Comparative Preclinical Evaluation of Three Recombinant Brucella Vaccine Formulations, BruVac-Pro, BruVac-CPX and BruVac-FLU, in a Murine Brucella melitensis Challenge Model

Background/Objectives: Brucellosis remains a widespread zoonosis, yet no licensed human vaccine exists and the live attenuated vaccines used in animals are unsuitable for people because of residual virulence and the risk of accidental infection. Recombinant platforms are attractive alternatives, but direct head-to-head comparisons within a single study are scarce. We compared the safety, immunogenicity and protective efficacy of three recombinant candidates: the AddaS03-adjuvanted subunit vaccine BruVac-Pro, the capripoxvirus-vectored BruVac-CPX and the influenza virus-vectored BruVac-FLU. Methods: Inbred BALB/c mice (n = 355) received a prime-boost immunization, except for the single-dose Rev-1 reference group. A subgroup of 10 animals per group was monitored serologically from day 0 and subsequently challenged intraperitoneally with virulent Brucella melitensis 16M, and the remaining animals were assigned to safety, vector-detection and cellular-immunity read-outs. The timing and prospective nature of allocation to the challenge cohort could not be documented; selection bias in the efficacy endpoint therefore cannot be excluded. Safety was assessed from clinical observation, body weight and early serum clinical chemistry; antigen-specific IgG to Omp16, Omp19, L7/L12 and SodC was measured by indirect ELISA; and protection was quantified from splenic bacterial load (Y), the protective index (PI) and the proportion of animals with no splenic recovery (n = 10 per group). Immunogenicity was assessed as humoral (IgG) responses only; antigen-specific cellular responses were measured in separate subsets of the same experiment and are reported separately. Results: Mortality during the immunization phase was confined to the three sublingually dosed viral-vector and stabilizer groups (CPX/CPX 4/50, CCFLU/CCFLU 3/55, FLU/FLU 1/55), with no deaths in the Pro/Pro, AddaS03/AddaS03, Rev-1 or PBS/PBS groups (8/160 versus 0/195); surviving animals showed no overt clinical signs. Influenza-vector RNA was not detected in serum or spleens at days 3 and 6 by a qualitative assay of undetermined sensitivity. Seroconversion to Omp16 and Omp19 was confined to the Pro-containing group; no animal in any group seroconverted to SodC, and responses to L7/L12 were confined to isolated single animals without a group-level pattern. Splenic burden differed across groups (Kruskal–Wallis, p = 4 × 10−7). BruVac-Pro gave the strongest protection (Y 2.32; PI 2.62; 70.0% with no recovery; p = 0.028 for splenic burden and adjusted p = 0.015 for the proportion of animals with no recovery, both versus the phosphate-buffered saline (PBS) control). BruVac-CPX showed only a non-significant trend towards reduced burden (PI 1.54; p = 0.57 after correction for multiple comparisons) without increasing clearance (20.0%), and BruVac-FLU did not prevent colonization. Rev-1 confirmed model validity (PI 3.15; 80.0%). Conclusions: Under the non-equivalent, formulation-specific regimens tested, BruVac-Pro was the only experimental formulation with no mortality and no overt clinical signs, produced the most consistent antigen-specific IgG response, and was the only experimental candidate to reduce day-14 splenic burden significantly relative to the PBS and adjuvant-only controls. These findings support further dose, mechanism and durability studies of this formulation, but do not establish platform superiority, sterile protection or suitability for human use.