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Early initiation of antiretroviral therapy (ART) alters viral rebound kinetics after analytic treatment interruption (ATI) and may play a role in promoting HIV-1 remission. Autologous neutralizing antibodies (aNAbs) represent a key adaptive immune response in people living with HIV-1. We aimed to investigate the role of aNAbs in shaping post-ATI HIV-1 rebound variants. We performed single-genome amplification of HIV-1 env from pre-ART and post-ATI plasma samples of 12 individuals who initiated ART early after infection. aNAb activity was quantified using pseudoviruses derived from the most common plasma variant, and the serum dilution that inhibited 50% of viral infections was determined. aNAb responses matured while participants were on suppressive ART, because on-ART plasma and purified immunoglobulin G (IgG) demonstrated improved neutralizing activity against pre-ART HIV-1 strains when compared with pre-ART plasma or purified IgG. Post-ATI aNAb responses exerted selective pressure on the rebounding viruses, because the post-ATI HIV-1 strains were more resistant to post-ATI plasma neutralization compared with the pre-ART virus. Several pre-ATI features distinguished post-treatment controllers from noncontrollers, including an infecting HIV-1 sequence that was more similar to consensus HIV-1 subtype B, more restricted proviral diversity, and a stronger aNAb response. Post-treatment control was also associated with the evolution of distinct N-glycosylation profiles in the HIV-1 envelope. In summary, aNAb responses appeared to mature after early initiation of ART and applied selective pressure on rebounding viruses. The combination of aNAb activity with select HIV-1 sequence and reservoir features identified individuals with a greater chance of post-treatment control.

The recently emerged SARS-CoV-2 virus has led to a prolonged pandemic characterized by ongoing viral evolution. Vaccines have been an important piece in the strategy to combat the virus but have been insufficient to contain it as the virus continues to evolve to evade immunity developed by vaccination and infection. A consistent argument is that vaccination or prior immunity will lead to less severe infections. In this review, we address the question of whether the virus can evolve to become more virulent, despite prior infection. We describe the intrinsic characteristics of the virus and their relationship to altered virulence. We show that it is likely that viral evolution is subject to evolutionary drift, and it cannot be assumed that the virus will necessarily evolve to be less virulent, or that prior immunity will offer durable protection against severe disease. This has strong implications for public health strategies to confront the ongoing challenges presented by SARS-CoV-2 and implies that there are significant risks to a strategy based on the assumption of waning virulence.

High autoantibody levels are found in individuals hospitalized for COVID-19. The temporal trajectories and levels of these autoantibodies months into convalescence after SARS-CoV-2 infection are unclear. It is also unknown if the composite autoantibody signatures of convalescent SARS-CoV-2-infected individuals resemble those with diagnosed autoimmune diseases. We measured the circulating levels of 17 autoantibodies associated with autoimmune connective tissue diseases from SARS-CoV-2 hospitalized and outpatient participants, as well as from individuals with scleroderma (SSc), systemic lupus erythematosus (SLE), and uninfected pre-pandemic controls. Seven of the 17 autoantibodies measured were higher in hospitalized and/or outpatient SARS-CoV-2 individuals an average of six months after symptom onset compared with controls, with multivariate analyses revealing links between SARS-CoV-2 infection and positivity of SSB-La, Sm, Proteinase 3, Myleoperoxidase, Jo-1, and Ku reactive IgG six months post-symptom onset. Autoantibody levels from SARS-CoV-2 infected individuals were followed over time from initial symptom onset for an average of six months, and different temporal autoantibody trajectories were classified. A ‘negative, then positive’ expression pattern was found for at least one autoantibody in 18% of the outpatient and 53% of the hospitalized participants, indicating initiation and durable expression of self-reactive immune responses post-infection, particularly with severe acute illness. Analysis of individual participant autoantibody expression patterns revealed similar patterns between pre-pandemic and convalescent SARS-CoV-2 infected groups that are distinct from participants with both the SSc and SLE. As autoantibody positivity can occur years prior to autoimmune disease onset, the possibility that SARS-CoV-2-associated autoantibodies are a herald of future autoimmune disorders requires further investigation.One Sentence SummaryAutoantibody levels rise after acute SARS-CoV-2 infection and remain elevated for at least six months after symptom onset in participants with mild or severe COVID-19.

BACKGROUND: The efficacy of interleukin-6 receptor blockade in hospitalized patients with coronavirus disease 2019 (Covid-19) who are not receiving mechanical ventilation is unclear. METHODS: We performed a randomized, double-blind, placebo-controlled trial involving patients with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, hyperinflammatory states, and at least two of the following signs: fever (body temperature >38°C), pulmonary infiltrates, or the need for supplemental oxygen in order to maintain an oxygen saturation greater than 92%. Patients were randomly assigned in a 2:1 ratio to receive standard care plus a single dose of either tocilizumab (8 mg per kilogram of body weight) or placebo. The primary outcome was intubation or death, assessed in a time-to-event analysis. The secondary efficacy outcomes were clinical worsening and discontinuation of supplemental oxygen among patients who had been receiving it at baseline, both assessed in time-to-event analyses. RESULTS: We enrolled 243 patients; 141 (58%) were men, and 102 (42%) were women. The median age was 59.8 years (range, 21.7 to 85.4), and 45% of the patients were Hispanic or Latino. The hazard ratio for intubation or death in the tocilizumab group as compared with the placebo group was 0.83 (95% confidence interval [CI], 0.38 to 1.81; P = 0.64), and the hazard ratio for disease worsening was 1.11 (95% CI, 0.59 to 2.10; P = 0.73). At 14 days, 18.0% of the patients in the tocilizumab group and 14.9% of the patients in the placebo group had had worsening of disease. The median time to discontinuation of supplemental oxygen was 5.0 days (95% CI, 3.8 to 7.6) in the tocilizumab group and 4.9 days (95% CI, 3.8 to 7.8) in the placebo group (P = 0.69). At 14 days, 24.6% of the patients in the tocilizumab group and 21.2% of the patients in the placebo group were still receiving supplemental oxygen. Patients who received tocilizumab had fewer serious infections than patients who received placebo. CONCLUSIONS: Tocilizumab was not effective for preventing intubation or death in moderately ill hospitalized patients with Covid-19. Some benefit or harm cannot be ruled out, however, because the confidence intervals for efficacy comparisons were wide. (Funded by Genentech; ClinicalTrials.gov number, NCT04356937.).

