Compartmentalised mucosal and blood immunity to SARS-CoV-2 associated with high seroprevalence before Delta wave in Africa

doi:10.21203/rs.3.rs-4873950/v1

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Compartmentalised mucosal and blood immunity to SARS-CoV-2 associated with high seroprevalence before Delta wave in Africa

https://doi.org/10.21203/rs.3.rs-4873950/v1

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Background

The reported number of SARS-CoV-2 cases and deaths are lower in Africa compared to many high-income countries. However, in African cohorts, detailed characterisation of SARS-CoV-2 mucosal and T cell immunity are limited. We assessed SARS-CoV-2-specific immune landscape in The Gambia pre-Delta variant in July 2021.

Methods

A cross-sectional assessment of SARS-CoV-2 immunity in 349 unvaccinated individuals from 52 Gambian households was performed between March - June 2021. SARS-CoV-2 spike (S) and nucleocapsid (N) specific binding antibodies were measured by ELISA, variant-specific serum neutralizing-antibodies (NAb) by viral pseudotype assays and nasal fluid IgA by mesoscale discovery assay. SARS-CoV-2 T-cell responses were evaluated using ELISpot assay.

Results

We show that adjusted seroprevalence of anti-Spike antibodies was 56.7% (95% confidence interval (CI) 49.0–64.0), which was lower in children < 5 years (26.2%, 13.9–43.8) and 5–17 years (46.4%, 36.2–56.7) compared to adults 18–49 years (78.4%, 68.8–85.8). In spike-seropositive individuals, NAb titres were highest to Alpha variant (median IC50 110), with 27% showing pre-existing Delta variant titres > 1:50. Whilst T-cell responses were significantly higher in spike-seropositive individuals, 34% of spike-seronegative showed reactivity to one or more T-cell antigen pools. Strong correlations within SARS-CoV-2 T-cell, mucosal IgA, and serum NAb responses was observed.

Conclusion

High SARS-CoV-2 seroprevalence in The-Gambia induced mucosal and blood immunity, reducing Delta and Omicron impact. Children were relatively protected from infection. T-cell responses in seronegative individuals may indicate either pre-pandemic cross-reactivity or individuals with a T-cell dominated response to SARS-CoV-2 infection with absent or poor humoral responses.

Biological sciences/Immunology/Adaptive immunity/Humoral immunity/Antibodies

Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Immunological memory

Age

pre-pandemic

Mucosal IgA

Neutralizing antibodies to ancestral and B.1.1.7 SARS-Cov-2 strains

Globally, high mortality and morbidity rates were associated with the COVID-19 pandemic. In Africa, the reported number of SARS-CoV-2 cases remained low and while under-reporting may have played a role, no excess mortality was seen in The Gambia during the first year of the COVID-19 pandemic. There is limited data on the detailed immune response to SARS-CoV-2 in African populations. We therefore did a cross-sectional household study in The Gambia to assess SARS-CoV-2 immune responses using blood and nasal swabs collected from the participants. Our results demonstrate that SARS- CoV-2 infection led to the induction of T cell, mucosal and systemic antibody responses but these responses where different in each of the immune compartments. We also found that more adults were infected compared to children and immune responses induced by infection with the earlier SARS-CoV-2 variants may have led to protection against infection with other subsequent variants.

SARS-CoV-2 infection was declared a pandemic by the World Health Organization (WHO) on March 11th, 2020, and as of 07th of July 2024, an estimated 775,673,955 confirmed cases and a total of 7,053,524 related deaths have been recorded worldwide¹. Africa, which accounts for 16% of the world population, has recorded the lowest rates of SARS-CoV-2 infections with only 9,580,532 million reported cases and 175,510 related deaths so far in a total of 54 countries accounting for only 1.2% of total cases worldwide¹.

In The Gambia, the first case of COVID-19 was recorded on the 17th of March 2020 and as of 07th of July 2024, 12,627 individuals have been confirmed infected with the virus with 372 related deaths recorded^1,2. As seen with other countries, a series of nationwide lockdowns were implemented to curb the spread of the virus. A state of emergency which included lockdown of the border between Gambia and Senegal, all schools, some workplaces, places of worship and nonessential shops was announced on 27th March 2020, and this was further extended multiple times^3,4.

