Immune responses and protection against Streptococcus pneumoniae elicited by recombinant Bordetella pertussis adenylate cyclase (CyaA) carrying fragments of pneumococcal surface protein A, PspA.

Streptococcus pneumoniae is a common agent of important human diseases such as otitis media, pneumonia, meningitis and sepsis. Current available vaccines that target capsular polysaccharides induce protection against invasive disease and nasopharyngeal colonization in children, yet their efficacy is limited to the serotypes included in the formulations. The virulence factor Pneumococcal Surface Protein A (PspA) interacts with host immune system and helps the bacteria to evade phagocytosis. Due to its essential role in virulence, PspA is an important vaccine candidate. Here we have tested a delivery system based on the adenylate cyclase toxin of Bordetella pertussis (CyaA) to induce immune responses against PspA in mice. CyaA was engineered to express fragments of the N-terminal region of PspAs from clades 2 and 4 (A2 and A4) and the resulting proteins were used in immunization experiments in mice. The recombinant CyaA-A2 and CyaA-A4 proteins were able to induce high levels of anti-PspA antibodies that reacted with pneumococcal strains expressing either PspA2 or PspA4. Moreover, reactivity of the antibodies against pneumococcal strains that express PspAs from clades 3 and 5 (PspA3 and PspA5) was also observed. A formulation containing CyaA-A2 and CyaA-A4 was able to protect mice against invasive pneumococcal challenges with isolates that express PspA2, PspA4 or PspA5. Moreover, a CyaA-A2-A4 fusion protein induced antibodies at similar levels and with similar reactivity as the formulation containing both proteins, and protected mice against the invasive challenge. Our results indicate that CyaA-PspA proteins are good candidates to induce broad protection against pneumococcal isolates.

Systems analysis of subjects acutely infected with the Chikungunya virus.

The largest ever recorded epidemic of the Chikungunya virus (CHIKV) broke out in 2004 and affected four continents. Acute symptomatic infections are typically associated with the onset of fever and often debilitating polyarthralgia/polyarthritis. In this study, a systems biology approach was adopted to analyze the blood transcriptomes of adults acutely infected with the CHIKV. Gene signatures that were associated with viral RNA levels and the onset of symptoms were identified. Among these genes, the putative role of the Eukaryotic Initiation Factor (eIF) family genes and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC3A) in the CHIKV replication process were displayed. We further compared these signatures with signatures induced by the Dengue virus infection and rheumatoid arthritis. Finally, we demonstrated that the CHIKV in vitro infection of murine bone marrow-derived macrophages induced IL-1 beta production in a mechanism that is significantly dependent on the inflammasome NLRP3 activation. The observations provided valuable insights into virus-host interactions during the acute phase and can be instrumental in the investigation of new and effective therapeutic interventions.

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.

Nasal immunization of mice with Lactobacillus casei expressing the pneumococcal surface protein C primes the immune system and decreases pneumococcal nasopharyngeal colonization in mice.

Streptococcus pneumoniae colonizes the upper respiratory tract of healthy individuals, from where it can be transmitted to the community. Occasionally, bacteria invade sterile niches, causing diseases. The pneumococcal surface protein C (PspC) is a virulence factor that is important during colonization and the systemic phases of the diseases. Here, we have evaluated the effect of nasal or sublingual immunization of mice with Lactobacillus casei expressing PspC, as well as prime-boosting protocols using recombinant PspC, on nasopharyngeal pneumococcal colonization. None of the protocols tested was able to elicit significant levels of anti-PspC antibodies before challenge. However, a significant decrease in pneumococcal recovery from the nasopharynx was observed in animals immunized through the nasal route with L. casei-PspC. Immune responses evaluated after colonization challenge in this group of mice were characterized by an increase in mucosal anti-PspC immunoglobulin A (IgA) 5 days later, a time point in which the pneumococcal loads were already low. A negative correlation between the concentrations of anti-PspC IgA and pneumococcal recovery from the nasopharynx was observed, with animals with the lowest colonization levels having higher IgA concentrations. These results show that nasal immunization with L. casei-PspC primes the immune system of mice, prompting faster immune responses that result in a decrease in pneumococcal colonization.

