A replicating RNA vaccine confers protection in a rhesus macaque model of Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne febrile illness with a wide geographic distribution. In recent years the geographic range of Crimean-Congo hemorrhagic fever virus (CCHFV) and its tick vector have increased, placing an increasing number of people at risk of CCHFV infection. Currently, there are no widely available vaccines, and although the World Health Organization recommends ribavirin for treatment, its efficacy is unclear. Here we evaluate a promising replicating RNA vaccine in a rhesus macaque (Macaca mulatta) model of CCHF. This model provides an alternative to the established cynomolgus macaque model and recapitulates mild-to-moderate human disease. Rhesus macaques infected with CCHFV consistently exhibit viremia, detectable viral RNA in a multitude of tissues, and moderate pathology in the liver and spleen. We used this model to evaluate the immunogenicity and protective efficacy of a replicating RNA vaccine. Rhesus macaques vaccinated with RNAs expressing the CCHFV nucleoprotein and glycoprotein precursor developed robust non-neutralizing humoral immunity against the CCHFV nucleoprotein and had significant protection against the CCHFV challenge. Together, our data report a model of CCHF using rhesus macaques and demonstrate that our replicating RNA vaccine is immunogenic and protective in non-human primates after a prime-boost immunization.

Antibodies targeting the Crimean-Congo Hemorrhagic Fever Virus nucleoprotein protect via TRIM21.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a negative-sense RNA virus spread by Hyalomma genus ticks across Europe, Asia, and Africa. CCHF disease begins as a non-specific febrile illness which may progress into a severe hemorrhagic disease with no widely approved or highly efficacious interventions currently available. Recently, we reported a self-replicating, alphavirus-based RNA vaccine that expresses the CCHFV nucleoprotein and is protective against lethal CCHFV disease in mice. This vaccine induces high titers of non-neutralizing anti-NP antibodies and we show here that protection does not require Fc-gamma receptors or complement. Instead, vaccinated mice deficient in the intracellular Fc-receptor TRIM21 were unable to control the infection despite mounting robust CCHFV-specific immunity. We also show that passive transfer of NP-immune sera confers significant TRIM21-dependent protection against lethal CCHFV challenge. Together our data identifies TRIM21-mediated mechanisms as the Fc effector function of protective antibodies against the CCHFV NP and provides mechanistic insight into how vaccines against the CCHFV NP confer protection.

Single dose, dual antigen RNA vaccines protect against lethal Crimean-Congo haemorrhagic fever virus infection in mice.

BACKGROUND: Crimean-Congo Haemorrhagic Fever Virus is a tick-borne bunyavirus prevalent across Asia, Africa, the Middle East, and Europe. The virus causes a non-specific febrile illness which may develop into severe haemorrhagic disease. To date, there are no widely approved therapeutics. Recently, we reported an alphavirus-based replicon RNA vaccine which expresses the CCHFV nucleoprotein (repNP) or glycoprotein precursor (repGPC) and is protective against lethal disease in mice. METHODS: Here, we evaluated engineered GPC constructs to find the minimal enhancing epitope of repGPC and test two RNA vaccine approaches to express multiple antigens in vivo to optimize protective efficacy of our repRNA. FINDINGS: Vaccination with repNP and a construct expressing just the Gc antigen (repGc-FL) resulted in equivalent immunogenicity and protective efficacy compared to original repNP + repGPC vaccination. This vaccine was protective when prepared in either of two vaccine approaches, a mixed synthesis reaction producing two RNAs in a single tube and a single RNA expressing two antigens. INTERPRETATION: Overall, our data illustrate two vaccine approaches to deliver two antigens in a single immunization. Both approaches induced protective immune responses against CCHFV in this model. These approaches support their continued development for this and future vaccine candidates for CCHFV and other vaccines where inclusion of multiple antigens would be optimal. FUNDING: This work was supported by the Intramural Research Program, NIAID/NIH, HDT Bio and MCDC Grant #MCDC2204-011.

Replicating RNA vaccination elicits an unexpected immune response that efficiently protects mice against lethal Crimean-Congo hemorrhagic fever virus challenge.

