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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. Graphical abstract: [Figure not available: see fulltext.]

Conjugate vaccines against encapsulated pathogens like Streptococcus pneumoniae face many challenges, including the existence of multiple serotypes with a diverse global distribution that constantly requires new formulations and higher coverage. Multivalency is usually achieved by combining capsular polysaccharide-protein conjugates from invasive serotypes, and for S. pneumoniae, this has evolved from 7- up to 20-valent vaccines. These glycoconjugate formulations often contain high concentrations of carrier proteins, which may negatively affect glycoconjugate immune response. This work broadens the scope of an efficient multicomponent strategy, leading to multivalent pneumococcal glycoconjugates assembled in a single synthetic operation. The bioconjugation method, based on the Ugi four-component reaction, enables the one-pot incorporation of two different polysaccharide antigens to a tetanus toxoid carrier, thus representing the fastest approach to achieve multivalency. The reported glycoconjugates incorporate three combinations of capsular polysaccharides 1, 6B, 14, and 18C from S. pneumoniae. The glycoconjugates were able to elicit functional specific antibodies against pneumococcal strains comparable to those shown by mixtures of the two monovalent glycoconjugates.

To evaluate a heterologous vaccination scheme in children 3-18 y/o combining two SARS-CoV-2 r-RBD protein vaccines.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 3-18y/o in Havana Cuba. Primary outcomes were safety (phase I) and safety/immunogenicity (phase II) measured by anti-RBD IgG ELISA, molecular and live-virus neutralization titers and specific T-cells response. A comparison with adult´s immunogenicity and predictions of efficacy were made based on immunological results RESULTS: Local pain was the unique adverse event with frequency10%, and none was serious or 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 GMT neutralizing titer was 173.8 (CI 95% 131.7; 229.5) vs. alpha, 142 (CI 95% 101.3; 198.9) vs. delta, 24.8 (CI 95% 16.8; 36.6) vs. beta and 99.2 (CI 95% 67.8; 145.4) vs. omicron.The heterologous scheme was safe and immunogenic in children 3-18 y/o.https://rpcec.sld.cu/trials/RPCEC00000374.

This study evaluates safety of FINLAY-FR-02, a vaccine candidate against SARS-CoV-2 based on the recombinant receptor binding domain conjugated to tetanus toxoid, in a preclinical, repeat-dose toxicity and local tolerance study. Sprague Dawley rats were randomly allocated to three experimental groups: control (receiving physiological saline solution); placebo (receiving all vaccine components except antigens) and vaccine group (receiving three doses of the vaccine candidate, 37.5 µg of RBD) administered intramuscularly in hind limbs at 24 h intervals during three days. We evaluated physiological condition, pain, food and water consumption, body temperature, dermal irritability, injection site temperature and inflammation, immunological response, blood chemistry, relative organ weight, histopathology and immunotoxicology. The product was well tolerated; no clinically relevant changes, pain, local effects or adverse systemic toxicological changes or deaths were observed. These preliminary results permitted the Cuban regulatory authorities to authorize clinical trials in humans.

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.

BackgroundSOBERANA 02 is a COVID-19 conjugate vaccine candidate based on SARS-CoV-2 recombinant RBD conjugated to tetanus toxoid. SOBERANA Plus antigen is dimeric-RBD. Here we report safety, reactogenicity and immunogenicity from phase I and IIa clinical trials using two-doses SOBERANA 02 (homologous protocol) and three-doses (homologous) or heterologous (with SOBERANA Plus) protocols.MethodWe performed an open-label, monocentric, sequential and adaptive phase I for evaluating safety, reactogenicity and exploring immunogenicity of SOBERANA 02 in two formulations (15 and 25 μg) in 40 subjects, 19–59 years old. Phase IIa was open-label including 100 volunteers 19–80 years, receiving two doses of SOBERANA 02-25 μg. In both trials, half of volunteers received a third dose of SOBERANA 02, half received a heterologous dose of SOBERANA Plus-50 μg. Primary outcomes were safety and reactogenicity. The secondary outcome was vaccine immunogenicity evaluated by anti-RBD IgG ELISA, molecular neutralization test of RBD:hACE2 interaction, live-virus neutralization test and specific T-cells response.ResultsThe most frequent AE was local pain, other AEs had frequencies ≤ 5%. No serious related AEs were reported. Phase IIa confirmed the safety results in 60–80 years subjects. In phase-I SOBERANA 02-25µg elicited higher immune response than SOBERANA 02-15 µg; in consequence, the higher dose progressed to phase IIa. Phase IIa results confirmed the immunogenicity of SOBERANA 02-25 μg even in 60–80 age range. Two doses of SOBERANA02-25 μg elicited an immune response similar to that of the Cuban Convalescent Serum Panel; it was higher after both the homologous and heterologous third doses; the heterologous scheme showing a higher immunological response.ConclusionsSOBERANA 02 was safe and immunogenic in persons aged 19–80 years, eliciting neutralizing antibodies and specific T cell response. Highest immune responses were obtained in the heterologous three doses protocol. Trial registry: https://rpcec.sld.cu/trials/RPCEC00000340 and https://rpcec.sld.cu/trials/RPCEC00000347

