Recombinant Bacillus Calmette-Guérin Expressing SARS-CoV-2 Chimeric Protein Protects K18-hACE2 Mice against Viral Challenge.

COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette-Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.

SARS-CoV-2 vaccine development and how Brazil is contributing.

The SARS-CoV-2 coronavirus pandemic calls for coordinated efforts by the scientific community for the development of vaccines. The most advanced strategies have focused on modifications of technologies that were already under development for other viruses, such as SARS, MERS, and even Influenza. Classic and new technologies, such as inactivated and attenuated viruses (non-replicative and replicative), DNA and mRNA vaccines, and nanoparticles containing SARS-CoV-2 antigens, are some of the strategies currently investigated. Although there is a very high expectation for the effectiveness of the most advanced vaccine candidates, there are still no established correlates of protection. Previous experience in vaccine development for other pathogens shows that differences in vaccine formulation can result in diverse immune responses and consequently, different protective properties. Therefore the importance of continuing investigations on a broad range of strategies. Expertise in vaccine development in Brazil was refocused to the new coronavirus. Impressive collaboration between institutions will support further developments until we have available a safe, effective, and economically viable vaccine. Established competence and collaborations will allow preparedness for future challenges and can also be used to address local issues as neglected infectious diseases.

Process intensification for production of Streptococcus pneumoniae whole-cell vaccine.

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.

New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

Schistosoma mansoni venom allergen-like protein 4 (SmVAL4) is a novel lipid-binding SCP/TAPS protein that lacks the prototypical CAP motifs.

Schistosomiasis is a parasitic disease that affects over 200 million people. Vaccine candidates have been identified, including Schistosoma mansoni venom allergen-like proteins (SmVALs) from the SCP/TAPS (sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. The first SmVAL structure, SmVAL4, was refined to a resolution limit of 2.16 Å. SmVAL4 has a unique structure that could not be predicted from homologous structures, with longer loops and an unusual C-terminal extension. SmVAL4 has the characteristic α/ß-sandwich and central SCP/TAPS cavity. Furthermore, SmVAL4 has only one of the signature CAP cavity tetrad amino-acid residues and is missing the histidines that coordinate divalent cations such as Zn(2+) in other SCP/TAPS proteins. SmVAL4 has a cavity between α-helices 1 and 4 that was observed to bind lipids in tablysin-15, suggesting the ability to bind lipids. Subsequently, SmVAL4 was shown to bind cholesterol in vitro. Additionally, SmVAL4 was shown to complement the in vivo sterol-export phenotype of yeast mutants lacking their endogenous CAP proteins. Expression of SmVAL4 in yeast cells lacking endogenous CAP function restores the block in sterol export. These studies suggest an evolutionarily conserved lipid-binding function shared by CAP proteins such as SmVAL4 and yeast CAP proteins such as Pry1.

Schistosoma mansoni vaccine candidates identified by unbiased phage display screening in self-cured rhesus macaques.

Schistosomiasis, a challenging neglected tropical disease, affects millions of people worldwide. Developing a prophylactic vaccine against Schistosoma mansoni has been hindered by the parasite's biological complexity. In this study, we utilized the innovative phage-display immunoprecipitation followed by a sequencing approach (PhIP-Seq) to screen the immune response of 10 infected rhesus macaques during self-cure and challenge-resistant phases, identifying vaccine candidates. Our high-throughput S. mansoni synthetic DNA phage-display library encoded 99.6% of 119,747 58-mer peptides, providing comprehensive coverage of the parasite's proteome. Library screening with rhesus macaques' antibodies, from the early phase of establishment of parasite infection, identified significantly enriched epitopes of parasite extracellular proteins known to be expressed in the digestive tract, shifting towards intracellular proteins during the late phase of parasite clearance. Immunization of mice with a selected pool of PhIP-Seq-enriched phage-displayed peptides from MEG proteins, cathepsins B, and asparaginyl endopeptidase significantly reduced worm burden in a vaccination assay. These findings enhance our understanding of parasite-host immune responses and provide promising prospects for developing an effective schistosomiasis vaccine.

Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni.

Schistosoma mansoni is the primary causative agent of schistosomiasis, which affects 200 million individuals in 74 countries. We generated 163,000 expressed-sequence tags (ESTs) from normalized cDNA libraries from six selected developmental stages of the parasite, resulting in 31,000 assembled sequences and 92% sampling of an estimated 14,000 gene complement. By analyzing automated Gene Ontology assignments, we provide a detailed view of important S. mansoni biological systems, including characterization of metazoa-specific and eukarya-conserved genes. Phylogenetic analysis suggests an early divergence from other metazoa. The data set provides insights into the molecular mechanisms of tissue organization, development, signaling, sexual dimorphism, host interactions and immune evasion and identifies novel proteins to be investigated as vaccine candidates and potential drug targets.

Antigenic epitope targets of rhesus macaques self-curing from Schistosoma mansoni infection.

The self-cure of rhesus macaques from a schistosome infection and their subsequent strong immunity to a cercarial challenge should provide novel insights into the way these parasites can be eliminated by immunological attack. High-density arrays comprising overlapping 15-mer peptides from target proteins printed on glass slides can be used to screen sera from host species to determine antibody reactivity at the single epitope level. Careful selection of proteins, based on compositional studies, is crucial to encompass only those exposed on or secreted from the intra-mammalian stages and is intended to focus the analysis solely on targets mediating protection. We report the results of this approach using two pools of sera from hi- and lo-responder macaques undergoing self-cure, to screen arrays comprising tegument, esophageal gland, and gastrodermis proteins. We show that, overall, the target epitopes are the same in both groups, but the intensity of response is twice as strong in the high responders. In addition, apart from Sm25, tegument proteins elicit much weaker responses than those originating in the alimentary tract, as was apparent in IFNγR KO mice. We also highlight the most reactive epitopes in key proteins. Armed with this knowledge, we intend to use multi-epitope constructs in vaccination experiments, which seek to emulate the self-cure process in experimental animals and potentially in humans.

Fusion of PspA to detoxified pneumolysin enhances pneumococcal vaccine coverage.

Despite the implementation of conjugate vaccines in several countries, S. pneumoniae continues to pose a great burden worldwide, causing around 1 million annual deaths. Pneumococcal proteins have long been investigated as serotype-independent vaccines against this pathogen, with promising results. However, it is a consensus that one antigen alone will not be sufficient to provide long-term protection with wide coverage. Amongst the most well studied pneumococcal proteins are PspA and pneumolysin (Ply), two major virulence factors required by the bacterium for successful invasion of host tissues. PspA is highly immunogenic and protective, but it is structurally variable; pneumolysin is conserved among different pneumococci, but it is toxic to the host. To overcome these limitations, N-terminal PspA fragments have been genetically fused to non-toxic pneumolysin derivatives (PlD) to create PspA_PlD chimeras. Mouse immunization with these fusions confers protection against pneumococcal strains expressing heterologous PspAs, which correlates with antibody-induced complement C3 deposition on the surface of multiple pneumococcal strains. Analysis of mutant strains lacking PspA or Pneumolysin shows that both proteins contribute to the antibody-mediated enhancement in complement deposition induced by the fusion. These results expand previous data evaluating PspA_PlD and demonstrate that the fusion combines the protective traits of both proteins, inducing antibodies that efficiently promote complement deposition on multiple strains and cross-protection.

Recombinant BCG Expressing the Subunit 1 of Pertussis Toxin Induces Innate Immune Memory and Confers Protection against Non-Related Pathogens.

