HIV-1 Gag co-localizes with euchromatin histone marks at the nuclear periphery.

IMPORTANCE: The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.

Virus update for the M2 "mac-in-touch".

While HIV-1 infection of macrophages plays a major role in viral persistence and pathogenesis, how HIV-1 transfers from infected T cells to macrophages remains elusive. In this issue, Mascarau et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202205103) demonstrate how macrophage polarization drives their ability to fuse with HIV-1 infected T cells via the CD81/RhoA-ROCK/Myosin axis.

FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.

A Morbillivirus Infection Shifts DC Maturation Toward a Tolerogenic Phenotype to Suppress T Cell Activation.

Viruses have evolved numerous strategies to impair immunity so that they can replicate more efficiently. Among those, the immunosuppressive effects of morbillivirus infection can be particularly problematic, as they allow secondary infections to take hold in the host, worsening disease prognosis. In the present work, we hypothesized that the highly contagious morbillivirus peste des petits ruminants virus (PPRV) could target monocytes and dendritic cells (DC) to contribute to the immunosuppressive effects produced by the infection. Monocytes isolated from healthy sheep, a natural host of the disease, were able be infected by PPRV and this impaired the differentiation and phagocytic ability of immature monocyte-derived DC (MoDC). We also assessed PPRV capacity to infect differentiated MoDC. Ovine MoDC could be productively infected by PPRV, and this drastically reduced MoDC capacity to activate allogeneic T cell responses. Transcriptomic analysis of infected MoDC indicated that several tolerogenic DC signature genes were upregulated upon PPRV infection. Furthermore, PPRV-infected MoDC could impair the proliferative response of autologous CD4+ and CD8+ T cell to the mitogen concanavalin A (ConA), which indicated that DC targeting by the virus could promote immunosuppression. These results shed new light on the mechanisms employed by morbillivirus to suppress the host immune responses. IMPORTANCE Morbilliviruses pose a threat to global health given their high infectivity. The morbillivirus peste des petits ruminants virus (PPRV) severely affects small-ruminant-productivity and leads to important economic losses in communities that rely on these animals for subsistence. PPRV produces in the infected host a period of severe immunosuppression that opportunistic pathogens exploit, which worsens the course of the infection. The mechanisms of PPRV immunosuppression are not fully understood. In the present work, we demonstrate that PPRV can infect professional antigen-presenting cells called dendritic cells (DC) and disrupt their capacity to elicit an immune response. PPRV infection promoted a DC activation profile that favored the induction of tolerance instead of the activation of an antiviral immune response. These results shed new light on the mechanisms employed by morbilliviruses to suppress the immune responses.

Avatar Mice Underscore the Role of the T Cell-Dendritic Cell Crosstalk in Ebola Virus Disease and Reveal Mechanisms of Protection in Survivors.

Ebola virus disease (EVD) is a complex infectious disease characterized by high inflammation, multiorgan failure, the dysregulation of innate and adaptive immune responses, and coagulation abnormalities. Evidence accumulated over the last 2 decades indicates that, during fatal EVD, the infection of antigen-presenting cells (APC) and the dysregulation of T cell immunity preclude a successful transition between innate and adaptive immunity, which constitutes a key disease checkpoint. In order to better understand the contribution of the APC-T cell crosstalk to EVD pathophysiology, we have developed avatar mice transplanted with human, donor-specific APCs and T cells. Here, we show that the transplantation of T cells and APCs from Ebola virus (EBOV)-naive individuals into avatar mice results in severe disease and death and that this phenotype is dependent on T cell receptor (TCR)-major histocompatibility complex (MCH) recognition. Conversely, avatar mice were rescued from death induced by EBOV infection after the transplantation of both T cells and plasma from EVD survivors. These results strongly suggest that protection from EBOV reinfection requires both cellular and humoral immune memory responses. IMPORTANCE The crosstalk between dendritic cells and T cells marks the transition between innate and adaptive immune responses, and it constitutes an important checkpoint in EVD. In this study, we present a mouse avatar model in which T cell and dendritic cell interactions from a specific donor can be studied during EVD. Our findings indicate that T cell receptor-major histocompatibility complex-mediated T cell-dendritic cell interactions are associated with disease severity, which mimics the main features of severe EVD in these mice. Resistance to an EBOV challenge in the model was achieved via the transplantation of both survivor T cells and plasma.

