Biology subjects

Alvarez, X.

Publications and source records attributed to Alvarez, X..

11 recordsLinked to original sources

bioRxiv2026

Bronchus associated lymphoid tissue induced by an attenuated Mycobacterium tuberculosis vaccine prevents tuberculosis from heterologous TB challenge

Tuberculosis (TB) remains a leading cause of death from infectious disease worldwide, underscoring the urgent need for vaccines with greater and more consistent efficacy than Bacille Calmette-Guerin (BCG). We previously showed that mucosal vaccination with an isogenic Mycobacterium tuberculosis (Mtb) mutant lacking the stress-response transcription factor SigH ({Delta}sigH) prevents pulmonary TB in two macaque species. In the absence of SigH, Mtb is unable to effectively counter host oxidative stress. Vaccinated macaques were notably protected from disease, exhibiting an absence of granulomatous pathology together with the formation of lymphoid follicles and robust antigen-specific CD4+ and CD8+ T cell responses, identifying{Delta} sigH as a promising live-attenuated TB vaccine candidate. The{Delta} sigH mutant used in prior studies was generated in the Mtb CDC1551 background, a commonly used laboratory strain for challenge studies. Here, we evaluated whether{Delta} sigH-mediated protection extends to heterologous challenge with the more virulent Mtb Erdman strain. Mucosal{Delta} sigH vaccination conferred significant protection against heterologous challenge, markedly reducing pulmonary bacterial burden and TB-associated pathology. Longitudinal high-resolution PET/CT imaging demonstrated that aerosol{Delta} sigH vaccination induced robust inducible bronchus-associated lymphoid tissue (iBALT) responses in the lung. Unlike granulomas, these iBALT structures resolved over time while remaining associated with protection against subsequent Mtb challenge. Protection of highly susceptible rhesus macaques against virulent heterologous Mtb challenge following aerosol{Delta} sigH vaccination supports the further preclinical development of{Delta} sigH-based live-attenuated TB vaccines and highlights iBALT induction as a potential correlate and mechanistic driver of protective immunity against TB.

immunology↗

bioRxiv2025

Increased vaccine efficacy against tuberculosis with a recombinant BCG overexpressing the STING agonist cyclic di AMP

Tuberculosis (TB) remains the leading cause of death due to infection globally. Bacillus Calmette Guerin (BCG), a live attenuated bacterial strain, is the only available TB vaccine, but it has poor efficacy in preventing pulmonary TB in adults. There are advantages associated with the BCG platform however, including a remarkable safely profile, billions of administered doses and a public health ecosystem associated with its production, administration and care. A recombinant/modified BCG (rBCG/modBCG) anti-TB vaccine candidate would be able to leverage these advantages while improving on efficacy. BCG-STING is a recombinant BCG strain which overexpresses the mycobacterial diadenylate cyclase disA gene leading to the release of [~]15-fold greater levels of the endogenous small molecule STING agonist cyclic di-AMP. We evaluated vaccination with BCG-STING compared to its parental BCG-WT strain in rhesus macaques challenged with virulent Mycobacterium tuberculosis (Mtb). BCG-STING given intradermally was well-tolerated, and during life serial BAL samples showed that BCG-STING vaccinated animals had lower Mtb CFU counts than those receiving BCG-WT. At necropsy, BCG-STING vaccinated animals had significantly lower Mtb lung CFU counts, and a higher percentage of sterile lobes and granuloma lesions than the BCG-WT recipients. The enhanced protection observed in BCG-STING vaccinated animals was associated with significant elevations of antigen-specific CD4+ and CD8+ T cells. Modifying BCG to overexpress a small molecule recognized by the innate immune system significantly improves protection and cell-mediated immune responses against TB the non-human primate (NHP) model.

microbiology↗

bioRxiv2025

Using Imaris to rigorously track PET-defined sites of lung inflammation in Mycobacterium tuberculosis-exposed non-human primates

