Anti-Nucleocapsid Protein Immune Responses Counteract Pathogenic Effects of Rift Valley Fever Virus Infection in Mice

The known virulence factor of Rift Valley fever virus (RVFV), the NSs protein, counteracts the antiviral effects of the type I interferon response. In this study we evaluated the expression of several genes in the liver and spleen involved in innate and adaptive immunity of mice immunized with a RVFV recombinant nucleocapsid protein (recNP) combined with Alhydrogel adjuvant and control animals after challenge with wild type RVFV. Mice immunized with recNP elicited an earlier IFNβ response after challenge compared to non-immunized controls. In the acute phase of liver infection in non-immunized mice there was a massive upregulation of type I and II interferon, accompanied by high viral titers, and the up- and downregulation of several genes involved in the activation of B- and T-cells, indicating that both humoral and cellular immunity is modulated during RVFV infection. Various genes involved in pro-inflammatory responses and with pro-apoptotic effects were strongly upregulated and anti-apoptotic genes were downregulated in liver of non-immunized mice. Expression of many genes involved in B- and T-cell immunity were downregulated in spleen of non-immunized mice but normal in immunized mice. A strong bias towards apoptosis and inflammation in non-immunized mice at an acute stage of liver infection associated with suppression of several genes involved in activation of humoral and cellular immunity in spleen, suggests that RVFV evades the host immune response in more ways than only by inhibition of type I interferon, and that immunopathology of the liver plays a crucial role in RVF disease progression

Cellular expression and crystal structure of the murine cytomegalovirus MHC-Iv glycoprotein, m153

Mouse cytomegalovirus (MCMV), a β-herpesvirus that establishes latent and persistent infections in mice, is a valuable model for studying complex virus-host interactions. MCMV encodes the m145 family of putative immunoevasins with predicted MHC-I structure. Functions attributed to some family members include downregulation of host MHC-I (m152) and NKG2D ligands (m145, m152, m155) and interaction with inhibitory or activating NK receptors (m157). We present the cellular, biochemical and structural characterization of m153, which is a heavily glycosylated homodimer, that does not require β2m or peptide, and is expressed at the surface of MCMV-infected cells. Its 2.4 Å crystal structure confirms that this compact molecule preserves an MHC-I-like fold and reveals a novel mode of dimerization, confirmed by site-directed mutagenesis, and a distinctive disulfide-stabilized extended amino terminus. The structure provides a useful framework for comparative analysis of the divergent members of the m145 family

Chronic HIV-1 Infection Alters the Cellular Distribution of FcγRIIIa and the Functional Consequence of the FcγRIIIa-F158V Variant

Chronic HIV-infection modulates the expression of Fc gamma receptors (FcγRs) on immune cells and their antibody-dependent effector function capability. Given the increasingly recognized importance of antibody-dependent cellular cytotoxicity (ADCC) in HIV-specific immunity, we investigated the cellular distribution of FcγRIIIa on cytotoxic lymphocytes—natural killer cells and CD8+ T cells—and the effect of the FcγRIIIa-F158V variant on ADCC capacity in HIV-infected individuals (n = 23) and healthy controls (n = 23). Study participants were matched for F158V genotypes, carried two copies of the FCGR3A gene and were negative for FcγRIIb expression on NK cells. The distribution of CD56dimFcγRIIIabright and CD56negFcγRIIIabright NK cell subsets, but not FcγRIIIa surface expression, differed significantly between HIV-1 negative and HIV-1 positive donors. NK cell-mediated ADCC responses negatively correlated with the proportion of the immunoregulatory CD56brightFcγRIIIadim/neg cells and were lower in the HIV-1 positive group. Intriguingly, the FcγRIIIa-F158V variant differentially affected the NK-mediated ADCC responses for HIV-1 negative and HIV-1 positive donors. Healthy donors bearing at least one 158V allele had higher ADCC responses compared to those homozygous for the 158F allele (48.1 vs. 34.1%), whereas the opposite was observed for the HIV-infected group (26.4 vs. 34.6%), although not statistically significantly different. Furthermore, FcγRIIIa+CD8bright and FcγRIIIa+CD8dim T cell subsets were observed in both HIV-1 negative and HIV-1 positive donors, with median proportions that were significantly higher in HIV-1 positive donors compared to healthy controls (15.7 vs. 8.3%; P = 0.016 and 18.2 vs. 14.1%; P = 0.038, respectively). Using an HIV-1-specific GranToxiLux assay, we demonstrate that CD8+ T cells mediate ADCC through the delivery of granzyme B, which was overall lower compared to that of autologous NK cells. In conclusion, our findings demonstrate that in the presence of an HIV-1 infection, the cellular distribution of FcγRIIIa is altered and that the functional consequence of FcγRIIIa variant is affected. Importantly, it underscores the need to characterize FcγR expression, cellular distribution and functional consequences of FcγR genetic variants within a specific environment or disease state