Dominant Negative Mutants of Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) Disrupt Core-Binding Factor Beta-Vif Interaction.

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFß) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFß to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFß affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFß target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFß to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFß interaction may be promising in the development of a new AIDS therapeutic strategy.

Endocytosed HIV-1 Envelope Glycoprotein Traffics to Rab14+ Late Endosomes and Lysosomes to Regulate Surface Levels in T-Cell Lines.

Production of infectious HIV-1 particles requires incorporation of the viral envelope glycoprotein (Env) at the plasma membrane (PM) of infected CD4+ T cells. Env trafficking to the PM exposes viral epitopes that can be exploited by the host immune system; however, HIV-1 can evade this response by endocytosis of excess Env from the PM. The fate of Env after internalization remains unclear, with evidence suggesting several different vesicular trafficking steps may be involved, including recycling pathways. To date, there have been very few studies documenting the trafficking pathways of native Env in infected T cells. Furthermore, it remains unclear whether there are T-cell-specific endosomal pathways regulating the fate of endocytic Env. Here, we use a pulse-labeling approach with a monovalent anti-Env Fab probe to characterize the trafficking of internalized Env within infected CD4+ T-cell lines, together with CRISPR/Cas9-mediated endogenous protein tagging, to assess the role of host cell Rab GTPases in Env trafficking. We show that endocytosed Env traffics to Rab14+ compartments that possess hallmarks of late endosomes and lysosomes. We also demonstrate that Env can recycle back to the PM, although we find that recycling does not occur at high rates when compared to the model recycling protein transferrin. These results help to resolve open questions about the fate and relevance of endocytosed Env in HIV-infected cells and suggest a novel role for Rab14 in a cell-type-specific late-endosomal/lysosomal trafficking pathway in T cells. IMPORTANCE HIV-1 envelope glycoprotein (Env) evades immune neutralization through many mechanisms. One immune evasion strategy may result from the internalization of excess surface-exposed Env to prevent antibody-dependent cellular cytotoxicity or neutralization. Characterization of the fate of endocytosed Env is critical to understand which vesicular pathways could be targeted to promote display of Env epitopes to the immune system. In this study, we characterize the endocytic fate of native Env, expressed from infected human T-cell lines. We demonstrate that Env is rapidly trafficked to a late-endosome/lysosome-like compartment and can be recycled to the cell surface for incorporation into virus assembly sites. This study implicates a novel intracellular compartment, marked by host-cell Rab14 GTPases, for the sequestration of Env. Therapeutic approaches aimed at mobilizing this intracellular pool of Env could lead to stronger immune control of HIV-1 infection via antibody-dependent cell-mediated cytotoxicity.

T Cell-Intrinsic Interleukin 17 Receptor A Signaling Supports the Establishment of Chronic Murine Gammaherpesvirus 68 Infection.

