Maria Leonor S. Oliveira

Characterization of Protective Mucosal and Systemic Immune Responses Elicited by Pneumococcal Surface Protein PspA and PspC Nasal Vaccines against a Respiratory Pneumococcal Challenge in Mice

ABSTRACT Pneumococcal surface protein A (PspA) and PspC are virulence factors that are involved in the adhesion of Streptococcus pneumoniae to epithelial cells and/or evasion from the immune system. Here, the immune responses induced by mucosal vaccines composed of both antigens as recombinant proteins or delivered by Lactobacillus casei were evaluated. None of the PspC vaccines protected mice against an invasive challenge with pneumococcal strain ATCC 6303. On the other hand, protection was observed for immunization with vaccines composed of PspA from clade 5 (PspA5 or L. casei expressing PspA5) through the intranasal route. The protective response was distinguished by a Th1 profile with high levels of immunoglobulin G2a production, efficient complement deposition, release of proinflammatory cytokines, and infiltration of neutrophils. Intranasal immunization with PspA5 elicited the highest level of protection, characterized by increased levels of secretion of interleukin-17 and gamma interferon by lung and spleen cells, respectively, and low levels of tumor necrosis factor alpha in the respiratory tract.

Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A.

Abstract Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus helveticus. After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3days and produced a similar amount of PsaA protein (150–250ng per 109 CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments.

Production of Human Papillomavirus Type 16 L1 Virus-Like Particles by Recombinant Lactobacillus casei Cells

ABSTRACT Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.

Immunization of Mice with Single PspA Fragments Induces Antibodies Capable of Mediating Complement Deposition on Different Pneumococcal Strains and Cross-Protection

ABSTRACT PspA is an important candidate for a vaccine with serotype-independent immunity against pneumococcal infections. Based on sequence relatedness, PspA has been classified into three families comprising six clades. We have previously addressed the cross-reactivity of antibodies against PspA fragments containing the N-terminal and proline-rich regions of PspA from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis and reported that anti-PspA4 and anti-PspA5 were able to recognize pneumococci expressing PspA proteins from all of the clades analyzed. We have now analyzed the functional capacity of these antibodies to bind and to mediate complement deposition on intact bacteria in vitro. Our results show that both PspA4 and PspA5 elicit antibodies that are able to bind and to mediate complement deposition efficiently on pneumococcal strains bearing PspA proteins from clades 1 to 5. Moreover, mice immunized with PspA4 and PspA5 were protected against an intranasal lethal challenge with strains expressing PspA proteins from the two major families. PspA4 and PspA5 are thus able to induce antibodies with a high degree of cross-reactivity in vitro, which is reflected in cross-protection of mice. We have also analyzed the contribution of the nonproline (NonPro) block within the conserved proline-rich region to the reactivity of anti-PspA antibodies, and the results indicate that N-terminal α-helical region, the blocks of proline repeats, and the NonPro region can influence the degree of cross-reactivity of antibodies to PspA.

Serotype-independent pneumococcal vaccines

Streptococcus pneumoniae remains an important cause of disease with high mortality and morbidity, especially in children and in the elderly. The widespread use of the polysaccharide conjugate vaccines in some countries has led to a significant decrease in invasive disease caused by vaccine serotypes, but an increase in disease caused by non-vaccine serotypes has impacted on the overall efficacy of these vaccines on pneumococcal disease. The obvious solution to overcome such shortcomings would be the development of new formulations that provide serotype-independent immunity. This review focuses on the most promising approaches, including protein antigens, whole cell pneumococcal vaccines, and recombinant bacteria expressing pneumococcal antigens. The protective capacity of these vaccine candidates against the different stages of pneumococcal infection, including colonization, mucosal disease, and invasive disease in animal models is reviewed. Some of the human trials that have already been performed or that are currently ongoing are presented. Finally, the feasibility and the possible shortcomings of these candidates in relation to an ideal vaccine against pneumococcal infections are discussed.

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface‐exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.

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.

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade 1 PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed.

Intranasal Immunization with the Cholera Toxin B Subunit-Pneumococcal Surface Antigen A Fusion Protein Induces Protection against Colonization with Streptococcus pneumoniae and Has Negligible Impact on the Nasopharyngeal and Oral Microbiota of Mice

ABSTRACT One of the candidate proteins for a mucosal vaccine antigen against Streptococcus pneumoniae is PsaA (pneumococcal surface antigen A). Vaccines targeting mucosal immunity may raise concerns as to possible alterations in the normal microbiota, especially in the case of PsaA, which was shown to have homologs with elevated sequence identity in other viridans group streptococci. In this work, we demonstrate that intranasal immunization with a cholera toxin B subunit-PsaA fusion protein is able to protect mice against colonization with S. pneumoniae but does not significantly alter the natural oral or nasopharyngeal microbiota of mice.

Lactic acid bacteria in the prevention of pneumococcal respiratory infection: Future opportunities and challenges

Lactic acid bacteria (LAB) are technologically and commercially important and have various beneficial effects on human health. Several studies have demonstrated that certain LAB strains can exert their beneficial effect on the host through their immunomudulatory activity. Although most research concerning LAB-mediated enhanced immune protection is focused on gastrointestinal tract pathogens, recent studies have centered on whether these immunobiotics might sufficiently stimulate the common mucosal immune system to provide protection to other mucosal sites as well. In this sense, LAB have been used for the development of probiotic foods with the ability to stimulate respiratory immunity, which would increase resistance to infections, even in immunocompromised hosts. On the other hand, the advances in the molecular biology of LAB have enabled the development of recombinant strains expressing antigens from respiratory pathogens that have proved effective to induce protective immunity. In this review we examine the current scientific literature concerning the use of LAB strains to prevent respiratory infections. In particular, we have focused on the works that deal with the capacity of probiotic and recombinant LAB to improve the immune response against Streptococcus pneumoniae. Research from the last decade demonstrates that LAB represent a promising resource for the development of prevention strategies against respiratory infections that could be effective tools for medical application.