A Thermostable mRNA Vaccine against COVID-19.

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.

The infectivity and pathogenicity of hepatitis A virus live-attenuated vaccine strain H2 in type I interferon receptor-deficient mice.

Hepatitis A virus (HAV) live-attenuated vaccine H2 strain has been approved for clinical use for decades with ideal safety profiles in nonhuman primate models and humans. Recently, type I interferon (IFN) receptor-deficient mice were shown to be susceptible to HAV infection. Herein, we sought to determine the infection and replication dynamics of the H2 in Ifnar-/- mice that lack type I IFN receptor. Following intravenous injection, the H2 failed to cause obvious clinical symptoms in Ifnar-/- mice, and no significant upregulation in serum alanine aminotransferase (ALT) levels was observed. Notably, the histopathological examination showed that there were significant focal infiltrations of lymphocytes and neutrophils in the portal area, but no focal necrosis was observed in liver tissues. Viral RNAs sustained in the liver, and the infectious virus could be recovered from the liver tissue until 42 days post-infection. More importantly, H2 infection induced obvious viremia and persistent viral shedding in feces. In addition, robust HAV-specific humoral immune responses were induced in Ifnar-/- mice. Overall, our study revealed the safety profile of H2 in Ifnar-/- mice, which not only helps understand the attenuation mechanism of H2, but also expands the application of the Ifnar-/- mouse model for HAV studies.

A highly immunogenic live-attenuated vaccine candidate prevents SARS-CoV-2 infection and transmission in hamsters.

The highly pathogenic and readily transmissible SARS-CoV-2 has caused a global coronavirus pandemic, urgently requiring effective countermeasures against its rapid expansion. All available vaccine platforms are being used to generate safe and effective COVID-19 vaccines. Here, we generated a live-attenuated candidate vaccine strain by serial passaging of a SARS-CoV-2 clinical isolate in Vero cells. Deep sequencing revealed the dynamic adaptation of SARS-CoV-2 in Vero cells, resulting in a stable clone with a deletion of seven amino acids (N679SPRRAR685) at the S1/S2 junction of the S protein (named VAS5). VAS5 showed significant attenuation of replication in multiple human cell lines, human airway epithelium organoids, and hACE2 mice. Viral fitness competition assays demonstrated that VAS5 showed specific tropism to Vero cells but decreased fitness in human cells compared with the parental virus. More importantly, a single intranasal injection of VAS5 elicited a high level of neutralizing antibodies and prevented SARS-CoV-2 infection in mice as well as close-contact transmission in golden Syrian hamsters. Structural and biochemical analysis revealed a stable and locked prefusion conformation of the S trimer of VAS5, which most resembles SARS-CoV-2-3Q-2P, an advanced vaccine immunogen (NVAX-CoV2373). Further systematic antigenic profiling and immunogenicity validation confirmed that the VAS5 S trimer presents an enhanced antigenic mimic of the wild-type S trimer. Our results not only provide a potent live-attenuated vaccine candidate against COVID-19 but also clarify the molecular and structural basis for the highly attenuated and super immunogenic phenotype of VAS5.

Aerosolized Zika virus infection in Guinea pigs.

Zika virus (ZIKV) is primarily transmitted through mosquito bites and sexual contact, and vertical transmission of ZIKV has also been observed in humans. In addition, ZIKV infection via unknown transmission routes has been frequently reported in clinical settings. However, whether ZIKV can be transmitted via aerosol routes remains unknown. In this study, we demonstrated that aerosolized ZIKV is fully infectious in vitro and in vivo. Remarkably, intratracheal (i.t.) inoculation with aerosolized ZIKV led to rapid viremia and viral secretion in saliva, as well as robust humoral and innate immune responses in guinea pigs. Transcriptome analysis further revealed that the expression of genes related to viral processes, biological regulation and the immune response was significantly changed. Together, our results confirm that aerosolized ZIKV can result in systemic infection and induce both innate and adaptive immune responses in guinea pigs, highlighting the possibility of ZIKV transmission via aerosols.

Rational development of a combined mRNA vaccine against COVID-19 and influenza.

As the world continues to experience the COVID-19 pandemic, seasonal influenza remain a cause of severe morbidity and mortality globally. Worse yet, coinfection with SARS-CoV-2 and influenza A virus (IAV) leads to more severe clinical outcomes. The development of a combined vaccine against both COVID-19 and influenza is thus of high priority. Based on our established lipid nanoparticle (LNP)-encapsulated mRNA vaccine platform, we developed and characterized a novel mRNA vaccine encoding the HA antigen of influenza A (H1N1) virus, termed ARIAV. Then, ARIAV was combined with our COVID-19 mRNA vaccine ARCoV, which encodes the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, to formulate the final combined vaccine, AR-CoV/IAV. Further characterization demonstrated that immunization with two doses of AR-CoV/IAV elicited robust protective antibodies as well as antigen-specific cellular immune responses against SARS-CoV-2 and IAV. More importantly, AR-CoV/IAV immunization protected mice from coinfection with IAV and the SARS-CoV-2 Alpha and Delta variants. Our results highlight the potential of the LNP-mRNA vaccine platform in preventing COVID-19 and influenza, as well as other respiratory diseases.