The low number of reported SARS-CoV-2 cases and deaths in The Gambia is consistent with existing data from most African countries showing lower rates of infection and mortality in Africa compared to many high-income countries. The reported COVID-19 related deaths remained low in most African countries even during SARS-CoV-2 Alpha and Delta variant waves, despite the higher disease severity observed in many high-income countries. Although under-reporting and the young population structure may have played a role in the number of recorded cases seen, the resource limited care systems in many African countries were not overwhelmed, suggesting that many cases may have been mild or asymptomatic⁴. No excess mortality was observed in The Gambia during the first year of the COVID-19 pandemic⁵. This milder disease may also be due higher prevalence of cross-reactive SARS-CoV-2 antibodies and T-cell responses from previous exposure to other coronaviruses⁶.

The objective of our study was to describe the seroprevalence and immunity to SARS-CoV-2 in The Gambia after the first two waves, prior to the introduction of any SARS-CoV-2 vaccines and the emergence of the Delta and Omicron variants. We hypothesised that substantial humoral and cellular immunity would have been present in the population due to high attack rates with earlier SARS-CoV-2 variants, potentially limiting the impact of Delta and Omicron variants that followed in the third and fourth SARS-CoV-2 waves in The Gambia. While many studies from Africa have measured binding and neutralizing antibody responses, very few have established the extent of mucosal antibodies and T-cell immunity, which may both play a significant role in protection from future infections and severe disease.

Participant recruitment and data collection

Following community and household sensitization, interested families were invited to the MRCG clinical site for consenting and screening. Recruited households ranged in size from 6 to 10 household members, including at least one adult and one child. Household participants with travel planned during the study follow-up period were excluded. Written consent or a thumb print was obtained from all participants above 18 whilst assent was obtained from participants aged 12–18 years. Consent was obtained from the parents or guardians for participants under 12 years. Study participants were not financially compensated but provided with free transportation during clinical visits.

Study approval was given by the joint Gambia Government and Medical Research Council Unit, The Gambia (MRCG) Ethics committee, and the London School of Hygiene and Tropical Medicine ethics committee (LEO project ID 22556). The protocol was registered at clinicaltrials.gov (NCT05952336) and has been published (ref to add).

Sample collection and processing

During the baseline visit, venous blood was collected for peripheral blood mononuclear cell isolation and cryopreservation, and serum separation and storage. A synthetic absorptive matrix (SAM) strip was used to collect nasal lining fluid sample. In addition, a combined throat and nose swab (TNS) using flocked swabs was collected and transferred into RNAprotect cell reagent (Qiagen) as previously described ⁷.

Pre-pandemic samples

Following ethical approval (SCC 1269, SCC 1449, SCC 1185), 825 biobanked sera collected from unrelated studies conducted in 2016 in three villages of West Kiang Lower River Region by the MRC Unit, The Gambia were used to determine pre-pandemic cross-reactivity with SARS-CoV-2 antigens and establish the specificity of the antibody-ELISA. The samples allowed for a representative spread across age groups (1–75 years), seasons, and sex. Twenty samples were randomly selected from males and females for each month for two years to account for potential seasonal variations to exposure to human corona viruses.

SARS-CoV-2 ELISA

SARS-CoV-2 spike- (S) and N-specific immunoglobulin G (IgG) was measured using a previously described in-house ELISA, shown to hav99.5% sensitivity and 98.8% specificity for anti-S IgG, and 99.5% sensitivity and 84.1% specificity for anti-N IgG ³⁹. Immunolon 4 HBX microplates (Thermo Scientific, USA) were coated with either the full-length extracellular domain of the SARS-CoV-2 spike glycoprotein produced in mammalian cells or a full-length N protein produced in Escherichia coli ⁴⁰. A standard curve calibrated to the WHO International Standard for anti-SARS-CoV-2Immunoglobulin (NIBSC 20/136) was used to quantify S and N antibody titres. For each antigen, serostatus was determined using previously defined thresholds based on the ratio between the sample and control well optical densities.

