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The pathogenesis of COVID-19 is associated with a hyperinflammatory immune response. Monocytes and macrophages play a central role in this hyperinflammatory response to SARS-CoV-2. NLRP3 inflammasome activation has been observed in monocytes of patients with COVID-19, but the mechanism and consequences of inflammasome activation require further investigation. In this study, we inoculated a macrophage-like THP-1 cell line, primary differentiated human nasal epithelial cell (hNEC) cultures, and primary monocytes with SARS-CoV-2. We found that the activation of the NLRP3 inflammasome in macrophages does not rely on viral replication, receptor-mediated entry, or actin-dependent entry. SARS-CoV-2 productively infected hNEC cultures without triggering the production of inflammasome cytokines IL-18 and IL-1β. Importantly, these cytokines did not inhibit viral replication in hNEC cultures. SARS-CoV-2 inoculation of primary monocytes led to inflammasome activation and induced a macrophage phenotype in these cells. Monocytic cells from bronchoalveolar lavage (BAL) fluid, but not from peripheral blood, of patients with COVID-19, showed evidence of inflammasome activation, expressed the proinflammatory marker CD11b, and displayed oxidative burst. These findings highlight the central role of activated macrophages, as a result of direct viral sensing, in COVID-19 and support the inhibition of IL-1β and IL-18 as potential therapeutic strategies to reduce immunopathology without increasing viral replication., Importance: Inflammasome activation is associated with severe COVID-19. The impact of inflammasome activation on viral replication and mechanistic details of this activation are not clarified. This study advances our understanding of the role of inflammasome activation in macrophages by identifying TLR2, NLRP3, ASC, and caspase-1 as dependent factors in this activation. Further, it highlights that SARS-CoV-2 inflammasome activation is not a feature of nasal epithelial cells but rather activation of bystander macrophages in the airway. Finally, we demonstrate that two pro inflammatory cytokines produced by inflammasome activation, IL-18 and IL-1β, do not restrict viral replication and are potential targets to ameliorate pathological inflammation in severe COVID-19.

Pregnant patients are at greater risk of hospitalization with severe COVID-19 than non-pregnant people. This was a retrospective observational cohort study of remnant clinical specimens from patients who visited acute care hospitals within the Johns Hopkins Health System in the Baltimore, MD-Washington DC, area between October 2020 and May 2022. Participants included confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected pregnant people and matched non-pregnant people (the matching criteria included age, race/ethnicity, area deprivation index, insurance status, and vaccination status to ensure matched demographics). The primary dependent measures were clinical COVID-19 outcomes, infectious virus recovery, viral RNA levels, and mucosal anti-spike (S) IgG titers from upper respiratory tract samples. A total of 452 individuals (117 pregnant and 335 non-pregnant) were included in the study, with both vaccinated and unvaccinated individuals represented. Pregnant patients were at increased risk of hospitalization (odds ratio [OR] = 4.2; confidence interval [CI] = 2.0-8.6), intensive care unit admittance (OR = 4.5; CI = 1.2-14.2), and being placed on supplemental oxygen therapy (OR = 3.1; CI = 1.3-6.9). Individuals infected during their third trimester had higher mucosal anti-S IgG titers and lower viral RNA levels ( P < 0.05) than those infected during their first or second trimesters. Pregnant individuals experiencing breakthrough infections due to the Omicron variant had reduced anti-S IgG compared to non-pregnant patients ( P < 0.05). The observed increased severity of COVID-19 and reduced mucosal antibody responses particularly among pregnant participants infected with the Omicron variant suggest that maintaining high levels of SARS-CoV-2 immunity through booster vaccines may be important for the protection of this at-risk population.IMPORTANCEIn this retrospective observational cohort study, we analyzed remnant clinical samples from non-pregnant and pregnant individuals with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections who visited the Johns Hopkins Hospital System between October 2020 and May 2022. Disease severity, including intensive care unit admission, was greater among pregnant than non-pregnant patients. Vaccination reduced recovery of infectious virus and viral RNA levels in non-pregnant patients, but not in pregnant patients. In pregnant patients, increased nasopharyngeal viral RNA levels and recovery of infectious virus were associated with reduced mucosal IgG antibody responses, especially among women in their first trimester of pregnancy or experiencing breakthrough infections from Omicron variants. Taken together, this study provides insights into how pregnant patients are at greater risk of severe COVID-19. The novelty of this study is that it focuses on the relationship between the mucosal antibody response and its association with virus load and disease outcomes in pregnant people, whereas previous studies have focused on serological immunity. Vaccination status, gestational age, and SARS-CoV-2 omicron variant impact mucosal antibody responses and recovery of infectious virus from pregnant patients., Competing Interests: The authors declare no conflict of interest.

