Epidemiology and outcomes of SARS-CoV-2 infection associated with anti-nucleocapsid seropositivity in Cape Town, South Africa

BackgroundIn low- and middle-income countries where SARS-CoV-2 testing is limited, seroprevalence studies can characterise the scale and determinants of the pandemic, as well as elucidate protection conferred by prior exposure. MethodsWe conducted repeated cross-sectional serosurveys (July 2020 - November 2021) using residual plasma from routine convenient blood samples from patients with non-COVID-19 conditions from Cape Town, South Africa. SARS-CoV-2 anti-nucleocapsid antibodies and linked clinical information were used to investigate: (1) seroprevalence over time and risk factors associated with seropositivity, (2) ecological comparison of seroprevalence between subdistricts, (3) case ascertainment rates, and (4) the relative protection against COVID-19 associated with seropositivity and vaccination statuses, to estimate variant disease severity. FindingsAmong the subset sampled, seroprevalence of SARS-CoV-2 in Cape Town increased from 39.2% in July 2020 to 67.8% in November 2021. Poorer communities had both higher seroprevalence and COVID-19 mortality. Only 10% of seropositive individuals had a recorded positive SARS-CoV-2 test. Antibody positivity before the start of the Omicron BA.1 wave (28 November 2021) was strongly protective for severe disease (adjusted odds ratio [aOR] 0.15; 95%CI 0.05-0.46), with additional benefit in those who were also vaccinated (aOR 0.07, 95%CI 0.01-0.35). InterpretationThe high population seroprevalence in Cape Town was attained at the cost of substantial COVID-19 mortality. At the individual level, seropositivity was highly protective against subsequent infections and severe COVID-19. FundingWellcome Trust, National Health Laboratory Service, the Division of Intramural Research, NIAID, NIH (ADR) and Western Cape Government Health. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSIn low- and middle-income countries where SARS-CoV-2 testing is limited, seroprevalence studies can help describe the true extent of the pandemic. Infection from Omicron was associated with less severe disease in South Africa, but it is unclear whether this was due to a decrease in virulence of the variant or if prior infection provided protection. Added value of this studyThe seroprevalence data nested within a population cohort enabled us to assess differential case ascertainment rates, as well as to examine the contribution of both natural and vaccine-induced immunity in protecting communities against infections and severe disease with different SARS-CoV-2 variants. Implications of the available evidenceInequality and differential access to resources resulted in poorer communities having higher seroprevalence and COVID-19 death rates, with lower case ascertainment rates. Antibody positivity provided strong protection against an immune escape variant like Omicron but came at a high mortality cost.

O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Contribute to the Mutational Trajectory in Variants of Concern

The emergence of a polybasic cleavage motif for the protease furin in the SARS-CoV-2 spike protein has been established as a major factor for enhanced viral transmission in humans. The peptide region N-terminal to that motif is extensively mutated in major variants of concern including Alpha, Delta and Omicron. Besides furin, spike proteins from these variants appear to rely on other proteases for maturation, including TMPRSS2 that may share the same cleavage motif. Glycans found near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, with a suite of chemical tools, we establish O-linked glycosylation as a major determinant of SARS-CoV-2 spike cleavage by the host proteases furin and TMPRSS2, and as a likely driving force for the emergence of common mutations in variants of concern. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, and this glycosylation event is suppressed by many, but not all variant mutations. A novel strategy for rapid bioorthogonal modification of Thr678-containing glycopeptides revealed that introduction of a negative charge completely abrogates furin activity. In a panel of synthetic glycopeptides containing elaborated O-glycans, we found that the sole incorporation of N-acetylgalactosamine did not substantially impact furin activity, but the presence of sialic acid in elaborated O-glycans reduced furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on spike cleavage by TMPRSS2. With a chemistry-centered approach, we firmly establish O-glycosylation as a major determinant of spike maturation and propose that a disruption of O-GalNAc glycosylation is a substantial driving force for the evolution of variants of concern. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/508093v3_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@71b54eorg.highwire.dtl.DTLVardef@1361b4aorg.highwire.dtl.DTLVardef@139bdd9org.highwire.dtl.DTLVardef@1df192b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Outcomes of laboratory-confirmed SARS-CoV-2 infection during resurgence driven by Omicron lineages BA.4 and BA.5 compared with previous waves in the Western Cape Province, South Africa

ObjectiveWe aimed to compare clinical severity of Omicron BA.4/BA.5 infection with BA.1 and earlier variant infections among laboratory-confirmed SARS-CoV-2 cases in the Western Cape, South Africa, using timing of infection to infer the lineage/variant causing infection. MethodsWe included public sector patients aged [≥]20 years with laboratory-confirmed COVID-19 between 1-21 May 2022 (BA.4/BA.5 wave) and equivalent prior wave periods. We compared the risk between waves of (i) death and (ii) severe hospitalization/death (all within 21 days of diagnosis) using Cox regression adjusted for demographics, comorbidities, admission pressure, vaccination and prior infection. ResultsAmong 3,793 patients from the BA.4/BA.5 wave and 190,836 patients from previous waves the risk of severe hospitalization/death was similar in the BA.4/BA.5 and BA.1 waves (adjusted hazard ratio [aHR] 1.12; 95% confidence interval [CI] 0.93; 1.34). Both Omicron waves had lower risk of severe outcomes than previous waves. Prior infection (aHR 0.29, 95% CI 0.24; 0.36) and vaccination (aHR 0.17; 95% CI 0.07; 0.40 for boosted vs. no vaccine) were protective. ConclusionDisease severity was similar amongst diagnosed COVID-19 cases in the BA.4/BA.5 and BA.1 periods in the context of growing immunity against SARS-CoV-2 due to prior infection and vaccination, both of which were strongly protective.

Assessing the clinical severity of the Omicron variant in the Western Cape Province, South Africa, using the diagnostic PCR proxy marker of RdRp target delay to distinguish between Omicron and Delta infections: a survival analysis

BackgroundEmerging data suggest that SARS-CoV-2 Omicron variant of concern (VOC)is associated with reduced risk of severe disease. The extent to which this reflects a difference in the inherent virulence of Omicron, or just higher levels of population immunity, is currently not clear. MethodsRdRp target delay (RTD: a difference in cycle threshold value of RdRp - E > 3.5) in the Seegene Allplex 2019-nCoV PCR assay is a proxy marker for the Delta VOC. The absence of this proxy marker in the period of transition to Omicron was used to identify suspected Omicron VOC infections. Cox regression was performed for the outcome of hospital admission in those who tested positive for SARS-CoV-2 on the Seegene Allplex assay from 1 November to 14 December 2021 in the Western Cape Province, South Africa, public sector. Vaccination status at time of diagnosis, as well as prior diagnosed infection and comorbidities, were adjusted for. Results150 cases with RTD (proxy for Delta) and 1486 cases without RTD (proxy for Omicron) were included. Cases without RTD had a lower hazard of admission (adjusted Hazard Ratio [aHR] of 0.56, 95% confidence interval [CI] 0.34-0.91). Complete vaccination was protective of admission with an aHR of 0.45 (95%CI 0.26-0.77). ConclusionOmicron has resulted in a lower risk of hospital admission, compared to contemporaneous Delta infection in the Western Cape Province, when using the proxy marker of RTD. Under-ascertainment of reinfections with an immune escape variant like Omicron remains a challenge to accurately assessing variant virulence.

Selection analysis identifies unusual clustered mutational changes in Omicron lineage BA.1 that likely impact Spike function

Among the 30 non-synonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (i) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (ii) interactions of Spike with ACE2 receptors, and (iii) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any genomes within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron over all previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

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

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 Omicrons 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.

Higher mortality associated with the SARS-CoV-2 Delta variant in the Western Cape, South Africa, using RdRp target delay as a proxy

A novel proxy for the Delta variant, RNA-dependent RNA polymerase target delay in the Seegene Allplex 2019-nCoV PCR assay, was associated with higher mortality (adjusted Odds Ratio 1.45 [95%CI 1.13-1.86]), compared to presumptive Beta infection, in the Western Cape, South Africa (April-July 2021). Prior diagnosed infection and vaccination were protective.

Communicable and non-communicable co-morbidities and the presentation of COVID-19 in an African setting of high HIV-1 and tuberculosis prevalence

