Search

We developed a 4-parameter clinical assay using Electric Field Induced Release and Measurement (EFIRM) technology to simultaneously assess SARS-CoV-2 RNA (vRNA), nucleocapsid antigen, host binding (BAb) and neutralizing antibody (NAb) levels from a drop of saliva with performance that equals or surpasses current EUA-approved tests. The vRNA and antigen assays achieved lower limit of detection (LOD) of 100 copies/reaction and 3.5 TCID₅₀/mL, respectively. The vRNA assay differentiated between acutely infected (n=10) and infection-naïve patients (n=33) with an AUC of 0.9818, sensitivity of 90%, and specificity of 100%. The antigen assay similarly differentiated these patient populations with an AUC of 1.000. The BAb assay detected BAbs with an LOD of 39 pg/mL and distinguished acutely infected (n=35), vaccinated with prior infection (n=13), and vaccinated infection-naïve patients (n=13) from control (n=81) with AUC of 0.9481, 1.000, and 0.9962, respectively. The NAb assay detected NAbs with an LOD of 31.6 Unit/mL and differentiated between COVID-19 recovered or vaccinated patients (n=31) and pre-pandemic controls (n=60) with an AUC 0.923, sensitivity of 87.10%, and specificity of 86.67%. Our multiparameter assay represents a significant technological advancement to simultaneously address SARS-CoV-2 infection and immunity, and it lays the foundation for tackling potential future pandemics.

BackgroundThere are limited data on how coronavirus disease 2019 (COVID-19) severity, timing of infection, and subsequent vaccination impact transplacental transfer and persistence of maternal and infant antibodies.MethodsIn a longitudinal cohort of pregnant women with polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, maternal/infant sera were collected at enrollment, delivery/birth, and 6 months. Anti-SARS-CoV-2 spike immunoglobulin (Ig)G, IgM, and IgA were measured by enzyme-linked immunosorbent assay.ResultsTwo-hundred fifty-six pregnant women and 135 infants were enrolled; 148 maternal and 122 neonatal specimens were collected at delivery/birth; 45 maternal and 48 infant specimens were collected at 6 months. Sixty-eight percent of women produced all anti-SARS-CoV-2 isotypes at delivery (IgG, IgM, IgA); 96% had at least 1 isotype. Symptomatic disease and vaccination before delivery were associated with higher maternal IgG at labor and delivery. Detectable IgG in infants dropped from 78% at birth to 52% at 6 months. In the multivariate analysis evaluating factors associated with detectable IgG in infants at delivery, significant predictors were 3rd trimester infection (odds ratio [OR] = 4.0), mild/moderate disease (OR = 4.8), severe/critical disease (OR = 6.3), and maternal vaccination before delivery (OR = 18.8). No factors were significant in the multivariate analysis at 6 months postpartum.ConclusionsVaccination in pregnancy post-COVID-19 recovery is a strategy for boosting antibodies in mother-infant dyads.

IntroductionThe COVID-19 pandemic has highlighted the need to identify mechanisms of antiviral host defense against SARS-CoV-2. One such mediator is interferon-g (IFN-γ), which, when administered to infected patients, is reported to result in viral clearance and resolution of pulmonary symptoms. IFN-γ treatment of a human lung epithelial cell line triggered an antiviral activity against SARS-CoV-2, yet the mechanism for this antiviral response was not identified.MethodsGiven that IFN-γ has been shown to trigger antiviral activity via the generation of nitric oxide (NO), we investigated whether IFN-γ induction of antiviral activity against SARS-CoV-2 infection is dependent upon the generation of NO in human pulmonary epithelial cells. We treated the simian epithelial cell line Vero E6 and human pulmonary epithelial cell lines, including A549-ACE2, and Calu-3, with IFN-γ and observed the resulting induction of NO and its effects on SARS-CoV-2 replication. Pharmacological inhibition of inducible nitric oxide synthase (iNOS) was employed to assess the dependency on NO production. Additionally, the study examined the effect of interleukin-1b (IL-1β) on the IFN-g-induced NO production and its antiviral efficacy.ResultsTreatment of Vero E6 cells with IFN-γ resulted in a dose-responsive induction of NO and an inhibitory effect on SARS-CoV-2 replication. This antiviral activity was blocked by pharmacologic inhibition of iNOS. IFN-γ also triggered a NO-mediated antiviral activity in SARS-CoV-2 infected human lung epithelial cell lines A549-ACE2 and Calu-3. IL-1β enhanced IFN-γ induction of NO, but it had little effect on antiviral activity.DiscussionGiven that IFN-g has been shown to be produced by CD8+ T cells in the early response to SARS-CoV-2, our findings in human lung epithelial cell lines, of an IFN-γ-triggered, NO-dependent, links the adaptive immune response to an innate antiviral pathway in host defense against SARS-CoV-2. These results underscore the importance of IFN-γ and NO in the antiviral response and provide insights into potential therapeutic strategies for COVID-19.

BackgroundAlthough mRNA vaccines have overall efficacy preventing morbidity/mortality from SARS-CoV-2 infection, immunocompromised persons remain at risk. Antibodies mostly prevent early symptomatic infection, but cellular immunity, particularly the virus-specific CD8+ T cell response, is protective against disease. Defects in T cell responses to vaccination have not been well characterized in immunocompromised hosts; persons with lung transplantation are particularly vulnerable to vaccine failure with severe illness.MethodsComparison groups included persons with lung transplantation and no history of COVID-19 (21 and 19 persons after initial mRNA vaccination and a third booster vaccination respectively), 8 lung transplantation participants recovered from COVID-19, and 22 non-immunocompromised healthy control individuals after initial mRNA vaccination (without history of COVID-19). Anti-spike T cell responses were assayed by stimulating peripheral blood mononuclear cells (PBMCs) with pooled small overlapping peptides spanning the SARS-CoV-2 spike protein, followed by intracellular cytokine staining (ICS) and flow cytometry for release of cytokines in response to stimulation, including negative controls (no peptide stimulation) and positive controls (phorbol myristate acetate [PMA] and ionomycin stimulation). To evaluate for low frequency memory responses, PBMCs were cultured in the presence of the mRNA-1273 vaccine for 14 days before this evaluation.ResultsIonophore stimulation of PBMCs revealed a less inflammatory milieu in terms of interleukin (IL)-2, IL-4, and IL-10 profiling in lung transplantation individuals, reflecting the effect of immunosuppressive treatments. Similar to what we previously reported in healthy vaccinees, spike-specific responses in lung transplantation recipients were undetectable (