Excessive weight gain associated with integrase strand transfer inhibitor (InSTI) antiretrovirals is an emerging issue; however, the metabolic consequences of this effect have not been established. Our objective was to evaluate for InSTI-emergent weight gain and potential associated type 2 diabetes mellitus (T2DM) among a diverse HIV patient cohort. For this retrospective cohort study, we obtained clinical warehouse data for HIV+ patients between fiscal years 2007–17. We compared patients initiated on an InSTI with those started on an alternate regimen. Our primary outcome was percentage weight change from baseline to 24 months postinitiation using the linear mixed-effects model fit by restricted maximum likelihood. Our secondary outcome was incident T2DM as defined by a new prescription for antihyperglycemic medication within 18 months after antiretroviral therapy (ART) start. Diabetes-free survival was estimated using the Kaplan–Meier method, log-rank test, and Cox proportional-hazards model. The cohort included 1,235 individuals initiating ART, 136 (11.0%) with an InSTI. InSTI use in women was significantly associated with greater weight gain compared with non-InSTIs (11.0%, 95% confidence interval, CI: 5.22 to 16.8, p

Despite the worldwide availability of antiretroviral therapy (ART), approximately 150,000 pediatric HIV infections continue to occur annually. ART can dramatically reduce HIV mother-to-child transmission (MTCT), but inconsistent drug access and adherence, as well as primary maternal HIV infection during pregnancy and lactation are major barriers to eliminating vertical HIV transmission. Thus, immunologic strategies to prevent MTCT, such as an HIV vaccine, will be required to attain an HIV-free generation. A primary goal of HIV vaccine research has been to elicit broadly neutralizing antibodies (bnAbs) given the ability of passive bnAb immunization to protect against sensitive strains, yet we previously observed that HIV-transmitting mothers have more plasma neutralization breadth than non-transmitting mothers. Additionally, we have identified infant transmitted/founder (T/F) viruses that escape maternal bnAb responses. In this study, we examine a cohort of postpartum HIV-transmitting women with neutralization breadth to determine if certain maternal bnAb specificities drive the selection of infant T/F viruses. Using HIV pseudoviruses that are resistant to neutralizing antibodies targeting common bnAb epitopes, we mapped the plasma bnAb specificities of this cohort. Significantly more transmitting women with plasma bnAb activity had a mappable plasma bnAb specificity (six of seven, or 85.7%), compared to that of non-transmitting women with plasma bnAb activity (seven of twenty-one, or 33.3%, p=0.029 by 2-sided Fisher exact test). Our study suggests that having multispecific broad activity and/or uncommon epitope-specific bnAbs in plasma may be associated with protection against the vertical HIV transmission in the setting of maternal bnAb responses.ImportanceAs mother to child transmission (MTCT) of HIV plays a major part in the persistence of the HIV/AIDS epidemic and bnAb-based passive and active vaccines are a primary strategy for HIV prevention, research in this field is of great importance. While previous MTCT research has investigated the neutralizing antibody activity of HIV-infected women, this is to our knowledge the largest study identifying differences in bnAb specificity of maternal plasma between transmitting and non-transmitting women. Here, we show that among HIV-infected women with broad and potent neutralization activity, more postpartum-transmitting women had a mappable plasma broadly neutralizing antibody (bnAb) specificity, compared to that of non-transmitting women, suggesting that the non-transmitting women more often have multi-specific bnAb responses or bnAb responses that target uncommon epitopes. Such responses may be required for protection against vertical HIV transmission in the setting of maternal bnAb responses.

The increasing frequency of pathogenic coronaviruses in the human population has raised public health concerns about possible future pandemics. It is critical to understand whether immune responses to the current circulating coronaviruses provide protection against related viruses or those that may emerge in the future. In this issue of the JCI, Dangi, Palacio, and co-authors detail the extent of coronavirus cross-protection following both vaccination and natural infection and ultimately used murine models to highlight the mechanism behind this heterotypic immunity. This study provides insight into the possibility of a pan-coronavirus vaccine that could protect humans against future coronavirus outbreaks.

SummaryIn humans, pre-existing anti-HIV-1 neutralizing antibodies (nAbs) have not been associated with decreased HIV-1 acquisition. We evaluated antibody-dependent cellular cytotoxicity (ADCC) present in pre-transmission infant and maternal plasma and breast milk (BM) against the contemporaneous maternal HIV-1 variants. HIV-1 exposed uninfected as compared to HIV-1 exposed infected infants had higher ADCC and a combination of ADCC and nAb responses against their corresponding mother’s strains. ADCC did not correlate with nAbs suggesting they are independent activities. The infected infants with high as compared to low ADCC, but not those with higher ADCC plus nAbs, had lower morbidity and mortality up to 1 year after birth. ADCC was lower in BM supernatants as compared to BM isolated IgG, but was not significantly different in the transmitting versus non-transmitting mothers’ BM or plasma. A high IgA to IgG ratio, as in BM supernatants, was also more prevalent in the infected infants, and it was also associated with lower infant ADCC. Overall, ADCC, more so than nAbs, against the exposure strains associates with lower mother-to-child-transmission (MTCT) and decreased post-infection infant morbidity. Future strategies should aim to elicit robust ADCC activity to prevent MTCT and to improve infant outcomes.