SARS-CoV-2 Lentiviral Pseudotype Neutralisation Assays

Serum neutralizing antibodies were evaluated using Pseudotype neutralisation assays based on the protocol described by Di Genova et al. (2021). Briefly, human serum was diluted with Hams F-12 at a 1:20 dilution and serially diluted 3-fold to 1: 4860. Fifty (50)µL of the dilution series was added to the wells of a flat-bottomed white 96-well plate in duplicate, pseudoviruses were added at a titre of around 5x10⁶ RLU per well. Plates were incubated for one hour at 37°C and 5% CO2, followed by addition of CHO cells expressing ACE2/TMPRSS2 at 20,000 cells per well. The plates were incubated for 48 hours prior to assaying with Bright-Glo luciferase assay reagent. Each experiment was performed with cell only well, serum-free wells and internal calibrants generated from pools of neutralizing or non-neutralizing serum samples from participants. NT₅₀ values below 1:100 dilution was considered negative.

SAM strips processing for mucosal IgA measurements

SAM strips were thawed in their containers on ice for 30 minutes. The strips were then detached using disposable forceps and placed into a tube containing 300µl of elution buffer made up of 1% Bovine Serum Albumin (BSA) (MELFORD, 9048-46-8), 1 x protease inhibitor cocktail (Calbiochem, 539131) and the required volume of PBS (Gibco, 10010031). The tube containing the buffer is then vortexed for 30 seconds. The SAM strip and its elution buffer were then transferred into spin columns and centrifuged at 16,000 × g, for 20 minutes at 4°C. The eluate contained within the spin tube was transferred into labelled cryotubes and heat-inactivated at 56°C for 30 min. Samples were then used to measure mucosal IgA antibodies.

Meso scale discovery assay to measure mucosal IgA responses in nasal lining fluid samples

Mucosal anti-spike IgA responses to different SARS-CoV-2 variants (Ancestral, B.1.1.7, B.1.617.2, BA.1, BA.2) were measured using a V-PLEX SARS-CoV-2 Panel 25 (IgA) Kit (K15585U, MSD, Maryland, USA) according to the manufacturer's instructions. Nasal mucosal lining fluid samples obtained from the SAM strips were diluted 1:25 − 1:800 for detection of IgA antibody responses.

SARS-CoV-2 T-cell IFN-gamma ELISpots

S, N and M-specific T-cell responses were quantified using a Human IFN-gamma ELISPOT kit (Mabtech) following the manufacturer’s instructions and published protocols ⁴¹. Briefly, 50 µl of 2.5 × 10⁵ PBMCs/well were stimulated overnight (~ 18 h) with peptides representing S1, S2, M and N proteins (15-mers overlapping by 10 amino acids) at a final concentration of 2 µg/ml, a peptide pool of CMV, EBV and Influenza peptides (CEF) Stem Cell Technologies, USA). Staphylococcal Enterotoxin B/ Phytohemagglutinin-L (SEB/PHA) at 2 µg/ml (Sigma-Aldrich, USA) and medium alone were used as positive and negative controls all in triplicates. The numbers of SARS-CoV-2-specific IFN-gamma-secreting T-cells were determined using an AID Elispot Reader (AID, Strasberg, Germany) and analysed using ELISpot 7.0 Software (AID).

Results were expressed as IFN-gamma spot forming units (SFU)/10⁶ PBMC, after subtracting the responses from the medium control well. The results for a sample were considered for further analysis when responses in the positive control well were higher than 200 IFN-gamma SFU/10⁶ PBMCs and negative control lower than 50 IFN-gamma SFU/10⁶ PBMC cells. Antigen wells with responses higher than mean plus 2 standard deviations (SD) of negative control wells were considered to have a positive T-cell response.

Statistical analysis

All data management, graphical representations and statistical analyses were done in R (version 4.3.1) and RStudio (2023.09.1) using the utils (v1.4.3), dplyr (v1.1.3), ggplot2 (v3.4.4), reshape2 (v1.4.4), ggpubr (v0.6.0), ggbreak (v.0.1.2), FSA (v0.9.5), corrplot (v.0.92), dunn.test (v1.3.5), forcats (v1.0.0), wesanderson (v0.3.6), tidyverse (v2.0.0), scales (v1.2.1), stringr (v1.5.0), ggsignif (v0.6.4), cowplot (v1.1.1), viridis (v0.6.5), Hmisc (v5.1-2), patchwork (v1.1.3) and stats (v.4.3.1) packages. Seroprevalence estimates were adjusted for household clustering and enumerated by calculating the baseline odds from a mixed effects logistic regression model with household as a random effect. The Wilcoxon-test was used to compare responses between the different serogroups and between the different SARS-CoV-2 antigens for both antibody and T-cell data. Nonparametric Spearman correlations were used for the comparison between, T-cell, neutralizing antibody as well as mucosal antibody responses. The adjusted P values used in the correlation plot were calculated using the Holm-Sidak method. For comparison of cellular and T cell responses in the different age groups, the nonparametric Kruskal-Wallis test was used. In cases in which Kruskal–Wallis testing indicated significant differences, post hoc testing using Dunn’s test was performed. Correction for multiple comparisons was performed using the Bonferroni-Holm method. A threshold for significance for these tests was set at P-value < 0.05.

