Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo.

Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2-9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow-liver-thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal-in combination with appropriate tools for systemic clearance of persistent HIV infection-greatly increases opportunities for HIV eradication.

Essential Role of Human T Cell Leukemia Virus Type 1 orf-I in Lethal Proliferation of CD4+ Cells in Humanized Mice.

Human T cell leukemia virus type 1 (HTLV-1) is the ethological agent of adult T cell leukemia/lymphoma (ATLL) and a number of lymphocyte-mediated inflammatory conditions, including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 orf-I encodes two proteins, p8 and p12, whose functions in humans are to counteract innate and adaptive responses and to support viral transmission. However, the in vivo requirements for orf-I expression vary in different animal models. In macaques, the ablation of orf-I expression by mutation of its ATG initiation codon abolishes the infectivity of the molecular clone HTLV-1p12KO In rabbits, HTLV-1p12KO is infective and persists efficiently. We used humanized mouse models to assess the infectivity of both wild-type HTLV-1 (HTLV-1WT) and HTLV-1p12KO We found that NOD/SCID/γC-/- c-kit+ mice engrafted with human tissues 1 day after birth (designated NSG-1d mice) were highly susceptible to infection by HTLV-1WT, with a syndrome characterized by the rapid polyclonal proliferation and infiltration of CD4+ CD25+ T cells into vital organs, weight loss, and death. HTLV-1 clonality studies revealed the presence of multiple clones of low abundance, confirming the polyclonal expansion of HTLV-1-infected cells in vivo HTLV-1p12KO infection in a bone marrow-liver-thymus (BLT) mouse model prone to graft-versus-host disease occurred only following reversion of the orf-I initiation codon mutation within weeks after exposure and was associated with high levels of HTLV-1 DNA in blood and the expansion of CD4+ CD25+ T cells. Thus, the incomplete reconstitution of the human immune system in BLT mice may provide a window of opportunity for HTLV-1 replication and the selection of viral variants with greater fitness.IMPORTANCE Humanized mice constitute a useful model for studying the HTLV-1-associated polyclonal proliferation of CD4+ T cells and viral integration sites in the human genome. The rapid death of infected animals, however, appears to preclude the clonal selection typically observed in human ATLL, which normally develops in 2 to 5% of individuals infected with HTLV-1. Nevertheless, the expansion of multiple clones of low abundance in these humanized mice mirrors the early phase of HTLV-1 infection in humans, providing a useful model to investigate approaches to inhibit virus-induced CD4+ T cell proliferation.

In Vivo Models of Human Immunodeficiency Virus Persistence and Cure Strategies.

Current HIV therapy is not curative regardless of how soon after infection it is initiated or how long it is administered, and therapy interruption almost invariably results in robust viral rebound. Human immunodeficiency virus persistence is therefore the major obstacle to a cure for AIDS. The testing and implementation of novel yet unproven approaches to HIV eradication that could compromise the health status of HIV-infected individuals might not be ethically warranted. Therefore, adequate in vitro and in vivo evidence of efficacy is needed to facilitate the clinical implementation of promising strategies for an HIV cure. Animal models of HIV infection have a strong and well-documented history of bridging the gap between laboratory discoveries and eventual clinical implementation. More recently, animal models have been developed and implemented for the in vivo evaluation of novel HIV cure strategies. In this article, we review the recent progress in this rapidly moving area of research, focusing on the two most promising model systems: humanized mice and nonhuman primates.

HIV pre-exposure prophylaxis for women and infants prevents vaginal and oral HIV transmission in a preclinical model of HIV infection.

