Discovery of Highly Potent Small Molecule Pan-Coronavirus Fusion Inhibitors.

The unprecedented pandemic of COVID-19, caused by a novel coronavirus, SARS-CoV-2, and its highly transmissible variants, led to massive human suffering, death, and economic devastation worldwide. Recently, antibody-evasive SARS-CoV-2 subvariants, BQ and XBB, have been reported. Therefore, the continued development of novel drugs with pan-coronavirus inhibition is critical to treat and prevent infection of COVID-19 and any new pandemics that may emerge. We report the discovery of several highly potent small-molecule inhibitors. One of which, NBCoV63, showed low nM potency against SARS-CoV-2 (IC50: 55 nM), SARS-CoV-1 (IC50: 59 nM), and MERS-CoV (IC50: 75 nM) in pseudovirus-based assays with excellent selectivity indices (SI > 900), suggesting its pan-coronavirus inhibition. NBCoV63 showed equally effective antiviral potency against SARS-CoV-2 mutant (D614G) and several variants of concerns (VOCs) such as B.1.617.2 (Delta), B.1.1.529/BA.1 and BA.4/BA.5 (Omicron), and K417T/E484K/N501Y (Gamma). NBCoV63 also showed similar efficacy profiles to Remdesivir against authentic SARS-CoV-2 (Hong Kong strain) and two of its variants (Delta and Omicron), SARS-CoV-1, and MERS-CoV by plaque reduction in Calu-3 cells. Additionally, we show that NBCoV63 inhibits virus-mediated cell-to-cell fusion in a dose-dependent manner. Furthermore, the absorption, distribution, metabolism, and excretion (ADME) data of NBCoV63 demonstrated drug-like properties.

Environmental enrichment alters splenic immune cell composition and enhances secondary influenza vaccine responses in mice.

Chronic stress has deleterious effects on immune function, which can lead to adverse health outcomes. However, studies investigating the impact of stress reduction interventions on immunity in clinical research have yielded divergent results, potentially stemming from differences in study design and genetic heterogeneity, among other clinical research challenges. To test the hypothesis that reducing glucocorticoid levels enhances certain immune functions, we administered influenza vaccine once (prime) or twice (boost) to mice housed in either standard control caging or environmental enrichment (EE) caging. We have shown that this approach reduces mouse corticosterone production. Compared with controls, EE mice had significantly lower levels of fecal corticosterone metabolites (FCMs) and increased splenic B and T lymphocyte numbers. Corticosterone levels were negatively associated with the numbers of CD19(+) (r(2) = 0.43, p = 0.0017), CD4(+) (r(2) = 0.28, p = 0.0154) and CD8(+) cells (r(2) = 0.20, p = 0.0503). Vaccinated mice showed nonsignificant differences in immunoglobulin G (IgG) titer between caging groups, although EE mice tended to exhibit larger increases in titer from prime to boost than controls; the interaction between the caging group (control versus EE) and vaccine group (prime versus boost) showed a strong statistical trend (cage-group*vaccine-group, F = 4.27, p = 0.0555), suggesting that there may be distinct effects of EE caging on primary versus secondary IgG vaccine responses. Vaccine-stimulated splenocytes from boosted EE mice had a significantly greater frequency of interleukin 5 (IL-5)-secreting cells than boosted controls (mean difference 7.7, IL-5 spot-forming units/10(6) splenocytes, 95% confidence interval 0.24-135.1, p = 0.0493) and showed a greater increase in the frequency of IL-5-secreting cells from prime to boost. Our results suggest that corticosterone reduction via EE caging was associated with enhanced secondary vaccine responses, but had little effect on primary responses in mice. These findings help identify differences in primary and secondary vaccine responses in relationship to stress mediators that may be relevant in clinical studies.

CD8 T cell memory recall is enhanced by novel direct interactions with CD4 T cells enabled by MHC class II transferred from APCs.

Protection against many intracellular pathogens is provided by CD8 T cells, which are thought to need CD4 T cell help to develop into effective memory CD8 T cells. Because murine CD8 T cells do not transcribe MHC class II (MHC-II) genes, several models have proposed antigen presenting cells (APCs) as intermediaries required for CD4 T cells to deliver their help to CD8 T cells. Here, we demonstrate the presence of MHC-II molecules on activated murine CD8 T cells in vitro as well as in vivo. These MHC-II molecules are acquired via trogocytosis by CD8 T cells from their activating APCs, particularly CD11c positive dendritic cells (DCs). Transferred MHC-II molecules on activated murine CD8 T cells were functionally competent in stimulating specific indicator CD4 T cells. CD8 T cells that were "helped" in vitro and subsequently allowed to rest in vivo showed enhanced recall responses upon challenge compared to "helpless" CD8 T cells; in contrast, no differences were seen upon immediate challenge. These data indicate that direct CD8:CD4 T cell interactions may significantly contribute to help for CD8 T cells. Furthermore, this mechanism may enable CD8 T cells to communicate with different subsets of interacting CD4 T cells that could modulate immune responses.

CD8+ T cell-derived IFN-γ prevents infection by a second heterologous virus.

