Early pneumococcal clearance in mice induced by systemic immunization with recombinant BCG PspA-PdT prime and protein boost correlates with cellular and humoral immune response in bronchoalveolar fluids (BALF)

© 2019 The Author(s) An effective immunological response in the lungs during a pneumococcal infection is a key factor to the bacteria clearance and prevention of sepsis. In order to develop broad-range pneumococcal vaccines several pneumococcal proteins and strong adjuvants have been investigated. Previously, we constructed a recombinant BCG (rBCG) strain expressing a fragment of PspA (Pneumococcal surface protein A) fused to PdT (detoxified form of pneumolysin). Immunization of mice with a priming dose of rBCG PspA-PdT followed by a booster dose of rPspA-PdT fused protein induced a high antibody response in the serum and protected mice against lethal challenge. Here, we investigated the humoral and cellular immune response in the Bronchoalveolar lavage fluid (BALF). Immunization of mice with rBCG PspA-PdT / rPspA-PdT induced rapid clearance of bacteria after challenge, an early control of the cellular influx and reduced inflammatory cytokine levels in the BALF. In addition, rBCG PspA-PdT / rPspA-PdT induced higher lymphocyte recruitment to the lungs at 48 h, showing an increased percentage of CD4+ T cells. Furthermore, BALF samples from mice immunized with rBCG PspA-PdT / PspA-PdT showed high binding of IgG2c and enhanced complement deposition on the pneumococcal surface; antibody binding was specific to PspA as no binding was observed to a PspA-knockout strain. Taken together, our results show that the immunization with rBCG PspA-PdT / rPspA-PdT induces humoral and cellular immune responses in the lungs, promotes an early clearance of pneumococci and protects against the systemic dissemination of pneumococci

A bivalent recombinant mycobacterium bovis BCG expressing the S1 subunit of the pertussis toxin induces a polyfunctional CD4 ⁺ T cell immune response

© 2019 Alex I. Kanno et al. Background. A recombinant BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (rBCG-S1PT), previously constructed by our research group, demonstrated the ability to develop high protection in mouse models of pertussis challenge which correlated with the induction of a Th1 immune response pattern. The Th1 immune response induced by rBCG-S1PT treatment was also confirmed in the murine orthotopic bladder cancer model, in which the intravesical instillation of rBCG-S1PT resulted in an improved antitumor effect. Based on these observations, we hypothesize that the reengineering of the S1PT expression in BCG could increase the efficiency of the protective Th1 immune response in order to develop a new alternative of immunotherapy in bladder cancer treatment. Objectives. To construct rBCG strains expressing S1PT from extrachromosomal (rBCG-S1PT) and integrative vectors (rBCG-Sli), or their combination, generating the bivalent strain (rBCG-S1+S1i), and to evaluate the respective immunogenicity of rBCG strains in mice. Methods. Mycobacterial plasmids were constructed by cloning the s1pt gene under integrative and extrachromosomal vectors and used to transform BCG, individually or in combination. Antigen expression and localization were confirmed by Western blot. Mice were immunized with wild-type BCG or the rBCG strains, and cytokines quantification and flow cytometry analysis were performed in splenocytes culture stimulated with mycobacterial-specific proteins. Findings. S1PT expression was confirmed in all rBCG strains. The extrachromosomal vector directs S1PT to the cell wall-associated fraction, while the integrative vector directs its expression mainly to the intracellular fraction. Higher levels of IFN-γ were observed in the splenocytes culture from the group immunized with rBCG-S1i in comparison to BCG or rBCG-S1PT. rBCG-S1+S1i showed higher levels of CD4 + IFN-γ + and double-positive CD4 + IFN-γ + TNF-α + T cells. Conclusions. rBCG-S1+S1i was able to express the two forms of S1PT and elicited higher induction of polyfunctional CD4 + T cells, indicating enhanced immunogenicity and suggesting its use as immunotherapy for bladder cancer

New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response

A Combination of Recombinant Mycobacterium bovis BCG Strains Expressing Pneumococcal Proteins Induces Cellular and Humoral Immune Responses and Protects against Pneumococcal Colonization and Sepsis.