Gammaherpesviruses, such as human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68), are species-specific, ubiquitous pathogens that are associated with multiple cancers, including B cell lymphomas. These viruses have a natural tropism for B cells and usurp B cell differentiation to drive a unique and robust polyclonal germinal center response to establish a long-term latent reservoir in memory B cells. The robust polyclonal germinal center response driven by gammaherpesvirus infection increases the risk for B cell transformation. Unsurprisingly, many gammaherpesvirus cancers are derived from germinal center or post-germinal center B cells. The viral and host factors that influence the gammaherpesvirus-driven germinal center response are not clearly defined. We previously showed that host interleukin 17 receptor A (IL-17RA) signaling promotes the establishment of chronic MHV68 infection and the MHV68-driven germinal center response. In this study, we found that T cell-intrinsic IL-17RA signaling recapitulates some proviral aspects of global IL-17RA signaling during MHV68 infection. Specifically, we found that T cell-intrinsic IL-17RA signaling supports the MHV68-driven germinal center response, the establishment of latency in the spleen, and viral reactivation in the spleen and peritoneal cavity. Our study unveils an unexpected finding where the T cell-specific IL-17RA signaling supports the establishment of a latent reservoir of a B cell-tropic gammaherpesvirus. IMPORTANCE Gammaherpesviruses, such as human EBV, establish lifelong infection in >95% of adults and are associated with B cell lymphomas. Gammaherpesviruses usurp the germinal center response to establish latent infection, and the germinal center B cells are thought to be the target of viral transformation. We previously found that global expression of IL-17RA promotes the establishment of chronic MHV68 infection and the MHV68-driven germinal center response. In this study, we showed that T cell-intrinsic IL-17RA signaling is necessary to promote the MHV68-driven germinal center response by supporting CD4+ T follicular helper cell expansion. We also found that T cell-intrinsic IL-17RA signaling contributes to but is not solely responsible for the systemic proviral role of IL-17RA signaling, highlighting the multifaceted function of IL-17RA signaling during MHV68 infection.

A Novel Pathway for Porcine Epidemic Diarrhea Virus Transmission from Sows to Neonatal Piglets Mediated by Colostrum.

The mechanisms of colostrum-mediated virus transmission are difficult to elucidate because of the absence of experimental animal models and the difficulties in tissue sample collection from mothers in the peripartum period. Porcine epidemic diarrhea virus (PEDV) is a reemerging enteropathogenic coronavirus that has catastrophic impacts on the global pig industry. PEDV primarily infects neonatal piglets by multiple routes, especially 1- to 2-day-old neonatal piglets. Here, our epidemiological investigation and animal challenge experiments revealed that PEDV could be vertically transmitted from sows to neonatal piglets via colostrum, and CD3+ T cells in the colostrum play an important role in this process. The results showed that PEDV colonizing the intestinal epithelial cells (IECs) of orally immunized infected sows could be transferred to CD3+ T cells located just beneath the IECs. Next, PEDV-carrying CD3+ T cells, with the expression of integrin α4ß7 and CCR10, migrate from the intestine to the mammary gland through blood circulation. Arriving in the mammary gland, PEDV-carrying CD3+ T cells could be transported across mammary epithelial cells (MECs) into the lumen (colostrum), as illustrated by an autotransfusion assay and an MECs/T coculture system. The PEDV-carrying CD3+ T cells in colostrum could be interspersed between IECs of neonatal piglets, causing intestinal infection via cell-to-cell contact. Our study demonstrates for the first time that colostrum-derived CD3+ T cells comprise a potential route for the vertical transmission of PEDV. IMPORTANCE The colostrum represents an important infection route for many viruses. Here, we demonstrate the vertical transmission of porcine epidemic diarrhea virus (PEDV) from sows to neonatal piglets via colostrum. PEDV colonizing the intestinal epithelial cells could transfer the virus to CD3+ T cells located in the sow intestine. The PEDV-carrying CD3+ T cells in the sow intestine, with the expression of integrin α4ß7 and CCR10, arrive at the mammary gland through blood circulation and are transported across mammary epithelial cells into the lumen, finally leading to intestinal infection via cell-to-cell contact in neonatal piglets. Our study not only demonstrates an alternative route of PEDV infection but also provides an animal model of vertical transmission of human infectious disease.

Epstein-Barr Virus Genome Deletions in Epstein-Barr Virus-Positive T/NK Cell Lymphoproliferative Diseases.

