Distinct Cellular Tropism and Immune Responses to Alphavirus Infection.

Alphaviruses are emerging and reemerging viruses that cause disease syndromes ranging from incapacitating arthritis to potentially fatal encephalitis. While infection by arthritogenic and encephalitic alphaviruses results in distinct clinical manifestations, both virus groups induce robust innate and adaptive immune responses. However, differences in cellular tropism, type I interferon induction, immune cell recruitment, and B and T cell responses result in differential disease progression and outcome. In this review, we discuss aspects of immune responses that contribute to protective or pathogenic outcomes after alphavirus infection.

Vaccine elicitation and structural basis for antibody protection against alphaviruses.

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups. Cryo-EM structures revealed that broad VLP binding inversely correlated with sequence and conformational variability. One triple-specific antibody, SKT05, bound proximal to the fusion peptide and neutralized all three Env-pseudotyped encephalitic alphaviruses by using different symmetry elements for recognition across VLPs. Neutralization in other assays (e.g., chimeric Sindbis virus) yielded variable results. SKT05 bound backbone atoms of sequence-diverse residues, enabling broad recognition despite sequence variability; accordingly, SKT05 protected mice against Venezuelan equine encephalitis virus, chikungunya virus, and Ross River virus challenges. Thus, a single vaccine-elicited antibody can protect in vivo against a broad range of alphaviruses.

Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection.

Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.

A SARS-CoV-2 Infection Model in Mice Demonstrates Protection by Neutralizing Antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of human infections. One limitation to the evaluation of potential therapies and vaccines to inhibit SARS-CoV-2 infection and ameliorate disease is the lack of susceptible small animals in large numbers. Commercially available laboratory strains of mice are not readily infected by SARS-CoV-2 because of species-specific differences in their angiotensin-converting enzyme 2 (ACE2) receptors. Here, we transduced replication-defective adenoviruses encoding human ACE2 via intranasal administration into BALB/c mice and established receptor expression in lung tissues. hACE2-transduced mice were productively infected with SARS-CoV-2, and this resulted in high viral titers in the lung, lung pathology, and weight loss. Passive transfer of a neutralizing monoclonal antibody reduced viral burden in the lung and mitigated inflammation and weight loss. The development of an accessible mouse model of SARS-CoV-2 infection and pathogenesis will expedite the testing and deployment of therapeutics and vaccines.

A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2.

The coronavirus disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of neutralizing antibodies, promotes systemic and mucosal immunoglobulin A (IgA) and T cell responses, and almost entirely prevents SARS-CoV-2 infection in both the upper and lower respiratory tracts. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission and curtailing pandemic spread.

Epigenetic scars of CD8+ T cell exhaustion persist after cure of chronic infection in humans.

T cell exhaustion is an induced state of dysfunction that arises in response to chronic infection and cancer. Exhausted CD8+ T cells acquire a distinct epigenetic state, but it is not known whether that chromatin landscape is fixed or plastic following the resolution of a chronic infection. Here we show that the epigenetic state of exhaustion is largely irreversible, even after curative therapy. Analysis of chromatin accessibility in HCV- and HIV-specific responses identifies a core epigenetic program of exhaustion in CD8+ T cells, which undergoes only limited remodeling before and after resolution of infection. Moreover, canonical features of exhaustion, including super-enhancers near the genes TOX and HIF1A, remain 'epigenetically scarred.' T cell exhaustion is therefore a conserved epigenetic state that becomes fixed and persists independent of chronic antigen stimulation and inflammation. Therapeutic efforts to reverse T cell exhaustion may require new approaches that increase the epigenetic plasticity of exhausted T cells.

SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function.

Although animal models have been evaluated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, none have fully recapitulated the lung disease phenotypes seen in humans who have been hospitalized. Here, we evaluate transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2) as a model of SARS-CoV-2 infection. Intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice results in high levels of viral infection in lungs, with spread to other organs. A decline in pulmonary function occurs 4 days after peak viral titer and correlates with infiltration of monocytes, neutrophils and activated T cells. SARS-CoV-2-infected lung tissues show a massively upregulated innate immune response with signatures of nuclear factor-κB-dependent, type I and II interferon signaling, and leukocyte activation pathways. Thus, the K18-hACE2 model of SARS-CoV-2 infection shares many features of severe COVID-19 infection and can be used to define the basis of lung disease and test immune and antiviral-based countermeasures.

Publisher Correction: SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Modified mRNA Vaccines Protect against Zika Virus Infection.

