key: cord-1043421-qjobsg96 authors: Ndhlovu, Z. M.; Nkosi, T.; Mbatha, A.; Nsimbi, M.; Papadopoulos, A. O.; Nguni, T.; Karim, F.; Moosa, M. Y. S.; Gazy, I.; Jambo, K.; COMMIT-KZN,; Hanekom, W.; Sigal, A. title: Unsuppressed HIV infection impairs T cell responses to SARS-CoV-2 infection and abrogates T cell cross-recognition date: 2022-04-06 journal: nan DOI: 10.1101/2022.04.05.22273453 sha: 997909d71e3bce0fa5eabdcf5e9cd54476c668f5 doc_id: 1043421 cord_uid: qjobsg96 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. Despite measures to contain the spread of SARS-CoV-2 infection, the pandemic is persisting, with a devastating impact on healthcare systems and the world economy (1). The research community rapidly mobilized and developed vaccines and therapeutics at unprecedented speed (2, 3). COVID-19 vaccines have prevented serious illness and death and have in some cases interrupted chains of transmission at community level (4). However, the COVID-19 pandemic remains a major concern in Africa due to dismal vaccine coverage (5) and the emergence of variants of concern that may be more transmissible, cause more severe illness, or have the potential to evade immunity from prior infection or vaccination (6). The interaction of HIV-1 infection, common in sub-Saharan Africa, (7), with COVID-19 remains understudied. Initial small studies reported that PLWH had similar or better COVID-19 outcomes (8, 9) . Larger epidemiological studies have demonstrated increased hospitalization and higher rates of COVID-19-related deaths among PLWH compared with HIV negative individuals (10-13). Other studies have linked HIV mediated CD4⁺ T cell depletion to suboptimal T cell and humoral immune responses to . A recent study showed prolonged shedding of high titre SARS-CoV-2 and emergence of multiple mutations in an individual with advanced HIV and antiretroviral treatment (ART) failure (15). Although B cells have repeatedly been shown to play a pivotal role in immune protection against SARS-CoV-2 infection and antibody responses and are typically used to evaluate immune responses to currently licensed 17) , mounting evidence suggest that T cell responses are equally important. For instance, strong SARS-CoV-2-specific T cell responses are associated with milder disease (14, (18) (19) (20) (21) . Moreover, T cell responses can confer protection even in the absence of humoral responses, given that, patients with inherited B cell deficiencies or hematological malignancies are able to fully recover from SARS-CoV-2 infection (22) . In some instances, COVID-19 disease severity has been attributed to poor SARS-CoV-2-specific CD4⁺ T cell polyfunctionality potential, reduced proliferation capacity and enhanced . Importantly, a recent study identified nonsynonymous mutations in known MHC-1-restricted CD8 + T cell epitopes following deep sequencing of SARS-CoV-2 viral isolates from patients, demonstrating the capacity of SARS-CoV-2 to escape from CTL recognition (23). Regarding vaccine induced T cell responses, it was recently shown that mRNA vaccines can stimulate Th1 and Th2 CD4 + T cell responses that correlate with post-boost CD8 + T cell responses and neutralizing antibodies (24). The cited examples herein, highlight the need to gain more insight into T cell mediated protection against . This study used a cohort of PLWH and HIV-seronegative individuals diagnosed with COVID-19 during the first wave dominated by the wildtype D614G virus (26), and the second wave dorminated by the Beta variant. PBMCs were used to determine the impact of HIV infection on SARS-CoV-2 specific T cell responses and to assess T cell cross-recognition. Our data showed impaired SARS-CoV-2 specific T cell responses in individuals with unsuppressed HIV infection and highlighted poor cellular cross-recognition between variants, which was more pronounced than those with unsuppressed HIV. The muted responses in unsuppressed HIV infection may be attributable to low absolute CD4 count and immune activation. Importantly, we identified mutations in the Beta variant that could potentially reduce T cell Immunity to SARS-CoV-2 typically induces robust T cell responses but the impact of HIV infection on these responses has not been fully elucidated (22, 28, 29) . Thus, we sought to determine the impact of HIV infection on SARS-CoV-2-specific CD4 + and CD8 + T cell responses. PBMCs were stimulated with PepTivoter 15 mer megapools purchased from Miltenyi Biotec. The pools contained predicated CD4 and CD8 epitopes spanning the entire Spike coding sequence (aa5-1273). Intracellular cytokine staining of peptide stimulated PBMCs was followed by flowcytometric analyses described in the methods section. The samples used for these analyses were collected between two to four weeks after COVID-19 PCR positive diagnosis. Representative flow plots for each group and aggregate data show viremic PLWH had significantly lower frequencies of SARS-CoV-2 