Discovery of a Protective Rickettsia prowazekii Antigen Recognized by CD8+ T Cells, RP884, Using an In Vivo Screening Platform

Abstract

Rickettsia prowazekii has been tested for biological warfare due to the high mortality that it produces after aerosol transmission of very low numbers of rickettsiae. Epidemic typhus, the infection caused by these obligately intracellular bacteria, continues to be a threat because it is difficult to diagnose due to initial non-specific symptoms and the lack of commercial diagnostic tests that are sensitive and specific during the initial clinical presentation. A vaccine to prevent epidemic typhus would constitute an effective deterrent to the weaponization of R. prowazekii; however, an effective and safe vaccine is not currently available. Due to the cytoplasmic niche of Rickettsia, CD8+ T-cells are critical effectors of immunity; however, the identification of antigens recognized by these cells has not been systematically addressed. To help close this gap, we designed an antigen discovery strategy that uses cell-based vaccination with antigen presenting cells expressing microbe's proteins targeted to the MHC class I presentation pathway. We report the use of this method to discover a protective T-cell rickettsial antigen, RP884, among a test subset of rickettsial proteins.

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

“…Rickettsial proteins were further analyzed using RANKPEP (http://imed.med.ucm.es/Tools/rankpep.html), which combines MHC class-I-binding affinity and proteasome processing [19]. We used RANKPEP to evaluate the likelihood of peptides predicted by NetMHCpan, IEBD-ANN and SYFPEITHI to be generated via proteasome-processing as we previously described [12]. Analysis of similarity to human and mouse proteins, as well as HLA class-I binding, were performed using Vaxign and Vaxitope, respectively; these programs are available through the Vaccine Investigation and Online Information Network (VIOLIN, http://www.violinet.org/) [20].…”

“…C3H/HeN mice were housed in an animal biosafety level-3 (ABSL3). For immunization, we followed a short immunization protocol that enhances CD8 + T cell responses [21] as we did in our previous report [12]. Generation of antigen presenting (APCs) cell lines expressing rickettsial proteins is described in the supplementary methods section.…”

“…Animals were monitored for clinical symptoms and mortality for 21 days or euthanized after 7 days for rickettsia load assessment in the lungs. Rickettsiae were measured by quantitative real-time PCR using a validated assay previously described [12]. We followed the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.…”

“…No other antigens that trigger T-cell-mediated protective immunity have been identified since. To address this gap, we recently reported the proof-of-principle of an in vivo screening approach to identify antigens recognized by CD8 + T cells [12]. Herein, we extend this work through refinement of our screening platform and the identification of new antigens of R. prowazekii that stimulate a cross-protective response against the closely related R. typhi , the agent of flea-borne murine typhus, which is the most prevalent and neglected of the rickettsioses [13].…”

The role of CD8 T lymphocytes in rickettsial infections

“…A novel vaccine discovery strategy involving nucleofection of antigen-presenting cells targeting the MHC class I pathway with clones expressing R. prowazekii genes was used to immunize mice [15]. Rickettsial challenge with an ordinarily lethal dose of R. typhi revealed that R. prowazekii gene RP884 stimulated cross protection reducing the bacterial load in liver.…”