The emergence of ZIKV infection has prompted a global effort to develop safe and effective vaccines. We engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding wild-type or variant ZIKV structural genes and tested immunogenicity and protection in mice. Two doses of modified mRNA LNPs encoding prM-E genes that produced virus-like particles resulted in high neutralizing antibody titers (∼1/100,000) that protected against ZIKV infection and conferred sterilizing immunity. To offset a theoretical concern of ZIKV vaccines inducing antibodies that cross-react with the related dengue virus (DENV), we designed modified prM-E RNA encoding mutations destroying the conserved fusion-loop epitope in the E protein. This variant protected against ZIKV and diminished production of antibodies enhancing DENV infection in cells or mice. A modified mRNA vaccine can prevent ZIKV disease and be adapted to reduce the risk of sensitizing individuals to subsequent exposure to DENV, should this become a clinically relevant concern.

Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

Recombinant OC43 SARS-CoV-2 spike replacement virus: An improved BSL-2 proxy virus for SARS-CoV-2 neutralization assays.

We generated a replication-competent OC43 human seasonal coronavirus (CoV) expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in place of the native spike (rOC43-CoV2 S). This virus is highly attenuated relative to OC43 and SARS-CoV-2 in cultured cells and animals and is classified as a biosafety level 2 (BSL-2) agent by the NIH biosafety committee. Neutralization of rOC43-CoV2 S and SARS-CoV-2 by S-specific monoclonal antibodies and human sera is highly correlated, unlike recombinant vesicular stomatitis virus-CoV2 S. Single-dose immunization with rOC43-CoV2 S generates high levels of neutralizing antibodies against SARS-CoV-2 and fully protects human ACE2 transgenic mice from SARS-CoV-2 lethal challenge, despite nondetectable replication in respiratory and nonrespiratory organs. rOC43-CoV2 S induces S-specific serum and airway mucosal immunoglobulin A and IgG responses in rhesus macaques. rOC43-CoV2 S has enormous value as a BSL-2 agent to measure S-specific antibodies in the context of a bona fide CoV and is a candidate live attenuated SARS-CoV-2 mucosal vaccine that preferentially replicates in the upper airway.

Potently neutralizing and protective human antibodies against SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health1 and the medical countermeasures available so far are limited2,3. Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-24. Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein5, and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD, as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents.

The effect of Omicron breakthrough infection and extended BNT162b2 booster dosing on neutralization breadth against SARS-CoV-2 variants of concern.

COVID-19 vaccines are playing a vital role in controlling the COVID-19 pandemic. As SARS-CoV-2 variants encoding mutations in the surface glycoprotein, Spike, continue to emerge, there is increased need to identify immunogens and vaccination regimens that provide the broadest and most durable immune responses. We compared the magnitude and breadth of the neutralizing antibody response, as well as levels of Spike-reactive memory B cells, in individuals receiving a second dose of BNT162b2 at a short (3-4 week) or extended interval (8-12 weeks) and following a third vaccination approximately 6-8 months later. We show that whilst an extended interval between the first two vaccinations can greatly increase the breadth of the immune response and generate a higher proportion of Spike reactive memory B cells, a third vaccination leads to similar levels between the two groups. Furthermore, we show that the third vaccine dose enhances neutralization activity against omicron lineage members BA.1, BA.2 and BA.4/BA.5 and this is further increased following breakthrough infection during the UK omicron wave. These findings are relevant for vaccination strategies in populations where COVID-19 vaccine coverage remains low.

HIV outcomes during the COVID-19 pandemic in people of Black ethnicities living with HIV in England.

OBJECTIVES: To describe HIV care outcomes in people of Black ethnicities living in England during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; coronavirus disease 2019 [COVID-19]) pandemic. METHODS: This was an observational cohort study of people of self-reported Black ethnicities attending for HIV care at nine HIV clinics across England. The primary outcome was a composite of antiretroviral therapy (ART) interruption and HIV viraemia (HIV RNA ≥200 copies/mL) ascertained via self-completed questionnaires and review of medical records. We used multivariable logistic regression to explore associations between ART interruption/HIV viraemia and demographic factors, pre-pandemic HIV immunovirological control, comorbidity status, and COVID-19 disease and vaccination status. RESULTS: We included 2290 people (median age 49.3 years; 56% female; median CD4 cell count 555 cells/mm3; 92% pre-pandemic HIV RNA <200 copies/mL), of whom 302 (13%) reported one or more ART interruption, 312 (14%) had documented HIV viraemia ≥200 copies/mL, and 401 (18%) experienced the composite endpoint of ART interruption/HIV viraemia. In multivariable analysis, a pre-pandemic HIV RNA <200 copies/mL (odds ratio [OR] 0.21; 95% confidence interval [CI] 0.15-0.30) and being vaccinated against SARS-CoV-2 (OR 0.41; 95% CI 0.30-0.55) were associated with reduced odds of ART interruption/HIV viraemia; pandemic-related disruptions to HIV care were common self-reported additional factors. CONCLUSIONS: During the COVID-19 pandemic, one in six people of Black ethnicities in this HIV cohort experienced an ART interruption/HIV viraemia. Some of these episodes resulted from pandemic-related healthcare disruptions. Associations with suboptimal engagement in HIV care pre-pandemic and not being vaccinated against SARS-CoV-2 suggest that wider health beliefs and/or poor healthcare access may have been contributory factors.

