Validation and application of an ELISA for the quantification of IgG antibodies against Salmonella Typhi Vi capsular polysaccharide

An indirect ELISA for the quantification of IgG antibodies against the Vi polysaccharide of this bacteria was developed as a part of the stages of Research and Development of a conjugate vaccinal candidate against Salmonella Typhi. The results of the validation process are presented in this paper, in which the interval and linearity of the curve, the intra- and inter-assay precision, accuracy, specificity, limit of detection and robustness were determined. The calibration curve generated with an internal standard serum provided a good fit to a polynomial function and an interval between 1/100 and 1/3200 dilutions. The coefficients of variation in the precision and robustness tests and the percentages of recovered were in intervals established for each one (≤10%, ≤20% and 90-110%, respectively). The assay presented an optimal specificity, obtaining OD signals above 1.3 for positive sera against Vi and low for sera against unrelated antigens. The results support the use of this quantitative ELISA in immunogenicity assays for batch release of Vi conjugates. Likewise, they support their use for the immunogenicity evaluation of Vi polysaccharide formulations and Vi polysaccharide conjugates to proteins in phases of research and developmen

Bioengineered polyester beads co-displaying protein and carbohydrate-based antigens induce protective immunity against bacterial infection

Abstract The efficacy of protein and carbohydrate antigens as vaccines can be improved via particulate delivery strategies. Here, protein and carbohydrate antigens used in formulations of vaccines against Neisseria menigitidis were displayed on in vivo assembled polyester beads using a combined bioengineering and conjugation approach. An endotoxin-free mutant of Escherichia coli was engineered to produce translational fusions of antigens (Neisseria adhesin A (NadA) and factor H binding protein (fHbp) derived from serogroup B) to the polyhydroxybutyrate synthase (PhaC), in order to intracellularly assemble polyester beads displaying the respective antigens. Purified beads displaying NadA showed enhanced immunogenicity compared to soluble NadA. Both soluble and particulate NadA elicited functional antibodies with bactericidal activity associated with protective immunity. To expand the antigen repertoire and to design a more broadly protective vaccine, NadA-PhaC beads were additionally conjugated to the capsular polysaccharide from serogroup C. Co-delivery of surface displayed NadA and the capsular polysaccharide induced a strong and specific Th1/Th17 mediated immune response associated with functional bactericidal antibodies. Our findings provide the foundation for the design of multivalent antigen-coated polyester beads as suitable carriers for protein and polysaccharide antigens in order to induce protective immunity

Comparative Immune Response after Vaccination with SOBERANA^® 02 and SOBERANA^® plus Heterologous Scheme and Natural Infection in Young Children

(1) Background: In children, SARS-CoV-2 infection is mostly accompanied by mild COVID-19 symptoms. However, multisystem inflammatory syndrome (MIS-C) and long-term sequelae are often severe complications. Therefore, the protection of the pediatric population against SARS-CoV-2 with effective vaccines is particularly important. Here, we compare the humoral and cellular immune responses elicited in children (n = 15, aged 5–11 years) vaccinated with the RBD-based vaccines SOBERANA® 02 and SOBERANA® Plus combined in a heterologous scheme with those from children (n = 10, aged 4–11 years) who recovered from mild symptomatic COVID-19. (2) Methods: Blood samples were taken 14 days after the last dose for vaccinated children and 45–60 days after the infection diagnosis for COVID-19 recovered children. Anti-RBD IgG and ACE2-RBD inhibition were assessed by ELISA; IgA, cytokines, and cytotoxic-related proteins were determined by multiplex assays. Total B and T cell subpopulations and IFN-γ release were measured by multiparametric flow cytometry using a large panel of antibodies after in vitro stimulation with S1 peptides. (3) Results: Significant higher levels of specific anti-RBD IgG and IgA and ACE2-RBD inhibition capacity were found in vaccinated children in comparison to COVID-19 recovered children. Th1-like and Th2-like CD4+ T cells were also significantly higher in vaccinated subjects. IFN-γ secretion was higher in central memory CD4+ T cells of COVID-19 recovered children, but no differences between both groups were found in the CD4+ and CD8+ T cell effector, terminal effector, and naïve T cell subpopulations. In contrast to low levels of IL-4, high levels of IL-2, IL-6, IFN-γ, and IL-10 suggest a predominant Th1 cell polarization. Cytotoxic-related proteins granzyme A and B, perforin, and granulin were also found in the supernatant after S1 stimulation in both vaccinated and recovered children. (4) Conclusions: Vaccination with the heterologous scheme of SOBERANA® 02/SOBERANA® Plus induces a stronger antibody and cellular immune response compared to natural infections in young children

Safety and immunogenicity of the FINLAY-FR-1A vaccine in COVID-19 convalescent participants: an open-label phase 2a and double-blind, randomised, placebo-controlled, phase 2b, seamless, clinical trial