Cohort and participants

Between 2nd March 2021 and 13th June 2022, 349 participants from 52 households were recruited into the study, prior to the Delta wave³. The data reported here represent immunological data generated from the baseline sample collected in the study, which was a prospective, longitudinal, observational household cohort study of SARS-CoV-2 incidence and immunity (clinicaltrials.gov NCT05952336). The longitudinal SARS-CoV-2 incidence data from the study is reported elsewhere⁷. The Gambia is a small country in West Africa with a sub-tropical climate, ranked 174th by the United Nations Human Development Index in 2021⁸. The study was conducted at two urban sites, the West Coast Region, and Kanifing Municipality of The Gambia. A median of 6 individuals were recruited from each household (IQR 5–8), with 41 participants under 5 years old, 153 between 5–17 years old, 130 adults aged 18–49 years old, and 25 adults aged ≥ 50 years old. Two hundred and one (57.6%) participants were female. No participants had received SARS-CoV-2 vaccines at the time of study recruitment.

Pre-pandemic serological cross-reactivity and SARS-CoV-2 seroprevalence in 2021

Biobanked sera (n = 825) from unrelated studies conducted in 2016 in three villages of West Kiang, Lower River Region, The Gambia was used to determine pre-pandemic cross-reactivity to SARS-CoV-2 antigens. These included 69 samples from children younger than 5 years of age, 240 sera from 5–17-year-olds, 239 in the age group 18–49 and 277 for those 50 years of age and above. Analysis of binding antibody responses to SARS-CoV-2 spike (S) and nucleocapsid (N) in pre-pandemic serum showed that 630/825 (76.4%) were negative for both antigens (S-N-, Fig. 1a). A high degree of cross-reactivity to N alone was seen, with 158/825 (19.2%) classified as S-N+. Spike cross-reactivity was less pronounced, with 28 (3.4%) S + N- and only nine samples reactive to both antigens (S + N+, 1.1%). A statistical increase in cross-reactivity to nucleocapsid antibody responses with age was noted, with 4.3% of children < 5 years having N-specific antibody responses, compared to 9.6% aged 5–17 years, 23.8% aged 18–49 years and 30.3% 50 + years (Figure S1; <5years and 18-49years; p = 0.0005; 5-17years and 18-49years- p = 0.0002; 5–17 years and 50 + years, p = 2.56^e − 08 ; <5 years and 50 + years, p = 2.95^e − 06 ). Age was not associated with spike cross-reactivity in our cohort.

Following the first two-pandemic waves, 150 (43.1%) participants were found to be seropositive to both antigens (S + N+). Seropositivity to spike alone (S + N-) was seen in 46 (13.2%), with 138 (39.4%) participants seronegative to both antigens (Fig. 1b). Spike antibody titres was significantly higher in S + N + participants compared to those who were seropositive to spike alone (S + N-, p < 0.0001, Fig. 1b). Similarly, N antibody titres were higher in S + N + participants than those who were seropositive to nucleocapsid alone (p = 0.0007, Fig. 1b). In contrast, no differences were observed in pre-pandemic sera between antibody titres in dual-positive (S + N+) and single-positive (S + N- or S-N+) samples (Fig. 1a).

