HIV-Positive Liver Transplant Does not Alter the Latent Viral Reservoir in Recipients With Antiretroviral Therapy-Suppressed HIV.

The latent viral reservoir (LVR) remains a major barrier to HIV-1 curative strategies. It is unknown whether receiving a liver transplant from a donor with HIV might lead to an increase in the LVR because the liver is a large lymphoid organ. We found no differences in intact provirus, defective provirus, or the ratio of intact to defective provirus between recipients with ART-suppressed HIV who received a liver from a donor with (n = 19) or without HIV (n = 10). All measures remained stable from baseline by 1 year posttransplant. These data demonstrate that the LVR is stable after liver transplantation in people with HIV. Clinical Trials Registration. NCT02602262 and NCT03734393.

Absence of pathogenic viruses in COVID-19 convalescent plasma.

BACKGROUND: It is important to maintain the safety of blood products by avoiding the transfusion of units with known and novel viral pathogens. It is unknown whether COVID-19 convalescent plasma (CCP) may contain pathogenic viruses (either newly acquired or reactivated) that are not routinely screened for by blood centers. METHODS: The DNA virome was characterized in potential CCP donors (n = 30) using viral genome specific PCR primers to identify DNA plasma virome members of the Herpesviridae [Epstein Barr Virus (EBV), cytomegalovirus (CMV), human herpesvirus 6A/B, human herpesvirus 7] and Anelloviridae [Torque teno viruses (TTV), Torque teno mini viruses (TTMV), and Torque teno midi viruses (TTMDV)] families. In addition, the RNA plasma virome was characterized using unbiased metagenomic sequencing. Sequencing was done on a HiSeq2500 using high output mode with a read length of 2X100 bp. The sequencing reads were taxonomically classified using Kraken2. CMV and EBV seroprevalence were evaluated using a chemiluminescent immunoassay. RESULTS: TTV and TTMDV were detected in 12 (40%) and 4 (13%) of the 30 study participants, respectively; TTMDV was always associated with infection with TTV. We did not observe TTMV DNAemia. Despite CMV and EBV seroprevalences of 33.3% and 93.3%, respectively, we did not detect Herpesviridae DNA among the study participants. Metagenomic sequencing did not reveal any human RNA viruses in CCP, including no evidence of circulating SARS-CoV-2. DISCUSSION: There was no evidence of pathogenic viruses, whether newly acquired or reactivated, in CCP despite the presence of non-pathogenic Anelloviridae. These results confirm the growing safety data supporting CCP.

The effect of induction immunosuppression for kidney transplant on the latent HIV reservoir.

The HIV latent viral reservoir (LVR) remains a major challenge in the effort to find a cure for HIV. There is interest in lymphocyte-depleting agents, used in solid organ and bone marrow transplantation to reduce the LVR. This study evaluated the LVR and T cell receptor repertoire in HIV-infected kidney transplant recipients using intact proviral DNA assay and T cell receptor sequencing in patients receiving lymphocyte-depleting or lymphocyte-nondepleting immunosuppression induction therapy. CD4+ T cells and intact and defective provirus frequencies decreased following lymphocyte-depleting induction therapy but rebounded to near baseline levels within 1 year after induction. In contrast, these biomarkers were relatively stable over time in the lymphocyte-nondepleting group. The lymphocyte-depleting group had early TCRß repertoire turnover and newly detected and expanded clones compared with the lymphocyte-nondepleting group. No differences were observed in TCRß clonality and repertoire richness between groups. These findings suggest that, even with significant decreases in the overall size of the circulating LVR, the reservoir can be reconstituted in a relatively short period of time. These results, while from a relatively unique population, suggest that curative strategies aimed at depleting the HIV LVR will need to achieve specific and durable levels of HIV-infected T cell depletion.

Toll-Like Receptor 3 Is Critical to the Pancreatic Islet Milieu That Is Required for Coxsackievirus B4-Induced Type 1 Diabetes in Female Nonobese Diabetic Mice.