Background The factors associated with severe acute respiratory coronavirus 2 (SARS-CoV-2) reinfection remain poorly defined. Methods We identified patients with SARS-CoV-2 infection and at least 1 repeat reverse transcription polymerase chain reaction result a minimum of 90 days after the initial positive test and before 21 January 2021. Those with a repeat positive test were deemed to have reinfection (n = 75), and those with only negative tests were classified as convalescents (n = 1594). Demographics, coronavirus disease 2019 (COVID-19) severity, and treatment histories were obtained from the Boston Medical Center electronic medical record. Humoral responses were analyzed using SARS-CoV-2–specific enzyme-linked immunosorbent assays and pseudovirus neutralizations in a subset of reinfection (n = 16) and convalescent samples (n = 32). Univariate, multivariate, and time to event analyses were used to identify associations. Results Individuals with reinfection had more frequent testing at shorter intervals compared with the convalescents. Unstable housing was associated with more than 2-fold greater chance of reinfection. Preexisting comorbidities and COVID-19 severity after the initial infection were not associated with reinfection. SARS-CoV-2 immunoglobulin G levels and pseudovirus neutralization were not different within the early weeks after primary infection and at a timepoint at least 90 days later in the 2 groups. In the convalescents, but not in those with reinfection, the late as compared with early humoral responses were significantly higher. Conclusions Reinfection associates with unstable housing, which is likely a marker for virus exposure, and reinfection occurs in the presence of SARS-CoV-2 antibodies., SARS-CoV-2 reinfection occurred at greater frequency among individuals with housing instability, and it did not associate with preexisting comorbidities or COVID-19 severity after first infection. Antibodies were detectable among the subset of individuals with documented reinfection examined in this study.

BACKGROUND: Mycobacterium tuberculosis (TB) infection induces systemic inflammation that could impact HIV-1 persistence. SETTING: HIV-1–seropositive individuals either with or without pulmonary TB disease were recruited in Kampala, Uganda. METHODS: Plasma cytokines, HIV-1 DNA, and cell-associated (ca)-RNA were compared among those coinfected with TB (cases) to those without TB (controls). TB-coinfected cases and controls were compared at presentation (n = 15 and n = 16, respectively) and at around 6 months after HIV-1 treatment initiation among those who had achieved virologic suppression (n = 6 and n = 8, respectively). At follow-up, the TB-coinfected cases had also finished TB treatment. RESULTS: Before treatment, the TB-coinfected cases as compared to the controls had higher levels of soluble(s)-CD163 (P = 0.0002) and interleukin-6 (P = 0.006) but lower levels of macrophage chemoattractant protein-1 (P = 0.04). After treatment, the TB-coinfected cases as compared to controls still had higher plasma s-CD163 levels (P = 0007). Controls as compared to the coinfected cases had higher ca-RNA per DNA template both at baseline (P = 0.03) and at follow-up (P = 0.07). Levels of ca-RNA per DNA copy at follow-up showed a negative correlation with baseline plasma s-CD163 (P = 0.008) and interleukin-6 (P = 0.05) levels. CONCLUSIONS: TB disease is associated with inflammation and decreased HIV-1 RNA expression relative to the number of infected cells, both before and after viral suppression. Infections present before antiretroviral initiation impact HIV-1 latency.

Detection of diverse respiratory viruses in Boston was approximately 80% lower after practices were instituted to limit coronavirus disease 2019 (COVID-19) spread compared with the same time period during the previous 5 years. Continuing the strategies that lower COVID-19 dissemination may be useful in decreasing the incidence of other viral respiratory infections.

Increasing evidence suggests that autoimmunity may play a role in the pathophysiology of SARS-CoV-2 infection during both the acute and ‘long COVID’ phases of disease. However, an assessment of autoimmune antibodies in convalescent SARS-CoV-2 patients has not yet been reported.MethodologyWe compared the levels of 18 different IgG autoantibodies (AABs) between four groups: (1) unexposed pre-pandemic subjects from the general population (n = 29); (2) individuals hospitalized with acute moderate-severe COVID-19 (n = 20); (3) convalescent SARS-COV-2-infected subjects with asymptomatic to mild viral symptoms during the acute phase with samples obtained between 1.8 and 7.3 months after infection (n = 9); and (4) unexposed pre-pandemic subjects with systemic lupus erythematous (SLE) (n = 6). Total IgG and IgA levels were also measured from subjects in groups 1-3 to assess non-specific pan-B cell activation.ResultsAs expected, in multivariate analysis, AABs were detected at much higher odds in SLE subjects (5 of 6, 83%) compared to non-SLE pre-pandemic controls (11 of 29, 38%) [odds ratio (OR) 19.4,95% CI, 2.0 – 557.0, p = 0.03]. AAB detection (percentage of subjects with one or more autoantibodies) was higher in SARS-CoV-2 infected convalescent subjects (7 of 9, 78%) [OR 17.4, 95% CI, 2.0 – 287.4, p = 0.02] and subjects with acute COVID-19 (12 of 20, 60%) compared with non-SLE pre-pandemic controls, but was not statistically significant among the latter [OR 1.8,95% CI, 0.6 – 8.1, p = 0.23]. Within the convalescent subject group, AABs were detected in 5/5 with reported persistent symptoms and 2/4 without continued symptoms (p = 0.17). The multivariate computational algorithm Partial Least Squares Determinant Analysis (PLSDA) was used to determine if distinct AAB signatures distinguish subject groups 1-3. Of the 18 autoantibodies measured, anti-Beta 2-Glycoprotein, anti-Proteinase 3-ANCA, anti-Mi-2 and anti-PM/Scl-100 defined the convalescent group; anti-Proteinase 3-ANCA, anti-Mi-2, anti-Jo-1 and anti-RNP/SM defined acute COVID-19 subjects; and anti-Proteinase 3-ANCA, anti-Mi-2, anti-Jo-1, anti-Beta 2-Glycoprotein distinguished unexposed controls. The AABs defining SARS-COV-2 infected from pre-pandemic subjects are widely associated with myopathies, vasculitis, and antiphospholipid syndromes, conditions with some similarities to COVID-19. Compared to pre-pandemic non-SLE controls, subjects with acute COVID-19 had higher total IgG concentration (p-value=0.006) but convalescent subjects did not (p-value=0.08); no differences in total IgA levels were found between groups.ConclusionsOur findings support existing studies suggesting induction of immune responses to self-epitopes during acute, severe COVID-19 with evidence of general B cell hyperactivation. Also, the preponderance of AAB positivity among convalescent individuals up to seven months after infection indicates potential initiation or proliferation, and then persistence of self-reactive immunity without severe initial disease. These results underscore the importance of further investigation of autoimmunity during SARS-CoV-2 infection and its role in the onset and persistence of post-acute sequelae of COVID-19.

