Bacterial-induced or passively administered interferon gamma conditions the lung for early control of SARS-CoV-2.

Type-1 and type-3 interferons (IFNs) are important for control of viral replication; however, less is known about the role of Type-2 IFN (IFNγ) in anti-viral immunity. We previously observed that lung infection with Mycobacterium bovis BCG achieved though intravenous (iv) administration provides strong protection against SARS-CoV-2 in mice yet drives low levels of type-1 IFNs but robust IFNγ. Here we examine the role of ongoing IFNγ responses to pre-established bacterial infection on SARS-CoV-2 disease outcomes in two murine models. We report that IFNγ is required for iv BCG induced reduction in pulmonary viral loads, an outcome dependent on IFNγ receptor expression by non-hematopoietic cells. Importantly, we show that BCG infection prompts pulmonary epithelial cells to upregulate IFN-stimulated genes with reported anti-viral activity in an IFNγ-dependent manner, suggesting a possible mechanism for the observed protection. Finally, we confirm the anti-viral properties of IFNγ by demonstrating that the recombinant cytokine itself provides strong protection against SARS-CoV-2 challenge when administered intranasally. Together, our data show that a pre-established IFNγ response within the lung is protective against SARS-CoV-2 infection, suggesting that concurrent or recent infections that drive IFNγ may limit the pathogenesis of SARS-CoV-2 and supporting possible prophylactic uses of IFNγ in COVID-19 management.

SARS-CoV-2 infection of human lung epithelial cells induces TMPRSS-mediated acute fibrin deposition.

Severe COVID-associated lung injury is a major confounding factor of hospitalizations and death with no effective treatments. Here, we describe a non-classical fibrin clotting mechanism mediated by SARS-CoV-2 infected primary lung but not other susceptible epithelial cells. This infection-induced fibrin formation is observed in all variants of SARS-CoV-2 infections, and requires thrombin but is independent of tissue factor and other classical plasma coagulation factors. While prothrombin and fibrinogen levels are elevated in acute COVID BALF samples, fibrin clotting occurs only with the presence of viral infected but not uninfected lung epithelial cells. We suggest a viral-induced coagulation mechanism, in which prothrombin is activated by infection-induced transmembrane serine proteases, such as ST14 and TMPRSS11D, on NHBE cells. Our finding reveals the inefficiency of current plasma targeted anticoagulation therapy and suggests the need to develop a viral-induced ARDS animal model for treating respiratory airways with thrombin inhibitors.

Exposure to lung-migrating helminth protects against murine SARS-CoV-2 infection through macrophage-dependent T cell activation.

Helminth endemic regions report lower COVID-19 morbidity and mortality. Here, we show that lung remodeling from a prior infection with a lung-migrating helminth, Nippostrongylus brasiliensis, enhances viral clearance and survival of human-ACE2 transgenic mice challenged with SARS-CoV-2 (SCV2). This protection is associated with a lymphocytic infiltrate, including increased accumulation of pulmonary SCV2-specific CD8+ T cells, and anti-CD8 antibody depletion abrogated the N. brasiliensis-mediated reduction in viral loads. Pulmonary macrophages with a type 2 transcriptional and epigenetic signature persist in the lungs of N. brasiliensis-exposed mice after clearance of the parasite and establish a primed environment for increased CD8+ T cell recruitment and activation. Accordingly, depletion of macrophages ablated the augmented viral clearance and accumulation of CD8+ T cells driven by prior N. brasiliensis infection. Together, these findings support the concept that lung-migrating helminths can limit disease severity during SCV2 infection through macrophage-dependent enhancement of antiviral CD8+ T cell responses.

Helminth exposure protects against murine SARS-CoV-2 infection through macrophage dependent T cell activation.

Helminth endemic regions report lower COVID-19 morbidity and mortality. Here, we show that lung remodeling from a prior infection with a lung migrating helminth, Nippostrongylus brasiliensis , enhances viral clearance and survival of human-ACE2 transgenic mice challenged with SARS-CoV-2 (SCV2). This protection is associated with a lymphocytic infiltrate including an increased accumulation of pulmonary SCV2-specific CD8+ T cells and anti-CD8 antibody depletion abrogated the N. brasiliensis -mediated reduction in viral loads. Pulmonary macrophages with a type-2 transcriptional signature persist in the lungs of N. brasiliensis exposed mice after clearance of the parasite and establish a primed environment for increased antigen presentation. Accordingly, depletion of macrophages ablated the augmented viral clearance and accumulation of CD8+ T cells driven by prior N. brasiliensis infection. Together, these findings support the concept that lung migrating helminths can limit disease severity during SCV2 infection through macrophage-dependent enhancement of anti-viral CD8+ T cell responses.