SOBERANA 02 has been evaluated in phase I and IIa studies comparing homologous versus heterologous schedule (this one, including SOBERANA Plus). Here, we report results of immunogenicity, safety, and reactogenicity of SOBERANA 02 in a two- or three-dose heterologous scheme in adults.Phase IIb was a parallel, multicenter, adaptive, double-blind, randomized, and placebo-controlled trial. Subjects (n = 810) aged 19-80 years were randomized to receive two doses of SARS-CoV-2 RBD conjugated to tetanus toxoid (SOBERANA 02) and a third dose of dimeric RBD (SOBERANA Plus) 28 days apart; two production batches of active ingredients of SOBERANA 02 were evaluated. Primary outcome was the percentage of seroconverted subjects with ≥4-fold the anti-RBD immunoglobulin G (IgG) concentration. Secondary outcomes were safety, reactogenicity, and neutralizing antibodies.Seroconversion rate in vaccinees was 76.3% after two doses and 96.8% after the third dose of SOBERANA Plus (7.3% in the placebo group). Neutralizing IgG antibodies were detected against D614G and variants of concern (VOCs) Alpha, Beta, Delta, and Omicron. Specific, functional antibodies were detected 7-8 months after the third dose. The frequency of serious adverse events (AEs) associated with vaccination was very low (0.1%). Local pain was the most frequent AE.Two doses of SOBERANA 02 were safe and immunogenic in adults. The heterologous combination with SOBERANA Plus increased neutralizing antibodies, detectable 7-8 months after the third dose.https://rpcec.sld.cu/trials/RPCEC00000347 FUNDING: This work was supported by Finlay Vaccine Institute, BioCubaFarma, and the Fondo Nacional de Ciencia y Técnica (FONCI-CITMA-Cuba, contract 2020-20).

Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular polysaccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical challenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the polysaccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR results were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.

SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer

Vaccination represents the most effective way to prevent invasive pneumococcal diseases. The glycoconjugate vaccines licensed so far are obtained from capsular polysaccharides (CPSs) of the most virulent serotypes. Protection is largely limited to the specific vaccine serotypes, and the continuous need for broader coverage to control the outbreak of emerging serotypes is pushing the development of new vaccine candidates. Indeed, the development of efficacious vaccine formulation is complicated by the high number of bacterial serotypes with different CPSs. In this context, to simplify vaccine composition, we propose the design of new saccharide fragments containing chemical structures shared by different serotypes as cross-reactive and potentially cross-protective common antigens. In particular, we focused on Streptococcus pneumoniae (Sp) 19A and 19F. The CPS repeating units of Sp 19F and 19A are very similar and share a common structure, the disaccharide ManNAc-beta-(1 -> 4)-Glc (A-B). Herein, we describe the synthesis of a small library of compounds containing different combinations of the common 19F/19A disaccharide. The six new compounds were tested with a glycan array to evaluate their recognition by antibodies in reference group 19 antisera and factor reference antisera (reacting against 19F or 19A). The disaccharide A-B, phosphorylated at the upstream end, emerged as a hit from the glycan array screening because it is strongly recognized by the group 19 antisera and by the 19F and 19A factor antisera, with similar intensity compared with the CPSs used as controls. Our data give a strong indication that the phosphorylated disaccharide A-B can be considered a common epitope among different Sp 19 serotypes.