Clinical epidemiology of COVID-19 in people of black ethnicity living with HIV in the UK.

OBJECTIVES: To describe the clinical epidemiology of COVID-19 in people of black ethnicity living with HIV in the UK. METHODS: We investigated the incidence and factors associated with COVID-19 in a previously established and well-characterized cohort of black people with HIV. Primary outcomes were COVID-19 acquisition and severe COVID-19 disease (requiring hospitalization and/or resulting in death). Cumulative incidence was analysed using Nelson-Aalen methods, and associations between demographic, pre-pandemic immune-virological parameters, comorbidity status and (severe) COVID-19 were identified using Cox regression analysis. RESULTS: COVID-19 status was available for 1847 (74%) of 2495 COVID-AFRICA participants (median age 49.6 years; 56% female; median CD4 cell count = 555 cells/µL; 93% HIV RNA <200 copies/mL), 573 (31%) of whom reported at least one episode of COVID-19. The cumulative incidence rates of COVID-19 and severe COVID-19 were 31.0% and 3.4%, respectively. Region of ancestry (East/Southern/Central vs. West Africa), nadir CD4 count and kidney disease were associated with COVID-19 acquisition. Diabetes mellitus [adjusted hazard ratio (aHR) = 2.39, 95% confidence interval (CI): 1.26-4.53] and kidney disease (aHR = 2.53, 95% CI: 1.26-4.53) were associated with an increased risk, and recent CD4 count >500 cells/µL (aHR = 0.49, 95% CI: 0.25-0.93) with a lower risk of severe COVID-19. CONCLUSIONS: Region of ancestry was associated with COVID-19 acquisition, and immune and comorbidity statuses were associated with COVID-19 disease severity in people of black ethnicity living with HIV in the UK.

Mxra8 is a receptor for multiple arthritogenic alphaviruses.

Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease 1 . The host factors required for alphavirus entry remain poorly characterized 2 . Here we use a genome-wide CRISPR-Cas9-based screen to identify the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses, including chikungunya, Ross River, Mayaro and O'nyong nyong viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced levels of viral infection of cells and, reciprocally, ectopic expression of these genes resulted in increased infection. Mxra8 bound directly to chikungunya virus particles and enhanced virus attachment and internalization into cells. Consistent with these findings, Mxra8-Fc fusion protein or anti-Mxra8 monoclonal antibodies blocked chikungunya virus infection in multiple cell types, including primary human synovial fibroblasts, osteoblasts, chondrocytes and skeletal muscle cells. Mutagenesis experiments suggest that Mxra8 binds to a surface-exposed region across the A and B domains of chikungunya virus E2 protein, which are a speculated site of attachment. Finally, administration of the Mxra8-Fc protein or anti-Mxra8 blocking antibodies to mice reduced chikungunya and O'nyong nyong virus infection as well as associated foot swelling. Pharmacological targeting of Mxra8 could form a strategy for mitigating infection and disease by multiple arthritogenic alphaviruses.

Post-hospitalization remote monitoring for patients with heart failure or chronic obstructive pulmonary disease in an accountable care organization.

BACKGROUND: Post-hospitalization remote patient monitoring (RPM) has potential to improve health outcomes for high-risk patients with chronic medical conditions. The purpose of this study is to determine the extent to which RPM for patients with congestive heart failure (CHF) and chronic obstructive pulmonary disease (COPD) is associated with reductions in post-hospitalization mortality, hospital readmission, and ED visits within an Accountable Care Organization (ACO). METHODS: Nonrandomized prospective study of patients in an ACO offered enrollment in RPM upon hospital discharge between February 2021 and December 2021. RPM comprised of vital sign monitoring equipment (blood pressure monitor, scale, pulse oximeter), tablet device with symptom tracking software and educational material, and nurse-provided oversight and triage. Expected enrollment was for at least 30-days of monitoring, and outcomes were followed for 6 months following enrollment. The co-primary outcomes were (a) the composite of death, hospital admission, or emergency care visit within 180 days of eligibility, and (b) time to occurrence of this composite. Secondary outcomes were each component individually, the composite of death or hospital admission, and outpatient office visits. Adjusted analyses involved doubly robust estimation to address confounding by indication. RESULTS: Of 361 patients offered remote monitoring (251 with CHF and 110 with COPD), 140 elected to enroll (106 with CHF and 34 with COPD). The median duration of RPM-enrollment was 54 days (IQR 34-85). Neither the 6-month frequency of the co-primary composite outcome (59% vs 66%, FDR p-value = 0.47) nor the time to this composite (median 29 vs 38 days, FDR p-value = 0.60) differed between the groups, but 6-month mortality was lower in the RPM group (6.4% vs 17%, FDR p-value = 0.02). After adjustment for confounders, RPM enrollment was associated with nonsignificantly decreased odds for the composite outcome (adjusted OR [aOR] 0.68, 99% CI 0.25-1.34, FDR p-value 0.30) and lower 6-month mortality (aOR 0.41, 99% CI 0.00-0.86, FDR p-value 0.20). CONCLUSIONS: RPM enrollment may be associated with improved health outcomes, including 6-month mortality, for selected patient populations.

