Toll-like receptor-2 (TLR2) serves as an innate immune sensor that recognizes specific viral proteins, thereby initiating signaling pathways that can either enhance antiviral host defenses or induce pathological inflammatory responses. In this forum, we discuss the role of TLR2 and its agonists in enhancing antiviral and vaccine-induced immune responses.

Immune modulation in the male genital tract during HIV infection is not well understood, despite its role in viral persistence and transmission. In a study using an SIV rhesus macaque model, we assessed viral RNA and 61 cytokines, chemokines, and growth factors in peripheral plasma (PP) and seminal plasma (SP). SIV RNA was found in both plasma samples, but the SP viral loads were more variable and decreased over time, independent of the systemic viremia. Baseline immune mediator profiles exhibited compartment-specific patterns, and principal component analysis demonstrated distinct clustering between PP and SP samples. Unlike the extensive systemic immune activation in PP, SIV infection in SP results in selective modulation involving CXCL1, IL-7, CCL4, and YKL-40, with these changes not correlating with the local viral loads. Our findings suggest that the seminal compartment is immunologically distinct, with selective inflammatory responses, highlighting the mechanisms that may influence viral persistence and transmission.

Development of candidate countermeasures against human pathogens frequently includes nonhuman animal experimentation. Preclinical animal pathogen exposure studies are conducted to model diseases and accumulate preliminary and hypothetically translatable data to inform and justify the design of clinical trial evaluation of countermeasure safety and efficacy. In addition to frequent ethical critiques, challenges associated with animal experimentation include considerable resources needed to achieve statistical power and robustness, replicability and reproducibility concerns, potentially compromised objectivity through lack of blinding, fundamental species-specific biological differences, and risk of unpredictable pathogen adaptation to the experimental animal. Recent U.S. and U.K. government initiatives aim to reduce animal experimentation by complementing or potentially replacing them with new approach methodologies (NAMs), i.e., increasingly sophisticated in silico, in chemico, and in vitro approaches. We piloted development of one type of NAM, organ-on-chips (OOCs), in the highly challenging environment of a maximum (biosafety level 4) containment laboratory. Using a Risk Group 4 virus, Nipah virus (NiV), and two types of lung OOCs seeded with human or porcine cells, we demonstrated the recapitulation of key features of NiV lung infection, including viral infection, replication, and translocation, that are associated with proinflammatory cytokine secretion, immune cell recruitment, and disruption of the air-liquid interface barrier. We reproduced the known anti-NiV activity of remdesivir and evaluated that of another potential antiviral, zotatifin. Our results pave the way for similar applications of advanced microphysiological systems for modeling infections caused by high-consequence viruses.

New approach methodologies, such as high-complexity in vitro systems, are increasingly prioritized in biomedical research as potential alternatives to animal experimentation. We show that cerebral organoids derived from human induced pluripotent stem cells can be leveraged to (i) investigate isolate-specific replication dynamics of Nipah virus and (ii) model key histopathological lesions found in the brain tissue of infected human patients. Furthermore, we discuss the importance of organoid models for the study of Risk Group 4 viruses. IMPORTANCE:Advanced development of medical countermeasures against Risk Group 4 viruses, such as the Nipah virus, historically required testing in mammals under the FDA Animal Rule and translation of data to inform clinical trials in humans. Because the application of human organoids in research on viruses pathogenic for humans is conspecific, it bears the potential to reduce, refine, or replace animal studies where unnecessary. Human cerebral organoids are three-dimensional cell aggregates that resemble the developing human brain functionally and structurally. Brain organoids may be valuable in investigating the replication, neuroinvasion, pathogenesis, virulence, and persistence of neurotropic viruses and provide scientific discernment when developing medical countermeasures destined for the human end-user.

We developed a 29-color spectral cytometry panel to enhance nonhuman primate (NHP) models for cross-reactive immunophenotyping. This panel is suitable for biosafety level 4 (BSL-4) viruses and can be used with both human and NHP samples in BSL-2 research settings. Tissues from humans, rhesus monkeys (RhMs), crab-eating macaques (CEMs), and green monkeys (GMs) were stained with a 29-color immunophenotyping panel requiring only 2 clone substitutions. Comparable staining was observed for all samples. Unbiased analysis showed acceptable overlap in T cell phenotypes across samples, with differences in human and NHP B cells and granulocytes. In CEMs, most circulating CD8+ T cells were from effector memory cells, with significantly higher levels than in humans, RhMs, and GMs. Analysis of samples from various anatomical sites revealed distinct location-specific phenotypes. In Nipah virus-exposed animals, splenocytes showed a substantial increase in IgM+ B cells and a reduction in effector memory CD8+ T cells compared with unexposed controls. Lymph nodes from Ebola virus-exposed animals showed a loss of CXCR3+CD8+ T cells versus unexposed controls. This panel may guide the development of additional multicolor panels in preclinical and clinical settings and may increase understanding of the pathogenesis of diseases caused by emerging and reemerging viruses.

