Adolescent BCG revaccination induces a phenotypic shift in CD4+ T cell responses to Mycobacterium tuberculosis.

A recent clinical trial demonstrated that Bacille Calmette-Guérin (BCG) revaccination of adolescents reduced the risk of sustained infection with Mycobacterium tuberculosis (M.tb). In a companion phase 1b trial, HVTN 602/Aeras A-042, we characterize in-depth the cellular responses to BCG revaccination or to a H4:IC31 vaccine boost to identify T cell subsets that could be responsible for the protection observed. High-dimensional clustering analysis of cells profiled using a 26-color flow cytometric panel show marked increases in five effector memory CD4+ T cell subpopulations (TEM) after BCG revaccination, two of which are highly polyfunctional. CITE-Seq single-cell analysis shows that the activated subsets include an abundant cluster of Th1 cells with migratory potential. Additionally, a small cluster of Th17 TEM cells induced by BCG revaccination expresses high levels of CD103; these may represent recirculating tissue-resident memory cells that could provide pulmonary immune protection. Together, these results identify unique populations of CD4+ T cells with potential to be immune correlates of protection conferred by BCG revaccination.

HIV BG505 SOSIP.664 trimer with 3M-052-AF/alum induces human autologous tier-2 neutralizing antibodies.

Stabilized trimers preserving the native-like HIV envelope structure may be key components of a preventive HIV vaccine regimen to induce broadly neutralizing antibodies (bnAbs). We evaluated trimeric BG505 SOSIP.664 gp140, formulated with a novel TLR7/8 signaling adjuvant, 3M-052-AF/Alum, for safety, adjuvant dose-finding and immunogenicity in a first-in-healthy adult (n=17), randomized, placebo-controlled trial (HVTN 137A). The vaccine regimen appeared safe. Robust, trimer-specific antibody, B-cell and CD4+ T-cell responses emerged post-vaccination. Five vaccinees developed serum autologous tier-2 nAbs (ID50 titer, 1:28-1:8647) after 2-3 doses targeting C3/V5 and/or V1/V2/V3 Env regions by electron microscopy and mutated pseudovirus-based neutralization analyses. Trimer-specific, B-cell-derived monoclonal antibody activities confirmed these results and showed weak heterologous neutralization in the strongest responder. Our findings demonstrate the clinical utility of the 3M-052-AF/alum adjuvant and support further improvements of trimer-based Env immunogens to focus responses on multiple broad nAb epitopes. KEY TAKEAWAY/TAKE-HOME MESSAGES: HIV BG505 SOSIP.664 trimer with novel 3M-052-AF/alum adjuvant in humans appears safe and induces serum neutralizing antibodies to matched clade A, tier 2 virus, that map to diverse Env epitopes with relatively high titers. The novel adjuvant may be an important mediator of vaccine response.

Human immunoglobulin gene allelic variation impacts germline-targeting vaccine priming.

Vaccine priming immunogens that activate germline precursors for broadly neutralizing antibodies (bnAbs) have promise for development of precision vaccines against major human pathogens. In a clinical trial of the eOD-GT8 60mer germline-targeting immunogen, higher frequencies of vaccine-induced VRC01-class bnAb-precursor B cells were observed in the high dose compared to the low dose group. Through immunoglobulin heavy chain variable (IGHV) genotyping, statistical modeling, quantification of IGHV1-2 allele usage and B cell frequencies in the naive repertoire for each trial participant, and antibody affinity analyses, we found that the difference between dose groups in VRC01-class response frequency was best explained by IGHV1-2 genotype rather than dose and was most likely due to differences in IGHV1-2 B cell frequencies for different genotypes. The results demonstrate the need to define population-level immunoglobulin allelic variations when designing germline-targeting immunogens and evaluating them in clinical trials.

Persistent serum protein signatures define an inflammatory subcategory of long COVID.

