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.

Molecular Epidemiology and Diversity of SARS-CoV-2 in Ethiopia, 2020-2022 (preprint)

Ethiopia is the second most populous country in Africa and the sixth most affected by COVID-19 on the continent. Despite having experienced five infection waves, >499 000 cases, and ~7 500 COVID-19-related deaths as of January 2023, there is still no detailed genomic epidemiological report on the introduction and spread of SARS-CoV-2 in Ethiopia. In this study, we reconstructed and elucidated the COVID-19 epidemic dynamics. Specifically, we investigated the introduction, local transmission, ongoing evolution, and spread of SARS-CoV-2 during the first four infection waves using 353 high-quality near-whole genomes sampled in Ethiopia. Our results show that whereas viral introductions seeded the first wave, subsequent waves were seeded by local transmission. The B.1.480 lineage emerged in the first wave and notably remained in circulation even after the emergence of the Alpha variant. The B.1.480 was out-competed by the Delta variant. Notably, Ethiopia lack of local sequencing capacity was further limited by sporadic, uneven, and insufficient sampling that limited the incorporation of genomic epidemiology in the epidemic public health response in Ethiopia. These results highlight Ethiopia role in SARS-CoV-2 dissemination and the urgent need for balanced, near-real-time genomic sequencing.

Global Expansion of SARS-CoV-2 Variants of Concern: Dispersal Patterns and Influence of Air Travel (preprint)

In many regions of the world, the Alpha, Beta and Gamma SARS-CoV-2 Variants of Concern (VOCs) co-circulated during 2020-21 and fueled waves of infections. During 2021, these variants were almost completely displaced by the Delta variant, causing a third wave of infections worldwide. This phenomenon of global viral lineage displacement was observed again in late 2021, when the Omicron variant disseminated globally. In this study, we use phylogenetic and phylogeographic methods to reconstruct the dispersal patterns of SARS-CoV-2 VOCs worldwide. We find that the source-sink dynamics of SARS-CoV-2 varied substantially by VOC, and identify countries that acted as global hubs of variant dissemination, while other countries became regional contributors to the export of specific variants. We demonstrate a declining role of presumed origin countries of VOCs to their global dispersal: we estimate that India contributed <15% of all global exports of Delta to other countries and South Africa <1-2% of all global Omicron exports globally. We further estimate that >80 countries had received introductions of Omicron BA.1 100 days after its inferred date of emergence, compared to just over 25 countries for the Alpha variant. This increased speed of global dissemination was associated with a rebound in air travel volume prior to Omicron emergence in addition to the higher transmissibility of Omicron relative to Alpha. Our study highlights the importance of global and regional hubs in VOC dispersal, and the speed at which highly transmissible variants disseminate through these hubs, even before their detection and characterization through genomic surveillance.

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.

Comparing SARS-CoV-2 sequencing methodologies during early phase detection of the Delta variant in South Africa (preprint)

Background: Genomic surveillance, with the aid of next-generation sequencing (NGS) technologies, revolutionized the SARS-CoV-2 pandemic. Coupled with high-performance analysis software, methodologies such as the Ion Torrent S5 and Illumina MiSeq dramatically improved the genomic surveillance capacity within South Africa during the height of the pandemic. Using de-identified remnant samples collected from Eastern Cape and analysis software, Genome Detective and NextClade, we compared the sequencing process, genomic coverage, quantification of mutations, and clade classification from sequence data generated by these two common “benchtop” NGS platforms. Results: Sequence data analysis revealed success rates of 175/183 (96%) and 172/183 (94%) on the Ion Torrent S5 and Illumina MiSeq, respectively. Internal quality metrics were assessed in terms of genomic coverage (>80%) and the number of mutations identified (<100). A greater number of higher-genomic coverage sequences were generated on the Ion Torrent S5 (99%) than on the Illumina MiSeq (80%) and <100 mutations was obtained by both platforms. Ion Torrent S5 generated high coverage sequences from samples having a broader range of viral loads (VL) compared to the Illumina MiSeq, which was less successful in sequencing samples with lower viral loads. Clade assignments were comparable across platforms which accurately differentiated between Beta (<45%) and Delta (≤30%) VOCs. A disparity in clade assignment was observed in <10% of sequences due to poor coverage obtained on the Illumina MiSeq, followed by a failure rate of ≤6% across the two platforms. Manual library preparation found both methods similar in terms of sample processing, handling of larger sample quantities, and clade assignment for SARS-CoV-2. Variability between the Ion Torrent S5 and Illumina MiSeq was observed in sequencing run duration (3,5 hrs vs 36 hrs), sequencing process (semi-automation vs manual), genomic coverage (99% vs 80%), and viral load requirements (broad range vs high VL). Conclusion: The Illumina MiSeq and Ion Torrent S5 are both reliable platforms capable of performing WGS with the use of amplicons and providing specific, accurate, and high throughput analysis of the SARS-CoV-2 whole viral genomes. Both sequencing platforms are feasible platforms for the genomic surveillance of SARS-CoV-2, each with its specific advantages and trade-offs.

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 (preprint)

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.

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.

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

Investment in Africa over the past year with regards to SARS-CoV-2 genotyping has led to a massive increase in the number of sequences, exceeding 100,000 genomes generated to track the pandemic on the continent. Our results show an increase in the number of African countries able to sequence within their own borders, coupled with a decrease in sequencing turnaround time. Findings from this genomic surveillance underscores the heterogeneous nature of the pandemic but we observe repeated dissemination of SARS-CoV-2 variants within the continent. Sustained investment for genomic surveillance in Africa is needed as the virus continues to evolve, particularly in the low vaccination landscape. These investments are very crucial for preparedness and response for future pathogen outbreaks.

