Cross-reactive immunity against SARS-CoV-2 N protein in Central and West Africa precedes the COVID-19 pandemic

Overall, under our experimental conditions, cross-reactive antibodies against SARS-CoV-2 N protein do not significantly reduce the viral load in the lungs. Whether the presence of these antibodies correlates with decreased pathology or moderation of symptoms remains to be investigated. In the mouse model of infection, the Fc mediated antibody functions of the purified human antibodies are limited, hence underestimating their therapeutic contributions10. Furthermore, our work is limited to the humoral response against SARS-CoV-2. Individuals from Gabon and Senegal likely also have cross-reactive T cells against SARS-CoV-2 N protein. Upon exposure to SARS-CoV-2, these individuals would mount a recall response resulting in a sharp increase in the magnitude of both cellular and humoral response. Therefore, our results from the mouse model of infection probably underestimate the protective efficacy of cross-reactive immunity against SARS-CoV-2 N protein. It is worth noting that recent studies have demonstrated the protective role of the adaptive immunity against the SARS-CoV-2 N protein. First, immunizing mice with both SARS-CoV-2 N and S proteins improved vaccine efficacy, notably viral control within the brain of SARS-CoV-2 challenged mice11. Second, a peptide vaccine encoding for T cells epitopes from the N protein was able to prevent lung abnormalities and reduce viral shedding in the majority of non-human primates challenged with SARS-CoV-212. Finally, a N specific antibody, isolated from a recovering individuals, was demonstrated to inhibit SARS-CoV-2 N mediated complement hyperactivation. The latter is a risk factor for morbidity and mortality in SARS-CoV-2 infected individuals13. The above studies suggest that N specific T cells and antibodies can both impact the severity of COVID-19.

Cross-reactive cellular and humoral immunity against additional SARS-CoV-2 protein may also contribute to SARS-CoV-2 protection. It is worth noting that younger demographic alone cannot explain the lower number of reported COVID-19 cases in Africa. Several countries in South America and Asia, have young populations but still reported significantly larger SARS-CoV-2 circulation compared with African countries.

Coronaviruses circulate widely in both the animal and human populations6. Spill-over events from susceptible animals to individuals can lead to human exposures. SARS-CoV-2 is closely related to the bat coronavirus, Bat Cov RatG13. The current COVID-19 pandemic may therefore have originated from a spill-over event from an infected bat1. Similar spill-over events of less pathogenic coronaviruses into the human populations may have occurred previously, or maybe located on an ongoing basis.

Individuals from Gabon and Senegal exhibited high levels of antibodies against the SARS-CoV-2 N and/or S protein. In contrast, individuals from the other tested locations, including Brazil, Canada and Denmark, had no or few antibodies against either protein. This low level of cross-reactive antibodies against SARS-CoV-2 are in line with previous studies. Low levels of pre-existing humoral against the SARS-CoV-2 S protein in uninfected individuals has also been documented in France, England and the US14,15,16.

The causes underlying for the high seropositivity against SARS-CoV-2 in the Gabonese and Senegalese populations remain elusive. The common HCoVs, 229E, OC43, NL-63 and HKU1, are ubiquitous throughout the globe6. Others have hypothesized that infections with these common HCoVs could lead to cross-reactive antibodies against SARS-CoV-217,18. However, a recent serological study demonstrated that antibodies against the N protein of common HCoVs are equally present in Africa, Europe and South Africa and their presence do not correlate with cross-reactive antibodies against SARS-CoV-2 N protein18. It is therefore highly unlikely that common HCoVs are the main contributor to the higher cross-reactive immunity observed in sera from Gabon and Senegal. In these pre-pandemic samples, high seropositivity against SARS-CoV-2 N could be indicative of the undocumented circulation of other as yet unrecognized coronaviruses. While the S proteins of coronaviruses are more diverse, their N proteins are far more conserved6,9. At the amino acid level, SARS-CoV-2 N protein shows between 93 and 100% homology with the N protein of several coronaviruses from bats (QHR63308.1; AVP78038.1; AVP78049.1) and pangolin origin (including QIG55953.1 , QIA48630.1, QIQ54056.1, QIA486481.1) (Fig. S2). Both animals are present in Central and West Africa; and several coronaviruses from both the alpha and betacoronavirus genus, have been isolated from bats in Gabon and Guinea19,20. These coronaviruses were however distinct from SARS-CoV-2. It is also worth noting that the possibility of different pathogens able to induce cross-reactive antibodies to SARS-CoV-2 N protein cannot be excluded. However, the co-occurrence of antibodies against SARS-CoV-2 and other pathogens is not sufficient to conclude that these pathogens are responsible for cross-reactive antibodies, especially in endemic areas for Plasmodium and Dengue virus21.

The above findings are restricted to Gabon and Senegal. Of note, separate studies also found high pre-existing immunity against SARS-CoV-2 N and to a lower extend S protein in samples from Nigeria, Ghana, Benin (West Africa), Gabon (Central Africa), as well as Tanzania ( East Africa) and Zambia (Southern Africa) indicating that this phenomenon is wide spread in Africa17,18,22,23. Existing serological assays relying on the detection of antibodies against either SARS-CoV-2 N or S protein. These assays, especially the ones monitoring SARS-CoV-2 N antibodies, will overestimate SARS-CoV-2 prevalence in Africa due to the significant number of false positives. For accurate evaluation of SARS-CoV-2 seroprevalence in Africa, the specificity of the serological assays used should first be confirmed using local pre-pandemic samples. Furthermore, a high proportion of BSA reactive samples were detected in Gabon and Senegal. Such samples exhibiting non-specific binding should also be excluded from seroprevalence studies.

Overall, the evidence presented herein supports the idea that cross-reactive humoral immunity against SARS-CoV-2 N protein is common in Gabon and Senegal. Individually, antibodies targeting SARS-CoV-2 N protein did not impact viral load in the mouse model of infection, in which Fc mediated functions are not fully recapitulated and which does not take into account the sharp rise in adaptive immunity following a recall response. Additional work will be required to determine whether the presence of both cellular and humoral responses against the SARS-CoV-2 N protein could contribute to the lower COVID-19 disease prevalence observed in Africa.