specific IFNγ/TNF-a-produding CD4 + T cells compared to suppressed PLWH (p=0.002) and HIVseronegative individuals (p=0.0006) ( Figure 1A) . Similarly, viremic PLWH had significantly lower frequencies of SARS-CoV-2 specific IFN-γ/TNF-a-producing CD8 + T cells than HIV-seronegative individuals (p=0.02) ( Figure 1B ). But no significant differences in SARS-CoV-2 specific CD4 + T cell or CD8 + T cell frequencies was observed between the suppressed PLWH and HIV seronegative individuals ( Figure 1A & 1B). Simultaneous production of cytokines, commonly referred to as polyfunctionality, which is regarded as a measure of the quality of the T cell response, has been shown to correlate with viral control (30). Thus, we evaluated the quality of the CD4 + and CD8 + T cell responses among the groups by enumerating cells co-producing IFN-γ, TNF-α and IL-2. Cells producing all three cytokines were very rare regardless of HIV status ( Figure 1C & 1D) . The patterns of cytokine production differed in viremic PLWH compared to HIV-seronegative individuals and suppressed PLWH. HIV-seronegative individuals and suppressed PLWH predominantly exhibited IFN-γ responses whereas viremic PLWH predominantly displayed TNF-α responses for both CD8 + and CD4 + T cells ( Figure 1C & 1D) . Comparing frequencies of polyfunctional responses between groups confirmed that HIV-seronegative individuals had significantly more dual cytokine producing CD8 + and CD4 + T cells compared to viremic PLWH (p=0.0330 for CD4 Figure 1C ; p=0.0368 for CD8 Figure 1D ). Viremic PLWH had significantly lower frequencies of monofunctional IFN-γ producing CD8 + and CD4 + T cells than suppressed PLWH and HIV seronegative individuals (p=0.0263) ( Figure 1C & 1D) . Together, these data show that uncontrolled HIV infection lowers the magnitude and alters the quality of SARS-CoV-2 T cells. Importantly, complete plasma HIV suppression preserves the capacity to mount high magnitude dual-functional SARS-CoV-2 specific T cell responses. Having observed difference in magnitude and quality of SARS-CoV-2 spike specific T responses, we next measured responses directed against major structural proteins, the nucleocapsid (N) and the membrane (M), again using PepTivoter peptide pools from Miltenyi biotec. Our data show that, although all three major SARS-CoV-2 proteins are targetted, there was a preponderance for T cells to target the S, particularly by HIV-seronegative individuals (Figure 2A & 2B) . These data suggest that HIV diminishes Spike specific T cell responses. To evaluate the impact of uncontrolled HIV infection on cross reactive T cell responses between wt and the Beta variant, we compared the breadth of responses and the ability to cross-recognize SARS-CoV-2 Beta variant peptides among the three study groups. These studies were conducted using two sets of 15mer overlapping peptides. Set 1 was comprised of 16 wild type (wt) peptides, spanning the receptor binding domain (RBD) and non RBD regions of spike (S) that are known hotspots for mutations (31). Set 2 consisted of corresponding peptides that included all the major mutations that define the Beta variant lineage (32). A detailed description of the peptides is contained in (Supplementary Table 1 ). We first sought to determine cross reactivity of SARS-CoV-2 specific CD4 + and CD8 + T cells induced following infection with the wild type (D614G, Wave 1) and Beta variant (Wave 2), between each other. We found that wave 1 donors had significantly lower CD8 + (p=0.0312) and CD4 + T cell responses (p=0.0078) to Beta variant relative to corresponding wt responses ( Figure 3A ). Wave 2 donors had no significant differences in T cells responses to Beta and wt ( Figure 3B ). Using a 12 days cultured stimulation assay, we were able to massively expand the magnitude of SARS-CoV-2 specific CD4 + and CD8 + T cells ( Figure Table 1) . Representative data for a wave 1 donor shows three CD8 + and two CD4 + positive wt responses (red circles), that did not cross-recognize corresponding Beta variants (blue bars) ( Figure 3D ). Contrariwise, a representative wave 2 donor had one CD8 + and one CD4 + T cell response to the Beta variant that did not cross-react to the wt version of the peptide ( Figure 3E ). Intra-donor comparison revealed significantly more CD8 + (p=0.0156) and CD4 + T cell responses (p=0.0312) to wt peptides compared to the corresponding Beta variant peptides in wave 1 donors ( Figure 3F ). Conversely, unlike the ex vivo data ( Figure 3B ), wave 2 donors had significantly more CD8 + T cell responses to Beta variant peptides relative to wt peptides (p=0.0312), and a trend towards increased CD4 + T cells against Beta peptides (p=0.0625), highlighting the increased sensitivity of expanded cells ( Figure 3G ). Together, these data show poor cross-recognition of wt and Beta variant epitopes. We then assessed the impact of HIV infection on cross recognition of wt and Beta