Limited Weight Impact After Switching From Boosted Protease Inhibitors to Dolutegravir in Persons With Human Immunodeficiency Virus With High Cardiovascular Risk: A Post Hoc Analysis of the 96-Week NEAT-022 Randomized Trial.

BACKGROUND: In the NEAT022 trial, virologically suppressed persons with human immunodeficiency virus (HIV) at high cardiovascular risk switching from protease inhibitors to dolutegravir either immediately (DTG-I) or after 48 weeks (DTG-D) showed noninferior virological suppression and significant lipid and cardiovascular disease risk reductions on switching to dolutegravir relative to continuing protease inhibitors. METHODS: In post hoc analysis, major endpoints were 48-week and 96-week weight and body mass index (BMI) changes. Factors associated with weight/BMI changes within the first 48 weeks of DTG exposure, proportion of participants by category of percentage weight change, proportions of BMI categories over time, and impact on metabolic outcomes were also assessed. RESULTS: Between May 2014 and November 2015, 204 (DTG-I) and 208 (DTG-D) participants were included. Weight significantly increased (mean, +0.810 kg DTG-I arm, and +0.979 kg DTG-D arm) in the first 48 weeks postswitch, but remained stable from 48 to 96 weeks in DTG-I arm. Switching from darunavir, White race, total to high-density lipoprotein cholesterol ratio <3.7, and normal/underweight BMI were independently associated with higher weight/BMI gains. The proportion of participants with ≥5% weight change increased similarly in both arms over time. The proportions of BMI categories, use of lipid-lowering drugs, diabetes and/or use of antidiabetic agents, and hypertension and/or use of antihypertensive agents did not change within or between arms at 48 and 96 weeks. CONCLUSIONS: Switching from protease inhibitors to dolutegravir in persons with HIV with high cardiovascular risk led to modest weight gain limited to the first 48 weeks, which involved preferentially normal-weight or underweight persons and was not associated with negative metabolic outcomes. CLINICAL TRIALS REGISTRATION: NCT02098837 and EudraCT 2013-003704-39.

Incidence of Hypertension and Blood Pressure Changes in Persons With Human Immunodeficiency Virus at High Risk for Cardiovascular Disease Switching From Boosted Protease Inhibitors to Dolutegravir: A Post-hoc Analysis of the 96-week Randomised NEAT-022 Trial.

BACKGROUND: Integrase inhibitors have been recently linked to a higher risk for hypertension. In NEAT022 randomized trial, virologically suppressed persons with human immunodeficiency virus (HIV, PWH) with high cardiovascular risk switched from protease inhibitors to dolutegravir either immediately (DTG-I) or after 48 weeks (DTG-D). METHODS: Primary endpoint was incident hypertension at 48 weeks. Secondary endpoints were changes in systolic (SBP) and diastolic (DBP) blood pressure; adverse events and discontinuations associated with high blood pressure; and factors associated with incident hypertension. RESULTS: At baseline, 191 (46.4%) participants had hypertension and 24 persons without hypertension were receiving antihypertensive medications for other reasons. In the 197 PWH (n = 98, DTG-I arm; n = 99, DTG-D arm) without hypertension or antihypertensive agents at baseline, incidence rates per 100 person-years were 40.3 and 36.3 (DTG-I) and 34.7 and 52.0 (DTG-D) at 48 (P = .5755) and 96 (P = .2347) weeks. SBP or DBP changes did not differed between arms. DBP (mean, 95% confidence interval) significantly increased in both DTG-I (+2.78 mmHg [1.07-4.50], P = .0016) and DTG-D (+2.29 mmHg [0.35-4.23], P = .0211) arms in the first 48 weeks of exposure to dolutegravir. Four (3 under dolutegravir, 1 under protease inhibitors) participants discontinued study drugs due to adverse events associated with high blood pressure. Classical factors, but not treatment arm, were independently associated with incident hypertension. CONCLUSIONS: PWH at high risk for cardiovascular disease showed high rates of hypertension at baseline and after 96 weeks. Switching to dolutegravir did not negatively impact on the incidence of hypertension or blood pressure changes relative to continuing protease inhibitors.