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.

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

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 virus 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, in combination with other mutations, 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.

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 (preprint)

Background Emerging 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. Methods RdRp 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. Results 150 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). Conclusion Omicron 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.

Outcomes of laboratory-confirmed SARS-CoV-2 infection in the Omicron-driven fourth wave compared with previous waves in the Western Cape Province, South Africa (preprint)

Objectives: We aimed to compare COVID-19 outcomes in the Omicron-driven fourth wave with prior waves in the Western Cape, the contribution of undiagnosed prior infection to differences in outcomes in a context of high seroprevalence due to prior infection, and whether protection against severe disease conferred by prior infection and/or vaccination was maintained. Methods: In this cohort study, we included public sector patients aged [≥]20 years with a laboratory confirmed COVID-19 diagnosis between 14 November-11 December 2021 (wave four) and equivalent prior wave periods. We compared the risk between waves of the following outcomes using Cox regression: death, severe hospitalization or death and any hospitalization or death (all [≤]14 days after diagnosis) adjusted for age, sex, comorbidities, geography, vaccination and prior infection. Results: We included 5,144 patients from wave four and 11,609 from prior waves. Risk of all outcomes was lower in wave four compared to the Delta-driven wave three (adjusted Hazard Ratio (aHR) [95% confidence interval (CI)] for death 0.27 [0.19; 0.38]. Risk reduction was lower when adjusting for vaccination and prior diagnosed infection (aHR:0.41, 95% CI: 0.29; 0.59) and reduced further when accounting for unascertained prior infections (aHR: 0.72). Vaccine protection was maintained in wave four (aHR for outcome of death: 0.24; 95% CI: 0.10; 0.58). Conclusions: In the Omicron-driven wave, severe COVID-19 outcomes were reduced mostly due to protection conferred by prior infection and/or vaccination, but intrinsically reduced virulence may account for an approximately 25% reduced risk of severe hospitalization or death compared to Delta.

SARS-CoV-2 Genetic diversity and lineage dynamics of in Egypt (preprint)

COVID-19 was first diagnosed in Egypt on 14 February 2020. By the end of November 2021, over 333,840 cases and 18,832 deaths had been reported. As part of national genomic surveillance, 1,027 SARS-CoV-2 near whole-genomes had been generated and published by the end of May 2021. Here we describe the genomic epidemiology of SARS-CoV-2 in Egypt over this period using a subset of 976 high-quality Egyptian genomes analysed together with a representative set of global sequences within a phylogenetic framework. We show that a single lineage, C.36, introduced early in the pandemic was responsible for most cases in Egypt. Furthermore, we show that to remain dominant in the face of mounting immunity from previous infection and vaccination, this lineage evolved into various sub-lineages acquiring several mutations known to confer adaptive advantage and pathogenic properties. These results highlight the value of continuous genomic surveillance in regions where VOCs are not predominant and enforcement of public health measures to prevent expansion of existing lineages.

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.

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

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.

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.

Rapid replacement of the Beta variant by the Delta variant in South Africa (preprint)

The Beta variant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in South Africa in late 2020 and rapidly became the dominant variant, causing over 95% of infections in the country during and after the second epidemic wave. Here we show rapid replacement of the Beta variant by the Delta variant, a highly transmissible variant of concern (VOC) that emerged in India and subsequently spread around the world. The Delta variant was imported to South Africa primarily from India, spread rapidly in large monophyletic clusters to all provinces, and became dominant within three months of introduction. This was associated with a resurgence in community transmission, leading to a third wave which was associated with a high number of deaths. We estimated a growth advantage for the Delta variant in South Africa of 0.089 (95% confidence interval [CI] 0.084-0.093) per day which corresponds to a transmission advantage of 46% (95% CI 44-48) compared to the Beta variant. These data provide additional support for the increased transmissibility of the Delta variant relative to other VOC and highlight how dynamic shifts in the distribution of variants contribute to the ongoing public health threat.

Co-Creation of HIVST Delivery Approaches for Improving Urban Men’s Engagement with HIV Services in eThekwini District, KwaZulu-Natal: Nominal Group Technique (preprint)

Background: HIV self-testing (HIVST) is one of the recommended approaches for HIV testing services, particularly for helping reach populations who would not normally access facility-based HIV testing. HIVST must be tailored to different populations to ensure uptake. Objective: The main objective of this study was to develop an acceptable HIVST delivery strategy to help improve urban men’s engagement with HIV services. Methods: : We invited key stakeholders for urban men’s HIV services to participate in a co-creation workshop aimed at developing HIVST delivery approaches for urban men, using eThekwini municipality as a study setting. We conducted purposive sampling to include health care users and health care providers, representing a range of views across the public sector and voluntary sector. We employed the Nominal Group Technique (NGT) method for data collection. The NGT workshop was conducted in two consecutive phases: phase one was focused on determining barriers for men’s engagement with the current/facility-based HIV testing services; phase two was aimed at determining HIVST delivery strategies. We used the results of the NGT to design a tailored HIVST strategy for urban men in eThekwini District. Results: : Participants identified the following psychological factors as the most important barriers to uptake of HIV testing services by urban men: stigma, ignorance about the importance of testing and testing process as well as fear of positive test results. Key stakeholders suggested internal motivation strategies as a potentially effective approach to support HIVST delivery strategy. Guided by the NGT results, we designed a HIVST delivery strategy that is supported by a risk communication approach. Conclusion: We designed an evidence-based risk communication mobile health (mHealth) strategy coupled with SARS COV-2 self-testing tailored to improve men’s uptake of HIVST. A follow-up study to evaluate the feasibility of implementing these approaches is recommended.