BACKGROUND: A phase 1, clinical trial to evaluate FINLAY-FR-1A vaccine in COVID-19 convalescent individuals was completed. Here, we report results of the phase 2, clinical trial. METHODS: We studied 450 convalescent participants with a history of asymptomatic, mild, or moderate COVID-19 at the National Institute of Hematology and Immunology and the National Centre for Sexual Education in Havana, Cuba. The study included adults aged 19-78 years who had recovered from COVID-19 and had had a negative PCR test at least 2 months before the initiation of the study. Phase 2 was done sequentially in two stages. The first stage to assess safety comprised an open, non-controlled phase 2a study in participants aged 60-78 years who received a single dose of the FINLAY-FR-1A vaccine (50 μg of recombinant dimeric receptor binding domain [RBD]). The second stage comprised the placebo-controlled, double-blind, phase 2b trial in participants aged 19-78 years, where participants were randomly assigned (4:1) into two groups: an experimental group vaccinated with a single dose of the FINLAY-FR-1A vaccine, and a control (placebo) group injected with vaccine excipient. The primary outcomes were safety, evaluated 28 days after vaccination by the occurrence of serious adverse events in all participants, and successful immune response, assessed by neutralising antibody ELISA, and defined as half-maximal surrogate virus neutralisation titres of 250 or more. Secondary endpoints included vaccine immunogenicity assessed by ELISA anti-RBD and live-virus neutralisation test. All randomly assigned participants were included in the safety analysis (safety population), and immunogenicity was evaluated in participants without study interruptions (per-protocol population). The trial is registered with the Cuban Public Registry of Clinical Trials, RPCEC00000366-En and WHO-ICTRP and is complete. FINDINGS: From April 9, 2021, to April 17, 2021, 663 COVID-19 convalescent participants were enrolled in the study; 213 participants did not meet the selection criteria and 450 volunteers were recruited. 20 participants aged 60-78 years were included in the open, single-group, phase 2a study and 430 participants were randomly assigned to the experimental (n=344) or control groups (n=86) in the phase 2b study of participants aged 19-78 years. 19 (95%) of 20 phase 2a volunteers achieved a successful immune response after vaccination. No vaccine-associated serious adverse events were reported in the whole study population. Minor adverse events were found, the most common being pain at the injection site (105 [29%] of 364 in the intervention group; 13 [15%] of 86 in the placebo group). A successful immune response was found in 289 (81%) of 358 participants 28 days after vaccination. The vaccine elicited a greater than 31-times increase in anti-RBD-IgG antibodies compared with prevaccination rates, and the seroconversion rate was 302 (84%) of 358 on day 28 after vaccination; the geometric mean titres of live-virus neutralisation test increased from 15·4 (95% CI 10·3-23·2) to 400·3 (272·4-588·1) and high response was found against alpha, beta, and delta variants of concern. INTERPRETATION: A single dose of the FINLAY-FR-1A vaccine against SARS-CoV-2 strengthened the pre-existing natural immunity, with excellent safety profile. FUNDING: Cuba's Ministry of Science, Technology, and Environment

Diseño, desarrollo y evaluación preclínica de SOBERANA®02: una vacuna cubana contra COVID-19

International audienc

Open-label phase I/II clinical trial of SARS-CoV-2 receptor binding domain-tetanus toxoid conjugate vaccine (FINLAY-FR-2) in combination with receptor binding domain-protein vaccine (FINLAY-FR-1A) in children

Objectives: To evaluate a heterologous vaccination scheme in children 3-18 years old (y/o) combining two SARS-CoV-2r- receptor binding domain (RBD)protein vaccines. Methods: A phase I/II open-label, adaptive, and multicenter trial evaluated the safety and immunogenicity of two doses of FINLAY-FR-2 (subsequently called SOBERANA 02) and the third heterologous dose of FINLAY-FR-1A (subsequently called SOBERANA Plus) in 350 children 3-18 y/o in Havana Cuba. Primary outcomes were safety (phase I) and safety/immunogenicity (phase II) measured by anti-RBD immunoglobulin (Ig)G enzyme-linked immunoassay (ELISA), molecular and live-virus neutralization titers, and specific T-cells response. A comparison with adult immunogenicity and predictions of efficacy were made based on immunological results. Results: Local pain was the unique adverse event with frequency >10%, and none was serious neither severe. Two doses of FINLAY-FR-2 elicited a humoral immune response similar to natural infection; the third dose with FINLAY-FR-1A increased the response in all children, similar to that achieved in vaccinated young adults. The geometric mean (GMT) neutralizing titer was 173.8 (95% confidence interval [CI] 131.7; 229.5) vs Alpha, 142 (95% CI 101.3; 198.9) vs Delta, 24.8 (95% CI 16.8; 36.6) vs Beta and 99.2 (95% CI 67.8; 145.4) vs Omicron. Conclusion: The heterologous scheme was safe and immunogenic in children 3-18 y/o. Trial registry: https://rpcec.sld.cu/trials/RPCEC0000037

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

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