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

Abstract

Brucella is a genus of gram-negative bacteria that cause brucellosis. B. abortus and B. melitensis infect domestic ruminants while B. suis (biovars 1-3) infect swine, and all these bacteria but B. suis biovar 2 are zoonotic. Live attenuated B. abortus S19 and B. melitensis Rev1 are effective vaccines in domestic ruminants, though both can infect humans. However, there is no swine brucellosis vaccine. Here, we investigated the potential use as vaccines of B. suis biovar 2 rough (R) lipopolysaccharide (LPS) mutants totally lacking O-chain (Bs2ΔwbkF) or only producing internal O-chain precursors (Bs2Δwzm) and mutants with a smooth (S) LPS defective in the core lateral branch (Bs2ΔwadB and Bs2ΔwadD). We also investigated mutants in the pyruvate phosphate dikinase (Bs2ΔppdK) and phosphoenolpyruvate carboxykinase (Bs2ΔpckA) genes encoding enzymes bridging phosphoenolpyruvate and the tricarboxylic acid cycle. When tested in the OIE mouse model at the recommended R or S vaccine doses (10 8 and 10 5 CFU, respectively), CFU/spleen of all LPS mutants were reduced with respect to the wild type and decreased faster for the R than for the S mutants. At those doses, protection against B. suis was similar for Bs2ΔwbkF, Bs2Δwzm, Bs2ΔwadB and the Rev1 control (10 5 CFU). As described before for B. abortus, B. suis biovar 2 carried a disabled pckA so that a double mutant Bs2ΔppdKΔpckA had the same metabolic phenotype as Bs2ΔppdK and ppdK mutation was enough to generate attenuation. At 10 5 CFU, Bs2ΔppdK also conferred the same protection as Rev1. As compared to other B. suis vaccine candidates described before, the mutants described here simultaneously carry irreversible deletions easy to identify as vaccine markers, lack antibiotic-resistance markers and were obtained in a non-zoonotic background. Since R vaccines should not elicit antibodies to the S-LPS and wzm mutants carry immunogenic O-chain precursors and did not improve Bs2ΔwbkF, the latter seems a better R vaccine candidate than Bs2Δwzm. However, taking into account that all R vaccines interfere in ELISA and other widely used assays, whether Bs2ΔwbkF is advantageous over Bs2ΔwadB or Bs2ΔppdK requires experiments in the natural host.

The Phospholipid N-Methyltransferase and Phosphatidylcholine Synthase Pathways and the ChoXWV Choline Uptake System Involved in Phosphatidylcholine Synthesis Are Widely Conserved in Most, but Not All Brucella Species

“…We used the mouse model ( Grilló et al, 2012 ; Aragón-Aranda et al, 2020 ) to examine the contribution of PC to B. suis bv2 virulence. We observed that impairment of PC synthesis in Bs2WT by deletion of both pcs and pmtA resulted in a low degree of attenuation that only became significant at late stages of the infection ( Figure 7 ).…”

“…The characteristics of the bacterial strains and plasmids are shown in Supplementary Table 1 . The virulent B. suis biovar 2 (bv2) strain CITA 198 (henceforth Bs2WT) was selected for this study because the reference bv2 strain B. suis Thomsen is attenuated in mice ( Aragón-Aranda et al, 2020 ). The choline-containing media used were tryptic soy broth (TSB, Scharlau, Barcelona, Spain), the same medium with 15% agar (TSA) or Blood Agar Base (BAB; Oxoid, United Kingdom), supplemented when necessary with 50 μg/ml kanamycin (Km), or/and 5% sucrose or/and 0.2% activated charcoal (Sigma) (see below).…”

When the Going Gets Rough: The Significance of Brucella Lipopolysaccharide Phenotype in Host–Pathogen Interactions

“…variations depending on strain, in which "A-dominant" strains exhibit a linear α-1,2-linked polymer with approximately 2% α-1,3 linkages while the O-PS from "M-dominant" strains is a linear polymer of tetrasaccharide repeating units containing one α-1,3 linkage and three α-1,2-linked monosaccharide residues (Meikle et al, 1989;Kubler-Kielb and Vinogradov, 2013;Mancilla, 2015). Although not sufficient for speciation, serotyping using monoclonal antibodies directed against A or M epitopes can allow for distinction of different biovars within a particular smooth Brucella species (Alton et al, 1988). Further discussion of O-PS in this review will focus on the classical smooth Brucella spp.…”

“…RB51's reduced efficacy against B. ovis and its smooth counterparts in the natural host may be associated with its attenuation and/or alterations to its outer membrane or LPS outside of its lack of O-PS. Several studies have demonstrated that targeted mutagenesis can produce rough vaccines that are superior in protection to RB51 and comparable to protection by S19 or Rev.1 in mice (Monreal et al, 2003Kahl-McDonagh andAragón-Aranda et al, 2020). One prominent finding is that for a rough vaccine to be effective, the core must be intact although lack of the aforementioned lateral side branch may actually enhance vaccine efficacy, presumably due to enhanced recognition of surface molecules in the absence of this "shield" (Monreal et al, 2003;Soler-Lloréns et al, 2014).…”

“…First, certain rough LAV candidates such as B. melitensis B115 still produce partial or complete O-PS that remains within the cytoplasm and the possibility that this internal O-PS assists in mediating protection cannot be ruled out. Second, some studies administered rough vaccine candidates at a higher dose than smooth candidates, as is the case with the recommended doses of RB51 vs. S19 in mice (10 8 CFU vs. 10 5 CFU) (Adone et al, 2008(Adone et al, , 2011Aragón-Aranda et al, 2020). Despite this, these studies, although still few in number, indicate that a rough vaccine has the potential to protect against both smooth and rough Brucella spp.…”