Pneumococcal diseases remain a substantial cause of mortality in young children in developing countries. The development of potentially serotype-transcending vaccines has been extensively studied; ideally, such a vaccine should include antigens that are able to induce protection against colonization (likely mediated by interleukin-17A [IL-17A]) and invasive disease (likely mediated by antibody). The use of strong adjuvants or alternative delivery systems that are able to improve the immunological response of recombinant proteins has been proposed but poses potential safety and practical concerns in children. We have previously constructed a recombinant Mycobacterium bovis BCG strain expressing a pneumococcal surface protein A (PspA)-PdT fusion protein (rBCG PspA-PdT) that was able to induce an effective immune response and protection against sepsis in a prime-boost strategy. Here, we constructed two new rBCG strains expressing the pneumococcal proteins SP 0148 and SP 2108, which confer IL-17A-dependent protection against pneumococcal colonization in mouse models. Immunization of mice with rBCG 0148 or rBCG 2108 in a prime-boost strategy induced IL-17A and gamma interferon (IFN-γ) production. The combination of these rBCG strains with rBCG PspA-PdT (rBCG Mix), followed by a booster dose of the combined recombinant proteins (rMix) induced an IL-17A response against SP 0148 and SP 2108 and a humoral response characterized by increased levels of IgG2c against PspA and functional antibodies against pneumolysin. Furthermore, immunization with the rBCG Mix prime/rMix booster (rBCG Mix/rMix) provides protection against pneumococcal colonization and sepsis. These results suggest the use of combined rBCG strains as a potentially serotype-transcending pneumococcal vaccine in a prime-boost strategy, which could provide protection against pneumococcal colonization and sepsis

A protein chimera including PspA in fusion with PotD is protective against invasive pneumococcal infection and reduces nasopharyngeal colonization in mice

© 2017 Elsevier Ltd Despite the success of the available polysaccharide-based vaccines against Streptococcus pneumoniae in preventing invasive diseases, this bacterium remains a major cause of death in many parts of the world. New vaccine strategies are needed in order to increase protection. Thus, the utilization of fusion proteins is being investigated as an alternative to the current formulations. In the present work, we demonstrate that a chimeric protein, composed of PspA and PotD in fusion is able to maintain the protective characteristics of both parental proteins, providing protection against systemic infection while reducing nasal colonization. The hybrid was not able to improve the response against invasive disease elicited by PspA alone, but the inclusion of PotD was able to reduce colonization, an effect never observed using subcutaneous immunization with PspA. The mechanisms underlying the protective efficacy of the rPspA-PotD hybrid protein were investigated, revealing the production of antibodies with an increased binding capacity to pneumococcal strains of diverse serotypes and genetic backgrounds, enhanced opsonophagocytosis, and secretion of IL-17 by splenocytes. These findings reinforce the use of chimeric proteins based on surface antigens as an effective strategy against pneumococcal infections

DIFFERENT LETHAL EFFECTS BY ENZYME-GENERATED TRIPLET INDOLE-3-ALDEHYDE IN DIFFERENT ESCHERICHIA-COLI STRAINS

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PEROXIDASE-HYDROGEN PEROXIDE SYSTEM ACTING ON LIGNIN .1.

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Systemic immunization with rPotD reduces Streptococcus pneumoniae nasopharyngeal colonization in mice

© 2016 Elsevier Ltd Streptococcus pneumoniae (pneumococcus) is a human pathogen that can cause otitis media, pneumonia and, in severe cases, meningitis and bacteremia. The pneumococcus expresses PotD, a protein belonging to the polyamines transporter complex called PotABCD. PotD is a membrane-associated protein that binds polyamines and has been shown to be important for virulence. In this work we demonstrate that subcutaneous immunization with rPotD reduces the bacterial load in the nasal tissue of mice, following intranasal challenge with a type 6B pneumococcus. The protective effect correlated with the induction of high levels of antibodies in the immunized group; the antibodies were able to increase bacterial phagocytosis by mouse peritoneal cells. The cellular immune response was characterized by the production of gamma-interferon, IL-2 and IL-17 by splenocytes and nitric oxide by peritoneal cells of immunized mice, upon stimulation with rPotD. Taken together our results suggest that PotD is a promising candidate to be included in a protein based pneumococcal vaccine, able to induce phagocytic antibodies, a Th1 cellular immune response and production of IL-17, reducing nasopharyngeal colonization, the main event responsible for transmission of pneumococci in humans