Relevance of the viral Spike protein/cellular Estrogen Receptor-α interaction for endothelial-based coagulopathy induced by SARS-CoV-2

Severe coagulopathy has been observed at the level of the microcirculation in several organs including lungs, heart and kidneys in patients with COVID-19, and in a minority of subjects receiving the SARS-CoV-2 vaccine. Various mechanisms have been implicated in these effects, including increases in circulating neutrophil extracellular traps, excessive inflammation, and endothelial dysfunction. Even if a correlation between infection by SARS-CoV-2 and upregulation of coagulation cascade components has been established in the lung, no direct proofs have been yet provided about the transcriptional machinery controlling the expression of these factors. Recent results obtained by us reported a novel transcriptional function of the SARS-CoV-2 Spike (S) viral protein involving a direct protein-protein interaction with the human Estrogen Receptor- (ER). Given the implications of ER in the control of key effectors in the coagulation cascade, we hypothesized that S-protein might increase the pro-coagulation activity of endothelial cells via the transcriptional activity of the ER, thus justifying the enhanced risk of thrombosis. To assess this, we tested the effects of S-protein on the expression of Tissue Factor (TF) and the overall procoagulation activity in a human endothelial cell line and confirmed this finding by overexpressing S-protein by gene transfer in mice. We then designed and tested two-point mutations in the S2 S-protein sequence that abolished the pro-coagulation function of S-protein in vitro and in vivo, without compromising its immunogenicity. In addition to reveal a new potential transcriptional function of S-protein, these results inspire the design of new vaccines with lower risk of thrombogenesis. Indeed, while the benefit/risk ratio remains overwhelming in favor of COVID-19 vaccination, our results shed light on the causal mechanisms of some rare anti-SARS-CoV-2 vaccine adverse events, and are thus essential for current and future vaccination and booster campaigns.

A linear SARS-CoV-2 DNA vaccine candidate reduces virus shedding in ferrets

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused more than 600 million cases and over 6 million deaths worldwide. Vaccination has been the main strategy used to contain the spread of the virus, and to avoid hospitalizations and deaths. Currently, there are two mRNA-based and one adenovirus vectored vaccines approved and available for use in the U.S. population. The versatility, low cost and rapid-to-manufacture attributes of DNA vaccines are important advantages over other platforms. However, DNA vaccination must meet higher efficiency levels for use in humans. Importantly, in vivo DNA delivery combined with electroporation (EP) has been successfully used in the veterinary field. Here we evaluated the safety, immunogenicity and protective efficacy of a novel linear SARS-CoV-2 DNA vaccine candidate for delivered by intramuscular injection followed by electroporation (Vet-ePorator) in ferrets. The results demonstrated that the linear SARS-CoV-2 DNA vaccine candidate did not cause unexpected side effects, and was able to elicit neutralizing antibodies and T cell responses using a low dose of the linear DNA construct in prime-boost regimen, and significantly reduced shedding of infectious SARS-CoV-2 through oral and nasal secretions in a ferret model.

A linear DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain elicits protective immunity in domestic cats

Since its first detection in China in late 2019, SARS-CoV-2, the etiologic agent of COVID-19 pandemic, has infected a wide range of animal species, especially mammals, all over the world. Indeed, as reported by the American Veterinary Medical Association, besides human-to-human transmission, human-to-animal transmission has been observed in some wild animals and pets, especially in cats. With animal models as an invaluable tool in the study of infectious diseases combined with the fact that the intermediate animal source of SARS-CoV-2 is still unknown, researchers have demonstrated that cats are permissive to COVID-19 and are susceptible to airborne infections. Given the high transmissibility potential of SARS-CoV-2 to different host species and the close contact between humans and animals, it is crucial to find mechanisms to prevent the transmission chain and reduce the risk of spillover to susceptible species. Here, we show results from a randomized Phase I/II clinical study conducted in domestic cats to assess safety and immunogenicity of a linear DNA ("linDNA") vaccine encoding the RBD domain of SARS-CoV-2. No significant adverse events occurred and both RBD-specific binding/neutralizing antibodies and T cells were detected. These findings demonstrate the safety and immunogenicity of a genetic vaccine against COVID-19 administered to cats and strongly support the development of vaccines for preventing viral spread in susceptible species, especially those in close contact with humans.

COVID-eVax, an electroporated plasmid DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain, elicits protective immune responses in animal models of COVID-19

The COVID-19 pandemic caused by the {beta}-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax - a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD - induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.