Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant.

Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen.

Production of native flagellin from Salmonella Typhimurium in a bioreactor and purification by tangential ultrafiltration

Flagellin is the structural protein and most abundant component of bacterial flagella. The flagellum filament contains around 20,000 – 100,000 subunits of 50 kDa flagellin that can have diversebiotechnological applications such as vaccine adjuvant and cellular protector during chemo- and radiotherapy.The main aim of this work was to study a production process of purified native FliC flagellin of Salmonella Typhimurium. The culture conditions in shakers were established with medium devoid of animal-derived components. In bioreactors, culture conditions were established in order to obtain flagellin from the culture supernatant by tangential ultrafiltration (TUF). The concentrated 750 kDa cut-off TUF fraction had a purification factor of 1.5 and a recovery yield of 52.2% for flagellin. The volumetric production of flagellin using the described procedure achieved around 307 mg/L of culture, which represented a significant improvement over previously reported methods. These results permit the development of production and purification processes that can be easily scaled up.

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157: H7 / Resposta de anticorpos obtidas em camundongos imunizados com linhagens vacinais de Bacillus subtilis expressando a subunidade B da Stx2 de Escherichia coli O157: H7 enterohemorrágica

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) producedbyenterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.

Até o presente o momento, não há vacina ou imunoterapia disponível para pacientes com Síndrome Hemolítica Urêmica (SHU) induzida pela toxina Shiga-like (Stx) produzida por linhagens de Escherichia coli entero-hemorragica (EHEC), tais como as pertencentes ao sorotipo O157:H7. Neste trabalho, avaliamos a performance de Bacillus subtilis, uma espécie bacteriana gram-positiva não-patogênica formadora de esporos, como veículo vacinal para a expressão da subunidade B da Stx2B (Stx2B). Uma linhagem vacinal recombinante de B. subtilis expressando Stx2B, sob o controle de um promoter induzível por estresse, foi administrada a camundongos BALB/c por via oral, nasal ou subcutânea usando células vegetativas e esporos. Camundongos imunizados com células vegetativas e esporos pela via oral desenvolveram títulos anti-Stx2B baixos, mas específicos, de IgG sérico e IgA fecal, enquanto camundongos imunizados com esporos recombinates desenvolveram resposta anti-Stx2B apenas após a administração pela via parenteral. No entanto, anticorpos produzidos em camundongos imunizados com a linhagem recombinante de B. subtilis não inibiram os efeitos tóxicos da toxina nativa em condições in vitro e in vivo, sugerindo que a quantidade e/ou a qualidade da resposta imune gerada não suportam uma neutralização efetiva da Stx2 produzidas por linhagens de EHEC.

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7.

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) produced by enterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.

Anti-flagellin antibody responses elicited in mice orally immunized with attenuated Salmonella enterica serovar Typhimurium vaccine strains.

In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.

Anti-flagellin antibody responses elicited in mice orally immunized with attenuated Salmonella enterica serovar Typhimurium vaccine strains

In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.

Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium.

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

Cytotoxic T cell adjuvant effects of three Salmonella enterica flagellins

Bacterial flagellins are important virulence-associated factors and strong inducers of inflammatory responses in mammalian hosts. Flagellins have also been investigated as potential vaccine adjuvants, either for induction of humoral or cellular immune responses, to different target antigens. In this study we investigated the adjuvant properties of three Salmonella enterica flagellins types (FliCd, FliCi and FljB) to an ovalbumin-derived CD8+ T cell-restricted epitope (OVA257264). Although mice immunized with the three tested flagellins elicited antigen-specific activated CD8+ T cells, only animals immunized with FliCi and FliCd flagellins admixed with ovalbumin mounted specific in vivo cytotoxic responses to peptide-pulsed target cells. The present results indicate that Salmonella flagellins are endowed with type-specific adjuvant effects toward murine CD8+ T cells, a feature that may impact their use as adjuvants for prophylatic or therapeutic vaccines.

As flagelinas bacterianas são importantes fatores associados à virulência e potentes indutores de resposta inflamatória em mamíferos. Estas moléculas são também investigadas como potencial adjuvante para uso em vacinas na indução de resposta imune humoral e celular para diferentes antígenos alvo. No presente estudo investigamos as propriedades adjuvantes de três tipos de flagelinas de Salmonella enterica (FliCd, FliCi e FljB) para um epítopo derivado da ovalbumina específico para células T CD8+. As três flagelinas testadas induziram respostas de células T CD8+ específicas em camundongos imunizados, porém, somente animais imunizados com as flagelinas FliCi e FliCd co-administradas com ovalbumina montaram resposta citotóxica específica in vivo para células-alvo pulsadas com peptídeo OVA. Os resultados apresentados indicam que flagelinas de Salmonella são dotadas de efeitos adjuvantes tipo-específico frente a células T CD8+ in vivo, uma característica que pode gerar impactos no uso dessas proteínas como adjuvantes em vacinas profiláticas ou terapêuticas.

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans.

The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT(+) (25 strains) only or LT(+)/ST(+) (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

Cytotoxic T cell adjuvant effects of three Salmonella enterica flagellins.