The impact of preexisting antibodies to the four endemic human coronaviruses (ehCoV) (229E, OC43, NL63, and HKU1) on severe (hospitalization) coronavirus disease 2019 (COVID-19) outcomes has been described in small cohorts. Many studies have measured ehCoV 229E, OC43, NL63, and HKU1 antibody levels weeks after recovery rather than in the first weeks of illness, which is more relevant to early hospitalizations. Antibody levels to the spike protein of the four coronaviruses (229E, OC43, NL63, and HKU1), as well as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were measured both before and immediately after convalescent or control plasma transfusion in 51 participants who were hospitalized and 250 who were not hospitalized, as well as in 71 convalescent and 50 control plasma donors as a subset from a completed randomized controlled trial. In COVID-19 convalescent plasma donors, the ehCoV spike antibodies were 1.2 to 2 times greater than the control donor unit levels, while donor COVID-19 convalescent plasma (CCP) SARS-CoV-2 spike antibodies were more than 600 times the control plasma units. Plasma transfusion, whether COVID-19 convalescent or control, did not alter the post-transfusion antibody levels for the endemic human coronaviruses (229E, OC43, NL63, and HKU1) in those hospitalized and not hospitalized, despite the 1.2- to 2-fold elevation in donor COVID-19 convalescent plasma. There was no influence of prior antibody levels to 229E, OC43, NL63, and HKU1 or post-transfusion antibody levels on subsequent hospitalization. These data, from a well-controlled prospective randomized clinical trial, add evidence that antibodies to ehCoV do not significantly impact COVID-19 outcomes, despite the apparent back-boosting of some ehCoV after SARS-CoV-2 infection. IMPORTANCE The relevance of preexisting immunity to the four endemic human coronaviruses in the first week of COVID-19 illness on the outcome of COVID-19 progression stems from the high prevalence of the ehCoV and SARS-CoV-2 coronaviruses. The question has been raised of whether therapeutic convalescent plasma or control plasma containing ehCoV antibodies might alter the outcome of COVID-19 progression to hospitalization. Here, we observed that plasma transfusion did not significantly change the preexisting ehCoV antibody levels. In over 50 hospitalized participants and 250 nonhospitalized participants, ehCoV antibody levels were comparable, without statistical differences. Antibody levels were stable over the more than 12 months of the intervention trial, with individual heterogeneity similar in hospitalized and nonhospitalized participants. The ehCoV antibodies in plasma transfusion did not alter the recipient preexisting antibody levels nor hasten the COVID-19 progression to hospitalization in this clinical trial data.

Emergency departments (EDs) can serve as surveillance sites for infectious diseases. The objective of this study was to determine the burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and to monitor the prevalence of vaccination against coronavirus disease 2019 (COVID-19) among patients attending an urban ED in Baltimore City. Using 1,914 samples of known exposure status, we developed an algorithm to differentiate previously infected, vaccinated, and unexposed individuals using a combination of antibody assays. We applied this testing algorithm to 4,360 samples from ED patients obtained in the spring of 2020 and 2021. Using multinomial logistic regression, we determined factors associated with infection and vaccination. For the algorithm, sensitivity and specificity for identifying vaccinated individuals were 100% and 99%, respectively, and 84% and 100% for previously infected individuals. Among the ED subjects, seroprevalence to SARS-CoV-2 increased from 2% to 24% between April 2020 and March 2021. Vaccination prevalence rose to 11% by mid-March 2021. Marked differences in burden of disease and vaccination coverage were seen by sex, race, and ethnicity. Hispanic patients, though accounting for 7% of the study population, had the highest relative burden of disease (17% of total infections) but with similar vaccination rates. Women and white individuals were more likely to be vaccinated than men or Black individuals. Individuals previously infected with SARS-CoV-2 can often be differentiated from vaccinated individuals using a serologic testing algorithm. The utility of this algorithm can aid in monitoring SARS-CoV-2 exposure and vaccination uptake frequencies and can potentially reflect gender, race, and ethnic health disparities.