Four different endemic coronaviruses (eCoVs) are etiologic agents for the seasonal common cold, and these eCoVs share extensive sequence homology with human SARS coronavirus 2 (SARS-CoV-2). Here, we show that individuals with, as compared with those without, a recent documented infection with eCoV were tested at greater frequency for respiratory infections but had a similar rate of SARS-CoV-2 acquisition. Importantly, the patients with a previously detected eCoV had less-severe coronavirus disease 2019 (COVID-19) illness. Our observations suggest that preexisting immune responses against endemic human coronaviruses can mitigate disease manifestations from SARS-CoV-2 infection.

Background The effectiveness of interleukin-6 inhibitors (IL-6i) in ameliorating coronavirus disease 2019 (COVID-19) remains uncertain. Methods We analyzed data for patients aged ≥18 years admitted with a positive severe acute respiratory syndrome coronavirus 2 polymerase chain reaction test at 4 safety-net hospital systems with diverse populations and high rates of medical comorbidities in 3 US regions. We used inverse probability of treatment weighting via machine learning for confounding adjustment by demographics, comorbidities, and disease severity markers. We estimated the average treatment effect, the odds of IL-6i effect on in-hospital mortality from COVID-19, using a logistic marginal structural model. Results Of 516 patients, 104 (20.1%) received IL-6i. Estimate of the average treatment effect adjusted for confounders suggested a 37% reduction in odds of in-hospital mortality in those who received IL-6i compared with those who did not, although the confidence interval included the null value of 1 (odds ratio = 0.63; 95% confidence interval, .29–1.38). A sensitivity analysis suggested that potential unmeasured confounding would require a minimum odds ratio of 2.55 to nullify our estimated IL-6i effect size. Conclusions Despite low precision, our findings suggested a relatively large effect size of IL-6i in reducing the odds of COVID-19–related in-hospital mortality.

Objective The aim of this observational study was to determine the optimal timing of interleukin-6 receptor inhibitor (IL6ri) administration for coronavirus disease 2019 (COVID-19). Methods Patients with COVID-19 were given an IL6ri (sarilumab or tocilizumab) based on iteratively reviewed guidelines. IL6ri were initially reserved for critically ill patients, but after review, treatment was liberalized to patients with lower oxygen requirements. Patients were divided into two groups: those requiring ≤45% fraction of inspired oxygen (FiO2) (termed stage IIB) and those requiring >45% FiO2 (termed stage III) at the time of IL6ri administration. The main outcomes were all-cause mortality, discharge alive from hospital, and extubation. Results A total of 255 COVID-19 patients were treated with IL6ri (149 stage IIB and 106 stage III). Patients treated in stage IIB had lower mortality than those treated in stage III (adjusted hazard ratio (aHR) 0.24, 95% confidence interval (CI) 0.08–0.74). Overall, 218 (85.5%) patients were discharged alive. Patients treated in stage IIB were more likely to be discharged (aHR 1.43, 95% CI 1.06–1.93) and were less likely to be intubated (aHR 0.43, 95% CI 0.24–0.79). Conclusions IL6ri administration prior to >45% FiO2 requirement was associated with improved COVID-19 outcomes. This can guide clinical management pending results from randomized controlled trials.

The COVID-19 pandemic has significantly impacted work, economy, and way of life. The SARS-CoV-2 virus displays unique features including widely varying symptoms and outcomes between infected individuals. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into virus transmission dynamics, pre-existing cross-reactive immunity, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. This ‘BU ELISA’ method exhibits very low signal from plasma or serum samples added to uncoated wells at as low as a 1:5 dilution. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (NP) reactive antibodies from blood samples drawn prior to May 2019. Of our prepandemic cohort, no SARS-CoV-2 RBD-reactive IgG antibodies were detected in subjects over 70 years of age, and SARS-CoV-2 NP-reactive antibodies were present at similar levels to infected subjects in some individuals and very low in others. Also, samples drawn in May 2020 from two individuals with no symptoms or no known virus exposure contained SARS-CoV-2 RBD-reactive antibodies at intermediate amounts compared with other subject groups (higher than pre-pandemic and lower than confirmed SARS-CoV-2 infected). The one asymptomatic SARS-CoV-2 convalescent subject in our study possessed comparable amounts of SARS-CoV-2 NP-specific IgM and IgG but drastically lower IgA than the symptomatic counterparts. Also, our assay detected positive signal from samples that gave negative results in a commercially available Lateral Flow Device (LFD) and the EUA approved Abbott IgG chemiluminescent microparticle immunoassay for SARS-CoV-2 antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and has promising implications for improved detection of all analytes measurable by this platform.

Individuals infected with human immunodeficiency virus (HIV) 1 have increased inflammation, which has been associated with age-associated diseases. Plasma markers, cell-associated virus levels, and ability to stimulate RNA transcription in latently infected cell lines was examined in younger and older HIV-1–infected individuals with suppressed virus. Cell-associated RNA, but not intact provirus level, had positive correlation with plasma D-dimer levels. Compared with the younger group, the older group had higher D-dimer levels and a trend toward more cell-associated RNA but similar levels of intact proviruses. Even though all measured inflammatory markers were relatively higher in the older group, this greater inflammation did not induce more HIV-1 transcription in latently infected cell lines. Inflammation and HIV-1 RNA expression increase with age despite similar levels of intact infectious HIV DNA. While plasma inflammation is correlated with HIV-1 RNA expression in peripheral blood mononuclear cells, it does not induce HIV-1 transcription in latently infected cell lines.