Aerosol exposure of non-human primates (NHPs) to Mycobacterium tuberculosis (Mtb) typically results in discrete sites of inflammation of the lung that is detectable by 2-deoxy-2-[fluorine-18]fluoro-D-glucose (18F-FDG)-based PET/CT scans. Such scans are often analyzed using software such as Invicro VivoQuant or OsiriX as 3D images by manual labeling sites of PET signal using 2D slices and by reporting maximal standardized uptake value (SUVmax) either of the whole lung or of individual lesions. Here we propose a pipeline for analysis of the same PET/CT scans using Imaris, a proprietary software typically used for analysis of fluorescent microscopy data. We show that by using locations of spine vertebra (denoted as "landmarks") we can align serials scans of the same animal, and by using automated (with some manual corrections) image segmentation of PET scans in 3D as "surfaces", we can accurately define location of all sites of inflammation in the lung and lung-associated thoracic lymph nodes (LNs). We show that there is an excellent correlation between individual lesions SUVmax determined by VivoQuant and maximum intensity determined by Imaris suggesting utility of this approach. Imaris also provides wealth of additional information for each of the identified lesions such as volume, location, shape, surface area, and others, and each lesion can be exported in Virtual Reality file format (.wrl) allowing for detailed and rigorous analyses of how features of these PET-defined lesions evolve over time and correlate with the outcome of infection and/or treatment.

immunology↗

bioRxiv2025

Resolving Inflammation: The Impact of Antiretroviral Therapy on Macrophage Traffic In and Out of the CNS.

The effect of antiretroviral therapy (ART) and therapy interruption on myeloid cell traffic out of the central nervous system (CNS) with human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection are understudied. Using intracisternal (i.c.) injection of dual-colored fluorescent superparamagnetic iron oxide nanoparticles (SPION) in SIV-infected macaques early (12-14 dpi) and late (30 days before sacrifice) we studied CNS macrophage viral infection, turnover, and traffic out. SPION are preferentially taken up by perivascular, meningeal, and choroid plexus macrophages. In non-infected macaques, SPION+ macrophages can traffic out of the CNS to the periphery (deep cervical lymph node (dcLN), spleen, and dorsal root ganglia (DRG)), but accumulate in the CNS with SIV infection. ART reduces the accumulation of CNS SPION+ perivascular macrophages but not meningeal or choroid plexus macrophages. ART interruption does not affect the number of SPION+ perivascular and choroid plexus but the number of SPION+ meningeal macrophages increase. ART eliminates SIV-RNA perivascular macrophages, but few scattered RNA+ macrophages in the meninges and choroid plexus remain. With ART interruption, perivascular macrophages remain SIV-but scattered SIV+ meningeal and choroid plexus macrophages exist. In non-infected animals SPION+ macrophages traffic to dcLN, spleen, and DRG at a rate that is decreased with SIV infection and AIDS. SIV- RNA+ SPION+ macrophages that traffic out of the CNS are eliminated by ART and do not rebound with ART interruption. Using two different colored SPION to study the establishment of CNS SIV viral reservoirs, we find greater numbers of early SPION+ macrophages within and outside of the CNS with SIVE, ART, and ART interruption. These data are consistent with SIV-infected perivascular macrophages establishing an early viral reservoir, and continual viral seeding of the meninges and choroid plexus during infection. These findings are consistent with ART reducing traffic of infected macrophages out of the CNS, clearing the CNS perivascular macrophage viral reservoir but not SIV-RNA+ macrophages in meninges and choroid plexus that can rebound with ART interruption.