The main target cells for Epstein-Barr virus (EBV) infection and persistence are B lymphocytes, although T and NK cells can also become infected. In this paper, we characterize the EBV present in 21 pediatric and adult patients who were treated in France for a range of diseases that involve infection of T or NK cells. Of these 21 cases, 5 pediatric patients (21%) and 11 adult patients (52%) were of Caucasian origin. In about 30% of the cases, some of the EBV genomes contain a large deletion. The deletions are different in every patient but tend to cluster near the BART region of the viral genome. Detailed investigation of a family in which several members have persistent T or NK cell infection by EBV indicates that the virus genome deletions arise or are selected independently in each individual patient. Genome sequence polymorphisms in the EBV in these T or NK cell diseases reflect the geographic origin of the patient and not a distinct type of EBV (the 21 cases studied included examples of both type 1 and type 2 EBV infection). Using virus produced from type 1 or type 2 EBV genomes cloned in bacterial artificial chromosome (BAC) vectors, we demonstrate infection of T cells in cord blood from healthy donors. Our results are consistent with transient infection of some T cells being part of normal asymptomatic infection by EBV in young children. IMPORTANCE EBV contributes to several types of human cancer. Some cancers and nonmalignant lymphoproliferative diseases involving T or NK cells contain EBV. These diseases are relatively frequent in Japan and China and have been shown sometimes to have deletions in the EBV genome in the disease cells. We identify further examples of deletions within the EBV genome associated with T or NK cell diseases, and we provide evidence that the virus genomes with these deletions are most likely selected in the individual cases, rather than being transmitted between people during infection. We demonstrate EBV infection of cord blood T cells by highly characterized, cloned EBV genomes and suggest that transient infection of T cells may be part of normal asymptomatic infection by EBV in young children.

In-depth analysis of SARS-CoV-2-specific T cells reveals diverse differentiation hierarchies in vaccinated individuals.

SARS-CoV-2 vaccines pose as the most effective approach for mitigating the COVID-19 pandemic. High-degree efficacy of SARS-CoV-2 vaccines in clinical trials indicates that vaccination invariably induces an adaptive immune response. However, the emergence of breakthrough infections in vaccinated individuals suggests that the breadth and magnitude of vaccine-induced adaptive immune response may vary. We assessed vaccine-induced SARS-CoV-2 T cell response in 21 vaccinated individuals and found that SARS-CoV-2-specific T cells, which were mainly CD4+ T cells, were invariably detected in all individuals but the response was varied. We then investigated differentiation states and cytokine profiles to identify immune features associated with superior recall function and longevity. We identified SARS-CoV-2-specific CD4+ T cells were polyfunctional and produced high levels of IL-2, which could be associated with superior longevity. Based on the breadth and magnitude of vaccine-induced SARS-CoV-2 response, we identified 2 distinct response groups: individuals with high abundance versus low abundance of SARS-CoV-2-specific T cells. The fractions of TNF-α- and IL-2-producing SARS-CoV-2 T cells were the main determinants distinguishing high versus low responders. Last, we identified that the majority of vaccine-induced SARS-CoV-2 T cells were reactive against non-mutated regions of mutant S-protein, suggesting that vaccine-induced SARS-CoV-2 T cells could provide continued protection against emerging variants of concern.

Induction of tier-2 neutralizing antibodies in mice with a DNA-encoded HIV envelope native like trimer.

HIV Envelope (Env) is the main vaccine target for induction of neutralizing antibodies. Stabilizing Env into native-like trimer (NLT) conformations is required for recombinant protein immunogens to induce autologous neutralizing antibodies(nAbs) against difficult to neutralize HIV strains (tier-2) in rabbits and non-human primates. Immunizations of mice with NLTs have generally failed to induce tier-2 nAbs. Here, we show that DNA-encoded NLTs fold properly in vivo and induce autologous tier-2 nAbs in mice. DNA-encoded NLTs also uniquely induce both CD4 + and CD8 + T-cell responses as compared to corresponding protein immunizations. Murine neutralizing antibodies are identified with an advanced sequencing technology. The structure of an Env-Ab (C05) complex, as determined by cryo-EM, identifies a previously undescribed neutralizing Env C3/V5 epitope. Beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.