IntroductionSevere COVID-19 illness is characterized by an overwhelming immune hyperactivation. Autoantibodies against vascular, tissue, and cytokine antigens have been detected across the spectrum of COVID-19. How these autoantibodies correlate with COVID-19 severity is not fully defined.MethodsWe performed an exploratory study to investigate the expression of vascular and non-HLA autoantibodies in 110 hospitalized patients with COVID-19 ranging from moderate to critically ill. Relationships between autoantibodies and COVID- 19 severity and clinical risk factors were examined using logistic regression analysis.ResultsThere were no absolute differences in levels of expression of autoantibodies against angiotensin II receptor type 1 (AT1R) or endothelial cell proteins between COVID-19 severity groups. AT1R autoantibody expression also did not differ by age, sex, or diabetes status. Using a multiplex panel of 60 non- HLA autoantigens we did identify seven autoantibodies that differed by COVID-19 severity including myosin (myosin; p=0.02), SHC-transforming protein 3 (shc3; p=0.07), peroxisome proliferator-activated receptor gamma coactivator 1-beta (perc; p=0.05), glial-cell derived neurotrophic factor (gdnf; p=0.07), enolase 1 (eno1; p=0.08), latrophilin-1 (lphn1; p=0.08), and collagen VI (coll6; p=0.05) with greater breadth and higher expression levels seen in less severe COVID-19.DiscussionOverall, we found that patients hospitalized with COVID-19 demonstrate evidence of auto-reactive antibodies targeting endothelial cells, angiotensin II receptors, and numerous structural proteins including collagens. Phenotypic severity did not correlate with specific autoantibodies. This exploratory study underscores the importance of better understanding of the role of autoimmunity in COVID-19 disease and sequelae.

IntroductionWhile antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined.MethodsInterferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD).ResultsIn two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination.DiscussionOverall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

A hallmark of HIV-1 infection is chronic inflammation, even in patients treated with antiretroviral therapy (ART). Chronic inflammation drives HIV-1 pathogenesis, leading to loss of CD4+ T cells and exhaustion of antiviral immunity. Therefore, strategies to safely reduce systematic inflammation are needed to halt disease progression and restore defective immune responses. Autophagy is a cellular mechanism for disposal of damaged organelles and elimination of intracellular pathogens. Autophagy is pivotal for energy homeostasis and plays critical roles in regulating immunity. However, how it regulates inflammation and antiviral T cell responses during HIV infection is unclear. Here, we demonstrate that autophagy is directly linked to IFN-I signaling, which is a key driver of immune activation and T cell exhaustion during chronic HIV infection. Impairment of autophagy leads to spontaneous IFN-I signaling, and autophagy induction reduces IFN-I signaling in monocytic cells. Importantly, in HIV-1-infected humanized mice, autophagy inducer rapamycin treatment significantly reduced persistent IFN-I-mediated inflammation and improved antiviral T cell responses. Cotreatment of rapamycin with ART led to significantly reduced viral rebound after ART withdrawal. Taken together, our data suggest that therapeutically targeting autophagy is a promising approach to treat persistent inflammation and improve immune control of HIV replication.

In an exploratory trial treating "long COVID" with the CCR5-binding antibody leronlimab, we observed significantly increased blood cell surface CCR5 in treated symptomatic responders but not in nonresponders or placebo-treated participants. These findings suggest an unexpected mechanism of abnormal immune downmodulation in some persons that is normalized by leronlimab. Clinical Trials Registration. NCT04678830.

