Production and characterization of a chimeric antigen, based on nucleocapsid of SARS-CoV-2 fused to the extracellular domain of human CD154 in HEK-293 cells as a vaccine candidate against COVID-19.

Despite that more than one hundred vaccines against SARS-CoV-2 have been developed and that some of them were evaluated in clinical trials, the latest results revealed that these vaccines still face great challenges. Among the components of the virus, the N-protein constitutes an attractive target for a subunit vaccine because it is the most abundant, highly conserved and immunogenic protein. In the present work, a chimeric protein (N-CD protein) was constructed by the fusion of the N-protein to the extracellular domain of human CD154 as the molecular adjuvant. HEK-293 cells were transduced with lentiviral vector bearing the N-CD gene and polyclonal cell populations were obtained. The N-CD protein was purified from cell culture supernatant and further characterized by several techniques. Immunogenicity studies in mice and non-human primates showed the N-CD protein induced high IgG titers in both models after two doses. Moreover, overall health monitoring of non-human primates demonstrated that animals were healthy during 228 days after first immunization. Data obtained support further investigation in order to develop this chimeric protein as vaccine candidate against COVID-19 and other coronavirus diseases.

Prognosis and Personalized In Silico Prediction of Treatment Efficacy in Cardiovascular and Chronic Kidney Disease: A Proof-of-Concept Study.

(1) Background: Kidney and cardiovascular diseases are responsible for a large fraction of population morbidity and mortality. Early, targeted, personalized intervention represents the ideal approach to cope with this challenge. Proteomic/peptidomic changes are largely responsible for the onset and progression of these diseases and should hold information about the optimal means of treatment and prevention. (2) Methods: We investigated the prediction of renal or cardiovascular events using previously defined urinary peptidomic classifiers CKD273, HF2, and CAD160 in a cohort of 5585 subjects, in a retrospective study. (3) Results: We have demonstrated a highly significant prediction of events, with an HR of 2.59, 1.71, and 4.12 for HF, CAD, and CKD, respectively. We applied in silico treatment, implementing on each patient's urinary profile changes to the classifiers corresponding to exactly defined peptide abundance changes, following commonly used interventions (MRA, SGLT2i, DPP4i, ARB, GLP1RA, olive oil, and exercise), as defined in previous studies. Applying the proteomic classifiers after the in silico treatment indicated the individual benefits of specific interventions on a personalized level. (4) Conclusions: The in silico evaluation may provide information on the future impact of specific drugs and interventions on endpoints, opening the door to a precision-based medicine approach. An investigation into the extent of the benefit of this approach in a prospective clinical trial is warranted.

Chimeric Antigen by the Fusion of SARS-CoV-2 Receptor Binding Domain with the Extracellular Domain of Human CD154: A Promising Improved Vaccine Candidate.

COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor-binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host's cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immunogenicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose.

Transient production of receptor-binding domain of SARS-CoV-2 in Nicotiana benthamiana plants induces specific antibodies in immunized mice.

BACKGROUND: The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has currently affected millions of people around the world. To combat the rapid spread of COVID-19 there is an urgent need to implement technological platforms for the production of vaccines, drugs and diagnostic systems by the scientific community and pharmaceutical companies. The SARS-CoV-2 virus enters the cells by the interaction between the receptor-binding domain (RBD) present in the viral surface spike protein and its human receptor ACE2. The RBD protein is therefore considered as the target for potential subunit-based vaccines. METHODS AND RESULTS: We evaluate the use of Nicotiana benthamiana plants as the host to transiently-producing recombinant RBD (RBDr) protein. The identity of the plant-produced RBDr was confirmed by immune assays and mass spectrometry. Immunogenicity was confirmed through the specific antibodies generated in all of the immunized mice compared to the PBS treated group. CONCLUSIONS: In conclusions, the immunogenicity of the RBDr produced in N. benthamiana was confirmed. These findings support the use of plants as an antigen expression system for the rapid development of vaccine candidates.

In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI-MS spectrum.

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs.