Seasonal quadrivalent mRNA vaccine prevents and mitigates influenza infection.

Annually, seasonal influenza is responsible for millions of infections and hundreds of thousands of deaths. The current method for managing influenza is vaccination using a standardized amount of the influenza virus' primary surface antigen, hemagglutinin (HA), as the intended target of the immune response. This vaccination strategy results in vaccines with variable efficacy year to year due to antigenic drift of HA, which can be further exacerbated by manufacturing processes optimizing growth of vaccine virus in eggs. Due to these limitations, alternative vaccine platforms are actively being explored to improve influenza vaccine efficacy, including cell-based, recombinant protein, and mRNA vaccines. mRNA's rapid, in vitro production makes it an appealing platform for influenza vaccination, and the success of SARS-CoV-2 mRNA vaccines in the clinic has encouraged the development of mRNA vaccines for other pathogens. Here, the immunogenicity and protective efficacy of a quadrivalent mRNA vaccine encoding HA from four seasonal influenza viruses, A/California/07/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2), B/Brisbane/60/2008 (B-Victoria lineage), and B/Phuket/3073/2013 (B-Yamagata lineage), was evaluated. In mice, a 120 µg total dose of this quadrivalent mRNA vaccine induced robust antibody titers against each subtype that were commensurate with titers when each antigen was administered alone. Following A/California/04/2009 challenge, mice were fully protected from morbidity and mortality, even at doses as low as 1 µg of each antigen. Additionally, a single administration of 10 µg of quadrivalent mRNA was sufficient to prevent weight loss caused by A/California/04/2009. These results support the promise of this mRNA vaccine for prevention and mitigation of influenza vaccine.

mRNA Vaccine Mitigates SARS-CoV-2 Infections and COVID-19.

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in December of 2019 and is responsible for millions of infections and deaths across the globe. Vaccination against SARS-CoV-2 has proven effective to contain the spread of the virus and reduce disease. The production and distribution of these vaccines occurred at a remarkable pace, largely through the employment of the novel mRNA platform. However, interruptions in supply chain and high demand for clinical grade reagents have impeded the manufacture and distribution of mRNA vaccines at a time when accelerated vaccine deployment is crucial. Furthermore, the emergence of SARS-CoV-2 variants across the globe continues to threaten the efficacy of vaccines encoding the ancestral virus spike protein. Here, we report results from preclinical studies on mRNA vaccines developed using a proprietary mRNA production process developed by GreenLight Biosciences. Two mRNA vaccines encoding the full-length, nonstabilized SARS-CoV-2 spike protein, GLB-COV2-042 and GLB-COV2-043, containing uridine and pseudouridine, respectively, were evaluated in rodents for their immunogenicity and protection from SARS-CoV-2 challenge with the ancestral strain and the Alpha (B.1.1.7) and Beta (B.1.351) variants. In mice and hamsters, both vaccines induced robust spike-specific binding and neutralizing antibodies, and in mice, vaccines induced significant T cell responses with a clear Th1 bias. In hamsters, both vaccines conferred significant protection following challenge with SARS-CoV-2 as assessed by weight loss, viral load, and virus replication in the lungs and nasopharynx. These results support the development of GLB-COV2-042 and GLB-COV2-043 for clinical use. IMPORTANCE SARS-CoV-2 continues to disrupt everyday life and cause excess morbidity and mortality worldwide. Vaccination has been key to quelling the impact of this respiratory pathogen, and mRNA vaccines have led the charge on this front. However, the emergence of SARS-CoV-2 variants has sparked fears regarding vaccine efficacy. Furthermore, SARS-CoV-2 vaccines continue to be unevenly distributed across the globe. For these reasons and despite the success of emergency authorized and licensed SARS-CoV-2 vaccines, additional vaccines are needed to meet public health demands. The studies presented here are significant as they demonstrate robust protective efficacy of mRNA vaccines developed by GreenLight Biosciences against not only wild-type SARS-CoV-2, but also Alpha and Beta variants. These results support the progression of GreenLight Biosciences SARS-CoV-2 mRNA vaccines to clinical trials as another defense against SARS-CoV-2.

Self-Amplifying RNA Vaccines for Venezuelan Equine Encephalitis Virus Induce Robust Protective Immunogenicity in Mice.

