Validation of two multiplex platforms to quantify circulating markers of inflammation and endothelial injury in severe infection

Biomarkers can prognosticate outcome and enable risk-stratification. In severe infection, focusing on multiple markers reflecting pathophysiological mechanisms of organ injury could enhance management and pathway-directed therapeutics. Limited data exist on the performance of multiplex biomarker platforms. Our goal was to compare endothelial and immune activation biomarkers in severe pediatric infections using two multiplex platforms. Frozen plasma from 410 children presenting to the Jinja Regional Hospital in Uganda with suspected infection was used to measure biomarkers of endothelial (Angiopoietin-2, sFlt-1, sVCAM-1, sICAM-1) and immune (IL-6, IP-10, sTNFR-1, CHI3L1) activation. Two multiplex platforms (Luminex®, EllaTM) based on monoclonal antibody sandwich immunoassays using biotin-streptavidin conjugate chemistry were selected with reagents from R&D Systems. The two platforms differed in ease and time of completion, number of samples per assay, and dynamic concentration range. Intra-assay variability assessed using a coefficient of variation (CV%) was 2.2-3.4 for Luminex® and 1.2-2.9 for EllaTM. Correlations for biomarker concentrations within dynamic range of both platforms were best for IL-6 (ρ = 0.96, p<0.0001), IP-10 (ρ = 0.94, p<0.0001) and sFlt-1 (ρ = 0.94, p<0.0001). Agreement between concentrations obtained by both methods assessed by the Bland-Altman test varied, with best agreement for CHI3L1. Our data suggest that biomarkers of endothelial and immune activation can be readily measured with multiplex platforms. Luminex® and EllaTM produced reliable results with excellent CV% values. The EllaTM platform was more automated and completed in 75 minutes, potentially compatible with near-patient use. Trends in concentrations obtained by these methods were highly correlated, although absolute values varied, suggesting caution is required when comparing data from different multiplex platforms

Blackwater fever and acute kidney injury in children hospitalized with an acute febrile illness: pathophysiology and prognostic significance

Background: Acute kidney injury (AKI) and blackwater fever (BWF) are related but distinct renal complications of acute febrile illness in East Africa. The pathogenesis and prognostic signifcance of BWF and AKI are not well understood. Methods: A prospective observational cohort study was conducted to evaluate the association between BWF and AKI in children hospitalized with an acute febrile illness. Secondary objectives were to examine the association of AKI and BWF with (i) host response biomarkers and (ii) mortality. AKI was defned using the Kidney Disease: Improving Global Outcomes criteria and BWF was based on parental report of tea-colored urine. Host markers of immune and endothelial activation were quantifed on admission plasma samples. The relationships between BWF and AKI and clinical and biologic factors were evaluated using multivariable regression. Results: We evaluated BWF and AKI in 999 children with acute febrile illness (mean age 1.7 years (standard deviation 1.06), 55.7% male). At enrollment, 8.2% of children had a history of BWF, 49.5% had AKI, and 11.1% had severe AKI. A history of BWF was independently associated with 2.18-fold increased odds of AKI (95% CI 1.15 to 4.16). When examining host response, severe AKI was associated with increased immune and endothelial activation (increased CHI3L1, sTNFR1, sTREM-1, IL-8, Angpt-2, sFlt-1) while BWF was predominantly associated with endothelial activation (increased Angpt-2 and sFlt-1, decreased Angpt-1). The presence of severe AKI, not BWF, was associated with increased risk of in-hospital death (RR, 2.17 95% CI 1.01 to 4.64) adjusting for age, sex, and disease severity. Conclusions: BWF is associated with severe AKI in children hospitalized with a severe febrile illness. Increased awareness of AKI in the setting of BWF, and improved access to AKI diagnostics, is needed to reduce disease progression and in-hospital mortality in this high-risk group of children through early implementation of kidney-protective measures

The presence of prolines in the flanking region of an immunodominant HIV-2 gag epitope influences the quality and quantity of the epitope generated

Both the recognition of HIV‐infected cells and the immunogenicity of candidate CTL vaccines depend on the presentation of a peptide epitope at the cell surface, which in turn depends on intracellular antigen processing. Differential antigen processing maybe responsible for the differences in both the quality and the quantity of epitopes produced, influencing the immunodominance hierarchy of viral epitopes. Previously, we showed that the magnitude of the HIV‐2 gag‐specific T‐cell response is inversely correlated with plasma viral load, particularly when responses are directed against an epitope, 165DRFYKSLRA173, within the highly conserved Major Homology Region of gag‐p26. We also showed that the presence of three proline residues, at positions 119, 159 and 178 of gag‐p26, was significantly correlated with low viral load. Since this proline motif was also associated with stronger gag‐specific CTL responses, we investigated the impact of these prolines on proteasomal processing of the protective 165DRFYKSLRA173 epitope. Our data demonstrate that the 165DRFYKSLRA173 epitope is most efficiently processed from precursors that contain two flanking proline residues, found naturally in low viral‐load patients. Superior antigen processing and enhanced presentation may account for the link between infection with HIV‐2 encoding the “PPP‐gag” sequence and both strong gag‐specific CTL responses as well as lower viral load