KIR-HLA and Maternal-Infant HIV-1 Transmission in Sub-Saharan Africa

Numerous studies have suggested a role for natural killer (NK) cells in attenuation of HIV-1 disease progression via recognition by killer-cell immunoglobulin-like receptors (KIRs) of specific HLA class I molecules. The role of KIR and HLA class I has not been addressed in the context of maternal-infant HIV-1 transmission. KIR and HLA class I B and C genes from 224 HIV-1-infected mothers and 222 infants (72 infected and 150 uninfected) from South Africa were characterized. Although a number of significant associations were determined in both the total group and in the nevirapine (NVP) exposed group, the most significant findings involved KIR2DL2 and KIR2DL3 and HLA-C. KIR2DL2/KIR2DL3 was underrepresented in intrapartum (IP)-transmitting mothers compared to non-transmitting (NT) mothers (P = 0.008) and remained significant (P = 0.036) after correction for maternal viral load (MVL). Homozygosity for KIR2DL3 alone and in combination with HLA-C allotype heterozygosity (C1C2) was elevated in IP-transmitting mothers compared to NT mothers (P = 0.034 and P = 0.01 respectively), and after MVL correction (P = 0.033 and P = 0.027, respectively). In infants, KIR2DL3 in combination with its HLA-C1 ligand (C1) as well as homozygosity for KIR2DL3 with C1C2, were both found to be underrepresented in infected infants compared to exposed uninfected infants in the total group (P = 0.06 and P = 0.038, respectively) and in the sub-group of infants whose mothers received NVP (P = 0.007 and P = 0.03, respectively). These associations were stronger post MVL adjustment (total group: P = 0.02 and P = 0.009, respectively; NVP group: P = 0.004 and P = 0.02, respectively). Upon stratification according to low and high MVL, all significant associations fell within the low MVL group, suggesting that with low viral load, the effects of genotype can be more easily detected. In conclusion this study has identified a number of significant associations that suggest an important role for NK cells in maternal-to-infant HIV-1 transmission