BackgroundBaricitinib and dexamethasone have randomised trials supporting their use for the treatment of patients with COVID-19. We assessed the combination of baricitinib plus remdesivir versus dexamethasone plus remdesivir in preventing progression to mechanical ventilation or death in hospitalised patients with COVID-19.MethodsIn this randomised, double-blind, double placebo-controlled trial, patients were enrolled at 67 trial sites in the USA (60 sites), South Korea (two sites), Mexico (two sites), Singapore (two sites), and Japan (one site). Hospitalised adults (≥18 years) with COVID-19 who required supplemental oxygen administered by low-flow (≤15 L/min), high-flow (>15 L/min), or non-invasive mechanical ventilation modalities who met the study eligibility criteria (male or non-pregnant female adults ≥18 years old with laboratory-confirmed SARS-CoV-2 infection) were enrolled in the study. Patients were randomly assigned (1:1) to receive either baricitinib, remdesivir, and placebo, or dexamethasone, remdesivir, and placebo using a permuted block design. Randomisation was stratified by study site and baseline ordinal score at enrolment. All patients received remdesivir (≤10 days) and either baricitinib (or matching oral placebo) for a maximum of 14 days or dexamethasone (or matching intravenous placebo) for a maximum of 10 days. The primary outcome was the difference in mechanical ventilation-free survival by day 29 between the two treatment groups in the modified intention-to-treat population. Safety analyses were done in the as-treated population, comprising all participants who received one dose of the study drug. The trial is registered with ClinicalTrials.gov, NCT04640168.FindingsBetween Dec 1, 2020, and April 13, 2021, 1047 patients were assessed for eligibility. 1010 patients were enrolled and randomly assigned, 516 (51%) to baricitinib plus remdesivir plus placebo and 494 (49%) to dexamethasone plus remdesivir plus placebo. The mean age of the patients was 58·3 years (SD 14·0) and 590 (58%) of 1010 patients were male. 588 (58%) of 1010 patients were White, 188 (19%) were Black, 70 (7%) were Asian, and 18 (2%) were American Indian or Alaska Native. 347 (34%) of 1010 patients were Hispanic or Latino. Mechanical ventilation-free survival by day 29 was similar between the study groups (Kaplan-Meier estimates of 87·0% [95% CI 83·7 to 89·6] in the baricitinib plus remdesivir plus placebo group and 87·6% [84·2 to 90·3] in the dexamethasone plus remdesivir plus placebo group; risk difference 0·6 [95% CI -3·6 to 4·8]; p=0·91). The odds ratio for improved status in the dexamethasone plus remdesivir plus placebo group compared with the baricitinib plus remdesivir plus placebo group was 1·01 (95% CI 0·80 to 1·27). At least one adverse event occurred in 149 (30%) of 503 patients in the baricitinib plus remdesivir plus placebo group and 179 (37%) of 482 patients in the dexamethasone plus remdesivir plus placebo group (risk difference 7·5% [1·6 to 13·3]; p=0·014). 21 (4%) of 503 patients in the baricitinib plus remdesivir plus placebo group had at least one treatment-related adverse event versus 49 (10%) of 482 patients in the dexamethasone plus remdesivir plus placebo group (risk difference 6·0% [2·8 to 9·3]; p=0·00041). Severe or life-threatening grade 3 or 4 adverse events occurred in 143 (28%) of 503 patients in the baricitinib plus remdesivir plus placebo group and 174 (36%) of 482 patients in the dexamethasone plus remdesivir plus placebo group (risk difference 7·7% [1·8 to 13·4]; p=0·012).InterpretationIn hospitalised patients with COVID-19 requiring supplemental oxygen by low-flow, high-flow, or non-invasive ventilation, baricitinib plus remdesivir and dexamethasone plus remdesivir resulted in similar mechanical ventilation-free survival by day 29, but dexamethasone was associated with significantly more adverse events, treatment-related adverse events, and severe or life-threatening adverse events. A more individually tailored choice of immunomodulation now appears possible, where side-effect profile, ease of administration, cost, and patient comorbidities can all be considered.FundingNational Institute of Allergy and Infectious Diseases.

The lower efficacy of the COVID-19 mRNA vaccines in 5-11 year old children was unexpected. Neutralizing antibody titers elicited by the vaccines in children, adolescents, and young adults suggest that the lower efficacy is not due to the lower dosage. Confirming the efficacy of these vaccines in children, determining if mRNA vaccination strategies are less effective in younger children, as well as optimizing the dosage, dosing intervals, and number of doses needed in children, adolescents, and young adults are critical to improve vaccination strategies for these populations going forward.

This post hoc analysis of the Adaptive Coronavirus Disease 2019 (COVID-19) Treatment Trial-1 (ACTT-1) shows a treatment effect of remdesivir (RDV) on progression to invasive mechanical ventilation (IMV) or death. Additionally, we create a risk profile that better predicts progression than baseline oxygen requirement alone. The highest risk group derives the greatest treatment effect from RDV.

BackgroundA comprehensive understanding of the SARS-CoV-2 infection dynamics and the ensuing host immune responses is needed to explain the pathogenesis as it relates to viral transmission. Knowledge gaps exist surrounding SARS-CoV-2 in vivo kinetics, particularly in the earliest stages after exposure.MethodsAn ongoing, workplace clinical surveillance study was used to intensely sample a small cohort longitudinally. Nine study participants who developed COVID-19 between November, 2020 and March, 2021 were monitored at high temporal resolution for three months in terms of viral loads as well as associated inflammatory biomarker and antibody responses. CD8 + T cells targeting SARS-CoV-2 in blood samples from study participants were evaluated.ResultsHere we show that the resulting datasets, supported by Bayesian modeling, allowed the underlying kinetic processes to be described, yielding a number of unexpected findings. Early viral replication is rapid (median doubling time, 3.1 h), providing a narrow window between exposure and viral shedding, while the clearance phase is slow and heterogeneous. Host immune responses different widely across participants.ConclusionsResults from our small study give a rare insight into the life-cycle of COVID-19 infection and hold a number of important biological, clinical, and public health implications.

CD8+ T cells have key protective roles in many viral infections. While an overall Th1-biased cellular immune response against SARS-CoV-2 has been demonstrated, most reports of anti-SARS-CoV-2 cellular immunity have evaluated bulk T cells using pools of predicted epitopes, without clear delineation of the CD8+ subset and its magnitude and targeting. In recently infected persons (mean 29.8 days after COVID-19 symptom onset), we confirm a Th1 bias (and a novel IL-4-producing population of unclear significance) by flow cytometry, which does not correlate to antibody responses against the receptor binding domain. Evaluating isolated CD8+ T cells in more detail by IFN-γ ELISpot assays, responses against spike, nucleocapsid, matrix, and envelope proteins average 396, 901, 296, and 0 spot-forming cells (SFC) per million, targeting 1.4, 1.5, 0.59, and 0.0 epitope regions respectively. Nucleocapsid targeting is dominant in terms of magnitude, breadth, and density of targeting. The magnitude of responses drops rapidly post-infection; nucleocapsid targeting is most sustained, and vaccination selectively boosts spike targeting. In SARS-CoV-2-naïve persons, evaluation of the anti-spike CD8+ T cell response soon after vaccination (mean 11.3 days) yields anti-spike CD8+ T cell responses averaging 2,463 SFC/million against 4.2 epitope regions, and targeting mirrors that seen in infected persons. These findings provide greater clarity on CD8+ T cell anti-SARS-CoV-2 targeting, breadth, and persistence, suggesting that nucleocapsid inclusion in vaccines could broaden coverage and durability.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve in humans. Spike protein mutations increase transmission and potentially evade antibodies raised against the original sequence used in current vaccines. Our evaluation of serum neutralizing activity in both persons soon after SARS-CoV-2 infection (in April 2020 or earlier) or vaccination without prior infection confirmed that common spike mutations can reduce antibody antiviral activity. However, when the persons with prior infection were subsequently vaccinated, their antibodies attained an apparent biologic ceiling of neutralizing potency against all tested variants, equivalent to the original spike sequence. These findings indicate that additional antigenic exposure further improves antibody efficacy against variants. IMPORTANCE As SARS-CoV-2 evolves to become better suited for circulating in humans, mutations have occurred in the spike protein it uses for attaching to cells it infects. Protective antibodies from prior infection or vaccination target the spike protein to interfere with its function. These mutations can reduce the efficacy of antibodies generated against the original spike sequence, raising concerns for reinfections and vaccine failures, because current vaccines contain the original sequence. In this study, we tested antibodies from people infected early in the pandemic (before spike variants started circulating) or people who were vaccinated without prior infection. We confirmed that some mutations reduce the ability of antibodies to neutralize the spike protein, whether the antibodies were from past infection or vaccination. Upon retesting the previously infected persons after vaccination, their antibodies gained the same ability to neutralize mutated spike as the original spike, suggesting that the combination of infection and vaccination drove the production of enhanced antibodies to reach a maximal level of potency. Whether this can be accomplished by vaccination alone remains to be determined, but the results suggest that booster vaccinations may help improve efficacy against spike variants through improving not only antibody quantity, but also quality.