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 use 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 the immunization and correlates well with viral neutralization. Here we show that macromolecular constructs with recombinant RBD conjugated to tetanus toxoid induce a potent immune response in laboratory animals. Some advantages of the immunization with the viral antigen coupled to tetanus toxoid have become evident such as predominant IgG immune response due to affinity maturation and long-term specific B-memory cells. This paper demonstrates that subunit conjugate vaccines can be an alternative for COVID-19, paving the way for other viral conjugate vaccines based on the use of small viral proteins involved in the infection process.

Capsular polysaccharides of Streptococcus pneumoniae are key components of commercially available anti-pneumococcal vaccines; meanwhile C-polysaccharide is considered an impurity. World Health Organization recommends a strict control over the presence of this biomolecule due to the possibility of introducing an undesired response. An alternative way for assessing this impurity is focused on detect the phosphocholine residues by means of quantitative

Streptococcus pneumoniae can cause life-threatening infections mostly in infants, children, and elderly people. Capsular polysaccharide conjugate vaccines provide serotype-dependent protection against S. pneumoniae infections but fail to protect against new emerging serotypes. To overcome these limitations, pneumolysin (Ply), a serotype-independent and conserved protein was selected. As such subunit vaccines lack immunogenicity, we engineered Ply to be attached to self-assembled polyester beads in order to boost immunogenicity. To display Ply at the surface of these polyester beads, it was translationally fused to the N-terminus of the polyhydroxybutyrate (PHB) synthase (PhaC), which mediates PHB bead assembly inside recombinant Escherichia coli. We also chemically conjugated the capsular polysaccharide (CPS) 19F to isolated PHB beads to further assess their antigen carrier properties. CPS conjugated to soluble tetanus toxoid served as control. Balb/c mice immunized with Ply-PhaC beads and 19F-PhaC beads induced specific and higher IgG levels than the respective soluble counterparts. The induced IgG antibodies recognized Ply in whole cell lysates of six different serotypes of S. pneumoniae. Additionally, restimulated splenocytes from animals immunized with Ply-PhaC beads produced a balanced INF-γ/IL-17A profile unlike animals immunized with soluble Ply. The 19F-PhaC beads induced production of antibodies showing high opsonophagocytic titers against the homologous strain, serotype 19F, while CPS 19F only mixed with PhaC beads did not elicit any detectable immune response. This study provided insight into the design of PHB beads as a carrier of proteinaceous antigens and CPS in order to induce immune responses for the prevention of pneumococcal infections.

A new synthetic strategy for the development of multivalent antibacterial glycoconjugate vaccines is described. The approach comprises the utilization of an isocyanide-based multicomponent process for the conjugation of functionalized capsular polysaccharides of S. pneumoniae and S. Typhi to carrier proteins such as diphtheria and tetanus toxoids. For the first time, oxo- and carboxylic acid-functionalized polysaccharides could be either independently or simultaneously conjugated to immunogenic proteins by means of the Ugi-multicomponent reaction, thus leading to mono- or multivalent unimolecular glycoconjugates as vaccine candidates. Despite the high molecular weight of the two or three reacting biomolecules, the multicomponent bioconjugation proved highly efficient and reproducible. The Ugi-derived glycoconjugates showed notable antigenicity and elicited good titers of functional specific antibodies. To our knowledge, this is the only bioconjugation method that enables the incorporation of two different polysaccharidic antigens to a carrier protein in a single step. Applications in the field of self-adjuvanting, eventually anticancer, multicomponent vaccines are foreseeable.

A new pneumococcal conjugate vaccine is currently undergoing advanced clinical evaluation prior to its planned introduction in Cuba. The implementation of the pneumococcal vaccination strategy has been designed with consideration of the need to maximize both its direct and indirect effects. A novel approach is suggested, which addresses preschool children as the first-line target group to generate herd immunity in infants and to have an impact on transmission at the community level. The clinical evaluation pipeline is described herein, including evaluations of effectiveness, cost-effectiveness, and impact. The scientific contribution of the Cuban strategy could support a paradigm shift from individual protection to a population effect based on a rigorous body of scientific evidence.

