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Mayra M F Barbosa,1,2 Alex I Kanno,1 Giovana C Barazzone,1 Dunia Rodriguez,1 Violeta Pancakova,1,3 Monalisa Trentini,1 Eliana L Faquim-Mauro,4 Amanda P Freitas,4 Mariana I Khouri,5 Jessica Lobo-Silva,5 Viviane M Goncalves,1 Rocilda P F Schenkman,1 Martha M Tanizaki,1 Diana Boraschi,6– 8 Richard Malley,9 Leonardo P Farias,5 Luciana C C Leite1 1Laboratório de Desenvolvimento de Vacinas, Instituto Butantan, São Paulo, Brazil; 2Programa de Pós-Graduação Interunidades em Biotecnologia, Universidade de São Paulo, São Paulo, Brazil; 3Université Claude Bernard Lyon 1 (UCBL1), Villeurbanne, 69100, France; 4Laboratório de Imunopatologia, Instituto Butantan, São Paulo, Brazil; 5Laboratório de Biomarcadores e Inflamação, Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil; 6Istituto di Biochimica e Biologia Cellulare, Consiglio Nazionale delle Ricerche, Napoli, Italy; 7Stazione Zoologica Anton Dohrn, Napoli, Italy; 8Shenzhen Institute of Advanced Technologies (SIAT), Chinese Academy of Sciences (CAS), Shenzhen, China Division of Infectious Diseases, Boston Children’s Hospital, Boston, MA, USA; 9Division of Infectious Diseases, Boston Children’s Hospital, Boston, MA, USACorrespondence: Luciana C C LeiteLaboratório de Desenvolvimento de Vacinas, Instituto Butantan, Av. Vital Brasil, São Paulo, 1500, SP, BrazilTel +55-11-2627-9816Email luciana.leite@butantan.gov.brPurpose: 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.Keywords: Schistosoma mansoni, vaccine, TSP-2 antigen, outer membrane vesicles, OMV, biotin-avidin coupling, nanoparticle

Serogroup B outer membrane vesicles (OMV) with iron regulated proteins (IRP) from Neisseria meningitidis constitute the antigen for the vaccine against the disease caused by this bacterium. Aiming to enhance final OMV concentration, seven batch experiments were carried out under four different conditions: (i) with original Catlin medium; (ii) with original Catlin medium and lactate and amino acids pulse at the 6th cultivation hour; (iii) with Catlin medium with double initial concentrations of lactate and amino acids and (iv) Catlin medium without glycerol and with double initial concentrations of lactate and amino acids. The cultivation experiments were carried out in a 7-L bioreactor under the following conditions: 36°C, 0.5atm, overlay air 1L/min, agitation: 250-850 rpm, and O(2) control at 10%, 20 h. After lactate and amino acids exhaustion, cell growth reached stationary phase and a significant release increase of OMV was observed. According to the Luedeking & Piret model, OMV liberation is non-growth associated. Glycerol was not consumed during cultivation. The maximum OMV concentration value attained was 162 mg/L with correspondent productivity of 8.1mg/(Lh) employing Catlin medium with double initial concentrations of lactate and amino acids. The obtained OMV satisfied constitution and protein pattern criteria and were suitable for vaccine production., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A conjugate meningococcal vaccine against serogroup B/C consisting of capsular PS (polysaccharide) from serogroup C conjugated to OMV (outer membrane vesicle) from serogroup B would be a very useful vaccine in regions where there is a prevalence of both serogroups, for example in Brazil. For this purpose, the conjugation method that uses ADHy (adipic acid dihydrazide) as spacer and a carbodi-imide derivative, EDAC [1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide], as catalyser was optimized looking for synthesis yield and maintenance of the antigenicity of both components. The best synthesis conditions preserving the vaccine immunogenicity resulted in a final yield of approx. 17%. Immunogenicity of the vaccine was highest when 10% of the sialic acid residues of the PS were occupied by the ADHy spacer. Sterilization of the conjugate by filtration through a 0.22-microm-pore-size membrane resulted in a low recovery of protein and PS (approximately 50%), although the vaccine immunogenicity was maintained. Using gamma irradiation on freeze-dried sample, it was possible to maintain the integrity of OMV structure and, consequently, its ability to induce bactericidal antibodies.

Meningococcal outer membrane vesicle (OMV) vaccines are weak antigens in infants. This study aimed at investigating alternative adjuvants for induction of functional antibodies in newborn mice. Serogroup B/C anti-meningococcal vaccines, consisting of capsular polysaccharide from serogroup C (PSC) conjugated to OMV from one serogroup B serosubtype prevalent in Brazil, combined with OMV from another prevalent serosubtype, were tested in newborn and adult mice with the following adjuvants: aluminum hydroxide, MPL (monophosphoryl lipid A), Titermax and MF59. Total IgG, IgG avidity index determination and bactericidal assay were performed with sera from immunized mice. Antibodies induced against PSC in newborn mice showed avidity and bactericidal titers, similar to those obtained in adult mice, independently of the adjuvant. Evidence is presented that the inclusion of MF59 enhanced the immune response against OMV in newborn mice.