Long COVID or post-acute sequelae of SARS-CoV-2 (PASC) is a clinical syndrome featuring diverse symptoms that can persist for months following acute SARS-CoV-2 infection. The aetiologies may include persistent inflammation, unresolved tissue damage or delayed clearance of viral protein or RNA, but the biological differences they represent are not fully understood. Here we evaluate the serum proteome in samples, longitudinally collected from 55 PASC individuals with symptoms lasting ≥60 days after onset of acute infection, in comparison to samples from symptomatically recovered SARS-CoV-2 infected and uninfected individuals. Our analysis indicates heterogeneity in PASC and identified subsets with distinct signatures of persistent inflammation. Type II interferon signaling and canonical NF-κB signaling (particularly associated with TNF), appear to be the most differentially enriched signaling pathways, distinguishing a group of patients characterized also by a persistent neutrophil activation signature. These findings help to clarify biological diversity within PASC, identify participants with molecular evidence of persistent inflammation, and highlight dominant pathways that may have diagnostic or therapeutic relevance, including a protein panel that we propose as having diagnostic utility for differentiating inflammatory and non-inflammatory PASC.

A first-in-human germline-targeting HIV nanoparticle vaccine induced broad and publicly targeted helper T cell responses.

The engineered outer domain germline targeting version 8 (eOD-GT8) 60-mer nanoparticle was designed to prime VRC01-class HIV-specific B cells that would need to be matured, through additional heterologous immunizations, into B cells that are able to produce broadly neutralizing antibodies. CD4 T cell help will be critical for the development of such high-affinity neutralizing antibody responses. Thus, we assessed the induction and epitope specificities of the vaccine-specific T cells from the IAVI G001 phase 1 clinical trial that tested immunization with eOD-GT8 60-mer adjuvanted with AS01B. Robust polyfunctional CD4 T cells specific for eOD-GT8 and the lumazine synthase (LumSyn) component of eOD-GT8 60-mer were induced after two vaccinations with either the 20- or 100-microgram dose. Antigen-specific CD4 T helper responses to eOD-GT8 and LumSyn were observed in 84 and 93% of vaccine recipients, respectively. CD4 helper T cell epitope "hotspots" preferentially targeted across participants were identified within both the eOD-GT8 and LumSyn proteins. CD4 T cell responses specific to one of these three LumSyn epitope hotspots were observed in 85% of vaccine recipients. Last, we found that induction of vaccine-specific peripheral CD4 T cells correlated with expansion of eOD-GT8-specific memory B cells. Our findings demonstrate strong human CD4 T cell responses to an HIV vaccine candidate priming immunogen and identify immunodominant CD4 T cell epitopes that might improve human immune responses either to heterologous boost immunogens after this prime vaccination or to other human vaccine immunogens.

Human immunoglobulin gene allelic variation impacts germline-targeting vaccine priming.

Vaccine priming immunogens that activate germline precursors for broadly neutralizing antibodies (bnAbs) have promise for development of precision vaccines against major human pathogens. In a clinical trial of the eOD-GT8 60mer germline-targeting immunogen, higher frequencies of vaccine-induced VRC01-class bnAb-precursor B cells were observed in the high dose compared to the low dose group. Through immunoglobulin heavy chain variable (IGHV) genotyping, statistical modeling, quantification of IGHV1-2 allele usage and B cell frequencies in the naive repertoire for each trial participant, and antibody affinity analyses, we found that the difference between dose groups in VRC01-class response frequency was best explained by IGHV1-2 genotype rather than dose and was most likely due to differences in IGHV1-2 B cell frequencies for different genotypes. The results demonstrate the need to define population-level immunoglobulin allelic variations when designing germline-targeting immunogens and evaluating them in clinical trials. One-Sentence Summary: Human genetic variation can modulate the strength of vaccine-induced broadly neutralizing antibody precursor B cell responses.

Vaccination induces HIV broadly neutralizing antibody precursors in humans.