Due to the high pre-pandemic anti-N cross-reactivity observed, adjusted seroprevalence estimates were based on reactivity to spike alone (a combination of S + N + and S + N- groups). The overall seroprevalence in the cohort prior to the Delta wave, adjusted for household clustering, was 56.7% (95% CI 49.0–64.0). This estimate varied by age; seroprevalence was only 26.2% (95% CI 13.9–43.8) in children aged under 5 years, 46.4% (95% CI 36.2–56.7) in participants aged 5–17 years, 78%, (95% CI 68.8–85.8) in those aged 18–49, and 52.8% (95% CI 31.4–73.3) in participants aged ≥50 years.

Neutralizing antibody responses to SARS-CoV-2 variants

Serum neutralizing antibodies (NAb) to SARS-CoV-2 variants that had circulated by the time of sampling (ancestral and Alpha) and that emerged after (Delta, Omicron BA.1 and BA.2) were measured using a pseudovirus neutralization (PtNA) assay⁹ (Fig. 2a). Spike seropositive participants (S + N + and S + N-) had significantly higher NAb responses against ancestral and Alpha variants than spike seronegative (S-N + and S-N-) participants, with S + N + participants demonstrating the highest titres (Fig. 2b). In spike seropositive participants, NAb were highest to Alpha (median IC50 S + N+: 211.1, S + N-96.5), which were significantly higher than NAb against the ancestral strain (median IC50 S + N+:117.0, S + N:32.9). Neutralizing antibody responses were independent of age for all variants tested except for the ancestral variant were NAb responses was significantly higher in participants aged 5–17 years (median IC50 = 118.0, 95% CI = 3.8–974.0) compared to those aged 18–49 years (median IC50 = 69.0, 95% CI = 10.0–480.0) (adjusted p value = 0.014; Figure S2).

Mucosal IgA responses to SARS-CoV-2 variants

SARS-CoV-2 variant specific binding IgA was measured in nasal lining fluid, focused on the same variants as above. Spike-specific mucosal IgA titres were highest in participants who were seropositive to both spike and nucleocapsid (S + N+) across all variants (Fig. 3a). Participants who were only seropositive to spike (S + N-) had slightly lower titres than those S + N + that were still significantly higher for ancestral, Alpha and Delta variants compared to seronegative (S-N-) individuals (Fig. 3b). Mucosal IgA responses were independent of age for all variants (Figure S3).

T-cell responses to SARS-CoV-2 antigens

Interferon-gamma ELISpot assays were performed on participants with adequate peripheral blood mononuclear cell (PBMC) recovery (n = 289) to assess T-cell responses to SARS-CoV-2 S, N and M proteins. Following exclusion due to high background (n = 23), data from 266 participants were analysed. T-cell responses to all pools tested (S1, S2, N, M) were significantly higher in spike-seropositive (S + N + or S + N-) individuals compared to those seronegative for both spike and nucleocapsid (S-N-; Fig. 4). Interestingly, 33% of the S + N + group and 43% of the S + N- had no reactivity to any T-cell pools. However, and unlike the low serum NAb activity observed in the S-N- group (Fig. 2), several spike and nucleocapsid seronegative individuals had high T-cell responses, in some cases to multiple antigens (Fig. 4a and c). T-cell responses in seropositive participants aged 5–17 years were significantly lower than those observed in participants aged 18–49 years for several SARS CoV-2 antigen pools (S1 - adjusted p value = 0.0103, M - adjusted p value = 0.007, N - adjusted p value = 0.0006; Figure S4).

Correlation between different immune responses

To assess whether blood and mucosal immune responses were correlated with each other or compartmentalised, a correlation matrix was constructed comparing T-cell, serum NAb, mucosal and IgA responses (Fig. 5). A weak but significant positive correlation was observed between age and SARS-CoV-2 T-cell responses, and mucosal specific responses to the ancestral, Alpha and Delta strains.

Strong significant positive correlations were observed between all T-cell pools and between IgA responses to different SARS-CoV-2 variants (Fig. 5). Furthermore, significant positive correlations were present between NAb responses to ancestral, Alpha, and Delta variants. Correlations between immune compartments were absent or weak, showing compartmentalised immune memory to SARS-CoV-2.

Within this comprehensive household study conducted in a peri-urban setting in The Gambia, we have established the high seroprevalence present early in the SARS-CoV-2 pandemic, and neutralising antibody, mucosal and T-cell responses to SARS-CoV-2 in adults and children. In prepandemic samples, there was a higher seroprevalence of nucleocapsid specific SARS-CoV-2 antibody responses in adults compared to young children. Whilst strong correlation was noted within each immune parameter, the correlations between the different immune parameters were weak suggesting differences in the cellular, systemic and mucosal immune responses elicited against SARS-CoV-2.

Following the first two waves of SARS-CoV-2 and prior to the emergence of the Delta variant, we reported high seroprevalence among unvaccinated and mostly asymptomatic adult individuals living in a peri-urban setting in The Gambia⁷. These findings are similar to those reported in an earlier study, which was conducted in Farafenni, a partly rural and partly urban settlement in The Gambia around the same time ¹⁰, but the previous study was limited to pregnant women and only reported SARS-CoV-2 total receptor binding domain (RBD) immunoglobulin (Ig) M/IgG.

Our study revealed noticeable age-specific differences in seroprevalence rates ¹¹. When adjusted for spike protein only, we found that just 25% of children under 5 years old were seropositive, compared to over 70% of participants in the 18–49-year age group. Higher SARS-CoV-2 seroprevalence has been observed among younger and middle-aged adults in various African settings, albeit with significant variations, ^12–15 and in high income settings such as the UK ^16,17. The differences in SARS-CoV-2 seroprevalence across Africa, may be influenced by country-specific and regional factors, along with variations in behaviour and pandemic measures, which likely impacted transmission dynamics and may explain the observed regional disparities ¹⁸.

Neutralizing serum antibody responses were higher in 5-17-year age group compared to adults aged 18–49 years for the ancestral variant. Increased neutralizing antibody titres in young children compared to adults has been previously described¹⁹. The high neutralizing antibodies in these young children may arise from repeated viral infections especially to seasonal coronaviruses which are cross-reactive against SARS-CoV-2²⁰. Given the wide circulation of coronaviruses, especially amongst children, we accessed pre-pandemic samples to measure potential cross-reactive antibody to spike and nucleocapsid protein. The nucleocapsid specific responses to human coronaviruses are conserved and highly cross- reactive whilst the spike specific responses are more diverse. Our findings of high nucleocapsid specific responses to SARS-CoV-2, in prepandemic samples has also been reported in other African countries ²¹ and has been attributed to exposure to other seasonal coronavirus but also with the high burden of malaria ²² and dengue fever ²³ in this region.

Mucosal IgA responses have been shown to protect against viral infections. An induction of broad and persistent mucosal antibody response after primary SARS-CoV-2 infection against the spike and receptor binding domain of the virus has already been described ²⁴. In these SARS-CoV-2 infected individuals, spike and receptor binding domain mucosal antibodies increased within 7–9 days after the start of symptoms and correlated with a lower viral load and faster resolution of systemic symptoms. We showed that participants infected with SARS-CoV-2 (S + N+) had higher mucosal responses compared to those that were seronegative for all variants tested. Whilst we did not see any significant differences in mucosal antibody responses with age in our cohort, previous studies have reported an earlier and more robust induction of IgA responses in asymptomatic children compared to symptomatic children and adults ²⁵. The early induction of mucosal IgA in asymptomatic may possibly control viral replication leading to mild infections. A significant correlation in mucosal responses for Ancestral, Alpha, Delta and Omicron variants was noted in our cohort, suggesting that mucosal responses could be more cross-reactive compared to serum antibody responses. Although we have not assessed durability of the mucosal antibody responses, detection of mucosal antibodies up to 9 months post infection has been reported ²⁴. However, results from a human challenge model of SARS-CoV-2, showed that in adults, whilst both mucosal and systemic antibodies are detected within 10 days after infection, mucosal antibodies plateau at day 14 post infection whilst systemic antibodies continue to rise²⁶.

We were able to assess T-cell responses to SARS-CoV-2 antigens in a subset of participants and found that among participants who were seropositive for both spike and nucleocapsid, 67.5% had a T-cell response to at least one antigen whilst 49.6% responded to two or more antigens. Interestingly, we also observed that 50% of (S-N+) and 34.3% of (S-N-) individuals had a T-cell response to at least one SARS-CoV-2 antigen. SARS-CoV-2 specific T cell responses in spike seronegative individuals has also been reported in Kenya with 70% of these individuals responding to multiple SARS-CoV-2 specific peptide pool as opposed to responses in single peptide pools when T cell responses were assessed in prepandemic samples ²⁷. The difference in magnitude and breadth of SARS-CoV-2 specific T cells between prepandemic and exposed seronegative healthcare workers in the UK has also been reported ²⁸. Whilst the observed T-cell responses in seronegative participants in our study cohort may warrant further investigations, the observation that these responses were not limited to a single peptide similar to the Kenyan study ²⁷ suggests that they are genuine responses.

