Parenteral Adjuvant Effects of an Enterotoxigenic Escherichia coli Natural Heat-Labile Toxin Variant.

Native type I heat-labile toxins (LTs) produced by enterotoxigenic Escherichia coli (ETEC) strains exert strong adjuvant effects on both antibody and T cell responses to soluble and particulate antigens following co-administration via mucosal routes. However, inherent enterotoxicity and neurotoxicity (following intra-nasal delivery) had reduced the interest in the use of these toxins as mucosal adjuvants. LTs can also behave as powerful and safe adjuvants following delivery via parenteral routes, particularly for activation of cytotoxic lymphocytes. In the present study, we evaluated the adjuvant effects of a new natural LT polymorphic form (LT2), after delivery via intradermal (i.d.) and subcutaneous (s.c.) routes, with regard to both antibody and T cell responses. A recombinant HIV-1 p24 protein was employed as a model antigen for determination of antigen-specific immune responses while the reference LT (LT1), produced by the ETEC H10407 strain, and a non-toxigenic LT form (LTK63) were employed as previously characterized LT types. LT-treated mice submitted to a four dose-base immunization regimen elicited similar p24-specific serum IgG responses and CD4(+) T cell activation. Nonetheless, mice immunized with LT1 or LT2 induced higher numbers of antigen-specific CD8(+) T cells and in vivo cytotoxic responses compared to mice immunized with the non-toxic LT derivative. These effects were correlated with stronger activation of local dendritic cell populations. In addition, mice immunized with LT1 and LT2, but not with LTK63, via s.c. or i.d. routes developed local inflammatory reactions. Altogether, the present results confirmed that the two most prevalent natural polymorphic LT variants (LT1 or LT2) display similar and strong adjuvant effects for subunit vaccines administered via i.d. or s.c. routes.

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.

Salmonella enterica Serovar Typhimurium Vaccine Strains Expressing a Nontoxic Shiga-Like Toxin 2 Derivative Induce Partial ProtectiveImmunity to the Toxin Expressed by Enterohemorrhagic Escherichia coli

Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strainsare the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and highmortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeuticanti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2 AB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ inthe expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. Thevaccine strains expressed Stx2 AB, either cell bound or secreted into the extracellular environment, andshowed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions.Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B(IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) andconferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2 AB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.

Salmonella enterica serovar Typhimurium vaccine strains expressing a nontoxic Shiga-like toxin 2 derivative induce partial protective immunity to the toxin expressed by enterohemorrhagic Escherichia coli.

Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2DeltaAB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2DeltaAB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2DeltaAB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.

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.

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 vaccineadjuvant 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 H2d-restricted CD8+ T cell-specific epitope (CS280–288) 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 CS280–288 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 CS280–288 peptide. The results showed that CS280–288-specific cytotoxic CD8+ T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280–288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280–288 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 adaptativeimmune 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.

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.

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.

Optimizing expression of Streptococcus pneumoniae surface protein a, PspA: serocross-reactivity within families of antisera induced against clades 1 and 3.

Streptococcus pneumoniae is the agent responsible for infections such as pneumonia, otitis media, and meningitis. Among virulence factors, the Pneumococcal surface protein A (PspA) has been shown to be immunogenic and protective in mice, and is thus a good vaccine candidate. PspA has been classified into 6 clades and 3 families. Initially, pspA fragments, clades 1 and 3, were cloned into the pAE-6His expression vector. Proteins were expressed in Escherichia coli BL21(DE3) and purified by affinity and anion exchange chromatographies, with a yield of 11 mg/l of culture. Due to plasmid instability in E. coli, another construct using pspA1 was obtained based on pET-37b(+), which was shown to be stable in E. coli and increased the yield approximately 3-fold. Our results show good conditions for scale-up. Sera from immunized mice recognized PspA in total extracts of S. pneumoniae strains: anti-rPspA1p sera recognized native PspA clades 1 (+++), 2 (++) and 4 (+) and anti-rPspA3p sera recognized PspA clades 1 (+), 2 (+), 3 (+++) and 4 (+). The cross-reactivity pattern obtained confirms the notion that proteins from both families should be included for development of a broad-coverage vaccine; lower-cross reactivity between rPspAs of family 2 indicates that it may be necessary to include 2 proteins from this family.

Evaluation of different promoter sequences and antigen sorting signals on the immunogenicity of Bacillus subtilis vaccine vehicles.