Bacterial flagellins are important virulence-associated factors and strong inducers of inflammatory responses in mammalian hosts. Flagellins have also been investigated as potential vaccine adjuvants, either for induction of humoral or cellular immune responses, to different target antigens. In this study we investigated the adjuvant properties of three Salmonella enterica flagellins types (FliCd, FliCi and FljB) to an ovalbumin-derived CD8(+) T cell-restricted epitope (OVA257-264). Although mice immunized with the three tested flagellins elicited antigen-specific activated CD8(+) T cells, only animals immunized with FliCi and FliCd flagellins admixed with ovalbumin mounted specific in vivo cytotoxic responses to peptide-pulsed target cells. The present results indicate that Salmonella flagellins are endowed with type-specific adjuvant effects toward murine CD8(+) T cells, a feature that may impact their use as adjuvants for prophylatic or therapeutic vaccines.

Prime-boost vaccine regimen confers protective immunity to human-derived enterotoxigenic Escherichia coli.

Development of effective vaccines against diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains is still a priority for those living at or traveling to endemic regions. In this work, we evaluated the protective role of an anti-ETEC vaccine regimen based on parenteral priming with a DNA vaccine, pRECFA, followed by oral boosting with a recombinant attenuated Salmonella Typhimurium vaccine strain, HG3, both encoding the same antigen, the structural subunit (CfaB) of the ETEC CFA/I fimbriae. The DNA-priming Salmonella-boosting protocol enhanced both murine anti-CfaB serum IgG and fecal IgA antibody responses and increased the ability of serum antibodies to inhibit the adhesive properties of the CFA/I fimbriae expressed by live bacteria, as compared to mice immunized with only one vaccine type. Addition of a mucosal adjuvant (LTR192G) to the Salmonella vaccine strain further enhanced the synergic effects of the vaccine regimen on the induced CfaB-specific antibody responses. DBA/2 dams submitted to the prime-boost regimen transferred complete passive protection to suckling neonates challenged with a virulent ETEC strain. Detection of milk anti-CfaB IgA antibodies and protection conferred by vaccinated dams to neonates born from non-vaccinated dams indicated that secretion of antigen-specific IgA is the immune response induced by the protective vaccine regimen. These results demonstrate that priming with a DNA vaccine and boosting with a Salmonella strain enhances both quantitatively and qualitatively the antibody responses to the CfaB antigen and represents an alternative for either active or passive immunization approach to ETEC-associated diarrhea.

Host and bacterial factors affecting induction of immune responses to flagellin expressed by attenuated Salmonella vaccine strains.

Previous observations demonstrated that the delivery of recombinant Salmonella enterica serovar Dublin strains to mice via mucosal routes did not efficiently activate systemic and secreted antibody responses to either type d flagellin or genetically fused heterologous B-cell epitopes, thus reducing the usefulness of the protein as a carrier of epitopes for vaccine purposes. In this work, we investigated murine systemic and mucosal flagellin immunogenicity after oral immunization with attenuated Salmonella strains. The reduced anti-type d flagellin antibody responses in mice immunized via mucosal routes with three doses of flagellated S. enterica serovar Dublin strains were not caused by oral tolerance and could not be restored by coadministration of a mucosal adjuvant. The induction of antibody responses to Salmonella flagellins was shown to differ according to the genetic background, but not the haplotype, of the mouse lineage. Moreover, BALB/c mice orally immunized with S. enterica serovar Typhimurium strains developed anti-type i flagellin sera and secreted antibody responses, which indicated that the serovar of the Salmonella vaccine strain also affected flagellin immunogenicity. Analyses of cytokine responses of BALB/c mice immunized with three oral doses of flagellated S. enterica serovar Dublin vaccine strains showed that, in spite of the lack of antibody responses, elevated type d flagellin-specific CD4-cell-activation-dependent gamma interferon (IFN-gamma) and interleukin-10 responses were elicited after the administration of the vaccine strains via either parenteral or mucosal routes. Similar cytokine production patterns were detected to a T-cell heterologous epitope, derived from the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC), in mice orally immunized with a Salmonella vaccine strain expressing hybrid flagella. These results indicate that the immunogenicities of Salmonella flagellins can differ significantly, depending on the murine host and on the bacterial vector used, and demonstrate that the induction of CD4-cell-activation-dependent IFN-gamma production represents a major immune response triggered by flagellin and in-frame fused heterologous T-cell epitopes after the oral administration of recombinant S. enterica serovar Dublin vaccine strains.

Flagellin expressed by live Salmonella vaccine strains induces distinct antibody responses following delivery via systemic or mucosal immunization routes.

Salmonella flagellin, expressed as flagella in live attenuated vaccine strains, elicits distinct systemic (IgG) and secreted (IgA) antibody responses in mice following delivery via mucosal (nasal/oral) or parenteral (intraperitoneal (i.p.)) immunization routes. Reduced flagellin-specific antibodies were detected either systemically or locally following delivery of flagellated derivatives of aroA Salmonella enterica serovar Dublin SL1438 via the nasal route, the most effective mucosal site for activation of immune responses in mice. In contrast, flagellin represents the most potent Salmonella antigen for the generation of specific serum antibody (IgG) responses following i.p. inoculations. The distinct immunogenic properties of Salmonella flagellin could not be ascribed to deficient colonization, reduced invasive ability or loss of the flagellin expression by the flagellated vaccine strains.