A cell-based ELISA as surrogate of virus neutralization assay for RBD SARS-CoV-2 specific antibodies. Pi-Estopiñan F, Pérez MT, Fraga A, Bergado G, Díaz GD, Orosa I, et al. Vaccine. 2022 Mar 18;40(13):1958-67. https://doi.org/10.1016/j.vaccine.2022.02.044. Epub 2022 Feb 15.
SARS-CoV-2, the cause of the COVID-19 pandemic, has provoked a global crisis and death of millions of people. Several serological assays to determine the quality of the immune response against SARS-CoV-2 and the efficacy of vaccines have been developed, among them the gold standard conventional virus neutralization assays. However, these tests are time consuming, require biosafety level 3 (BSL3), and are low throughput and expensive. This has motivated the development of alternative methods, including molecular inhibition assays. Herein, we present a safe cell-based ELISA-virus neutralization test (cbE-VNT) as a surrogate for the conventional viral neutralization assays that detects the inhibition of SARS-CoV-2 RBD binding to ACE2-bearing cells independently of species. Our test shows a very good correlation with the conventional and molecular neutralization assays and achieves 100% specificity and 95% sensitivity. cbE-VNT is cost-effective, fast and enables a large-scale serological evaluation that can be performed in a BSL2 laboratory, allowing its use in pre-clinical and clinical investigations.

A COVID-19 vaccine candidate composed of SARS-CoV-2 RBD dimer and Neisseria meningitidis outer membrane Vesicles. Santana D, Pérez-Nicado R, Climent Y, Rodríguez L, Sánchez Ramírez B, Perez-Rodríguez S, et al. RSC Chem Biol. 2022 Feb 9;3(2):242–9. Epub 2021 Dec 8. https://dx.doi.org/10.1039/d1cb00200g
SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host’s cellular receptor. Vaccines seek to block this interaction by eliciting neutralizing antibodies, most of which are directed toward the RBD. Many protein subunit vaccines require powerful adjuvants to generate a potent antibody response. Here, we report on the use of a SARS-CoV-2 dimeric recombinant RBD combined with Neisseria meningitidis outer membrane vesicles (OMVs), adsorbed on alum, as a promising COVID-19 vaccine candidate. This formulation induces a potent and neutralizing immune response in laboratory animals, which is higher than that of the dimeric RBD alone adsorbed on alum. Sera of people vaccinated with this vaccine candidate, named Soberana01, show a high inhibition level of the RBD-ACE2 interaction using RBD mutants corresponding to SARS-CoV-2 variants of concern and wild-type expressed using the phage display technology. To our knowledge, this is the first time that the immunostimulation effect of N. meningitidis OMVs is evaluated in vaccine candidates against SARS-CoV-2.

A randomized, double-blind phase I clinical trial of two recombinant dimeric RBD COVID-19 vaccine candidates: Safety, reactogenicity and immunogenicity. Pérez-Rodríguez S, de la Caridad Rodríguez-González M, Ochoa-Azze R, Climent-Ruiz Y, Alberto González-Delgado C, Paredes-Moreno B, et al. Vaccine. 2022 Mar 18;40(13):2068–75. Epub 2022 Feb 8. https://doi.org10.1016/j.vaccine.2022.02.029
Background The Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein is the target for many COVID-19 vaccines. Here we report results for phase I clinical trial of two COVID-19 vaccine candidates based on recombinant dimeric RBD (d-RBD). Methods We performed a randomized, double-blind, phase I clinical trial in the National Centre of Toxicology in Havana. Sixty Cuban volunteers aged 19-59 years were randomized into three groups (20 subjects each): 1) FINLAY-FR-1 (50 µg d-RBD plus outer membrane vesicles from N. meningitidis); 2) FINLAY-FR-1A-50 (50 µg d-RBD, three doses); 3) FINLAY-FR-1A-25 (25 µg d-RDB, three doses). The FINLAY-FR-1 group was randomly divided to receive a third dose of the same vaccine candidate (homologous schedule) or FINLAY-FR-1A-50 (heterologous schedule). The primary outcomes were safety and reactogenicity. The secondary outcome was vaccine immunogenicity. Humoral response at baseline and following each vaccination was evaluated using live-virus neutralization test, anti-RBD IgG ELISA and in-vitro neutralization test of RBD:hACE2 interaction. Results Most adverse events were of mild intensity (63.5%), solicited (58.8%), and local (61.8%); 69.4% with causal association with vaccination. Serious adverse events were not found. The FINLAY-FR-1 group reported more subjects with adverse events than the other two groups. After the third dose, anti-RBD seroconversion was 100%, 94.4% and 90% for the FINLAY-FR-1, FINLAY-FR-1A-50 and FINLAY-FR-1A-25 respectively. The in-vitro inhibition of RBD:hACE2 interaction increased after the second dose in all formulations. The geometric mean neutralizing titres after the third dose rose significantly in the group vaccinated with FINLAY-FR-1 with respect to the other formulations and the COVID-19 Convalescent Serum Panel. No differences were found between FINLAY-FR-1 homologous or heterologous schedules. Conclusions Vaccine candidates were safe and immunogenic, and induced live-virus neutralizing antibodies against SARS-CoV-2. The highest values were obtained when outer membrane vesicles were used as adjuvant. Trial registry https://rpcec.sld.cu/en/trials/RPCEC00000338-En.