Recombinant Bacillus subtilis strains, either in the form of spores or vegetative cells, may be employed as safe and low-cost vaccine vehicles. In this study, we studied the role of promoter sequences and antigen-sorting signals on the immunogenicity based on previously constructed B. subtilis episomal expression systems. Mice orally immunized with spores or cells encoding the B subunit of the heat labile toxin (LTB), originally expressed by some enterotoxigenic Escherichia coli (ETEC) strains, under control of the stress-inducible gsiB promoter developed higher anti-LTB serum IgG and fecal IgA responses with regard to vaccine strains transformed with plasmids encoding the antigen under control of IPTG-inducible (Pspac) or constitutive (PlepA) promoters. Moreover, surface expression of the vaccine antigen under the control of the PgsiB promoter enhanced the immunogenicity of vegetative cells, while intracellular accumulation of LTB led to higher antibody responses in mice orally immunized with recombinant B. subtilis spores. Specific anti-LTB antibodies raised in vaccinated mice recognized and neutralized in vitro the native toxin produced by ETEC strains. Nonetheless, only mice orally immunized with recombinant B. subtilis strains, either as vegetative cells or spores, expressing intracellular LTB under the control of the gsiB promoter conferred partial protection to lethal challenges with purified LT. The present report further demonstrates that B. subtilis plasmid-based heterologous protein expression systems are adequate for antigen delivery via the oral route.

Stable episomal expression system under control of a stress inducible promoter enhances the immunogenicity of Bacillus subtilis as a vector for antigen delivery.

Bacillus subtilis has been successfully engineered to express heterologous antigens genetically fused to surface-exposed spore coat proteins as a vaccine vehicle endowed with remarkable heat resistance and probiotic effects for both humans and animals. Nonetheless, the immunogenicity of passenger antigens expressed by B. subtilis spores is low particularly following oral delivery. In this work, we describe a new episomal expression system promoting enhanced immunogenicity of heterologous antigens carried by B. subtilis strains, either in the form of spores or vegetative cells, following oral or parenteral delivery to mice. Based on a bi-directional replicating multicopy plasmid, the gene encoding the B subunit of the heat-labile toxin (LTB), produced by enterotoxigenic Escherichia coli (ETEC) strains, was cloned under the control of the B. subtilis glucose starvation inducible (gsiB) gene promoter, active in vegetative cells submitted to heat and other stress conditions. The recombinant plasmid proved to be structurally and segregationally stable in both cells and spores under in vitro and in vivo conditions. Moreover, BALB/c mice orally immunized with B. subtilis cells or spores elicited enhanced anti-LTB systemic (serum IgG) and secreted (fecal IgA) antibody responses, thus, suggesting that antigen expression occurred during in vivo transit. These results indicate that the new episomal expression system may improve the performance of B. subtilis as a live orally-delivered vaccine carrier.

Combined vaccine regimen based on parenteral priming with a DNA vaccine and administration of an oral booster consisting of a recombinant Salmonella enterica serovar Typhimurium vaccine strain for immunization against infection with human-derived enterotoxigenic Escherichia coli strains.

Repeated evidence has demonstrated that combined primer-booster immunization regimens can improve both secreted and humoral immune responses to antigens derived from viral, bacterial, and parasitic pathogens. For the present work, we evaluated the synergic serum immunoglobulin G (IgG) and fecal IgA antibody responses elicited in BALB/c mice who were intramuscularly primed with a DNA vaccine, pRECFA, followed by oral boosting with an attenuated Salmonella enterica serovar Typhimurium vaccine (HG3) strain, with both vaccines encoding the structural subunit (CfaB) of the CFA/I fimbriae produced by human-derived enterotoxigenic Escherichia coli (ETEC) strains. The immunological properties of the vaccine regimen were evaluated according to the order of the administered vaccines, the nature of the oral antigen carrier, the age of the vaccinated animals, the interval between the priming and boosting doses, and the amount of injected DNA. The production of gamma interferon and the IgG2a subclass in serum indicated that mice immunized with the primer-booster regimen developed prevailing type 1 T-cell-dependent immune responses. The synergic effect of the vaccine regimen on the induced antibody responses was also revealed by its ability to block the adhesive properties of CFA/I fimbriae expressed by live bacteria, as shown by the inhibition of Caco-2 cell and human erythrocyte binding. Moreover, DBA2 newborn mice were protected from lethal challenges with a CFA/I+ ETEC strain after the incubation of live bacteria with serum samples harvested from mice who were subjected to the primer-booster regimen. We propose, therefore, that the DNA primer-Salmonella booster regimen represents an alternative for the development of vaccines requiring both mucosal and systemic antibody responses for immunological protection.