The T cell immune response against SARS-CoV-2.

The adaptive immune response is a major determinant of the clinical outcome after SARS-CoV-2 infection and underpins vaccine efficacy. T cell responses develop early and correlate with protection but are relatively impaired in severe disease and are associated with intense activation and lymphopenia. A subset of T cells primed against seasonal coronaviruses cross reacts with SARS-CoV-2 and may contribute to clinical protection, particularly in early life. T cell memory encompasses broad recognition of viral proteins, estimated at around 30 epitopes within each individual, and seems to be well sustained so far. This breadth of recognition can limit the impact of individual viral mutations and is likely to underpin protection against severe disease from viral variants, including Omicron. Current COVID-19 vaccines elicit robust T cell responses that likely contribute to remarkable protection against hospitalization or death, and novel or heterologous regimens offer the potential to further enhance cellular responses. T cell immunity plays a central role in the control of SARS-CoV-2 and its importance may have been relatively underestimated thus far.

Post-mortem Findings of Inflammatory Cells and the Association of 4-Hydroxynonenal with Systemic Vascular and Oxidative Stress in Lethal COVID-19.

A recent comparison of clinical and inflammatory parameters, together with biomarkers of oxidative stress, in patients who died from aggressive COVID-19 and survivors suggested that the lipid peroxidation product 4-hydroxynonenal (4-HNE) might be detrimental in lethal SARS-CoV-2 infection. The current study further explores the involvement of inflammatory cells, systemic vascular stress, and 4-HNE in lethal COVID-19 using specific immunohistochemical analyses of the inflammatory cells within the vital organs obtained by autopsy of nine patients who died from aggressive SAR-CoV-2 infection. Besides 4-HNE, myeloperoxidase (MPO) and mitochondrial superoxide dismutase (SOD2) were analyzed alongside standard leukocyte biomarkers (CDs). All the immunohistochemical slides were simultaneously prepared for each analyzed biomarker. The results revealed abundant 4-HNE in the vital organs, but the primary origin of 4-HNE was sepsis-like vascular stress, not an oxidative burst of the inflammatory cells. In particular, inflammatory cells were often negative for 4-HNE, while blood vessels were always very strongly immunopositive, as was edematous tissue even in the absence of inflammatory cells. The most affected organs were the lungs with diffuse alveolar damage and the brain with edema and reactive astrocytes, whereas despite acute tubular necrosis, 4-HNE was not abundant in the kidneys, which had prominent SOD2. Although SOD2 in most cases gave strong immunohistochemical positivity similar to 4-HNE, unlike 4-HNE, it was always limited to the cells, as was MPO. Due to their differential expressions in blood vessels, inflammatory cells, and the kidneys, we think that SOD2 could, together with 4-HNE, be a potential link between a malfunctioning immune system, oxidative stress, and vascular stress in lethal COVID-19.

Visualization of Marek's Disease Virus Genomes in Living Cells during Lytic Replication and Latency.

Visualization of the herpesvirus genomes during lytic replication and latency is mainly achieved by fluorescence in situ hybridization (FISH). Unfortunately, this technique cannot be used for the real-time detection of viral genome in living cells. To facilitate the visualization of the Marek's disease virus (MDV) genome during all stages of the virus lifecycle, we took advantage of the well-established tetracycline operator/repressor (TetO/TetR) system. This system consists of a fluorescently labeled TetR (TetR-GFP) that specifically binds to an array of tetO sequences. This tetO repeat array was first inserted into the MDV genome (vTetO). Subsequently, we fused TetR-GFP via a P2a self-cleaving peptide to the C-terminus of the viral interleukin 8 (vIL8), which is expressed during lytic replication and latency. Upon reconstitution of this vTetO-TetR virus, fluorescently labeled replication compartments were detected in the nucleus during lytic replication. After validating the specificity of the observed signal, we used the system to visualize the genesis and mobility of the viral replication compartments. In addition, we assessed the infection of nuclei in syncytia as well as lytic replication and latency in T cells. Taken together, we established a system allowing us to track the MDV genome in living cells that can be applied to many other DNA viruses.