KIR-HLA and Maternal-Infant HIV-1 Transmission in Sub-Saharan Africa

Numerous studies have suggested a role for natural killer (NK) cells in attenuation of HIV-1 disease progression via recognition by killer-cell immunoglobulin-like receptors (KIRs) of specific HLA class I molecules. The role of KIR and HLA class I has not been addressed in the context of maternal-infant HIV-1 transmission. KIR and HLA class I B and C genes from 224 HIV-1-infected mothers and 222 infants (72 infected and 150 uninfected) from South Africa were characterized. Although a number of significant associations were determined in both the total group and in the nevirapine (NVP) exposed group, the most significant findings involved KIR2DL2 and KIR2DL3 and HLA-C. KIR2DL2/KIR2DL3 was underrepresented in intrapartum (IP)-transmitting mothers compared to non-transmitting (NT) mothers (P = 0.008) and remained significant (P = 0.036) after correction for maternal viral load (MVL). Homozygosity for KIR2DL3 alone and in combination with HLA-C allotype heterozygosity (C1C2) was elevated in IP-transmitting mothers compared to NT mothers (P = 0.034 and P = 0.01 respectively), and after MVL correction (P = 0.033 and P = 0.027, respectively). In infants, KIR2DL3 in combination with its HLA-C1 ligand (C1) as well as homozygosity for KIR2DL3 with C1C2, were both found to be underrepresented in infected infants compared to exposed uninfected infants in the total group (P = 0.06 and P = 0.038, respectively) and in the sub-group of infants whose mothers received NVP (P = 0.007 and P = 0.03, respectively). These associations were stronger post MVL adjustment (total group: P = 0.02 and P = 0.009, respectively; NVP group: P = 0.004 and P = 0.02, respectively). Upon stratification according to low and high MVL, all significant associations fell within the low MVL group, suggesting that with low viral load, the effects of genotype can be more easily detected. In conclusion this study has identified a number of significant associations that suggest an important role for NK cells in maternal-to-infant HIV-1 transmission

Age at antiretroviral therapy initiation and cell-associated HIV-1 DNA levels in HIV-1-infected children

Background The latent viral reservoir is the major obstacle to achieving HIV remission and necessitates life-long antiretroviral therapy (ART) for HIV-infected individuals. Studies in adults and children have found that initiating ART soon after infection is associated with a reduction in the size of the HIV-1 reservoir. Here we quantified cell-associated HIV-1 DNA in early-treated but currently older HIV-infected children suppressed on ART. Methods The study participants comprised of a cohort of 146 early-treated children with HIV-1 RNA <50 copies/ml enrolled as part of a clinical trial in Johannesburg, South Africa. A stored buffy coat sample collected after a median 4.3 years on ART and where HIV-1 RNA was <50 copies/ml was tested for cell-associated HIV-1 DNA levels. An in-house, semi-nested real-time quantitative hydrolysis probe PCR assay to detect total HIV-1 subtype C proviral DNA was used. Children were followed prospectively for up to 3 years after this measurement to investigate subsequent HIV-1 RNA rebound/failure while remaining on ART. Age at ART initiation, HIV-1 RNA decline prior to HIV-1 DNA measurement and other factors were investigated. Results A gradient between age at ART initiation and later HIV-1 DNA levels was observed. When ART was started 50 copies/ml whilst on ART within 3 years after the DNA measurement was 2.07 (95% CI: 1.352–3.167) times greater if the HIV-1 DNA level was above the median of 55 copies/106 cells. Conclusions Cell-associated HIV-1 DNA levels measured after more than 4 years on ART were lower the younger the age of the child when ART was initiated. This marker of the size of the viral reservoir also predicted subsequent viral rebound/treatment failure while ART was sustained. The results provide additional evidence of the benefits of prompt diagnosis and early ART initiation in newborns and infants

Deep sequencing of the HIV‑1 polymerase gene for characterisation of cytotoxic T‑lymphocyte epitopes during early and chronic disease stages

