Juliana Falcão Rodrigues

Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant

The dengue virus non-structural 1 (NS1) protein contributes to evasion of host immune defenses and represents a target for immune responses. Evidences generated in experimental models, as well as the immune responses elicited by infected individuals, showed that induction of anti-NS1 immunity correlates with protective immunity but may also result in the generation of cross-reactive antibodies that recognize platelets and proteins involved in the coagulation cascade. In the present work, we evaluated the immune responses, protection to type 2 dengue virus (DENV2) challenges and safety parameters in BALB/c mice vaccinated with a recombinant NS1 protein in combination with three different adjuvants: aluminum hydroxide (alum), Freunds adjuvant (FA) or a genetically detoxified derivative of the heat-labile toxin (LT(G33D)), originally produced by some enterotoxigenic Escherichia coli (ETEC) strains. Mice were subcutaneously (s.c.) immunized with different vaccine formulations and the induced NS1-specific responses, including serum antibodies and T cell responses, were measured. Mice were also subjected to lethal challenges with the DENV2 NGC strain. The results showed that maximal protective immunity (50%) was achieved in mice vaccinated with NS1 in combination with LT(G33D). Analyses of the NS1-specific immune responses showed that the anti-virus protection correlated mainly with the serum anti-NS1 antibody responses including higher avidity to the target antigen. Mice immunized with LT(G33D) elicited a prevailing IgG2a subclass response and generated antibodies with stronger affinity to the antigen than those generated in mice immunized with the other vaccine formulations. The vaccine formulations were also evaluated regarding induction of deleterious side effects and, in contrast to mice immunized with the FA-adjuvanted vaccine, no significant hepatic damage or enhanced C-reactive protein levels were detected in mice immunized with NS1 and LT(G33D.) Similarly, no detectable alterations in bleeding time and hematological parameters were detected in mice vaccinated with NS1 and LT(G33D). Altogether, these results indicate that the combination of a purified recombinant NS1 and a nontoxic LT derivative is a promising alternative for the generation of safe and effective protein-based anti-dengue vaccine.

Diarrheagenic Escherichia coli

Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

Functional Diversity of Heat-labile Toxins (LT) Produced by Enterotoxigenic Escherichia coli DIFFERENTIAL ENZYMATIC AND IMMUNOLOGICAL ACTIVITIES OF LT1 (hLT) AND LT4 (pLT)

Heat-labile toxins (LTs) have ADP-ribosylation activity and induce the secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains in different mammalian hosts. LTs also act as adjuvants following delivery via mucosal, parenteral, or transcutaneous routes. Previously we have shown that LT produced by human-derived ETEC strains encompass a group of 16 polymorphic variants, including the reference toxin (LT1 or hLT) produced by the H10407 strain and one variant that is found mainly among bacterial strains isolated from pigs (LT4 or pLT). Herein, we show that LT4 (with six polymorphic sites in the A (K4R, K213E, and N238D) and B (S4T, A46E, and E102K) subunits) displays differential in vitro toxicity and in vivo adjuvant activities compared with LT1. One in vitro generated LT mutant (LTK4R), in which the lysine at position 4 of the A subunit was replaced by arginine, showed most of the LT4 features with an ∼10-fold reduction of the cytotonic effects, ADP-ribosylation activity, and accumulation of intracellular cAMP in Y1 cells. Molecular dynamic studies of the A subunit showed that the K4R replacement reduces the N-terminal region flexibility and decreases the catalytic site crevice. Noticeably, LT4 showed a stronger Th1-biased adjuvant activity with regard to LT1, particularly concerning activation of cytotoxic CD8+ T lymphocytes when delivered via the intranasal route. Our results further emphasize the relevance of LT polymorphism among human-derived ETEC strains that may impact both the pathogenicity of the bacterial strain and the use of these toxins as potential vaccine adjuvants.

Distinctive Immunomodulatory and Inflammatory Properties of the Escherichia coli Type II Heat-Labile Enterotoxin LT-IIa and Its B Pentamer following Intradermal Administration

ABSTRACT The type I and type II heat-labile enterotoxins (LT-I and LT-II) are strong mucosal adjuvants when they are coadministered with soluble antigens. Nonetheless, data on the parenteral adjuvant activities of LT-II are still limited. Particularly, no previous study has evaluated the adjuvant effects and induced inflammatory reactions of LT-II holotoxins or their B pentameric subunits after delivery via the intradermal (i.d.) route to mice. In the present report, the adjuvant and local skin inflammatory effects of LT-IIa and its B subunit pentamer (LT-IIaB5) were determined. When coadministered with ovalbumin (OVA), LT-IIa and, to a lesser extent, LT-IIaB5 exhibited serum IgG adjuvant effects. In addition, LT-IIa but not LT-IIaB5 induced T cell-specific anti-OVA responses, particularly in respect to induction of antigen-specific cytotoxic CD8+ T cell responses. LT-IIa and LT-IIaB5 induced differential tissue permeability and local inflammatory reactions after i.d. injection. Of particular interest was the reduced or complete lack of local reactions, such as edema and tissue induration, in mice i.d. inoculated with LT-IIa and LT-IIaB5, respectively, compared with mice immunized with LT-I. In conclusion, the present results show that LT-IIa and, to a lesser extent, LT-IIaB5 exert adjuvant effects when they are delivered via the i.d. route. In addition, the low inflammatory effects of LT-IIa and LT-IIaB5 in comparison to those of LT-I support the usefulness of LT-IIa and LT-IIaB5 as parenterally delivered vaccine adjuvants.