Nucleic acid amplification tests (NAATs), which amplify and detect pathogen nucleic acids, are vital methods to diagnose diseases, particularly in cases where patients exhibit low levels of infection. For many blood-borne pathogens such as HIV or Plasmodium, it is necessary to first extract pathogen RNA or DNA from patient blood prior to analysis with NAATs. Traditional nucleic acid extraction methods are expensive, resource-intensive and are often difficult to deploy to resource-limited areas where many blood-borne infections are widespread. Here, we describe a portable, paper-and-plastic device for instrument-free nucleic acid extraction from whole blood, which we call SNAPflex, that builds upon our previous work extracting RNA in a 2D platform from nasopharyngeal swabs. We demonstrated improved extraction of HIV RNA from simulated patient samples compared to traditional extraction methods and long-term stability of extracted RNA without the need for cold storage. We further demonstrated successful extraction and recovery of Plasmodium falciparum DNA from simulated patient samples with superior recovery compared to existing extraction methods. The SNAPflex device extracts and purifies DNA and RNA from whole blood which can be amplified with traditional NAATs, and was designed to easily manufacture and integrate into existing health systems.

In clinical trials, HIV-1 broadly neutralizing antibodies (bnAbs) effectively lower plasma viremia and delay virus reemergence. The presence of less neutralization-susceptible strains prior to treatment decreases the efficacy of these antibody-based treatments, but neutralization sensitivity often cannot be predicted by sequence analysis alone. We found that phenotypically confirmed CXCR4-utilizing strains are less neutralization sensitive, especially to variable loop 3 (V3 loop)-directed bnAbs, than exclusively CCR5-utilizing strains in some, but not all, cases. Homology modeling suggested that the primary V3 loop bnAb epitope is equally accessible among CCR5- and CXCR4-using strains, although variants that exclusively use CXCR4 have V3 loop protrusions that interfere with CCR5 receptor interactions. Homology modeling also showed that among some, but not all, envelopes with decreased neutralization sensitivity, V1 loop orientation interfered with V3 loop-directed bnAb binding. Thus, there are likely different structural reasons for the coreceptor usage restriction and the different bnAb susceptibilities. Importantly, we show that individuals harboring envelopes with higher likelihood of using CXCR4 or greater predicted V1 loop interference have faster virus rebound and a lower maximum decrease in plasma viremia, respectively, after treatment with a V3 loop bnAb. Knowledge of receptor usage and homology models may be useful in developing future algorithms that predict treatment efficacy with V3 loop bnAbs. IMPORTANCE The efficacy of HIV-1 broadly neutralizing antibody (bnAb) therapies may be compromised by the preexistence of less susceptible variants. Sequence-based methods are needed to predict pretreatment variants’ neutralization sensitivities. HIV-1 strains that exclusively use the CXCR4 receptor rather than the CCR5 receptor are less neutralization susceptible, especially to variable loop 3 (V3 loop) bnAbs in some, but not all, instances. While the inability to utilize the CCR5 receptor maps to a predicted protrusion in the envelope V3 loop, this viral determinant does not directly influence V3 loop bnAb sensitivity. Homology modeling predicts that contact between the envelope V1 loop and the antibody impacts V3 loop bnAb susceptibility in some cases. Among pretreatment envelopes, increased probability of using CXCR4 and greater predicted V1 interference are associated with faster virus rebound and a smaller decrease in the plasma virus level, respectively, after V3 loop bnAb treatment. Receptor usage information and homology models may be useful for predicting V3 loop bnAb therapy efficacy.

Objectives Patients with Covid-19 and obesity have worse clinical outcomes which may be driven by increased inflammation. This study aimed to characterize the association between clinical outcomes in patients with obesity and inflammatory markers. Methods We analyzed data for patients aged ≥18 years admitted with a positive SARS-CoV-2 PCR test. We used multivariate logistic regression to determine the association between BMI and intensive care unit (ICU) transfer and all-cause mortality. Inflammatory markers (C-reactive protein [CRP], lactate dehydrogenase [LDH], ferritin, and D-dimer) were compared between patients with and without obesity (body mass index [BMI] ≥30 kg/m2). Results Of 791 patients with Covid-19, 361 (45.6%) had obesity. In multivariate analyses, BMI ≥35 was associated with a higher odds of ICU transfer (adjusted odds ratio [aOR] 2.388 (95% confidence interval [CI]: 1.074–5.310) and hospital mortality (aOR = 4.3, 95% CI: 1.69–10.82). Compared to those with BMI Conclusions Patients with obesity were more likely to have poor outcomes even without increased inflammation.

Background: The impact of interleukin-6 receptor inhibitors (IL6ri) on Coronavirus disease 2019 (Covid-19) outcomes remains uncertain. Methods: Patients with severe and critical Covid-19 were given an IL6ri (sarilumab or tocilizumab) based on pragmatic and iteratively reviewed guidelines at a safety net hospital with a diverse population. IL6ri were initially reserved for critically ill patients, but after review, treatment was liberalized to patients requiring >4 liters/minute of oxygen or having an alveolar-arterial gradient >50 mmHg and elevated inflammatory markers (C-reactive protein >100 mg/L or lactate dehydrogenase >450 units/L). Patients were divided into 2 groups, those requiring ≤ 45% fraction of inspired oxygen (FiO2) (termed stage IIB) and those requiring >45% FiO2 (termed stage III) at the time of IL6ri administration. Main outcomes were all-cause mortality, discharge alive from hospital, and extubation. Findings: Prior to treatment, 255 consecutive Covid-19 patients treated with IL6ri (149 stage IIB and 106 stage III) had hypoxemia requiring supplemental oxygen and had elevated inflammatory markers. Patients treated in stage IIB had lower mortality than the stage III group (adjusted hazard ratio [aHR]: 0.20; 95% confidence interval [CI] 0.07-0.60). Overall, 218 (85.5%) patients were discharged alive. Patients treated in stage IIB were more likely to be discharged (aHR: 2.04; 95% CI 1.53 – 2.73) and to have intubation-free survival (HR: 0.39; 95% CI: 0.20-0.74). Interpretation: Our finding that early IL6ri were associated with reduced mortality from severe Covid-19 can guide current clinical management while we await definitive results from randomized controlled trials. Funding Statement: This work was supported by National Institutes of Health grants K24-AI145661 and 5T32AI052074-13. Declaration of Interests: None to declare Ethics Approval Statement: All activities associated with this project were approved by the Boston University Medical Center Institutional Review Board (IRB)