Persistent viral infection is often associated with dysfunctional immune responses against unrelated pathogens. Lymphocytic choriomeningitis virus (LCMV) can establish acute or chronic infections in mice and is widely used as a model for persistent virus infections in humans. Mice infected with LCMV develop a transient defect in Ag-specific immunity against heterologous viral infection. Although it has been proposed that LCMV infection induces an immunosuppressed state within the host, our data show that infected mice successfully clear vaccinia virus through a mechanism that involves CD8(+) T cell-derived IFN-γ. This observation demonstrates that chronic LCMV infection does not impair protective immunity against heterologous viral challenge. Rather, a natural sterilizing immunity is induced following a primary infection that prevents a secondary infection. Our findings suggest a need to re-evaluate current thoughts about the immune suppression that might occur during a persistent infection.

Upregulation of retinal dehydrogenase 2 in alternatively activated macrophages during retinoid-dependent type-2 immunity to helminth infection in mice.

Although the vitamin A metabolite retinoic acid (RA) plays a critical role in immune function, RA synthesis during infection is poorly understood. Here, we show that retinal dehydrogenases (Raldh), required for the synthesis of RA, are induced during a retinoid-dependent type-2 immune response elicited by Schistosoma mansoni infection, but not during a retinoid-independent anti-viral immune response. Vitamin A deficient mice have a selective defect in T(H)2 responses to S. mansoni, but retained normal LCMV specific T(H)1 responses. A combination of in situ imaging, intra-vital imaging, and sort purification revealed that alternatively activated macrophages (AAMφ) express high levels of Raldh2 during S. mansoni infection. IL-4 induces Raldh2 expression in bone marrow-derived macrophages in vitro and peritoneal macrophages in vivo. Finally, in vivo derived AAMφ have an enhanced capacity to induce Foxp3 expression in CD4+ cells through an RA dependent mechanism, especially in combination with TGF-ß. The regulation of Raldh enzymes during infection is pathogen specific and reflects differential requirements for RA during effector responses. Specifically, AAMφ are an inducible source of RA synthesis during helminth infections and T(H)2 responses that may be important in regulating immune responses.

The calm mouse: an animal model of stress reduction.

Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, deleterious effects on innate and adaptive immunity, and central nervous system neuropathology. Although stress management is commonly advocated clinically, there is insufficient mechanistic understanding of how decreasing stress affects disease pathogenesis. Therefore, we have developed a "calm mouse model" with caging enhancements designed to reduce murine stress. Male BALB/c mice were divided into four groups: control (Cntl), standard caging; calm (Calm), large caging to reduce animal density, a cardboard nest box for shelter, paper nesting material to promote innate nesting behavior, and a polycarbonate tube to mimic tunneling; control exercise (Cntl Ex), standard caging with a running wheel, known to reduce stress; and calm exercise (Calm Ex), calm caging with a running wheel. Calm, Cntl Ex and Calm Ex animals exhibited significantly less corticosterone production than Cntl animals. We also observed changes in spleen mass, and in vitro splenocyte studies demonstrated that Calm Ex animals had innate and adaptive immune responses that were more sensitive to acute handling stress than those in Cntl. Calm animals gained greater body mass than Cntl, although they had similar food intake, and we also observed changes in body composition, using magnetic resonance imaging. Together, our results suggest that the Calm mouse model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with existing disease models.

An intermediate dose of LCMV clone 13 causes prolonged morbidity that is maintained by CD4+ T cells.

Wasting is a sign of various underlying disorders and is a common feature of cancer, sepsis, and AIDS. We have developed an in vivo model to study the various stages of wasting following infection of mice with lymphocytic choriomeningitis virus cl-13. Using this model we have identified four distinct stages of wasting and have discovered that all stages occur in the different groups of mice regardless of whether the virus is cleared or persists. However, the degree and extent of wasting vary between groups of mice, depending upon the dose of virus administered. Blocking IFNγ or TNFα, which are believed to take part in the wasting process, did not affect the wasting state. Finally, we found that CD4+ T cells control the maintenance stage of wasting. We believe this model will be useful in studying the regulation of wasting during a persistent viral infection, hopefully leading to improved therapies to ameliorate the disorder.

Transient FTY720 treatment promotes immune-mediated clearance of a chronic viral infection.

For a wide variety of microbial pathogens, the outcome of the infection is indeterminate. In some individuals the microbe is cleared, but in others it establishes a chronic infection, and the factors that tip this balance are often unknown. In a widely used model of chronic viral infection, C57BL/6 mice clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV), but the clone 13 strain persists. Here we show that the Armstrong strain induces a profound lymphopenia at days 1-3 after infection, but the clone 13 strain does not. If we transiently augment lymphopenia by treating the clone-13-infected mice with the drug FTY720 at days 0-2 after infection, the mice successfully clear the infection by day 30. Clearance does not occur when CD4 T cells are absent at the time of treatment, indicating that the drug is not exerting direct antiviral effects. Notably, FTY720 treatment of an already established persistent infection also leads to viral clearance. In both models, FTY720 treatment preserves or augments LCMV-specific CD4 and CD8 T-cell responses, a result that is counter-intuitive because FTY720 is generally regarded as a new immunosuppressive agent. Because FTY720 targets host pathways that are completely evolutionarily conserved, our results may be translatable into new immunotherapies for the treatment of chronic microbial infections in humans.

Maternal antibody inhibits both cellular and humoral immunity in response to measles vaccination at birth.

Maternal antibody prevents the use of live, attenuated measles vaccine (LAV) before 6-9 months of age, but vaccinated 6-month-old infants can mount a T cell response. An infant macaque model was used to study the immune response to LAV in the newborn in the presence or absence of maternal antibody. Four newborn monkeys without detectable maternal antibody and 9 newborns with passive measles antibody were vaccinated with LAV. Only the infants without passive antibody seroconverted after vaccination and 3 of 4 of these infants also developed measles-specific interferon gamma+ T cells. The monkeys were challenged with wild-type measles virus at 5 months of age, and 7 of 9 infants vaccinated in the presence of passive antibody had systemic infection and skin rash, while 3 of the 4 infants vaccinated in the absence of passive antibody were protected from viremia and rash. This suggests that the newborn can respond to LAV but that maternal antibody suppresses the priming of both humoral and cellular immunity at birth.

Protection against challenge with measles virus (MV) in infant macaques by an MV DNA vaccine administered in the presence of neutralizing antibody.

Measles virus (MV) infection is the major cause of vaccine-preventable death in infants and children worldwide. It is difficult to achieve immunity to MV infection by use of vaccines in infants during the first 6-9 months of life because of the presence of maternal antibody. Morbidity and mortality due to MV infection would decrease substantially if a vaccine administered at birth could prime immunity in the presence of maternal antibody. We demonstrate here that an MV DNA vaccine administered to infant macaques in the presence of maternal antibody primes MV-specific T cell responses but not de novo neutralizing antibody. This vaccine protected 80% of the infant macaques from skin rash and MV-induced immunosuppression. A molecular interleukin-2 adjuvant was required for protection with this vaccine. This macaque model shows that infants can be vaccinated against MV in the presence of maternal antibody. These results suggest that it is possible to develop an MV DNA vaccine that could protect infants in developing countries during the first months of life.

Prior DNA vaccination does not interfere with the live-attenuated measles vaccine.

The currently used live-attenuated measles vaccine is very effective although maternal antibody prevents its administration prior to 6 months of age. We are investigating the ability of a DNA vaccine encoding the measles viral hemagglutinin, fusion and nucleoprotein to protect newborn infants from measles. Here, we show that a measles DNA vaccine protects juvenile macaques from pathogenic measles virus challenge and that macaques primed and boosted with this DNA vaccine have anemnestic antibody and cell-mediated responses after vaccination with a live-attenuated canine distemper-measles vaccine. Therefore, this DNA vaccine administered to newborn infants may not hinder the subsequent use of live-attenuated measles vaccine.

DNA vaccination of infants in the presence of maternal antibody: a measles model in the primate.

To eradicate measles in developing nations a vaccine capable of being administered at birth may be necessary. We immunized newborn rhesus macaques with naked DNA encoding the measles virus hemagglutinin, fusion and nucleoprotein genes. Prior to vaccination we passively transferred measles immunoglobulin to mimic maternal antibody. In the presence or absence of measles immunoglobulin, 23 of 25 infant macaques had detectable cell mediated immunity and 16 had protective levels of neutralizing antibody. The co-administration of an IL-2/IgG plasmid augmented the vaccine, increasing cell mediated immunity in all infants and increasing the antibody response in infants vaccinated without immunoglobulin. We show for the first time that DNA vaccination can protect a newborn primate from the high-level viremia that correlates with severe measles, even in the presence of maternal antibody. Further, the addition of a molecular IL-2 adjuvant augments this DNA vaccine.

Detection of measles virus RNA in whole blood stored on filter paper.

The purpose of this study was to evaluate the use of dried blood spots stored on filter paper as a means to provide specimens for virologic surveillance for measles virus (MV) in situations when the reverse cold chain is not available. Two single-step RT-PCR assays were evaluated for sensitivity of detection of MV nucleoprotein gene RNA. The more sensitive assay was then used to assess the stability of MV RNA in dried whole blood stored on filter paper. MV RNA was found to be stable in dried blood spots for up to 2 months at room temperature or 1 month at 37 degrees C. As few as 100 infected human peripheral blood mononuclear cells (PBMC) per blood spot could be detected using a single-step RT-PCR reaction and ethidium bromide detection. MV RNA was also detected in dried blood spots obtained from rhesus macaques after challenge with wild-type MV. In the rhesus samples, the single-step RT-PCR reaction could detect approximately 10(3) TCID(50) per blood spot, while nested PCR detected 3 TCID(50) per blood spot. The results of this laboratory-based study suggest that the use of dried blood spots stored on filter has the potential to improve virologic surveillance for MV in some areas, and they emphasize the need for continued testing under field conditions.