Characterisation of alternative expression vectors for recombinant Bacillus Calmette-Guérin as live bacterial delivery systems

Abstract

BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (P AN , P αAg, P Hsp60 , P BlaF* and P L5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: P AN was the weakest in both Mycobacterium smegmatis and BCG and P BlaF* was higher than P Hsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that P BlaF* and P Hsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the P L5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.

Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil

“…1A), we focused ongoing SARS-CoV-2 genomic surveillance (2,(18)(19)(20)(21)(22) on recently collected samples from the city (supplementary materials, materials and methods, and table S2). Before this, only seven SARS-CoV-2 genome sequences from Amazonas were publicly available (SARS-CoV-2 was first detected in Manaus on 13 March 2020) (19,23). We sequenced SARS-CoV-2 genomes from 184 samples from patients seeking COVID-19 testing in two diagnostic laboratories in Manaus between November and December 2020, using the ARTIC V3 multiplexed amplicon scheme (24) and the MinION sequencing platform.…”

CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate

“…Here, we evaluated two different constructs using lysA complementation either in tandem with ltak63 [pAN71- ltak63-lysA (t)] or each gene with its own expression cassette [pAN71- ltak63-lysA (c)]. The construct in tandem is driven by the pAN promoter, which is considered a weak mycobacterial promoter ( 28 ). If the expression of LysA is too low, then it could also affect BCG’s fitness and the protection induced against Mtb .…”

COVID-19 vaccine development based on recombinant viral and bacterial vector systems: combinatorial effect of adaptive and trained immunity

“…A number of lines of evidence have indicated that childhood vaccination with some live-attenuated vaccines, such as the tuberculosis vaccine, bacilli Calmette-Guerin (BCG), smallpox and polio vaccines, can induce beneficial, nonspecific, heterologous vaccine effects against unrelated pathogens by eliciting innate cell-mediated immune responses (Nascimento et al, 2020;Pasco and Anguita, 2020). These innate cell-based heterologous effects are referred to as trained immunity.…”

“…Compared to other platforms, vaccines that use viral or bacterial delivery systems have a distinct benefit in that they can induce off-target effects to defend against unrelated pathogens via an innate immune memory system termed trained immunity. In epidemiological studies, some live viral or bacterial vaccines have been reported to induce heterologous vaccine effects via trained innate immune cells, resulting in a reduction in all-cause mortality from infectious agents (Agrawal, 2019;Nascimento et al, 2020;Marín-Hernández et al, 2021). The enhanced nonspecific immune response of trained immunity can further enhance both specific humoral and cell-mediated immune responses to defend against SARS-CoV-2, providing better protection against even vaccine escape variants (Covián et al, 2021).…”