BCG has shown the ability to induce protection against unrelated pathogens, which likely depends on an immune mechanism known as innate immune memory or trained immunity. In this study, we evaluated the induction of innate memory by a recombinant BCG strain expressing the genetically detoxified S1 subunit of the pertussis toxin (rBCG-S1PT). In vitro pre-exposure of naïve murine macrophages to rBCG-S1PT increased their innate/inflammatory response (IL-6, TNF-α, and IL-10) to a subsequent challenge with unrelated pathogens, as compared to pre-exposure to wild-type BCG. Following LPS challenge, mice immunized with rBCG-S1PT produced higher levels of IFN-γ, while the release of other inflammatory cytokines was comparable to that measured after BCG immunization. SCID mice previously immunized with rBCG-S1PT and challenged with pathogenic Candida albicans displayed a similar survival curve as BCG-immunized mice but a lower CFU burden in the kidneys, suggesting an innate memory-dependent control of C. albicans infection. This study highlights the potential of recombinant BCG to increase innate immune memory and, ultimately, non-specific protection, more effectively than wild-type BCG. To our knowledge, this is the first report describing the potential of a recombinant BCG strain to strengthen innate immune memory responses.

100 Years of BCG Immunization: Past, Present, and Future.

The 100th anniversary of the introduction of Bacille-Calmette-Guérin (BCG) as a tuberculosis (TB) vaccine is an occasion warranting further investigation of the early attempts which culminated in the introduction of BCG as a TB vaccine, as well as of subsequent recognition of failures, new findings that broaden its applications, outstanding questions, and approaches towards the development of novel vaccine candidates [...].

Immune response induced in mice by a hybrid rPotD-PdT pneumococcal protein.

Streptococcus pneumoniae is a human pathogen that colonizes the naso and/or oropharynx and can cause otitis, pneumonia, bacteremia and meningitis. To broaden the protection against pneumococcus, several pneumococcal proteins have been investigated as vaccine candidates. In this study we analyzed the immunological response induced by mouse subcutaneous immunization with a fusion of the Polyamine transport protein D (PotD) and a pneumolysin derivative (PdT), resulting in a hybrid rPotD-PdT protein. Immunization of mice with rPotD-PdT induced increased production of nitric oxide, indicating a higher innate immune response. In agreement, immunization of mice with the hybrid protein was more immunogenic than the individual proteins or their combination, eliciting higher antibody levels. The anti-rPotD-PdT IgG displayed increased binding onto the pneumococcal surface. Furthermore, the anti-rPotD-PdT antisera promoted superior opsonophagocytosis as compared with the other tested formulations. However, despite that the encouraging results in vitro, immunization with the hybrid was not sufficient to induce protection against sepsis with a highly virulent pneumococcal strain. taken together, the results suggest that hybrid proteins are an interesting strategy, able to promote improved immune responses, but the inclusion of other antigens may be necessary to promote protection against invasive infections caused by this bacterium.

Recombinant BCG-LTAK63 Vaccine Candidate for Tuberculosis Induces an Inflammatory Profile in Human Macrophages.

Tuberculosis (TB) is one of the top 10 leading causes of death worldwide. The recombinant BCG strain expressing the genetically detoxified A subunit of the thermolabile toxin from Escherichia coli (LTAK63) adjuvant (rBCG-LTAK63) has previously been shown to confer superior protection and immunogenicity compared to BCG in a murine TB infection model. To further investigate the immunological mechanisms induced by rBCG-LTAK63, we evaluated the immune responses induced by rBCG-LTAK63, BCG, and Mycobacterium tuberculosis (Mtb) H37Rv strains in experimental infections of primary human M1 and M2 macrophages at the transcriptomic and cytokine secretion levels. The rBCG-LTAK63-infected M1 macrophages more profoundly upregulated interferon-inducible genes such as IFIT3, OAS3, and antimicrobial gene CXCL9 compared to BCG, and induced higher levels of inflammatory cytokines such as IL-12(p70), TNF-ß, and IL-15. The rBCG-LTAK63-infected M2 macrophages more extensively upregulated transcripts of inflammation-related genes, TAP1, GBP1, SLAMF7, TNIP1, and IL6, and induced higher levels of cytokines related to inflammation and tissue repair, MCP-3 and EGF, as compared to BCG. Thus, our data revealed an important signature of immune responses induced in human macrophages by rBCG-LTAK63 associated with increased inflammation, activation, and tissue repair, which may be correlated with a protective immune response against TB.

