RESUMEN
Streptococcus pneumoniae is a pathogen responsible for high morbidity and mortality worldwide. The polysaccharide capsule confers protection against phagocytosis and influences many aspects of pneumococcal pathogenesis. The capsular polysaccharides (CPS) are highly immunogenic and exhibit great structural variability, with more than 100 serotypes described so far. Antimicrobial peptides (AMPs) are an important part of the innate defense mechanisms against many pathogens. Indolicidin is a cationic AMP produced by bovine neutrophils, with bactericidal effects against several bacteria. CPS has been shown to interfere with the ability of AMPs to kill pneumococci, but the effects of capsule variability on susceptibility to indolicidin have not been explored. The present work determined the effects of capsule on resistance to indolicidin in vitro. Using a bactericidal plate assay, we observed that different pneumococcal serotypes exhibited variable resistance to indolicidin, which correlated with the capsule net charge. Interestingly, the effect of capsule expression on resistance to indolicidin was dependent on the serotype; bacteria with lower zeta potential were more resistant to indolicidin when capsule was present, while those with less negative surface charge were more resistant in the absence of capsule. The addition of purified CPS partially rescued the bacteria from the bactericidal effects of indolicidin, while the addition of anticapsular antibodies accentuated the peptide’s bactericidal action, suggesting a possible new protective mechanism induced by polysaccharide-based pneumococcal vaccines.
RESUMEN
Human vaccination against leptospirosis has been relatively unsuccessful in clinical applications despite an expressive amount of vaccine candidates has been tested over years of research. Pathogenic Leptospira encompass a great number of serovars, most of which do not cross-react, and there has been a lack of genetic tools for many years. These obstacles have hampered the understanding of the bacteria’s biology and, consequently, the identification of an effective antigen. Thus far, many approaches have been used in an attempt to find a cost-effective and broad-spectrum protective antigen(s) against the disease. In this extensive review, we discuss several strategies that have been used to develop an effective vaccine against leptospirosis, starting with Leptospira-inactivated bacterin, proteins identified in the genome sequences of pathogenic Leptospira, including reverse vaccinology, plasmid DNA, live vaccines, chimeric multi-epitope, and toll- and nod-like receptors agonists. This overview should be able to guide scientists working in the field to select potential antigens and to choose the appropriate formulation to administer the candidates.
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Biopharmaceutical production is currently a multibillion-dollar industry with high growth perspectives. The research and development of biologically sourced pharmaceuticals are extremely important and a reality in our current healthcare system. Interferon alpha consensus (cIFN) is a non-natural synthetic antiviral molecule that comprises all the most prevalent amino acids of IFN-α into one consensus protein sequence. For clinical use, cIFN is produced in E. coli in the form of inclusion bodies. Here, we describe the use of two solubility tags (Fh8 and DsbC) to improve soluble cIFN production. Furthermore, we analyzed cIFN production in different culture media and temperatures in order to improve biopharmaceutical production. Our results demonstrate that Fh8-cIFN yield was improved when bacteria were cultivated in autoinduction culture medium at 30 °C. After hydrolysis, the recovery of soluble untagged cIFN was 58% from purified Fh8-cIFN molecule, fourfold higher when compared to cIFN recovered from the DsbC-cIFN, which achieved 14% recovery. The biological activity of cIFN was tested on in vitro model of antiviral effect against Zika, Mayaro, Chikungunya and SARS-CoV-2 virus infection in susceptible VERO cells. We show, for the first time, that cIFN has a potent activity against these viruses, being very low amounts of the molecule sufficient to inhibit virus multiplication. Thus, this molecule could be used in a clinical approach to treat Arboviruses and SARS-CoV-2.