variant epitopes. Representative data for a HIV-seronegative individual from the first wave had 8 wt and 5 Beta variant CD8 + T cell responses, one was cross-recognized (circled) ( Figure 4A ). The same individual had 5 wt and 5 Beta variant CD4 + T responses, none was cross-recognized ( Figure 4B ). Similarly, a representative suppressed wave 1 donor had 5 wt and 2 Beta variant responses one of which was cross recognized ( Figure 4C ). This same donor had 6 wt and zero Beta variant CD4 + T cell responses ( Figure 4D ). A representative viremic individual had 4 weak wt CD8 + T cell responses and 3 borderline CD4 responses, none of which were crossrecognized ( Figure 4E & 4F) . Summary data showed viremic PLWH had significantly narrow breadth of SARS-CoV-2 specific CD8 + (p=0.039) and CD4 + T cell responses (p=0.033) compared to suppressed PLWH and HIV seronegative individuals ( Figure 4G & 4H). Collectively, these data show that SARS-CoV-2 specific T cell responses in viremic PLWH have limited breadth and subsequently poor cross-recognition potential. Having shown poor T cell cross-recognition of SARS-CoV-2 epitopes between wt and Beta variant, we next sought to identify mutations that might be responsible for the loss of recognition. We combined all the T cell data for the 12 donors used for cultured epitope screening studies. This analysis identified four Beta variant peptides (listed in Table 2 ) that had significant reduction in CD8 + T cell recognition relative to wt peptides ( Figure 5A ). Three of these peptides were also poorly recognized by CD4 + T cells ( Figure 5B ). The amino acid sequences for wt and corresponding mutations include the E484K mutation, a key Beta variant spike residual change also associated with loss antibody binding (33). Together, these data identified mutations in the Beta variant that may abrogate T cell recognition, suggesting that they may be potential T cell escape mutations and warrant further investigation. Immunodominance hierarchy of SARS-CoV-2 CD8 + and CD4 + T cell responses targeting the spike protein. Virus specific CD8 + and CD4 + T cells typically target viral epitopes in a distinct hierarchical order (34, 35) . Identifying SARS-CoV-2 epitopes that are most frequently targeted by T cells is important for the design of vaccines that can induce protective T cell responses. To determine the immunodominance hierarchy of SAR-CoV-2 specific T cell responses targeting the spike protein, OLPs were ranked based on magnitude and frequency of recognition. This analysis revealed the most immunodominant wt peptides targeted by CD8 + T cell responses ( Figure 6A To gain more insight into why viremic PLWH responded poorly to SARS-CoV-2 infection, we investigated if T cell activation defined here as co-expression of CD38 and HLA-DR, absolute CD4 count and plasma viral load, impacted immune responses (36). The proportion of activated (CD38/HLA-DR) SARS-CoV-2 specific CD4 + T cells was higher in viremic PLWH compared to suppressed (p=0.02) and HIV seronegative individuals (p=0.002) ( Figure 7A ). Moreover, proportion of activated (CD38/HLA-DR) SARS-CoV-2 specific CD4 + T cells among viremic PLWH negatively correlated with absolute CD4 counts (r=-0.7, p=0.04: Figure 7B ), and positively correlated with HIV plasma viral loads (r=0.9, p=0.0004: Figure 7C ). Similarly, proportion of activated (CD38/HLA-DR) SARS-CoV-2 specific CD8 + T cells were significantly higher in viremic PLWH relative to suppressed PLWH (p=0.04) and HIV seronegative individuals (p=0.0008; Figure 7D ). The negative relationship between proportion of activated (CD38/HLA-DR) SARS-CoV-2 specific CD8 + T cells and CD4 counts did not reach statistical significance ( Figure 7E ), but proportion of activated (CD38/HLA-DR) SARS-CoV-2 specific CD8 + T cells were positively correlated with HIV plasma viral loads among viremic PLWH (r=0.8, p=0.0006; Figure 7F ). Together, these data suggest that hyper immune activation driven by uncontrolled HIV infection impacts SARS-CoV-2 specific CD4 + and CD8 + T cell responses. Finally, we interrogated the relationship between SARS-CoV-2 specific responses and disease severity, stratified into asymptomatic, mild and severe disease requiring oxygen supplementation, as previously defined (27). We found no significant differences between the magnitude of CD4 + or CD8 + T cell responses and diseases severity among the groups ( Figure S2 A, B) . We next, examined sex differences and found no difference in CD4 + and CD8 + T cell responses to SARS-CoV-2 infection ( Figure S2 C, D). Age is a risk factor for severe COVID-19 (5), thus, we examined the relationship between age and T cell responses. There was a negative relationship between age and magnitude of CD8 + T cell responses (CD8 r=-0.6, p=0.002: Figure S2 The greater burden of HIV in sub-Saharan Africa, makes investigating the impact of HIV infection on COVID-19 immunity and disease outcomes