Restriction of Viral Replication, Rather than T Cell Immunopathology, Drives Lethality in Murine Norovirus CR6-Infected STAT1-Deficient Mice.

Recent evidence indicates that viral components of the microbiota can contribute to intestinal homeostasis and protection from local inflammatory or infectious insults. However, host-derived mechanisms that regulate the virome remain largely unknown. In this study, we used colonization with the model commensal murine norovirus (MNV; strain CR6) to interrogate host-directed mechanisms of viral regulation, and we show that STAT1 is a central coordinator of both viral replication and antiviral T cell responses. In addition to restricting CR6 replication to the intestinal tract, we show that STAT1 regulates antiviral CD4+ and CD8+ T cell responses and prevents systemic viral-induced tissue damage and disease. Despite altered T cell responses that resemble those that mediate lethal immunopathology in systemic viral infections in STAT1-deficient mice, depletion of adaptive immune cells and their associated effector functions had no effect on CR6-induced disease. However, therapeutic administration of an antiviral compound limited viral replication, preventing virus-induced tissue damage and death without impacting the generation of inflammatory antiviral T cell responses. Collectively, our data show that STAT1 restricts MNV CR6 replication within the intestinal mucosa and that uncontrolled viral replication mediates disease rather than the concomitant development of dysregulated antiviral T cell responses in STAT1-deficient mice. IMPORTANCE The intestinal microbiota is a collection of bacteria, archaea, fungi, and viruses that colonize the mammalian gut. Coevolution of the host and microbiota has required development of immunological tolerance to prevent ongoing inflammatory responses against intestinal microbes. Breakdown of tolerance to bacterial components of the microbiota can contribute to immune activation and inflammatory disease. However, the mechanisms that are necessary to maintain tolerance to viral components of the microbiome, and the consequences of loss of tolerance, are less well understood. Here, we show that STAT1 is integral for preventing escape of a commensal-like virus, murine norovirus CR6 (MNV CR6), from the gut and that in the absence of STAT1, mice succumb to infection-induced disease. In contrast to the case with other systemic viral infections, mortality of STAT1-deficient mice is not driven by immune-mediated pathology. Our data demonstrate the importance of host-mediated geographical restriction of commensal-like viruses.

Three doses of prototypic SARS-CoV-2 inactivated vaccine induce cross-protection against its variants of concern.

Variants are globally emerging very quickly following pandemic prototypic SARS-CoV-2. To evaluate the cross-protection of prototypic SARS-CoV-2 vaccine against its variants, we vaccinated rhesus monkeys with three doses of prototypic SARS-CoV-2 inactivated vaccine, followed by challenging with emerging SARS-CoV-2 variants of concern (VOCs). These vaccinated animals produced neutralizing antibodies against Alpha, Beta, Delta, and Omicron variants, although there were certain declinations of geometric mean titer (GMT) as compared with prototypic SARS-CoV-2. Of note, in vivo this prototypic vaccine not only reduced the viral loads in nasal, throat and anal swabs, pulmonary tissues, but also improved the pathological changes in the lung infected by variants of Alpha, Beta, and Delta. In summary, the prototypic SARS-CoV-2 inactivated vaccine in this study protected against VOCs to certain extension, which is of great significance for prevention and control of COVID-19.