HIV-1 acquisition occurs most commonly after sexual contact. To establish infection, HIV-1 must infect cells that support high-level replication, namely CD4+ T cells, which are absent from the outermost genital epithelium. Dendritic cells (DCs), present in mucosal epithelia, potentially facilitate HIV-1 acquisition. We show that vaginal epithelial DCs, termed CD1a+ VEDCs, are unlike other blood- and tissue-derived DCs because they express langerin but not DC-SIGN, and unlike skin-based langerin+ DC subset Langerhans cells (LCs), they do not harbor Birbeck granules. Individuals primarily acquire HIV-1 that utilizes the CCR5 receptor (termed either R5 or R5X4) during heterosexual transmission, and the mechanism for the block against variants that only use the CXCR4 receptor (classified as X4) remains unclear. We show that X4 as compared with R5 HIV-1 shows limited to no replication in CD1a+ VEDCs. This differential replication occurs after fusion, suggesting that receptor usage influences postentry steps in the virus life cycle. Furthermore, CD1a+ VEDCs isolated from HIV-1-infected virologically suppressed women harbor HIV-1 DNA. Thus, CD1a+ VEDCs are potentially infected early during heterosexual transmission and also retain virus during treatment. Understanding the interplay between HIV-1 and CD1a+ VEDCs is important for future prevention and cure strategies.

Background Excessive weight gain associated with integrase strand transfer inhibitor (InSTI) antiretrovirals is an emerging issue in the current antiretroviral treatment (ART) era. Despite the known association between excess weight and impaired glucose tolerance in the general population, the metabolic implications of InSTI-associated weight gain have not been established. The objective of this study was to evaluate the presence of InSTI-associated weight gain amongst a diverse, urban population and to investigate potential risk factors and metabolic implications. Methods We obtained demographic, pharmacy and laboratory data from the hospital clinical data warehouse for ART-naïve adult HIV+ patients at Boston Medical Center between fiscal year (FY) 2007-2017. We compared patients who initiated on an InSTI to those with an alternate regimen (i.e. PI, NNRTI) who remained on their initial regimen for at least 18 months. Individuals with diabetes mellitus (DM) prescription or ICD 9/10 code prior to ART initiation were excluded. Our primary outcome was percent weight change in the first 24 months of ART estimated by linear mixed effects model fit by restricted maximum likelihood. Our secondary outcome was incident DM diagnosis in the 18 months after ART using progression-free survival (PFS). PFS rates were estimated by the Kaplan-Meier method and log-rank test, and Cox proportional-hazards model were determined, all using R v3.6.2. Results Between FY 2007-2017, 139 patients were initiated on InSTIs and 1117 were initiated on alternative anchor regimens. Approximately, one third of the cohort was female and more than 75% were non-white. InSTI use in women was associated with increased weight gain in the first 24 months of ART compared to non-InSTIs (+9.57%, p = 0.002, Figure 1). InSTI use was associated with more incident DM diagnoses in the first 18 months of ART compared to non-InSTI regimens (HR = 3.27, p = 0.014). Figure 1: Percentage weight change from baseline at ART initiation between InSTI and non-InSTI regimens Conclusion Females have higher InSTI-associated weight gain which suggests they may be more susceptible to adverse metabolic issues. InSTI use appears to be associated with an increased incidence of DM diagnoses following ART initiation. Further prospective and controlled studies will be necessary to describe the mechanism of this effect and refine HIV management strategies. Disclosures Archana Asundi, MD, Gilead (Scientific Research Study Investigator)Merck (Scientific Research Study Investigator)ViiV (Scientific Research Study Investigator) Nina H. Lin, MD, Gilead Sciences (Scientific Research Study Investigator)ViiV (Scientific Research Study Investigator)

In clinical trials, HIV-1 broadly neutralizing antibodies (bnAbs) effectively lower plasma viremia and delay virus reemergence after antiretroviral treatment is stopped among infected individuals that have undetectable virus levels. Presence of less neutralization susceptible strains prior to treatment, however, decreases the efficacy of these antibody-based treatments. The HIV-1 envelope glycoprotein harbors extensive genetic variation, and thus, neutralization sensitivity often cannot be predicted by sequence analysis alone. Sequence-based prediction methods are needed because phenotypic-based assays are labor intensive and not sensitive. Based on the finding that phenotypically confirmed CXCR4- as compared to exclusive CCR5-utilizing strains are less neutralization sensitive, especially to variable loop 1 and 2 (V1-V2) and V3 loop bnAbs, we show that an algorithm that predicts receptor usage identifies envelopes with decreased V3 loop bnAb susceptibility. Homology modeling suggests that the primary V3 loop bnAb epitope is equally accessible among CCR5- and CXCR4-using strains although variants that exclusively use CXCR4 have V3 loop protrusions that interfere with CCR5 receptor interactions. On the other hand, homology modeling also shows that envelope V1 loop orientation interferes with V3 loop directed bnAb binding, and this accounts for decreased neutralization sensitivity in some but not all cases. Thus, there are likely different structural reasons for the co-receptor usage restriction and the differential bnAb susceptibility. Algorithms that use sequence data to predict receptor usage and antibody-envelope homology models can be used to identify variants with decreased sensitivity to V3 loop and potentially other bnAbs.AUTHOR SUMMARYHIV-1 broadly neutralizing antibody (bnAb) therapies are effective, but the pre-existence of less susceptible variants may lead to therapeutic failure. Sequence-based methods are needed to predict pre-treatment variants’ neutralization sensitivity. HIV-1 strains that use the CXCR4 as compared to the CCR5 receptor are less neutralization susceptible, especially to V1-V2 and V3 loop bnAbs. A sequence-based algorithm that predicts receptor usage can identify envelope variants with decreased V3 loop bnAb susceptibility. While the inability to utilize the CCR5 receptor maps to a predicted protrusion in the envelope V3 loop, this viral determinant does not directly influence V3 loop bnAb sensitivity. Furthermore, homology modeling predicted contact between the envelope V1 loop and an antibody also impact V3 loop bnAb susceptibility in some but not all cases. An algorithm that predicts receptor usage and homology modeling can be used to predict sensitivity to bnAbs that target the V3 loop and potentially other envelope domains. These sequence-based methods will be useful as HIV-1 bnAbs enter the clinical arena.