BACKGROUND: Approximately 1.5 million HIV-positive women become pregnant annually. Without treatment, up to 45% will transmit HIV to their infants, primarily through breastfeeding. These numbers highlight that HIV acquisition is a major health concern for women and children globally. They also emphasize the urgent need for novel approaches to prevent HIV acquisition that are safe, effective and convenient to use by women and children in places where they are most needed. METHODS: 4'-Ethynyl-2-fluoro-2'-deoxyadenosine, a potent NRTI with low cytotoxicity, was administered orally to NOD/SCID/γc-/- mice and to bone marrow/liver/thymus (BLT) humanized mice, a preclinical model of HIV infection. HIV inhibitory activity in serum, cervicovaginal secretions and saliva was evaluated 4 h after administration. 4'-Ethynyl-2-fluoro-2'-deoxyadenosine's ability to prevent vaginal and oral HIV transmission was evaluated using highly relevant transmitted/founder viruses in BLT mice. RESULTS: Strong HIV inhibitory activity in serum, cervicovaginal secretions and saliva obtained from animals after a single oral dose of 4'-ethynyl-2-fluoro-2'-deoxyadenosine (10 mg/kg) demonstrated efficient drug penetration into relevant mucosal sites. A single daily oral dose of 4'-ethynyl-2-fluoro-2'-deoxyadenosine resulted in efficient prevention of vaginal and oral HIV transmission after multiple high-dose exposures to transmitted/founder viruses in BLT humanized mice. CONCLUSIONS: Our data demonstrated that 4'-ethynyl-2-fluoro-2'-deoxyadenosine efficiently prevents both vaginal and oral HIV transmission. Together with 4'-ethynyl-2-fluoro-2'-deoxyadenosine's relatively low toxicity and high potency against drug-resistant HIV strains, these data support further clinical development of 4'-ethynyl-2-fluoro-2'-deoxyadenosine as a potential pre-exposure prophylaxis agent to prevent HIV transmission in women and their infants.

HIV-1 infection of hematopoietic progenitor cells in vivo in humanized mice.

HIV infection has been associated with defective hematopoiesis since the earliest days of the HIV/AIDS epidemic. Generation of all hematopoietic lineages suffers in the face of infection. The mechanisms by which HIV impairs normal blood cell development remain unclear, and direct infection of intermediate hematopoietic progenitors has not been established as a source of HIV-associated hematopoietic pathology. Here, we demonstrate infection of multiple subsets of highly purified intermediate hematopoietic progenitors by wild-type HIV both in vitro and in vivo. Although direct infection is clearly cytotoxic, we find that some infected progenitors can survive and harbor proviral DNA. We report intermediate hematopoietic progenitors to be a novel target of infection and their permissivity to infection increases with development. Further, the nonobese diabetic severe combined immunodeficiency common γ chain knockout-bone marrow-liver-thymus humanized mouse provides a unique model for studying the impact of HIV infection on bone marrow-based human hematopoiesis.

Antitumor activity from antigen-specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells.

The goal of cancer immunotherapy is the generation of an effective, stable, and self-renewing antitumor T-cell population. One such approach involves the use of high-affinity cancer-specific T-cell receptors in gene-therapy protocols. Here, we present the generation of functional tumor-specific human T cells in vivo from genetically modified human hematopoietic stem cells (hHSC) using a human/mouse chimera model. Transduced hHSC expressing an HLA-A*0201-restricted melanoma-specific T-cell receptor were introduced into humanized mice, resulting in the generation of a sizeable melanoma-specific naïve CD8(+) T-cell population. Following tumor challenge, these transgenic CD8(+) T cells, in the absence of additional manipulation, limited and cleared human melanoma tumors in vivo. Furthermore, the genetically enhanced T cells underwent proper thymic selection, because we did not observe any responses against non-HLA-matched tumors, and no killing of any kind occurred in the absence of a human thymus. Finally, the transduced hHSC established long-term bone marrow engraftment. These studies present a potential therapeutic approach and an important tool to understand better and to optimize the human immune response to melanoma and, potentially, to other types of cancer.

First-in-human immunoPET imaging of HIV-1 infection using 89Zr-labeled VRC01 broadly neutralizing antibody.

