HIV Viral Load (HIV-VL) and Early Infant Diagnosis (EID) play a pivotal role in the laboratory surveillance, monitoring of HIV/AIDS, and its elimination as a public concern. However, sample rejection due to sample nonconformity (SNC) resulting from inadequate collection, transportation, and management, especially during the pre-analytical phase, negatively affects laboratory performance. This study aimed to characterize errors observed during the pre-analytical phase of HIV-VL and EID testing across national reference laboratories in Cameroon and to identify factors associated with rejection. A descriptive and quantitative study of the nonconformities (NC) identified was collected from 11/01/2024 to 08/12/2024 in seventeen HIV reference laboratories, which constitute the national network of HIV-VL and EID testing coverage. For this study, the number of rejected samples, the reason for rejection, and the type of test ordered were recorded monthly. During the study period, 326,885 and 38,354 specimens received for HIV viral load and EID. Of those 12,748 (3.9%) and 2.7% (1,039) were rejected. The SNC analysis indicates the presence of multiple errors or NC in some samples. For HIV viral load, our results indicate that specimen identification errors for viral load were the most common NC (63.14%; n = 8049; P = 0.031), followed by insufficient specimen volume (43.7%; n = 5571; P = 0.049) and quality errors, including hemolyzed specimens (27.8%; n = 3543; P = 0.054), and specimen transport packaging errors (9.1%; n = 1160; P = 0.069). For HIV EID, specimen rejections were primarily attributed to missing or mismatched identification on the request forms (37.12%, n = 386; P = 0.042), sample unavailability (13.4%; n = 139; P = 0.056), and information discrepancies (44.2%; n = 459; P = 0.033). The observed significant rejection rates for both HIV viral load and EID exceeded the established national rejection rate of <2% of errors. Our results suggest that corrective action is critical, along with the establishment of policies to detect and resolve preanalytical errors in Cameroon. Our findings highlight the high magnitude of preanalytical errors for HIV-VL and EID tests used in the testing and management of people living with HIV/AIDS in Cameroon. Therefore, the laboratory system should be strengthened to ensure high-quality patient services and support optimization. Suggestions for improvement include distributing a validated specimen-collection manual, creating electronic test-request forms, providing staff training, and regularly on-site supervising the use of available resources, all of which are necessary in this country.
Clonally expanded CD4+ T cells harbouring rebound-competent HIV persist lifelong during antiretroviral therapy1-5. Latency is considered the principal barrier to viral eradication and has resisted pharmacological reversal6,7, yet sustained immune pressure appears to erode reservoirs8-15. Recent advances have yielded glimpses into exceptionally rare reservoir-harbouring cells, implicating prosurvival properties in persistence16-18. Here we isolate and characterize authentic reservoir clones (ARCs) that robustly proliferate and accumulate while producing infectious virus, without overtly succumbing to cytopathicity. At any moment, only small fractions of ARCs expressed HIV proteins, a state associated with conserved host transcriptional programs but remarkably refractory to potent T cell stimulation. Nevertheless, sustained co-culture with a CD8+ cytotoxic T lymphocyte clone substantially culled proliferating ARCs, revealing time-integrated vulnerability to immune pressure. The corresponding ex vivo CD8+ T cell response was poorly cytotoxic, and in vivo erosion of ARCs occurred only slowly. A regulatory T cell ARC displayed pronounced cell-intrinsic resistance to cytotoxic T cells - a longstanding hypothesis now directly demonstrated - linked to low oxidative stress and reversed with deferoxamine19, a hypoxic stress inducer and FDA-approved therapeutic. Overall, we provide insights into the vulnerabilities of reservoir clones to potent, sustained cytotoxic T cell pressure and highlight intrinsic resistance pathways as actionable therapeutic targets, opening opportunities for advancing immune-based HIV cure strategies.