A limited number of human immunodeficiency virus type 1 (HIV-1) variants initially infect HIV-1–naive individuals. Recent studies imply that this may occur because generally inefficient transcytosis across intact mucosal surfaces could be enhanced for specific viruses with bound antibodies and in the presence of acidic pH. We found that transcytosis of both cell-free and cell-associated viruses with diverse envelopes was significantly decreased in the presence of either antibodies or plasma from chronically infected transmitting partners regardless of pH. Transmitted variants also did not have greater transmigration as compared to chronic-infection strains. Enhanced translocation capacity is unlikely to influence which HIV-1 variant establishes infection.

Human immunodeficiency virus type 1 (HIV-1) studies suggest that antibody-dependent cellular cytotoxicity (ADCC) influences both virus acquisition and subsequent disease outcome. Technical issues with currently available assays, however, have limited the ability to comprehensively assess the impact of ADCC on transmission and disease progression. Commonly used ADCC assays use a target cell line, CEM.NKr-CCR5-Luc, that often does not support replication of relevant HIV-1 variants. Thus, the extent of ADCC responses against a large panel of HIV-1 strains often cannot be assessed using the currently available methods. We developed two new reporter cell-lines (MT4-CCR5-Luc and PM1-CCR5-Luc) to overcome these issues. MT4-CCR5-Luc cells are resistant, whereas PM1-CCR5-Luc cells are susceptible, to killing by a natural killer cell line, CD16(+)KHYG-1, in the absence of antibody. Polyclonal HIVIG gave similar ADCC estimates against HIV-1 isolate, NL4–3, regardless of which of the three cell lines were used as the targets. In contrast to CEM.NKr-CCR5-Luc and PM1-CCR5-Luc, however, MT4-CCR5-Luc target cells produce significantly higher luciferase after exposure to various HIV-1 strains, including transmitted founder variants and viruses incorporating specific envelopes of interest. This higher luciferase expression does not yield spurious results because ADCC estimates are similar when killing is assessed by both reporter protein expression and flow cytometry. Furthermore, ADCC estimates derived from MT4-CCR5-Luc cells are not skewed by non-antibody contents present in human plasma. In aggregate, the MT4-CCR5-Luc cell line can be used to estimate monoclonal antibody or plasma-induced ADCC responses against a diverse range of HIV-1 envelopes relevant for transmission and disease progression studies.

Background. Metabolic effects following combination antiretroviral therapy (cART) vary by regimen type. Changes in metabolic effects were assessed following cART in the AIDS Clinical Trials Group (ACTG) A5257 study, and correlated with plasma ritonavir trough concentrations (C24). Methods. Treatment-naive adult subjects were randomized to ritonavir-boosted atazanavir or darunavir, or raltegravir-based cART. Changes in lipids and other metabolic outcomes over time were estimated. Differences between arms were estimated with 97.5% confidence intervals and compared using pairwise Student t tests. Associations between ritonavir C24 and lipid changes at week 48 were evaluated via linear regression. Results. Analyses included 1797 subjects with baseline fasting data. Baseline lipid profiles and metabolic syndrome rates (approximately 21%) were similar across arms. Comparable increases occurred in total cholesterol, triglycerides, and low-density lipoprotein cholesterol with the boosted protease inhibitors (PIs); each PI had greater increases relative to raltegravir (all P ≤ .001 at week 96). Metabolic syndrome incident rates by week 96 (approximately 22%) were not different across arms. Ritonavir C24 was not different by arm (P = .89) (median, 69 ng/mL and 74 ng/mL in the atazanavir and darunavir arms, respectively) and were not associated with changes in lipid measures (all P > .1). Conclusions. Raltegravir produced the most favorable lipid profile. Metabolic syndrome rates were high at baseline and increased to the same degree in all arms. Ritonavir C24 was not different in the PI arms and had no relationship with the modest but comparable increases in lipids observed with either atazanavir or darunavir. The long-term clinical significance of the lipid changes noted with the PIs relative to raltegravir deserves further evaluation. Clinical Trials Registration. {"type":"clinical-trial","attrs":{"text":"NCT 00811954","term_id":"NCT00811954"}}NCT 00811954.

Recent studies suggest that single genome amplification (SGA) as compared to standard bulk PCR and virus stocks from 293T transfection versus short term passage in peripheral blood mononuclear cells (PBMC) yield a less biased representation of HIV-1 envelope characteristics. In 9 different subjects, genetic diversity, divergence, and population structure were not significantly different among SGA or bulk PCR amplified envelope V1-V3 segments. In these subjects, 293T transfection derived virus stocks with SGA or bulk PCR amplified envelopes have similar infectivity, replication kinetics, co-receptor usage, and neutralization susceptibility. While PBMC passage as compared to the 293T derived virus stocks had similar co-receptor usage, PBMC viruses were less neutralization susceptible to some specific antibodies. Our results suggest that the method of envelope sequence amplification, either SGA or bulk PCR, does not have a significant impact on the genotypic and phenotypic properties of the virus envelope quasispecies.

All human immunodeficiency virus type 1 (HIV-1)–infected inocula, such as genital secretions, breast milk, and blood, contain both cell-free virus and infected cells. The relative contributions of cell-free and/or cell-associated virus in establishing an infection in a naive host during the different modes of HIV-1 acquisition remains unclear. Studies aim to elucidate the source of the acquired virus because strategies to prevent acquisition may have differential efficacy against the different modes of transmission. In this review, I will detail some of the challenges in identifying the source of the transmitted virus, genotypic and phenotypic differences among cell-free compared with cell-associated HIV-1, and implications on the efficacy for prevention strategies.

It has been postulated that HIV-1 envelope properties, such as shorter and less glycosylated V1-V2 loops commonly observed among non-subtype B early – transmitted viruses, promote utilization of the gut homing integrin α4β7. This property potentially confers an advantage to some HIV-1 variants early after acquisition. We found that replication competent recombinant viruses incorporating HIV-1 subtype A compact and less glycosylated early versus chronic phase V1-V2 loops demonstrated no significant difference in binding to α4β7 high CD8+ T cells or replication in α4β7 high CD4+ T cells. Integrin α4β7 usage does not select for shorter less glycosylated envelopes during transmission.

