bDespite the substantial beneficial effects of incorporating the 7-valent pneumococcal conjugate vaccine (PCV7) into immunization programs, serotype replacement has been observed after its widespread use. As there are many serotypes currently documented, the use of a conjugate vaccine relying on protective pneumococcal proteins as active carriers is a promising alternative to expand PCV coverage. In this study, capsular polysaccharide serotype 6B (PS6B) and recombinant pneumococcal surface protein A (rPspA), a well-known protective antigen from Streptococcus pneumoniae, were covalently attached by two conjugation methods. The conjugation methodology developed by our laboratory, employing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as an activating agent through carboxamide formation, was compared with reductive amination, a classical methodology. DMT-MM-mediated conjugation was shown to be more efficient in coupling PS6B to rPspA clade 1 (rPspA1): 55.0% of PS6B was in the conjugate fraction, whereas 24% was observed in the conjugate fraction with reductive amination. The influence of the conjugation process on the rPspA1 structure was assessed by circular dichroism. According to our results, both conjugation processes reduced the alpha-helical content of rPspA; reduction was more pronounced when the reaction between the polysaccharide capsule and rPspA1 was promoted between the carboxyl groups than the amine groups (46% and 13%, respectively). Regarding the immune response, both conjugates induced functional anti-rPspA1 and anti-PS6B antibodies. These results suggest that the secondary structure of PspA1, as well as its reactive groups (amine or carboxyl) involved in the linkage to PS6B, may not play an important role in eliciting a protective immune response to the antigens.

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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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 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 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 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.

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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.

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.