immunology↗

bioRxiv2023

AlphaFold2 structures template ligand discovery

AlphaFold2 (AF2) and RosettaFold have greatly expanded the number of structures available for structure-based ligand discovery, even though retrospective studies have cast doubt on their direct usefulness for that goal. Here, we tested unrefined AF2 models prospectively, comparing experimental hit-rates and affinities from large library docking against AF2 models vs the same screens targeting experimental structures of the same receptors. In retrospective docking screens against the {sigma}2 and the 5-HT2A receptors, the AF2 structures struggled to recapitulate ligands that we had previously found docking against the receptors experimental structures, consistent with published results. Prospective large library docking against the AF2 models, however, yielded similar hit rates for both receptors versus docking against experimentally-derived structures; hundreds of molecules were prioritized and tested against each model and each structure of each receptor. The success of the AF2 models was achieved despite differences in orthosteric pocket residue conformations for both targets versus the experimental structures. Intriguingly, against the 5-HT2A receptor the most potent, subtype-selective agonists were discovered via docking against the AF2 model, not the experimental structure. To understand this from a molecular perspective, a cryoEM structure was determined for one of the more potent and selective ligands to emerge from docking against the AF2 model of the 5-HT2A receptor. Our findings suggest that AF2 models may sample conformations that are relevant for ligand discovery, much extending the domain of applicability of structure-based ligand discovery.

pharmacology and toxicology↗

bioRxiv2023

Lymphoid tissues contribute to viral clonotypes present in plasma at early post-ATI in SIV-infected rhesus macaques

The rebound-competent viral reservoir (RCVR), comprised of virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after antiretroviral therapy interruption (ATI), remains the biggest obstacle to the eradication of HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop targeted therapeutic strategies for reducing the RCVR. In this study, barcoded SIVmac239M was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood, lymphoid tissues (LTs, spleen, mesenteric and inguinal lymph nodes), and non-lymphoid tissues (NLTs, colon, ileum, lung, liver, and brain) were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX/RNAscope/in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy although plasma viral RNA remained < 22 copies/mL. Among the tissues studied, mesenteric and inguinal lymph nodes, and spleen contained viral barcodes detected in plasma, and trended to have higher cell-associated viral loads, higher intact provirus levels, and greater diversity of viral barcodes. CD4+ T cells were the main cell type harboring viral RNA (vRNA) after ATI. Further, T cell zones in LTs showed higher vRNA levels than B cell zones for most animals. These findings are consistent with LTs contributing to virus present in plasma early after ATI. One Sentence SummaryThe reemerging of SIV clonotypes at early post-ATI are likely from the secondary lymphoid tissues.

immunology↗

bioRxiv2021

A tyrosine-based trafficking signal in the simian immunodeficiency virus envelope cytoplasmic domain is strongly selected for in pathogenic SIV infection

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-dependent trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting of Env. Despite extensive characterization, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which the Tyr-based signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239{Delta}GY) replicates to high levels acutely in pigtail macaques (PTM) but is rapidly controlled. We previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239{Delta}GY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the {Delta}GY deletion and a nine nucleotide deletion that encodes amino acids 734-736 ({Delta}QTH) and overlaps with the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected, which generated a new signal for polarized sorting but not endocytosis. This mutation by itself was sufficient to maintain high viral loads for several months when introduced into naive PTMs. These findings reveal, for the first time, strong selection pressure for Env endocytosis and, in particular, for polarized sorting during pathogenic SIV infection in vivo.

cell biology↗

bioRxiv2020

Lethality of SARS-CoV-2 infection in K18 human angiotensin converting enzyme 2 transgenic mice

ABSTRACTVaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease currently lacks a validated small animal model. Here, we show that transgenic mice expressing human angiotensin converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2-transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2-transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 4. K18 hACE2-transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