A fusion protein comprising pneumococcal surface protein A and a pneumolysin derivate confers protection in a murine model of pneumococcal pneumonia.

PspA and pneumolysin are two important vaccine candidates, able to elicit protection in different models of pneumococcal infection. The high immunogenic potential of PspA, combined with a possible adjuvant effect of pneumolysin derivatives (due to their ability to interact with TLR-4) could greatly improve the immunogenicity and coverage of a protein-based pneumococcal vaccine. A chimeric protein including the N-terminal region of PspA in fusion with the pneumolysin derivative, PlD1, has been shown to induce high antibody levels against each protein, and protect mice against invasive challenge. The aim of the present study was to investigate the cellular response induced by such vaccine, and to evaluate protection in a murine model of lobar pneumococcal pneumonia. Pneumococcal pneumonia was induced in BALB/c mice by nasal instillation of a high dose of a serotype 14 strain with low virulence. Airway inflammation was confirmed by total and differential cell counts in BAL and by histological analysis of the lungs, and bacterial loads were measured 7 days after challenge. Cytokine levels were determined in the bronchoalveolar fluid (BALF) of mice immunized with rPspA-PlD1 fusion after challenge, by flow cytometry and ELISA. After challenge, the mice developed lung inflammation with no invasion of other sites, as demonstrated by histological analysis. We detected significant production of TNF-α and IL-6 in the BALF, which correlated with protection against pneumonia in the group immunized with rPspA-PlD1. In conclusion, we found that the rPspA-PlD1fusion is protective against pneumococcal pneumonia in mice, and protection is correlated with an early and controlled local inflammatory response. These results are in agreement with previous data demonstrating the efficacy of the fusion protein against pneumococcal sepsis and reinforce the potential of the rPspA-PlD1 protein chimera as a promising vaccine strategy to prevent pneumococcal disease.

Optimization of Expression and Purification of Schistosoma mansoni Antigens in Fusion with Rhizavidin.

Schistosomiasis causes significant morbidity and mortality. Vaccine efforts to date indicate the need to increase the immunogenicity of Schistosoma antigens. The multiple antigen-presenting system, whereby proteins are genetically fused to rhizavidin and affinity linked to biotinylated templates, enables the generation of robust immune responses. The objective of this work was to express and purify the S. mansoni antigens, SmTSP-2 and SmCD59.2, in fusion with rhizavidin. The fusion with rhizavidin greatly decreased the expression level of rSmTSP-2, but not rSmCD59.2, and both were expressed in the insoluble fraction, requiring optimization of culture conditions. Evaluation of different E. coli strains and media showed that BL21-DE3 cultured in Terrific Broth provided the highest expression levels of both proteins. Investigation of a range of time and temperature of induction showed that E. coli strains expressing rRzv:SmTSP-2 and rRzv:SmCD59.2 showed the highest protein production at 23 °C for 15 h. Recombinant proteins were purified by a single step of affinity chromatography allowing isolation of these proteins in high concentration and purity. The optimization process increased final soluble protein yield of rRzv:SmTSP-2 by fourfold and rRzv:SmCD59.2 by tenfold, providing ~ 20 mg/L of each protein. Optimized fusion protein production will allow antigen use in biotin-rhizavidin affinity platforms.

Engineering a new vaccine platform for heterologous antigen delivery in live-attenuated Mycobacterium tuberculosis.