Streptococcus pneumoniae is one of the most important human pathogens and improvement of the currently used polysaccharide vaccines is being pursued. We constructed DNA vaccine vectors containing either the full-length psaA (pneumococcal surface adhesin A) or a truncated pspA (pneumococcal surface protein A--pspA') gene. Both constructs showed transient expression of the antigens in vertebrate cells and induced significant antibody response to the pneumococcal antigens in BALB/c mice injected intramuscularly (i.m.). Fusion with an N-terminal cytoplasmatic SV40 T-antigen (CT-Ag), which was previously shown to stabilize poorly expressed antigens through association with Hsp73, also induced anti-PspA antibody response. The induction of antibodies with a low IgG1:IgG2a ratio and elevated gamma interferon (IFN-gamma) production by spleen cells elicited by DNA vaccination indicate preferential priming of Th1 immunity. Since induction of antibodies against both PsaA and PspA was previously shown to correlate with protection against fatal infection with S. pneumoniae and cell-mediated immune responses could contribute to protection, further evaluation of PsaA and PspA as antigens for a DNA vaccine against S. pneumoniae could be promising.

Neisseria meningitidis serogroup C polysaccharide (PS C) was conjugated to serogroup B outer membrane vesicles (OMV) in order to test the possibility of obtaining a bivalent group B and C meningococcus vaccine. The conjugate and controls were injected intraperitoneally into groups of ten mice with boosters on days 14 and 28 after the primary immunization. The following groups were used as control: (i) PS C; (ii) PS C plus OMV; (iii) OMV; and (iv) saline. The serum collected on days 0, 14, 28 and 42 were tested by enzyme-linked immunosorbent assay (ELISA) for PS C and OMV, and by complement mediated bactericidal assay against serogroups B and C. ELISA for PS C as well as bactericidal titres against serogroup C meningococci of the conjugated vaccine increased eight-fold (ELISA) and 32 fold (bactericidal) after 42 days in comparison with the PS C control group. ELISA for OMV and bactericidal titre against serogroup B meningococci of the conjugate showed no significant difference in comparison with the OMV containing controls. Furthermore, Western Blot assay of the conjugate immune serum did not bind OMV class four protein which is related to the complement dependent antibody suppressor. The results indicate that the PS C-OMV conjugate could be a candidate for a bivalent vaccine toward serogroups B and C meningococci.

Trypanosoma cruzi trans-sialidase is encoded by a family of genes containing a conserved region, which corresponds to the catalytic and amino-terminal domain of the enzyme. Most, but not all genes, also encode a variable region formed by 12 amino acid repeats at the carboxy-terminus of the protein that are not required for enzymatic activity. To design gene knock-out strategies and understand how trans-sialidase expression is regulated, we have studied the genome organization of trans-sialidase genes. We show here that the different types of trans-sialidase genes are distributed in more than one chromosomal band with sizes ranging from 0.8 to 1.5 Mb pairs in several T. cruzi strains. In the Y-strain, all repeat-containing genes are localized in one chromosomal band of 1.1 Mb, while the repeat-minus genes are in two chromosomes of 0.82 and 0.79 Mb. The repeat-containing genes have similar catalytic and intergenic regions, but variable lengths of the repeated region. The trans-sialidase genes with the repeats are in tandem of up to 12 genes in at least four different clusters. Each cluster contains genes with different numbers of repeats, according to the physical maps of eight independent cosmids, and in the same cluster there are genes that code for active and inactive trans-sialidases. There are 80 +/- 30 copies of the repeat-containing genes grouped in two NotI fragments of 120 and 180 Kb. Therefore, in the Y-strain, the trans-sialidase genes containing repeats might be arranged in three to four clusters in two homologous chromosomes, each cluster having up to 12 genes with different repeat numbers.

Trans-sialidase catalyzes the transference of sialic acid from host to the Trypanosoma cruzi surface. Here, we characterize the sialic acid acceptors of this protozoan parasite as mucin-like molecules, which are anchored to the membrane by glycosylphosphatidylinositol. The mucins isolated from the insect stages differ from the mucins isolated from the mammalian stages in size and reactivity to monoclonal antibodies, suggesting that they are formed by variable polypeptide chains and/or O-linked carbohydrate structures.

We have used a Chinese hamster ovary cell mutant (Lec2) that express much less sialic acid on the surface than the parental cell line (Pro5) to investigate whether sialic acid plays a role during cell invasion by Trypanosoma cruzi. Trypomastigotes derived from a tissue culture (corresponding to bloodstream trypomastigotes) and metacyclic trypomastigotes (corresponding to infective stages of the insect vector) invaded the Lec2 mutant less efficiently than the parental cell line. Invasion of the Lec2 mutant cells could be restored to the Pro5 level by resialylation of the mutant cells with T. cruzi trans-sialidase and sialyllactose. Conversely, pretreatment of the Pro5 parental cells with bacterial neuraminidase decreased invasion. These results indicate that sialic acid associated with the host cell contributes to invasion by T. cruzi.