Overexpression of wbkF gene in Brucella abortus RB51WboA leads to increased O-polysaccharide expression and enhanced vaccine efficacy against B. abortus 2308, B. melitensis 16M, and B. suis 1330 in a murine brucellosis model

Abstract

Brucella abortus RB51 is an attenuated, stable, spontaneous rough mutant derived in the laboratory from the virulent strain B. abortus 2308. Previous studies discovered that the wboA gene, which encodes a glycosyltransferase required for synthesis of the O-polysaccharide, is disrupted in strain RB51 by an IS711 element. However, complementation of strain RB51 with a functional wboA gene (strain RB51WboA) does not confer it a smooth phenotype but results in low levels of cytoplasmic O-polysaccharide synthesis. In this study, we asked if increasing the potential availability of bactoprenol priming precursors in strain RB51WboA would increase the levels of O-polysaccharide synthesis and enhance the protective efficacy against virulent Brucella challenge. To achieve this, we overexpressed the wbkF gene, which encodes a putative undecaprenyl-glycosyltransferase involved in bactoprenol priming for O-polysaccharide polymerization, in strain RB51WboA to generate strain RB51WboAKF. In comparison to strain RB51WboA, strain RB51WboAKF expressed higher levels of O-polysaccharide, but was still attenuated and remained phenotypically rough. Mice immunized with strain RB51WboAKF developed increased levels of smooth LPS-specific serum antibodies, primarily of IgG2a and IgG3 isotype. Splenocytes from mice vaccinated with strain RB51WboAKF secreted higher levels of antigen-specific IFN-γ and TNF-α and contained more numbers of antigen-specific IFN-γ secreting CD4+ and CD8+ T lymphocytes when compared to those of the RB51 or RB51WboA vaccinated groups. Immunization with strain RB51WboAKF conferred enhanced protection against virulent B. abortus 2308, B. melitensis 16M and B. suis 1330 challenge when compared to the currently used vaccine strains. Our results suggest that strain RB51WboAKF has the potential to be a more efficacious vaccine than its parent strain in natural hosts.

Development of attenuated live vaccine candidates against swine brucellosis in a non-zoonotic B. suis biovar 2 background

“…The best construct (RB51 leuB /SOD/WboA) administered IP reduced B. suis 1330 spleen colonization but no reference vaccine control was included. In a more recent work [ 26 ], the expression of O-chain was further enhanced by over-expressing wboA and wbkF . The construct (RB51 WboAKF ) provided protection against B. suis 1330 similar to that of the wboA mutated R B. suis VTRS1 [ 26 ]), and improved VTRS1 when administered at a higher dose.…”

“…In a more recent work [ 26 ], the expression of O-chain was further enhanced by over-expressing wboA and wbkF . The construct (RB51 WboAKF ) provided protection against B. suis 1330 similar to that of the wboA mutated R B. suis VTRS1 [ 26 ]), and improved VTRS1 when administered at a higher dose. However, no reference vaccine was included in the experiments of protection against B. suis .…”

“…Whereas both types of mutants yielded some protection in mice (see "Discussion"), both are made in brucellae that are infectious for humans and do not fully explore the possibilities offered by our current understanding of the genetics and structure of Brucella LPS. RB51 carries also a disrupted wboA [19] and it has been manipulated in attempts to bolster its performance either by overexpressing proteins thought to be immunogenic or involved in virulence [25] and/or by restoring the ability to induce anti-S-LPS antibodies [25,26]. Yet, RB51 carries unknown genetic defects affecting LPS [19], which together with its poor performance in pigs [10] indicate that it is not an optimal background for developing a swine brucellosis vaccine.…”

Human brucellosis: recent advances and future challenges

“…The general methods to create a DIVA vaccine have been to remove an immunogenic antigen from the vaccine (such as the loss of O-antigen in RB51). This may reduce efficiency of the vaccine (hence the attempts to restore O-antigen production to RB51 [3]) and mean that accidental human infections are not detected [4]. A more efficient method could be to express an unrelated immunogenic protein in the vaccine strain that would induce a detectable serological response not seen in infected animals.…”

Design and Characterization of a Recombinant Brucella abortus RB51 Vaccine That Elicits Enhanced T Cell-Mediated Immune Response

“…In many countries, the B. abortus RB51 rough strain live attenuated vaccine is used instead of B. abortus S19 [ 8 ]. This vaccine is a spontaneous mutant obtained by sub-culturing the virulent strain B. abortus 2308 on a medium containing rifampicin and penicillin [ 9 ]. There are limitations to the immunization efficacy of RB51 depending on the challenge, with no absolute protection achieved with this vaccine [ 10 ].…”