Maria E. Sbrogio-Almeida

Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli.

Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni(2+)-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.

CD8+ T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (CS) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS(280-288) epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS(280-288) peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations.

Paracoccidioides brasiliensis vaccine formulations based on the gp43-derived P10 sequence and the Salmonella enterica FliC flagellin.

ABSTRACT Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43) or the derived P10 sequence containing a CD4+ T-cell-specific epitope have shown promising results. In the present study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P. brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innate immunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunized with gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) and IL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused with P10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10 peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secreted type 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeast cells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimeric flagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lung damage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P. brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.

Host and Bacterial Factors Affecting Induction of Immune Responses to Flagellin Expressed by Attenuated Salmonella Vaccine Strains

ABSTRACT 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-γ) 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-γ 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.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium.

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coli and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His(6)FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freunds adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP1(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity.

Antibody response against Escherichia coli heat-stable enterotoxin expressed as fusions to flagellin

The heat-stable toxin (ST) produced by enterotoxigenic Escherichia coli strains causes diarrhoea by altering the fluid secretion in intestinal epithelial cells. Here, the effectiveness of a flagellin fusion protein of Salmonella containing a 19-amino-acid sequence derived from the ST sequence (FLA--ST) in generating antibodies capable of neutralizing the toxic activity of ST was evaluated. This fusion protein, and an alternative construction where two cysteine residues in the ST sequence were substituted by alanines (ST(mt)), were delivered to the immune system by three distinct strategies: (i) orally, using an attenuated Salmonella strain expressing FLA--ST; (ii) intraperitoneally, by injection of purified FLA--ST; (iii) orally, using attenuated Salmonella carrying a eukaryotic expression plasmid (pCDNA3) with the gene encoding FLA-ST. The results showed that the flagellin system can be used as a carrier to generate ST-neutralizing antibodies. However, it should be mentioned that humoral immune response against ST was only obtained when the mutated ST sequence was employed. FLA-ST was found to be non-immunogenic when delivered via the oral route with attenuated Salmonella strains. However, a flagellin antibody response was obtained by immunizing mice with Salmonella carrying pCDNA3/FLA-ST(mt). Oral immunization with Salmonella carrying the eukaryotic expression plasmid (pCDNA3/FLA--ST(mt)) seems to be a promising method to elicit an appropriate response against fusions to flagellin.

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle.

Protection induced by pneumococcal surface protein A (PspA) is enhanced by conjugation to a Streptococcus pneumoniae capsular polysaccharide

The currently available anti-pneumococcal vaccines are based on capsular polysaccharide (PS), plain or conjugated to a carrier protein. Conjugated vaccines are expensive products, especially in the case of pneumococcus, in which reasonable coverage requires from 7 to 13 serotypes. To obtain increased coverage with fewer components, we evaluated the immunogenicity of the pneumococcal surface protein A (PspA), conjugated to capsular polysaccharide serotype 23F, aiming at induction of an immune response against both components. Mice immunized with PS23F-rPspA1 conjugate produced antibodies against both PS and rPspA1, comparable or slightly higher than that obtained by free PS. The immunized animals challenged with a lethal dose of a virulent strain bearing a homologous PspA, showed that the PS23F-rPspA1 conjugate induced higher survival than rPspA1 alone or in combination with PS. This increased protection was shown to correlate with the enhanced capacity of the antibodies to bind to the pneumococcal surface and to induce complement deposition. Our results indicate that the use of PS-PspA conjugates may be a way to increase coverage against pneumococci with fewer components.

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.

Development of a whole cell pneumococcal vaccine: BPL inactivation, cGMP production, and stability.

Pneumococcal infections impose a large burden of disease on the human population, mainly in developing countries, and the current pneumococcal vaccines offer serotype-specific protection, but do not cover all pathogenic strains, leaving populations vulnerable to disease caused by non-vaccine serotypes. The pneumococcal whole cell vaccine is a low-cost strategy based on non-capsular antigens common to all strains, inducing serotype-independent immunity. Therefore, we developed the process for the cGMP production of this cellular vaccine. Initially, three engineering runs and two cGMP runs were performed in 60-L bioreactors, demonstrating the consistency of the production process, as evaluated by the growth curves, glucose consumption and metabolite formation (lactate and acetate). Cell recovery by tangential filtration was 92 ± 13 %. We optimized the conditions for beta-propiolactone (BPL) inactivation of the bacterial suspensions, establishing a maximum cell density of OD600 between 27 and 30, with a BPL concentration of 1:4000 (v/v) at 150 rpm and 4 °C for 30 h. BPL was hydrolyzed by heating for 2h at 37 °C. The criteria and methods for quality control were defined using the engineering runs and the cGMP Lots passed all specifications. cGMP vaccine Lots displayed high potency, inducing between 80 and 90% survival in immunized mice when challenged with virulent pneumococci. Sera from mice immunized with the cGMP Lots recognized several pneumococcal proteins in the extract of encapsulated strains by Western blot. The cGMP whole cell antigen bulk and whole cell vaccine product lots were shown to be stable for up to 12 and 18 months, respectively, based upon survival assays following i.p. challenge. Our results show the consistency and stability of the cGMP whole cell pneumococcal vaccine lots and demonstrate the feasibility of production in a developing country setting.