Characterization of T cell responses to the non-structural proteins of the M segment in survivors of Crimean-Congo haemorrhagic fever
Maotoana, Makgotso Golda
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Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is one of the most widely distributed arboviruses globally. The disease caused by the virus, Crimean- Congo haemorrhagic fever (CCHF), continues to emerge and re-emerge across the globe. There are currently various vaccines under development for CCHF prevention. The non-structural M protein (NSM), GP38, highly variable mucin-like domain and N-terminus of GC regions in CCHFV have proved to be immunogenic in vaccine studies. Furthermore, both arms of the immune system have been found to be fundamental for protection in mice. However, there is limited information about immunity in patients following natural infection. The aim of the study was to characterize T cell immune responses against the NSM, GP38 and the highly variable mucin-like domain of CCHFV in survivors of CCHF. This was achieved by first identifying immunogenic peptides in the regions of interest and determining the amino acid conservation of the identified peptides. An overlapping peptide library spanning the NSM, GP38 and highly variable mucin-like domain was designed using the South African CCHFV isolate SPU 103/87. The secretion of interferon-gamma (IFN-γ) by peripheral blood mononuclear cells isolated from 12 participants was screened using 24 peptide pools in an IFN-γ enzyme linked immunospot (ELISpot) assay. IFN-γ secretion was detected in eight of the twelve participants. Two participants showed no detectable IFN-γ responses to any of the peptide pools, and another two were excluded from the analysis due to a high background in the negative controls indicating non-specific reactivity. Nine peptides were identified with the IFN-γ ELISpot, including five peptides in the GP38 region and four in the NSM region, thus confirming the immunogenic potential of these regions during natural infection. No immunogenic peptides were identified in the highly variable mucin-like domain, which is possibly because of the high genetic diversity in the region. The identified immunogenic peptides were used to stimulate T cells of participants and a flow cytometry assay was performed to characterize the immune responses, with the focus on detecting the presence of the T cell memory subsets, the expression of CD107a, which is a cytotoxic marker, and the secretion of IFN-γ and tumour necrosis factor-alpha (TNF-α), which are antiviral cytokines. Cytotoxic CD8+ T cells were detected in six participants in response to nine peptides. IFN-γ and TNF-α secretion within the CD4 and CD8 populations were comparable; thus, highlighting the ability of the CD8+ T cell population to secrete antiviral cytokines, even though the population is known to be predominantly cytotoxic. The secretion of IFN-γ was more frequent than TNF-α secretion in both the CD4 and CD8 populations. Polyfunctional T cells were detected with the phenotypes IFN- γ+CD107a+ and IFN-γ+ TNF-α+, in both the CD4 and CD8 populations. Therefore, the results indicate the possibility of efficient antiviral responses upon stimulation with viral epitopes in survivors of infection. Heterogeneous functionality of the T cell memory subsets was observed, however the terminally differentiated (TEMRA) subset was the most dominant and abundant, followed by the naïve (TN), effector memory (TEM), with the least abundant being the central memory (TCM) T cell memory subset. The IFN-γ secretion detected with the IFN-γ ELISpot and the flow cytometry assay was used as basis for comparing the sensitivity of the two techniques. The IFN-γ ELISpot proved to be comparable to the flow cytometry assay. The ELISpot is suited for screening purposes, while the flow cytometry allowed further characterization of the T cell responses. Therefore, it is recommended that these complementary assays be used in combination. In conclusion, T cell epitopes were identified in the NSM and GP38 regions of CCHFV. Polyfunctional T cells were found in both the CD4 and CD8 populations, thus suggesting the presence of effective long-term memory T cells responses in survivors of CCHF.