Live vaccines are attractive vehicles for antigen delivery as a strategy to immunize against heterologous pathogens. The live vaccine MTBVAC is based on rational attenuation of Mycobacterium tuberculosis with the objective of improving BCG protection against pulmonary tuberculosis. However, the development of recombinant mycobacteria as antigen-presenting microorganisms has been hindered due to their fastidious genetic manipulation. In this study, we used MTBVAC as a genetic platform to deliver diphtheria, tetanus, or pertussis toxoids, which are the immunogenic constituents of the DTP vaccine. When using nonoptimal genetic conditions, the expression of these immunogens was barely detectable. Accordingly, we pursued a rational, step-by-step optimization of the genetic components to achieve the expression and secretion of these toxoids. We explored variants of the L5 mycobacteriophage promoter to ensure balanced antigen expression and plasmid stability. Optimal signal sequences were identified by comparative proteomics of MTBVAC and its parental strain. It was determined that proteins secreted by the Twin Arginine Translocation pathway displayed higher secretion in MTBVAC, and the Ag85A secretion sequence was selected as the best candidate. Because the coding regions of diphtheria, tetanus, and pertussis toxoids significantly differ in G + C content relative to mycobacterial genes, their codon usage was optimized. We also placed a 3xFLAG epitope in frame with the C-terminus of these toxoids to facilitate protein detection. Altogether, these optimizations resulted in the secretion of DTP antigens by MTBVAC, as demonstrated by western blot and MRM-MS. Finally, we examined specific antibody responses in mice vaccinated with recombinant MTBVAC expressing DTP antigens.

Thirty years of recombinant BCG: new trends for a centenary vaccine.

Introduction: Global perception of the potential for Bacille Calmette-Guérin (BCG), and consequently recombinant BCG (rBCG), in a variety of prophylactic and therapeutic applications has been increasing. A century of information on BCG, and three decades of experience with rBCG, has generated solid knowledge in this field.Area covered: Here, we review the current state of knowledge of BCG and rBCG development. Molecular tools have facilitated the expression of a variety of molecules in BCG, with the aim of improving its efficacy as a tuberculosis vaccine, generating polyvalent vaccines against other pathogens, including viruses, bacteria, and parasites, and developing immunotherapy approaches against noninvasive bladder cancer. BCG's recently appraised heterologous effects and prospects for expanding its application to other diseases are also addressed.Expert opinion: There are high expectations for new tuberculosis vaccines currently undergoing advanced clinical trials, which could change the prospects of the field. Systems biology could reveal effective biomarkers of protection, which would greatly support vaccine development. The development of appropriate large-scale production processes would further support implementation of new vaccines and rBCG products. The next few years should consolidate the broader applications of BCG and produce insights into improvements using the recombinant BCG technology.

Primary and Memory Response of Human Monocytes to Vaccines: Role of Nanoparticulate Antigens in Inducing Innate Memory.

Innate immune cells such as monocytes and macrophages are activated in response to microbial and other challenges and mount an inflammatory defensive response. Exposed cells develop the so-called innate memory, which allows them to react differently to a subsequent challenge, aiming at better protection. In this study, using human primary monocytes in vitro, we have assessed the memory-inducing capacity of two antigenic molecules of Schistosoma mansoni in soluble form compared to the same molecules coupled to outer membrane vesicles of Neisseria lactamica. The results show that particulate challenges are much more efficient than soluble molecules in inducing innate memory, which is measured as the production of inflammatory and anti-inflammatory cytokines (TNFα, IL-6, IL-10). Controls run with LPS from Klebsiella pneumoniae compared to the whole bacteria show that while LPS alone has strong memory-inducing capacity, the entire bacteria are more efficient. These data suggest that microbial antigens that are unable to induce innate immune activation can nevertheless participate in innate activation and memory when in a particulate form, which is a notion that supports the use of nanoparticulate antigens in vaccination strategies for achieving adjuvant-like effects of innate activation as well as priming for improved reactivity to future challenges.

BCG vaccination improves DTaP immune responses in mice and is associated with lower pertussis incidence in ecological epidemiological studies.