BackgroundLeronlimab, a monoclonal antibody blocker of C-C chemokine receptor type 5 originally developed to treat human immunodeficiency virus infection, was administered as an open-label compassionate-use therapeutic for coronavirus disease 2019 (COVID-19).MethodsTwenty-three hospitalized severe/critical COVID-19 patients received 700 mg leronlimab subcutaneously, repeated after 7 days in 17 of 23 patients still hospitalized. Eighteen of 23 received other experimental treatments, including convalescent plasma, hydroxychloroquine, steroids, and/or tocilizumab. Five of 23 received leronlimab after blinded, placebo-controlled trials of remdesivir, sarilumab, selinexor, or tocilizumab. Outcomes and results were extracted from medical records.ResultsMean age was 69.5 ± 14.9 years; 20 had significant comorbidities. At baseline, 22 were receiving supplemental oxygen (3 high flow, 7 mechanical ventilation). Blood showed markedly elevated inflammatory markers (ferritin, D-dimer, C-reactive protein) and an elevated neutrophil-to-lymphocyte ratio. By day 30 after initial dosing, 17 were recovered, 2 were still hospitalized, and 4 had died. Of the 7 intubated at baseline, 4 were fully recovered off oxygen, 2 were still hospitalized, and 1 had died.ConclusionsLeronlimab appeared safe and well tolerated. The high recovery rate suggested benefit, and those with lower inflammatory markers had better outcomes. Some, but not all, patients appeared to have dramatic clinical responses, indicating that unknown factors may determine responsiveness to leronlimab. Routine inflammatory and cell prognostic markers did not markedly change immediately after treatment, although interleukin-6 tended to fall. In some persons, C-reactive protein clearly dropped only after the second leronlimab dose, suggesting that a higher loading dose might be more effective. Future controlled trials will be informative.

[This corrects the article DOI: 10.1371/journal.ppat.1009404.].

Public health practices and high vaccination rates currently represent the primary interventions for managing the spread of coronavirus disease 2019 (COVID-19). We initiated a clinical study based on frequent, longitudinal workplace disease surveillance to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission among employees and their household members. We hypothesized that the study would reduce the economic burden and loss of productivity of both individuals and small businesses resulting from standard isolation methods, while providing new insights into virus-host dynamics. Study participants (27 employees and 27 household members) consented to provide frequent nasal or oral swab samples that were analyzed by reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 RNA. Two study participants were found to be infected by SARS-CoV-2 during the study. One subject, a household member, was SARS-CoV-2 RNA positive for at least 71 days and had quantifiable serum virus-specific antibody concentrations for over 1 year. One unrelated employee became positive for SARS-CoV-2 RNA over the course of the study but remained asymptomatic, with low associated viral RNA copy numbers, no detectable serum IgM and IgG concentrations, and IgA concentrations that decayed rapidly (half-life: 1.3 days). A COVID-19 infection model was used to predict that without surveillance intervention, up to 7 employees (95% confidence interval [CI] = 3 to 10) would have become infected, with at most 1 of them requiring hospitalization. Our scalable and transferable surveillance plan met its primary objectives and represents a powerful example of an innovative public health initiative dovetailed with scientific discovery. IMPORTANCE The rapid spread of SARS-CoV-2 and the associated COVID-19 has precipitated a global pandemic heavily challenging our social behavior, economy, and health care infrastructure. In the absence of widespread, worldwide access to safe and effective vaccines and therapeutics, public health measures represent a key intervention for curbing the devastating impacts from the pandemic. We are conducting an ongoing clinical study based on frequent, longitudinal workplace disease surveillance to control SARS-CoV-2 transmission among employees and their household members. Our study was successful in surveying the viral and immune response dynamics in two participants with unusual infections: one remained positive for SARS-CoV-2 for 71 days, while the other was asymptomatic, with low associated viral RNA copy numbers. A COVID-19 infection model was used to predict that without surveillance intervention, up to 7 employees would have become infected, with at most 1 of them requiring hospitalization, underscoring the importance of our program.

CD8+ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E-restricted CD8+ T cell responses in HIV infection, however, remains unknown. In this study, CD8+ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E-restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8+ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8+ T cells. Importantly, bulk CD8+ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E-restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E-restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8+ T cell responses in HIV infection.

This post-hoc analysis of The Adaptive COVID-19 Treatment Trial-1 shows a treatment effect of remdesivir on progression to invasive mechanical ventilation (IMV) or death. Additionally, we create a risk profile that better predicts progression than baseline oxygen requirement alone. The highest risk group derives the greatest treatment effect from RDV.