Broadly neutralizing antibodies (bnAbs) can protect against HIV infection but have not been induced by human vaccination. A key barrier to bnAb induction is vaccine priming of rare bnAb-precursor B cells. In a randomized, double-blind, placebo-controlled phase 1 clinical trial, the HIV vaccine-priming candidate eOD-GT8 60mer adjuvanted with AS01B had a favorable safety profile and induced VRC01-class bnAb precursors in 97% of vaccine recipients with median frequencies reaching 0.1% among immunoglobulin G B cells in blood. bnAb precursors shared properties with bnAbs and gained somatic hypermutation and affinity with the boost. The results establish clinical proof of concept for germline-targeting vaccine priming, support development of boosting regimens to induce bnAbs, and encourage application of the germline-targeting strategy to other targets in HIV and other pathogens.

Characterization of a vaccine-elicited human antibody with sequence homology to VRC01-class antibodies that binds the C1C2 gp120 domain.

Broadly HIV-1-neutralizing VRC01-class antibodies bind the CD4-binding site of Env and contain VH1-2*02-derived heavy chains paired with light chains expressing five-amino acid-long CDRL3s. Their unmutated germline forms do not recognize HIV-1 Env, and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding naïve B cells by the vaccine immunogens. To address this point, we examined Env-specific B cell receptor sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed, only one displayed homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in the CDRH3 and CDRL3 were necessary. Only germ line-targeting Env immunogens efficiently activated VRC01 B cells, even in the presence of FH1 B cells. Our findings support the use of these immunogens to activate VRC01 B cells in humans.

Rectal tissue and vaginal tissue from intravenous VRC01 recipients show protection against ex vivo HIV-1 challenge.

BackgroundVRC01, a potent, broadly neutralizing monoclonal antibody, inhibits simian-HIV infection in animal models. The HVTN 104 study assessed the safety and pharmacokinetics of VRC01 in humans. We extend the clinical evaluation to determine intravenously infused VRC01 distribution and protective function at mucosal sites of HIV-1 entry.MethodsHealthy, HIV-1-uninfected men (n = 7) and women (n = 5) receiving VRC01 every 2 months provided mucosal and serum samples once, 4-13 days after infusion. Eleven male and 8 female HIV-seronegative volunteers provided untreated control samples. VRC01 levels were measured in serum, secretions, and tissue, and HIV-1 inhibition was determined in tissue explants.ResultsMedian VRC01 levels were quantifiable in serum (96.2 µg/mL or 1.3 pg/ng protein), rectal tissue (0.11 pg/ng protein), rectal secretions (0.13 pg/ng protein), vaginal tissue (0.1 pg/ng protein), and cervical secretions (0.44 pg/ng protein) from all recipients. VRC01/IgG ratios in male serum correlated with those in paired rectal tissue (r = 0.893, P = 0.012) and rectal secretions (r = 0.9643, P = 0.003). Ex vivo HIV-1Bal26 challenge infected 4 of 21 rectal explants from VRC01 recipients versus 20 of 22 from controls (P = 0.005); HIV-1Du422.1 infected 20 of 21 rectal explants from VRC01 recipients and 12 of 12 from controls (P = 0.639). HIV-1Bal26 infected 0 of 14 vaginal explants of VRC01 recipients compared with 23 of 28 control explants (P = 0.003).ConclusionIntravenous VRC01 distributes into the female genital and male rectal mucosa and retains anti-HIV-1 functionality, inhibiting a highly neutralization-sensitive but not a highly resistant HIV-1 strain in mucosal tissue. These findings lend insight into VRC01 mucosal infiltration and provide perspective on in vivo protective efficacy.FundingNational Institute of Allergy and Infectious Diseases and Bill & Melinda Gates Foundation.

Integrated analysis of multimodal single-cell data.

The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce "weighted-nearest neighbor" analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.

Innate immune signatures to a partially-efficacious HIV vaccine predict correlates of HIV-1 infection risk.