Lipid nanoparticle-encapsulated mRNA antibody provides long-term protection against SARS-CoV-2 in mice and hamsters.

Monoclonal antibodies represent important weapons in our arsenal to against the COVID-19 pandemic. However, this potential is severely limited by the time-consuming process of developing effective antibodies and the relative high cost of manufacturing. Herein, we present a rapid and cost-effective lipid nanoparticle (LNP) encapsulated-mRNA platform for in vivo delivery of SARS-CoV-2 neutralization antibodies. Two mRNAs encoding the light and heavy chains of a potent SARS-CoV-2 neutralizing antibody HB27, which is currently being evaluated in clinical trials, were encapsulated into clinical grade LNP formulations (named as mRNA-HB27-LNP). In vivo characterization demonstrated that intravenous administration of mRNA-HB27-LNP in mice resulted in a longer circulating half-life compared with the original HB27 antibody in protein format. More importantly, a single prophylactic administration of mRNA-HB27-LNP provided protection against SARS-CoV-2 challenge in mice at 1, 7 and even 63 days post administration. In a close contact transmission model, prophylactic administration of mRNA-HB27-LNP prevented SARS-CoV-2 infection between hamsters in a dose-dependent manner. Overall, our results demonstrate a superior long-term protection against SARS-CoV-2 conferred by a single administration of this unique mRNA antibody, highlighting the potential of this universal platform for antibody-based disease prevention and therapy against COVID-19 as well as a variety of other infectious diseases.

SARS-CoV-2 infection in the mouse olfactory system.

SARS-CoV-2 infection causes a wide spectrum of clinical manifestations in humans, and olfactory dysfunction is one of the most predictive and common symptoms in COVID-19 patients. However, the underlying mechanism by which SARS-CoV-2 infection leads to olfactory disorders remains elusive. Herein, we demonstrate that intranasal inoculation with SARS-CoV-2 induces robust viral replication in the olfactory epithelium (OE), not the olfactory bulb (OB), resulting in transient olfactory dysfunction in humanized ACE2 (hACE2) mice. The sustentacular cells and Bowman's gland cells in the OE were identified as the major target cells of SARS-CoV-2 before invasion into olfactory sensory neurons (OSNs). Remarkably, SARS-CoV-2 infection triggers massive cell death and immune cell infiltration and directly impairs the uniformity of the OE structure. Combined transcriptomic and quantitative proteomic analyses revealed the induction of antiviral and inflammatory responses, as well as the downregulation of olfactory receptor (OR) genes in the OE from the infected animals. Overall, our mouse model recapitulates olfactory dysfunction in COVID-19 patients and provides critical clues for understanding the physiological basis for extrapulmonary manifestations of COVID-19.

A proof of concept for neutralizing antibody-guided vaccine design against SARS-CoV-2.

Mutations and transient conformational movements of the receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable present immune escape routes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting the N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, a combination of RBD-targeting NAbs and NTD-binding NAbs, FC05, enhanced the neutralization potency in cell-based assays and an animal model. Results of competitive surface plasmon resonance assays and cryo-electron microscopy (cryo-EM) structures of antigen-binding fragments bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in the NTD and RBD of S, when immunized in rabbits and macaques, elicited potent protective immune responses against SARS-CoV-2. More importantly, two immunizations of this combination of NTD and RBD immunogens provided complete protection in macaques against a SARS-CoV-2 challenge, without observable antibody-dependent enhancement of infection. These results provide a proof of concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD.

Effects of Danshen Injection () on inhibiting proliferation and inducing apoptosis through down-regulation of mutant JAK2 gene and its protein phosphorylation in human erythroid leukemic cells.

OBJECTIVE: To explore the effects of Danshen Injection () on inhibition proliferation, inducing apoptosis and its possible mechanisms on human erythroid leukemic (HEL) cells. METHODS: The commercial Chinese patent medicine of Danshen Injection was extracted and isolated from Chinese herb of Salvia miltiorrhiza bung. The inhibition effects of proliferation were assayed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method in HEL cells treated by Danshen Injection at various concentrations for 48 h. The cellular apoptosis was observed in morphology, analyzed by flow cytometry with annexin V and propidium iodide (PI) staining, and examined by DNA degradation ladder on agarose gel electrophoresis. Meanwhile, the expression levels of mutant Janus kinasez (JAK2) gene and phosphorylation-JAK2 (P-JAK2) protein were detected by allele specific-polymerase chain reaction and Western blot. RESULTS: The proliferation of HEL cells was effectively inhibited by Danshen Injection in a dose-dependent manner, with suppression rates from 19.46±2.31% to 50.20±5.21%. Typical apoptosis cells was observed in Danshen Injection treated HEL cells, the rates of annexin V positive cells increased obviously in a dose-dependent manner, as well as the DNA degradation ladder of apoptosis revealed on gel electrophoresis. The expression levels of mutant JAK2 gene and P-JAK2 protein reduced gradually with increasing dosage of Danshen injection. CONCLUSION: Danshen Injection could not only significantly inhibit the proliferation, but also induce apoptosis in HEL cells; down-regulation of the mutant JAK2 gene and P-JAK2 protein expressions are probably one of its molecular mechanisms.