Studies of two SARS-CoV-2 mRNA vaccines suggested that they yield ∼95% protection from symptomatic infection at least short-term, but important clinical questions remain. It is unclear how vaccine-induced antibody levels quantitatively compare to the wide spectrum induced by natural SARS-CoV-2 infection. Vaccine response kinetics and magnitudes in persons with prior COVID-19 compared to virus-naı̈ve persons are not well-defined. The relative stability of vaccine-induced versus infection-induced antibody levels is unclear. We addressed these issues with longitudinal assessments of vaccinees with and without prior SARS-CoV-2 infection using quantitative enzyme-linked immunosorbent assay (ELISA) of anti-RBD antibodies. SARS-CoV-2-naı̈ve individuals achieved levels similar to mild natural infection after the first vaccination; a second dose generated levels approaching severe natural infection. In persons with prior COVID-19, one dose boosted levels to the high end of severe natural infection even in those who never had robust responses from infection, increasing no further after the second dose. Antiviral neutralizing assessments using a spike-pseudovirus assay revealed that virus-naı̈ve vaccinees did not develop physiologic neutralizing potency until the second dose, while previously infected persons exhibited maximal neutralization after one dose. Finally, antibodies from vaccination waned similarly to natural infection, resulting in an average of ∼90% loss within 90 days. In summary, our findings suggest that two doses are important for quantity and quality of humoral immunity in SARS-CoV-2-naı̈ve persons, while a single dose has maximal effects in those with past infection. Antibodies from vaccination wane with kinetics very similar to that seen after mild natural infection; booster vaccinations will likely be required.

BackgroundT lymphocyte-mediated acute rejection is a significant complication following solid organ transplantation. Standard methods of monitoring for acute rejection rely on assessing histological tissue damage but do not define the immunopathogenesis. Additionally, current therapies for rejection broadly blunt cellular immunity, creating a high risk for opportunistic infections. There is, therefore, a need to better understand the process of acute cellular rejection to help develop improved prognostic tests and narrowly targeted therapies.MethodsThrough next-generation sequencing, we characterized and compared the clonal T-cell receptor (TCR) repertoires of graft-infiltrating lymphocytes (GILs) and blood-derived lymphocytes from a hand transplant recipient over 420 days following transplantation. We also tracked the TCR clonal persistence and V beta (BV) gene usage, evaluating overlap between these 2 compartments.ResultsTCR repertoires of blood and GIL populations remained distinct throughout the sampling period, and differential BV usage was consistently seen between these compartments. GIL TCR clones persisted over time and were seen in only limited frequency in the blood T-lymphocyte populations.ConclusionsWe demonstrate that blood monitoring of TCR clones does not reveal the pathogenic process of acute cellular rejection in transplanted tissue. GILs show clonal persistence with biased BV usage, suggesting that tissue TCR clonal monitoring could be useful, although a deeper understanding is necessary to prognosticate rejection based on TCR clonal repertoires. Finally, the distinct TCR BV usage bias in GILs raises the possibility for prevention and therapy of acute cellular rejection based on targeting of specific TCR clones.

Due to the durability and persistence of reservoirs of HIV-1-infected cells, combination antiretroviral therapy (ART) is insufficient in eradicating infection. Achieving HIV-1 cure or sustained remission without ART treatment will require the enhanced and persistent effective antiviral immune responses. Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy and show promise in treating HIV-1 infection. Persistence, trafficking, and maintenance of function remain to be a challenge in many of these approaches, which are based on peripheral T cell modification. To overcome many of these issues, we have previously demonstrated successful long-term engraftment and production of anti-HIV CAR T cells in modified hematopoietic stem cells (HSCs) in vivo. Here we report the development and in vivo testing of second generation CD4-based CARs (CD4CAR) against HIV-1 infection using a HSCs-based approach. We found that a modified, truncated CD4-based CAR (D1D2CAR) allows better CAR-T cell differentiation from gene modified HSCs, and maintains similar CTL activity as compared to the full length CD4-based CAR. In addition, D1D2CAR does not mediate HIV infection or stimulation mediated by IL-16, suggesting lower risk of off-target effects. Interestingly, stimulatory domains of 4-1BB but not CD28 allowed successful hematopoietic differentiation and improved anti-viral function of CAR T cells from CAR modified HSCs. Addition of 4-1BB to CD4 based CARs led to faster suppression of viremia during early untreated HIV-1 infection. D1D2CAR 4-1BB mice had faster viral suppression in combination with ART and better persistence of CAR T cells during ART. In summary, our data indicate that the D1D2CAR-41BB is a superior CAR, showing better HSC differentiation, viral suppression and persistence, and less deleterious functions compared to the original CD4CAR, and should continue to be pursued as a candidate for clinical study.

Two mRNA vaccines (BNT162b2 and mRNA-1273) against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) are globally authorized as a two-dose regimen. Understanding the magnitude and duration of protective immune responses is vital to curbing the pandemic. We enrolled 461 high-risk health services workers at the University of California, Los Angeles (UCLA) and first responders in the Los Angeles County Fire Department (LACoFD) to assess the humoral responses in previously infected (PI) and infection naïve (NPI) individuals to mRNA-based vaccines (BNT162b2/Pfizer- BioNTech or mRNA-1273/Moderna). A chemiluminescent microparticle immunoassay was used to detect antibodies against SARS-CoV-2 Spike in vaccinees prior to (n = 21) and following each vaccine dose (n = 246 following dose 1 and n = 315 following dose 2), and at days 31-60 (n = 110) and 61-90 (n = 190) following completion of the 2-dose series. Both vaccines induced robust antibody responses in all immunocompetent individuals. Previously infected individuals achieved higher median peak titers (p = 0.002) and had a slower rate of decay (p = 0.047) than infection-naïve individuals. mRNA-1273 vaccinated infection-naïve individuals demonstrated modestly higher titers following each dose (p = 0.005 and p = 0.029, respectively) and slower rates of antibody decay (p = 0.003) than those who received BNT162b2. A subset of previously infected individuals (25%) required both doses in order to reach peak antibody titers. The biologic significance of the differences between previously infected individuals and between the mRNA-1273 and BNT162b2 vaccines remains uncertain, but may have important implications for booster strategies.