Avian influenza A viruses, especially highly pathogenic avian influenza (HPAI) viruses, pose a significant public health threat, and a multivalent vaccine is the primary prophylactic measure to control these viruses. To establish such a vaccine, we generated two multivalent vesicular stomatitis virus (VSV)-based vaccine candidates that simultaneously target H5N1 hemagglutinin (H505 or H522) and the ectodomain of matrix protein 2 (M2e) and characterized their ability to induce protective immune responses. Our results revealed that vaccine immunization induced high humoral immune responses against both the HPAI hemagglutinin (HA) protein and the M2 protein in mice. Intriguingly, vaccine-immunized mouse sera exhibited highly efficient neutralizing activity against the corresponding H5 pseudovirus and mediated potent and broad antibody-dependent cellular cytotoxicity (ADCC) activity against M2e derived from human and avian influenza H5, H1, H3, and H7 viruses. Furthermore, both intranasal (IN) and intramuscular immunization (IM) of V-EtM2e/H522 provided efficient protection against HPAI H5N1 virus challenge in mice, with a 100% survival rate and a nondetectable viral load in several tissues. Notably, noninvasive mucosal IN delivery achieved protection equal to that of IM delivery at a 100-fold lower immunizing dose. These findings provide strong evidence for the effectiveness of a multivalent VSV-based vaccine against human (avian) influenza A viruses. Avian influenza, especially highly pathogenic avian influenza (HPAI) viruses, can cause a highly contagious airborne disease that poses a significant public health threat. The development of a multivalent vaccine is very important to control these viral infections. In this study, we have developed a multivalent vaccine that simultaneously targets two critical HPAI H5N1 surface viral proteins and fully protects against highly pathogenic H5N1 infection in a mouse model. This study provides convincing evidence for the effectiveness of a multivalent vaccine against highly pathogenic influenza A viruses.
Defective HIV-1 proviruses harboring mutations and/or large internal deletions represent the majority of HIV-1 sequences found in circulating peripheral blood mononuclear cells of people living with HIV with viremia suppressed by combination antiretroviral therapy; indirect evidence suggests that such sequences are transcriptionally active and may contribute to immune activation. In this study, we present a new approach allowing for high-efficiency screening, immortalization, and targeted enrichment of HIV-positive CD4+ T-cells isolated from people living with HIV. Using this method, we were able to isolate and expand patient-derived cells, identify mutations and deletions via sequencing, and confirm that those proviruses were transcriptionally and translationally active in vitro. Moreover, our findings indicate that the majority of proviral sequences circulating in suppressed HIV-infected patients may undergo 3'-LTR deletions, suggesting that sequence diversity reported using LTR-to-LTR amplification and sequencing approaches may indeed be underscored.
The mechanisms by which latent HIV-1 reservoirs persist during antiretroviral therapy is incompletely understood. Here, we derive a model system to measure clonal expansion and viral latency in which populations of human memory CD4+ T cells, each bearing a single transcriptionally active HIV-1 provirus are engrafted into immunodeficient mice. Over ~2 months in vivo, clonal expansion and the establishment of latency occurred in subsets of engrafted infected cells. Clonal expansion in vivo was driven by T-cell receptor identity, but not by proviral insertional mutagenesis. The integration sites of proviruses that became latent in vivo were enriched on chromosome 19, in intergenic and centromeric satellite regions, and genes whose expression is atypically low. Pre-existing repressive epigenetic features were associated with latency for subsets of proviruses. Our findings suggest a confluency of genomic and epigenomic factors predispose certain genomic locations, including ZNF genes, to host proviruses that constitute the latent reservoir.