Venezuelan equine encephalitis virus (VEEV) is a known biological defense threat. A live-attenuated investigational vaccine, TC-83, is available, but it has a high non-response rate and can also cause severe reactogenicity. We generated two novel VEE vaccine candidates using self-amplifying mRNA (SAM). LAV-CNE is a live-attenuated VEE SAM vaccine formulated with synthetic cationic nanoemulsion (CNE) and carrying the RNA genome of TC-83. IAV-CNE is an irreversibly-attenuated VEE SAM vaccine formulated with CNE, delivering a TC-83 genome lacking the capsid gene. LAV-CNE launches a TC-83 infection cycle in vaccinated subjects but eliminates the need for live-attenuated vaccine production and potentially reduces manufacturing time and complexity. IAV-CNE produces a single cycle of RNA amplification and antigen expression without generating infectious viruses in subjects, thereby creating a potentially safer alternative to live-attenuated vaccine. Here, we demonstrated that mice vaccinated with LAV-CNE elicited immune responses similar to those of TC-83, providing 100% protection against aerosol VEEV challenge. IAV-CNE was also immunogenic, resulting in significant protection against VEEV challenge. These studies demonstrate the proof of concept for using the SAM platform to streamline the development of effective attenuated vaccines against VEEV and closely related alphavirus pathogens such as western and eastern equine encephalitis and Chikungunya viruses.

Dengue Virus Uses a Non-Canonical Function of the Host GBF1-Arf-COPI System for Capsid Protein Accumulation on Lipid Droplets.

Dengue viruses cause the most important human viral disease transmitted by mosquitoes. In recent years, a great deal has been learned about molecular details of dengue virus genome replication; however, little is known about genome encapsidation and the functions of the viral capsid protein. During infection, dengue virus capsid progressively accumulates around lipid droplets (LDs) by an unknown mechanism. Here, we examined the process by which the viral capsid is transported from the endoplasmic reticulum (ER) membrane, where the protein is synthesized, to LDs. Using different methods of intervention, we found that the GBF1-Arf1/Arf4-COPI pathway is necessary for capsid transport to LDs, while the process is independent of both COPII components and Golgi integrity. The transport was sensitive to Brefeldin A, while a drug resistant form of GBF1 was sufficient to restore capsid subcellular distribution in infected cells. The mechanism by which LDs gain or lose proteins is still an open question. Our results support a model in which the virus uses a non-canonical function of the COPI system for capsid accumulation on LDs, providing new ideas for antiviral strategies.

Uncoupling cis-Acting RNA elements from coding sequences revealed a requirement of the N-terminal region of dengue virus capsid protein in virus particle formation.

Little is known about the mechanism of flavivirus genome encapsidation. Here, functional elements of the dengue virus (DENV) capsid (C) protein were investigated. Study of the N-terminal region of DENV C has been limited by the presence of overlapping cis-acting RNA elements within the protein-coding region. To dissociate these two functions, we used a recombinant DENV RNA with a duplication of essential RNA structures outside the C coding sequence. By the use of this system, the highly conserved amino acids FNML, which are encoded in the RNA cyclization sequence 5'CS, were found to be dispensable for C function. In contrast, deletion of the N-terminal 18 amino acids of C impaired DENV particle formation. Two clusters of basic residues (R5-K6-K7-R9 and K17-R18-R20-R22) were identified as important. A systematic mutational analysis indicated that a high density of positive charges, rather than particular residues at specific positions, was necessary. Furthermore, a differential requirement of N-terminal sequences of C for viral particle assembly was observed in mosquito and human cells. While no viral particles were observed in human cells with a virus lacking the first 18 residues of C, DENV propagation was detected in mosquito cells, although to a level about 50-fold less than that observed for a wild-type (WT) virus. We conclude that basic residues at the N terminus of C are necessary for efficient particle formation in mosquito cells but that they are crucial for propagation in human cells. This is the first report demonstrating that the N terminus of C plays a role in DENV particle formation. In addition, our results suggest that this function of C is differentially modulated in different host cells.

Dengue virus capsid protein usurps lipid droplets for viral particle formation.

Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation.

A 5' RNA element promotes dengue virus RNA synthesis on a circular genome.

The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5' element named SLA. We demonstrated that RNA-RNA interactions between 5' and 3' end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5' end of the genome and reaches the site of initiation at the 3' end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.