BackgroundLeronlimab, a monoclonal antibody blocker of CCR5 originally developed to treat HIV-1 infection, was administered as an open label compassionate use therapeutic for COVID-19.Methods23 hospitalized severe/critical COVID-19 patients received 700mg leronlimab subcutaneously, repeated after seven days in 17/23 patients still hospitalized. 18/23 received other experimental treatments, including convalescent plasma, hydroxychloroquine, steroids, and/or tocilizumab. 5/23 received leronlimab after blinded placebo-controlled trials of remdesivir, sarilumab, selinexor, or tocilizumab. Outcomes and results were extracted from medical records.ResultsMean age was 69.5±14.9 years. 20/23 had significant co-morbidities. At baseline, 22/23 were receiving supplemental oxygen (3/23 high flow, 7/23 mechanical ventilation). Blood showed markedly elevated inflammatory markers (ferritin, D-dimer, C-reactive protein) and elevated neutrophil:lymphocyte ratio. By day 30 after initial dosing, 17/23 were recovered, 2/23 were still hospitalized, and 4/23 had died. Of the 7 intubated at baseline, 4/7 were fully recovered off oxygen, 2/7 were still hospitalized, and 1/7 had died.ConclusionsLeronlimab appeared safe and well tolerated. The high recovery rate suggested benefit, and those with lower inflammatory markers had better outcomes. Some but not all patients appeared to have dramatic clinical responses, indicating that unknown factors may determine responsiveness to leronlimab. Routine inflammatory and cell prognostic markers did not markedly change immediately after treatment, although IL-6 tended to fall. In some persons C-reactive protein clearly dropped only after the second leronlimab dose, suggesting that a higher loading dose might be more effective. Future controlled trials will be informative.

Human cytomegalovirus (CMV) is a ubiquitous pathogen that causes significant morbidity in some vulnerable populations. Individualized adoptive transfer of ex vivo expanded CMV-specific CD8+ T cells has provided proof-of-concept that immunotherapy can be highly effective, but a chimeric antigen receptor (CAR) approach would provide a feasible method for broad application. We created 8 novel CARs using anti-CMV neutralizing antibody sequences, which were transduced via lentiviral vector into primary CD8+ T cells. All CARs were expressed. Activity against CMV-infected target cells was assessed by release of cytokines (interferon-γ and tumor necrosis factor-α), upregulation of surface CD107a, proliferation, cytolysis of infected cells, and suppression of viral replication. While some CARs showed varying functional activity across these assays, 1 CAR based on antibody 21E9 was consistently superior in all measures. These results support development of a CMV-specific CAR for therapeutic use against CMV and potentially other applications harnessing CMV-driven immunotherapies.

HIV-1-induced cytopathicity of thymocytes is a major cause of reduced peripheral T cells and rapid disease progression observed in HIV-1-infected infants. Understanding the virulence factors responsible for thymocyte depletion has paramount importance in addressing the pathogenesis of disease progression in children. In this study, thymocyte depletion was analyzed following infection with two primary CXCR4-tropic HIV-1 pediatric isolates (PI), PI-2 and PI-2.1, which were serially derived from an in utero-infected infant. Although highly similar to each other, PI-2 showed markedly decreased thymocyte depletion in vitro compared with PI-2.1. Further analysis showed a novel deletion in the Nef protein (NefΔK7S) of PI-2, which was absent in PI-2.1. This deletion inhibited Nef-mediated major histocompatibility complex class I (MHC-I) downregulation in infected thymocytes in vitro and in vivo; in contrast, the mutated Nef continued to downregulate CD4 surface expression in vitro. These results suggest that HIV-1 Nef contributes to thymic damage in infants through selective functions.

"Vaults" are ubiquitously expressed endogenous ribonucleoprotein nanoparticles with potential utility for targeted drug delivery. Here, we show that recombinant human vault nanoparticles are readily engulfed by certain key human peripheral blood mononuclear cells (PBMC), predominately dendritic cells, monocytes/macrophages, and activated T cells. As these cell types are the primary targets for human immunodeficiency virus type 1 (HIV-1) infection, we examined the utility of recombinant human vaults for targeted delivery of antiretroviral drugs. We chemically modified three different antiretroviral drugs, zidovudine, tenofovir, and elvitegravir, for direct conjugation to vaults. Tested in infection assays, drug-conjugated vaults inhibited HIV-1 infection of PBMC with equivalent activity to free drugs, indicating vault delivery and drug release in the cytoplasm of HIV-1-susceptible cells. The ability to deliver functional drugs via vault nanoparticle conjugates suggests their potential utility for targeted drug delivery against HIV-1.

Almost 80% of viral transcripts during early HIV-1 infection encode the Nef protein, which has been implicated in altering expression of a number of genes. In this study, we infected primary human CD4+ T cells with pseudotyped Nef-containing or Nef-deleted (Δ-nef) NL4-3 virus and used RNA-Sequencing (RNA-Seq) for transcriptomic analysis. Our results showed that the interferon response, IL-15 and JAK/STAT signaling, as well as genes involved in metabolism, apoptosis, cell cycle regulation, and ribosome biogenesis were all altered in the presence of Nef. These early Nef-mediated transcriptional alterations may play a role in priming the host cell for cellular activation and viral replication.

