A COVID-19 vaccine candidate composed of the SARS-CoV-2 RBD dimer and Neisseria meningitidis outer membrane vesicles.

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.

SARS-CoV-2 RBD-Tetanus Toxoid Conjugate Vaccine Induces a Strong Neutralizing Immunity in Preclinical Studies.

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 result 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.

Molecular Aspects Concerning the Use of the SARS-CoV-2 Receptor Binding Domain as a Target for Preventive Vaccines.

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.

Outer membrane vesicles extracted from Neisseria meningitidis serogroup X for prevention of meningococcal disease in Africa.

Meningococcal disease is caused mainly by serogroups A, B, C, Y, W of N. meningitidis. However, numerous cases of meningitis caused by serogroup X N. meningitidis (MenX) have recently been reported in several African countries. Currently, there are no licensed vaccines against this pathogen and most of the MenX cases have been caused by meningococci from clonal complex (c.c) 181. Detergent extracted meningococcal outer membrane vesicle (dOMV) vaccines have previously shown to be safe and effective against epidemics of serogroup B meningococcal disease in all age groups. The aim of this work is therefore to obtain, characterize and evaluate the vaccine potential of dOMVs derived from a MenX strain (OMVx). Three experimental lots of OMVx were prepared by deoxycholate extraction from the MenX strain BF 2/97. Size and morphology of the vesicles was determined by Dynamic Light Scattering and electron microscopy, whereas the antigenic composition was characterized by gel electrophoresis and immunoblotting. OMVx were thereafter adsorbed to aluminium hydroxide (OMVx/AL) and two doses of OMVx were administered s.c. to groups of Balb/c mice three weeks apart. The immunogenicity and functional antibody activities in sera were evaluated by ELISA (anti-OMVx specific IgG responses) and serum bactericidal activity (SBA) assay. The size range of OMVx was shown to be between 90 and 120nm, whereas some of the antigens detected were the outer membrane proteins PorA, OpcA and RmpM. The OMVx/AL elicited high anti-OMVx antibody responses with bactericidal activity and no bactericidal activity was observed in the control group of no immunised mice. The results demonstrate that OMVx are immunogenic and could form part of a future vaccine to prevent the majority of meningococcal disease in the African meningitis belt.

Mycobacterium smegmatis proteoliposome induce protection in a murine progressive pulmonary tuberculosis model.

Tuberculosis (TB) remains an important cause of mortality and morbidity. The TB vaccine, BCG, is not fully protective against the adult form of the disease and is unable to prevent its transmission although it is still useful against severe childhood TB. Hence, the search for new vaccines is of great interest. In a previous study, we have shown that proteoliposomes obtained from Mycobacterium smegmatis (PLMs) induced cross reactive humoral and cellular response against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLMs, a murine model of progressive pulmonary TB was used. Animals immunized with PLMs with and without alum (PLMs/PLMsAL respectively) showed protection compared to non-immunized animals. Mice immunized with PLMsAL induced similar protection as that of BCG. Animals immunized with BCG, PLMs and PLMsAL showed a significant decrease in tissue damage (percentage of pneumonic area/lung) compared to non-immunized animals, with a more prominent effect in BCG vaccinated mice. The protective effect of the administration of PLMs in mice supports its future evaluation as experimental vaccine candidate against Mtb.

Protective capacity of proteoliposomes from Mycobacterium bovis BCG in a mouse model of tuberculosis.

Tuberculosis (TB) is one of the most important causes of mortality and morbidity due to infectious diseases. BCG, the vaccine in use, is not fully protective against TB. In a previous study, we have shown that proteoliposomes (outer membrane extracts), obtained from BCG (PLBCG) were able to induce humoral immune responses against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLBCG alone or as a booster with BCG, a murine model of progressive pulmonary TB was used. Animals immunized with PLBCG adjuvanted with alum (PLBCG-Al) showed similar protection to that conferred by BCG. The group immunized with PLBCG-Al as a booster to BCG gave superior protection than BCG as evidenced by a reduction of bacterial load in lungs 2 months after infection with Mtb. Animals immunized with BCG, PLBCG-Al and this formulation as a booster of BCG, showed a significant decrease of tissue damage (percentage of pneumonic area/lung) compared with non-immunized animals. These results demonstrate that immunization with PLBCG-Al alone or as a booster to BCG induce appropriate protection against challenge with Mtb in mice and support the future evaluation of PLBCG as a promising vaccine candidate against Mtb.