The ease with which emerging SARS-CoV-2 variants escape neutralizing antibodies limits the protection afforded by a prior exposure, be it infection or vaccination. While rare, broadly neutralizing antibodies with activity toward diverse sarbecoviruses have been detected in convalescent serum. Motivated by findings that plasma responses show increased neutralization breadth and potency with continued antigen exposure, we isolated monoclonal antibodies (mAbs) after a SARS-CoV-2 re-infection and compared them to those isolated 1 year prior, after the first breakthrough infection. Among clonal lineage members identified at both time points, mAbs from the later time point showed improved neutralization potency and breadth. One mAb isolated after re-infection, C68.490, targets a conserved region in the receptor binding domain and shows remarkable activity not only against SARS-CoV-2 variants, but also diverse sarbecoviruses from more distant clades present in animal reservoirs. These findings suggest that a focus on individuals with diverse and repeated antigen exposure could lead to the identification of antibodies with therapeutic utility not just toward current and future SARS-CoV-2 variants, but also distant sarbecoviruses in the event of a future spillover.IMPORTANCESpillover of SARS-related viruses (sarbecoviruses) from animal reservoirs into humans has occurred multiple times in the past few decades. The most recent spillover due to SARS-CoV-2 continues to cause significant disease burden, and treatment options are few, in part because of selection for new variants due to immune escape. Thus, discovering antibodies that can block infection with sarbecoviruses, including SARS-CoV-2 variants, remains critical for both the current pandemic as well as those to come. Our study shows that an individual who was vaccinated and then had repeated breakthrough infections with distinct SARS-CoV-2 variants generated more potent antibodies after the second infection compared to the first infection. Notably, we discovered an antibody in this individual that not only neutralized the dominant SARS-CoV-2 variants but also a range of diverse sarbecoviruses present in animal reservoirs. This antibody thus holds promise as a therapeutic for both the current pandemic and future spillover events.
Adult T-cell leukemia/lymphoma (ATL) is caused by chronic infection with human T-lymphotropic virus type 1 (HTLV-1). HTLV-1 contains highly immunogenic CD8 + T-cell epitopes that elicit high frequencies of virus-specific CD8 + T cells in most virus carriers. Despite the virus being present in the tumor, HTLV-1-specific CD8 + cells are often undetectable in ATL. To characterize HTLV-1-specific CD8 + T cells during ATL development, we studied a subgroup of people living with asymptomatic HTLV-1 infection at very high risk of developing ATL. These so-called "high-risk" carriers have suspected premalignant lesions: expanded, HTLV-1-infected "ATL-like" clones circulating in their peripheral blood. Compared to viral antigen-burden matched controls, high-risk carriers had significantly fewer Tax-specific IFN-γ + CD8 + cells in peripheral blood. Furthermore, ex vivo CD8 + T cells from high-risk carriers did not efficiently kill autologous HTLV-1-infected T cells, including premalignant ATL-like clones. We stained Tax11-19/HLA-A∗0201 pentamer + CD8 + T cells to test whether the low frequencies of functional CD8+ T cells resulted from phenotype or absolute frequency of HTLV-1-specific CD8 + T cells. High-risk carriers had significantly lower frequencies of Tax11-19/HLA-A∗0201 pentamer + CD8 + T cells than controls, but we observed no difference in effector function, memory phenotype, or expression of checkpoint control molecules. In contrast, there was no difference in the frequency of CD8 + T cells specific for other viruses (cytomegalovirus, Epstein-Barr virus, influenza virus) between high-risk carriers and controls. This is the first report of HTLV-1-specific immune dysregulation in the premalignant stage of ATL. Low frequencies of HTLV-1-specific CD8 + T cells may contribute to ATL development and may be a novel therapeutic target for ATL prevention.
The Practice Parameter for Inborn Errors of Immunity was revised in 2025 and published recently. We reviewed the most significant changes of this update regarding the previous parameter update in 2015. In contrast to previous revisions focused on individual genetic entities, this update was written with a new structure of recommendations divided into 2 parts: diagnosis and management. The diagnosis part includes discussions of newborn screening for severe combined immunodeficiency, genetic testing, and surveillance of comorbidities, in addition to reviewing clinically available testing for immunologic diagnosis. The management part includes recommendations for immunoglobulin replacement, antibiotic prophylaxis, hematopoietic stem cell transplantation, precision medicine, and quality-of-life assessment.
