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Abstract: The effect of successive cultures – undergoing or not cycles of freezing, storage and thawing – on the growth curves of the Mycobacterium bovis Bacille Calmette-Guerin (BCG) Moreau strain and a recombinant-BCG (rBCG) vaccine preparation were evaluated. The results showed that both strains going through three rounds of freezing and thawing were not able to grow efficiently in the third stage of liquid culture. This effect and also long-term frozen storage appeared to be more preeminent in cultures that had been harvested at 0.8 optical density (OD at 600nm) prior to freezing and storage, as in comparison to their 0.4OD counterparts. Altogether, the data suggest that cultures inoculated with samples harvested at lower OD are less sensitive to the limiting effects of serial cultivation, regardless of being BCG or rBCG. Successive cultivations without freezing and thawing also affect growth of BCG culture inoculated with cells at later exponential phase (0.8OD). Finally, macrophage infectivity with BCG cells from the third growth passage was significantly lower than from the first passage. These results draw attention to the importance of using fresh, low-passage and/or growth and infection capacity-controlled vaccine stocks for the evaluation of strains of BCG or rBCG. [Copyright &y& Elsevier]

This article discusses standard and new disruptive strategies in the race to develop an anti-COVID-19 vaccine. We also included new bioinformatic data from our group mapping immunodominant epitopes and structural analysis of the spike protein. Another innovative approach reviewed here is the use of BCG vaccine as priming strategy and/or delivery system expressing SARS-CoV-2 antigens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Bacillus Calmette-Guerin (BCG) continues to be employed as the most effective immunotherapy against superficial bladder cancer. We have developed an rBCG-S1PT strain that induces a stronger cellular immune response than BCG. This preclinical study was designed to test the potential of rBCG-S1PT as an immunotherapeutic agent for intravesical bladder cancer therapy. Materials and methods: A tumor was induced in C57BL/6 mice after chemical cauterization of the bladder and inoculation of the tumor cell line MB49. Next, mice were treated by intravesical instillation with BCG, rBCG-S1PT, or PBS once a week for 4 weeks. After 35 days, the bladders were removed and weighed, Th1 (IL-2, IL-12, INOS, INF-γ, TNF-α), and Th2 (IL-5, IL-6, IL-10, TGF-β) cytokine mRNA responses in individual mice bladders were measured by quantitative real time PCR, and the viability of MB49 cells in 18-hour coculture with splenocytes from treated mice was assessed. In an equivalent experiment, animals were observed for 60 days to quantify their survival. Results: Both BCG and rBCG-S1PT immunotherapy resulted in bladder weight reduction, and rBCG-S1PT increased survival time compared with the control group. There were increases in TNF-α in the BCG treated group, as well as increases in TNF-α and IL-10 mRNA in the rBCG-S1PT group. The viability of MB49 cells cocultured with splenocytes from rBCG-S1PT-treated mice was lower than in both the BCG and control groups. Conclusions: rBCG-S1PT therapy improved outcomes and lengthened survival times. These results indicate that rBCG could serve as a useful substitute for wild-type BCG. [Copyright &y& Elsevier]

Abstract: Mycobacterium bovis BCG has long been investigated as a candidate for heterologous antigen presentation. We have previously described an rBCG-Pertussis that confers protection against challenge with Bordetella pertussis in neonate and adult mice. In order to obtain stable expression in vivo, we constructed an unmarked BCG lysine auxotrophic and a complementation vector containing the lysine and the genetically detoxified S1 pertussis toxin genes, both under control of the same promoter. Complemented BCG-Δlysine growth and expression of the pertussis antigen were stable, without the use of an antibiotic marker. Our results show that the complemented rBCG-ΔlysA-S1PT-lysA+(kan−), which is now suitable to be evaluated in clinical trials, maintains similar characteristics of the original rBCG-pNL71S1PT strain, such as the antigen expression level, cellular immune response and protection against the same model challenge in neonatal-immunized mice. [Copyright &y& Elsevier]