Bacterial outer membrane vesicles and vaccine applications.

Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP) process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB) using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA), serogroup W (dOMVW), and serogroup X (dOMVX) were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC), Bordetella pertussis (dOMVBP), Mycobacterium smegmatis (dOMVSM), and BCG (dOMVBCG). The immunogenicity of the OMV has been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice has shown their protective potential. dOMVB has been evaluated with non-neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin, and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates.

Repeated dose toxicity study of Vibrio cholerae-loaded gastro-resistant microparticles.

CONTEXT: Microencapsulation of antigens has been extensively studied over the last decades aiming at improving the immunogenicity of vaccine candidates. OBJECTIVE: Addressing microparticles (MPs) toxicity in rats. MATERIAL AND METHODS: Spray-dried Eudragit® L 30 D-55 MPs and Eudragit® L 30 D-55 alginate MPs were elaborated and characterized. MPs obtained were administered to rats, three groups were defined: G1, control group; G2, administered with Vibrio cholerae (VC)-loaded MPs; G3, receiving VC-loaded alginate MPs. Animals received three vaccine doses. Body weight, food and water intake were controlled during the study. Haematological parameters, vibriocidal titres, organ weight and histology in necropsy were also analyzed. RESULTS: All animals grew healthy. Body weight gain, food and water intake and haematological parameters remained within physiological values, showing no treatment-related differences. Moreover, organ weight changes were not detected and animals developed protective vibriocidal titres. CONCLUSION: VC-loaded MPs and VC-loaded alginate MPs have proved to be safe and effective in the assessed conditions.

Immunogenicity and cross-reactivity against Mycobacterium tuberculosis of proteoliposomes derived from Mycobacterium bovis BCG.

The only currently available vaccine against tuberculosis (TB) is Mycobacterium bovis Bacille Calmette-Guerin (BCG), which has inconsistent efficacy to protect against the disease in adults. M. tuberculosis (MTB) cell wall components have been implicated in the pathogenicity of TB and therefore have been a prime target for the identification and characterization of cell wall proteins with potential application in vaccine development. In this regard, proteoliposomes (PLs) derived from mycobacteria containing lipids and cell wall proteins could be potential vaccine candidates against TB. In the present study PLs derived from BCG were prepared. These homogeneous population of spherical microparticles was then immunized into Balb/c mice. Sera of immunized animals showed high IgG response and strong cross-reactivity against different MTB antigens.These results showed that BCG PLs could be potential vaccine candidates against TB.

A proteoliposome formulation derived from Bordetella pertussis induces protection in two murine challenge models.

Whooping cough remains a health problem despite high vaccination coverage. It has been recommended that development of new strategies provide long-lasting immunity. The aim of this work was to evaluate the potential of proteoliposomes (PL) extracted from Bordetella pertussis as a vaccine candidate against whooping cough. The size of the B. pertussis PL was estimated to be 96.7 ± 50.9 nm by Scanning Correlation Spectroscopy and the polydispersity index was 0.268. Western blots using monoclonal antibodies revealed the presence of pertussis toxin, pertactin, and fimbriae 3. The Limulus Amebocyte Lisate (LAL) assay showed endotoxin levels lower than those reported for whole cell pertussis licensed vaccines, while the Pyrogen Test indicated 75 ng/mL/Kg. The PL showed high protection capacity in mouse challenge models. There was 89.7% survival in the intracerebral challenge and total reduction of the number of CFU in the intranasal challenge. No significant differences (p > 0.05) were observed between mice immunized with B. pertussis PL and the Cuban DTwP vaccine, whichever challenge model used. These results encouraged us to continue the development of the B. pertussis PL as a component of a new combined vaccine formulated with tetanus and diphtheria toxoids or as a booster dose for adolescents and adults.