A major obstacle to achieving long-term antiretroviral (ART) free remission or functional cure of HIV infection is the presence of persistently infected cells that establish a long-lived viral reservoir. HIV largely resides in anatomical regions that are inaccessible to routine sampling, however, and non-invasive methods to understand the longitudinal tissue-wide burden of HIV persistence are urgently needed. Positron emission tomography (PET) imaging is a promising strategy to identify and characterize the tissue-wide burden of HIV. Here, we assess the efficacy of using immunoPET imaging to characterize HIV reservoirs and identify anatomical foci of persistent viral transcriptional activity using a radiolabeled HIV Env-specific broadly neutralizing antibody, 89Zr-VRC01, in HIV-infected individuals with detectable viremia and on suppressive ART compared to uninfected controls (NCT03729752). We also assess the relationship between PET tracer uptake in tissues and timing of ART initiation and direct HIV protein expression in CD4 T cells obtained from lymph node biopsies. We observe significant increases in 89Zr-VRC01 uptake in various tissues (including lymph nodes and gut) in HIV-infected individuals with detectable viremia (N = 5) and on suppressive ART (N = 5) compared to uninfected controls (N = 5). Importantly, PET tracer uptake in inguinal lymph nodes in viremic and ART-suppressed participants significantly and positively correlates with HIV protein expression measured directly in tissue. Our strategy may allow non-invasive longitudinal characterization of residual HIV infection and lays the framework for the development of immunoPET imaging in a variety of other infectious diseases.

Characterization and Biomarker Analyses of Post-COVID-19 Complications and Neurological Manifestations.

As the SARS-CoV-2 pandemic continues, reports have demonstrated neurologic sequelae following COVID-19 recovery. Mechanisms to explain long-term neurological sequelae are unknown and need to be identified. Plasma from 24 individuals recovering from COVID-19 at 1 to 3 months after initial infection were collected for cytokine and antibody levels and neuronal-enriched extracellular vesicle (nEV) protein cargo analyses. Plasma cytokine IL-4 was increased in all COVID-19 participants. Volunteers with self-reported neurological problems (nCoV, n = 8) had a positive correlation of IL6 with age or severity of the sequalae, at least one co-morbidity and increased SARS-CoV-2 antibody compared to those COVID-19 individuals without neurological issues (CoV, n = 16). Protein markers of neuronal dysfunction including amyloid beta, neurofilament light, neurogranin, total tau, and p-T181-tau were all significantly increased in the nEVs of all participants recovering from COVID-19 compared to historic controls. This study suggests ongoing peripheral and neuroinflammation after COVID-19 infection that may influence neurological sequelae by altering nEV proteins. Individuals recovering from COVID-19 may have occult neural damage while those with demonstrative neurological symptoms additionally had more severe infection. Longitudinal studies to monitor plasma biomarkers and nEV cargo are warranted to assess persistent neurodegeneration and systemic effects.

Long-term SARS-CoV-2-specific immune and inflammatory responses in individuals recovering from COVID-19 with and without post-acute symptoms.

We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses, soluble markers of inflammation, and antibody levels and neutralization capacity longitudinally in 70 individuals with PCR-confirmed SARS-CoV-2 infection. Participants represent a spectrum of illness and recovery, including some with persistent viral shedding in saliva and many experiencing post-acute sequelae of SARS-CoV-2 infection (PASC). T cell responses remain stable for up to 9 months. Whereas the magnitude of early CD4+ T cell immune responses correlates with severity of initial infection, pre-existing lung disease is independently associated with higher long-term SARS-CoV-2-specific CD8+ T cell responses. Among participants with PASC 4 months following coronavirus disease 2019 (COVID-19) symptom onset, we observe a lower frequency of CD8+ T cells expressing CD107a, a marker of degranulation, in response to Nucleocapsid (N) peptide pool stimulation, and a more rapid decline in the frequency of N-specific interferon-γ-producing CD8+ T cells. Neutralizing antibody levels strongly correlate with SARS-CoV-2-specific CD4+ T cell responses.

Long-Term SARS-CoV-2-Specific Immune and Inflammatory Responses Across a Clinically Diverse Cohort of Individuals Recovering from COVID-19.