BACKGROUND: Crimean-Congo hemorrhagic fever virus is the cause of a severe hemorrhagic fever with cases reported throughout a wide-geographic region. Spread by the bite of infected ticks, contact with infected livestock or in the health care setting, disease begins as a non-specific febrile illness that can rapidly progress to hemorrhagic manifestations. Currently, there are no approved vaccines and antivirals such as ribavirin have unclear efficacy. Thus treatment is mostly limited to supportive care. METHODS: In this report we evaluated an alphavirus-based replicon RNA vaccine expressing either the CCHFV nucleoprotein or glycoprotein precursor in a stringent, heterologous lethal challenge mouse model. FINDINGS: Vaccination with the RNA expressing the nucleoprotein alone could confer complete protection against clinical disease, but vaccination with a combination of both the nucleoprotein and glycoprotein precursor afforded robust protection against disease and viral replication. Protection from lethal challenge required as little as a single immunization with 100ng of RNA. Unexpectedly, analysis of the immune responses elicited by the vaccine components showed that vaccination resulted in antibodies against the internal viral nucleoprotein and cellular immunity against the virion-exposed glycoproteins. INTERPRETATION: Cumulatively this vaccine conferred robust protection against Crimean-Congo hemorrhagic fever virus and supports continued development of this vaccine candidate. FUNDING: This research was supported by the Intramural Research Program of the NIAID/NIH and HDT Bio.

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease following challenge with heterologous variants of concern.

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late 2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoCs) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second-generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform can be updated to target emergent VoCs, elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.

Since 2019, the SARS-CoV-2 virus has spread worldwide and caused hundreds of millions of cases of COVID-19. Vaccines were rapidly developed to protect people from becoming severely ill from the virus and decrease the risk of death. However, new variants ­ such as Alpha, Beta and Omicron ­ have emerged that the vaccines do not work as well against, contributing to the ongoing spread of the virus. One way to overcome this is to create a vaccine that can be quickly and easily updated to target new variants, like the vaccine against influenza. Many of the vaccines made against COVID-19 use a new technology to introduce the RNA sequence of the spike protein on the surface of SARS-CoV-2 into our cells. Once injected, our cells use their own machinery to build the protein, or 'antigen', so the immune system can learn how to recognize and destroy the virus. Here, Hawman et al. have renovated an RNA vaccine they made in 2020 which provides immunity against the original strain of SARS-CoV-2 in monkeys and mice. In the newer versions of the vaccine, the RNA was updated with a sequence that matches the spike protein on the Beta or Alpha variant of the virus. Both the original and updated vaccines were then administered to mice and hamsters to see how well they worked against SARS-CoV-2 infections. The experiment showed that all three vaccines caused the animals to produce antibodies that can neutralize the original, Alpha and Beta strains of the virus. Vaccinated hamsters were then infected with one of the three variants ­ either matched or mismatched to their vaccination ­ to see how much protection each vaccine provided. All the vaccines reduced the amount of virus in the animals after infection and mitigated damage in their lungs. But animals that received a vaccine which corresponded to the SARS-CoV-2 strain they were infected with had slightly better protection. These findings suggest that these vaccines work best when their RNA sequence matches the strain responsible for the infection; however, even non-matched vaccines still provide a decent degree of protection. Furthermore, the data demonstrate that the vaccine platform created by Hawman et al. can be easily updated to target new strains of SARS-CoV-2 that may emerge in the future. Recently, the Beta variant of the vaccine entered clinical trials in the United States (led by HDT Bio) to evaluate whether it can be used as a booster in previously vaccinated individuals as well as unvaccinated participants.

Three-Week Old Pigs Are Not Susceptible to Productive Infection with SARS-COV-2.

