Development of REC1: A Reverse Vaccinology-Derived Multi-Epitope Vaccine Candidate Against Brucellosis

Background/Objectives: Brucellosis is a widespread intracellular zoonosis that continues to impose substantial public-health and economic burdens in endemic regions. No human brucellosis vaccine has yet been approved, and most existing multi-epitope candidates rely solely on computational predictions, without population-specific human leukocyte antigen (HLA) optimization or in vivo epitope validation. REC1 is therefore an early-stage, proof-of-concept, human-oriented multi-epitope candidate, with epitopes screened against high-frequency HLA alleles in the Chinese population. This study developed a multi-epitope vaccine candidate, REC1, using an integrated reverse-vaccinology workflow. Methods: We predicted Major Histocompatibility Complex (MHC) I and II epitopes against high-frequency HLA alleles representative of the Chinese population (AFND). Population coverage of the final incorporated epitope set was estimated with the IEDB Population Coverage tool under Hardy-Weinberg equilibrium, while paired HLA-DQ heterodimers, which the tool cannot model, were evaluated independently from Chinese DQA1-DQB1 haplotype frequencies. Epitopes supported by in vivo immunoreactivity were then assembled into the modular REC1 construct, which we characterized in silico for physicochemical properties, structural architecture, and simulated immune responses. Results: IFN-γ ELISpot shortlisted seven MHC-I and five MHC-II candidate immunoreactive epitopes derived from five Brucella antigens, of which five MHC-I and five MHC-II (one per antigen) were incorporated into REC1. The incorporated set was predicted to cover 91.17% of the China Oriental population on a conservative class I+II estimate (98.88% when HLA-DP was included; the HLA-DQ component alone covered 58-79%, 68.17% in Han). Epitope-level BLAST showed that all twelve shortlisted epitopes were 100% conserved across major Brucella species, except that the two Omp31 epitopes are absent from the Omp31-deleted B. abortus. The 35.86 kDa REC1 construct was predicted to be highly stable (instability index = 25.25) and hydrophilic, with a distinct architecture in which the carrier domain is ordered and the epitope region is disordered. Immune simulations suggested that REC1 can induce Th1-polarized humoral and cellular responses and may support long-term immunological memory. Molecular docking predicted that the L7/L12 carrier forms well-defined interfaces with TLR2, CD14, C1q, and DC-SIGN, and a representative peptide-MHC model confirmed canonical groove binding of a CTL epitope. Conclusions: REC1 exhibits favorable in silico properties, and its embedded peptides show IFN-γ immunogenicity in mice (candidate immunoreactive epitopes), lending preliminary support to its value as a multi-epitope vaccine candidate. HLA-matched validation and challenge studies are still needed to establish protective efficacy. Our study also offers a validated reverse vaccinology framework for future brucellosis vaccine development.