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RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans and the technology, is being leveraged for rapid emergency response. In this report, we assessed immunogenicity, and, for the first time, toxicity, biodistribution and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion stabilized Spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNP). In mice, one immunization with the SAM vaccine elicited a robust Spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of Spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to Phase-1 clinical evaluation (NCT04758962)., Graphical Abstract, Comprehensive preclinical development of a lipid nanoparticle (LNP)-formulated SAM vaccine encoding the prefusion stabilized SARS-CoV-2 Spike full-length antigen is reported. Protective efficacy, immunogenicity, toxicity, and biodistribution assessment of this candidate vaccine in mice, rats, and hamsters showed a favorable safety profile and robust immune responses against SARS-CoV-2 infection and disease.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the global coronavirus disease 2019 (COVID-19) pandemic, contains a unique, four amino acid (aa) "PRRA" insertion in the spike (S) protein that creates a transmembrane protease serine 2 (TMPRSS2)/furin cleavage site and enhances viral infectivity. More research into immunogenic epitopes and protective antibodies against this SARS-CoV-2 furin cleavage site is needed.MethodsCombining computational and experimental methods, we identified and characterized an immunogenic epitope overlapping the furin cleavage site that detects antibodies in COVID-19 patients and elicits strong antibody responses in immunized mice. We also identified a high-affinity monoclonal antibody from COVID-19 patient peripheral blood mononuclear cells; the antibody directly binds the furin cleavage site and protects against SARS-CoV-2 infection in a mouse model.FindingsThe presence of "PRRA" amino acids in the S protein of SARS-CoV-2 not only creates a furin cleavage site but also generates an immunogenic epitope that elicits an antibody response in COVID-19 patients. An antibody against this epitope protected against SARS-CoV-2 infection in mice.InterpretationThe immunogenic epitope and protective antibody we have identified may augment our strategy in handling COVID-19 epidemic.FundingThe National Natural Science Foundation of China (82102371, 91542201, 81925025, 82073181, and 81802870), the Chinese Academy of Medical Sciences Initiative for Innovative Medicine (2021-I2M-1-047 and 2022-I2M-2-004), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2020-PT310-006, 2019XK310002, and 2018TX31001), the National Key Research and Development Project of China (2020YFC0841700), US National Institute of Health (NIH) funds grant AI158154, University of California Los Angeles (UCLA) AI and Charity Treks, and UCLA DGSOM BSCRC COVID-19 Award Program. H.Y. is supported by Natural Science Foundation of Jiangsu Province (BK20211554 andBE2022728).

Summary Beginning in the summer of 2020, a variant of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the United Kingdom (UK). This B.1.1.7 variant increased rapidly in prevalence among sequenced strains, attributed to an increase in infection and/or transmission efficiency. The UK variant has 19 nonsynonymous mutations across its viral genome including 8 substitutions or deletions in the spike protein, which interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that, of the 8 individual spike protein substitutions, only N501Y exhibited consistent fitness gains for replication in the upper airway in the hamster model as well as primary human airway epithelial cells. The N501Y substitution recapitulated the phenotype of enhanced viral transmission seen with the combined 8 UK spike mutations, suggesting it is a major determinant responsible for increased transmission of this variant. Mechanistically, the N501Y substitution improved the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil and South Africa, our results indicate that N501Y substitution is a major adaptive spike mutation of major concern.

The rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as the Omicron variants that are highly transmissible and immune evasive, underscores the need to develop therapeutic antibodies with broad neutralizing activities. Here, we used the LIBRA-seq technology, which identified SARS-CoV-2–specific B cells via DNA barcoding and subsequently single-cell sequenced BCRs, to identify an antibody, SW186, which could neutralize major SARS-CoV-2 variants of concern, including Beta, Delta, and Omicron, as well as SARS-CoV-1. The cryo-EM structure of SW186 bound to the receptor binding domain (RBD) of the viral spike protein showed that SW186 interacted with an epitope of the RBD that is not at the interface of its binding to the ACE2 receptor but is highly conserved among SARS coronaviruses. This epitope encompasses a glycosylation site (N343) of the viral spike protein. Administration of SW186 in mice after they were infected with SARS-CoV-2 Alpha, Beta, or Delta variants reduced the viral loads in the lung. These results demonstrated that SW186 neutralizes diverse SARS coronaviruses by binding to a conserved RBD epitope, which could serve as a target for further antibody development.

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase–mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.

Emergence of SARS-CoV-2 variants of concern (VOCs), including the highly transmissible Omicron and Delta strains, has posed constant challenges to the current COVID-19 vaccines that principally target the viral spike protein (S). Here, we report a nucleoside-modified messenger RNA (mRNA) vaccine that expresses the more conserved viral nucleoprotein (mRNA-N) and show that mRNA-N vaccination alone can induce modest control of SARS-CoV-2. Critically, combining mRNA-N with the clinically proven S-expressing mRNA vaccine (mRNA-S+N) induced robust protection against both Delta and Omicron variants. In the hamster models of SARS-CoV-2 VOC challenge, we demonstrated that, compared to mRNA-S alone, combination mRNA-S+N vaccination not only induced more robust control of the Delta and Omicron variants in the lungs but also provided enhanced protection in the upper respiratory tract. In vivo CD8 + T cell depletion suggested a potential role for CD8 + T cells in protection conferred by mRNA-S+N vaccination. Antigen-specific immune analyses indicated that N-specific immunity, as well as augmented S-specific immunity, was associated with enhanced protection elicited by the combination mRNA vaccination. Our findings suggest that combined mRNA-S+N vaccination is an effective approach for promoting broad protection against SARS-CoV-2 variants.