BACKGROUND : Despite multiple attempts, there is still no effective HIV-1 vaccine available. The HIV-1 polymerase (pol) gene is highly conserved and encodes cytotoxic T-lymphocyte (CTL) epitopes. The aim of the study was to characterise HIV-1 Pol CTL epitopes in mostly sample pairs obtained during early and chronic stages of infection. METHODS : Illumina deep sequencing was performed for all samples while Sanger sequencing was only performed on baseline samples. Codons under immune selection pressure were assessed by computing nonsynonymous to synonymous mutation ratios using MEGA. Minority CTL epitope variants occurring at 5% were detected using lowfrequency variant tool in CLC Genomics. Los Alamos HIV database was used for mapping mutations to known HIV-1 CTL epitopes. RESULTS : Fifty-two participants were enrolled in the study. Their median age was 28 years (interquartile range: 24–32 years) and majority of participants (92.3%) were female. Illumina minority variant analysis identified a significantly higher number of CTL epitopes (n = 65) compared to epitopes (n = 8) identified through Sanger sequencing. Most of the identified epitopes mapped to reverse transcriptase (RT) and integrase (IN) regardless of sequencing method. There was a significantly higher proportion of minority variant epitopes in RT (n = 39, 60.0%) compared to IN (n = 17, 26.2%) and PR (n = 9, 13.8%), p = 0.002 and < 0.0001, respectively. However, no significant difference was observed between the proportion of minority variant epitopes in IN versus PR, p = 0.06. Some epitopes were detected in either early or chronic HIV-1 infection whereas others were detected in both stages. Different distribution patterns of minority variant epitopes were observed in sample pairs; with some increasing or decreasing over time, while others remained constant. Some of the identified epitopes have not been previously reported for HIV-1 subtype C. There were also variants that could not be mapped to reported CTL epitopes in the Los Alamos HIV database. CONCLUSION : Deep sequencing revealed many Pol CTL epitopes, including some not previously reported for HIV-1 subtype C. The findings of this study support the inclusion of RT and IN epitopes in HIV-1 vaccine candidates as these proteins harbour many CTL epitopes.Additional file 1. Supplementary Table 1: The in-house complete HIV pol nested PCR conditions and Sanger sequencing primers.Additional file 2. Supplementary Table 2: Pol CTL epitopes identified through Sanger sequencing and comparison by stage of infectionAdditional file 3. Supplementary Table 3: Minority variant proportions within Pol CTL epitopes by stage of infection.Additional file 4. Supplementary Figure 1: Neighbour-joining phylogenetic analysis of Illumina consensus and Sanger consensus sequences for baseline samples. Sanger and Illumina consensus of the same sample significantly clustered together. Majority of the sequences clustered with HIV-1 subtype C references. HIV group O was used for rooting the tree and a bootstrap value of 1000 was used for analysis. S = Sanger sequencing; D = Deep sequencing.The National Research Foundation of South Africa; Poliomyelitis Research Foundation (PRF); Discovery Foundation; National Health Laboratory Service Research Trust (NHLS-RT); South African Medical Research Council Self-Initiated Research (MRC-SIR); University of Pretoria Faculty of Health Sciences Research Committee; the South African Research Chairs Initiative of the Department of Science and Innovation and ADR—The Division of Intramural Research, NIAID, NIH.http://www.virologyj.comam2023Medical Virolog

Evaluation of the HIV-1 polymerase gene sequence diversity for prediction of recent HIV-1 infections using Shannon entropy analysis

SUPPLEMENTARY MATERIALS : TABLE S1: List of articles from which HIV-1 subtype C reference sequences were obtained; Supplementary TABLE S2: Nucleotide differences within amino acid mutations found to have a different distribution between recent and chronic infection sequences; FIGURE S1: Amino acid sequence alignment to show the difference in sequences between recent and chronic HIV-1 infection; FIGURE S2: Comparison of differences in amino acids E628 and R629 in study sequences and non-subtype C reference sequences, for recent and chronic sequences.DATA AVAILABILITY STATEMENT : All data generated or analysed during this study are included in this manuscript and its supplementary information files.HIV-1 incidence is an important parameter for assessing the impact of HIV-1 interventions. The aim of this study was to evaluate HIV-1 polymerase (pol) gene sequence diversity for the prediction of recent HIV-1 infections. Complete pol Sanger sequences obtained from 45 participants confirmed to have recent or chronic HIV-1 infection were used. Shannon entropy was calculated for amino acid (aa) sequences for the entire pol and for sliding windows consisting of 50 aa each. Entropy scores for the complete HIV-1 pol were significantly higher in chronic compared to recent HIV-1 infections (p < 0.0001) and the same pattern was observed for some sliding windows (p-values ranging from 0.011 to <0.001), leading to the identification of some aa mutations that could discriminate between recent and chronic infection. Different aa mutation groups were assessed for predicting recent infection and their performance ranged from 64.3% to 100% but had a high false recency rate (FRR), which was decreased to 19.4% when another amino acid mutation (M456) was included in the analysis. The pol-based molecular method identified in this study would not be ideal for use on its own due to high FRR; however, this method could be considered for complementing existing serological assays to further reduce FRR.The National Research Foundation (NRF), Poliomyelitis Research Foundation, Discovery Foundation, National Health Laboratory Service Research Trust (NHLS-RT), South African Medical Research Council Self-Initiated Research (MRC-SIR), University of Pretoria Faculty of Health Sciences Research Committee, the South African Research Chairs Initiative of the Department of Science and Innovation and National Research Foundation of South Africa and the Division of Intramural Research.https://www.mdpi.com/journal/virusesam2023Medical Virolog

