A phase 3, randomised, double-blind, placebo-controlled clinical trial for adult evaluation of the efficacy and safety of a SARS-CoV-2 recombinant spike RBD protein vaccine (ABDALA-3 Study).

BackgroundThe pandemic of COVID-19 raised the urgent need of safe and efficacious vaccines against SARS-CoV-2. We evaluated the efficacy and safety of a new SARS-CoV-2 virus receptor-binding domain (RBD) vaccine. MethodsA phase 3, multicentre, randomised, double-blind, placebo-controlled trial was carried out at 18 clinical sites in three provinces of the south-eastern region of Cuba. Subjects (healthy or those with controlled chronic diseases) aged between 19 and 80 years, who gave written informed consent were eligible. Subjects were randomly assigned (1:1, in blocks) to two groups: placebo, and 50 {micro}g RBD vaccine (Abdala). The product was administered intramuscularly, 0.5 mL in the deltoid region, in a three dose immunization schedule at 0-14-28 days. The organoleptic characteristics and presentations of vaccine and placebo were identical. All participants (subjects, clinical researchers, statisticians, laboratory technicians, and monitors) remained blinded during the study period. The main endpoint was to evaluate the efficacy of the Abdala vaccine in the prevention of symptomatic COVID-19. The trial is registered with the Cuban Public Registry of Clinical Trials, RPCEC00000359. FindingsBetween March 22 to April 03, 2021, 48290 subjects were included (24144 and 21146 in the placebo and Abdala groups, respectively). The product was well tolerated. No severe adverse events with demonstrated cause-effect relationship attributable to vaccine were reported. The incidence of adverse reactions in the placebo and Abdala vaccine arms were 446/24144 (1.9%) and 615/24146 (2.5%), respectively. Adverse reactions were mostly mild, and from the injection site, which resolved in the first 24-48 hours. The Abdala vaccine efficacy against symptomatic COVID-19 was 92.28% (95% CI 85.74-95.82). In the case of mild/moderate disease the vaccine efficacy was 91.96% (84.69-95.78) and 94.46% (58.52-99.28) for the severe forms (serious/critical disease). There were five critical patients (of which four died), all in the placebo group, indicating that Abdala vaccine efficacy for both conditions was of 100%. InterpretationThe Abdala vaccine was safe, well tolerated, and highly effective, fulfilling the WHO target product profile for COVID-19 vaccines. FundingCentre for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.

The SARS-CoV-2 receptor-binding domain expressed in Pichia pastoris as a candidate vaccine antigen

1.The effort to develop vaccines based on economically accessible technological platforms available by developing countries vaccine manufacturers is essential to extend the immunization to the whole world population and to achieve the desired herd immunity, necessary to end the COVID-19 pandemic. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed in yeast Pichia pastoris. The RBD was modified with addition of flexible N- and C-terminal amino acid extensions aimed to modulate the protein/protein interactions and facilitate protein purification. Fermentation with yeast extract culture medium yielded 30-40 mg/L. After purification by immobilized metal ion affinity chromatography and hydrophobic interaction chromatography, the RBD protein was characterized by mass-spectrometry, circular dichroism, and binding affinity to angiotensin-converting enzyme 2 (ACE2) receptor. The recombinant protein shows high antigenicity with convalescent human sera and also with sera from individuals vaccinated with the Pfizer-BioNTech mRNA or Sputnik V adenoviral-based vaccines. The RBD protein stimulates IFN{gamma}, IL-2, IL-6, IL-4, and TNF in mice secreting splenocytes from PBMC and lung CD3+ enriched cells. Immunogenicity studies with 50 {micro}g of the recombinant RBD formulated with alum, induce high levels of binding antibodies in mice and non-human primates, assessed by ELISA plates covered with RBD protein expressed in HEK293T cells. The mouse sera inhibited the RBD binding to ACE2 receptor in an in-vitro test and show neutralization of SARS-CoV-2 infection of Vero E6 cells. These data suggest that the RBD recombinant protein expressed in yeast P. pastoris is suitable as a vaccine candidate against COVID-19. HighlightsO_LIThe RBD protein (C-RBD-H6 PP) is expressed with high purity in P. pastoris. C_LIO_LIPhysico-chemical characterization confirms the right folding of the protein. C_LIO_LIThe recombinant protein shows high antigenicity with sera from convalescents. C_LIO_LIThe sera from animals inhibit the RBD-ACE2 binding and neutralize the virus. C_LIO_LIThe C-RBD-H6 protein stimulates IFN{gamma}, IL-2, IL-6, IL-4, and TNF in mice. C_LI

In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among 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), the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. 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 and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.

The influence of different peptide combinations to increase the immunogenicity of the Gonadotrophin Releasing Hormone Vaccine for prostate cancer treatment.

PURPOSE: Therapeutic vaccines, specifically the Gonadotrophin Releasing Hormone (GnRH) vaccine, are considered an additional therapeutic option for advanced stage prostate cancer. Our work showed amplification of the immune response when combining two peptides with and without the Very Small Size Proteoliposomes (VSSP). VSSP is a potent adjuvant for dendritic cells activation and Th1 differentiation. as enhanced immune response. METHODS: The test was carried out in Copenhagen rats as animal model. RESULTST: The use of both peptides and their combination with VSSP generated a potentiation of the immune response statistically superior, in term of generating anti GnRH antibody and effects on target organs, when it was compared with the effects which occurs with independent peptides and with and without the VSSP. These results can find application in the development of GnRH vaccine candidates and in peptide based vaccine strategies. CONCLUSIONS: Immunization with the peptide combination enhances the immune response when mixed with the VSSPs.

Very small size proteoliposomes (VSSP) and Montanide combination enhance the humoral immuno response in a GnRH based vaccine directed to prostate cancer.

Very small size proteoliposomes (VSSP) constitute a complex of very small size proteoliposomes that includes proteins, lipids, CpG and gangliosides tumor-associated that provides a potential target for cancer immunotherapy. This compound has been described to stimulate the humoral and cellular response, dendritic cells (DC) activation and differentiation of T-helper cells, specially, in immunocompromised patients with cancer status. This work deals with the stimulating capacity of the VSSP to reach a humoral response when they are used as a component in a peptidic vaccine based on the gonadotrophin releasing hormone (GnRH). This study was carried out in male Copenhagen rats, which were immunized with 750µg of the GnRH mimetic peptide (GnRHm1-TT) with or without the VSSP. The mixtures were always emulsified with the oil adjuvant Montanide ISA 51. The anti GnRH seroconversion analysis revealed that the group immunized with the peptide GnRHm1-TT/VSSP developed a strong anti GnRH seroconversion. These antibody levels proved to be significant superior to those reached by the use of the GnRHm1-TT peptide solely emulsified in Montanide. Post-mortem analysis on the Testosterone ablation target organs (prostate and testicles) yielded a sudden decrease in their size and weight in respect to the control group. On the other hand, the group submitted to the use of GnRHm1-TT/VSSP, showed a significant difference in the reduction of these target organs in comparison with the group only immunized with GnRHm1-TT adjuvated in Montanide ISA 51. These values turned to be of p=0.023 and p=0.009 in the prostate and testicles respectively. These findings foreground the VSSP as a useful immunopotentiator to be used as part of a GnRH based vaccine to treat prostate cancer.