The pox-protein regimen tested in the RV144 trial is the only vaccine strategy demonstrated to prevent HIV-1 infection. Subsequent analyses identified antibody and cellular immune responses as correlates of risk (CoRs) for HIV infection. Early predictors of these CoRs could provide insight into vaccine-induced protection and guide efforts to enhance vaccine efficacy. Using specimens from a phase 1b trial of the RV144 regimen in HIV-1-uninfected South Africans (HVTN 097), we profiled innate responses to the first ALVAC-HIV immunization. PBMC transcriptional responses peaked 1 day post-vaccination. Type I and II interferon signaling pathways were activated, as were innate pathways critical for adaptive immune priming. We then identified two innate immune transcriptional signatures strongly associated with adaptive immune CoR after completion of the 4-dose regimen. Day 1 signatures were positively associated with antibody-dependent cellular cytotoxicity and phagocytosis activity at Month 6.5. Conversely, a signature present on Days 3 and 7 was inversely associated with Env-specific CD4+ T cell responses at Months 6.5 and 12; rapid resolution of this signature was associated with higher Env-specific CD4+ T-cell responses. These are the first-reported early immune biomarkers of vaccine-induced responses associated with HIV-1 acquisition risk in humans and suggest hypotheses to improve HIV-1 vaccine regimens.

Mechanisms of Endogenous HIV-1 Reactivation by Endocervical Epithelial Cells.

Pharmacological HIV-1 reactivation to reverse latent infection has been extensively studied. However, HIV-1 reactivation also occurs naturally, as evidenced by occasional low-level viremia ("viral blips") during antiretroviral treatment (ART). Clarifying where blips originate from and how they happen could provide clues to stimulate latency reversal more effectively and safely or to prevent viral rebound following ART cessation. We studied HIV-1 reactivation in the female genital tract, a dynamic anatomical target for HIV-1 infection throughout all disease stages. We found that primary endocervical epithelial cells from several women reactivated HIV-1 from latently infected T cells. The endocervical cells' HIV-1 reactivation capacity further increased upon Toll-like receptor 3 stimulation with poly(I·C) double-stranded RNA or infection with herpes simplex virus 2 (HSV-2). Notably, acyclovir did not eliminate HSV-2-induced HIV-1 reactivation. While endocervical epithelial cells secreted large amounts of several cytokines and chemokines, especially tumor necrosis factor alpha (TNF-α), CCL3, CCL4, and CCL20, their HIV-1 reactivation capacity was almost completely blocked by TNF-α neutralization alone. Thus, immunosurveillance activities by columnar epithelial cells in the endocervix can cause endogenous HIV-1 reactivation, which may contribute to viral blips during ART or rebound following ART interruption.IMPORTANCE A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped.

Neutralization Takes Precedence Over IgG or IgA Isotype-related Functions in Mucosal HIV-1 Antibody-mediated Protection.

HIV-1 infection occurs primarily through mucosal transmission. Application of biologically relevant mucosal models can advance understanding of the functional properties of antibodies that mediate HIV protection, thereby guiding antibody-based vaccine development. Here, we employed a human ex vivo vaginal HIV-1 infection model and a rhesus macaque in vivo intrarectal SHIV challenge model to probe the protective capacity of monoclonal broadly-neutralizing (bnAb) and non-neutralizing Abs (nnAbs) that were functionally modified by isotype switching. For human vaginal explants, we developed a replication-competent, secreted NanoLuc reporter virus system and showed that CD4 binding site bnAbs b12 IgG1 and CH31 IgG1 and IgA2 isoforms potently blocked HIV-1JR-CSF and HIV-1Bal26 infection. However, IgG1 and IgA nnAbs, either alone or together, did not inhibit infection despite the presence of FcR-expressing effector cells in the tissue. In macaques, the CH31 IgG1 and IgA2 isoforms infused before high-dose SHIV challenge were completely to partially protective, respectively, while nnAbs (CH54 IgG1 and CH38 mIgA2) were non-protective. Importantly, in both mucosal models IgG1 isotype bnAbs were more protective than the IgA2 isotypes, attributable in part to greater neutralization activity of the IgG1 variants. These findings underscore the importance of potent bnAb induction as a primary goal of HIV-1 vaccine development.

Vpu-deficient HIV strains stimulate innate immune signaling responses in target cells.

