The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses.

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on α-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of α-dystroglycan (α-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of α-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant α-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant α-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.

Adverse effects following anti-COVID-19 vaccination with mRNA-based BNT162b2 are alleviated by altering the route of administration and correlate with baseline enrichment of T and NK cell genes.

Ensuring high vaccination and even booster vaccination coverage is critical in preventing severe Coronavirus Disease 2019 (COVID-19). Among the various COVID-19 vaccines currently in use, the mRNA vaccines have shown remarkable effectiveness. However, systemic adverse events (AEs), such as postvaccination fatigue, are prevalent following mRNA vaccination, and the underpinnings of which are not understood. Herein, we found that higher baseline expression of genes related to T and NK cell exhaustion and suppression were positively correlated with the development of moderately severe fatigue after Pfizer-BioNTech BNT162b2 vaccination; increased expression of genes associated with T and NK cell exhaustion and suppression reacted to vaccination were associated with greater levels of innate immune activation at 1 day postvaccination. We further found, in a mouse model, that altering the route of vaccination from intramuscular (i.m.) to subcutaneous (s.c.) could lessen the pro-inflammatory response and correspondingly the extent of systemic AEs; the humoral immune response to BNT162b2 vaccination was not compromised. Instead, it is possible that the s.c. route could improve cytotoxic CD8 T-cell responses to BNT162b2 vaccination. Our findings thus provide a glimpse of the molecular basis of postvaccination fatigue from mRNA vaccination and suggest a readily translatable solution to minimize systemic AEs.

Dysregulated metabolism underpins Zika-virus-infection-associated impairment in fetal development.

Zika virus (ZIKV) is an Aedes-mosquito-borne flavivirus that causes debilitating congenital and developmental disorders. Improved understanding of ZIKV pathogenesis could assist efforts to fill the therapeutic and vaccine gap. We use several ZIKV strains, including a pair differing by a single phenylalanine-to-leucine substitution (M-F37L) in the membrane (M) protein, coupled with unbiased genomics to demarcate the border between attenuated and pathogenic infection. We identify infection-induced metabolic dysregulation as a minimal set of host alterations that differentiates attenuated from pathogenic ZIKV strains. Glycolytic rewiring results in impaired oxidative phosphorylation and mitochondrial dysfunction that trigger inflammation and apoptosis in pathogenic but not attenuated ZIKV strains. Critically, pyruvate supplementation prevents cell death, in vitro, and rescues fetal development in ZIKV-infected dams. Our findings thus demonstrate dysregulated metabolism as an underpinning of ZIKV pathogenicity and raise the potential of pyruvate supplementation in expectant women as a prophylaxis against congenital Zika syndrome.

A mouse model of lethal respiratory dysfunction for SARS-CoV-2 infection.

The global spread of SARS-CoV-2 has made millions ill with COVID-19 and even more from the economic fallout of this pandemic. Our quest to test new therapeutics and vaccines require small animal models that replicate disease phenotypes seen in COVID-19 cases. Rodent models of SARS-CoV-2 infection thus far have shown mild to moderate pulmonary disease; mortality, if any, has been associated with prominent signs of central nervous system (CNS) infection and dysfunction. Here we describe the isolation of SARS-CoV-2 variants with propensity for either pulmonary or CNS infection. Using a wild-type SARS-CoV-2 isolated from a COVID-19 patient, we first found that infection was lethal in transgenic mice expressing the human angiotensin I-converting enzyme 2 (hACE2). Fortuitously, full genome sequencing of SARS-CoV-2 from the brain and lung of these animals showed genetic differences. Likewise, SARS-CoV-2 isolates from brains and lungs of these also showed differences in plaque morphology. Inoculation of these brain and lung SARS-CoV-2 isolates into new batch of hACE2 mice intra-nasally resulted in lethal CNS and pulmonary infection, respectively. Collectively, our study suggests that genetic variants of SARS-CoV-2 could be used to replicate specific features of COVID-19 for the testing of potential vaccines or therapeutics.

A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice.

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 µg and 10 µg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.

Aviremic organ transplant dengue virus transmission - A case report.

Dengue virus (DENV), a mosquito-borne pathogen, causes systemic infections. There are no clear guidelines regarding the screening of donor blood is used in endemic countries to prevent blood transfusion or transplant-associated dengue. DENV has been shown to be detected in urine samples even when DENV viremia is undetectable. We describe an incident of transplant-associated dengue where the donor tested negative for DENV viremia but positive for DENV viuria resulting in the transmission of DENV to our two kidney recipients. Both recipients resolved DENV infection uneventfully, with no adverse impact on the renal graft. Our findings raise the consideration for revised screening recommendations in endemic countries to include DENV RT-PCR in the urine.

Impact of immune enhancement on Covid-19 polyclonal hyperimmune globulin therapy and vaccine development.

The pandemic spread of a novel coronavirus - SARS coronavirus-2 (SARS-CoV-2) as a cause of acute respiratory illness, named Covid-19, is placing the healthcare systems of many countries under unprecedented stress. Global economies are also spiraling towards a recession in fear of this new life-threatening disease. Vaccines that prevent SARS-CoV-2 infection and therapeutics that reduces the risk of severe Covid-19 are thus urgently needed. A rapid method to derive antiviral treatment for Covid-19 is the use of convalescent plasma derived hyperimmune globulin. However, both hyperimmune globulin and vaccine development face a common hurdle - the risk of antibody-mediated disease enhancement. The goal of this review is to examine the body of evidence supporting the hypothesis of immune enhancement that could be pertinent to Covid-19. We also discuss how this risk could be mitigated so that both hyperimmune globulin and vaccines could be rapidly translated to overcome the current global health crisis.

Identification and in vivo Efficacy Assessment of Approved Orally Bioavailable Human Host Protein-Targeting Drugs With Broad Anti-influenza A Activity.

The high genetic variability of influenza A viruses poses a continual challenge to seasonal and pandemic vaccine development, leaving antiviral drugs as the first line of defense against antigenically different strains or new subtypes. As resistance against drugs targeting viral proteins emerges rapidly, we assessed the antiviral activity of already approved drugs that target cellular proteins involved in the viral life cycle and were orally bioavailable. Out of 15 candidate compounds, four were able to inhibit infection by 10- to 100-fold without causing toxicity, in vitro. Two of the drugs, dextromethorphan and ketotifen, displayed a 50% effective dose between 5 and 50 µM, not only for the classic H1N1 PR8 strain, but also for a pandemic H1N1 and a seasonal H3N2 strain. Efficacy assessment in mice revealed that dextromethorphan consistently resulted in a significant reduction of viral lung titers and also enhanced the efficacy of oseltamivir. Dextromethorphan treatment of ferrets infected with a pandemic H1N1 strain led to a reduction in clinical disease severity, but no effect on viral titer was observed. In addition to identifying dextromethorphan as a potential influenza treatment option, our study illustrates the feasibility of a bioinformatics-driven rational approach for repurposing approved drugs against infectious diseases.

Genomic signature of early T-cell response is associated with lower antibody titer threshold for sterilizing immunity.

Vaccination is an effective approach to reduce disease burden. High vaccination coverage blocks pathogen transmission to ensure herd immunity. However, the concept of herd immunity assumes that vaccinated individuals cannot be infected and mediate silent pathogen transmission. While the correlates of vaccine-mediated protection against disease have been examined, the correlates of sterilizing immunity that prevents infection have not been systematically defined. Here, we used full genome expression profiling to explore the molecular correlates of serological response and non-response to measles, mumps and rubella (MMR) vaccination as surrogates of infection and sterilizing immunity, respectively. We observed that the antibody titers needed to sterilize infection with the vaccine strains were higher than current WHO disease protection thresholds. In subjects with baseline antibodies below such sterilizing immunity thresholds, serological non-response to MMR vaccination was associated with gene expression profile indicative of early T-cell activation and signalling. Specifically, genes that regulate T-cell function and response were induced at day 1 post-vaccination in non-responders but not in responders. These findings suggest that rapid T-cell response prevented MMR vaccine infection to limit antigenic presentation and hence serological response. Collectively, our findings suggest an important role for T-cells in engendering sterilizing immunity.

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope.

Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods to engineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer. ZAb_FLEP neutralized a breadth of ZIKV strains and protected mice in distinct in vivo models, including resolving vertical transmission and fetal mortality in infected pregnant mice. Serial passaging of ZIKV in the presence of ZAb_FLEP failed to generate viral escape mutants, suggesting that its epitope is indeed mutationally constrained. A single-particle cryo-EM reconstruction of the Fab-ZIKV complex validated the structural model and revealed insights into ZAb_FLEP's neutralization mechanism. ZAb_FLEP has potential as a therapeutic in future outbreaks.

A systematic approach to the development of a safe live attenuated Zika vaccine.

Zika virus (ZIKV) is a flavivirus that can cause congenital disease and requires development of an effective long-term preventative strategy. A replicative ZIKV vaccine with properties similar to the yellow fever 17D (YF17D) live-attenuated vaccine (LAV) would be advantageous, as a single dose of YF17D produces lifelong immunity. However, a replicative ZIKV vaccine must also be safe from causing persistent organ infections. Here we report an approach to ZIKV LAV development. We identify a ZIKV variant that produces small plaques due to interferon (IFN)-restricted viral propagation and displays attenuated infection of endothelial cells. We show that these properties collectively reduce the risk of organ infections and vertical transmission in a mouse model but remain sufficiently immunogenic to prevent wild-type ZIKV infection. Our findings suggest a strategy for the development of a safe but efficacious ZIKV LAV.

Dengue virus activates cGAS through the release of mitochondrial DNA.

Cyclic GMP-AMP synthetase (cGAS) is a DNA-specific cytosolic sensor, which detects and initiates host defense responses against microbial DNA. It is thus curious that a recent study identified cGAS as playing important roles in inhibiting positive-sense single-stranded RNA (+ssRNA) viral infection, especially since RNA is not known to activate cGAS. Using a dengue virus serotype 2 (DENV-2) vaccine strain (PDK53), we show that infection creates an endogenous source of cytosolic DNA in infected cells through the release of mitochondrial DNA (mtDNA) to drive the production of cGAMP by cGAS. Innate immune responses triggered by cGAMP contribute to limiting the spread of DENV to adjacent uninfected cells through contact dependent gap junctions. Our result thus supports the notion that RNA virus indirectly activates a DNA-specific innate immune signaling pathway and highlights the breadth of the cGAS-induced antiviral response.

Dengue virus compartmentalization during antibody-enhanced infection.

Secondary infection with a heterologous dengue virus (DENV) serotype increases the risk of severe dengue, through a process termed antibody-dependent enhancement (ADE). During ADE, DENV is opsonized with non- or sub-neutralizing antibody levels that augment entry into monocytes and dendritic cells through Fc-gamma receptors (FcγRs). We previously reported that co-ligation of leukocyte immunoglobulin-like receptor-B1 (LILRB1) by antibody-opsonized DENV led to recruitment of SH2 domain-containing phosphatase-1 (SHP-1) to dephosphorylate spleen tyrosine kinase (Syk) and reduce interferon stimulated gene induction. Here, we show that LILRB1 also signals through SHP-1 to attenuate the otherwise rapid acidification for lysosomal enzyme activation following FcγR-mediated uptake of DENV. Reduced or slower trafficking of antibody-opsonized DENV to lytic phagolysosomal compartments, demonstrates how co-ligation of LILRB1 also permits DENV to overcome a cell-autonomous immune response, enhancing intracellular survival of DENV. Our findings provide insights on how antiviral drugs that modify phagosome acidification should be used for viruses such as DENV.