IL-10 suppresses T cell expansion while promoting tissue-resident memory cell formation during SARS-CoV-2 infection in rhesus macaques.

The pro- and anti-inflammatory pathways that determine the balance of inflammation and viral control during SARS-CoV-2 infection are not well understood. Here we examine the roles of IFNγ and IL-10 in regulating inflammation, immune cell responses and viral replication during SARS-CoV-2 infection of rhesus macaques. IFNγ blockade tended to decrease lung inflammation based on 18 FDG-PET/CT imaging but had no major impact on innate lymphocytes, neutralizing antibodies, or antigen-specific T cells. In contrast, IL-10 blockade transiently increased lung inflammation and enhanced accumulation of virus-specific T cells in the lower airways. However, IL-10 blockade also inhibited the differentiation of virus-specific T cells into airway CD69 + CD103 + T RM cells. While virus-specific T cells were undetectable in the nasal mucosa of all groups, IL-10 blockade similarly reduced the frequency of total T RM cells in the nasal mucosa. Neither cytokine blockade substantially affected viral load and infection ultimately resolved. Thus, in the macaque model of mild COVID-19, the pro- and anti-inflammatory effects of IFNγ and IL-10 have no major role in control of viral replication. However, IL-10 has a key role in suppressing the accumulation of SARS-CoV-2-specific T cells in the lower airways, while also promoting T RM at respiratory mucosal surfaces.

Expanding the antiviral potential of the mosquito lipid-transfer protein AEG12 against SARS-CoV-2 using hydrophobic antiviral ligands.

The mosquito protein AEG12 encompasses a large (~ 3800 Å3 ) hydrophobic cavity which binds and delivers unsaturated fatty acids into biological membranes, allowing it to lyse cells and neutralize a wide range of enveloped viruses. Herein, the lytic and antiviral activities are modified with non-naturally occurring lipid ligands. We generated novel AEG12 complexes in which the endogenous fatty acid ligands were replaced with hydrophobic viral inhibitors. The resulting compounds modulated cytotoxicity and infectivity against SARS-CoV-2, potentially reflecting additional mechanisms of action beyond membrane destabilization. These studies provide valuable insight into the design of novel broad-spectrum antiviral therapeutics centred on the AEG12 protein scaffold as a delivery vehicle for hydrophobic therapeutic compounds.

Intranasal immunization with avian paramyxovirus type 3 expressing SARS-CoV-2 spike protein protects hamsters against SARS-CoV-2.

Current vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are administered parenterally and appear to be more protective in the lower versus the upper respiratory tract. Vaccines are needed that directly stimulate immunity in the respiratory tract, as well as systemic immunity. We used avian paramyxovirus type 3 (APMV3) as an intranasal vaccine vector to express the SARS-CoV-2 spike (S) protein. A lack of pre-existing immunity in humans and attenuation by host-range restriction make APMV3 a vector of interest. The SARS-CoV-2 S protein was stabilized in its prefusion conformation by six proline substitutions (S-6P) rather than the two that are used in most vaccine candidates, providing increased stability. APMV3 expressing S-6P (APMV3/S-6P) replicated to high titers in embryonated chicken eggs and was genetically stable, whereas APMV3 expressing non-stabilized S or S-2P were unstable. In hamsters, a single intranasal dose of APMV3/S-6P induced strong serum IgG and IgA responses to the S protein and its receptor-binding domain, and strong serum neutralizing antibody responses to SARS-CoV-2 isolate WA1/2020 (lineage A). Sera from APMV3/S-6P-immunized hamsters also efficiently neutralized Alpha and Beta variants of concern. Immunized hamsters challenged with WA1/2020 did not exhibit the weight loss and lung inflammation observed in empty-vector-immunized controls; SARS-CoV-2 replication in the upper and lower respiratory tract of immunized animals was low or undetectable compared to the substantial replication in controls. Thus, a single intranasal dose of APMV3/S-6P was highly immunogenic and protective against SARS-CoV-2 challenge, suggesting that APMV3/S-6P is suitable for clinical development.