Acute virus infection induces a cell-intrinsic innate immune response comprising our first line of immunity to limit virus replication and spread, but viruses have developed strategies to overcome these defenses. HIV-1 is a major public health problem; however, the virus-host interactions that regulate innate immune defenses against HIV-1 are not fully defined. We have recently identified the viral protein Vpu to be a key determinant responsible for HIV-1 targeting and degradation of interferon regulatory factor 3 (IRF3), a central transcription factor driving host cell innate immunity. IRF3 plays a major role in pathogen recognition receptor (PRR) signaling of innate immunity to drive the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs), including a variety of HIV restriction factors, that serve to limit viral replication directly and/or program adaptive immunity. Here we interrogate the cellular responses to target cell infection with Vpu-deficient HIV-1 strains. Remarkably, in the absence of Vpu, HIV-1 triggers a potent intracellular innate immune response that suppresses infection. Thus, HIV-1 can be recognized by PRRs within the host cell to trigger an innate immune response, and this response is unmasked only in the absence of Vpu. Vpu modulation of IRF3 therefore prevents virus induction of specific innate defense programs that could otherwise limit infection. These observations show that HIV-1 can indeed be recognized as a pathogen in infected cells and provide a novel and effective platform for defining the native innate immune programs of target cells of HIV-1 infection.

Extracorporeal shock wave therapy for the treatment of plantar fasciitis.

One hundred fifty patients were enrolled in a multicenter, randomized, placebo-controlled, prospective, double-blind study to assess the clinical safety and effectiveness of extracorporeal shock wave therapy (ESWT) using the Dornier Epos Ultra for the treatment of plantar fasciitis. The Active Group was treated with electromagnetically generated shocks using ultrasound guidance during a single therapy session. The Control Group received a sham treatment under similar clinical conditions. The groups were demographically similar with respect to age, height, and weight. The average duration of symptoms was nearly 2 years in both groups. All patients were evaluated by the visual analog scale for pain, American Orthopaedic Foot and Ankle Society scores, Roles and Maudsley Score, SF-12 health status questionnaire, and physical examination. The Active Group reported 56% success at 3 months and 94% success at 12 months posttreatment. The Control Group reported 47% success at 3 months posttreatment. Twelve-month data were not collected for the Control Group as they were unblinded at 3 months and offered treatment. ESWT represents a safe treatment option for chronic proximal plantar fasciitis.

Achilles tendon rupture: an alternative treatment.

We have found percutaneous repair of the Achilles tendon to be a straightforward operation that can be performed under local anesthesia on an outpatient basis. Our experience leaves us with no doubt that the results in terms of wound complications and cosmesis are superior to those obtained with open operative treatment. Repeat rupture rates are comparable to the rates seen with open procedures, and patient satisfaction is excellent. Although this technique does not replace the more traditional open operative repair, it certainly has become an acceptable treatment option, and it is gaining more popularity among orthopedic surgeons. We recommend this technique without reservation.

Evaluation of the validity of the AOFAS Clinical Rating Systems by correlation to the SF-36.

This study evaluates the validity of the AOFAS Clinical Rating Systems by examining their level of correlation to the Medical Outcomes Study Short Form-36 (SF-36) in patients with foot and ankle complaints. The SF-36 is an extensively validated outcomes tool that has been used as a benchmark in examining the validity of outcomes instruments designed for the upper extremity, knee, shoulder, and general orthopaedic conditions. The study sample was 91 patients seen at the foot and ankle clinic of a university-based orthopaedic practice. Patients were administered both the AOFAS Clinical Rating Systems and SF-36 instruments. Pearson correlation coefficients of the AOFAS scores to the SF-36 sub-scales ranged from 0.02 to 0.36 in the overall study population. Correlation was higher for the sub-set of patients with ankle-hindfoot disorders (0.11 to 0.53) than patients with forefoot disorders (-0.05 to 0.25). The low levels of correlation seen in this study suggest poor construct validity of the AOFAS Clinical Rating Systems.