HIV-1 infection persists in humans despite expression of antiviral type 1 interferons (IFN). Even exogenous administration of IFNα only marginally reduces HIV-1 abundance, raising the hypothesis that people living with HIV-1 (PLWH) are refractory to type 1 IFN. We demonstrated type 1 IFN refractoriness in CD4+ and CD8+ T cells isolated from HIV-1 infected persons by detecting diminished STAT1 phosphorylation (pSTAT1) and interferon-stimulated gene (ISG) induction upon type 1 IFN stimulation compared to healthy controls. Importantly, HIV-1 infected people who were virologically suppressed with antiretrovirals also showed type 1 IFN refractoriness. We found that USP18 levels were elevated in people with refractory pSTAT1 and ISG induction and confirmed this finding ex vivo in CD4+ T cells from another cohort of HIV-HCV coinfected persons who received exogenous pegylated interferon-α2b in a clinical trial. We used a cell culture model to recapitulate type 1 IFN refractoriness in uninfected CD4+ T cells that were conditioned with media from HIV-1 inoculated PBMCs, inhibiting de novo infection with antiretroviral agents. In this model, RNA interference against USP18 partly restored type 1 IFN responses in CD4+ T cells. We found evidence of type 1 IFN refractoriness in PLWH irrespective of virologic suppression that was associated with upregulated USP18, a process that might be therapeutically targeted to improve endogenous control of infection. Importance People living with HIV-1 (PLWH) have elevated constitutive expression of type 1 interferons (IFN). However, it is unclear whether this impacts downstream innate immune responses. We identified refractory responses to type 1 IFN stimulation in T cells from PLWH, independent of antiretroviral treatment. Type 1 IFN refractoriness was linked to elevated USP18 levels in the same cells. Moreover, we found that USP18 levels predicted the anti-HIV-1 effect of type 1 IFN-based therapy on PLWH. In vitro , we demonstrated that refractory type 1 IFN responses were transferrable to HIV-1 uninfected target CD4+ T cells, and this phenomenon was mediated by type 1 IFN from HIV-1 infected cells. Type 1 IFN responses were partially restored by USP18 knockdown. Our findings illuminate a new mechanism by which HIV-1 contributes to innate immune dysfunction in PLWH, through the continuous production of type 1 IFN that induces a refractory state of responsiveness., (Copyright © 2021 American Society for Microbiology.)

Increasing evidence indicates that broadly neutralizing antibodies (bNAbs) play an important role in immune-mediated control of hepatitis C virus (HCV) infection, but the relative contribution of neutralizing antibodies targeting antigenic sites across the HCV envelope (E1 and E2) proteins is unclear. Here, we isolated thirteen E1E2-specific monoclonal antibodies (MAbs) from B cells of a single HCV-infected individual who cleared one genotype 1a infection and then became persistently infected with a second genotype 1a strain. These MAbs bound six distinct discontinuous antigenic sites on the E1 protein, the E2 protein, or the E1E2 heterodimer. Three antigenic sites, designated AS108, AS112 (an N-terminal E1 site), and AS146, were distinct from previously described antigenic regions (ARs) 1 to 5 and E1 sites. Antibodies targeting four sites (AR3, AR4-5, AS108, and AS146) were broadly neutralizing. These MAbs also displayed distinct patterns of relative neutralizing potency (i.e., neutralization profiles) across a panel of diverse HCV strains, which led to complementary neutralizing breadth when they were tested in combination. Overall, this study demonstrates that HCV bNAb epitopes are not restricted to previously described antigenic sites, expanding the number of sites that could be targeted for vaccine development. IMPORTANCE Worldwide, more than 70 million people are infected with hepatitis C virus (HCV), which is a leading cause of hepatocellular carcinoma and liver transplantation. Despite the development of potent direct acting antivirals (DAAs) for HCV treatment, a vaccine is urgently needed due to the high cost of treatment and the possibility of reinfection after cure. Induction of multiple broadly neutralizing antibodies (bNAbs) that target distinct epitopes on the HCV envelope proteins is one approach to vaccine development. However, antigenic sites targeted by bNAbs in individuals with spontaneous control of HCV have not been fully defined. In this study, we characterize 13 monoclonal antibodies (MAbs) from a single person who cleared an HCV infection without treatment, and we identify 3 new sites targeted by neutralizing antibodies. The sites targeted by these MAbs could inform HCV vaccine development., (Copyright © 2019 American Society for Microbiology.)