A single dose of SARS-CoV-2 FINLAY-FR-1A vaccine enhances neutralization response in COVID-19 convalescents, with a very good safety profile: An open-label phase 1 clinical trial. Chang-Monteagudo A, Ochoa-Azze R, Climent-Ruiz Y, Macías-Abraham C, Rodríguez-Noda L, Valenzuela-Silva C, et al. Lancet Reg Health Am. 2021 Dec;4:100079.
https://dx.doi.org/10.1016%2Fj.lana.2021.100079. Epub 2021 Sep 15. PMID: 34541571 Free PMC article.
Background As a first step towards a vaccine protecting COVID-19 convalescents from reinfection, we evaluated FINLAY-FR-1A vaccine in a clinical trial. Method: Thirty COVID-19 convalescents aged 22-57 years were studied: convalescents of mild COVID-19, asymptomatic convalescents, both with PCR-positive at the moment of diagnosis; and individuals with subclinical infection detected by viral-specific IgG. They received a single intramuscular injection of the FINLAY-FR-1A vaccine (50 µg of the recombinant dimeric receptor binding domain). The primary outcomes were safety and reactogenicity, assessed over 28 days after vaccination. The secondary outcome was vaccine immunogenicity. Humoral response at baseline and following vaccination was evaluated by ELISA and live-virus neutralization test. The effector T cellular response was also assessed. Cuban Public Registry of Clinical Trials, WHO-ICTRP: https://rpcec.sld.cu/en/trials/RPCEC00000349-En. Findings No serious adverse events were reported. Minor adverse events were found, the most common, local pain: 3 (10%) and redness: 2 (6·7%). The vaccine elicited a >21 fold increase in IgG anti-RBD antibodies 28 days after vaccination. The median of inhibitory antibody titres (94·0%) was three times greater than that of the COVID-19 convalescent panel. Virus neutralization titres higher than 1:160 were found in 24 (80%) participants. There was also an increase in RBD-specific T cells producing IFN-γ and TNF-α. Interpretation A single dose of the FINLAY-FR-1A vaccine against SARS-CoV-2 was an efficient booster of pre-existing natural immunity, with excellent safety profile. Funding Partial funding for this study was received from the Project-2020-20, Fondo de Ciencia e Innovación (FONCI), Ministry of Science, Technology and the Environment, Cuba. 

In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD protein allows a full sequence coverage and PMT characterization in a single ESI-MS spectrum. Espinosa LA, Ramos Y, Andújar I, Onill Torres E, Cabrera G, Martín A, et al. Anal Bioanal Chem. 2021 Dec;413(30):7559–85. https://doi.org/10.1007/s00216-021-03721-w
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.