Serum levels of inflammatory cytokines in Rift Valley fever patients are indicative of severe disease

BACKGROUND : Rift Valley fever (RVF) is a mosquito-borne viral zoonosis affecting domestic and wild ruminants, camels and humans. Outbreaks of RVF are characterized by a sudden onset of abortions and high mortality amongst domestic ruminants. Humans develop disease ranging from a mild flu-like illness to more severe complications including hemorrhagic syndrome, ocular and neurological lesions and death. During the RVF outbreak in South Africa in 2010/11, a total of 278 human cases were laboratory confirmed, including 25 deaths. The role of the host inflammatory response to RVF pathogenesis is not completely understood. METHODS : Virus load in serum from human fatal and non-fatal cases was determined by standard tissue culture infective dose 50 (TCID50) titration on Vero cells. Patient serum concentration of chemokines and cytokines involved in inflammatory responses (IL-8, RANTES, CXCL9, MCP-1, IP-10, IL-1β, IL-6, IL-10, TNF and IL-12p70) was determined using cytometric bead assays and flow cytometry. RESULTS : Fatal cases had a 1-log10 higher TCID50/ml serum concentration of RVF virus (RVFV) than survivors (p < 0.05). There were no significant sequence differences between isolates recovered from fatal and non-fatal cases. Chemokines and pro- and anti-inflammatory cytokines were detected at significantly increased (IL-8, CXCL9, MCP-1, IP-10, IL-10) or decreased (RANTES) levels when comparing fatal cases to infected survivors and uninfected controls, or when comparing combined infected patients to uninfected controls. CONCLUSIONS : The results suggest that regulation of the host inflammatory responses plays an important role in the outcome of RVFV infection in humans. Dysregulation of the inflammatory response contributes to a fatal outcome. The cytokines and chemokines identified in this study that correlate with fatal outcomes warrant further investigation as markers for disease severity.The Poliomyelitis Research Foundation (PRF), grant number 12/10. PJvV is further supported by a grant from the Incentive Funding for Rated Researchers program of the National Research Foundation (NRF), South Africa. This work is based on the research supported in part by the National Research Foundation of South Africa (Grant specific unique reference number UID 85544).http://www.virologyj.comam201

Cellular Expression and Crystal Structure of the Murine Cytomegalovirus Major Histocompatibility Complex Class I-like Glycoprotein, m153

Mouse cytomegalovirus (MCMV), a β-herpesvirus that establishes latent and persistent infections in mice, is a valuable model for studying complex virus-host interactions. MCMV encodes the m145 family of putative immunoevasins with predicted MHC-I structure. Functions attributed to some family members include downregulation of host MHC-I (m152) and NKG2D ligands (m145, m152, m155) and interaction with inhibitory or activating NK receptors (m157). We present the cellular, biochemical and structural characterization of m153, which is a heavily glycosylated homodimer, that does not require β2m or peptide, and is expressed at the surface of MCMV-infected cells. Its 2.4 Å crystal structure confirms that this compact molecule preserves an MHC-I-like fold and reveals a novel mode of dimerization, confirmed by site-directed mutagenesis, and a distinctive disulfide-stabilized extended amino terminus. The structure provides a useful framework for comparative analysis of the divergent members of the m145 family