Mild SARS-CoV-2 infection in rhesus macaques is associated with viral control prior to antigen-specific T cell responses in tissues.

SARS-CoV-2 primarily replicates in mucosal sites, and more information is needed about immune responses in infected tissues. Here, we used rhesus macaques to model protective primary immune responses in tissues during mild COVID-19. Viral RNA levels were highest on days 1-2 post-infection and fell precipitously thereafter. 18F-fluorodeoxyglucose (FDG)-avid lung abnormalities and interferon (IFN)-activated monocytes and macrophages in the bronchoalveolar lavage (BAL) were found on days 3-4 post-infection. Virus-specific effector CD8+ and CD4+ T cells became detectable in the BAL and lung tissue on days 7-10, after viral RNA, radiologic evidence of lung inflammation, and IFN-activated myeloid cells had substantially declined. Notably, SARS-CoV-2-specific T cells were not detectable in the nasal turbinates, salivary glands, and tonsils on day 10 post-infection. Thus, SARS-CoV-2 replication wanes in the lungs of rhesus macaques prior to T cell responses, and in the nasal and oral mucosa despite the apparent lack of antigen-specific T cells, suggesting that innate immunity efficiently restricts viral replication during mild COVID-19.

Intravenous administration of BCG protects mice against lethal SARS-CoV-2 challenge.

In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guérin (BCG) has been reported to confer nonspecific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here, we demonstrate that intravenous, but not subcutaneous, inoculation of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 (SCV2) and results in reduced viral loads in non-transgenic animals infected with an α variant. The observed increase in host resistance was associated with reductions in SCV2-induced tissue pathology, inflammatory cell recruitment, and cytokine production that multivariate analysis revealed as only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and ensuing immunopathology. While intravenous BCG vaccination is not a clinically acceptable practice, our findings provide an experimental model for identifying mechanisms by which nonspecific stimulation of the pulmonary immune response promotes host resistance to SCV2 lethality.

A single intranasal dose of a live-attenuated parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in hamsters.

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2-neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 104.5 50% tissue-culture infectious-dose (TCID50) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 106.6 TCID50/g in lungs and 107 TCID50/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P-immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.

Intravenous administration of BCG protects mice against lethal SARS-CoV-2 challenge.

Early events in the host response to SARS-CoV-2 are thought to play a major role in determining disease severity. During pulmonary infection, the virus encounters both myeloid and epithelioid lineage cells that can either support or restrict pathogen replication as well as respond with host protective versus detrimental mediators. In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guérin (BCG) has been reported to confer non-specific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here we demonstrate that prior intravenous, but not subcutaneous, administration of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 and results in reduced viral loads in non-transgenic animals infected with an alpha variant. The observed increase in host resistance was associated with reductions in SARS-CoV-2-induced tissue pathology, inflammatory cell recruitment and cytokine production that multivariate analysis revealed to be only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and the ensuing immunopathology.

Fe-S cofactors in the SARS-CoV-2 RNA-dependent RNA polymerase are potential antiviral targets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.

Simian Immunodeficiency Virus-Infected Memory CD4+ T Cells Infiltrate to the Site of Infected Macrophages in the Neuroparenchyma of a Chronic Macaque Model of Neurological Complications of AIDS.

Simian immunodeficiency virus (SIV)-infected nonhuman primates can serve as a relevant model for AIDS neuropathogenesis. Current SIV-induced encephalitis (SIVE)/neurological complications of AIDS (neuroAIDS) models are generally associated with rapid progression to neuroAIDS, which does not reflect the tempo of neuroAIDS progression in humans. Recently, we isolated a neuropathogenic clone, SIVsm804E-CL757 (CL757), obtained from an SIV-infected rhesus macaque (RM). CL757 causes a more protracted progression to disease, inducing SIVE in 50% of inoculated animals, with high cerebral spinal fluid viral loads, multinucleated giant cells (MNGCs), and perivascular lymphocytic cuffing in the central nervous system (CNS). This latter finding is reminiscent of human immunodeficiency virus (HIV) encephalitis in humans but not generally observed in rapid progressor animals with neuroAIDS. Here, we studied which subsets of cells within the CNS were targeted by CL757 in animals with neurological symptoms of SIVE. Immunohistochemistry of brain sections demonstrated infiltration of CD4+ T cells (CD4) and macrophages (MΦs) to the site of MNGCs. Moreover, an increase in mononuclear cells isolated from the brain tissues of RMs with SIVE correlated with increased cerebrospinal fluid (CSF) viral load. Subset analysis showed a specific increase in brain CD4+ memory T cells (Br-mCD4), brain-MΦs (Br-MΦs), and brain B cells (Br-B cells). Both Br-mCD4s and Br-MΦs harbored replication-competent viral DNA, as demonstrated by virus isolation by coculture. However, only in animals exhibiting SIVE/neuroAIDS was virus isolated from Br-MΦs. These findings support the use of CL757 to study the pathogenesis of AIDS viruses in the central nervous system and indicate a previously unanticipated role of CD4s cells as a potential reservoir in the brain.IMPORTANCE While the use of combination antiretroviral therapy effectively suppresses systemic viral replication in the body, neurocognitive disorders as a result of HIV infection of the central nervous system (CNS) remain a clinical problem. Therefore, the use of nonhuman primate models is necessary to study mechanisms of neuropathogenesis. The neurotropic, molecular clone SIVsm804E-CL757 (CL757) results in neuroAIDS in 50% of infected rhesus macaques approximately 1 year postinfection. Using CL757-infected macaques, we investigate disease progression by examining subsets of cells within the CNS that were targeted by CL757 and could potentially serve as viral reservoirs. By isolating mononuclear cells from the brains of SIV-infected rhesus macaques with and without encephalitis, we show that immune cells invade the neuroparenchyma and increase in number in the CNS in animals with SIV-induced encephalitis (SIVE). Of these cells, both brain macrophages and brain memory CD4+ T cells harbor replication-competent SIV DNA; however, only brain CD4+ T cells harbored SIV DNA in animals without SIVE. These findings support use of CL757 as an important model to investigate disease progression in the CNS and as a model to study virus reservoirs in the CNS.

Correction to: Nomenclature report 2019: major histocompatibility complex genes and alleles of great and small ape and old and new world monkey species.

The original version of this article contained a spelling error in the Acknowledgments regarding the name of the funding organisation supporting GM and JAH. UKRI-BBSCR should have been UKRI-BBSRC, as is now indicated correctly below.

Nomenclature report 2019: major histocompatibility complex genes and alleles of Great and Small Ape and Old and New World monkey species.

The major histocompatibility complex (MHC) is central to the innate and adaptive immune responses of jawed vertebrates. Characteristic of the MHC are high gene density, gene copy number variation, and allelic polymorphism. Because apes and monkeys are the closest living relatives of humans, the MHCs of these non-human primates (NHP) are studied in depth in the context of evolution, biomedicine, and conservation biology. The Immuno Polymorphism Database (IPD)-MHC NHP Database (IPD-MHC NHP), which curates MHC data of great and small apes, as well as Old and New World monkeys, has been upgraded. The curators of the database are responsible for providing official designations for newly discovered alleles. This nomenclature report updates the 2012 report, and summarizes important nomenclature issues and relevant novel features of the IPD-MHC NHP Database.

A SIV molecular clone that targets the CNS and induces neuroAIDS in rhesus macaques.

Despite effective control of plasma viremia with the use of combination antiretroviral therapies (cART), minor cognitive and motor disorders (MCMD) persist as a significant clinical problem in HIV-infected patients. Non-human primate models are therefore required to study mechanisms of disease progression in the central nervous system (CNS). We isolated a strain of simian immunodeficiency virus (SIV), SIVsm804E, which induces neuroAIDS in a high proportion of rhesus macaques and identified enhanced antagonism of the host innate factor BST-2 as an important factor in the macrophage tropism and initial neuro-invasion of this isolate. In the present study, we further developed this model by deriving a molecular clone SIVsm804E-CL757 (CL757). This clone induced neurological disorders in high frequencies but without rapid disease progression and thus is more reflective of the tempo of neuroAIDS in HIV-infection. NeuroAIDS was also induced in macaques co-inoculated with CL757 and the parental AIDS-inducing, but non-neurovirulent SIVsmE543-3 (E543-3). Molecular analysis of macaques infected with CL757 revealed compartmentalization of virus populations between the CNS and the periphery. CL757 exclusively targeted the CNS whereas E543-3 was restricted to the periphery consistent with a role for viral determinants in the mechanisms of neuroinvasion. CL757 would be a useful model to investigate disease progression in the CNS and as a model to study virus reservoirs in the CNS.