Capsular polysaccharides of Streptococcus pneumoniae are key components of commercially available anti-pneumococcal vaccines; meanwhile C-polysaccharide is considered an impurity. World Health Organization recommends a strict control over the presence of this biomolecule due to the possibility of introducing an undesired response. An alternative way for assessing this impurity is focused on detect the phosphocholine residues by means of quantitative 1H-NMR. This could be tricky due to the amounts of this substituent may vary generating two C-polysaccharides forms. In this work we propose an improved quantitative NMR methodology based on 31P-NMR for the quantification of C-polysaccharide on capsular polysaccharide preparations. The technique also focuses on phosphocholine but, conversely to above-mentioned methods, allows to discriminate between phosphocholine linked in different positions. The methodology was run on samples of eleven vaccine serotypes, including seven with phosphate groups. From a rational acceptance criterion of 10 wt%, the method allows to quantified from 30 μg of the impurity in 3 mg of total polysaccharide (1 wt%) with a signal/noise ratio of 16:1. Repeatability and intermediate precision evaluation showed a relative standard deviation of 3.33 % and 8.34 % respectively. Additionally, the method provides information about structural identity of phosphate contained in capsular polysaccharides and C-polysaccharide species. This constitutes a new contribution from the NMR that highlights the power of these techniques for assessing imperative parameters in carbohydrate-based vaccines.

Background Cuba has a new pneumococcal conjugate vaccine candidate (PCV7-TT). This study evaluates the safety and immunogenicity in healthy infants using 2p+1 vaccination schedule. Methods A phase I, controlled, randomized and double blind clinical trial was designed. 30 unvaccinated healthy infants were included. 20 subjects were assigned to study group (PCV7-TT) and 10 to control group (Synflorix®) to receive the vaccines at 7, 8 months of age (primary series) and 11 months (booster dose). Blood samples were collected 30 days after second dose and post booster for antibodies measure analysis by ELISA and OPA. The statistics analysis included the frequency of occurrence for adverse events and the immune response. Non-parametric tests were used to compare the immune response. The clinical trial was published in the Cuban Public Register of Clinical Trials with code RPCEC00000173 available at http://registroclinico.sld.cu . Results Overall, the safety profile of PCV7-TT was similar to Synflorix®. Local reactions were predominant and systemic events were mild in severity. Swelling and redness were frequently associated with PCV7-TT mainly after the first dose (50% and 40% respectively). 15% and 10% of subject reported severe swelling after first dose with PCV7-TT and after second dose with Synflorix®. Mild fever (≥38–≤39), vomiting and sleep disturb were the systemic events reported. 100% of infants achieved pneumococcal IgG antibody concentrations ≥0.35 µg/ml after booster dose for serotypes 1, 14, 18C and 19F in each vaccine group. For serotypes 5, 6B and 23F, more than 80% infants vaccinated with Synflorix® or PCV7-TT achieved protective IgG GMC ≥ 0.35 µg/ml after booster dose. OPA proportion’s responders to the seven common serotypes were 89.5% or more after the primary dose and 100% after booster dose in vaccinated with PCV7-TT. Conclusions The Cuban PCV7-TT is safe, well tolerated and immunogenic in healthy infants.

Finlay Vaccine Institute is developing a new heptavalent conjugate vaccine against Streptococcus pneumoniae. As infants are the target population, PCV7-TT will be necessarily co-administered with other vaccines, and then, the interactions represent a concern. The aim of this work is to evaluate the possible immunological interferences in rabbits as animal experimental model. Rabbits were immunized with Heberpenta®-L, VA-MENGOC-BC®, and PCV7-TT. Blood samples were taken fourteen days after final immunization for obtaining sera. Antibody responses to all antigens were evaluated by indirect ELISA. Functional responses against diphtheria and tetanus toxoid were done by in vivo seroneutralization assay. No interference was observed by PCV7-TT over the humoral response against diphtheria toxoid and meningococcal antigens (p > 0.05). A nonstatistically significant reduction (p > 0.05) was observed in the case of the humoral response against Haemophilus influenzae type b oligosaccharide. Concomitant administration of Heberpenta®-L and PCV7-TT increased twice the antibody titers as well as the protective activity against tetanus toxoid, but no statistical differences were found. The co-administration did not induce a reduction in the percent of responders against pneumococcal polysaccharides contained in PCV7-TT vaccine. Concomitant administration of PCV7-TT did not induce interferences over the evaluated antigens of Heberpenta®-L and VA-MENGOC-BC®. Also, no interference was observed on the immune response elicited by PCV7-TT. These preclinical results suggest that PCV7-TT will not result in a serious problem over the immune response elicited by the licensed vaccines Heberpenta®-L and VA-MENGOC-BC®. However, the clinical interference could be strictly studied during clinical trials in infants.

A new vaccine candidate against pneumococcus is being developed in Cuba, and it is a priority of the national health system. There is limited information on nasopharyngeal colonization burden, though it is essential for monitoring the impact of the vaccine. The study aims to estimate the prevalence of nasopharyngeal colonization in children 2-18 months of age and identify circulating serotypes, antimicrobial resistance and its association with selected risk factors.A cross-sectional study was conducted between October and December 2013 in Cienfuegos municipality. Inclusion criteria were evaluated, and informed consent was obtained from the parents. Clinical and epidemiologic data were collected through a semistructured questionnaire. Nasopharyngeal swabs according to established protocols were taken. Data analysis included frequency distributions and comparison of proportions. The association between colonization and selected risk factors was assessed by multivariate analysis.A total of 984 children (87.2% living in urban areas) were included. The overall prevalence of colonization was 21.6%. The most frequent serotypes isolated were 6A (23.1%), 23F (10.8%), 6B (10.3%), 19F (8.5%) and 14 (3.3%). We found no resistance to β-lactamases in circulating serotypes. Living with sibling younger than 5 years, previous respiratory infections, previous hospitalization and day-care attendance were determinants of nasopharyngeal carriage.The findings suggest that the burden of pneumococcal disease and colonization in Cuba could be significantly affected after vaccine introduction.

Streptococcus pneumoniae causes annually 826 000 deaths in children under five years. The serotype 6B, one of higher incidence, is targeted by the Cuban research and development project to develop a conjugate vaccine. There is limited data on how modifications caused by conjugation affect the physicochemical and antigenic characteristics of polysaccharides, particularly for serotype 6B capsular polysaccharide (PS6B), despite being the least immunogenic among S. pneumoniae polysaccharides. In this work, a conjugation procedure was established for PS6B comprising: fragmentation by acid hydrolysis, activation by periodate oxidation, and conjugation to tetanus toxoid (TT) by reductive amination to increase its immunogenicity. Reaction conditions were set to obtain the polysaccharide in three molecular size ranges (1-10, 10-30, 30-100 kDa) and levels of oxidation. PS6B fragmentation below 10 kDa and oxidation above 24 % of the repetitive units implied the loss of antigenicity. Polysaccharide length but not oxidation level had an impact on the physicochemical characteristics of the conjugates in the tested conditions. Unlike the native polysaccharide, conjugated 10-30 kDa and 30-100 kDa PS6B were immunogenic in rabbits, with evidence of thymus-dependent response. The procedure described supports obtaining PS6B-TT conjugates reproducibly in the 30-100 kDa and 10-30 kDa molecular size ranges and with 8-18 % oxidized repeat units, which are immunogenic.

Este trabajo resume el marco de evidencias que sustenta el actual uso de las vacunas antineumocócicas conjugadas en términos de seguridad, inmunogenicidad, eficacia y efectividad. Presenta una nueva alternativa para prevenir la enfermedad neumocócica en la población infantil basada en el nuevo candidato vacunal cubano contra neumococo. Expone los principios básicos que definen la estrategia para su introducción en Cuba, y los avances derivados de la aplicación de un modelo integrado para la evaluación clínica e impacto en salud en fase de implementación, el cual busca proveer las evidencias científicas que soporten la decisión del sistema nacional de salud para su introducción en el esquema nacional de inmunizaciones y su posterior comercialización en la era de las vacunas conjugadas antineumocócicas. Se presentan los resultados preliminares de estudios concluidos, y se elaboran conclusiones sobre los aportes científicos y prácticos de la estrategia de implementación y el modelo de evaluación para la nueva vacuna.