Introduction: The mammarenavirus Lassa virus (LASV) infects tens to hundreds of thousands of individuals in Western Africa annually. Approximately 20% of infections results in Lassa fever (LF), an acute disease with an estimated case-fatality rate of up to 26%. LF is characterized by high fever, malaise, myalgia, and gastrointestinal signs. In severe cases, central nervous system involvement and respiratory distress may manifest and progress to shock and death. Due to the potential to cause a public health emergency, the World Health Organization has listed LF as a Priority Disease of Concern. No vaccines or specific therapeutics are approved to prevent or treat LF. Methods: The LASV genome consists of a large (L) segment that encodes two proteins, L and Z, and a small (S) segment that also encodes two proteins, glycoprotein (GP) and nucleoprotein (NP). Genes in each genome segment are separated by a non-coding intergenic region (IGR). Previously, we generated recombinant LASVs (rLASVs) via traditional reverse genetics, in which the L IGR was replaced with the S IGR or in which the GP-encoding gene was codon-deoptimized and showed that both were less fit in vitro and were highly attenuated in vivo. Further, vaccination with these rLASVs protected domesticated guinea pigs from otherwise lethal LASV exposure. Here, we utilize the same approach and report the generation of an rLASV that combines both determinants of attenuation (rLASV/IGR-CD), which further enhances the vaccine safety of its predecessors. Results: rLASV/IGR-CD grew to high titers in Vero cells but did not cause signs of disease or pathological lesions in exposed domesticated guinea pigs. Additionally, and most importantly, animals vaccinated with rLASV/IGR-CD were completely protected from disease and death after a typically lethal exposure to wild-type LASV. Moreover, the animals developed high antibody titers after vaccination and significantly reduced proinflammatory cytokines after exposure. Discussion: The only currently approved effective vaccine against mammarenavirus infection in humans is Candid#1, which uses a live-attenuated strain of Junín virus; Candid#1 is approved for use in Argentina and has investigational new drug status in the U.S. LAVs frequently trigger lasting robust cell-mediated and humoral immune responses. However, concerns about reversion to virulence events (through mutation, recombination, or reassortment with wild-type strains) obligate careful preclinical evaluation of this risk, that, at minimum, establishes an extremely low likelihood of reversion. Conclusion: Our previous findings, together with the data presented here, indicate that the incorporation of genetic determinants of attenuation in both the S segment and L segment is an attractive approach to develop mammarenavirus LAV candidates. Our data support the development of rLASV/IGR-CD to solidify a foundation for the creation of a live-attenuated LF vaccine with unassailable safety features.

The dysregulated production of reactive oxygen species (ROS) during viral infections may lead to immune cell death and ineffective host responses. ROS dynamics have been under-investigated in severe Ebola virus disease (EVD), a condition in which hyperinflammation and excessive immune cell death are well described but poorly understood. Through ex vivo immunohistochemistry and in vivo ROS-sensitive magnetic resonance imaging (MRI) we demonstrate significant ROS-related oxidative changes in the spleens of domestic ferrets exposed to Ebola virus (EBOV). By immunohistochemistry or MRI, detection of splenic ROS was inversely correlated with the number of CD4+/CD8+ T lymphocytes and apoptotic CD8+ lymphocytes, but detection was positively correlated with the frequency of apoptotic CD4+ cells and the number and frequency of apoptotic B lymphocytes. These results suggest that ROS-induced apoptosis may contribute to the loss of splenic CD4+ T lymphocytes in EBOV-exposed ferrets and warrant further investigation of the role of ROS in severe EVD.