critical for bringing the epidemic under control in the region. Recent studies have documented strong cellular responses following SARS-CoV-2 infection and vaccination, but the effects of HIV on SARS-CoV-2 specific T cell responses is not well characterized. Here, we investigated the antigen-specific CD4 + and CD8 + T cell responses in a cohort of SARS-CoV-2 infection. We also show that low absolute CD4 count, and hyper immune activation are associated with diminution of SARS-CoV-2 specific T cell responses. Importantly, we identify spike mutations in the Beta variant that abrogate recognition by memory T cells raised against wt epitopes. Similarly, immune responses targeting Beta variant epitopes poorly cross recognize corresponding wt epitopes. These data reveal the potential for emerging SARS-CoV-2 variants to escape T cell recognition. Importantly, our data highlight the potential for unsuppressed HIV infection to attenuate vaccine induced T cell immunity. HIV induced immune dysregulation is well documented (37). Unsuppressed HIV infection is associated with profound dysfunction of virus-specific T cell immunity partly caused by immune activation (37, 38) . Our data show that individuals with unsuppressed HIV infection mount weak responses to SARS-CoV-2 infection and poorly recognize SARS-CoV-2 Beta variant mutations. In this study, HIV induced immune defects such as low CD4 + T cell counts, higher HIV plasma viral loads and elevated immune activation were invariably associated with diminished SARS-CoV-2 responses. This suggest that HIV induced immune dysregulation negatively impacts the potential to mount robust T cell responses to SARS-CoV-2 infection. Furthermore, although ART mediated HIV suppression rarely results in complete immune reconstitution (39), sustained complete plasma HIV suppression was associated with robust SARS-CoV-2 responses that were mostly similar in magnitude and quality to responses mounted by HIV-seronegative individuals. Given All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint reduced levels of CD38 and HLA-DR dual positive cells and near normal absolute CD4 counts in suppressed individuals, it is reasonable to speculate that reduced immune activation and superior CD4 + T helper function were partly responsible for improved immune responses in suppressed individuals. The emergence of several SARS-CoV-2 variants with mutations in the viral Spike (S) protein such as mutations in the receptor binding domain (RBD), N-terminal domain (NTD), and furin cleavage site region (40) continue to fuel the epidemic. These mutations have been shown to directly affect ACE2 receptor binding affinity, infectivity, viral load, and transmissibility (40-42). The variants of concern identified since the start of the COVID-19 pandemic include the Alpha (43), Beta (44), Gamma (45), and Delta (46) and now the Omicron variant. Most of these have been shown to attenuate neutralization but the impact of these mutations on T cell responses has not been extensively explored (47). However, a recent report demonstrating the potential for SARS-CoV-2 to evade cytolytic T lymphocyte (CTL) surveillance, highlight the need for more investigations regarding the potential CTL driven immune pressure to shape emerging variants (23). To this end, our study provides new evidence that SARS-CoV-2 has the potential to evade T cell recognition. Moreover, our data suggest that spike mutations in the Beta vatiant that were associated with antibody escape may also escape T cell recognition. Southern Africa, has had at least three epidemic waves of COVID-19. The first was a mixture of SARS-CoV-2 lineages (with D614G), the second wave was driven by the Beta variant (48) and the third by the Delta variant (49). The region is currently experiencing the fourth wave dominated by the highly mutated Omicron variant (50, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint 51). Intriguingly, there was some evidence that PLWH in South Africa had increased disease severity in the second wave compared to the first wave(27). The precise mechanisms responsible for increased severity are not fully understood, but low CD4 + T cell counts and high neutrophil to lymphocyte ratio (NLR) showed strong association with disease severity (27). Our data suggest that diminished T cell responses to the Beta variant even in previously exposed individuals may have contributed to severe disease in the second wave. Although, we repeatedly showed robust in vitro T cell expansion following ex vivo peptide stimulation but limited expansion against mutant versions of the peptides, there is need to identify optimal peptides that were targeted by CD8 + and CD4 + T cells in the context of restricting MHC class I and II alleles. SARS-CoV-2 responses are generally very broad (29), thus, it is not clear from these studies how loss of T cell cross recognition in Spike affects the overall protective