As the COVID-19 pandemic moves into its third year, there remains a need for additional animal models better recapitulating severe COVID to study SARS-CoV-2 pathogenesis and develop countermeasures, especially treatment options. Pigs are known intermediate hosts for many viruses with zoonotic potential and are susceptible to infection with alpha, beta and delta genera of coronaviruses. Herein, we infected young (3 weeks of age) pigs with SARS-CoV-2 using a combination of respiratory and parenteral inoculation routes. Pigs did not develop clinical disease, nor macroscopic or microscopic pathologic lesions upon SARS-CoV-2 infection. Despite occasional low levels of SARS-CoV-2 genomic RNA in the respiratory tract, subgenomic RNA and infectious virus were never found, and SARS-CoV-2-specific adaptive immune responses were not detectable over the 13-day study period. We concluded that pigs are not susceptible to productive SARS-CoV-2 infection and do not serve as a SARS-CoV-2 reservoir for zoonotic transmission.

Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine.

BACKGROUND: In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. METHODS: We rapidly developed a replicating RNA vaccine expressing the B.1.1.529 spike and evaluated immunogenicity in mice and hamsters. We also challenged hamsters with B.1.1.529 and evaluated whether vaccination could protect against viral shedding and replication within respiratory tissue. FINDINGS: We found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529-targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine. INTERPRETATION: Our data suggest that B.1.1.529-targeted vaccines may provide superior protection against B.1.1.529 but pre-existing immunity and timing of boosting may need to be considered for optimum protection. FUNDING: This research was supported in part by the Intramural Research Program, NIAID/NIH, Washington Research Foundation and by grants 27220140006C (JHE), AI100625, AI151698, and AI145296 (MG).

A Look into Bunyavirales Genomes: Functions of Non-Structural (NS) Proteins.

In 2016, the Bunyavirales order was established by the International Committee on Taxonomy of Viruses (ICTV) to incorporate the increasing number of related viruses across 13 viral families. While diverse, four of the families (Peribunyaviridae, Nairoviridae, Hantaviridae, and Phenuiviridae) contain known human pathogens and share a similar tri-segmented, negative-sense RNA genomic organization. In addition to the nucleoprotein and envelope glycoproteins encoded by the small and medium segments, respectively, many of the viruses in these families also encode for non-structural (NS) NSs and NSm proteins. The NSs of Phenuiviridae is the most extensively studied as a host interferon antagonist, functioning through a variety of mechanisms seen throughout the other three families. In addition, functions impacting cellular apoptosis, chromatin organization, and transcriptional activities, to name a few, are possessed by NSs across the families. Peribunyaviridae, Nairoviridae, and Phenuiviridae also encode an NSm, although less extensively studied than NSs, that has roles in antagonizing immune responses, promoting viral assembly and infectivity, and even maintenance of infection in host mosquito vectors. Overall, the similar and divergent roles of NS proteins of these human pathogenic Bunyavirales are of particular interest in understanding disease progression, viral pathogenesis, and developing strategies for interventions and treatments.

An Intramuscular DNA Vaccine for SARS-CoV-2 Decreases Viral Lung Load but Not Lung Pathology in Syrian Hamsters.

The 2019 novel coronavirus, SARS-CoV-2, first reported in December 2019, has infected over 102 million people around the world as of February 2021 and thus calls for rapid development of safe and effective interventions, namely vaccines. In our study, we evaluated a DNA vaccine against SARS-CoV-2 in the Syrian hamster model. Hamsters were vaccinated with a DNA-plasmid encoding the SARS-CoV-2 full length spike open reading frame (ORF) to induce host cells to produce spike protein and protective immune responses before exposure to infectious virus. We tested this vaccine candidate by both intranasal (IN) and intramuscular (IM) routes of administration and complexing with and without an in vivo delivery reagent. Hamsters receiving prime-boost-boost IM-only vaccinations recovered body weight quicker, had decreased lung viral loads, and increased SARS-CoV-2-specific antibody titers compared to control vaccinated animals but, surprisingly, lung pathology was as severe as sham vaccinated controls. The IM/IN combination group showed no efficacy in reducing lung virus titers or pathology. With increasing public health need for rapid and effective interventions, our data demonstrate that in some vaccine contexts, significant antibody responses and decreased viral loads may not be sufficient to prevent lung pathology.