Nipah virus (NiV), a highly pathogenic zoonotic paramyxovirus, causes severe respiratory and neurological disease in humans, with a case-fatality rate around 60%. Descriptions of cases in the clinical setting suggest that the two primary lineages of NiV cause disease with different presentations and outcomes. To define strain-specific differences in disease progression and host responses, African green monkeys were exposed to either the Malaysia (NiV-M) or Bangladesh (NiV-B) strain using a large-particle aerosol exposure. NiV-M infection resulted in a fatality rate of 27%, while NiV-B infection led to a 75% fatality rate characterized by rapid respiratory decline and systemic viral dissemination. Among survivors, NiV-M-infected animals mounted robust immunoglobulin M, immunoglobulin G, and neutralizing antibody responses, whereas NiV-B survivors exhibited weaker and delayed humoral responses. Non-survivors of both strains showed elevated pro-inflammatory cytokines, thrombocytopenia, and multi-organ dysfunction. Imaging showed that NiV-M infection was associated with neuroinflammation and systemic vasculitis, while NiV-B infection caused progressive pulmonary pathology. Histopathological analysis confirmed widespread vasculitis and encephalitis in animals with NiV-M infection and diffuse pulmonary hemorrhage and fibrin thrombi, consistent with vascular injury and coagulopathy, in animals with NiV-B infection. Cytokine profiling and flow cytometry showed a more intense and dysregulated immune response to NiV-B infection. Fatal outcomes in both groups were associated with thrombocytopenia, elevated pro-inflammatory cytokines, and multi-organ dysfunction. This study highlights fundamental differences in virulence, immune evasion, and pathogenesis between NiV strains and underscores the value of the African green monkey aerosol model for evaluating medical countermeasures under conditions that closely mimic natural human exposure.

AbstractLassa virus (LASV) is a rodent-borne mammarenavirus that causes tens to hundreds of thousands of human infections annually in Western Africa. Approximately 20% of these infections progress to Lassa fever (LF), an acute disease with case–fatality rates from ≈20–70%. Currently, there are no approved vaccines or specific therapeutics to prevent or treat LF. The LASV genome consists of a small (S) segment that has two genes, GP and NP, and a large (L) segment that has two genes, L and Z. In both segments, the two genes are separated by non-coding intergenic regions (IGRs). Recombinant LASVs (rLASVs), in which the L segment IGR was replaced with the S segment IGR or in which the GP gene was codon-deoptimized, lost fitness in vitro, were highly attenuated in vivo, and, when used as vaccines, protected domesticated guinea pigs from otherwise lethal LASV exposure. Here, we report the generation of rLASV/IGR-CD, which includes both determinants of attenuation and further enhances the safety of the vaccine compared with its predecessors. rLASV/IGR-CD grew to high titers in Vero cells, which are approved for human vaccine production, but did not cause signs of disease or pathology in guinea pigs. Importantly, guinea pigs vaccinated with rLASV/IGR-CD were completely protected from disease and death after a typically lethal exposure to wild-type LASV. Our data support the development of rLASV/IGR-CD as a live-attenuated LF vaccine with stringent safety features.

Abstract Guinea pigs serve as a valuable animal model for many aspects of infectious disease research. A primary limitation of the guinea pig model is the lack of a robust toolbox to probe the underlying immune responses in these animals. We developed an optimized 9-color flow cytometric assay for immunophenotyping peripheral and tissue-associated immune cells in the guinea pig. Using this assay, we can delineate CD45+ cells as well as monocyte and macrophage populations, in addition to further differentiating lymphocyte populations of B and T cells. Importantly, we found that this panel is able to detect these populations in both inbred Strain 13 and outbred Hartley guinea pigs in whole blood and from fresh and frozen cells isolated from several peripheral tissues. We utilized this panel to separately interrogate the immune responses in whole blood and spleen tissue samples after exposure of Hartley guinea pigs with Ebola virus (EBOV) and Strain 13 or Hartley guinea pigs with Lassa virus (LASV). We believe this flow cytometry panel will serve as an important tool to interrogate the underlying immune responses to risk group 4 pathogens in addition to numerous other infectious diseases with the guinea pig model.