Generation of polyclonal antibodies against recombinant human glucocerebrosidase produced in Escherichia coli.

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher's disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

Intranasal vaccines for protection against respiratory and systemic bacterial infections.

More than 4 million deaths per year are due to respiratory diseases. Although licensed vaccines are available, bacteria, such as Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Bordetella pertussis and Neisseria meningiditis, among others, continue to be the major agents of diseases in young children, the elderly and/or immunocompromized individuals. Following respiratory tract infection, some microorganisms may also invade the epithelial tissue, achieving systemic circulation and/or other organs. Nasal administration of different antigen formulations has shown promising results in the induction of immune responses and the defeat of the pathogens at the site of infection. This review will focus on the main nasal vaccine strategies and technologies being investigated against the most common infections caused by respiratory bacteria.

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.

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 3 days and produced a similar amount of PsaA protein (150-250 ng per 10(9) 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.

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.

Expression and characterization of cholera toxin B-pneumococcal surface adhesin A fusion protein in Escherichia coli: ability of CTB-PsaA to induce humoral immune response in mice.

Cholera toxin B subunit (CTB) is responsible for CT holotoxin binding to the cell and has been described as a mucosal adjuvant for vaccines. In this work, the ctxB gene was genetically fused to the psaA gene from Streptococcus pneumoniae, a surface protein involved in its colonization in the host that is also considered a vaccine antigen candidate against this pathogen. The CTB-PsaA fusion protein was expressed in Escherichia coli, and the purified protein was used for intranasal immunization experiments in Balb/C mice. CTB-PsaA was able to induce both systemic and mucosal antibodies evaluated in serum, saliva, and in nasal and bronchial wash samples, showing that CTB-PsaA is a promising molecule to be investigated as S. pneumoniae vaccine antigen candidate.

Suppression of AP-1 constitutive activity interferes with polyomavirus MT antigen transformation ability.

Polyomavirus (Py) encodes a potent oncogene, the middle T antigen (MT), that induces cell transformation by binding to and activating several cytoplasmic proteins which take part in transduction of growth factors-induced mitogenic signal to the nucleus. We have previously reported that the AP-1 transcriptional complex is a target for MT during cell transformation although, its activation was not sufficient for establishment of the transformed phenotype. Here we show that expression of a dominant-negative cJun mutant in MT transformed cell lines inhibits its transformation ability, indicating that constitutive AP-1 activity is necessary for cell transformation mediated by MT. Evidences also suggest that proliferation of MT transformed cells in low serum concentrations and their ability to form colonies in agarose are controlled by distinct mechanisms.

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.

Angiostatin-like molecules are generated by snake venom metalloproteinases.

Angiostatin is a plasminogen-derived anti-angiogenic factor composed of its first four kringle structures. This molecule is generated by proteolytic cleavage of plasminogen by some proteolytic enzymes in vitro. Since venoms of viper snakes are a rich source of both serine- and metalloproteinase, we hypothesized that angiostatin-like polypeptides could be generated during the envenomation after snake bites and play a pathophysiological role in the local tissue damage and regeneration. Our results showed that crude venoms from several species of Bothrops snakes were able to generate angiostatin-like polypeptides and purified metalloproteinases but not serine proteinases from Bothrops jararaca and Bothrops moojeni venoms were responsible for their generation in vitro. The putative plasminogen cleavage sites by the crude venoms and purified proteinases were determined by N-terminal amino acid sequencing of the angiostatin-like molecules. Angiostatin-like peptides derived from human plasminogen digestion by jararhagin, a metalloproteinase isolated from B. jararaca venom, inhibited endothelial cell proliferation in vitro. These results indicate that angiostatin-like molecules can be generated upon snakebite envenomations and may account for the poor and incomplete regenerative response observed in the damaged tissue.