutralization capacity is lower or wanes with time, protection against infection is likely to be low. However, protection against severe disease, requiring lower neutralization levels and involving T cell immunity, would likely be maintained.

A SARS-CoV-2 variant elicits an antibody response with a shifted immunodominance hierarchy (preprint)

Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the "class 3" epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.

Immunogenicity of the Ad26.CoV2.S vaccine in people living with HIV (preprint)

Background People living with HIV (PLWH) have been reported to have an increased risk of more severe Covid-19 disease outcome and an increased risk of death relative to HIV-uninfected individuals. Here we assessed the ability of the Johnson and Johnson Ad26.CoV2.S vaccine to elicit neutralizing antibodies to the Delta variant in PLWH relative to HIV-uninfected individuals. Methods We enrolled 26 PLWH and 73 HIV-uninfected participants from the SISONKE phase 3b open label South African clinical trial of the Ad26.CoV2.S vaccine in health care workers (HCW) in a prospective observational cohort study. Enrollment was a median 56 days (range 19-98 days) post-vaccination. HCW PLWH had well suppressed HIV viremia. As a comparison, we also enrolled unvaccinated participants previously infected with SARS-CoV-2. This group consisted of 34 PLWH and 28 HIV-uninfected individuals. We used the presence of SARS-CoV-2 nucleocapsid antibodies and any previous record of SARS-CoV-2 infection to differentiate the vaccinated participants into participants who were previously infected with SARS-CoV-2 and those not previously infected. Neutralization capacity was assessed using participant plasma in a live virus neutralization assay of the Delta SARS-CoV-2 variant currently dominating infections in South Africa. This study was approved by the Biomedical Research Ethics Committee at the University of KwaZulu-Natal (reference BREC/00001275/2020). Findings Unvaccinated PLWH showed 6-fold reduced neutralization of the Delta variant relative to HIV-uninfected participants (GMT=105 for HIV-uninfected, 15 for PLWH, p=0.001). The majority (68%) of Ad26.CoV2.S vaccinated HCW were found to be previously infected with SARS-CoV-2. In this group, Delta variant neutralization was 9-fold higher compared to the infected only group (GMT of 306 versus 36) and 26-fold higher relative to the vaccinated only group (GMT=12). There was no significant difference in Delta variant neutralization in vaccinated and previously SARS-CoV-2 infected PLWH relative to vaccinated and previously SARS-CoV-2 infected, HIV-uninfected participants (GMT of 300 for PLWH versus 307 for HIV-uninfected). Vaccinated only participants showed a low neutralization of the Delta variant, with a stronger response in PLWH (GMT=73, for PLWH, 6 for HIV-uninfected, p=0.02). Interpretation While PLWH showed reduced neutralization of the Delta variant following SARS-CoV-2 infection, the neutralization response following Ad26.CoV2.S vaccination was not inferior to HIV-uninfected study participants. Funding South African Medical Research Council, The Bill & Melinda Gates Foundation.

Divergence of delta and beta variants and SARS-CoV-2 evolved in prolonged infection into distinct serological phenotypes (preprint)

Characterizing SARS-CoV-2 evolution in specific geographies may help predict the properties of variants coming from these regions. We mapped neutralization of a SARS-CoV-2 strain that evolved over 6 months from the ancestral virus in a person with advanced HIV disease. Infection was before the emergence of the Beta variant first identified in South Africa, and the Delta variant. We compared early and late evolved virus to the ancestral, Beta, Alpha, and Delta viruses and tested against convalescent plasma from ancestral, Beta, and Delta infections. Early virus was similar to ancestral, whereas late virus was similar to Beta, exhibiting vaccine escape and, despite pre-dating Delta, strong escape of Delta-elicited neutralization. This example is consistent with the notion that variants arising in immune-compromised hosts, including those with advanced HIV disease, may evolve immune escape of vaccines and enhanced escape of Delta immunity, with implications for vaccine breakthrough and reinfections. HighlightsO_LIA prolonged ancestral SARS-CoV-2 infection pre-dating the emergence of Beta and Delta resulted in evolution of a Beta-like serological phenotype C_LIO_LISerological phenotype includes strong escape from Delta infection elicited immunity, intermediate escape from ancestral virus immunity, and weak escape from Beta immunity C_LIO_LIEvolved virus showed substantial but incomplete escape from antibodies elicited by BNT162b2 vaccination C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/21263564v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1194bfdorg.highwire.dtl.DTLVardef@1cbe318org.highwire.dtl.DTLVardef@aa74f8org.highwire.dtl.DTLVardef@e57969_HPS_FORMAT_FIGEXP M_FIG C_FIG

Persistent SARS-CoV-2 infection and intra-host evolution in association with advanced HIV infection (preprint)

While most people effectively clear SARS-CoV-2, there are several reports of prolonged infection in immunosuppressed individuals. Here we present a case of prolonged infection of greater than 6 months with the shedding of high titter SARS-CoV-2 in an individual with advanced HIV and antiretroviral treatment failure. Through whole-genome sequencing at multiple time points, we demonstrate the early emergence of the E484K substitution associated with escape from neutralizing antibodies, followed by other escape mutations and the N501Y substitution found in most variants of concern. This provides support to the hypothesis of intra-host evolution as one mechanism for the emergence of SARS-CoV-2 variants with immune evasion properties.