Evaluation of enteric-coated tablets as a whole cell inactivated vaccine candidate against Vibrio cholerae.

A vaccine candidate against cholera was developed in the form of oral tablets to avoid difficulties during application exhibited by current whole cell inactivated cholera vaccines. In this study, enteric-coated tablets were used to improve the protection of the active compound from gastric acidity. Tablets containing heat-killed whole cells of Vibrio cholerae strain C7258 as the active pharmaceutical compound was enteric-coated with the polymer Kollicoat(®) MAE-100P, which protected them efficiently from acidity when a disintegration test was carried out. Enzyme-linked immunosorbent assay (ELISA) anti-lipopolysaccharide (LPS) inhibition test and Western blot assay revealed the presence of V. cholerae antigens as LPS, mannose-sensitive haemagglutinin (MSHA) and outer membrane protein U (Omp U) in enteric-coated tablets. Immunogenicity studies (ELISA and vibriocidal test) carried out by intraduodenal administration in rabbits showed that the coating process of tablets did not affect the immunogenicity of V. cholerae-inactivated cells. In addition, no differences were observed in the immune response elicited by enteric-coated or uncoated tablets, particularly because the animal model and immunization route used did not allow discriminating between acid resistances of both tablets formulations in vivo. Clinical studies with volunteers will be required to elucidate this aspect, but the results suggest the possibility of using enteric-coated tablets as a final pharmaceutical product for a cholera vaccine.

Proteoliposomes from Mycobacterium smegmatis induce immune cross-reactivity against Mycobacterium tuberculosis antigens in mice.

Proteoliposomes (PL) obtained from Mycobacterium smegmatis (Ms) were evaluated for their capacity to elicit cross-reactive responses against Mycobacterium tuberculosis (Mtb) antigens in BALB/c mice. Animals immunized with PL adjuvanted with alum (PL-AL) or Freund's Incomplete Adjuvant (PL-IFA) showed significant IgG responses against the PL as well as total Ms lipids. Both groups of animals also showed significant IgG responses against BCG, but only animals immunized with PL-AL produced significant IgG responses against soluble cell wall proteins (SCWP) or whole cell lysate (WCL) of Mtb. Significant DTH responses against WCL were observed in both groups of animals after 24 h, but only PL-AL-immunized mice showed significant DTH responses after 48 h and 72 h. PL-Ms are capable of eliciting cross-reactive humoral and cellular responses against Mtb antigens and thus may be a potential vaccine strategy against tuberculosis.

A new oral vaccine candidate based on the microencapsulation by spray-drying of inactivated Vibrio cholerae.

The aim of this work was to evaluate the microencapsulation by spray-drying of inactivated Vibrio cholerae, using methacrylic copolymers Eudragit® L30D-55 and FS30D. The microparticles obtained presented a particle size around 3.0 µm. The preparation temperature affected the morphology and the antigenicity of microparticles, but it did not affect the V. cholerae content. In vitro release studies showed that in acid medium less than 5% of bacteria was released, and in neutral medium, Eudragit® L30D-55 microparticles released 86% after 24 h, whereas FS30D released less than 30%. Rats inoculated with microparticles exhibited vibriocidal antibody titres. Microencapsulation by spray-drying of inactivated V. cholerae could be proposed as a method to obtain an oral vaccine which provides controlled release of the bacteria.

Pharmacology and toxicology of an oral tablet whole cells inactivated cholera vaccine in Sprague Dawley rats.

Here we further investigate the pharmacological and toxicological properties of a cholera vaccine based on inactivated whole cells presented in either enteric coated (COA) or uncoated (U/C) tablet formulation from Vibrio cholerae C7258 strain. Tablets were dispersed in 2mL drinking water and administered orally to Sprague Dawley rats distributed in five groups (I COA7, II U/C7 immunized at 0, 7, 69days and III COA14, IV U/C14 immunized at 0, 14, 69days and V control group). Serum vibriocidal antibody response was measured after the administration of two doses with an interval of 7-14days. To further investigate the toxicological aspects a third dose was applied 10 weeks after the initial one. Animals were observed daily and water and food consumption was measured every other day. Periodic blood extractions were performed for hematology, biochemistry, and the titer of serum vibriocidal antibodies was determined. Anatomopathological analysis was performed at days 3 or 14 after the third dose. Results from clinical observations, as well as from water and food consumption and body weigh indicated no toxicity of the vaccine product. Meanwhile, no biological differences were found among different groups in hematological, hemo-chemistry, and anatomopathological studies. Moreover, enteric coated and uncoated tablets against human cholera were found to induce an immune response in rats.