ObjectiveTo assess whether weakly recognized epitope variants induce anergy in HIV-1-specific CD8 T lymphocyte (CTL) clones as a mechanism of dysfunction.DesignHIV-1-specific CTL clones were exposed to suboptimally recognized epitope variants, and screened for anergy and other T-cell dysfunction markers, and subsequent capability to kill target cells bearing index epitope.MethodsIn addition to the optimally recognized index epitope, two suboptimally recognized epitope variants were selected based on titration curves for killing of peptide-labeled target cells by three HIV-1-specific CTL clones targeting the epitopes SLYNTVATL (Gag 77-85, A02-restricted), RPAEPVPLQL (Rev 66-75, B07-restricted), and KRWIIMGLNK (Gag 263-272, B27-restricted). Consequences of suboptimal stimulation were assessed by cytokine secretion, gene expression, and capacity to kill index epitope-labeled target cells upon rechallenge.ResultsSuboptimal recognition of epitope variants reduced cytokine production by CTL similarly to reduction in killing of target cells. Gene expression profiles after suboptimal stimulation demonstrated no patterns consistent with T-cell dysfunction due to anergy, exhaustion, or apoptosis. Preexposure of CTL to epitope variants had no discernable impact on their subsequent capacity to kill index epitope-bearing target cells.ConclusionOur data explore the hypothesis that poorly recognized epitope variants not only facilitate HIV-1 evasion of CTL recognition, but also induce CTL dysfunction through suboptimal signaling causing anergy. However, the results do not suggest that suboptimal signaling induces anergy (or exhaustion or apoptosis), indicating that the major role of CTL epitope variation is likely viral escape.

A 48-year-old woman was infected with a vpr-defective human immunodeficiency virus (HIV)-1 molecular clone. Seroconversion was markedly delayed, and without treatment she had durably suppressed viremia and normal T-cell levels. Neutralizing antibody and CD8+ T-cell immune responses against HIV-1 were unremarkable. Viral sequences confirmed the source but evolved defective nef, suggesting an unknown mechanistic link to vpr. There were subtle qualitative defects in T and B cells. To our knowledge, this is the only case of human infection with a characterized defective HIV-1 molecular clone, which furthermore recapitulated live-attenuated vaccination in macaque models of HIV-1 vaccine research.

Epitope escape from HIV-1-targeted CD8+ cytotoxic T lymphocyte (CTL) responses occurs rapidly after acute infection and contributes to the eventual failure of effective immune control of HIV-1 infection. Because the early CTL response is key in determining HIV-1 disease outcome, studying the process of epitope escape is crucial for understanding what leads to failure of immune control in acute HIV-1 infection and will provide important implications for HIV-1 vaccine design. HIV-1-specific CD8+ T lymphocyte responses against viral epitopes were mapped in six acutely infected individuals, and the magnitudes of these responses were measured longitudinally during acute infection. The evolution of autologous circulating viral epitopes was determined in four of these subjects. In-depth testing of CD8+ T lymphocyte responses against index and all autologous-detected variant epitopes was performed in one subject. Among the four individuals examined, 10 of a total of 35 CD8+ T cell responses within Gag, Pol, and Nef showed evidence of epitope escape. CTL responses with viral epitope variant evolution were shown in one subject, and this evolution occurred with and without measurable CTL responses against epitope variants. These results demonstrate a dynamic period of viral epitope evolution in early HIV-1 infection due to CD8+ CTL response pressure.

Although a high level of promiscuity for heterologous epitopes is believed to exist for cellular immunity, limited data explore this issue for human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T lymphocyte (CTL) responses. Here, we found an unexpected degree of heterologous cross-reactivity against HIV-1 epitopes, in addition to the targeted index epitope. Most CTL clones screened cross-reacted against other known HIV-1 epitopes of the same major histocompatibility complex type I (MHC-I) restriction, up to 40% of tested nonindex epitopes in some cases. The observed cross-reactivity was universally lower avidity than recognition of the index epitope when examined for several A*02- and B*57-restricted CTL clones, demonstrating that the high concentrations of exogenous epitope typically used for screening of CTL responses are prone to detect such cross-reactivity spuriously. In agreement with this, we found that these cross-reactive responses do not appear to mediate CTL activity against HIV-1-infected cells. Overall, our data indicate that low-level cross-reactivity is remarkably common for HIV-1-specific CTLs. The role of this phenomenon is unclear, but low-avidity interactions have been shown to foster homeostatic proliferation of memory T cells.IMPORTANCE This study raises two issues related to HIV-1-specific CTL responses. These are key immune responses that retard disease progression in infected persons that are highly relevant to immunotherapies and vaccines for HIV-1. First, we make the novel observation that these responses are promiscuous and that CTLs targeting one epitope may cross-recognize other, completely distinct epitopes in the virus. While these are low-avidity interactions that do not appear to contribute directly to the antiviral activity of CTLs, this raises interesting biologic implications regarding the purpose of the phenomenon, such as providing a stimulus for these responses to persist long term. Second, the data raise a technical caveat to detection of CTL responses against particular epitopes, suggesting that some methodologies may unintentionally detect cross-reactivity and overestimate responses against an epitope.

BackgroundThe contributions of the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) axis to cardiovascular and bone disease in treated HIV-1 infection are not well defined.SettingProspective, observational, longitudinal study.MethodsIn a subset analysis of a prospective randomized clinical trial, 234 HIV-1-infected antiretroviral therapy-naive participants received tenofovir-emtricitabine plus either atazanavir/ritonavir, darunavir/ritonavir, or raltegravir and achieved plasma HIV-1 RNA

Prior crystal structures of the vault have provided clues of its structural variability but are non-conclusive due to crystal packing. Here, we obtained vaults by engineering at the N terminus of rat major vault protein (MVP) an HIV-1 Gag protein segment and determined their near-atomic resolution (∼4.8 Å) structures in a solution/non-crystalline environment. The barrel-shaped vaults in solution adopt two conformations, 1 and 2, both with D39 symmetry. From the N to C termini, each MVP monomer has three regions: body, shoulder, and cap. While conformation 1 is identical to one of the crystal structures, the shoulder in conformation 2 is translocated longitudinally up to 10 Å, resulting in an outward-projected cap. Our structures clarify the structural discrepancies in the body region in the prior crystallography models. The vault's drug-delivery potential is highlighted by the internal disposition and structural flexibility of its Gag-loaded N-terminal extension at the barrel waist of the engineered vault.