SARS-CoV-2 cell-to-cell spread occurs rapidly and is insensitive to antibody neutralization (preprint)

Viruses increase the efficiency of close-range transmission between cells by manipulating cellular physiology and behavior, and SARS-CoV-2 uses cell fusion as one mechanism for cell-to-cell spread. Here we visualized infection using time-lapse microscopy of a human lung cell line and used live virus neutralization to determine the sensitivity of SARS-CoV-2 cell-to-cell spread to neutralizing antibodies. SARS-CoV-2 infection rapidly led to cell fusion, forming multinucleated cells with clustered nuclei which started to be detected at 6h post-infection. To compare sensitivity of cell-to-cell spread to neutralization, we infected either with cell-free virus or with single infected cells expressing on their surface the SARS-CoV-2 spike protein. We tested two variants of SARS-CoV-2: B.1.117 containing only the D614G substitution, and the escape variant B.1.351. We used the much smaller area of single infected cells relative to infection foci to exclude any input infected cells which did not lead to transmission. The monoclonal antibody and convalescent plasma we tested neutralized cell-free SARS-CoV-2, with the exception of B.1.351 virus, which was poorly neutralized with plasma from non-B.1.351 infections. In contrast, cell-to-cell spread of SARS-CoV-2 showed no sensitivity to monoclonal antibody or convalescent plasma neutralization. These observations suggest that, once cells are infected, SARS-CoV-2 may be more difficult to neutralize in cell types and anatomical compartments permissive for cell-to-cell spread.

Escape of SARS-CoV-2 501Y.V2 variants from neutralization by convalescent plasma (preprint)

New SARS-CoV-2 variants with mutations in the spike glycoprotein have arisen independently at multiple locations and may have functional significance. The combination of mutations in the 501Y.V2 variant first detected in South Africa include the N501Y, K417N, and E484K mutations in the receptor binding domain (RBD) as well as mutations in the N-terminal domain (NTD). Here we address whether the 501Y.V2 variant could escape the neutralizing antibody response elicited by natural infection with earlier variants. We were the first to outgrow two variants of 501Y.V2 from South Africa, designated 501Y.V2.HV001 and 501Y.V2.HVdF002. We examined the neutralizing effect of convalescent plasma collected from six adults hospitalized with COVID-19 using a microneutralization assay with live (authentic) virus. Whole genome sequencing of the infecting virus of the plasma donors confirmed the absence of the spike mutations which characterize 501Y.V2. We infected with 501Y.V2.HV001 and 501Y.V2.HVdF002 and compared plasma neutralization to first wave virus which contained the D614G mutation but no RBD or NTD mutations. We observed that neutralization of the 501Y.V2 variants was strongly attenuated, with IC50 6 to 200-fold higher relative to first wave virus. The degree of attenuation varied between participants and included a knockout of neutralization activity. This observation indicates that 501Y.V2 may escape the neutralizing antibody response elicited by prior natural infection. It raises a concern of potential reduced protection against re-infection and by vaccines designed to target the spike protein of earlier SARS-CoV-2 variants.

HIV infection alters SARS-CoV-2 responsive immune parameters but not clinical outcomes in COVID-19 disease (preprint)

HIV infection alters the immune response and can compromise protective immunity to multiple pathogens following vaccination. We investigated the impact of HIV on the immune response to SARS-CoV-2 using longitudinal samples from 124 participants from KwaZulu-Natal, South Africa, an area of extremely high HIV prevalence. 44% of participants were people living with HIV (PLWH) and commonly had other co-morbidities, including obesity, hypertension, and diabetes. The majority of PLWH but not HIV negative participants showed CD8 T cell expansion above the normal range post-SARS-CoV-2. Yet, in participants with HIV suppressed by antiretroviral therapy (ART), CD8 expansion was associated with milder COVID-19 disease. There were multiple differences in T cell, B cell, and natural killer cell correlations in PLWH compared to HIV negative participants, including lower tissue homing CXCR3+ CD8 T cells in the presence of SARS-CoV-2 RNA in PLWH but not HIV negative and a pronounced early antibody secreting cell (ASC) expansion in HIV negative but not PLWH. These changes were COVID-19 associated: low CXCR3 correlated with increased COVID-19 disease severity across groups, and high ASC correlated with increased disease severity in HIV negative participants and waned when SARS-CoV-2 was cleared. Despite the altered response of immune cell subsets, COVID-19 disease in PLWH was mostly mild and similar to HIV negative participants. This likely reflects the heterogeneity of an effective COVID-19 immune response. Whether the differences in immune cell dynamics in PLWH will lead to different long-term consequences or compromise vaccination is yet to be determined.