A detailed understanding of long-term SARS-CoV-2-specific T cell responses and their relationship to humoral immunity and markers of inflammation in diverse groups of individuals representing the spectrum of COVID-19 illness and recovery is urgently needed. Data are also lacking as to whether and how adaptive immune and inflammatory responses differ in individuals that experience persistent symptomatic sequelae months following acute infection compared to those with complete, rapid recovery. We measured SARS-CoV-2-specific T cell responses, soluble markers of inflammation, and antibody levels and neutralization capacity longitudinally up to 9 months following infection in a diverse group of 70 individuals with PCR-confirmed SARS-CoV-2 infection. The participants had varying degrees of initial disease severity and were enrolled in the northern California Long-term Impact of Infection with Novel Coronavirus (LIINC) cohort. Adaptive T cell responses remained remarkably stable in all participants across disease severity during the entire study interval. Whereas the magnitude of the early CD4+ T cell immune response is determined by the severity of initial infection (participants requiring hospitalization or intensive care), pre-existing lung disease was significantly associated with higher long-term SARS-CoV2-specific CD8+ T cell responses, independent of initial disease severity or age. Neutralizing antibody levels were strongly correlated with SARS-CoV-2-specific CD4+ T but not CD8+ T cell responses. Importantly, we did not identify substantial differences in long-term virus-specific T cell or antibody responses between participants with and without COVID-19-related symptoms that persist months after initial infection.

SARS-CoV-2 antibody magnitude and detectability are driven by disease severity, timing, and assay.

Interpretation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurveillance studies is limited by poorly defined performance of antibody assays over time in individuals with different clinical presentations. We measured antibody responses in plasma samples from 128 individuals over 160 days using 14 assays. We found a consistent and strong effect of disease severity on antibody magnitude, driven by fever, cough, hospitalization, and oxygen requirement. Responses to spike protein versus nucleocapsid had consistently higher correlation with neutralization. Assays varied substantially in sensitivity during early convalescence and time to seroreversion. Variability was dramatic for individuals with mild infection, who had consistently lower antibody titers, with sensitivities at 6 months ranging from 33 to 98% for commercial assays. Thus, the ability to detect previous infection by SARS-CoV-2 is highly dependent on infection severity, timing, and the assay used. These findings have important implications for the design and interpretation of SARS-CoV-2 serosurveillance studies.

SARS-CoV-2 antibody magnitude and detectability are driven by disease severity, timing, and assay.

Serosurveillance studies are critical for estimating SARS-CoV-2 transmission and immunity, but interpretation of results is currently limited by poorly defined variability in the performance of antibody assays to detect seroreactivity over time in individuals with different clinical presentations. We measured longitudinal antibody responses to SARS-CoV-2 in plasma samples from a diverse cohort of 128 individuals over 160 days using 14 binding and neutralization assays. For all assays, we found a consistent and strong effect of disease severity on antibody magnitude, with fever, cough, hospitalization, and oxygen requirement explaining much of this variation. We found that binding assays measuring responses to spike protein had consistently higher correlation with neutralization than those measuring responses to nucleocapsid, regardless of assay format and sample timing. However, assays varied substantially with respect to sensitivity during early convalescence and in time to seroreversion. Variations in sensitivity and durability were particularly dramatic for individuals with mild infection, who had consistently lower antibody titers and represent the majority of the infected population, with sensitivities often differing substantially from reported test characteristics (e.g., amongst commercial assays, sensitivity at 6 months ranged from 33% for ARCHITECT IgG to 98% for VITROS Total Ig). Thus, the ability to detect previous infection by SARS-CoV-2 is highly dependent on the severity of the initial infection, timing relative to infection, and the assay used. These findings have important implications for the design and interpretation of SARS-CoV-2 serosurveillance studies.

Differentially stimulated CD4+ T cells display altered human immunodeficiency virus infection kinetics: implications for the efficacy of antiviral agents.

The activation state of CD4(+) T cells plays a crucial role in the establishment of a productive human immunodeficiency virus infection. Here, we show that T cells stimulated for 1 day demonstrated delayed kinetics of viral reverse transcription and integration compared to cells stimulated for 2 days prior to infection. As a result, the efficiency of reverse transcription and integration inhibitors differs in these differentially stimulated cells. These studies increase our understanding of how T cells support viral replication and provide insight regarding the efficiency of antiretroviral therapy in lymphoid compartments.

