Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human chagas disease.

A therapeutic vaccine for human Chagas disease is under development by the Sabin Vaccine Institute Product Development Partnership. The aim of the vaccine is to significantly reduce the parasite burden of Trypanosoma cruzi in humans, either as a standalone product or in combination with conventional chemotherapy. Vaccination of mice with Tc24 formulated with monophosphoryl-lipid A (MPLA) adjuvant results in a Th1 skewed immune response with elevated IgG2a and IFNγ levels and a statistically significant decrease in parasitemia following T. cruzi challenge. Tc24 was therefore selected for scale-up and further evaluation. During scale up and downstream process development, significant protein aggregation was observed due to intermolecular disulfide bond formation. To prevent protein aggregation, cysteine codons were replaced with serine codons which resulted in the production of a non-aggregated and soluble recombinant protein, Tc24-C4. No changes to the secondary structure of the modified molecule were detected by circular dichroism. Immunization of mice with wild-type Tc24 or Tc24-C4, formulated with E6020 (TLR4 agonist analog to MPLA) emulsified in a squalene-oil-in-water emulsion, resulted in IgG2a and antigen specific IFNγ production levels from splenocytes that were not significantly different, indicating that eliminating putative intermolecular disulfide bonds had no significant impact on the immunogenicity of the molecule. In addition, vaccination with either formulated wild type Tc24 or Tc24-C4 antigen also significantly increased survival and reduced cardiac parasite burden in mice. Investigations are now underway to examine the efficacy of Tc24-C4 formulated with other adjuvants to reduce parasite burden and increase survival in pre-clinical studies.

Investigation of the essentiality of glutamate racemase in Mycobacterium smegmatis.

The mycobacterial cell wall frequently has been used as a target for drug development, and d-glutamate, synthesized by glutamate racemase (MurI), is an important component of peptidoglycan. While the essentiality of the murI gene has been shown in several bacterial species, including Escherichia coli, Bacillus anthracis, and Streptococcus pneumoniae, studies in mycobacteria have not yet provided definitive results. This study aimed to determine whether murI is indeed essential and can serve as a possible target for structure-aided drug design. We have achieved this goal by creating a ΔmurI strain of Mycobacterium smegmatis, a close relative of Mycobacterium tuberculosis. The deletion of the murI gene in M. smegmatis could be achieved only in minimal medium supplemented with D-glutamate, demonstrating that MurI is essential for growth and that glutamate racemase is the only source of D-glutamate for peptidoglycan synthesis in M. smegmatis.

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli.

BACKGROUND: Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. RESULTS: Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. CONCLUSIONS: The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.