Immunogenicity and In Vitro and In Vivo Protective Effects of Antibodies Targeting a Recombinant Form of the Streptococcus mutans P1 Surface Protein

ABSTRACT Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P139–512), produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro. In the present study, we further investigated the immunological features of P139–512 in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P139–512 preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P139–512-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P139–512 antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P139–512 as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1.

Intradermal Administration of the Type II Heat-Labile Enterotoxins LT-IIb and LT-IIc of Enterotoxigenic Escherichia coli Enhances Humoral and CD8+ T Cell Immunity to a Co-Administered Antigen

Vaccinations are extremely effective at combating infectious diseases. Many conserved antigen (Ag) targets, however, are poorly immunogenic. Protein subunit vaccines frequently elicit only humoral immune responses and fail to confer protection against serious intracellular pathogens. These barriers to vaccine development are often overcome by the use of appropriate adjuvants. Heat-labile enterotoxins (HLT) produced by enterotoxigenic strains of Escherichia coli are potent adjuvants when administered by mucosal or systemic routes. The efficacy of the type II HLT, however, has not been well-defined when administered by the intradermal (ID) route. Using a murine ID immunization model, the adjuvant properties of LT-IIb and LT-IIc, two type II HLTs, were compared with those of LT-I, a prototypical type I HLT. While all three HLT adjuvants enhanced Ag-specific humoral responses to similar levels, LT-IIb and LT-IIc, in contrast to LT-I, induced a more vigorous Ag-specific CD8+ T cell response and proffered faster clearance of Listeria monocytogenes in a challenge model. Additionally, LT-IIb and LT-IIc induced distinct differences in the profiles of the Ag-specific CD8+ T cell responses. While LT-IIc stimulated a robust and rapid primary CD8+ T cell response, LT-IIb exhibited slower CD8+ T cell expansion and contraction kinetics with the formation of higher percentages of effector memory cells. In comparison to LT-I and LT-IIc, LT-IIb evoked better long-term protection after immunization. Furthermore, LT-IIb and LT-IIc enhanced the total number of dendritic cells (DC) in the draining lymph node (DLN) and expression of costimulatory molecules CD80, CD86, and CD40 on DCs. In contrast to LT-I, LT-IIb and LT-IIc induced less edema, cellular infiltrates, and general inflammation at the site of ID injection. Thus, LT-IIb and LT-IIc are attractive comprehensive ID adjuvants with unique characteristic that enhance humoral and cellular immunity to a co-administered protein Ag.

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.

Evaluation of experimental conditions for quantification of LT produced by human derived enterotoxigenic Escherichia coli strains

The heat-labile toxin (LT) is a key virulence-associated factor associated with the non-invasive secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains either in humans or domestic animals. Several LT detection methods have been reported but quantification of the toxin produced by wild-type ETEC strains is usually performed by the GM1 ganglyoside enzyme-linked immunosorbent assay (GM1 ELISA). In this study we conducted the optimization of an alternative LT-quantification method, the antibody-capture ELISA (cELISA). Detailed analysis of the appropriate dilutions of capture and detecting LT-specific antibodies significantly improved the sensitivity of the method. Additionally, testing of different LT extraction techniques indicated that sonic disruption of the bacterial cells enhanced LT recovery yields, in contrast to the usual procedure based on addition of polymyxin B to the culture medium as well as extraction methods based on chloroform or Triton X-100. Moreover, the present data indicate that performance of the LT extraction method based on polymyxin B treatment can vary among wild ETEC strains.

Adjuvant-Mediated Epitope Specificity and Enhanced Neutralizing Activity of Antibodies Targeting Dengue Virus Envelope Protein

The heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB) in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV) envelope glycoprotein domain III (EDIII), which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.

Impact of Toxin-Specific Antibodies on the Adjuvanticity and Inflammatory Effects Induced by Parenterally Administered Escherichia coli heat-Labile Toxin

Introduction: Heat-labile toxins (LT), produced by enterotoxigenic Escherichia coli (ETEC) strains, exert potent adjuvant effects when admixed or linked to antigens delivered via mucosal, transcutaneous or parenteral routes. There is limited information regarding the impact of preexisting immunity on the immunomodulatory properties of LT, which is frequently observed among people infected with ETEC. Aims: In the present study, we evaluated the effect of anti-LT antibodies on the adjuvant and inflammatory activities triggered by LT admixed with a specific vaccine antigen following subcutaneous administration to mice. Material and Methods/Results: Animals were immunized with dengue virus nonstructural protein (NS1), as model antigen, in combination with native LT in the presence of LT-specific antibodies. Exposure to anti-LT antibodies did not impair the humoral adjuvanticity of LT regarding to the magnitude of the serum anti-NS1 IgG titers. In addition, anti-toxin antibodies did not reduce neutrophil migration nor edema formation after s.c. administration of LT. Nonetheless, administration of LT admixed with anti-LT antibodies changed the local cytokine production profile and modulated the NS1specific T cell responses to a Th1-type pattern. Conclusion: These results indicate that preexisting immunity does not affect the humoral adjuvant activities but may modulate different aspects of both innate and adaptive immune responses induced by parenterally administered LT.