OBJECTIVE: Genetic and environmental influences play a role as triggers of type 1 diabetes mellitus (T1DM). Female nonobese diabetic (NOD) mice are useful for studying T1DM as they spontaneously develop T1DM, which can be accelerated by some viruses. Toll-like receptor 3 (TLR3) is believed to play a critical role in viral-induced T1DM and ß-cell destruction, because female Tlr3 knockout (Tlr3-/-) NOD mice are protected from Coxsackievirus B4 (CVB4)-induced acceleration of T1DM. However, the exact role(s) TLR3 plays in the pathogenesis of CVB4-induced T1DM remain unknown. METHODS: This longitudinal study used immunostaining, laser capture microdissection, and reverse transcription real-time polymerase chain reaction of islets from female uninfected and CVB4-infected Tlr3+/+ and Tlr3-/- NOD mice. RESULTS: Islets isolated from female Tlr3+/+ NOD mice 4 to 8 weeks of age had higher amounts of insulitis, Cxcl10, Il1b, Tnfa, and Tgfb1 expression compared with Tlr3-/- NOD mice. After CVB4 infection, Tlr3+/+ NOD mice had higher amounts of insulitis and T-cell infiltration at 3 days after infection compared with Tlr3-/- CVB4-infected NOD mice. CONCLUSIONS: Toll-like receptor 3 is necessary for establishment of a pancreatic islet inflammatory microenvironment by increasing insulitis and cytokine expression that facilitates CVB4-induced T1DM in female NOD mice.

Antibody attributes that predict the neutralization and effector function of polyclonal responses to SARS-CoV-2.

BACKGROUND: While antibodies can provide significant protection from SARS-CoV-2 infection and disease sequelae, the specific attributes of the humoral response that contribute to immunity are incompletely defined. METHODS: We employ machine learning to relate characteristics of the polyclonal antibody response raised by natural infection to diverse antibody effector functions and neutralization potency with the goal of generating both accurate predictions of each activity based on antibody response profiles as well as insights into antibody mechanisms of action. RESULTS: To this end, antibody-mediated phagocytosis, cytotoxicity, complement deposition, and neutralization were accurately predicted from biophysical antibody profiles in both discovery and validation cohorts. These models identified SARS-CoV-2-specific IgM as a key predictor of neutralization activity whose mechanistic relevance was supported experimentally by depletion. CONCLUSIONS: Validated models of how different aspects of the humoral response relate to antiviral antibody activities suggest desirable attributes to recapitulate by vaccination or other antibody-based interventions.

Higher Proinflammatory Cytokines Are Associated With Increased Antibody Titer After a Third Dose of SARS-CoV-2 Vaccine in Solid Organ Transplant Recipients.

BACKGROUND: Solid organ transplant recipients (SOTRs) are at increased risk for severe COVID-19 and exhibit lower antibody responses to SARS-CoV-2 vaccines. This study aimed to determine if prevaccination cytokine levels are associated with antibody response to SARS-CoV-2 vaccination. METHODS: A cross-sectional study was performed among 58 SOTRs before and after two-dose mRNA vaccine series, 35 additional SOTRs before and after a third vaccine dose, and comparison to 16 healthy controls (HCs). Antispike antibody was assessed using the IgG Euroimmun ELISA. Electrochemiluminescence detection-based multiplexed sandwich immunoassays (Meso Scale Diagnostics) were used to quantify plasma cytokine and chemokine concentrations (n = 20 analytes) and compare concentrations between SOTRs and HCs, stratified by ultimate antibody response to the vaccine using Wilcoxon-rank-sum test with false discovery rates computed to correct for multiple comparisons. RESULTS: In the study population, 100% of HCs, 59% of SOTRs after 2 doses and 63% of SOTRs after 3 doses had a detectable antibody response. Multiple baseline cytokines were elevated in SOTRs versus HCs. There was no significant difference in baseline cytokine levels between SOTRs with high versus low-titer antibodies after 2 doses of vaccine. However, as compared with poor antibody responders, SOTRs who went on to develop a high-titer antibody response to a third dose of vaccine had significantly higher prethird dose levels of several innate immune cytokines including IL-17, IL-2Ra, IL-6, IP-10, MIP-1α, and TNF-α (false discovery rates < 0.05). CONCLUSIONS: A specific inflammatory profile may be associated with developing higher antibodies in response to a third dose of SARS-CoV-2 vaccine in SOTRs.

Antibody Attributes that Predict the Neutralization and Effector Function of Polyclonal Responses to SARS-CoV-2.

While antibodies provide significant protection from SARS-CoV-2 infection and disease sequelae, the specific attributes of the humoral response that contribute to immunity are incompletely defined. In this study, we employ machine learning to relate characteristics of the polyclonal antibody response raised by natural infection to diverse antibody effector functions and neutralization potency with the goal of generating both accurate predictions of each activity based on antibody response profiles as well as insights into antibody mechanisms of action. To this end, antibody-mediated phagocytosis, cytotoxicity, complement deposition, and neutralization were accurately predicted from biophysical antibody profiles in both discovery and validation cohorts. These predictive models identified SARS-CoV-2-specific IgM as a key predictor of neutralization activity whose mechanistic relevance was supported experimentally by depletion. Validated models of how different aspects of the humoral response relate to antiviral antibody activities suggest desirable attributes to recapitulate by vaccination or other antibody-based interventions.