Advances in clinical immunology have led to substantial developments in the diagnosis and treatment of patients with inborn errors of immunity. Still, publications between 2023 and 2025 reveal several persistent needs. First, ongoing reports from the International Union of Immunological Societies demonstrate the need for continued gene discoveries. Second, growing data illustrate the important need for phenotype improvement in not only new diseases but also established ones. Third, recognition of the potentially pleiomorphic nature of heterozygous variants exposes the critical need to challenge prior hypotheses about accepted disease models of inborn errors of immunity. Next, continued pursuit of genetic etiologies for inborn errors of immunity in unsolved cases portends the need to embrace new disease mechanisms, such as somatic mosaicism and monoallelic expression. Furthermore, the rising number of variants of uncertain significance underscores the vital need to investigate multiple variant pathogenicity at scale. Also, recent studies reflect avid realization of the constant need for biomarkers and enhanced detection, such as through bioassays, specific clinically available tests, or even artificial intelligence. Last, key reports support the further need to study innovative treatment approaches and disease outcomes. Overall, the field of clinical immunology must continue to push forward to address these needs.
DAVID is a widely used bioinformatics resource that provides functional annotation and functional enrichment analysis for gene and protein lists derived from high-throughput studies. It integrates a comprehensive gene-centered knowledgebase with a suite of web-accessible analytical tools. Since its initial release in 2003, DAVID developments have been published in 12 papers and cited >80 000 times. Here, we report updates made since the previous NAR Web Server Issue publication in 2022. This update introduces two new tools: DAVID Ortholog for cross-species functional analysis and DAVID Gene Search for identifier-agnostic gene exploration, modernizes the web interface, and implements a new backend architecture that decouples the frontend from the legacy Java processing engine. A new Servlet layer and REST APIs enable asynchronous processing and support integration of a Neo4j graph database for relationship-based queries. Major existing tools have been redesigned with modern, interactive interfaces, and multiformat result export. The pathway viewer has been redesigned with interactive drag-and-zoom navigation, animated user gene highlighting, and publication-quality downloads. Collectively, these updates enhance performance and usability, and the new backend architecture enables independent evolution of frontend and backend components while maintaining continuity with legacy analyses. DAVID remains freely available at https://davidbioinformatics.nih.gov without login.
Here we describe a damaging heterozygous variant in CDC45 in an individual with common variable immunodeficiency (CVID) and recurrent viral infections. This individual has variably decreased number of circulating NK cells, disruption in the ratio of CD56bright to CD56dim cells, and consistently decreased NK cell function. Interestingly, the inherited CDC45 variant is also present in a sibling with less severe clinical manifestations; we determined that allelic bias of the damaging allele accounts for this differential expressivity. As previously reported for other helicase variants that cause inborn errors of immunity (IEI), we found cell cycle defects in immune cells from the proband that lead to reduced survival of NK cells. Together, these findings link another member of the core replicative helicase complex to inborn errors of immunity and highlight the sensitivity of NK cells to these variants. They also define another case of allelic bias contributing to variable expressivity of an IEI gene.
Orthohantaviruses (hantaviruses) are emerging rodent-borne pathogens that can cause severe human disease. They are present on multiple continents and are responsible for thousands of human cases per year. Despite this, no licenced therapeutics are available, vaccines for most strains are lacking, and the immunological response to infection is poorly characterised. This study aimed to analyse the humoral immune response to Puumala virus (PUUV) infection to inform future studies focussing on the production of therapeutic monoclonal antibodies and vaccination strategies. Serum was obtained from a cohort of 24 patients hospitalised with PUUV infection at four time points, covering the early acute, late acute, early convalescent, and late convalescent stages of the disease. The humoral immune responses at each time point were quantified, and cross-binding, cross-neutralising antibody responses were investigated. Serum cytokine levels were also interrogated, and expression was correlated with humoral outputs. PUUV infection elicited a robust anti-PUUV neutralising antibody response. However, cross-reactive antibodies that were capable of binding diverse hantaviruses were also induced in late convalescence. Modulations in the abundance of IgG subclasses were evident following infection, with significant differences present months after infection. This study demonstrates that broadly reactive anti-hantavirus antibodies are produced in response to Old-World hantavirus infection, but predominantly months after recovery. As this is concomitant with changes in IgG subtypes, our results suggest that PUUV infection promotes prolonged class-switching and somatic hypermutation, favouring conserved epitopes long after exposure. Work at Mount Sinai was supported by Institutional Funds, work at the Medical University of Vienna was supported by Institutional Funds. The study was supported in part by the Styrian government, Austria (project no. ABT12-106729/2022-13) and the Austrian Science Fund (FWF) (number J 4737-B).
