Discovery of novel cross-protective Rickettsia prowazekii T-cell antigens using a combined reverse vaccinology and in vivo screening approach

Abstract

Rickettsial agents are some of the most lethal pathogens known to man. Among them, Rickettsia prowazekii is a select agent with potential use for bioterrorism; yet, there is no anti-Rickettsia vaccine commercially available. Owing to the obligate intracellular lifestyle of rickettsiae, CD8+ T cells are indispensable for protective cellular immunity. Furthermore, T cells can mediate cross-protective immunity between different pathogenic Rickettsia, a finding consistent with the remarkable similarity among rickettsial genomes. However, Rickettsia T cell antigens remain unidentified. In the present study, we report an algorithm that allowed us to identify and validate four novel R. prowazekii vaccine antigen candidates recognized by CD8+ T cells from a set of twelve in silico-defined protein targets. Our results highlight the importance of combining proteasome-processing as well as MHC class-I-binding predictions. The novel rickettsial vaccine candidate antigens, RP778, RP739, RP598, and RP403, protected mice against a lethal challenge with R. typhi, which is indicative of cross-protective immunity within the typhus group rickettsiae. Together, our findings validate a reverse vaccinology approach as a viable strategy to identify protective rickettsial antigens and highlight the feasibility of a subunit vaccine that triggers T-cell-mediated cross-protection among diverse rickettsiae.

Phenotype of the anti-Rickettsia CD8+ T cell response suggests cellular correlates of protection for the assessment of novel antigens

“…Our study supports this correlation since RP778, our strongest antigen as assessed by survival and rickettsial load [24], produced the largest increase in IFN-γ expression and generated the largest number of memory-like CD8 + T cells. Moreover, when we pooled RP884, RP739, RP403 and RP598, which includes antigens that were not as strong at inducing CD8 + T cell activation individually [23, 24], they provided an overall superior protection in terms of reduction of the rickettsial load (supplementary figure 9) and the induction of memory-type CD8 + T cells (Fig. 3 and 4), suggesting that some degree of synergy was achieved.…”

“…Our findings provide useful paradigms that could assist in the assessment of the quality of the immune response induced by novel vaccine targets and, together with other in vivo protection measurements such as survival, could provide a rationale for the selection of rickettsial antigens for a subunit vaccine. The present results together with in silico predictions and in vivo protection testing as reported by us [23, 24] are being combined to identify and select relevant vaccine candidates for further characterization. In consequence, the best candidate antigens will encompass MHC class-I binding peptides (both mouse and human), provide protection against a rickettsial lethal challenge, and stimulate protective CD8 + T cell responses.…”

“…These antigens stimulate CD8 + T cell responses and result in cross-protection against a lethal challenge with R. typhi, the other member of the typhus group Rickettsia (supplementary table 1)[23, 24]. We decided to use the phenotype of the natural primary anti-rickettsial CD8 + T cell response on 7 dpi as a paradigm against which to compare the response against the newly discovered protective antigens for four reasons: 1) both memory and effector-type CD8+ T cells are present at this time (Figure 2); 2) our antigen screening method involves a rapid prime and boost immunization followed by lethal challenge, which is known to generate memory-type CD8 + T cells within 5 to 7 days after immunization [32, 33]; 3) this screening method does not allow for a resting period (i.e., primary T cells are continuously stimulated); and 4) the natural anti-rickettsial memory CD8 + T cell response, as characterized with our ex vivo methodology, is indistinct (supplementary figures 6–8).…”

“…We first studied the kinetics of primary and memory CD8 + T cell responses with the aim of identifying a combination of markers of cellular protective immunity that could be used for the validation of antigens for vaccine candidates against Rickettsia . We then asked whether this marker combination could serve as an immune correlate of protection in mice immunized with antigens that we recently identified as protective [23, 24]. We immunized mice using a cell-based system in which the APCs are derived from endothelial cells, which is relevant since the main targets of infection in vivo are endothelial cells.…”

“…For immunization with protective antigens (RP884, RP778, RP739, RP403 and RP598) we used a cell-based system as reported by us [23, 24]. Briefly, the endothelial cell line SVEC4-10, modified to express the costimulatory molecules CD80 and CD137L through lentiviral transduction, was nucleofected with a PiggyBac transposon and a plasmid encoding the PiggyBac transposase in order to produce cell lines stably expressing R. prowazekii antigens in which the expression of the rickettsial protein is directly coupled to that of a fusion protein encoding resistance to hygromycin and GFP (see supplementary methods and supplementary figures 1 and 11).…”