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

Background: The pandemic of COVID-19 raised the urgent need for 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. Methods: A 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 µg 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 the 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. Findings: Between March 22 to April 03, 2021, 48,290 subjects were included (24,144 and 24,146 in the placebo and Abdala groups, respectively) in the context of predominant D614G variant circulation. The evaluation of the main efficacy outcomes occurred during May-June 2021, starting at May 3rd, in the context of high circulation of mutant viruses, predominantly VOC Beta. The incidence of adverse reactions for individuals in the placebo and Abdala vaccine groups were 1227/24,144 (5.1%) and 1621/24,146 (6.7%), respectively. Adverse reactions were mostly mild, and from the injection site, which resolved in the first 24-48 h. No severe adverse events with demonstrated cause-effect relationship attributable to the vaccine were reported. Symptomatic COVID-19 disease was confirmed in 142 participants in the placebo group (78.44 incidence per 1000 person-years, 95% confidence interval [CI], 66.07-92.46) and in 11 participants in Abdala vaccine group (6.05 incidence per 1000 person years; 95% CI 3.02-10.82). The Abdala vaccine efficacy against symptomatic COVID-19 was 92.28% (95% CI 85.74-95.82). Moderate/serious forms of COVID-19 occurred in 30 participants (28 in the placebo group and only 2 in the Abdala vaccine group) for a vaccine efficacy of 92.88% (95% CI 70.12-98.31). There were five critical patients (of which four died), all in the placebo group. Interpretation: The Abdala vaccine was safe, well tolerated, and highly effective, fulfilling the WHO target product profile for COVID-19 vaccines. Those results, along with its immunization schedule and the advantage of easy storage and handling conditions at 2-8 °C, make this vaccine an option for the use in immunization strategies as a key tool for the control of the pandemic. Funding: Centre for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.

Safety, tolerability, and immunogenicity of a SARS-CoV-2 recombinant spike RBD protein vaccine: A randomised, double-blind, placebo-controlled, phase 1-2 clinical trial (ABDALA Study).

Background: Multiple vaccine candidates against COVID-19 are currently being evaluated. We evaluate the safety and immunogenicity protein of a novel SARS-CoV-2 virus receptor-binding domain (RBD) vaccine. Methods: A phase 1-2, randomised, double-blind, placebo-controlled trial was carried out in "Saturnino Lora" Hospital, Santiago de Cuba, 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 (1:1:1, in blocks) to three groups: placebo, 25 µg and 50 µg RBD vaccine (Abdala). The product was administered intramuscularly, 0·5 mL in the deltoid region. During the first phase, two immunization schedules were studied: 0-14-28 days (short) and 0-28-56 days (long). In phase 2, only the short schedule was evaluated. The organoleptic characteristics and presentations of vaccine and placebo were identical. All participants (subjects, clinical researchers, statisticians, laboratory technicians, and monitors) remained masked during the study period. The main endpoints were safety and the proportion of subjects with seroconversion of anti-RBD IgG antibodies, analysed by intention to treat and per protocol, respectively. The trial is registered with the Cuban Public Registry of Clinical Trials, RPCEC00000346. Findings: Between Dec 7, 2020, and Feb 9, 2021, 792 subjects were included; 132 (66 in each vaccination schedule, divided into 22 for each group) in phase 1, and 660 (220 in each group plus 66 from the short scheme of phase 1) in phase 2. The product was well tolerated. No severe adverse events were reported. During phase 1, the incidence of adverse events in the 25 µg, 50 µg, and placebo arms for the short schedule were 6/22 (27·3%), 6/22 (27·3%), 3/22 (13·6%), respectively, and for the long schedule were 8/22 (36·4%), 9/22 (40·9%), 4/22 (18·2%), respectively. In phase 2, adverse reactions were reported by 53/242 (21·9%), 75/242 (31·0%) and 41/242 (16·9%) participants in the 25 µg, 50 µg, and placebo group, respectively. Adverse reactions were minimal, mostly mild, and from the injection site, which resolved in the first 24-48 hours. In phase 1, seroconversion at day 56 was seen in 95·2% of the participants (20/21) in the 50 µg group, 81% (17/21) in the 25 µg group, and none in the placebo group (0/22). For the long schedule, seroconversion at day 70 was seen in 100% of the participants (21/21) in the 50 µg group, 94·7% (18/19) in the 25 µg group, and none in the placebo group (0/22). In phase 2, seroconversion of anti-RBD IgG antibodies at day 56 was seen in 89·2% of the participants in the 50 µg group (214/240; 95% CI 84·5-92·82), 77·7% in the 25 µg group (185/238; 72·0-82·9) and 4·6% in the placebo group (11/239; 2·3-8·1). Compared with the placebo arm, the differences in the proportion of participants with seroconversion were 73·1% (95% CI 66·8-79·5) and 84·6% (79·4-89·7) in the 25 µg and 50 µg groups, respectively. The seroconversion rate in the 50 µg group was significantly higher than in the 25 µg group (p=0·0012). Interpretation: The Abdala vaccine was safe, well tolerated, and induced humoral immune responses against SARS-CoV-2. These results, in the context of the emergency COVID-19 pandemic, support the 50 µg dose, applied in a 0-14-28 days schedule, for further clinical trials to confirm vaccine efficacy. Funding: Centre for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.

Generation and characterization of murine monoclonal antibodies against immunoreactive trypsinogen for newborn screening of cystic fibrosis.

