Optimization of Burkholderia Glycoconjugate Vaccines: A Reverse Vaccinology Approach

Abstract

Burkholderia pseudomallei is a Gram-negative, intracellular pathogen and the etiological agent of melioidosis. Because of intrinsic multi-drug resistance, lack of effective treatment and high case-fatality rates, this organism is classified as a Tier 1 Select Agent and considered a priority for vaccine development. Previous studies have shown that glycoconjugate vaccines can provide enhanced protection against lethal B. pseudomallei challenge. However, the limited pool of Burkholderia antigens hinders continued optimization of these vaccines. In this study, we used a reverse vaccinology approach to identify outer membrane and secreted Burkholderia proteins. These proteins were ranked according to predicted immunogenicity, and top vaccine candidates were selected based on the number and affinity of Major Histocompatibility Complex (MHC) epitopes. To confirm the in silico immunogenicity predictions, the top seven proteins were purified and evaluated for seroreactivity against convalescent human and experimental murine melioidosis sera. All proteins were shown to exhibit varying reactivity with convalescent sera. To evaluate immunogenicity in vivo, a series of vaccination studies were performed in mice. Recombinant proteins were shown to be immunogenic in mice, generating high antibody titers irrespective of administration route, concentration or adjuvant. Despite the ability to induce a strong humoral immune response, vaccination did not protect animals from lethal B. pseudomallei challenge. To evaluate whether immunogenic proteins could enhance the immunogenicity of a glycoconjugate vaccine, we optimized a method for the construction of a gold nanoparticle (AuNP) glycoconjugate vaccines and evaluated immunogenicity in mice. Subcutaneous administration of AuNP-glycoconjugate vaccines resulted in high anti-lipopolysaccharide (LPS) responses, a correlate of protection in human and animal melioidosis. Additionally, immune sera were shown to facilitate uptake of B. pseudomallei by murine macrophages in vitro. While AuNP-glycoconjugate vaccination did not afford protection against lethal challenge, the ability to induce high antibody titers confirms immunogenicity and provides a strong rationale for continued optimization of this platform.