Zika virus (ZIKV), a mosquito-borne flavivirus, can cause severe eye disease and even blindness in newborns. However, ZIKV-induced retinal lesions have not been studied in a comprehensive way, mechanisms of ZIKV-induced retinal abnormalities are unknown, and no therapeutic intervention is available to treat or minimize the degree of vision loss in patients. Here, we developed a novel mouse model of ZIKV infection to evaluate its impact on retinal structure. ZIKV (20 plaque-forming units) was inoculated into neonatal wild type C57BL/6J mice at postnatal day (P) 0 subcutaneously. Retinas of infected mice and age-matched controls were collected at various ages, and retinal structural alterations were analyzed. We found that ZIKV induced progressive neuronal and vascular damage and retinal inflammation starting from P8. ZIKV-infected retina exhibited dramatically decreased thickness with loss of neurons, initial neovascular tufts followed by vessel dilation and degeneration, increased microglia and leukocyte recruitment and activation, degeneration of astrocyte network and gliosis. The above changes may involve inflammation and endoplasmic reticulum stress-mediated cell apoptosis and necroptosis. Moreover, we evaluated the efficacy of preclinical drugs and the safety of ZIKV vaccine candidate in this mouse model. We found that ZIKV-induced retinal abnormalities could be blocked by a selective flavivirus inhibitor NITD008 and a live-attenuated ZIKV vaccine candidate could potentially induce retinal abnormalities. Overall, we established a novel mouse model and provide a direct causative link between ZIKV and retinal lesion in vivo, which warrants further investigation of the underlying mechanisms of ZIKV-induced retinopathy and the development of effective therapeutics. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01195-6.

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD–foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD–foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD ‘down’, one-RBD ‘up’ state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime–boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2–18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1–3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728). BNT162b1 and BNT162b2 are two candidate mRNA vaccines against COVID-19 that elicit high virus-entry inhibition titres in mice, elicit high virus-neutralizing titres in rhesus macaques and protect macaques from SARS-CoV-2 challenge.

Zika virus (ZIKV) emerged from obscurity in 2013 to spread from Asia to the South Pacific and the Americas, where millions of people were infected, accompanied by severe disease including microcephaly following congenital infections. Phylogenetic studies have shown that ZIKV evolved in Africa and later spread to Asia, and that the Asian lineage is responsible for the recent epidemics in the South Pacific and Americas. However, the reasons for the sudden emergence of ZIKV remain enigmatic. Here we report evolutionary analyses that revealed four mutations, which occurred just before ZIKV introduction to the Americas, represent direct reversions of previous mutations that accompanied earlier spread from Africa to Asia and early circulation there. Our experimental infections of Aedes aegypti mosquitoes, human cells, and mice using ZIKV strains with and without these mutations demonstrate that the original mutations reduced fitness for urban, human-amplifed transmission, while the reversions restored fitness, increasing epidemic risk. These findings include characterization of three transmission-adaptive ZIKV mutations, and demonstration that these and one identified previously restored fitness for epidemic transmission soon before introduction into the Americas. The initial mutations may have followed founder effects and/or drift when the virus was introduced decades ago into Asia., The trajectory of the emergence of Zika virus (ZIKV) into the Americas remains unclear. Here, the authors find that four mutations originated before ZIKV introduction to the Americas are direct reversions of previous mutations that accompanied spread many decades ago from ZIKV’s native Africa to Asia, and show in experimental infections of mosquitoes, human cells, and mice that the original mutations reduced fitness for urban transmission, while the reversions restored fitness, likely increasing epidemic risk.

Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn’t escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19., Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. Here, Ku et al., identify SARS-CoV-2 neutralizing antibodies from a phage library and identify an antibody combination that prevents viral escape and protects mice from viral challenge.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.

Arboviruses maintain high mutation rates due to lack of proofreading ability of their viral polymerases, in some cases facilitating adaptive evolution and emergence. Here we show that, just before its 2013 spread to the Americas, Zika virus (ZIKV) underwent an envelope protein V473M substitution (E-V473M) that increased neurovirulence, maternal-to-fetal transmission, and viremia to facilitate urban transmission. A preepidemic Asian ZIKV strain (FSS13025 isolated in Cambodia in 2010) engineered with the V473M substitution significantly increased neurovirulence in neonatal mice and produced higher viral loads in the placenta and fetal heads in pregnant mice. Conversely, an epidemic ZIKV strain (PRVABC59 isolated in Puerto Rico in 2015) engineered with the inverse M473V substitution reversed the pathogenic phenotypes. Although E-V473M did not affect oral infection of Aedes aegypti mosquitoes, competition experiments in cynomolgus macaques showed that this mutation increased its fitness for viremia generation, suggesting adaptive evolution for human viremia and hence transmission. Mechanistically, the V473M mutation, located at the second transmembrane helix of the E protein, enhances virion morphogenesis. Overall, our study revealed E-V473M as a critical determinant for enhanced ZIKV virulence, intrauterine transmission during pregnancy, and viremia to facilitate urban transmission.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced following infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases (NIAID) within the National Institutes of Health (NIH) established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) program. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants potentially impacting transmission, virulence, and resistance to convalescent and vaccine-induced immunity. The SAVE program serves as a critical data-generating component of the United States Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines, and therapeutics and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity, and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models, and pivotal findings facilitated by this collaborative approach and identify future challenges. This program serves as a template for the response against rapidly evolving pandemic pathogens by monitoring viral evolution in the human population to identify variants that could erode the effectiveness of countermeasures.

Development of optimal SARS-CoV-2 vaccines to induce potent, long-lasting immunity and provide cross-reactive protection against emerging variants remains a high priority. Here, we report that a modified porous silicon microparticle (mPSM) adjuvant to SARS-CoV-2 receptor-binding domain (RBD) vaccine activated dendritic cells and generated more potent and durable systemic humoral and type 1 helper T (Th) cell- mediated immune responses than alum-formulated RBD following parenteral vaccination, and protected mice from SARS-CoV-2 and Beta variant challenge. Notably, mPSM facilitated the uptake of SARS-CoV-2 RBD antigens by nasal and airway epithelial cells. Parenteral and intranasal prime and boost vaccinations with mPSM-RBD elicited stronger lung resident T and B cells and IgA responses compared to parenteral vaccination alone, which led to markedly diminished viral loads and inflammation in the lung following SARS-CoV-2 Delta variant challenge. Overall, our results suggest that mPSM is effective adjuvant for SARS-CoV-2 subunit vaccine in both systemic and mucosal vaccinations.

We report a live-attenuated SARS-CoV-2 vaccine candidate with (i) re-engineered viral transcription regulator sequences and (ii) deleted open-reading-frames (ORF) 3, 6, 7, and 8 (∆3678). The ∆3678 virus replicates about 7,500-fold lower than wild-type SARS-CoV-2 on primary human airway cultures, but restores its replication on interferon-deficient Vero-E6 cells that are approved for vaccine production. The ∆3678 virus is highly attenuated in both hamster and K18-hACE2 mouse models. A single-dose immunization of the ∆3678 virus protects hamsters from wild-type virus challenge and transmission. Among the deleted ORFs in the ∆3678 virus, ORF3a accounts for the most attenuation through antagonizing STAT1 phosphorylation during type-I interferon signaling. We also developed an mNeonGreen reporter ∆3678 virus for high-throughput neutralization and antiviral testing. Altogether, the results suggest that ∆3678 SARS-CoV-2 may serve as a live-attenuated vaccine candidate and a research tool for potential biosafety level-2 use.