Peptide-Dependent Recognition of HLA-B*57:01 by KIR3DS1.

UNLABELLED: Killer cell immunoglobulin-like receptors (KIRs) play an important role in the activation of natural killer (NK) cells, which in turn contribute to the effective immune control of many viral infections. In the context of HIV infection, the closely related KIR3DL1 and KIR3DS1 molecules, in particular, have been associated with disease outcome. Inhibitory signals via KIR3DL1 are disrupted by downregulation of HLA class I ligands on the infected cell surface and can also be impacted by changes in the presented peptide repertoire. In contrast, the activatory ligands for KIR3DS1 remain obscure. We used a structure-driven approach to define the characteristics of HLA class I-restricted peptides that interact with KIR3DL1 and KIR3DS1. In the case of HLA-B*57:01, we used this knowledge to identify bona fide HIV-derived peptide epitopes with similar properties. Two such peptides facilitated productive interactions between HLA-B*57:01 and KIR3DS1. These data reveal the presence of KIR3DS1 ligands within the HIV-specific peptide repertoire presented by a protective HLA class I allotype, thereby enhancing our mechanistic understanding of the processes that enable NK cells to impact disease outcome. IMPORTANCE: Natural killer (NK) cells are implicated as determinants of immune control in many viral infections, but the precise molecular mechanisms that initiate and control these responses are unclear. The activating receptor KIR3DS1 in combination with HLA-Bw4 has been associated with better outcomes in HIV infection. However, evidence of a direct interaction between these molecules is lacking. In this study, we demonstrate that KIR3DS1 recognition of HLA-Bw4 is peptide dependent. We also identify HIV-derived peptide epitopes presented by the protective HLA-B*57:01 allotype that facilitate productive interactions with KIR3DS1. Collectively, these findings suggest a mechanism whereby changes in the peptide repertoire associated with viral infection provide a trigger for KIR3DS1 engagement and NK cell activation.

Mutational and structural analysis of KIR3DL1 reveals a lineage-defining allotypic dimorphism that impacts both HLA and peptide sensitivity.

Killer Ig-like receptors (KIRs) control the activation of human NK cells via interactions with peptide-laden HLAs. KIR3DL1 is a highly polymorphic inhibitory receptor that recognizes a diverse array of HLA molecules expressing the Bw4 epitope, a group with multiple polymorphisms incorporating variants within the Bw4 motif. Genetic studies suggest that KIR3DL1 variation has functional significance in several disease states, including HIV infection. However, owing to differences across KIR3DL1 allotypes, HLA-Bw4, and associated peptides, the mechanistic link with biological outcome remains unclear. In this study, we elucidated the impact of KIR3DL1 polymorphism on peptide-laden HLA recognition. Mutational analysis revealed that KIR residues involved in water-mediated contacts with the HLA-presented peptide influence peptide binding specificity. In particular, residue 282 (glutamate) in the D2 domain underpins the lack of tolerance of negatively charged C-terminal peptide residues. Allotypic KIR3DL1 variants, defined by neighboring residue 283, displayed differential sensitivities to HLA-bound peptide, including the variable HLA-B*57:01-restricted HIV-1 Gag-derived epitope TW10. Residue 283, which has undergone positive selection during the evolution of human KIRs, also played a central role in Bw4 subtype recognition by KIR3DL1. Collectively, our findings uncover a common molecular regulator that controls HLA and peptide discrimination without participating directly in peptide-laden HLA interactions. Furthermore, they provide insight into the mechanics of interaction and generate simple, easily assessed criteria for the definition of KIR3DL1 functional groupings that will be relevant in many clinical applications, including bone marrow transplantation.