Persistence of human immunodeficiency virus (HIV) reservoirs prevents viral eradication, and consequently HIV-infected patients require lifetime treatment with antiretroviral therapy (ART) [1–5]. Currently, there are no effective therapeutics to prevent HIV rebound upon ART cessation. Here we describe an HIV/SIV Rev-dependent lentiviral particle that can be administered to inhibit viral rebound [6–9]. Using simian immunodeficiency virus (SIV)-infected rhesus macaques as a model, we demonstrate that the administration of pre-assembled SIV Rev-dependent lentiviral particles into SIVmac239-infected Indian rhesus macaques can lead to reduction of viral rebound upon ART termination. One of the injected animals, KC50, controlled plasma and CNS viremia to an undetectable level most of the time for over two years after ART termination. Surprisingly, detailed molecular and immunological characterization revealed that viremia control was concomitant with the induction of neutralizing antibodies (nAbs) following the administration of the Rev-dependent vectors. This study emphasizes the importance of neutralizing antibodies (nAbs) for viremia control [10–15], and also provides proof of concept that the Rev-dependent vector can be used to target viral reservoirs, including the CNS reservoirs, in vivo. However, future large-scale in vivo studies are needed to understand the potential mechanisms of viremia control induced by the Rev-dependent vector.

Abstract Despite the critical importance of emerging infectious disease research, the underlying immune responses to risk group 4 pathogens remain largely understudied relative to many other pathogens. Spectral flow cytometry has dramatically increased the capacity of fluorescence-based cell analyses, allowing for the development of comprehensive panels across a variety of cell types. We have developed a 33-color phenotypic panel that incorporates a plethora of markers to simultaneously identify numerous distinct cellular populations within circulation and in tissue with a deliberate and extensive characterization of monocytes, dendritic cells, and tissue-resident T cells. This panel utilizes multiple approaches to accurately identify appropriate cell markers that can be easily distinguished with minimal to no compensation, despite containing fluorochromes with similar excitation and emission spectra. Importantly, this high-complexity panel was implemented on both fresh and frozen samples of whole blood, peripheral blood mononuclear cells (PBMCs), and cells isolated from multiple peripheral tissues across several nonhuman primate (NHP) models and on frozen human PBMCs, making this a broad cross-reactive panel enabling direct comparisons among NHP species and with humans. This new phenotypic flow panel allows for broad insight into underlying immune responses to infections across species while remaining amenable to additional potential markers of interest.

Cytokine and chemokine levels remain one of the significant predictive factors of HIV pathogenesis and disease outcome. Understanding the impact of cytokines and chemokines during early acute infection will help to recognize critical changes during HIV pathogenesis and might assist in establishing improved HIV treatment and prevention methods. Sixty-one cytokines and chemokines were evaluated in the plasma of an SIV-infected rhesus macaque model. A substantial change in 11 cytokines/growth factors and 9 chemokines were observed during acute infection. Almost all the cytokines/chemokines were below the baseline values for an initial couple of days of infection. We detected six important cytokines/chemokines, such as IL-18, IP-10, FLT3L, MCP-1, MCP-2, and MIP-3β, that can be used as biomarkers to predict the peripheral CD4+ T cell loss and increased viral replication during the acute SIV/HIV infection. Hence, regulating IL-18, IP-10, FLT3L, MCP-1, MCP-2, and MIP-3β expression might provide an antiviral response to combat acute SIV/HIV infection.

BACKGROUND:Ebola virus (EBOV) disease (EVD) is one of the most severe and fatal viral hemorrhagic fevers and appears to mimic many clinical and laboratory manifestations of hemophagocytic lymphohistiocytosis syndrome (HLS), also known as macrophage activation syndrome. However, a clear association is yet to be firmly established for effective host-targeted, immunomodulatory therapeutic approaches to improve outcomes in patients with severe EVD. METHODS:Twenty-four rhesus monkeys were exposed intramuscularly to the EBOV Kikwit isolate and euthanized at prescheduled time points or when they reached the end-stage disease criteria. Three additional monkeys were mock-exposed and used as uninfected controls. RESULTS:EBOV-exposed monkeys presented with clinicopathologic features of HLS, including fever, multiple organomegaly, pancytopenia, hemophagocytosis, hyperfibrinogenemia with disseminated intravascular coagulation, hypertriglyceridemia, hypercytokinemia, increased concentrations of soluble CD163 and CD25 in serum, and the loss of activated natural killer cells. CONCLUSIONS:Our data suggest that EVD in the rhesus macaque model mimics pathophysiologic features of HLS/macrophage activation syndrome. Hence, regulating inflammation and immune function might provide an effective treatment for controlling the pathogenesis of acute EVD.