Markers of Polyfunctional SARS-CoV-2 Antibodies in Convalescent Plasma.

Convalescent plasma is a promising therapy for coronavirus disease 2019 (COVID-19), but the antibody characteristics that contribute to efficacy remain poorly understood. This study analyzed plasma samples from 126 eligible convalescent blood donors in addition to 15 naive individuals, as well as an additional 20 convalescent individuals as a validation cohort. Multiplexed Fc Array binding assays and functional antibody response assays were utilized to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody composition and activity. Donor convalescent plasma samples contained a range of antibody cell- and complement-mediated effector functions, indicating the diverse antiviral activity of humoral responses observed among recovered individuals. In addition to viral neutralization, convalescent plasma samples contained antibodies capable of mediating such Fc-dependent functions as complement activation, phagocytosis, and antibody-dependent cellular cytotoxicity against SARS-CoV-2. Plasma samples from a fraction of eligible donors exhibited high activity across all activities evaluated. These polyfunctional plasma samples could be identified with high accuracy with even single Fc Array features, whose correlation with polyfunctional activity was confirmed in the validation cohort. Collectively, these results expand understanding of the diversity of antibody-mediated antiviral functions associated with convalescent plasma, and the polyfunctional antiviral functions suggest that it could retain activity even when its neutralizing capacity is reduced by mutations in variant SARS-CoV-2.IMPORTANCE Convalescent plasma has been deployed globally as a treatment for COVID-19, but efficacy has been mixed. Better understanding of the antibody characteristics that may contribute to its antiviral effects is important for this intervention as well as offer insights into correlates of vaccine-mediated protection. Here, a survey of convalescent plasma activities, including antibody neutralization and diverse effector functions, was used to define plasma samples with broad activity profiles. These polyfunctional plasma samples could be reliably identified in multiple cohorts by multiplex assay, presenting a widely deployable screening test for plasma selection and investigation of vaccine-elicited responses.

Cytokine and Chemokine Levels in Coronavirus Disease 2019 Convalescent Plasma.

BACKGROUND: The efficacy of coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) is primarily ascribed as a source of neutralizing anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. However, the composition of other immune components in CCP and their potential roles remain largely unexplored. This study aimed to describe the composition and concentrations of plasma cytokines and chemokines in eligible CCP donors. METHODS: A cross-sectional study was conducted among 20 prepandemic healthy blood donors without SARS-CoV-2 infection and 140 eligible CCP donors with confirmed SARS-CoV-2 infection. Electrochemiluminescence detection-based multiplexed sandwich immunoassays were used to quantify plasma cytokine and chemokine concentrations (n = 35 analytes). A SARS-CoV-2 microneutralization assay was also performed. Differences in the percentage of detection and distribution of cytokine and chemokine concentrations were examined by categorical groups using Fisher's exact and Wilcoxon rank-sum tests, respectively. RESULTS: Among CCP donors (n = 140), the median time since molecular diagnosis of SARS-CoV-2 was 44 days (interquartile range = 38-50) and 9% (n = 12) were hospitalized due to COVID-19. Compared with healthy blood donor controls, CCP donors had significantly higher plasma levels of interferon (IFN)-γ, interleukin (IL)-10, IL-15, IL-21, and macrophage-inflammatory protein-1, but lower levels of IL-1RA, IL-8, IL-16, and vascular endothelial growth factor-A (P < .0014). The distributions of plasma levels of IL-8, IL-15, and IFN-inducible protein-10 were significantly higher among CCP donors with high (≥160) versus low (<40) anti-SARS-CoV-2 neutralizing antibody titers (P < .0014). The median levels of IL-6 were significantly higher among CCP donors who were hospitalized versus nonhospitalized (P < .0014). CONCLUSIONS: Heterogeneity in cytokine and chemokine composition of CCP suggests there is a different inflammatory state among the CCP donors compared with SARS-CoV-2 naive, healthy blood donors.

Antibody responses to endemic coronaviruses modulate COVID-19 convalescent plasma functionality.