The global health emergency posed by the outbreak of Zika virus (ZIKV), an arthropod-borne flavivirus causing severe neonatal neurological conditions, has subsided, but there continues to be transmission of ZIKV in endemic regions. As such, there is still a medical need for discovering and developing therapeutical interventions against ZIKV. To identify small-molecule compounds that inhibit ZIKV disease and transmission, we screened multiple small-molecule collections, mostly derived from natural products, for their ability to inhibit wild-type ZIKV. As a primary high-throughput screen, we used a viral cytopathic effect (CPE) inhibition assay conducted in Vero cells that was optimized and miniaturized to a 1536-well format. Suitably active compounds identified from the primary screen were tested in a panel of orthogonal assays using recombinant Zika viruses, including a ZIKV Renilla luciferase reporter assay and a ZIKV mCherry reporter system. Compounds that were active in the wild-type ZIKV inhibition and ZIKV reporter assays were further evaluated for their inhibitory effects against other flaviviruses. Lastly, we demonstrated that wild-type ZIKV is able to infect a 3D-bioprinted outer-blood-retina barrier tissue model and disrupt its barrier function, as measured by electrical resistance. One of the identified compounds (3-Acetyl-13-deoxyphomenone, NCGC00380955) was able to prevent the pathological effects of the viral infection on this clinically relevant ZIKV infection model.

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce emergence of antibody resistance. Here we engineer two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv)2 design (14-H-06) but not the CrossMAb design (14-crs-06) shows increased antigen-binding and virus-neutralizing activities against multiple SARS-CoV-2 variants as well as increased breadth of neutralizing activity compared to the cocktail. X-ray crystallography and cryo-EM reveal distinct binding models for individual cocktail antibodies, and computational simulations suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and multiple variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth. Published version This work was supported in part by a Welch Foundation grant AU-0042- 20030616 and Cancer Prevention and Research Institute of Texas (CPRIT) Grants RP150551 and RP190561 (Z.A.); NIH grants HHSN272201600013C, AI134907, AI145617, and UL1TR001439, and awards from the Sealy Smith Foundation, Kleberg Foundation, John S. Dunn Foundation, Amon G. Carter Foundation, Gillson Longenbaugh Foundation, and Summerfield Robert Foundation (P-Y.S.); and AcRF Tier 1 RG105/20 (J.L.).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

Zika virus (ZIKV) and dengue virus (DENV) are arthropod-borne pathogenic flaviviruses that co-circulate in many countries. To understand some of the pressures that influence ZIKV evolution, we mimic the natural transmission cycle by repeating serial passaging of ZIKV through cultured mosquito cells and either DENV-naive or DENV-immune mice. Compared with wild-type ZIKV, the strains passaged under both conditions exhibit increased pathogenesis in DENV-immune mice. Application of reverse genetics identifies an isoleucine-to-valine mutation (I39V) in the NS2B proteins of both passaged strains that confers enhanced fitness and escape from pre-existing DENV immunity. Introduction of I39V or I39T, a naturally occurring homologous mutation detected in recent ZIKV isolates, increases the replication of wild-type ZIKV in human neuronal precursor cells and laboratory-raised mosquitoes. Our data indicate that ZIKV strains with enhanced transmissibility and pathogenicity can emerge in DENV-naive or -immune settings, and that NS2B-I39 mutants may represent ZIKV variants of interest.

Zika virus (ZIKV) caused millions of infections during its rapid and expansive spread from Asia to the Americas from 2015 to 2017. Here, we compared the infectivity of ZIKV mutants with individual stable substitutions which emerged throughout the Asian ZIKV lineage and were responsible for the explosive outbreaks in the Americas. A threonine (T) to alanine (A) mutation at the 106th residue of the ZIKV capsid (C) protein facilitated the transmission by its mosquito vector, as well as infection in both human cells and immunodeficient mice. A mechanistic study showed that the T106A substitution rendered the C a preferred substrate for the NS2B-NS3 protease, thereby facilitating the maturation of structural proteins and the formation of infectious viral particles. Over a complete “mosquito-mouse-mosquito” cycle, the ZIKV C-T106A mutant showed a higher prevalence of mosquito infection than did the preepidemic strain, thus promoting ZIKV dissemination. Our results support the contribution of this evolutionary adaptation to the occasional widespread reemergence of ZIKV in nature.

This work was supported by Amon G. Carter Foundation; Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico: [Grant Number 303999/2016-0,440405/2016-5]; Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior: [Grant Number Zika fast track project]; John S. Dunn Foundation; Gilson Longenbaugh Foundation; NIH: [Grant Number AI127744,AI136126,AI142759];PAHO: [Grant Number SCON2016-01353]; Summerfield Robert Foundation; Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation; Sealy & Smith Foundation. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Health Sciences Institute. Belem, PA, Brazil. Department of Biochemistry & Molecular Biology. Galveston, TX, USA. Department of Biochemistry & Molecular Biology. Galveston, TX, USA. Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Department of Microbiology & Immunology. Galveston, TX, USA. Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Health Sciences Institute. Belem, PA, Brazil. Health Sciences Institute. Belem, PA, Brazil / Federal University of Par?. Biological Sciences Institute. Belem, PA, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Par? State University. Department of Pathology. Bel?m, PA, Brazil. Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Institute for Human Infections & Immunity. Galveston, TX, USA / Institute for Translational Science. Galveston, TX, USA / Sealy Institute of Vaccine Sciences. Galveston, TX, USA / Texas Medical Branch. Sealy Center for Structural Biology & Molecular Biophysics. Galveston, TX, USA. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Programa de P?s-Gradua??o em Virologia. Ananindeua, PA, Brasil / Health Sciences Institute. Bel?m, PA, Brazil. The Asian lineage of Zika virus (ZIKV) is responsible for the recent epidemics in the Americas and severe disease, whereas the African lineage of ZIKV has not been reported to cause epidemics or severe disease. We constructed a cDNA infectious clone (IC) of an African ZIKV strain, which, together with our previously developed Asian ZIKV strain IC, allowed us to engineer chimeric viruses by swapping the structural and non-structural genes between the two lineages. Recombinant parental and chimeric viruses were analyzed in A129 and newborn CD1 mouse models. In the A129 mice, the African strain developed higher viremia, organ viral loading, and mortality rate. In CD1 mice, the African strain exhibited a higher neurovirulence than the Asian strain. A chimeric virus containing the structural genes from the African strain is more virulent than the Asian strain, whereas a chimeric virus containing the non-structural genes from the African strain exhibited a virulence comparable to the Asian strain. These results suggest that (i) African strain is more virulent than Asian strain and (ii) viral structural genes primarily determine the virulence difference between the two lineages in mouse models. Other factors may contribute to the discrepancy between the mouse and epidemic results.

Infections with mosquito-borne flaviviruses, such as Dengue virus, ZIKV virus, and West Nile virus, pose significant threats to public health. Flaviviruses cause up to 400 million infections each year, leading to many forms of diseases, including fatal hemorrhagic, encephalitis, congenital abnormalities, and deaths. Currently, there are no clinically approved antiviral drugs for the treatment of flavivirus infections. The non-structural protein NS4B is an emerging target for drug discovery due to its multiple roles in the flaviviral life cycle. In this review, we summarize the latest knowledge on the structure and function of flavivirus NS4B, as well as the progress on antiviral compounds that target NS4B.

The genus Flavivirus comprises numerous emerging and re‐emerging arboviruses causing human illness. Vaccines are the best approach to prevent flavivirus diseases. But pathogen diversities are always one of the major hindrances for timely development of new vaccines when confronting unpredicted flavivirus outbreaks. We used West Nile virus (WNV) as a model to develop a new live‐attenuated vaccine (LAV), WNV‐poly(A), by replacing 5ʹ portion (corresponding to SL and DB domains in WNV) of 3ʹ‐UTR with internal poly(A) tract. WNV‐poly(A) not only propagated efficiently in Vero cells, but also was highly attenuated in mouse model. A single‐dose vaccination elicited robust and long‐lasting immune responses, conferring full protection against WNV challenge. Such “poly(A)” vaccine strategy may be promising for wide application in the development of flavivirus LAVs because of its general target regions in flaviviruses., West Nile virus (WNV) is one of the many members of the genus Flavivirus known to cause human diseases. Here we present a novel live attenuated WNV vaccine, WNV‐poly(A), that protects against WNV infections in a mouse model.

Summary SARS-CoV-2 variants that attenuate antibody neutralization could jeopardize vaccine efficacy. We recently reported the protective activity of an intranasally administered spike protein-based chimpanzee adenovirus-vectored vaccine (ChAd-SARS-CoV-2-S) in animals, which has advanced to human trials. Here, we assessed its durability, dose response, and cross-protective activity in mice. A single intranasal dose of ChAd-SARS-CoV-2-S induced durably high neutralizing and Fc effector antibody responses in serum and S-specific IgG and IgA secreting long-lived plasma cells in the bone marrow. Protection against a historical SARS-CoV-2 strain was observed across a 100-fold vaccine dose range and over a 200-day period. At 6 weeks or 9 months after vaccination, serum antibodies neutralized SARS-CoV-2 strains with B.1.351, B.1.1.28, and B.1.617.1 spike proteins and conferred almost complete protection in the upper and lower respiratory tracts after challenge with variant viruses. Thus, in mice, intranasal immunization with ChAd-SARS-CoV-2-S provides durable protection against historical and emerging SARS-CoV-2 strains., Graphical abstract, Highlights • Immunization with ChAd-SARS-CoV-2 S induces durable immunity • Intranasal ChAd-SARS-CoV-2 S induces inhibitory IgG and IgA Abs • Abs induced by intranasal ChAd-SARS-CoV-2 S have robust Fc effector functions • Intranasal ChAd-SARS-CoV-2 S confers cross-protection against variants of concern, Hassan et al. show that immunization with ChAd-SARS-CoV-2-S is durably immunogenic and protects against SARS-CoV-2 challenge in a dose-dependent manner. Many months after single-dose intranasal immunization, ChAd-SARS-CoV-2 confers protection against variants of concerns of SARS-CoV-2 in both the upper and lower respiratory tracts of mice.

There is a great need for the development of vaccines that induce potent and long-lasting protective immunity against SARS-CoV-2. Multimeric display of the antigen combined with potent adjuvant can enhance the potency and longevity of the antibody response. The receptor binding domain (RBD) of the spike protein is a primary target of neutralizing antibodies. Here, we developed a trimeric form of the RBD and show that it induces a potent neutralizing antibody response against live virus with diverse effector functions and provides protection against SARS-CoV-2 challenge in mice and rhesus macaques. The trimeric form induces higher neutralizing antibody titer compared to monomer with as low as 1μg antigen dose. In mice, adjuvanting the protein with a TLR7/8 agonist formulation alum-3M-052 induces 100-fold higher neutralizing antibody titer and superior protection from infection compared to alum. SARS-CoV-2 infection causes significant loss of innate cells and pathology in the lung, and vaccination protects from changes in innate cells and lung pathology. These results demonstrate RBD trimer protein as a suitable candidate for vaccine against SARS-CoV-2., Efficient vaccines for SARS-CoV-2 are needed. Here, the authors show that a trimeric form of the receptor-binding domain of SARS-CoV-2 spike adjuvanted with alum-3M-052 protects non-human primates from disease and inhibits infection.

Rapidly-emerging variants jeopardize antibody-based countermeasures against SARS-CoV-2. While recent cell culture experiments have demonstrated loss of potency of several anti-spike neutralizing antibodies against SARS-CoV-2 variant strains1-3, the in vivo significance of these results remains uncertain. Here, using a panel of monoclonal antibodies (mAbs) corresponding to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron, and Lilly we report the impact on protection in animals against authentic SARS-CoV-2 variants including WA1/2020 strains, a B.1.1.7 isolate, and chimeric strains with South African (B.1.351) or Brazilian (B.1.1.28) spike genes. Although some individual mAbs showed reduced or abrogated neutralizing activity against B.1.351 and B.1.1.28 viruses with E484K spike protein mutations in cell culture, low prophylactic doses of mAb combinations protected against infection in K18-hACE2 transgenic mice, 129S2 immunocompetent mice, and hamsters without emergence of resistance. Two exceptions were mAb LY-CoV555 monotherapy which lost all protective activity in vivo, and AbbVie 2B04/47D11, which showed partial loss of activity. When administered after infection as therapy, higher doses of mAb cocktails protected in vivo against viruses displaying a B.1.351 spike gene. Thus, many, but not all, of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing SARS-CoV-2 variant strains.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuing to evolve around the world, generating new variants that are of concern on the basis of their potential for altered transmissibility, pathogenicity, and coverage by vaccines and therapeutic agents1–5. Here we show that serum samples taken from twenty human volunteers, two or four weeks after their second dose of the BNT162b2 vaccine, neutralize engineered SARS-CoV-2 with a USA-WA1/2020 genetic background (a virus strain isolated in January 2020) and spike glycoproteins from the recently identified B.1.617.1, B.1.617.2, B.1.618 (all of which were first identified in India) or B.1.525 (first identified in Nigeria) lineages. Geometric mean plaque reduction neutralization titres against the variant viruses—particularly the B.1.617.1 variant—seemed to be lower than the titre against the USA-WA1/2020 virus, but all sera tested neutralized the variant viruses at titres of at least 1:40. The susceptibility of the variant strains to neutralization elicited by the BNT162b2 vaccine supports mass immunization as a central strategy to end the coronavirus disease 2019 (COVID-19) pandemic globally. Samples of serum from individuals immunized with the BNT162b2 vaccine show neutralization activity against engineered SARS-CoV-2s bearing the spike mutations from B.1.617 and other variants.

The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of Coronavirus Disease 2019 (COVID-19) have been hampered by the lack of robust mouse models. To overcome this barrier, we used a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARS-CoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Coupled with the incorporation of mutations found in variants of concern, CMA3p20 offers several advantages over other mouse-adapted SARS-CoV-2 strains. Using this model, we demonstrate that SARS-CoV-2–infected mice are protected from lethal challenge with the original Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), suggesting immunity from heterologous Coronavirus (CoV) strains. Together, the results highlight the use of this mouse model for further study of SARS-CoV-2 infection and disease., Studying cross-protection from different coronaviruses is important to inform the research for a universal vaccine. This study uses a mouse-adapted SARS-CoV-2 strain to show that it confers protection from SARS-CoV challenge, suggesting possible immunity from heterologous challenge following natural infection.

Co-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses has been reported. We evaluated cell lines commonly used to isolate viruses and diagnose related diseases for their susceptibility to SARS-CoV-2. Although multiple kidney cell lines from monkeys were susceptible to SARS-CoV-2, we found many cell types derived from humans, dogs, minks, cats, mice, and chicken were not. We analyzed MDCK cells, which are most commonly used for surveillance and study of influenza viruses, and found that they were not susceptible to SARS-CoV-2. The low expression level of the angiotensin converting enzyme 2 receptor and lower receptor affinity to SARS-CoV-2 spike, which could be overcome by overexpression of canine angiotensin converting enzyme 2 in trans, strengthened the cellular barrier to productive infection. Moreover, a D614G mutation in the spike protein did not appear to affect SARS-CoV-2 cell tropism. Our findings should help avert inadvertent propagation of SARS-CoV-2 from diagnostic cell lines.

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern., Graphical abstract, SARS-CoV-2 variants with increased transmissibility are a public health threat. Schmitz et al. characterize 2C08, a human monoclonal antibody derived from a SARS-CoV-2 vaccine-induced germinal center B cell. 2C08 possesses a broad and potent neutralization capacity and protects hamsters against challenge with D614G, B.1.351, or B.1.617.2 strains. Public 2C08-like clones can be elicited by both SARS-CoV-2 infection and vaccination.

SARS-CoV-2 mRNA-based vaccines are about 95% effective in preventing COVID-191–5. The dynamics of antibody-secreting plasmablasts and germinal centre B cells induced by these vaccines in humans remain unclear. Here we examined antigen-specific B cell responses in peripheral blood (n = 41) and draining lymph nodes in 14 individuals who had received 2 doses of BNT162b2, an mRNA-based vaccine that encodes the full-length SARS-CoV-2 spike (S) gene1. Circulating IgG- and IgA-secreting plasmablasts that target the S protein peaked one week after the second immunization and then declined, becoming undetectable three weeks later. These plasmablast responses preceded maximal levels of serum anti-S binding and neutralizing antibodies to an early circulating SARS-CoV-2 strain as well as emerging variants, especially in individuals who had previously been infected with SARS-CoV-2 (who produced the most robust serological responses). By examining fine needle aspirates of draining axillary lymph nodes, we identified germinal centre B cells that bound S protein in all participants who were sampled after primary immunization. High frequencies of S-binding germinal centre B cells and plasmablasts were sustained in these draining lymph nodes for at least 12 weeks after the booster immunization. S-binding monoclonal antibodies derived from germinal centre B cells predominantly targeted the receptor-binding domain of the S protein, and fewer clones bound to the N-terminal domain or to epitopes shared with the S proteins of the human betacoronaviruses OC43 and HKU1. These latter cross-reactive B cell clones had higher levels of somatic hypermutation as compared to those that recognized only the SARS-CoV-2 S protein, which suggests a memory B cell origin. Our studies demonstrate that SARS-CoV-2 mRNA-based vaccination of humans induces a persistent germinal centre B cell response, which enables the generation of robust humoral immunity. Analysis of antigen-specific B cells in lymph nodes of individuals vaccinated with BNT162b2 reveals lasting germinal centre responses, explaining the robust humoral immunity induced by SARS-CoV-2 mRNA-based vaccines.

A combination of vaccination approaches will likely be necessary to fully control the SARS-CoV-2 pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane anchored pre-fusion stabilized spike (MVA/S), but not secreted S1, induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and showed protection from SARS-CoV-2 infection and virus replication in the lung as early as day 2 following intranasal or intratracheal challenge. Single-cell RNA sequencing analysis of lung cells at day 4 post-infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities, and lowered induction of interferon stimulated genes. These results demonstrate that MVA/S vaccination induces both neutralizing antibodies and CD8+ T cells in the blood and lung and serves as a potential vaccine candidate against SARS-CoV-2., Graphical Abstract, Modified vaccinia Ankara (MVA) vector-based vaccines are attractive for their excellent safety and ability to induce long-lived humoral and cellular immunity in humans. Routhu et al. show that an MVA-based COVID-19 vaccine encoding prefusion-stabilized spike MVA/S induces strong neutralizing antibody and CD8+ T cell responses and protects macaques from SARS-CoV2 infection, immunopathology and infection-induced B cell abnormalities in the lung.

SUMMARYDespite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to rise. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 receptor binding domain. Here, we investigated the capacity of modified versions of one lead binder, LCB1, to protect against SARS-CoV-2-mediated lung disease in human ACE2-expressing transgenic mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung even when given as many as five days before or two days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has infected over 260 million people over the past 2 years. Remdesivir (RDV, VEKLURY®) is currently the only antiviral therapy fully approved by the FDA for the treatment of COVID-19. The parent nucleoside of RDV, GS-441524, exhibits antiviral activity against numerous respiratory viruses including SARS-CoV-2, although at reduced in vitro potency compared to RDV in most assays. Here we find in both human alveolar and bronchial primary cells, GS-441524 is metabolized to the pharmacologically active GS-441524 triphosphate (TP) less efficiently than RDV, which correlates with a lower in vitro SARS-CoV-2 antiviral activity. In vivo, African green monkeys (AGM) orally dosed with GS-441524 yielded low plasma levels due to limited oral bioavailability of10%. When GS-441524 was delivered via intravenous (IV) administration, although plasma concentrations of GS-441524 were significantly higher, lung TP levels were lower than observed from IV RDV. To determine the required systemic exposure of GS-441524 associated with in vivo antiviral efficacy, SARS-CoV-2 infected AGMs were treated with a once-daily IV dose of either 7.5 or 20 mg/kg GS-441524 or IV RDV for 5 days and compared to vehicle control. Despite the reduced lung TP formation compared to IV dosing of RDV, daily treatment with IV GS-441524 resulted in dose-dependent efficacy, with the 20 mg/kg GS-441524 treatment resulting in significant reductions of SARS-CoV-2 replication in the lower respiratory tract of infected animals. These findings demonstrate the in vivo SARS-CoV-2 antiviral efficacy of GS-441524 and support evaluation of its orally bioavailable prodrugs as potential therapies for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic infecting more than 106 million people and causing 2.3 million deaths. The rapid deployment of antibody-based countermeasures has provided hope for curtailing disease and ending the pandemic1. However, the emergence of rapidly-spreading SARS-CoV-2 variants in the United Kingdom (B.1.1.7), South Africa (B.1.351), and elsewhere with mutations in the spike protein has raised concern for escape from neutralizing antibody responses and loss of vaccine efficacy based on preliminary data with pseudoviruses2–4. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera, and human sera from recipients of the Pfizer-BioNTech (BNT162b2) mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, a chimeric Washington strain with a South African spike gene (Wash SA-B.1.351), and isogenic recombinant variants with designed mutations or deletions at positions 69–70, 417, 484, 501, and/or 614 of the spike protein. Several highly neutralizing mAbs engaging the receptor binding domain (RBD) or N-terminal domain (NTD) lost inhibitory activity against Wash SA-B.1.351 or recombinant variants with an E484K spike mutation. Most convalescent sera and virtually all mRNA vaccine-induced immune sera tested showed markedly diminished neutralizing activity against the Wash SA-B.1.351 strain or recombinant viruses containing mutations at position 484 and 501. We also noted that cell line selection used for growth of virus stocks or neutralization assays can impact the potency of antibodies against different SARS-CoV-2 variants, which has implications for assay standardization and congruence of results across laboratories. As several antibodies binding specific regions of the RBD and NTD show loss-of-neutralization potency in vitro against emerging variants, updated mAb cocktails, targeting of highly conserved regions, enhancement of mAb potency, or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.

The mRNA-1273 vaccine is effective against SARS-CoV-2 and was granted emergency use authorization by the FDA. Clinical studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibits severe SARS-CoV-2 disease similar to that in hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and nonhuman primates, low-level virus replication in mRNA-1273-vaccinated hamsters coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high-resolution analysis that is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a 2-dose schedule and provide insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

The biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development., Zhang et al. develop a trans-complementation system to produce single-round infectious SARS-CoV-2 that recapitulates authentic viral infection and replication. They then show the system can be safely used at biosafety level-2 for high-throughput antiviral testing.

Glioblastoma multiforme (GBM) is a fatal human cancer in part because GBM stem cells are resistant to therapy and recurrence is inevitable. Previously, we demonstrated Zika virus (ZIKV) targets GBM stem cells and prevents death of mice with gliomas. Here, we evaluated the immunological basis of ZIKV-mediated protection against GBM. Introduction of ZIKV into the brain tumor increased recruitment of CD8+ T and myeloid cells to the tumor microenvironment. CD8+ T cells were required for ZIKV-dependent tumor clearance because survival benefits were lost with CD8+ T cell depletion. Moreover, while anti-PD-1 antibody monotherapy moderately improved tumor survival, when coadministered with ZIKV, survival increased. ZIKV-mediated tumor clearance also resulted in durable protection against syngeneic tumor rechallenge, which also depended on CD8+ T cells. To address safety concerns, we generated an immune-sensitized ZIKV strain, which was effective alone or in combination with immunotherapy. Thus, oncolytic ZIKV treatment can be leveraged by immunotherapies, which may prompt combination treatment paradigms for adult patients with GBM.

SARS-CoV-2 is a recently emerged human coronavirus that has escalated to a pandemic. There are currently no approved vaccines for SARS-CoV-2, which causes severe respiratory illness or death. Defining the antibody response to SARS-CoV-2 will be essential for understanding disease progression, long-term immunity, and vaccine efficacy. Here we describe two methods for evaluating the neutralization capacity of SARS-CoV-2 antibodies. The basic protocol is a focus reduction neutralization test (FRNT), which involves immunostaining infected cells with a chromogen deposit readout. The alternate protocol is a modification of the FRNT that uses an infectious clone-derived SARS-CoV-2 virus expressing a fluorescent reporter. These protocols are adapted for use in a high-throughput setting, and are compatible with large-scale vaccine studies or clinical testing. © 2020 Wiley Periodicals LLC Basic Protocol: Focus reduction neutralization test Alternate Protocol: mNeonGreen-based focus reduction neutralization test (FRNT-mNG).

Monophosphate prodrug analogs of 2′-deoxy-2′-fluoro-2′-C-methylguanosine have been reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These prodrugs also display potent anti-dengue virus activities in cellular assays although their prodrug moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood mononuclear cells (PBMCs) are among the major targets of dengue virus, different prodrug moieties were designed to effectively deliver 2′-deoxy-2′-fluoro-2′-C-methylguanosine monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral administration. We identified a cyclic phosphoramidate, prodrug 17, demonstrating well-balanced anti-dengue virus cellular activity and in vitro stability profiles. We further determined the PBMC concentration of active triphosphate needed to inhibit virus replication by 50% (TP(50)). Compound 17 was assessed in an AG129 mouse model and demonstrated 1.6- and 2.2-log viremia reductions at 100 and 300 mg/kg twice a day (BID), respectively. At 100 mg/kg BID, the terminal triphosphate concentration in PBMCs exceeded the TP(50) value, demonstrating TP(50) as the target exposure for efficacy. In dogs, oral administration of compound 17 resulted in high PBMC triphosphate levels, exceeding the TP(50) at 10 mg/kg. Unfortunately, 2-week dog toxicity studies at 30, 100, and 300 mg/kg/day showed that “no observed adverse effect level” (NOAEL) could not be achieved due to pulmonary inflammation and hemorrhage. The preclinical safety results suspended further development of compound 17. Nevertheless, present work has proven the concept that an efficacious monophosphate nucleoside prodrug could be developed for the potential treatment of dengue virus infection.

Mosquito-transmitted flaviviruses cause widespread disease across the world and medical countermeasures have yet to be proven effective. To provide better molecular tools for drug screens and pathogenesis studies, we report a new approach to produce stable NanoLuc-tagged flaviviruses, including dengue virus serotypes 1-4, Japanese encephalitis virus, yellow fever virus, West Nile virus, and Zika virus. Since the reporter gene is often engineered at the flaviviral capsid gene region, the capsid sequence must be duplicated to flank the reporter gene; such capsid duplication is essential for viral replication. The conventional approach for stabilizing reporter flaviviruses has been to shorten or modify the duplicated capsid sequence to minimize homologous recombination. No study has examined the effects of capsid duplication length on reporter virus stability. Here we report an optimal length of capsid duplication (34 or 38 amino acids without any other modifications) that can stabilize reporter flaviviruses. These NanoLuc-tagged flaviviruses were stable after ten rounds of cell culture passaging. In the case of NanoLuc-tagged Zika virus with a 38-amino acid capsid duplication (ZIKV C38), the reporter virus replicated to 107 FFU/ml in cell culture and produced robust luciferase signal after mosquitoes were intrathoracically injected or bloodmeal fed with the virus. Mechanistically, the optimal length of capsid duplication may contain all the cis-acting RNA elements required for viral RNA replication, thus reducing the selection pressure for recombination. Together, these data describe an improved method of constructing optimal reporter flaviviruses.

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients. IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.

Zika virus (ZIKV) infection is a serious public threat with cases reported in about 70 countries and territories. One of the most serious consequences of ZIKV infection is congenital microcephaly in babies. Congenital microcephaly has been suggested to result from infection of neural progenitor cells (NPCs) in the developing fetal brain. However, the molecular and cellular mechanisms underlying microcephaly development remains to be fully elucidated. In this study, we employed quantitative proteomics to determine protein expression profile that occur during viral replication in NPCs. Bioinformatics analysis of the protein expression changes resulted in the identification of a wide range of cell signaling pathways. Specifically, pathways involved in neurogenesis and embryonic development were markedly altered, along with those associated with cell cycle, apoptosis, lipid metabolism and oxidative stress. Notably, the differential regulation of Ephrin Receptor and PPAR signaling pathways, as revealed by quantitative proteomics and validated by qPCR array, underscores the need to explore these pathways in disease development. Collectively, these results indicate that ZIKV-induced pathogenesis involves complex virus-host reactions; the findings reported here could help shed light on the mechanisms underlying ZIKV-induced microcephaly and ZIKV replication in NPCs.

SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site (ΔPRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the ΔPRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the ΔPRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT50) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays., Article Summary: A deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro.

The nonstructural protein 1 (NS1) of several flaviviruses, including West Nile, dengue, and yellow fever viruses, is capable of inducing variable degrees of protection against flavivirus infection in animal models. However, the immunogenicity of NS1 protein of Zika virus (ZIKV) is less understood. Here, we determined the efficacy of ZIKV NS1-based vaccine candidates using two delivery platforms, methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV) and a DNA vaccine. We first show that expression of ZIKV NS1 could be significantly enhanced by optimizing the signal peptide. A single dose of mtdVSV-NS1-based vaccine or two doses of DNA vaccine induced high levels of NS1-specfic antibody and T cell immune responses but provided only partial protection against ZIKV viremia in BALB/c mice. In Ifnar1(−/−) mice, neither NS1-based vaccine provided protection against a lethal high dose (10(5) PFU) ZIKV challenge, but mtdVSV-NS1-based vaccine prevented deaths from a low dose (10(3) PFU) challenge, though they experienced viremia and body weight loss. We conclude that ZIKV NS1 alone conferred substantial, but not complete, protection against ZIKV infection. Nevertheless, these results highlight the value of ZIKV NS1 for vaccine development. IMPORTANCE Most Zika virus (ZIKV) vaccine research has focused on the E or prM-E proteins and the induction of high levels of neutralizing antibodies. However, these ZIKV neutralizing antibodies cross-react with other flaviviruses, which may aggravate the disease via an antibody-dependent enhancement (ADE) mechanism. ZIKV NS1 protein may be an alternative antigen for vaccine development, since antibodies to NS1 do not bind to the virion, thereby eliminating the risk of ADE. Here, we show that recombinant VSV and DNA vaccines expressing NS1, alone, confer partial protection against ZIKV infection in both immunocompetent and immunodeficient mice, highlighting the value of NS1 as a potential vaccine candidate.

Virus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. Here, we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS-CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to plaque reduction neutralizing assay, the gold standard of serological testing. The fluorescence-based neutralization assay is specific to measure COVID-19 neutralizing antibodies without cross reacting with patient specimens with other viral, bacterial, or parasitic infections. Collectively, our approach offers a rapid platform that can be scaled to screen people for antibody protection from COVID-19, a key parameter necessary to safely reopen local communities., Neutralizing antibody titers in SARS-CoV-2 infected or vaccinated people are an important measure for vaccine development and public health decision-making. Here, the authors develop a fluorescence based SARS-CoV-2 assay to determine neutralizing antibody titers in COVID-19 patient sera in a high throughput set-up.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication and host immune response determine coronavirus disease 2019 (COVID-19), but studies evaluating viral evasion of immune response are lacking. Here, we use unbiased screening to identify SARS-CoV-2 proteins that antagonize type I interferon (IFN-I) response. We found three proteins that antagonize IFN-I production via distinct mechanisms: nonstructural protein 6 (nsp6) binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, nsp13 binds and blocks TBK1 phosphorylation, and open reading frame 6 (ORF6) binds importin Karyopherin α 2 (KPNA2) to inhibit IRF3 nuclear translocation. We identify two sets of viral proteins that antagonize IFN-I signaling through blocking signal transducer and activator of transcription 1 (STAT1)/STAT2 phosphorylation or nuclear translocation. Remarkably, SARS-CoV-2 nsp1 and nsp6 suppress IFN-I signaling more efficiently than SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Thus, when treated with IFN-I, a SARS-CoV-2 replicon replicates to a higher level than chimeric replicons containing nsp1 or nsp6 from SARS-CoV or MERS-CoV. Altogether, the study provides insights on SARS-CoV-2 evasion of IFN-I response and its potential impact on viral transmission and pathogenesis., Graphical Abstract, Highlights • SARS-CoV-2 proteins antagonize IFN-I production and signaling • Different SARS-CoV-2 proteins inhibit IFN-I response through distinct mechanisms • SARS-CoV, SARS-CoV-2, and MERS-CoV proteins inhibit IFN-I at different efficacies • A reporter replicon of SARS-CoV-2 allows experiments at biosafety level 2, Xia et al. perform an unbiased screening to identify SARS-CoV-2 proteins that antagonize the IFN-I response. The identified viral proteins inhibit IFN-I production and signaling through distinct mechanisms. Compared with SARS-CoV and MERS-CoV, the IFN-I signaling is more efficiently suppressed by the SARS-CoV-2 nsp1 and nsp6 proteins.

Global Zika virus (ZIKV) outbreaks and their link to microcephaly have raised major public health concerns. However, the mechanism of maternal-fetal transmission remains largely unknown. In this study, we determined the role of yolk sac (YS) microglial progenitors in a mouse model of ZIKV vertical transmission. We found that embryonic (E) days 6.5-E8.5 were a critical window for ZIKV infection that resulted in fetal demise and microcephaly, and YS microglial progenitors were susceptible to ZIKV infection. Ablation of YS microglial progenitors significantly reduced the viral load in both the YS and the embryonic brain. Taken together, these results support the hypothesis that YS microglial progenitors serve as “Trojan horses,” contributing to ZIKV fetal brain dissemination and congenital brain defects., Author summary ZIKV is more likely to cause fetal demise and brain malformations when the mother is infected at an early stage of pregnancy, which is the critical time window when a special type of immune cells called microglia appear in the YS and migrate to the fetal brain. YS-derived microglia are susceptible to ZIKV infection and can act as “Trojan horses” to bring ZIKV from the mother to the fetal brain.

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2., A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. To address this, Shi and colleagues present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV2-Nluc), and show that it has potential for large-scale vaccine evaluation and neutralizing antibody testing.

Zika virus (ZIKV) infects pregnant women and causes devastating congenital zika syndrome (CZS). How the virus is vertically transmitted to the fetus and induces neuronal loss remains unclear. We previously reported that Pellino (Peli)1, an E3 ubiquitin ligase, promotes p38MAPK activation in microglia and induction of lethal encephalitis by facilitating the replication of West Nile virus (WNV), a closely related flavivirus. Here, we found that Peli1 expression was induced on ZIKV-infected human monocytic cells, peripheral blood mononuclear cells, human first-trimester placental trophoblasts, and neural stem cell (hNSC)s. Peli1 mediates ZIKV cell attachment, entry and viral translation and its expression is confined to the endoplasmic reticulum. Moreover, Peli1 mediated inflammatory cytokine and chemokine responses and induced cell death in placental trophoblasts and hNSCs. ZIKV-infected pregnant mice lacking Peli1 signaling had reduced placental inflammation and tissue damage, which resulted in attenuated congenital abnormalities. Smaducin-6, a membrane-tethered Smad6-derived peptide, blocked Peli1-mediated NF-κB activation but did not have direct effects on ZIKV infection. Smaducin-6 reduced inflammatory responses and cell death in placental trophoblasts and hNSCs, and diminished placental inflammation and damage, leading to attenuated congenital malformations in mice. Collectively, our results reveal a novel role of Peli1 in flavivirus pathogenesis and suggest that Peli1 promotes ZIKV vertical transmission and neuronal loss by mediating inflammatory cytokine responses and induction of cell death. Our results also identify Smaducin-6 as a potential therapeutic candidate for treatment of CZS., Author summary We previously reported that Pellino (Peli)1, an E3 ubiquitin ligase mediates p38MAPK activation in microglia and induces lethal encephalitis by facilitating replication of a mosquito -borne flavivirus, West Nile virus (WNV). Zika virus (ZIKV), a closely related flavivirus, causes devastating congenital zika syndrome (CZS) in pregnant women. How ZIKV is vertically transmitted to the fetus and induces neuronal loss remains unclear. Here, we found that Peli1 expression was enhanced in human monocytic cells, peripheral blood mononuclear cells, first-trimester placental trophoblasts and neural stem cell (hNSC)s following ZIKV infection. Peli1 expression colocalized with the endoplasmic reticulum and double-stranded RNA in ZIKV-infected cells and was required for ZIKV cell attachment and replication. Peli1 knockdown in placental trophoblasts inhibited ZIKV replication and decreased inflammatory cytokine responses and cell death. ZIKV-infected pregnant mice lacking Peli1 signaling showed reduced placental inflammation and tissue damage, which resulted in attenuated congenital abnormalities. Furthermore, Smaducin-6, a membrane-tethered Smad6-derived peptide, blocked Peli1-mediated NF-κB activation, but not ZIKV replication. Smaducin 6 inhibited Peli1-mediated inflammatory cytokine responses and cell death in placental trophoblasts and hNSCs, and attenuated congenital malformations in mice. Collectively, our results reveal a novel role of Peli1 in flavivirus pathogenesis and suggest that Peli1 promotes ZIKV vertical transmission and neuronal loss by mediating inflammatory cytokine responses and induction of cell death.