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Structure based design, synthesis and evaluation of a series of potent SARS-CoV-2 main protease inhibitors are described. We designed conformationally constrained tetrahydroisoquinoline-3-carboxamides as P2 ligands. The studies involved structure-activity relationship studies by varying P1, P2, P3, and P4 ligands, including five- and six-membered lactam rings as P1 ligands as well as nitrile and thiazole heterocycles as P1' ligands. These new inhibitors exhibited potent SARS-CoV-2 Mpro inhibitory activity. Several inhibitors also blocked the replication of SARS-CoV-2 in VeroE6 cells with low nanomolar EC50 values, more potent than the approved drug, nirmatrelvir. Inhibitor 5j displayed an Mpro inhibitory K i of 1.2 nM and antiviral EC50 value of 360 nM. We determined several high-resolution X-ray structures of inhibitor-SARS-CoV-2 Mpro complexes which provided important insights into the ligand-binding site interactions in the active site.
The SARS-CoV-2 main protease (Mpro) remains a prime antiviral target because its inhibition halts viral replication. To probe how subtle atomic changes influence drug performance, we carried out a systematic halogen scan on a potent ketoamide scaffold, replacing a single fluorine with chlorine, bromine, or iodine. Enzymatic assays revealed that the F- and Cl-substituted analogues inhibit Mpro at nanomolar levels, whereas Br and I variants are 10- to 20-fold weaker. Cell-based antiviral tests mirrored this trend, yet uptake studies showed the opposite: iodine markedly enhances intracellular accumulation. High-resolution X-ray structures (1.6-1.8 Å) explain the dichotomy: small halogens fit snugly in the S1' σ-hole pocket, maximizing hydrogen-bond geometry, while bulkier atoms distort binding but create a lipophilic patch that boosts permeability. These data yield the first fluorine-to-iodine structure-activity map for SARS-CoV-2 Mpro inhibitors. These findings highlight the critical role of halogen selection in antiviral inhibitor design.
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A central problem in achieving vaccine-based protection against viral infections is eliciting antibodies that are resilient to viral variation. Successive waves of SARS-CoV-2 infection during the COVID19 pandemic were driven by variants that acquired resistance to neutralizing antibodies elicited by prior SARS-CoV-2 variants. To the extent that serum neutralization breadth occurs in individuals with multiple exposures to SARS-CoV-2 antigens, we and others find that it is largely comprised of antibodies that target the variable receptor binding domain (RBD), rather than more conserved spike protein domains. By designing synthetic dimeric RBD immunogens we show that limiting divergence in heterodimeric components favors the generation of cross-reactive B cells and antibodies. We thus devised a vaccine approach based on a two-dose immunization with a pool of five overlapping heterodimeric synthetic RBD variants. Collectively, the RBD heterodimer pool was designed to cover 10% sequence variation and elicited greater antibody cross-reactivity and neutralization breadth than homodimers or heterodimers with highly divergent components. Using an unconventional 'prospective' challenge model in mice, we demonstrate the effectiveness of the RBD heterodimer pool in inducing antibody responses that attenuate infection by future SARS-CoV-2 variants, as well as protection in a challenge model based on a chimeric vesicular stomatitis virus bearing a spike protein from SARS-CoV-1.