Goulart, C, Rodriguez, D, Kanno, AI, Silva, JLSC & Leite, LCC 2020, '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)', Vaccine: X, vol. 4.View/Download from: Publisher's site
© 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.
Kanno, AI, Goulart, C, Leite, LCC, Pagliarone, AC & Nascimento, IP 2019, 'A Bivalent Recombinant Mycobacterium bovis BCG Expressing the S1 Subunit of the Pertussis Toxin Induces a Polyfunctional CD4+ T Cell Immune Response.', BioMed research international, vol. 2019.View/Download from: Publisher's site
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+S1...
Rodriguez, D, Goulart, C, Pagliarone, AC, Silva, EP, Cunegundes, PS, Nascimento, IP, Borra, RC, Dias, WO, Tagliabue, A, Boraschi, D & Leite, LCC 2019, 'In vitro Evidence of Human Immune Responsiveness Shows the Improved Potential of a Recombinant BCG Strain for Bladder Cancer Treatment', Frontiers in Immunology, vol. 10.View/Download from: Publisher's site
Zhang, F, Ledue, O, Jun, M, Goulart, C, Malley, R & Lu, Y-J 2018, 'Protection against Staphylococcus aureus Colonization and Infection by B- and T-Cell-Mediated Mechanisms.', mBio, vol. 9, no. 5.View/Download from: Publisher's site
Staphylococcus aureus is a major cause of morbidity and mortality worldwide. S. aureus colonizes 20 to 80% of humans at any one time and causes a variety of illnesses. Strains that are resistant to common antibiotics further complicate management. S. aureus vaccine development has been unsuccessful so far, largely due to the incomplete understanding of the mechanisms of protection against this pathogen. Here, we studied the role of different aspects of adaptive immunity induced by an S. aureus vaccine in protection against S. aureus bacteremia, dermonecrosis, skin abscess, and gastrointestinal (GI) colonization. We show that, depending on the challenge model, the contributions of vaccine-induced S. aureus-specific antibody and Th1 and Th17 responses to protection are different: antibodies play a major role in reducing mortality during S. aureus bacteremia, whereas Th1 or Th17 responses are essential for prevention of S. aureus skin abscesses and the clearance of bacteria from the GI tract. Both antibody- and T-cell-mediated mechanisms contribute to prevention of S. aureus dermonecrosis. Engagement of all three immune pathways results in the most robust protection under each pathological condition. Therefore, our results suggest that eliciting multipronged humoral and cellular responses to S. aureus antigens may be critical to achieve effective and comprehensive immune defense against this pathogen.IMPORTANCE S. aureus is a leading cause of healthcare- and community-associated bacterial infections. S. aureus causes various illnesses, including bacteremia, meningitis, endocarditis, pneumonia, osteomyelitis, sepsis, and skin and soft tissue infections. S. aureus colonizes between 20 and 80% of humans; carriers are at increased risk for infection and transmission to others. The spread of multidrug-resistant strains limits antibiotic treatment options. Vaccine development against S. aureus has been unsuccessful to date, likely due to an inadequate understanding about ...
Converso, TR, Goulart, C, Darrieux, M & Leite, LCC 2017, 'A protein chimera including PspA in fusion with PotD is protective against invasive pneumococcal infection and reduces nasopharyngeal colonization in mice', VACCINE, vol. 35, no. 38, pp. 5140-5147.View/Download from: Publisher's site
Converso, TR, Goulart, C, Rodriguez, D, Darrieux, M & Leite, LCC 2017, 'Rational selection of broadly cross-reactive family 2 PspA molecules for inclusion in chimeric pneumococcal vaccines.', Microbial Pathogenesis, vol. 109, pp. 233-238.View/Download from: Publisher's site
Pneumococcal surface protein A (PspA) is a widely studied pneumococcal protein, exposed at the surface of all strains. It is an important virulence factor, preventing complement deposition as well as inhibiting the lytic effects of lactoferrin over pneumococci. Several studies have investigated the use of PspA as a candidate in alternative pneumococcal vaccines, with great success. However, PspA presents sequence variability - there are six clades, grouped in three families - and PspAs within the same clade exhibit different levels of cross-reactivity. Therefore, the aim of this work was to select, from a panel of eight pneumococcal isolates expressing family 2 PspAs, the molecule with the broadest reactivity within this family. Antisera to these PspA fragments were initially screened by immunoblot against thirteen pneumococcal extracts; the three most cross-reactive antisera were tested for their ability to enhance the deposition of complement factor C3b on the bacterial surface and to promote their phagocytosis in vitro. PspA from strain P490 was the most effective, increasing phagocytosis of all but one pneumococcal isolate. Thus, this molecule was selected for inclusion in chimeric protein-based pneumococcal vaccines. In conclusion, the rational selection of cross-reactive molecules is an important step in the development of vaccines with broad coverage.
Converso, TR, Goulart, C, Rodriguez, D, Darrieux, M & Leite, LCC 2017, 'Systemic immunization with rPotD reduces Streptococcus pneumoniae nasopharyngeal colonization in mice.', Vaccine, vol. 35, no. 1, pp. 149-155.View/Download from: Publisher's site
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.
