Broad and strong memory CD4(+)and CD8(+)T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide–MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design

The role of Nef protein in the pathogenesis of HIV infection

Human Immunodeficiency virus (HIV-1) uses various mechanisms to evade immune recognition by cytotoxic T cells. Down-regulation of Human Leucocyte Antigen class I (HLA-I) by the viral protein Nef is one such important mechanism. HLA-A and B are believed to be down-regulated by Nef while HLA-C and E are not. The differences in down-regulation were due to sequence differences between alleles. I evaluated the functional significance of polymorphisms in the cytoplasmic domains of HLA-A and B in Nef mediated HIV-1 pathogenesis. HLA-B cytoplasmic alleles resisted down-regulation mediated by nef compared to HLA-A cytoplasmic alleles and these differences were seen to play a crucial role in maintaining CTL response as shown by live virus ELISPOT assays, viral suppression assays and CD107a secretion. Therefore, we propose that this relative resistance to Nef mediated down-regulation by HLA-B cytoplasmic allele contributes to the high efficacy and better control of HIV infection by HLA-B restricted CTLs. The partial resistance of HLA-B cytoplasmic domain was maintained in HIV-2 Nef alleles and sub group O HIV-1 viruses. The variability of the HLA-A and B susceptibility appeared to depend on the Nef allele while resistance of HLA-C was global. Substitution of 315DR316 to GG and truncation of C terminal amino acids play a role in mediating resistance to Nef. Possibly these amino acid changes lead to different molecular trafficking profiles as observed in wet lab methods, and changes to secondary protein structures as predicted by in silico methods resulting in partial resistance. In addition, the functional significance of CD4 down-regulation by Nef, another important effect was also explored in this thesis. Nef was found to be ineffective in down-regulating CD4 molecules truncated at cytoplasmic domain and the viruses which were produced in cell lines expressing truncated CD4, were found to be defective in terms of replication and infection. Evaluating the potential of these CD4 molecules in gene therapy is highlighted in my in vitro studies. In summary this thesis provides insight into important but unexplored areas of Nef mediated pathogenesis of HIV infection.</p

The role of Nef protein in the pathogenesis of HIV infection

Human Immunodeficiency virus (HIV-1) uses various mechanisms to evade immune recognition by cytotoxic T cells. Down-regulation of Human Leucocyte Antigen class I (HLA-I) by the viral protein Nef is one such important mechanism. HLA-A and B are believed to be down-regulated by Nef while HLA-C and E are not. The differences in down-regulation were due to sequence differences between alleles. I evaluated the functional significance of polymorphisms in the cytoplasmic domains of HLA-A and B in Nef mediated HIV-1 pathogenesis. HLA-B cytoplasmic alleles resisted down-regulation mediated by nef compared to HLA-A cytoplasmic alleles and these differences were seen to play a crucial role in maintaining CTL response as shown by live virus ELISPOT assays, viral suppression assays and CD107a secretion. Therefore, we propose that this relative resistance to Nef mediated down-regulation by HLA-B cytoplasmic allele contributes to the high efficacy and better control of HIV infection by HLA-B restricted CTLs. The partial resistance of HLA-B cytoplasmic domain was maintained in HIV-2 Nef alleles and sub group O HIV-1 viruses. The variability of the HLA-A and B susceptibility appeared to depend on the Nef allele while resistance of HLA-C was global. Substitution of 315DR316 to GG and truncation of C terminal amino acids play a role in mediating resistance to Nef. Possibly these amino acid changes lead to different molecular trafficking profiles as observed in wet lab methods, and changes to secondary protein structures as predicted by in silico methods resulting in partial resistance. In addition, the functional significance of CD4 down-regulation by Nef, another important effect was also explored in this thesis. Nef was found to be ineffective in down-regulating CD4 molecules truncated at cytoplasmic domain and the viruses which were produced in cell lines expressing truncated CD4, were found to be defective in terms of replication and infection. Evaluating the potential of these CD4 molecules in gene therapy is highlighted in my in vitro studies. In summary this thesis provides insight into important but unexplored areas of Nef mediated pathogenesis of HIV infection.This thesis is not currently available in ORA

A Comprehensive Analysis of the Impact of HIV on HCV Immune Responses and Its Association with Liver Disease Progression in a Unique Plasma Donor Cohort - Fig 4