Treatment with soluble CD24 attenuates COVID-19-associated systemic immunopathology.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) blunts the broad inflammatory response induced by damage-associated molecular patterns via binding to extracellular high mobility group box 1 and heat shock proteins, as well as regulating the downstream Siglec10-Src homology 2 domain-containing phosphatase 1 pathway. A recent randomized phase III trial evaluating CD24Fc for patients with severe COVID-19 (SAC-COVID; NCT04317040) demonstrated encouraging clinical efficacy. METHODS: Using a systems analytical approach, we studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial to discern the impact of CD24Fc treatment on immune homeostasis. We performed high dimensional spectral flow cytometry and measured the levels of a broad array of cytokines and chemokines to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. RESULTS: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found that patients with severe COVID-19 had systemic hyper-activation of multiple cellular compartments, including CD8+ T cells, CD4+ T cells, and CD56+ natural killer cells. Treatment with CD24Fc blunted this systemic inflammation, inducing a return to homeostasis in NK and T cells without compromising the anti-Spike protein antibody response. CD24Fc significantly attenuated the systemic cytokine response and diminished the cytokine coexpression and network connectivity linked with COVID-19 severity and pathogenesis. CONCLUSIONS: Our data demonstrate that CD24Fc rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19.

Key considerations and common adverse events for HIV-positive patients with cancer treated with immune checkpoint inhibitors.

HIV-infected patients are more susceptible to cancer due to their immune-compromised condition and HIV infection. Chronic inflammation and immune dysregulation are the main causes of cancer development in these patients. Because of lymphopenia and an immune-compromised condition, most HIV-infected patients with cancer were not considered for cytotoxic therapies, such as chemotherapy and radiotherapy. Immune checkpoint inhibitors (ICIs) have become a game-changer in many cancer types. However, not enough prospective data is available regarding the use of ICIs in HIV-infected patients with cancer. Retrospective data from case reports/series showed that ICIs are safe in HIV-infected patients with cancer.

CD46 Isoforms Influence the Mode of Entry by Human Herpesvirus 6A/B in T Cells.

CD46 is a receptor for human herpesvirus 6A (HHV-6A) and is in some cells also important for infection with HHV-6B. CD46 has several isoforms of which the most commonly expressed can be distinguished by expression of a BC domain or a C domain in a serine-threonine-proline-rich (STP) extracellular region. Using a SupT1 CD46 CRISPR-Cas9 knockout model system reconstituted with specific CD46 isoforms, we demonstrated that HHV-6A infection was more efficient when BC isoforms were expressed as opposed to C isoforms, measured by higher levels of intracellular viral transcripts and recovery of more progeny virus. Although the B domain contains several O-glycosylations, mutations of Ser and Thr residues did not prevent infection with HHV-6A. The HHV-6A infection was blocked by inhibitors of clathrin-mediated endocytosis. In contrast, infection with HHV-6B was preferentially promoted by C isoforms mediating fusion-from-without, and this infection was less affected by inhibitors of clathrin-mediated endocytosis. Taken together, HHV-6A preferred BC isoforms, mediating endocytosis, whereas HHV-6B preferred C isoforms, mediating fusion-from-without. This demonstrates that the STP region of CD46 is important for regulating the mode of infection in SupT1 cells and suggests an epigenetic regulation of the host susceptibility to HHV-6A and HHV-6B infection. IMPORTANCE CD46 is the receptor used by human herpesvirus 6A (HHV-6A) during infection of T cells, but it is also involved in infection of certain T cells by HHV-6B. The gene for CD46 allows expression of several variants of CD46, known as isoforms, but whether the isoforms matter for infection of T cells is unknown. We used a genetic approach to delete CD46 from T cells and reconstituted them with separate isoforms to study them individually. We expressed the isoforms known as BC and C, which are distinguished by the potential inclusion of a B domain in the CD46 molecule. We demonstrate that HHV-6A prefers the BC isoform to infect T cells, and this occurs predominantly by clathrin-mediated endocytosis. In contrast, HHV-6B prefers the C isoform and infects predominantly by fusion-from-without. Thus, CD46 isoforms may affect susceptibility of T cells to infection with HHV-6A and HHV-6B.