Induction of an early IFN-γ cellular response and high plasma levels of SDF-1α are inversely associated with COVID-19 severity and residence in rural areas in Kenyan patients

Abstract

Introduction: COVID-19 was less severe in Sub-Saharan Africa (SSA) compared with Europe and North America. It is unclear whether these differences could be explained immunologically. Here we determined levels of ex vivo SARS-CoV-2 peptide-specific IFN-γ producing cells, and plasma cytokines and chemokines over the first month of COVID-19 diagnosis among Kenyan COVID-19 patients from urban and rural areas.

Methods: Between June 2020 and August 2022, we recruited and longitudinally monitored 188 COVID-19 patients from two regions in Kenya, Nairobi (urban, n = 152) and Kilifi (rural, n = 36), with varying disease severity - severe, mild/moderate, and asymptomatic. IFN-γ secreting cells were enumerated at 0-, 7-, 14- and 28-days post diagnosis by an ex vivo enzyme-linked immunospot (ELISpot) assay following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with overlapping peptides from several SARS-CoV-2 proteins. A multiplexed binding assay was used to measure levels of 22 plasma cytokines and chemokines.

Results: Higher frequencies of IFN-γ-secreting cells against SARS-CoV-2 spike peptides were observed on the day of diagnosis among asymptomatic compared to patients with severe COVID-19. Higher concentrations of 17 of the 22 cytokines and chemokines measured were positively associated with severe disease, particularly interleukin (IL)-8, IL-18 and IL-1ra (p < 0.0001), while a lower concentration of SDF-1α was associated with severe disease (p < 0.0001). Concentrations of 8 and 16 cytokines and chemokines including IL-18 were higher among Nairobi asymptomatic and mild patients compared to their respective Kilifi counterparts. Conversely, concentrations for SDF-1α were higher in rural Kilifi compared to Nairobi (p = 0.012).

Conclusion: In Kenya, as seen elsewhere, pro-inflammatory cytokines and chemokines were associated with severe COVID-19, while an early IFN-γ cellular response to overlapping SARS-CoV-2 spike peptides was associated with reduced risk of disease. Living in urban Nairobi (compared with rural Kilifi) was associated with increased levels of pro-inflammatory cytokines and chemokines.

Fig 1. Higher IFN-γ responses to spike protein were observed in asymptomatic infection at the day diagnosis.

Frequencies of ex-vivo IFN-γ secreting cells against SARS-CoV-2 peptide pools spanning (a) spike responses, (b) M responses, (c) NP responses, (d) NSP responses and (e) ORF responses. Bars represent geometric mean and 95% CI. Linear mixed effects model with Tukey’s multiple comparisons, was used, * P < 0.05. Number of samples analysed for: spike responses = 171, M responses = 136, NP responses = 162, NSP responses = 100 and ORF responses = 90.

Fig 2. Higher IFN-γ responses to spike protein were observed in asymptomatic infections from Kilifi than Nairobi at the day diagnosis.

Comparison of IFN-γ cellular responses between Kilifi and Nairobi COVID-19 patients with: asymptomatic disease for (a) Spike responses, (b) M responses, (c) NP responses; Mild disease for (d) Spike responses, (e) M responses, (f) NP responses; and Severe disease for (g) Spike responses, (h) M responses, (i) NP responses. Kilifi didn’t have severe patients. Bars represent geometric mean and 95% CI. Kruskal–Wallis one-way ANOVA, with Dunn’s multiple comparisons test, was performed. * P < 0.05, **P < 0.01.

Fig 3. High plasma levels of pro-inflammatory cytokines and SDF-1α are positively and inversely, respectively, associated with COVID-19 severity.

Mean of log10-transformed cytokine/chemokine concentrations plotted over time for (a) Asymptomatic participants, (b) Mild/Moderate participants, and (c) Severe participants. * - Levels for all participants were below detectable levels.

Fig 4. Higher Cytokine and Chemokine concentrations in Asymptomatic and Mild Patients from Nairobi Compared to Kilifi, and in Mild/Moderate and Severe Patients Compared to Asymptomatic Individuals.

ns – not-significant, p value >0.05, empty means it was significant at p < 0.05 – < 0.0001. * - Levels for all participants were below detectable levels.

Fig 5. Asymptomatic patients cluster together in scatter plots by Severity at day of diagnosis.

(a) PC1 vs PC2; (b) PC2 vs PC3.