RESUMEN
Purpose: The use of adjuvants can significantly strengthen a vaccine’s efficacy. We sought to explore the immunization efficacy of bacterial outer membrane vesicles (OMVs) displaying the Schistosoma mansoni antigen, SmTSP-2, through a biotin-rhizavidin coupling approach. The rationale is to exploit the nanoparticulate structure and the adjuvant properties of OMVs to induce a robust antigen-specific immune response, in light of developing new vaccines against S. mansoni. Materials and Methods: OMVs were obtained from Neisseria lactamica and conjugated with biotin. The recombinant SmTSP-2 in fusion with the biotin-binding protein rhizavidin (rRzvSmTSP-2) was produced in E. coli and coupled to biotinylated OMVs to generate an OMV complex displaying SmTSP-2 on the membrane surface (OMV:rSmTSP-2). Transmission electron microscopy (TEM) and dynamic light scattering analysis were used to determine particle charge and size. The immunogenicity of the vaccine complex was evaluated in C57BL/6 mice. Results: The rRzvSmTSP-2 protein was successfully coupled to biotinylated OMVs and purified by size-exclusion chromatography. The OMV:rSmTSP-2 nanoparticles showed an average size of 200 nm, with zeta potential around – 28 mV. Mouse Bone Marrow Dendritic Cells were activated by the nanoparticles as determined by increased expression of the co-stimulatory molecules CD40 and CD86, and the proinflammatory cytokines (TNF-α, IL-6 and IL-12) or IL-10. Splenocytes of mice immunized with OMV:rSmTSP-2 nanoparticles reacted to an in vitro challenge with SmTSP-2 with an increased production of IL-6, IL-10 and IL-17 and displayed a higher number of CD4+ and CD8+ T lymphocytes expressing IFN-γ, IL-4 and IL-2, compared to mice immunized with the antigen alone. Immunization of mice with OMV:rSmTSP-2 induced a 100-fold increase in specific anti-SmTSP-2 IgG antibody titers, as compared to the group receiving the recombinant rSmTSP-2 protein alone or even co-administered with unconjugated OMV. Conclusion: Our results demonstrate that the SmTSP-2 antigen coupled with OMVs is highly immunogenic in mice, supporting the potential effectiveness of this platform for improved antigen delivery in novel vaccine strategies.
RESUMEN
Biopharmaceutical production is currently a multibillion-dollar industry with high growth perspectives. The research and development of biologically sourced pharmaceuticals are extremely important and a reality in our current healthcare system. Interferon alpha consensus (cIFN) is a non-natural synthetic antiviral molecule that comprises all the most prevalent amino acids of IFN-α into one consensus protein sequence. For clinical use, cIFN is produced in E. coli in the form of inclusion bodies. Here, we describe the use of two solubility tags (Fh8 and DsbC) to improve soluble cIFN production. Furthermore, we analyzed cIFN production in different culture media and temperatures in order to improve biopharmaceutical production. Our results demonstrate that Fh8-cIFN yield was improved when bacteria were cultivated in autoinduction culture medium at 30 °C. After hydrolysis, the recovery of soluble untagged cIFN was 58% from purified Fh8-cIFN molecule, fourfold higher when compared to cIFN recovered from the DsbC-cIFN, which achieved 14% recovery. The biological activity of cIFN was tested on in vitro model of antiviral effect against Zika, Mayaro, Chikungunya and SARS-CoV-2 virus infection in susceptible VERO cells. We show, for the first time, that cIFN has a potent activity against these viruses, being very low amounts of the molecule sufficient to inhibit virus multiplication. Thus, this molecule could be used in a clinical approach to treat Arboviruses and SARS-CoV-2.
RESUMEN
The granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%-98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity.
RESUMEN
Pathogenic Leptospira spp. is the etiological agent of leptospirosis. The high diversity among Leptospira species provides an array to look for important mediators involved in pathogenesis. Toxin-antitoxin (TA) systems represent an important survival mechanism on stress conditions. vapBC modules have been found in nearly one thousand genomes corresponding to about 40% of known TAs. In the present study, we investigated TA profiles of some strains of Leptospira using a TA database and compared them through protein alignment of VapC toxin sequences among Leptospira spp. genomes. Our analysis identified significant differences in the number of putative vapBC modules distributed in pathogenic, saprophytic, and intermediate strains: four in L. interrogans, three in L. borgpetersenii, eight in L. biflexa, and 15 in L. licerasiae. The VapC toxins show low identity among amino acid sequences within the species. Some VapC toxins appear to be exclusively conserved in unique species, others appear to be conserved among pathogenic or saprophytic strains, and some appear to be distributed randomly. The data shown here indicate that these modules evolved in a very complex manner, which highlights the strong need to identify and characterize new TAs as well as to understand their regulation networks and the possible roles of TA systems in pathogenic bacteria.