BACKGROUND: The Bacillus Calmette-Guérin (BCG), the only vaccine against tuberculosis (TB) currently in use, has shown beneficial effects against unrelated infections and to enhance immune responses to vaccines. However, there is little evidence regarding the influence of BCG vaccination on pertussis. METHODS: Here, we studied the ability of BCG to improve the immune responses to diphtheria, tetanus, and acellular (DTaP) or whole-cell pertussis (DTwP) vaccination in a mouse model. We included MTBVAC, an experimental live-attenuated vaccine derived from Mycobacterium tuberculosis, in our studies to explore if it presents similar heterologous immunity as BCG. Furthermore, we explored the potential effect of routine BCG vaccination on pertussis incidence worldwide. FINDINGS: We found that both BCG and MTBVAC when administered before DTaP, triggered Th1 immune responses against diphtheria, tetanus, and pertussis in mice. Immunization with DTaP alone failed to trigger a Th1 response, as measured by the production of IFN-γ. Humoral responses against DTaP antigens were also enhanced by previous immunization with BCG or MTBVAC. Furthermore, exploration of human epidemiological data showed that pertussis incidence was 10-fold lower in countries that use DTaP and BCG compared to countries that use only DTaP. INTERPRETATION: BCG vaccination may have a beneficial impact on the protection against pertussis conferred by DTaP. Further randomized controlled trials are needed to properly define the impact of BCG on pertussis incidence in a controlled setting. This could be a major finding that would support changes in immunization policies. FUNDING: This work was supported by the Ministry of "Economía y Competitividad"; European Commission H2020 program, "Gobierno de Aragón"; CIBERES; "Fundação Butantan"; Instituto de Salud Carlos III and "Fondo FEDER".

Robust Immune Response Induced by Schistosoma mansoni TSP-2 Antigen Coupled to Bacterial Outer Membrane Vesicles.

PURPOSE: The use of adjuvants can significantly strengthen a vaccine's efficacy. We sought to explore the immunization efficacy of bacterial outer membrane vesicles (OMVs) displaying the Schistosoma mansoni antigen, SmTSP-2, through a biotin-rhizavidin coupling approach. The rationale is to exploit the nanoparticulate structure and the adjuvant properties of OMVs to induce a robust antigen-specific immune response, in light of developing new vaccines against S. mansoni. MATERIALS AND METHODS: OMVs were obtained from Neisseria lactamica and conjugated with biotin. The recombinant SmTSP-2 in fusion with the biotin-binding protein rhizavidin (rRzvSmTSP-2) was produced in E. coli and coupled to biotinylated OMVs to generate an OMV complex displaying SmTSP-2 on the membrane surface (OMV:rSmTSP-2). Transmission electron microscopy (TEM) and dynamic light scattering analysis were used to determine particle charge and size. The immunogenicity of the vaccine complex was evaluated in C57BL/6 mice. RESULTS: The rRzvSmTSP-2 protein was successfully coupled to biotinylated OMVs and purified by size-exclusion chromatography. The OMV:rSmTSP-2 nanoparticles showed an average size of 200 nm, with zeta potential around - 28 mV. Mouse Bone Marrow Dendritic Cells were activated by the nanoparticles as determined by increased expression of the co-stimulatory molecules CD40 and CD86, and the proinflammatory cytokines (TNF-α, IL-6 and IL-12) or IL-10. Splenocytes of mice immunized with OMV:rSmTSP-2 nanoparticles reacted to an in vitro challenge with SmTSP-2 with an increased production of IL-6, IL-10 and IL-17 and displayed a higher number of CD4+ and CD8+ T lymphocytes expressing IFN-γ, IL-4 and IL-2, compared to mice immunized with the antigen alone. Immunization of mice with OMV:rSmTSP-2 induced a 100-fold increase in specific anti-SmTSP-2 IgG antibody titers, as compared to the group receiving the recombinant rSmTSP-2 protein alone or even co-administered with unconjugated OMV. CONCLUSION: Our results demonstrate that the SmTSP-2 antigen coupled with OMVs is highly immunogenic in mice, supporting the potential effectiveness of this platform for improved antigen delivery in novel vaccine strategies.