<p><b>A)</b> Reduced percentage of CD4+ specific T cell responses in HIV co-infected patients. The percentage of HCV specific CD4+ or CD8+ T cell responses were measured in a subset of HIV/HCV co-infected (n = 14) and HCV mono-infected (n = 7) individuals following <i>ex-vivo</i> stimulation of PBMC and IFNγ ELISPOT assay after CD8+ T cell depletion (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158037#sec006" target="_blank">Materials and methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158037#pone.0158037.s006" target="_blank">S5 Fig</a>). The <i>p</i> values calculated by Mann-Whitney U test. Data are presented as median with interquartile range. <b>B)</b> Representative data from HCV NS3 peptide stimulated enriched short-term T cell lines from three patients each of HCV mono-infected (Group HCVc) and HIV/HCV co-infected (group HIV/HCVc) to demonstrate HCV specific IFNγ response. CD4⁺ and CD8⁺ dependency of IFNγ responses were analysed after flow cytometry based sorting. <b>C and D</b> The antiviral activity of enriched HCV NS3 specific T cells. HCV suppression by peptide stimulated <b>(C)</b> bulk CD4+ T cells (n = 4 replicates) and (<b>D)</b> bulk CD8+ T cells (n = 4 replicates). Solid lines represent HCV mono-infected patients; dash lines represent HIV/HCV co-infected patients. The percentage suppression was calculated by measuring luciferase activity after 48 hours of co-culture of enriched HCV NS3 specific T cells with HLA-A2 transfected Huh7.5 HCV replicon cells. Bars represent mean + SD. <b>E)</b> HIV has no impact on HCV neutralizing antibodies. Serum from a subgroup of patients co-infected with HCV and HIV (group HIV/HCVc, HIV/HCVr, HIVt/HCVc & HIVt/HCVr, n = 40) and patients with HCV mono-infection (group HCVc & HCVr, n = 49) were used to neutralize the infectivity of H77 HCVpp. The percentage of neutralization by patient serum was determined for patients positive of HCV viral load (blue symbols) and negative for HCV viral load (red symbols). The data were analyzed by Kruskal-Wallis test, and followed with Dunn’s multiple comparisons test. Each point is representative of the mean of one patient from two independent experiments. Data are presented as mean with SEM.</p

Patient characteristics of 151 individuals infected with HCV and /or HIV.

<p>Patients in Group 1, 2 and 3 are considered infected with HIV/HCV for over 15 years before sampling. Group HCVc was mono-infected, HCV chronic. Group HCVr was mono-infected, HCV spontaneously resolved. Group HIV/HCVc was HIV-1+, HCV chronic, HAART naïve. Group HIV/HCVr was HIV-1+, HCV spontaneously resolved, HAART naïve. Group HIVt/HCVc was HIV-1+, HCV chronic, HAART treated. Group HIVt/HCVr was HIV-1+, HCV spontaneously resolved, HAART naïve. ALT, Alanine transaminase; Tbil, Total bilirubin; ALP, Alkaline phosphatase: γ-GT, gamma glutamyl transferase; VL, viral load; IU, international units; HAART, highly active anti retroviral therapy. Values represent mean ± standard deviation.</p

HIV/HCV Co-infection affects the progression of chronic liver disease.

<p><b>A)</b> The progression of liver disease was assessed by measuring the degree of liver fibrosis. The progression of liver disease in chronic HIV/HCV co-infected patients (Group HIV/HCVc+ HIVt/HCVc (n = 40)) was faster than chronic HCV mono-infected patients (Group HCVc (n = 12)). <b>B)</b> The ALB/GLB ratio was higher in Group HCVc (n = 24) than chronic HIV/HCV co-infected patients (Group HIV/HCVc + HIVt/HCVc (n = 67)). <b>C)</b> Gamma GT levels were higher in Group HIV/HCV co-infected patients (Group HIV/HCVc + HIVt/HCVc (n = 69) than HCVc (n = 24). <b>D)</b> HCV virus load was lower in Group HCVc (n = 24) than chronic HIV/HCV co-infected patients (Group HIV/HCVc +HIVt/HCVc (n = 70)). <b>E)</b> A higher HCV viral loads were observed in patients with low CD4⁺ T cell counts in HIV/HCV Co-infection (CD4+T<200 n = 12, CD4+T>200 n = 55). The <i>p</i> values calculated by Mann-Whitney U test. Data are presented as median with interquartile range.</p

HCV specific T cell responses are important in the control of HCV viral load and are higher in HCV mono infected patients.

<p><b>A)</b> HCV core and NS protein specific T cell responses were compared between a sub group of HCV mono-infected (Group HCVc (n = 21) and HAART naïve HIV/HCV co-infected patients (group HIV/HCVc (n = 24)). Median is shown. <b>B)</b> HCV specific T cell responses were compared between CD4>200 and CD4<200 in HCV/HIV co-infection group (n = 60). Data are presented as median with interquartile range. The <i>p</i> values calculated by Mann-Whitney U test.</p

Short term HAART has no effect on the progression of chronic liver disease in HIV/HCV Co-infection group.

<p><b>A)</b> The degree of liver fibrosis (Group HIV/HCVc (n = 21) vs Group HIVt/HCVc (n = 19) <b>B)</b> ALB/GLB ratio (Group HIV/HCVc (n = 25) vs Group HIVt/HCVc (n = 43) and <b>C)</b> HCV virus load (Group HIV/HCVc (n = 29) vs Group HIVt/HCVc (n = 42) were compared between subgroups of HIV/HCV co-infected treatment naive group and HIV/HCV co-infected group that received short term HAART. The <i>p</i> values calculated by Mann-Whitney U test. Data are presented as median with interquartile range.</p