RESUMEN
Pathogenic Leptospira spp. is the etiological agent of leptospirosis. The high diversity among Leptospira species provides an array to look for important mediators involved in pathogenesis. Toxin-antitoxin (TA) systems represent an important survival mechanism on stress conditions. vapBC modules have been found in nearly one thousand genomes corresponding to about 40% of known TAs. In the present study, we investigated TA profiles of some strains of Leptospira using a TA database and compared them through protein alignment of VapC toxin sequences among Leptospira spp. genomes. Our analysis identified significant differences in the number of putative vapBC modules distributed in pathogenic, saprophytic, and intermediate strains: four in L. interrogans, three in L. borgpetersenii, eight in L. biflexa, and 15 in L. licerasiae. The VapC toxins show low identity among amino acid sequences within the species. Some VapC toxins appear to be exclusively conserved in unique species, others appear to be conserved among pathogenic or saprophytic strains, and some appear to be distributed randomly. The data shown here indicate that these modules evolved in a very complex manner, which highlights the strong need to identify and characterize new TAs as well as to understand their regulation networks and the possible roles of TA systems in pathogenic bacteria.
RESUMEN
Current pneumococcal vaccines are composed of bacterial polysaccharides as antigens, plain or conjugated to carrier proteins. While efficacious against vaccine serotypes, epidemiologic data show an increasing incidence of infections caused by nonvaccine serotypes of Streptococcus pneumoniae. The use of pneumococcal surface protein A (PspA) as a carrier protein in a conjugate vaccine could help prevent serotype replacement by increasing vaccine coverage and reducing selective pressure of S. pneumoniae serotypes. PspA is present in all pneumococcal strains, is highly immunogenic, and is known to induce protective antibodies. Based on its sequence, PspA has been classified into three families and six clades. A PspA fragment derived from family 2, clade 4 (PspA4Pro), was shown to generate antibodies with a broad range of cross-reactivity, across clades and families. Here, PspA4Pro was modified and conjugated to capsular polysaccharide serotype 14 (PS14). We investigated the impact of conjugation on the immune response induced to PspA4Pro and PS14. Mice immunized with the PS14-mPspA4Pro conjugate produced higher titers of anti-PS14 antibodies than the animals that received coadministered antigens. The conjugate induced antibodies with opsonophagocytic activity against PS14-carrying strains, as well as against a panel of strains bearing PspAs from five clades (encompassing families 1 and 2) bearing a non-PS14 serotype. Furthermore, mice immunized with PS14-mPspA4Pro were protected against nasal colonization with a nonrelated S. pneumoniae strain bearing PspA from clade 1, serotype 6B. These results demonstrate that the cross-reactivity mediated by PspA4Pro is retained following conjugation, supporting the use of PspA4 as a carrier protein in order to enhance pneumococcal vaccine coverage and encourage its further investigation as a candidate in future vaccine designs
RESUMEN
Conjugate vaccines are being widely used since their introduction. Nowadays the interest in these vaccines is still growing and new antigens and conjugate chemistry are being studied and developed. Pneumococcal surface protein A (PspA) is one of the most studied pneumococcal antigens and is an important vaccine candidate. One approach to broaden the conjugate vaccine coverage could be the conjugation of the polysaccharide to a pneumococcal protein such as PspA. Previous results have shown that conjugated recombinant fragment of PspA (rPspA) not only maintained but also in some conjugates improved the induction of protective antibodies raised against the protein carrier. We describe here a characterization study to identify the domains of Streptococcus pneumoniae recombinant PspA (rPspA), from family 1 clade 1 and family 2 clade 3, involved in the conjugation with serotype 6B capsular polysaccharide.