Evaluación del potencial de Mycobacterium smegmatis como candidato vacunal contra la tuberculosis mediante estudios in sílico e in vivo / Evaluation of the potential of Mycobacterium smegmatis as vaccine Candidate against tuberculosis by in silico and in vivo studies

In this study, we scanned multiple published databases of gene expression in vivo of M tuberculosis at different phases of infection in animals and humans, to select 38 proteins that are highly expressed in the active, latent and reactivation phases. The selected proteins were predicted for T and B epitopes. For each proteins, the regions containing T and B epitopes were selected at the same time to look for identical epitopes on M smegmatis based on sequence alignments. Preliminary studies of humoral immunogenicity and cross-reactivity with M tuberculosis in mice using two M smegmatis-derived experimental vaccines were carried out, demonstrating the immunogenicity of M smegmatis proteoliposomes and the recognition of M tuberculosis proteins by the sera of animals immunized with this vaccine candidate. The conjunction of in silico and in vivo studies suggested the potential for future evaluation of M smegmatis as vaccine candidate against tuberculosis using different strategies.(AU)

En este estudio se revisaron múltiples bases de datos publicadas, relacionadas con experimentos de expresión de genes de M tuberculosis in vivo en diferentes estadios de la infeccción en humanos y animales. Se identificaron 38 proteínas con elevada expresión en las fases activa, latente y de reactivación de la infección. Se llevó a cabo la predicción de epítopes T y B en dichas proteínas. Las regiones de cada proteína que contenían simultàneamente epítopes T y B se seleccionaron y utilizaron para identificar regiones idénticas en M smegmatis mediante el alineamiento de secuencias. Se llevaron a cabo estudios de inmunogenicidad humoral y reactividad cruzada con M tuberculosis en ratones inmunizados con dos vacunas experimentales obtenidas a partir de M smegmatis, demostràndose la immunogenicidad de los proteoliposomas y el reconocimiento de proteínas de M tuberculosis por el suero de ratones vacunados con este candidato vacunal. Los resultados obtenidos con los estudios in sílico e in vivo sugieren la potencialidad para evaluación futura de candidatos vacunales obtenidos a partir de M smegmatis para la prevención de la tuberculosis(AU)

Intranasal administration of proteoliposome-derived cochleates from Vibrio cholerae O1 induce mucosal and systemic immune responses in mice.

Conservative estimates place the death toll from cholera at more than 100,000 persons each year. A particulate mucosal vaccine strategy combining antigens and immune stimulator molecules from Vibrio cholerae to overcome this problem is described. Proteoliposomes extracted from V. cholerae O1 were transformed into cochleates (AFCo2, Adjuvant Finlay cochleate 2) through a calcium inducible rotary dialysis method. Light microscopy was carried out and tubules of 16.25+/-4.57 microm in length were observed. Western blots were performed to verify the immunochemical properties of the main AFCo2 incorporated antigens, revealing full recognition of the outer membrane protein U (OmpU), lipopolysaccharide (LPS), and mannose-sensitive hemagglutinin (MSHA) antigens. AFCo2 were administered by the intranasal route using a two or three dose schedule and the immune response against V. cholerae antigens was assessed. Three AFCo2 doses were required to induce significant (p<0.05), antigen specific IgA in saliva (1.34+/-0.135) and feces (0.60+/-0.089). While, two or three doses of AFCo2 or proteoliposomes induce similar specific IgG and vibriocidal activity responses in sera. These results show for the first time that AFCo2 can be obtained from V. cholerae O1 proteoliposomes and have the potential to protect against the pathogen when administered intranasally.