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

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

Rational selection of broadly cross-reactive family 2 PspA molecules for inclusion in chimeric pneumococcal vaccines

© 2017 Elsevier Ltd 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

Immunodominance in T cell responses elicited against different domains of detoxified pneumolysin PlyD1

<div><p>Detoxified pneumolysin, PlyD1, is a protein vaccine candidate that induces protection against infections with <i>Streptococcus pneumoniae</i> in mouse models. Despite extensive knowledge on antibody responses against PlyD1, limited information is available about PlyD1 induced T cell recognition. Here we interrogated epitope breadth and functional characteristics of the T cell response to PlyD1 in two mouse strains. BALB/c (H-2^d) and C57BL/6 (H-2^b) mice were vaccinated with Al(OH)₃-adjuvanted or non-adjuvanted PlyD1, or placebo, on day 0, 21 and 42 and were sacrificed at day 56 for collection of sera and spleens. Vaccination with adjuvanted and non-adjuvanted PlyD1 induced anti-pneumolysin IgG antibodies with neutralizing capacity in both mouse strains. Adjuvantation of PlyD1 enhanced the serological responses in both strains. <i>In vitro</i> restimulation of splenocytes with PlyD1 and 18-mer synthetic peptides derived from pneumolysin revealed specific proliferative and cytokine responses. For both mouse strains, one immunodominant and three subdominant natural epitopes were identified. Overlap between H-2^d and H-2^b restricted T cell epitopes was limited, yet similarities were found between epitopes processed in mice and predicted to be immunogenic in humans. H-2^d restricted T cell epitopes were localized in pneumolysin domains 2 and 3, whereas H-2^b epitopes were scattered over the protein. Cytokine responses show mostly a Th2 profile, with low levels of Th1 cytokines, in both mouse strains. In conclusion, PlyD1 evokes T cell responses in mice directed against multiple epitope regions, that is dependent on Major Histocompatibility Complex (MHC) background. These results are important to understand human PlyD1 T cell immunogenicity, to guide cell mediated immunity studies in the context of vaccine development.</p></div

Measuring and interpreting the selectivity of protein kinase inhibitors

Protein kinase inhibitors are a well-established class of clinically useful drugs, particularly for the treatment of cancer. Achieving inhibitor selectivity for particular protein kinases often remains a significant challenge in the development of new small molecules as drugs or as tools for chemical biology research. This review summarises the methodologies available for measuring kinase inhibitor selectivity, both in vitro and in cells. The interpretation of kinase inhibitor selectivity data is discussed, particularly with reference to the structural biology of the protein targets. Measurement and prediction of kinase inhibitor selectivity will be important for the development of new multi-targeted kinase inhibitors