SARS-CoV-2 (CoV2) antibody therapies, including COVID-19 convalescent plasma (CCP), monoclonal antibodies, and hyperimmune globulin, are among the leading treatments for individuals with early COVID-19 infection. The functionality of convalescent plasma varies greatly, but the association of antibody epitope specificities with plasma functionality remains uncharacterized. We assessed antibody functionality and reactivities to peptides across the CoV2 and the 4 endemic human coronavirus (HCoV) genomes in 126 CCP donations. We found strong correlation between plasma functionality and polyclonal antibody targeting of CoV2 spike protein peptides. Antibody reactivity to many HCoV spike peptides also displayed strong correlation with plasma functionality, including pan-coronavirus cross-reactive epitopes located in a conserved region of the fusion peptide. After accounting for antibody cross-reactivity, we identified an association between greater alphacoronavirus NL63 antibody responses and development of highly neutralizing antibodies against CoV2. We also found that plasma preferentially reactive to the CoV2 spike receptor binding domain (RBD), versus the betacoronavirus HKU1 RBD, had higher neutralizing titer. Finally, we developed a 2-peptide serosignature that identifies plasma donations with high anti-spike titer, but that suffer from low neutralizing activity. These results suggest that analysis of coronavirus antibody fine specificities may be useful for selecting desired therapeutics and understanding the complex immune responses elicited by CoV2 infection.

SARS-CoV-2-specific CD8+ T cell responses in convalescent COVID-19 individuals.

Characterization of the T cell response in individuals who recover from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 coronavirus disease 2019 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis and from humoral and inflammatory responses. There were 132 SARS-CoV-2-specific CD8+ T cell responses detected across 6 different HLAs, corresponding to 52 unique epitope reactivities. CD8+ T cell responses were detected in almost all convalescent individuals and were directed against several structural and nonstructural target epitopes from the entire SARS-CoV-2 proteome. A unique phenotype for SARS-CoV-2-specific T cells was observed that was distinct from other common virus-specific T cells detected in the same cross-sectional sample and characterized by early differentiation kinetics. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem cell and transitional memory states (subsets), which may be key to developing durable protection.

Antibody responses to endemic coronaviruses modulate COVID-19 convalescent plasma functionality.

COVID-19 convalescent plasma, particularly plasma with high-titer SARS-CoV-2 (CoV2) antibodies, has been successfully used for treatment of COVID-19. The functionality of convalescent plasma varies greatly, but the association of antibody epitope specificities with plasma functionality remains uncharacterized. We assessed antibody functionality and reactivities to peptides across the CoV2 and the four endemic human coronavirus (HCoV) genomes in 126 COVID-19 convalescent plasma donations. We found strong correlation between plasma functionality and polyclonal antibody targeting of CoV2 spike protein peptides. Antibody reactivity to many HCoV spike peptides also displayed strong correlation with plasma functionality, including pan-coronavirus cross-reactive epitopes located in a conserved region of the fusion peptide. After accounting for antibody cross-reactivity, we identified an association between greater alphacoronavirus NL63 antibody responses and development of highly neutralizing antibodies to SARS-CoV-2. We also found that plasma preferentially reactive to the CoV2 receptor binding domain (RBD), versus the betacoronavirus HKU1 RBD, had higher neutralizing titer. Finally, we developed a two-peptide serosignature that identifies plasma donations with high anti-S titer but that suffer from low neutralizing activity. These results suggest that analysis of coronavirus antibody fine specificities may be useful for selecting therapeutic plasma with desired functionalities.

CD8+ T cell responses in convalescent COVID-19 individuals target epitopes from the entire SARS-CoV-2 proteome and show kinetics of early differentiation.

Characterization of the T cell response in individuals who recover from SARS-CoV-2 infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 COVID-19 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis, humoral and inflammatory responses. 132 distinct SARS-CoV-2-specific CD8+ T cell epitope responses across six different HLAs were detected, corresponding to 52 unique reactivities. T cell responses were directed against several structural and non-structural virus proteins. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem-cell and transitional memory states, subsets, which may be key to developing durable protection.

SARS-CoV-2 antibody signatures robustly predict diverse antiviral functions relevant for convalescent plasma therapy.

Convalescent plasma has emerged as a promising COVID-19 treatment. However, the humoral factors that contribute to efficacy are poorly understood. This study functionally and phenotypically profiled plasma from eligible convalescent donors. In addition to viral neutralization, convalescent plasma contained antibodies capable of mediating such Fc-dependent functions as complement activation, phagocytosis and antibody-dependent cellular cytotoxicity against SARS-CoV-2. These activities expand the antiviral functions associated with convalescent plasma and together with neutralization efficacy, could be accurately and robustly from antibody phenotypes. These results suggest that high-throughput profiling could be used to screen donors and plasma may provide benefits beyond neutralization.

SARS-CoV-2 Antibody Avidity Responses in COVID-19 Patients and Convalescent Plasma Donors.

BACKGROUND: Convalescent plasma therapy is a leading treatment for conferring temporary immunity to COVID-19-susceptible individuals or for use as post-exposure prophylaxis. However, not all recovered patients develop adequate antibody titers for donation and the relationship between avidity and neutralizing titers is currently not well understood. METHODS: SARS-CoV-2 anti-spike and anti-nucleocapsid IgG titers and avidity were measured in a longitudinal cohort of COVID-19 hospitalized patients (n = 16 individuals) and a cross-sectional sample of convalescent plasma donors (n = 130). Epidemiologic correlates of avidity were examined in donors by linear regression. The association of avidity and a high neutralizing titer (NT) were also assessed in donors using modified Poisson regression. RESULTS: Antibody avidity increased over duration of infection and remained elevated. In convalescent plasma donors, higher levels of anti-spike avidity were associated with older age, male sex, and hospitalization. Higher NTs had a stronger positive correlation with anti-spike IgG avidity (Spearman ρ = 0.386; P < .001) than with anti-nucleocapsid IgG avidity (Spearman ρ = 0.211; P = .026). Increasing levels of anti-spike IgG avidity were associated with high NT (≥160) (adjusted prevalence ratio = 1.58 [95% confidence interval = 1.19-2.12]), independent of age, sex, and hospitalization. CONCLUSIONS: SARS-CoV-2 antibody avidity correlated with duration of infection and higher neutralizing titers, suggesting a potential alternative screening parameter for identifying optimal convalescent plasma donors.

Sex, age, and hospitalization drive antibody responses in a COVID-19 convalescent plasma donor population.

Convalescent plasma is a leading treatment for coronavirus disease 2019 (COVID-19), but there is a paucity of data identifying its therapeutic efficacy. Among 126 potential convalescent plasma donors, the humoral immune response was evaluated using a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus neutralization assay with Vero-E6-TMPRSS2 cells; a commercial IgG and IgA ELISA to detect the spike (S) protein S1 domain (EUROIMMUN); IgA, IgG, and IgM indirect ELISAs to detect the full-length S protein or S receptor-binding domain (S-RBD); and an IgG avidity assay. We used multiple linear regression and predictive models to assess the correlations between antibody responses and demographic and clinical characteristics. IgG titers were greater than either IgM or IgA titers for S1, full-length S, and S-RBD in the overall population. Of the 126 plasma samples, 101 (80%) had detectable neutralizing antibody (nAb) titers. Using nAb titers as the reference, the IgG ELISAs confirmed 95%-98% of the nAb-positive samples, but 20%-32% of the nAb-negative samples were still IgG ELISA positive. Male sex, older age, and hospitalization for COVID-19 were associated with increased antibody responses across the serological assays. There was substantial heterogeneity in the antibody response among potential convalescent plasma donors, but sex, age, and hospitalization emerged as factors that can be used to identify individuals with a high likelihood of having strong antiviral antibody responses.

Coxsackievirus B4 Exposure Results in Variable Pattern Recognition Response in the Kidneys of Female Non-Obese Diabetic Mice Before Establishment of Diabetes.

End-stage renal disease (ESRD) is described by four primary diagnoses, diabetes, hypertension, glomerulonephritis, and cystic kidney disease, all of which have viruses implicated as causative agents. Enteroviruses, such as coxsackievirus (CV), are a common genus of viruses that have been implicated in both diabetes and cystic kidney disease; however, little is known about how CVs cause kidney injury and ESRD or predispose individuals with a genetic susceptibility to type 1 diabetes (T1D) to kidney injury. This study evaluated kidney injury resulting from coxsackievirus B4 (CVB4) inoculation of non-obese diabetic (NOD) mice to glean a better understanding of how viral exposure may predispose individuals with a genetic susceptibility to T1D to kidney injury. The objectives were to assess acute and chronic kidney damage in CVB4-inoculated NOD mice without diabetes. Results indicated the presence of CVB4 RNA in the kidney for at least 14 days post-CVB4 inoculation and a coordinated pattern recognition receptor response, but the absence of an immune response or cytotoxicity. CVB4-inoculated NOD mice also had a higher propensity to develop an increase in mesangial area 17 weeks post-CVB4 inoculation. These studies identified initial gene expression changes in the kidney resulting from CVB4 exposure that may predispose to ESRD. Thus, this study provides an initial characterization of kidney injury resulting from CVB4 inoculation of mice that are genetically susceptible to developing T1D that may one day provide better therapeutic options and predictive measures for patients who are at risk for developing kidney disease from T1D.