Mustard surgery three months after a COVID-19 infection: a case report. Corrales Arredondo I, Naranjo Ugalde CAM, Ceruto Ortíz LA, Escobar Bermúdez Y, López Rodríguez PR. MOJ Gero & Geri Med. 2022 Jan 6;7(1):1–3. https://doi.org/10.15406/mojgg.2022.07.00280
Introduction This past year, on a global scale, since 2019, public health warnings have gone off because of the recent epidemiological crisis set of the Covid-19 pandemic. This pandemic holds responsibility for millions of infections, manifesting broadly in its clinical presentation, which ranges from asymptomatic carriers to respiratory failure, myocardial pathology and death; increasing the rates of hospitalization. Pediatric patients are at high risk of contracting the disease including those with congenital cardiomyopathy that are in need of surgical intervention in order to survive. Objective Show that there exists an opportunity for elective surgical treatment and short term and medium term recovery in these patients in spite of respiratory and cardiovascular sequelae. Case presentation of an eleven-month infant diagnosed with Transposition of the Great Vessels, who after three months of idleness for having tested positive for Covid-19, received definitive surgical care for the initial diagnosis. Results The perioperative strategy was based in the probable sequelae due to the infection. There are not respiratory complications like consequence for the previous lung injury. The auriculoventricular dysfunctional immediate post-operative was related with the surgical technique. Conclusion A period no less than three months could be offer security for surgery using extracorporeal circulation in pediatric patients who suffered covid-19. Patient with favorable post-op prognosis resulting from the work of a multi-disciplinary team that met all challenges of the complications inherent in the post-operative period following a complex cardiovascular surgery along with those of a potentially fatal virus.

Molecular Aspects Concerning the Use of the SARS-CoV-2 Receptor Binding Domain as a Target for Preventive Vaccines.Valdes-Balbin Y, Santana-Mederos D, Paquet F, Fernandez S, Climent Y, Chiodo F, et al. ACS Cent Sci. 2021 May 26;7(5):757-67. https://doi.org/10.1021/acscentsci.1c00216. Epub 2021 Apr 19. PMID: 34075345 Free PMC article. Review.
The development of recombinant COVID-19 vaccines has resulted from scientific progress made at an unprecedented speed during 2020. The recombinant spike glycoprotein monomer, its trimer, and its recombinant receptor-binding domain (RBD) induce a potent anti-RBD neutralizing antibody response in animals. In COVID-19 convalescent sera, there is a good correlation between the antibody response and potent neutralization. In this review, we summarize with a critical view the molecular aspects associated with the interaction of SARS-CoV-2 RBD with its receptor in human cells, the angiotensin-converting enzyme 2 (ACE2), the epitopes involved in the neutralizing activity, and the impact of virus mutations thereof. Recent trends in RBD-based vaccines are analyzed, providing detailed insights into the role of antigen display and multivalence in the immune response of vaccines under development.

SARS-CoV-2 RBD-Tetanus Toxoid Conjugate Vaccine Induces a Strong Neutralizing Immunity in Preclinical Studies. Valdés-Balbin Y, Santana-Mederos D, Quintero L, Fernández S, Rodríguez L, Sánchez Ramírez B, et al. ACS Chem Biol. 2021 Jul 16;16(7):1223–33. https://doi.org/10.1021/acschembio.1c00272. Epub 2021 Jul 4. PMID: 34219448 Free article.
Controlling the global COVID-19 pandemic depends, among other measures, on developing preventive vaccines at an unprecedented pace. Vaccines approved for use and those in development intend to elicit neutralizing antibodies to block viral sites binding to the host’s cellular receptors. Virus infection is mediated by the spike glycoprotein trimer on the virion surface via its receptor binding domain (RBD). Antibody response to this domain is an important outcome of immunization and correlates well with viral neutralization. Here, we show that macromolecular constructs with recombinant RBD conjugated to tetanus toxoid (TT) induce a potent immune response in laboratory animals. Some advantages of immunization with RBD-TT conjugates include a predominant IgG immune response due to affinity maturation and long-term specific B-memory cells. These results demonstrate the potential of the conjugate COVID-19 vaccine candidates and enable their advance to clinical evaluation under the name SOBERANA02, paving the way for other antiviral conjugate vaccines.