Cystic fibrosis (CF) is a multisystem disorder that reduces quality of life and survival in affected individuals. In newborns, the release of pancreatic enzymes into the blood raises the levels of immunoreactive trypsinogen (IRT), the main marker for CF screening, which is detected in dried blood samples on filter paper by immunoenzymatic assays. In Cuba, CF has an estimated incidence of 1/9862 live births and should be included in the national basic newborn screening (NBS) panel given its benefits in terms of nutrition, lung function and survival. The Immunoassay Center develops and produces diagnostic kits allowing the establishment of large-scale NBS programs for inherited metabolic disorders in Cuba and other Latin American countries. IRT-specific monoclonal antibodies (MAbs) obtained at the Immunoassay Center are essential for developing an affordable immunoassay for IRT to support CF NBS in our low-income country. An immunization scheme with trypsinogen-1 originated two IgG1-producing murine hybridomas. 4C9C9 and 4C9E11 MAbs recognized different determinants on both trypsin-1 and trypsin-2 molecules. Both antibodies identified conformational epitopes on the molecule of trypsin-1 and of its zymogen. As 4C9E11 MAb cross-reacted with proteins structurally and functionally related to trypsinogen, it was used as revealing antibody in a sandwich-type UMELISA® assay for IRT determination with 4C9C9 MAb for capture. This combination, aside from detecting several commercially available trypsins, adequately quantified IRT from dried blood samples on filter paper of newborns. The evaluation of the assay's accuracy yielded percentage recoveries ranging 93.3-109.2% for commercial controls. The properties of the studied MAbs demonstrate their suitability for being used in a sandwich-type UMELISA® assay for the CF NBS in Cuba.

Generation and characterization of monoclonal antibodies against thyroid-stimulating hormone for newborn screening of congenital hypothyroidism.

Congenital hypothyroidism (CH) is one of the most frequent inherited-metabolic diseases in the world, and the main cause of treatable mental retardation in children. Because signs and symptoms of this disease are often scarce and not easily recognizable, newborns are screened for the early CH detection at birth. The Center of Immunoassay (CIE) has developed the UMELISA® TSH Neonatal and UMELISA® TSH to determine neonatal thyroid-stimulating hormone (TSH) levels in dried blood and serum samples. Both reagent kits use the same polystyrene plates coated with anti-ß-TSH monoclonal antibodies (MAbs), but one of these is commercially acquired. Obtaining appropriate anti-TSH MAbs at the CIE would guarantee economic independence and security in the production of these kits. Immunization of mice with TSH led to the generation of 7G11E3, an anti-ß-TSH IgG1-secreting hybridoma. The high affinity of 7G11E3 MAb and its characteristic epitopic recognition explain its better performance when adsorbed to UMELISA® plates for capturing low amounts of TSH in comparison with the studied MAbs. Performance of assays using polystyrene plates coated with 7G11E3 MAb was studied. Recovery percentages (100.0-106.7% for UMELISA® TSH NEONATAL and 97.3-99.0% for UMELISA® TSH) and intra (5.2-7.9% for UMELISA® TSH NEONATAL and 3.2-5.3% for UMELISA® TSH) and inter (6.6-7.7% for UMELISA® TSH NEONATAL and 5.2-8.0% for UMELISA® TSH) coefficients of variation were similar to the ones described for the commercial kits. Limits of detection and quantification were 1.0 and 3.8 mIU/L for UMELISA® TSH NEONATAL, and 0.3 and 0.6 mIU/L for UMELISA® TSH, respectively. The results also showed high overall concordance between assays (n = 2 019, ρc = 0.90 for UMELISA® TSH NEONATAL and n = 200, ρc = 0.94 for UMELISA® TSH). The 7G11E3 MAb meets the requirements for its use in the plates of UMELISA® TSH kits for CH newborn screening in Cuba. Abbreviations: CECMED, Center for the State Control of Medicaments and Medical Equipment and Devices; CH, congenital hypothyroidism; CIE, Center of Immunoassay; CLSI, Clinical and Laboratory Standards Institute; CV coefficient of variation; DBS, dried blood spots; LOB, limit of blank; LOD, limit of detection; LOQ, limit of quantitation; SD, standard deviation; Sr, repeatability standard deviation; SUMA, Ultra Micro Analytic System; UMELISA, ultramicro enzyme-linked immunosorbent assay.

Generation of monoclonal antibodies against 17α-hydroxyprogesterone for newborn screening of congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by the deficiency of one of the five enzymes involved in the biosynthesis of corticosteroids. The most common form of the disease is the lack of 21-hydroxylase which provokes an accumulation of high levels of 17α-hydroxyprogesterone (17-OHP), the main biochemical marker for illness detection. Given the significance of neonatal diagnosis for ensuring a timely treatment to patients suffering from CAH, newborn screening is worldwide performed for the determination of 17-OHP from dried blood spots on filter paper. The non-specificity of antisera employed in immunoassays and the cross-reaction with fetal adrenal hormones produce an overestimation in the 17-OHP quantification. Immunization of mice with 17-OHP-3-(O-carboxymethyl) oxime-bovine serum albumin led to the generation of 15 anti-17-OHP IgG1-and-IgG2b-secreting hybridomas. The 6E2G9 monoclonal antibody presents cross-reactivity values similar to those achieved by rabbit antibodies employed in the solid phase of UMELISA® 17-OH Progesterona Neonatal, assay for the newborn screening of CAH in Cuba. Additionally, the use of 6E2G9 in the evaluation of dried blood spots samples from newborns on filter paper showed a decrease in the mean 17-OHP levels, thus demonstrating it can replace the conventional rabbit antisera.