Unlabelled: Plasma microRNAs (miRNAs) change in abundance in response to disease and have been associated with liver fibrosis severity in chronic hepatitis C virus (HCV) infection. However, the early dynamics of miRNA release during acute HCV infection are poorly understood. In addition, circulating miRNA signatures have been difficult to reproduce among separate populations. We studied plasma miRNA abundance during acute HCV infection to identify an miRNA signature of early infection. We measured 754 plasma miRNAs by quantitative PCR array in a discovery cohort of 22 individuals before and during acute HCV infection and after spontaneous resolution (n = 11) or persistence (n = 11) to identify a plasma miRNA signature. The discovery cohort derived from the Baltimore Before and After Acute Study of Hepatitis. During acute HCV infection, increases in miR-122 (P < 0.01) and miR-885-5p (Pcorrected < 0.05) and a decrease in miR-494 (Pcorrected < 0.05) were observed at the earliest time points after virus detection. Changes in miR-122 and miR-885-5p were sustained in persistent (P < 0.001) but not resolved HCV infection. The circulating miRNA signature of acute HCV infection was confirmed in a separate validation cohort that was derived from the San Francisco-based You Find Out (UFO) Study (n = 28). As further confirmation, cellular changes of signature miRNAs were examined in a tissue culture model of HCV in hepatoma cells: HCV infection induced extracellular release of miR-122 and miR-885-5p despite unperturbed intracellular levels. In contrast, miR-494 accumulated intracellularly (P < 0.05). Collectively, these data are inconsistent with necrolytic release of hepatocyte miRNAs into the plasma during acute HCV infection of humans., Importance: MicroRNAs are small noncoding RNA molecules that emerging research shows can transmit regulatory signals between cells in health and disease. HCV infects 2% of humans worldwide, and chronic HCV infection is a major cause of severe liver disease. We profiled plasma miRNAs in injection drug users before, during, and (in the people with resolution) after HCV infection. We discovered miRNA signatures of acute and persistent viremia and confirmed these findings two ways: (i) in a separate cohort of people with newly acquired HCV infection and (ii) in an HCV cell culture system. Our results demonstrate that acute HCV infection induces early changes in the abundance of specific plasma miRNAs that may affect the host response to HCV infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Hepatitis C virus (HCV) research is hampered by the use of arbitrary representative isolates in cell culture and immunology. The most replicative isolate in vitro is a subtype 2a virus (JFH-1); however, genotype 1 is more prevalent worldwide and represents about 70% of infections in the United States, and genotypes differ from one another by 31% to 33% at the nucleotide level. For phylogenetic and immunologic analyses, viruses H77 and HCV-1 (both subtype 1a) are commonly used based on their historic importance. In an effort to rationally design a representative subtype 1a virus (Bole1a), we used Bayesian phylogenetics, ancestral sequence reconstruction, and covariance analysis on a curated set of 390 full-length human HCV 1a sequences from GenBank. By design, Bole1a contains variations present in widely circulating strains and matches more epitope-sized peptides in a full-genome comparison to subtype 1a isolates than any other sequence studied. Parallel analyses confirm that selected epitopes from the Bole1a genome were able to elicit a robust T cell response. In a proof of concept for infectivity, the envelope genes (E1 and E2) of Bole1a were expressed in an HIV pseudoparticle system containing HCV envelope genes and HIV nonenvelope genes with luciferase expression. The resulting Bole1a pseudoparticle robustly infected Hep3B cells. In this study, we demonstrate that a rationally designed, fully synthetic HCV genome contains representative epitopes and envelope genes that assemble properly and mediate entry into target cells.

During the transition from acute to chronic infection in individuals persistently infected with hepatitis C virus (HCV), cellular responses initiate within the first 6 months of primary infection and collapse thereafter, whereas humoral responses activate later during the chronic phase. Whether and how this deviation of immune responses specifically influences HCV evolution are unknown. To determine the pattern of HCV evolution during this critical period, we conducted extensive sequence analysis on annual clonal hemigenomic sequences for up to 3 years in six well-characterized subjects, using statistical methods that accounted for repeated measures. Significantly different evolutionary rates were observed in envelope versus nonenvelope genes, with an increasing rate of nonsynonymous change (dN) in envelope genes and a stable dN in nonenvelope genes (P = 0.006). The ratio of the envelope to nonenvelope nonsynonymous rate increased from 2 in year 1 to 5 in years 2 and 3. Centripetal changes (reversions toward matching of the worldwide subtype 1a consensus sequence) were frequently observed during the 3-year transition from acute infection to chronicity, even in the presence of neutralizing antibody (NAb) pressure. Remarkably, sequences of hypervariable region 1 (HVR1) remained stable for up to 21 months in the absence of NAb pressure in one subject, followed by rapid changes that were temporally associated with the detection of NAb responses, which strongly suggests that HVR1 evolution is shaped by NAb pressure. These data provide the first systematic estimates of HCV evolutionary rates in multiple genes during early infection in vivo and provide additional evidence for deterministic, rather than random, evolution of HCV.