The presence of prolines in the flanking region of an immunodominant HIV-2 gag epitope influences the quality and quantity of the epitope generated

Both the recognition of HIV‐infected cells and the immunogenicity of candidate CTL vaccines depend on the presentation of a peptide epitope at the cell surface, which in turn depends on intracellular antigen processing. Differential antigen processing maybe responsible for the differences in both the quality and the quantity of epitopes produced, influencing the immunodominance hierarchy of viral epitopes. Previously, we showed that the magnitude of the HIV‐2 gag‐specific T‐cell response is inversely correlated with plasma viral load, particularly when responses are directed against an epitope, 165DRFYKSLRA173, within the highly conserved Major Homology Region of gag‐p26. We also showed that the presence of three proline residues, at positions 119, 159 and 178 of gag‐p26, was significantly correlated with low viral load. Since this proline motif was also associated with stronger gag‐specific CTL responses, we investigated the impact of these prolines on proteasomal processing of the protective 165DRFYKSLRA173 epitope. Our data demonstrate that the 165DRFYKSLRA173 epitope is most efficiently processed from precursors that contain two flanking proline residues, found naturally in low viral‐load patients. Superior antigen processing and enhanced presentation may account for the link between infection with HIV‐2 encoding the “PPP‐gag” sequence and both strong gag‐specific CTL responses as well as lower viral load

Vaccinia Scars Associated with Improved Survival among Adults in Rural Guinea-Bissau

BACKGROUND: In urban Guinea-Bissau, adults with a vaccinia scar had better survival but also a higher prevalence of HIV-2 infection. We therefore investigated the association between vaccinia scar and survival and HIV infection in a rural area of Guinea-Bissau. METHODOLOGY/PRINCIPAL FINDINGS: In connection with a study of HIV in rural Guinea-Bissau, we assessed vaccinia and BCG scars in 193 HIV-1 or HIV-2 infected and 174 uninfected participants. Mortality was assessed after 2½–3 years of follow-up. The analyses were adjusted for age, sex, village, and HIV status. The prevalence of vaccinia scar was associated with age, village, and HIV-2 status but not with sex and schooling. Compared with individuals without any scar, individuals with a vaccinia scar had better survival (mortality rate ratio (MR) = 0.22 (95% CI 0.08–0.61)), the MR being 0.19 (95% CI 0.06–0.57) for women and 0.40 (95% CI 0.04–3.74) for men. Estimates were similar for HIV-2 infected and HIV-1 and HIV-2 uninfected individuals. The HIV-2 prevalence was higher among individuals with a vaccinia scar compared to individuals without a vaccinia scar (RR = 1.57 (95% CI 1.02–2.36)). CONCLUSION: The present study supports the hypothesis that vaccinia vaccination may have a non-specific beneficial effect on adult survival

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population

Evaluation of T lymphocytes in HIV-2 infection in West Africa: The role of antigen-specific immune responses in disease non-progression

Both HIV-1 and HIV-2 can cause the Acquired Immunodeficiency Syndrome (AIDS), yet the majority of patients infected with HIV-2 are clinically well and most never experience detrimental effects from the infection. Lessons learned from role of the immune system in the long-term non-progression characteristic of HIV-2 infection could offer insight into how a balanced immune response can protect from the immune system destruction associated with HIV-1 infection. The studies presented in this thesis were carried out in a community-based cohort of HIV-2 long-term non-progressors (LTNPs) in rural Guinea Bissau. The initial work (Chapter 3) outlines the first comprehensive analysis of HIV-2-specific immune responses. The data identified Gag as the most immunogenic HIV-2 protein. The magnitude of interferon gamma (IFN-y) generated against Gag was inversely related to virus load (VL). The most frequently recognised HIV-2 peptides clustered within a defined region of Gag with responses to a si~gle peptide associated with low VL. Given this information, the delineation of HIV-2-specific immune responses was focused on this immunodominant region of the HIV-2 proteome. Included in the detailed analysis was the identification of novel HIV-2 epitopes and ex vivo characterisation of HIV-2-specific T cell function (Chapter 4), in vitro characterisation of HIV-2-specific COB T cell clones (Chapter 5), as well as ex vivo phenotypic analysis of HIV-2-specific COB T cells (Chapter 6). Systemic immune activation is a hallmark of disease progression in both HIV-1 and HIV-2 infection. The final chapter (Chapter 7) examines the contribution of HIV-2 plasma antigenaemia to immune system activation which to date was incompletely defined. Cellular and plasma markers of immune activation were related to clinical (VL and CD4 T cell counts), immunological (HIV-2-specific IFN-y secretion) and virus sequence (p26 phenotype) correlates of protection from progression to AIDS. The aim of this work was to understand better what aspects of the host immune response in chronic HIV-2 infection contribute to the relative maintenance of the immune system during the course of this infection.EThOS - Electronic Theses Online ServiceGBUnited Kingdo