Toll-like receptors (TLRs) are crucial to the innate immune response. They regulate inflammatory reactions by initiating the production of pro-inflammatory cytokines and chemokines. TLRs also play a role in shaping the adaptive immune responses. While this protective response is important for eliminating infectious pathogens, persistent activation of TLRs may result in chronic immune activation, leading to detrimental effects. The role of TLR2 in regulating HIV-1 infection in vivo has yet to be well described. In this study, we used an SIV-infected rhesus macaque model to simulate HIV infection in humans. We evaluated the plasma of the macaques longitudinally and found a significant increase in the soluble TLR2 (sTLR2) level after SIV infection. We also observed an increase in membrane-bound TLR2 (mb-TLR2) in cytotoxic T cells, B cells, and NK cells in PBMC and NK cells in the gut after infection. Our results suggest that sTLR2 regulates the production of various cytokines and chemokines, including IL-18, IL-1RA, IL-15, IL-13, IL-9, TPO, FLT3L, and IL-17F, as well as chemokines, including IP-10, MCP-1, MCP-2, ENA-78, GRO-α, I-TAC, Fractalkine, SDF-1α, and MIP-3α. Interestingly, these cytokines and chemokines were also upregulated after the infection. The positive correlation between SIV copy number and sTLR2 in the plasma indicated the involvement of TLR2 in the regulation of viral replication. These cytokines and chemokines could directly or indirectly regulate viral replication through the TLR2 signaling pathways. When we stimulated PBMC with the TLR2 agonist in vitro, we observed a direct induction of various cytokines and chemokines. Some of these cytokines and chemokines, such as IL-1RA, IL-9, IL-15, GRO-α, and ENA-78, were positively correlated with sTLR2 in vivo, highlighting the direct involvement of TLR2 in the regulation of the production of these factors. Our findings suggest that TLR2 expression may be a target for developing new therapeutic strategies to combat HIV infection.

The pathophysiology of long-recognized hematologic abnormalities in Ebolavirus (EBOV) disease (EVD) is unknown. From limited human sampling (of peripheral blood), it has been postulated that emergency hematopoiesis plays a role in severe EVD, but the systematic characterization of the bone marrow (BM) has not occurred in human disease or in nonhuman primate models. In a lethal rhesus macaque model of EVD, 18 sternal BM samples exposed to the Kikwit strain of EBOV were compared to those from uninfected controls (n = 3). Immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy showed that EBOV infects BM monocytes/macrophages and megakaryocytes. EBOV exposure was associated with severe BM hypocellularity, including depletion of myeloid, erythroid, and megakaryocyte hematopoietic cells. These depletions were negatively correlated with cell proliferation (Ki67 expression) and were not associated with BM apoptosis during disease progression. In EBOV-infected rhesus macaques with terminal disease, BM showed marked hemophagocytosis, megakaryocyte emperipolesis, and the release of immature hematopoietic cells into the sinusoids. Collectively, these data demonstrate not only direct EBOV infection of BM monocytes/macrophages and megakaryocytes but also that disease progression is associated with hematopoietic failure, notably in peripheral cytopenia. These findings inform current pathophysiologic unknowns and suggest a crucial role for BM dysfunction and/or failure, including emergency hematopoiesis, as part of the natural history of severe human disease.

Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734-736 (ΔQTH) that overlaps 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 that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infection in vivo.

HIV vaccine mediated efficacy, using an expanded live attenuated recombinant varicella virus-vectored SIV rSVV-SIVgag/env vaccine prime with adjuvanted SIV-Env and SIV-Gag protein boosts, was evaluated in a female rhesus macaques (RM) model against repeated intravaginal SIV challenges. Vaccination induced anti-SIV IgG responses and neutralizing antibodies were found in all vaccinated RMs. Three of the eight vaccinated RM remained uninfected (vaccinated and protected, VP) after 13 repeated challenges with the pathogenic SIVmac251-CX-1. The remaining five vaccinated and infected (VI) macaques had significantly reduced plasma viral loads compared with the infected controls (IC). A significant increase in systemic central memory CD4+ T cells and mucosal CD8+ effector memory T-cell responses was detected in vaccinated RMs compared to controls. Variability in lymph node SIV-Gag and Env specific CD4+ and CD8+ T cell cytokine responses were detected in the VI RMs while all three VP RMs had more durable cytokine responses following vaccination and prior to challenge. VI RMs demonstrated predominately SIV-specific monofunctional cytokine responses while the VP RMs generated polyfunctional cytokine responses. This study demonstrates that varicella virus-vectored SIV vaccination with protein boosts induces a 37.5% efficacy rate against pathogenic SIV challenge by generating mucosal memory, virus specific neutralizing antibodies, binding antibodies, and polyfunctional T-cell responses.