ObjectivesTo describe the presentation and outcome of SARS-CoV2 infection in an African setting of high non-communicable co-morbidity and also HIV-1 and tuberculosis prevalence. DesignCase control analysis with cases stratified by HIV-1 and tuberculosis status. SettingA single-centre observational case-control study of adults admitted to a South African hospital with proven SARS-CoV-2 infection or alternative diagnosis. Participants104 adults with RT-PCR-proven SARS-CoV2 infection of which 55 (52.9%) were male and 31 (29.8%) HIV-1 co-infected. 40 adults (35.7% male, 30.9% HIV-1 co-infected) admitted during the same period with no RT-PCR or serological evidence of SARS-CoV2 infection and assigned alternative diagnoses. Additional in vitro data from prior studies of 72 healthy controls and 118 HIV-1 uninfected and infected persons participants enrolled to a prior study with either immune evidence of tuberculosis sensitization but no symptoms or microbiologically confirmed pulmonary tuberculosis. ResultsTwo or more co-morbidities were present in 57.7% of 104 RT-PCR proven COVID-19 presentations, the commonest being hypertension (48%), type 2 diabetes mellitus (39%), obesity (31%) but also HIV-1 (30%) and active tuberculosis (14%). Amongst patients dually infected by tuberculosis and SARS-CoV-2, clinical features could be dominated by either SARS-CoV-2 or tuberculosis: lymphopenia was exacerbated, and some markers of inflammation (D-dimer and ferritin) elevated in singly SARS-CoV-2 infected patients were even further elevated (p < 0.05). HIV-1 and SARS-CoV2 co-infection resulted in lower absolute number and proportion of CD4 lymphocytes, with those in the lowest peripheral CD4 percentage strata exhibiting absent or lower antibody responses against SARS-CoV2. Death occurred in 30/104 (29%) of all COVID-19 patients and in 6/15 (40%) of patients with coincident SARS-CoV-2 and tuberculosis. ConclusionsIn this South African setting, HIV-1 and tuberculosis are common co-morbidities in patients presenting with COVID-19. In environments in which tuberculosis is common, SARS-CoV-2 and tuberculosis may co-exist with clinical presentation being typical of either disease. Clinical suspicion of exacerbation of co-existent tuberculosis accompanying SARS-CoV-2 should be high. What is already known on this topic?It has been quite widely thought that Africa has been spared the worst effects of the COVID-19 pandemic. There are very few reported case series and no case-control studies comparing COVID-19 patients admitted to hospital to those admitted for other reasons. However several studies have indicated both HIV-1 and tuberculosis co-infection that are endemic in Africa constitute risk factors for poor outcome. In addition Africa is subject to demographic transition and the prevalence of non-communicable co-morbidities such as type 2 diabetes, hypertension and cardiovascular disease is rising rapidly. No study from Africa has described the clinical impact on the presentation of COVID-19 infection. What this study addsTwo or more co-morbidities were present in over half COVID-19 presentations, including HIV-1 (30%) and active tuberculosis (14%). Patients dually infected by tuberculosis and SARS-CoV-2, presented as either SARS-CoV-2 or tuberculosis. HIV-1 and SARS-CoV2 co-infection resulted in lower absolute number and proportion of CD4 lymphocytes, and those with low CD4 counts had absent or lower antibody responses against SARS-CoV2. Death occurred 29% of all COVID-19 patients and in 40% of patients with coincident SARS-CoV-2 and tuberculosis. Thus in environments in which tuberculosis is common, SARS-CoV-2 and tuberculosis may co-exist with clinical presentation being typical of either disease and clinical suspicion of exacerbation of co-existent tuberculosis accompanying SARS-CoV-2 should be high.

Spectrum of neurological manifestations and systematic evaluation of cerebrospinal fluid for SARS-CoV2 in patients admitted to hospital during the COVID-19 epidemic in South Africa

Neurological manifestations of COVID-19 are increasingly described in the literature. There is uncertainty whether these occur due to direct neuroinvasion of the virus, para-infectious immunopathology, as result of systemic complications of disease such as hypercoagulability or due to a combination of these mechanisms. Here we describe clinical and radiological manifestations in a sequential cohort of patients presenting to a district hospital in South Africa with neurological symptoms with and without confirmed COVID-19 during the first peak of the epidemic. In these patients, where symptoms suggestive of meningitis and encephalitis were most common, thorough assessment of presence in CSF via PCR for SARS-CoV2 did not explain neurological presentations, notwithstanding very high rates of COVID-19 admissions. Although an understanding of potential neurotropic mechanisms remains an important area of research, these results provide rationale for greater focus towards the understanding of para-immune pathogenic processes and the contribution of systemic coagulopathy and their interaction with pre-existing risk factors in order to better manage neurological disease in the context of COVID-19. These results also inform the clinician that consideration of an alternative diagnosis and treatment for neurological presentations in this context is crucial, even in the patient with a confirmed diagnosis COVID-19.

Profile of SARS-CoV-2-specific CD4 T cell response: Relationship with disease severity and impact of HIV-1 and active Mycobacterium tuberculosis co-infection

T cells are involved in control of COVID-19, but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we assessed the magnitude, function and phenotype of SARS-CoV-2-specific CD4 T cells in 95 hospitalized COVID-19 patients (38 of them being HIV-1 and/or tuberculosis (TB) co-infected) and 38 non-COVID-19 patients, using flow cytometry. We showed that SARS-CoV-2-specific CD4 T cell attributes, rather than magnitude, associates with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity and enhanced HLA-DR expression. Moreover, HIV-1 and TB co-infection skewed the SARS-CoV-2 T cell response. HIV-1 mediated CD4 T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2; and a decrease in the polyfunctional capacity of SARS-CoV-2-specific CD4 T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mtb-specific CD4 T cells, with possible implications for TB disease progression. There results corroborate the important role of SARS-CoV-2-specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease.