Recombinant measles virus expressing prefusion spike protein stabilized by six rather than two prolines is more efficacious against SARS-CoV-2 infection.

Measles virus (MeV) has been an excellent vector platform for delivering vaccines against many pathogens because of its high safety and efficacy, and induction of long-lived immunity. Early in the COVID-19 pandemic, a recombinant MeV (rMeV) expressing the prefusion full-length spike protein stabilized by two prolines (TMV-083) was developed and tested in phase 1 and 1/2 clinical trials but was discontinued because of insufficient immunogenicity and a low seroconversion rate in adults. Here, we compared the immunogenicity of rMeV expressing a soluble prefusion spike (preS) protein stabilized by two prolines (rMeV-preS-2P) with a rMeV expressing a soluble preS protein stabilized by six prolines (rMeV-preS-6P). We found that rMeV-preS-6P expressed approximately five times more preS than rMeV-preS-2P in cell culture. Importantly, rMeV-preS-6P induced 30-60 and six times more serum immunoglobulin G and neutralizing antibody than rMeV-preS-2P, respectively, in IFNAR-/- mice. IFNAR-/- mice immunized with rMeV-preS-6P were completely protected from challenge with a mouse-adapted SARS-CoV-2, whereas those immunized with rMeV-preS-2P were partially protected. In addition, hamsters immunized with rMeV-preS-6P were completely protected from the challenge with a Delta variant of SARS-CoV-2. Our results demonstrate that rMeV-preS-6P is significantly more efficacious than rMeV-preS-2P, highlighting the value of using preS-6P as the antigen for developing vaccines against SARS-CoV-2.

Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice.

Interferon-induced transmembrane protein 3 (IFITM3) is an antiviral protein that alters cell membranes to block fusion of viruses. Conflicting reports identified opposing effects of IFITM3 on SARS-CoV-2 infection of cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with SARS-CoV-2 experience extreme weight loss and lethality compared to mild infection in wild-type (WT) mice. KO mice have higher lung viral titers and increases in inflammatory cytokine levels, immune cell infiltration, and histopathology. Mechanistically, we observe disseminated viral antigen staining throughout the lung and pulmonary vasculature in KO mice, as well as increased heart infection, indicating that IFITM3 constrains dissemination of SARS-CoV-2. Global transcriptomic analysis of infected lungs shows upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections in vivo.

Next-generation RSV vaccines avoid flipping out.

In clinical trials, RSV prefusion F protein induced higher neutralizing antibodies and more activated memory B cells than postfusion F protein (Chang et al.).

Prime-Pull Immunization of Mice with a BcfA-Adjuvanted Vaccine Elicits Sustained Mucosal Immunity That Prevents SARS-CoV-2 Infection and Pathology.

Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. In this study, we demonstrate the efficacy of Bordetella colonization factor A (BcfA), a novel bacteria-derived protein adjuvant, in SARS-CoV-2 spike-based prime-pull immunizations. We show that i.m. priming of mice with an aluminum hydroxide- and BcfA-adjuvanted spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17-polarized CD4+ tissue-resident memory T cells and neutralizing Abs. Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 (MA10) and reduced viral replication in the respiratory tract. Histopathology showed a strong leukocyte and polymorphonuclear cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. Importantly, neutralizing Abs and tissue-resident memory T cells were maintained until 3 mo postbooster. Viral load in the nose of mice challenged with the MA10 virus at this time point was significantly reduced compared with naive challenged mice and mice immunized with an aluminum hydroxide-adjuvanted vaccine. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, provide sustained protection against SARS-CoV-2 infection.

Influenza A virus modulates ACE2 expression and SARS-CoV-2 infectivity in human cardiomyocytes.

Influenza A virus (IAV) and SARS-CoV-2 virus are both acute respiratory viruses currently circulating in the human population. This study aims to determine the impact of IAV infection on SARS-CoV-2 pathogenesis and cardiomyocyte function. Infection of human bronchial epithelial cells (HBEC), A549 cells, lung fibroblasts (HLF), monocyte derived macrophages (MDMs), cardiac fibroblasts (HCF) and hiPSC-derived cardiomyocytes with IAV enhanced the expression of ACE2, the SARS-CoV-2 receptor. Similarly, IAV infection increased levels of ACE2 in the lungs of mice and humans. Interestingly, we detected heavily glycosylated form of ACE2 in hiPSC-CMs and poorly glycosylated ACE2 in other cell types. Also, prior IAV infection enhances SARS-CoV-2 spike protein binding and viral entry in all cell types. However, efficient SARS-CoV-2 replication was uniquely inhibited in cardiomyocytes. Glycosylation of ACE2 correlated with enzymatic conversion of its substrate Ang II, induction of eNOS and nitric oxide production, may provide a potential mechanism for the restricted SARS-CoV-2 replication in cardiomyocytes.

SARS-CoV-2 prefusion spike protein stabilized by six rather than two prolines is more potent for inducing antibodies that neutralize viral variants of concern.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (104 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 103 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.

A highly efficacious live attenuated mumps virus-based SARS-CoV-2 vaccine candidate expressing a six-proline stabilized prefusion spike.

With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)-based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P-M or F-SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P-M gene junction was more efficient than from the F-SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1-/- mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.

Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and immunothrombosis.

Severe acute respiratory syndrome coronavirus 2 (SARS­CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS­CoV-2 infections and that CASP4 expression correlates with severity of SARS­CoV-2 infection in humans. SARS­CoV-2­infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS­CoV-2­infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1ß, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS­CoV-2­induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

Severe Acute Respiratory Syndrome Coronavirus 2 RNAemia and Clinical Outcomes in Children With Coronavirus Disease 2019.

The burden of coronavirus disease 2019 (COVID-19) in children represents a fraction of cases worldwide, yet a subset of those infected are at risk for severe disease. We measured plasma severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in a cohort of 103 children hospitalized with COVID-19 with diverse clinical manifestations. SARS-CoV-2 RNAemia was detected in 27 (26%) of these children, lasted for a median of 6 (interquartile range, 2-9) days, and was associated with higher rates of oxygen administration, admission to the intensive care unit, and longer hospitalization.

SARS-CoV-2 spreads through cell-to-cell transmission.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

Human Stem Cell Models of SARS-CoV-2 Infection in the Cardiovascular System.

The virus responsible for coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected over 190 million people to date, causing a global pandemic. SARS-CoV-2 relies on binding of its spike glycoprotein to angiotensin-converting enzyme 2 (ACE2) for infection. In addition to fever, cough, and shortness of breath, severe cases of SARS-CoV-2 infection may result in the rapid overproduction of pro-inflammatory cytokines. This overactive immune response is known as a cytokine storm, which leads to several serious clinical manifestations such as acute respiratory distress syndrome and myocardial injury. Cardiovascular disorders such as acute coronary syndrome (ACS) and heart failure not only enhance disease progression at the onset of infection, but also arise in hospitalized patients with COVID-19. Tissue-specific differentiated cells and organoids derived from human pluripotent stem cells (hPSCs) serve as an excellent model to address how SARS-CoV-2 damages the lungs and the heart. In this review, we summarize the molecular basis of SARS-CoV-2 infection and the current clinical perspectives of the bidirectional relationship between the cardiovascular system and viral progression. Furthermore, we also address the utility of hPSCs as a dynamic model for SARS-CoV-2 research and clinical translation.

A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike.

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR-/-mice, IFNAR-/--hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2-specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.