Background. The long-term impact of allogeneic hematopoietic stem cell transplantation (HSCT) on human immunodeficiency virus type 1 (HIV-1) reservoirs in patients receiving combination antiretroviral therapy (cART) is largely unknown. Methods. We studied the effects of a reduced-intensity conditioning allogeneic HSCT from donors with wildtype–CCR5 + cells on HIV-1 peripheral blood reservoirs in 2 patients heterozygous for the ccr5Δ32 mutation. Indepth analyses of the HIV-1 reservoir size in peripheral blood, coreceptor use, and specific antibody responses were performed on samples obtained before and up to 3.5 years after HSCT receipt. Results. Although HIV-1 DNA was readily detected in peripheral blood mononuclear cells (PBMCs) before and 2–3 months after HSCT receipt, HIV-1 DNA and RNA were undetectable in PBMCs, CD4 + T cells, or plasma up to 21 and 42 months after HSCT. The loss of detectable HIV-1 correlated temporally with full donor chimerism, development of graft-versus-host disease, and decreases in HIV-specific antibody levels. Conclusions. The ability of donor cells to engraft without evidence of ongoing HIV-1 infection suggests that HIV-1 replication may be fully suppressed during cART and does not contribute to maintenance of viral reservoirs in peripheral blood in our patients. HSCTs with wild-type–CCR5 + donor cells can lead to a sustained reduction in the size of the peripheral reservoir of HIV-1.

Objectives: We previously reported vicriviroc (VCV) resistance in an HIV-infected subject and used deep sequencing and clonal analyses to track the evolution of V3 sequence forms over 28 weeks of therapy. Here, we test the contribution of gp120 mutations to CCR5 antagonist resistance and investigate why certain minority V3 variants emerged as the dominant species under drug pressure. Methods: Nineteen site-directed HIV-1 mutants were generated that contained gp120 VCV resistance mutations. Viral sensitivities to VCV, maraviroc, TAK-779, and HGS004 were determined. Results: Three patterns of susceptibilities were observed as follows: sigmoid inhibition curves with 50% inhibitory concentration similar to pretreatment virus [07J-week 0 (W0)], single mutants with decreased 50% inhibitory concentrations compared with 07J-W0, and mutants that contained ≥5 of 7 VCV resistance mutations with flattened inhibition curves and decreased or negative percent maximal inhibition. Substitutions such as S306P, which sensitized virus to CCR5 antagonists when present as single mutations, were not detected in the baseline virus population but were necessary for maximal resistance when incorporated into V3 backbones that included preexisting VCV resistance mutations. Conclusions: CCR5 antagonist resistance was reproduced only when a majority of V3 mutations were present. Minority V3 loop variants may serve as a scaffold upon which additional mutations lead to complete VCV resistance.

Individuals with recent human immunodeficiency virus type 1 (HIV-1) acquisition are likely to be a major source for other new infections because they have a high level of plasma virus, and the circulating virions possess unique properties that are highly suited for transmission. The acute infection period, however, presents a unique "window of opportunity," because there are a limited number of genetic variants. Studies aim to elucidate the nature of the transmitted viruses and understand the mechanisms that inhibit the majority of variants present in the chronically infected partner from establishing a productive infection in the naive host. Greater understanding of these issues may open promising new ways to effectively block HIV-1 transmission.

Background—Newly infected subjects acquire a limited number of human immunodeficiency virus type 1 (HIV-1) variants with specific genotypic and phenotypic features from the array of viruses present in a chronically infected transmitting partner. Methods—We examined HIV-1 envelope sequences from the earliest available serum sample after HIV-1 acquisition in 13 newly infected subjects and from their epidemiologically linked HIV-1– infected heterosexual partner. Samples from both members were collected on the same day in the Rakai Community Cohort Study. Results—Ten couples were infected with subtype D HIV-1, and 3 pairs had subtype A HIV-1. Newly infected subjects acquired a subset of the viruses that were circulating in the transmitting partner; transmitted variants had less diversity and divergence and were more closely related to the ancestral sequences. The majority of signature amino acid differences among donor and recipient sequences were in and immediately following the V3 loop. Envelopes from recipients were significantly shorter and had a lower V3 charge than envelopes from donors, but there was no significant difference in the number of potential N-linked glycosylation sites. Conclusion—A minority subset of HIV-1 variants with signature genotypes is favored for transmission in this population.

Lamivudine therapy selects for the M184V mutation. Although this mutation reduces the replicative capacity of human immunodeficiency virus in vitro, its impact on viral fitness in vivo has not been well defined. We used quantitative allele-specific PCR to precisely calculate the fitness differences between the mutated M184V virus and one that had reverted to the wild type in a cohort of patients by selectively interrupting reverse transcriptase inhibitor therapy, and we found that the M184V variants were consistently 4 to 8% less fit than the wild type in the absence of drug. After a lag phase of variable duration, wild-type variants emerged due to continued evolution of pol and back mutation rather than through emergence of an archived wild-type variant.

Differences in the extent of genetic diversity have been observed in human immunodeficiency virus type-1 (HIV-1) envelope sequences early in infection, and this has been linked to gender and to modifiable exogenous factors such as hormonal contraceptive use and genital tract infections. But it is unclear whether envelope diversity is indicative of diversity in other regions of the viral genome, and thus whether it adequately reflects whether multiple or a single virus initiated the infection. Here we show that six women with homogeneous envelope V1-V3 sequences during primary infection also had homogeneous gag and polymerase (pol) sequences at the same time. On the other hand, six women with multiple envelope sequences had diverse gag and pol genotypes during a similar interval after infection. This suggests that envelope sequences reflect sequence diversity throughout the viral genomes present early in infection and thus provide an indication of whether a single virus or multiple viruses initiated the infection. Analysis of HIV-1 sequences from about 3 years after infection revealed that the level of diversity and diversification was similar between the women in the two groups.