immunity. Furthermore, investigating if the observed poor T cell cross-recognition between wave 1 and wave 2 is generalizable to the Delta and the Omicron variants is clearly warranted. Importantly, our data raises the question of whether CTL selection pressure plays a significant role in shaping emerging variants. This concept should be investigated using larger longitudinal studies with longer durations of follow-up. In conclusion, we show that uncontrolled HIV infection is associated with low magnitude, reduced polyfunctionality and diminished cross-recognition of SARS-CoV-2 specific CD4 + and CD8 + T cell responses. Importantly, fully suppressed PLWH had comparable SARS-CoV-2 specific T cell responses with HIVseronegative individuals. These findings may partly explain high propensity for All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint severe COVID-19 among PLWH and also highlights their vulnerability to emerging SARS-CoV-2 variants of concern, especially those with uncontrolled HIV infection. Hence, there is need to ensure uninterrupted access to ART for PLWH during the COVID-19 pandemic. Blood samples used in this study were collected between one to three weeks after COVID-19 PCR positive diagnosis. HIV testing was done using a rapid test and viral load quantification was performed from a 4ml EDTA by a commercial lab (Molecular Diagnostic Services, Durban, South Africa) using the Real Time HIV negative1 viral load test on an Abbott machine. CD4 counts were performed by a commecial lab (Ampath, Durban, South Africa). PLWH were categorised into suppressed and unsuppressed based on viral load measurements of <50 and > 1000 copies/ml respectively, at the time of sample collection. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density gradient method and cryopreserved in liquid nitrogen as previously described (Karim et al., 2020) . Frozen PBMCs were thawed, rested, and stimulated for 14 hours at 37 o C, 5% CO2 with either staphylococcal enterotoxin B (SEB, 0.5 µg/ml), SARS-CoV-2 wild type peptide pool (8 ug/ml), 501Y. Differences between groups were considered to be significant at a P-value of <0.05. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10. 1101 /2022 PBMCs at a concentration of 2 million cells per well in a 24-well plate in R10 medium were stimulated with 10 μg/ml of SARS-COV-2 OLPs peptide pools spanning the entire spike protein. The cells were incubated at 37°C in 5% CO2. After 2 days, the cells were washed and fresh R10 medium supplemented with 100 U/ml recombinant IL-2 was added. Cultured cells were fed twice weekly with regular medium replenishment. On day 14, the cells were washed three times with fresh R10 medium and rested at 37°C in 5% CO2 overnight in fresh R10 medium. On the following day, the cells were simultaneously assessed for their peptide specificity and functional activity by ICS. All statistical analyses were conducted with GraphPad Prism 9.3.1 (GraphPad Software, La Jolla, California, USA) and P values were considered significant if less than 0.05. Specifically, the Mann-Whitney U and Kruskal-Wallis H tests were used for group comparisons. Additional post hoc analyses were performed using the Dunn's multiple comparisons test. Correlations between variables were defined by the Spearman's rank correlation test. Categorical data was analysed using the Fisher's exact test. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. medRxiv, 2020 medRxiv, .2007 medRxiv, .2002 medRxiv, .20145185 (2020 . A All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint A. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. P values calculated using Wilcoxin matched -pairs signed rank T test. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. A. C. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. C. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint Figure S1: Cross-recognition of SARS-CoV-2 CD4 + T cell responses between wt and Beta variants in wave 1 and wave 2 COVID-19 patients: PBMC were expanded for 12 days in the presence of S1S2 SARS-CoV-2 peptide pools. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10. 1101 /2022 Expanded cells were tested against wt and corresponding Beta variants at single peptide level. (a) Intra-donor SARS-CoV-2 specific T cell responses to wt and corresponding Beta variant peptides by wave 1 participants. (b) Intra-donor SARS-CoV-2 specific T cell responses to wt and corresponding Beta variant peptides in wave 2 participants. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273453 doi: medRxiv preprint A Statistical Analysis of Impact of COVID19 on the Global Economy and Stock Index Returns We would like to thank our study participants, the laboratory and clinic staff at Africa Health Research Institute for collecting the samples and compiling the clinical demographic data for the study. We would like to thank Drs Wendy Burgers, All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.