Cross-reactivity to other human coronaviruses, malaria ²², and viral infections such as dengue fever ²³ and CMV ²⁹ has been implicated in the detection of SARS-CoV-2 specific T cells in both spike and nucleocapsid seronegative individuals. Higher frequencies of SARS-CoV-2-specific T cells were found in CMV seropositive donors compared to CMV seronegative individuals using PBMCs collected pre-pandemic. Further analysis of these cross-reactive T cells showed that HLA-B:35:01 which is the most common HLA-B epitope in The Gambian population³⁰ presents both the SARS-CoV-2 spike peptide FVSNGTHWF (FVS) and the unrelated CMV pp65 peptide IPSINVHHY (IPS)²⁹. CMV seropositivity is very high in The Gambia with over 85% of individuals being positive by the first year of life ³¹. It will therefore be difficult to ascertain the exact contribution of CMV infection to the SARS-CoV-2 cross-reactive T cells in seronegative donors in our setting.

Abortive infections may be another explanation for the presence of SARS-CoV-2 T cells in seronegative participants. In seronegative participants, Swadling et al showed that SARS-CoV-2 T cells that respond to replication–transcription complex (RTC) epitopes could also recognize human coronaviruses and these were associated with abortive infections in SARS-CoV-2 exposed seronegative donors ²⁸. This suggest that SARS-CoV-2 viruses that infect these seronegative individuals were most likely rapidly cleared by the highly conserved RTC specific T cells from prior infection with human coronaviruses. Swadling et al showed presence of T cell responses to both the structural and RTC region of SARS- CoV-2 in seronegative health care workers but a higher frequency of these T cells was directed against the RTC in seronegative healthcare workers compared to unexposed individuals from a prepandemic cohort and those infected with SARS-CoV-2. NSP12 and NSP13 of the RTC region were among the most conserved across SARS-CoV-2 clades and NSP-12 was shown to be the region that elicited the highest average magnitude and frequency of responders without triggering humoral immune responses ²⁸.

The presence of SARS-CoV-2-specific CD4 + and CD8 + T cells is linked to reduced severity of COVID-19 during active infection ³². Emergence of SARS-CoV-2 variants is of great concern as it can lead to immune escape but majority of SARS-CoV-2 specific CD4 + T cells (93%) and of CD8 + T cell (97%) in naturally infected or Pfizer/BioNTech and Moderna COVID-19 mRNA vaccinated individuals are conserved across variants including Alpha, Betta and Gamma strains ³³. Slower waning of SARS-CoV-2-specific T cells compared to antibodies had been previously described³⁴. Whilst sustained CD4 + T and CD8 + T cell responses up to 8 months post infection were reported in a study by Dan et al, a more recent study reported a decrease in the magnitude of these SARS-CoV-2 specific T cell 10 months post infection with the ancestral SARS-CoV-2 strain³⁵.

The findings that induction of CD4 + and CD8 + T cells in a cohort of seronegative SARS-CoV-2 exposed individuals was associated with time since exposure ³⁶, suggest that in our cohort, viral infection with SARS-CoV-2 may have occurred at a lower threshold without seroconversion in these seronegative individuals.

The assessment of systemic, mucosal, and T-cell responses in the same individuals is a strength of our study as our data has shown that systemic antibody responses to infection differ to that at the site of pathogen entry, the mucosa. Analysis of samples from both blood and mucosal sites should be incorporated in future studies to increase our understanding of compartmentalised immune responses to infection. Our study also strengthens the evidence that seroprevalence data based on antibody responses alone may underestimate the true burden of SARS-CoV-2 in populations. We reported that whilst 43.1% of the individuals in our study had detectable antibody responses to both spike and nucleocapsid, 67.5% had a T response to at least one antigen within these seropositive individuals, suggesting that T-cell responses may be more suitable in identifying seropositive individuals.