microbiology↗

bioRxiv2020

SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species

There are no known cures or vaccines for COVID-19, the defining pandemic of this era. Animal models are essential to fast track new interventions and nonhuman primate (NHP) models of other infectious diseases have proven extremely valuable. Here we compare SARS-CoV-2 infection in three species of experimentally infected NHPs (rhesus macaques, baboons, and marmosets). During the first 3 days, macaques developed clinical signatures of viral infection and systemic inflammation, coupled with early evidence of viral replication and mild-to-moderate interstitial and alveolar pneumonitis, as well as extra-pulmonary pathologies. Cone-beam CT scans showed evidence of moderate pneumonia, which progressed over 3 days. Longitudinal studies showed that while both young and old macaques developed early signs of COVID-19, both groups recovered within a two-week period. Recovery was characterized by low-levels of viral persistence in the lung, suggesting mechanisms by which individuals with compromised immune systems may be susceptible to prolonged and progressive COVID-19. The lung compartment contained a complex early inflammatory milieu with an influx of innate and adaptive immune cells, particularly interstitial macrophages, neutrophils and plasmacytoid dendritic cells, and a prominent Type I-interferon response. While macaques developed moderate disease, baboons exhibited prolonged shedding of virus and extensive pathology following infection; and marmosets demonstrated a milder form of infection. These results showcase in critical detail, the robust early cellular immune responses to SARS-CoV-2 infection, which are not sterilizing and likely impact development of antibody responses. Thus, various NHP genera recapitulate heterogeneous progression of COVID-19. Rhesus macaques and baboons develop different, quantifiable disease attributes making them immediately available essential models to test new vaccines and therapies.

immunology↗

bioRxiv2020

Control of Mycobacterium tuberculosis Infection in Lungs is Associated with Recruitment of Antigen-Specific Th1 and Th17 cells Co-expressing CXCR3 and CCR6.

Mycobacterium tuberculosis (Mtb)-specific T cell responses associated with immune control during asymptomatic latent tuberculosis infection (LTBI) remain poorly understood. Using a non-human primate (NHP) aerosol model, we studied the kinetics, phenotypes and functions of Mtb antigen-specific T cells in peripheral and lung compartments of Mtb-infected asymptomatic rhesus macaques by longitudinally sampling blood and bronchoalveolar lavage (BAL), for up to 24 weeks post-infection. We found significantly higher frequencies of Mtb-specific effector and memory CD4 and CD8 T cells producing IFN-{gamma} in the airways compared to peripheral blood, which were maintained throughout the study period. Moreover, Mtb-specific IL-17+ and IL-17/IFN-{gamma} double-positive T cells were present in the airways but were largely absent in the periphery, suggesting that balanced mucosal Th1/Th17 responses are associated with LTBI. The majority of Mtb-specific CD4 T cells that homed to the airways expressed the chemokine receptor CXCR3 and co-expressed CCR6. Notably, CXCR3+CD4+ cells were found in granulomatous and non-granulomatous regions of the lung and inversely correlated with Mtb burden. Our findings provide novel insights into antigen-specific T cell responses associated with asymptomatic Mtb infection that are relevant for developing better strategies to control TB.

immunology↗

bioRxiv2017

Preexisting antibodies can protect against congenital cytomegalovirus infection in monkeys

Human cytomegalovirus (HCMV) is the most common congenital infection and a known cause of microcephaly, sensorineural hearing loss, and cognitive impairment among newborns worldwide. Natural maternal HCMV immunity reduces the incidence of congenital infection, but does not prevent the disease altogether. We employed a nonhuman primate model of congenital CMV infection to investigate the ability of preexisting antibodies to protect against placental CMV transmission. Pregnant, CD4+ T cell-depleted, rhesus CMV (RhCMV)-seronegative rhesus monkeys were treated with either standardly-produced hyperimmune globulin (HIG) from RhCMV-seropositive macaques or dose-optimized, potently RhCMV-neutralizing HIG prior to intravenous challenge with an RhCMV swarm. HIG passive infusion provided complete protection against fetal loss in both groups, and the potently-neutralizing HIG additionally inhibited placental transmission of RhCMV. Our findings suggest that antibody alone at the time of primary infection can prevent congenital CMV and therefore could be a primary target of vaccines to eliminate this neonatal infection.

immunology↗