Certain "protective" major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8+ cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity.IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo Here we utilized our recently developed high-throughput fitness profiling method to quantitatively measure the fitness of mutations across the entirety of HIV-1 Gag. The data enabled us to integrate the results with in silico prediction of MHC-peptide binding affinity and analysis of intraperson virus evolution to systematically determine the differences in CTL escape mutations between epitopes targeted by protective HLA alleles and those targeted by nonprotective HLA alleles. We observed that the effects of Gag epitope mutations on HIV replicative fitness and MHC-I binding affinity are among the major determinants of CTL escape.

Immune control of human immunodeficiency virus type 1 (HIV) infection is typically associated with effective Gag-specific CD8+ T-cell responses. We here focus on HLA-B*14, which protects against HIV disease progression, but the immunodominant HLA-B*14-restricted anti-HIV response is Env specific (ERYLKDQQL, HLA-B*14-EL9). A subdominant HLA-B*14-restricted response targets Gag (DRYFKTLRA, HLA-B*14-DA9). Using HLA-B*14/peptide-saporin-conjugated tetramers, we show that HLA-B*14-EL9 is substantially more potent at inhibiting viral replication than HLA-B*14-DA9. HLA-B*14-EL9 also has significantly higher functional avidity (P < 0.0001) and drives stronger selection pressure on the virus than HLA-B*14-DA9. However, these differences were HLA-B*14 subtype specific, applying only to HLA-B*14:02 and not to HLA-B*14:01. Furthermore, the HLA-B*14-associated protection against HIV disease progression is significantly greater for HLA-B*14:02 than for HLA-B*14:01, consistent with the superior antiviral efficacy of the HLA-B*14-EL9 response. Thus, although Gag-specific CD8+ T-cell responses may usually have greater anti-HIV efficacy, factors independent of protein specificity, including functional avidity of individual responses, are also critically important to immune control of HIV.IMPORTANCE In HIV infection, although cytotoxic T lymphocytes (CTL) play a potentially critical role in eradication of viral reservoirs, the features that constitute an effective response remain poorly defined. We focus on HLA-B*14, unique among HLAs associated with control of HIV in that the dominant CTL response is Env specific, not Gag specific. We demonstrate that Env-specific HLA-B*14-restricted activity is substantially more efficacious than the subdominant HLA-B*14-restricted Gag response. Env immunodominance over Gag and strong Env-mediated selection pressure on HIV are observed only in subjects expressing HLA-B*14:02, and not HLA-B*14:01. This reflects the increased functional avidity of the Env response over Gag, substantially more marked for HLA-B*14:02. Finally, we show that HLA-B*14:02 is significantly more strongly associated with viremic control than HLA-B*14:01. These findings indicate that, although Gag-specific CTL may usually have greater anti-HIV efficacy than Env responses, factors independent of protein specificity, including functional avidity, may carry greater weight in mediating effective control of HIV.

Mutational escape of HIV-1 from HIV-1-specific CD8+ T lymphocytes (CTLs) is a major barrier for effective immune control. Each epitope typically is targeted by multiple clones with distinct T cell receptors (TCRs). While the clonal repertoire may be important for containing epitope variation, determinants of its composition are poorly understood. We investigate the clonal repertoire of 29 CTL responses against 23 HIV-1 epitopes longitudinally in nine chronically infected untreated subjects with plasma viremia of

Certain Major Histocompatibility-I (MHC-I) types are associated with superior immune containment of HIV-1 infection by CD8+ cytotoxic T lymphocytes (CTLs), but the mechanisms mediating this containment are difficult to elucidate in vivo. Here we provide controlled assessments of fitness landscapes and CTL-imposed constraints for immunodominant epitopes presented by two protective (B*57 and B*27) and one non-protective (A*02) MHC-I types. Libraries of HIV-1 with saturation mutagenesis of CTL epitopes are propagated with and without CTL selective pressure to define the fitness landscapes for epitope mutation and escape from CTLs via deep sequencing. Immunodominant B*57- and B*27- present epitopes are highly limited in options for fit mutations, with most viable variants recognizable by CTLs, whereas an immunodominant A*02 epitope-presented is highly permissive for mutation, with many options for CTL evasion without loss of viability. Generally, options for evasion overlap considerably between CTL clones despite highly distinct T cell receptors. Finally, patterns of variant recognition suggest population-wide CTL selection for the A*02-presented epitope. Overall, these findings indicate that these protective MHC-I types yield CTL targeting of highly constrained epitopes, and underscore the importance of blocking public escape pathways for CTL-based interventions against HIV-1.

Risk of HIV acquisition varies, and some individuals are highly HIV-1-exposed, yet, persistently seronegative (HESN). The immunologic mechanisms contributing to this phenomenon are an area of intense interest. As immune activation and inflammation facilitate disease progression in HIV-1-infected persons and gastrointestinal-associated lymphoid tissue is a highly susceptible site for transmission, we hypothesized that reduced gut mucosal immune reactivity may contribute to reduced HIV-1 susceptibility in HESN men with a history of numerous rectal sexual exposures. To test this, we used ex vivo mucosal explants from freshly acquired colorectal biopsies from healthy control and HESN subjects who were stimulated with specific innate immune ligands and inactivated whole pathogens. Immune reactivity was then assessed via cytokine arrays and proteomic analysis. Mucosal immune cell compositions were quantified via immunohistochemistry. We found that explants from HESN subjects produced less proinflammatory cytokines compared with controls following innate immune stimulation; while noninflammatory cytokines were similar between groups. Proteomic analysis identified several immune response proteins to be differentially expressed between HIV-1-stimulated HESN and control explants. Immunohistochemical examination of colorectal mucosa showed similar amounts of T cells, macrophages, and dendritic cells between groups. The results of this pilot study suggest that mucosal innate immune reactivity is dampened in HESN versus control groups, despite presence of similar densities of immune cells in the colorectal mucosa. This observed modulation of the rectal mucosal immune response may contribute to lower risk of mucosal HIV-1 transmission in these individuals.