Our study has some clear limitations. We could not assess SARS-CoV-2 specific T-cell responses in the pre-pandemic samples due to sample unavailability, but presence of SARS-CoV-2 spike, membrane, ORF3a, or ORF7 responses were detected in 31% of individuals tested using prepandemic samples ²⁷. We report the existence of pre-existing antibody and T-cell responses to coronaviruses, but the specific strain of coronavirus inducing this response cannot be ascertain within our study. To assess the role of pre-existing responses from other human coronavirus infections, future studies should assess antibody and T cell responses against the four major human coronaviruses species (a-hCCC-NL63, a-hCCC-229E, and b-hCCCHKU1, b-hCCC-OC43) in pandemic samples. The lack of flow cytometry data to assess polyfunctional T-cells which was shown to increase with repeated exposure to SARS-CoV-2 ^37,38 is also a limitation of our study. Finally, we were limited by the fact that we did not have a rural population with pandemic data to compare with our data. However, this has been addressed by the study done by Janha et al 2023, where data from both rural and urban settings in The Gambia have been reported¹⁰.

In summary, our data shows high seroprevalence of SARS-CoV-2 in The Gambia after two waves of the virus, especially in adults. Infection with SARS-CoV-2 induced systemic, mucosal and T-cell responses, with significant correlation observed with earlier variants as opposed to the later variants. Variant-specific responses were highly correlated at the mucosal level but not the serum antibody responses.

Data availability

All data supporting the findings of this study are available within the paper.

Code availability

All analyses were performed using the cited software and packages.

Competing interest

The authors declare no competing interests.

Correspondence and requests for materials should be addressed to

Beate Kampmann and Ya Jankey Jagne

Author Contributions

S.J, R.D.W, M.D, A.K.S, A.K, T.I.d.S and B.K conceived and design the study. Y.J.J, D.J, A.D, M.D, N.B, M.G, R.D.W, S.J, E.L.S, F.T, A.F.T, T.I.d.S and B.K collected and processed laboratory data. Y.J.J, D.J, A.D, M.D, M.K, N.B, R.D.W, S.J, D.Z, M.N, J.S, A.S, H.H, B.L, A.K.S, N.T, A.K, D.H, T.I.d.S and B.K analysed and interpreted data. Y.J.J, D.J, A.D, N.B, H.H, M.D, M.K, T.I.d.S and B.K assessed and verified underlying data. Y.J.J, A.D, D.J T.I.d.S and B.K drafted the article. All authors read and approved the final draft.

Acknowledgements

The study was funded by a United Kingdom Research and Innovation Grant (No. MC_PC_19084). We are extremely grateful to all the TransVir participants who took part in the study. We like to acknowledge the TransVir field team members Malang Mbenga, Fansu Dibba, Yusupha Fadera, Yusupha Faal, Dawda Jawara, Omar Jallow, Momodou Lamin Sanyang, Sarjo Wassa Koita, Hulaimatu Bangura, Mary Gibba, Maryama Jawara and Fatou Sanyang for their dedication and hard work during the field study. Our gratitude also goes to Nfamara Camara from the Research Support Office for all his support with the project and Ebrima Marenah and Ibrahim Lette for data entry.

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Compartmentalised mucosal and blood immunity to SARS-CoV-2 associated with high seroprevalence before Delta wave in Africa

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Abstract

Background

Methods

Results

Conclusion

Figures

Plain language summary

Introduction

Methods

Participant recruitment and data collection

Sample collection and processing

Pre-pandemic samples

SARS-CoV-2 ELISA

SARS-CoV-2 Lentiviral Pseudotype Neutralisation Assays

SAM strips processing for mucosal IgA measurements

Meso scale discovery assay to measure mucosal IgA responses in nasal lining fluid samples

SARS-CoV-2 T-cell IFN-gamma ELISpots

Statistical analysis

Results

Cohort and participants

Pre-pandemic serological cross-reactivity and SARS-CoV-2 seroprevalence in 2021

Neutralizing antibody responses to SARS-CoV-2 variants

Mucosal IgA responses to SARS-CoV-2 variants

T-cell responses to SARS-CoV-2 antigens

Correlation between different immune responses

Discussion

Declarations

Data availability

Code availability

Competing interest

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Author Contributions

Acknowledgements

References

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Supplementary Files

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