Goulart, C, Rodriguez, D, Kanno, AI, Converso, TR, Lu, Y-J, Malley, R & Leite, LCC 2017, 'A Combination of Recombinant Mycobacterium bovis BCG Strains Expressing Pneumococcal Proteins Induces Cellular and Humoral Immune Responses and Protects against Pneumococcal Colonization and Sepsis', CLINICAL AND VACCINE IMMUNOLOGY, vol. 24, no. 10.View/Download from: Publisher's site
Goulart, C, Rodriguez, D, Kanno, AI, Lu, Y-J, Malley, R & Leite, LCC 2017, 'Recombinant BCG expressing a PspA-PdT fusion protein protects mice against pneumococcal lethal challenge in a prime-boost strategy.', Vaccine, vol. 35, no. 13, pp. 1683-1691.View/Download from: Publisher's site
Pneumococcal proteins have been evaluated as genetically-conserved potential vaccine candidates. We have previously demonstrated that a fragment of PspA in fusion with PdT (rPspA-PdT) induced protective immune responses in mice. However, purified proteins have shown poor immunogenicity and often require the combination with strong adjuvants and booster doses. Here, we investigated the use of a Bacillus Calmette-Guérin (BCG) strain, a well-established prophylactic vaccine for tuberculosis with known adjuvant properties, for delivery of the PspA-PdT fusion protein. Immunization of mice in a prime-boost strategy, using rPspA-PdT as a boost, demonstrated that rBCG PspA-PdT/rPspA-PdT was able to induce an antibody response against both proteins, promoting an IgG1 to IgG2 antibody isotype shift. Sera from rBCG PspA-PdT/rPspA-PdT immunized mice showed antibodies able to bind to the pneumococcal surface and promoted higher complement deposition when compared with WT-BCG/rPspA-PdT or a single dose of rPspA-PdT. In addition, these antisera inhibited the cytolytic activity of Ply. Production of interleukin-6 (IL-6), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) was increased in splenocytes culture. Furthermore, a higher expression of CD69 early activation molecule was observed on splenic CD4+ T cells from mice immunized with rBCG PspA-PdT before and after the protein booster dose. Finally, immunization with rBCG PspA-PdT/rPspA-PdT protected mice against pneumococcal lethal challenge. These results support the further investigation of recombinant BCG strains to express pneumococcal proteins, which could be administered in early stages of life and lead to protective pneumococcal immunity in infants and children.
Kanno, AI, Goulart, C, Rofatto, HK, Oliveira, SC, Leite, LCC & McFadden, J 2016, 'New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters', APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 82, no. 8, pp. 2240-2246.View/Download from: Publisher's site
Darrieux, M, Goulart, C, Briles, D & de Cerqueira Leite, LC 2015, 'Current status and perspectives on protein-based pneumococcal vaccines', CRITICAL REVIEWS IN MICROBIOLOGY, vol. 41, no. 2, pp. 190-200.View/Download from: Publisher's site
De Amicis, KM, de Barros, SF, Alencar, RE, Postol, E, Martins, CDO, Arcuri, HA, Goulart, C, Kalil, J & Guilherme, L 2014, 'Analysis of the coverage capacity of the StreptInCor candidate vaccine against Streptococcus pyogenes', VACCINE, vol. 32, no. 32, pp. 4104-4110.View/Download from: Publisher's site
Goulart, C, da Silva, TR, Rodriguez, D, Politano, WR, Leite, LCC & Darrieux, M 2013, 'Characterization of Protective Immune Responses Induced by Pneumococcal Surface Protein A in Fusion with Pneumolysin Derivatives', PLOS ONE, vol. 8, no. 3.View/Download from: Publisher's site
Perciani, CT, Barazzone, GC, Goulart, C, Carvalho, E, Cabrera-Crespo, J, Goncalves, VM, Leite, LCC & Tanizaki, MM 2013, 'Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response', CLINICAL AND VACCINE IMMUNOLOGY, vol. 20, no. 6, pp. 858-866.View/Download from: Publisher's site
Goulart, C, Darrieux, M, Rodriguez, D, Pimenta, FC, Brandileone, MCC, de Andrade, ALSS & Leite, LCC 2011, 'Selection of family 1 PspA molecules capable of inducing broad-ranging cross-reactivity by complement deposition and opsonophagocytosis by murine peritoneal cells', VACCINE, vol. 29, no. 8, pp. 1634-1642.View/Download from: Publisher's site
Santamaria, R, Goulart, C, Perciani, CT, Barazzone, GC, Carvalhoa, R, Goncalves, VM, Leite, LCC & Tanizaki, MM 2011, 'Humoral immune response of a pneumococcal conjugate vaccine: Capsular polysaccharide serotype 14-Lysine modified PspA', VACCINE, vol. 29, no. 47, pp. 8689-8695.View/Download from: Publisher's site
Rajendran, E, Clark, M, Goulart, C, Steinhöfel, B, Tjhin, ET, Smith, NC, Kirk, K & van Dooren, GG, 'Substrate-mediated regulation of the arginine transporter ofToxoplasma gondii'.View/Download from: Publisher's site
ABSTRACTIntracellular parasites, such as the apicomplexanToxoplasma gondii, are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection.TgApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) inT. gondii. Here, we show that the abundance ofTgApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite's external environment, increasing in response to decreased [Arg]. Using a luciferase-based 'biosensor' strain ofT. gondii, we demonstrate that parasites vary the expression ofTgApiAT1 in different organs within their host, indicating that parasites are able to modulateTgApiAT1-dependent uptake of Arg as they encounter different nutrient environmentsin vivo. Finally, we show that Arg-dependent regulation ofTgApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in theTgApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient.
Barazzone, GC, Carvalho, R, Kraschowetz, S, Horta, ACL, Sargo, CR, Silva, AJ, Zangirolami, TC, Goulart, C, Leite, LCC, Tanizaki, MM, Goncalves, VM & Cabrera-Crespo, J 2010, 'Production and purification of recombinant fragment of pneumococcal surface protein A (PspA) in Escherichia coli', 4TH VACCINE AND ISV ANNUAL GLOBAL CONGRESS, 4th Annual Global Congress on Vaccine and International-Society-for-Vaccines (ISV), ELSEVIER SCIENCE BV, Vienna, AUSTRIA.View/Download from: Publisher's site