T cells establish and maintain CNS viral infection in HIV-infected humanized mice.

The human brain is an important site of HIV replication and persistence during antiretroviral therapy (ART). Direct evaluation of HIV infection in the brains of otherwise healthy individuals is not feasible; therefore, we performed a large-scale study of bone marrow/liver/thymus (BLT) humanized mice as an in vivo model to study HIV infection in the brain. Human immune cells, including CD4+ T cells and macrophages, were present throughout the BLT mouse brain. HIV DNA, HIV RNA, and/or p24+ cells were observed in the brains of HIV-infected animals, regardless of the HIV isolate used. HIV infection resulted in decreased numbers of CD4+ T cells, increased numbers of CD8+ T cells, and a decreased CD4+/CD8+ T cell ratio in the brain. Using humanized T cell-only mice (ToM), we demonstrated that T cells establish and maintain HIV infection of the brain in the complete absence of human myeloid cells. HIV infection of ToM resulted in CD4+ T cell depletion and a reduced CD4+/CD8+ T cell ratio. ART significantly reduced HIV levels in the BLT mouse brain, and the immune cell populations present were indistinguishable from those of uninfected controls, which demonstrated the effectiveness of ART in controlling HIV replication in the CNS and returning cellular homeostasis to a pre-HIV state.

In Vivo Suppression of HIV Rebound by Didehydro-Cortistatin A, a "Block-and-Lock" Strategy for HIV-1 Treatment.

HIV-1 Tat activates viral transcription and limited Tat transactivation correlates with latency establishment. We postulated a "block-and-lock" functional cure approach based on properties of the Tat inhibitor didehydro-Cortistatin A (dCA). HIV-1 transcriptional inhibitors could block ongoing viremia during antiretroviral therapy (ART), locking the HIV promoter in persistent latency. We investigated this hypothesis in human CD4+ T cells isolated from aviremic individuals. Combining dCA with ART accelerates HIV-1 suppression and prevents viral rebound after treatment interruption, even during strong cellular activation. We show that dCA mediates epigenetic silencing by increasing nucleosomal occupancy at Nucleosome-1, restricting RNAPII recruitment to the HIV-1 promoter. The efficacy of dCA was studied in the bone marrow-liver-thymus (BLT) mouse model of HIV latency and persistence. Adding dCA to ART-suppressed mice systemically reduces viral mRNA in tissues. Moreover, dCA significantly delays and reduces viral rebound levels upon treatment interruption. Altogether, this work demonstrates the potential of block-and-lock cure strategies.

Cocaine exposure impairs multilineage hematopoiesis of human hematopoietic progenitor cells mediated by the sigma-1 receptor [corrected].

Prenatal exposure to cocaine is a significant source of fetal and neonatal developmental defects. While cocaine associated neurological and cardiac pathologies are well-documented, it is apparent that cocaine use has far more diverse physiological effects. It is known that in some cell types, the sigma-1 receptor mediates many of cocaine's cellular effects. Here we present a novel and concise investigation into the mechanism that underlies cocaine associated hematopoietic pathology. Indeed, this is the first examination of the effects of cocaine on hematopoiesis. We show that cocaine impairs multilineage hematopoiesis from human progenitors from multiple donors and tissue types. We go on to present the first demonstration of the expression of the sigma-1 receptor in human CD34 + human hematopoietic stem/progenitor cells. Furthermore, we demonstrate that these cocaine-induced hematopoietic defects can be reversed through sigma-1 receptor blockade.

Quiescent T cells and HIV: an unresolved relationship.

The ability of HIV to infect quiescent CD4+ T cells has been a topic of intense debate. While early studies suggested that the virus could not infect this particular T cell subset, subsequent studies using more sensitive protocols demonstrated that these cells could inefficiently support HIV infection. Additional studies showed that the kinetics of infection in quiescent cells was delayed and multiple stages of the viral life cycle were marred by inefficiencies. Despite that, proviral DNA has been found in these cells presenting them as a potential viral reservoir. Therefore, a better understanding of the relationship between HIV and quiescent T cells may lead to further advances in the field of HIV.