Immunization with an Autotransporter Protein of <i>Orientia tsutsugamushi</i> Provides Protective Immunity against Scrub Typhus

<div><p>Background</p><p>Scrub typhus is an acute febrile disease caused by <i>Orientia tsutsugamushi</i> infection. Recently, the rapid increase of scrub typhus incidence in several countries within the endemic region has become a serious public health issue. Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine. Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of <i>O</i>. <i>tsutsugamushi</i>.</p><p>Methodology/Principal Findings</p><p>We examined the potential role of ScaA protein, an autotransporter protein of <i>O</i>. <i>tsutsugamushi</i>, in bacterial pathogenesis and evaluated the protective attributes of ScaA immunization in lethal <i>O</i>. <i>tsutsugamushi</i> infection in mice. Our findings demonstrate that ScaA functions as a bacterial adhesion factor, and anti-ScaA antibody significantly neutralizes bacterial infection of host cells. In addition, immunization with ScaA not only provides protective immunity against lethal challenges with the homologous strain, but also confers significant protection against heterologous strains when combined with TSA56, a major outer membrane protein of <i>O</i>. <i>tsutsugamushi</i>.</p><p>Conclusions/Significance</p><p>Immunization of ScaA proteins provides protective immunity in mice when challenged with the homologous strain and significantly enhanced protective immunity against infection with heterologous strains. To our knowledge, this is the most promising result of scrub typhus vaccination trials against infection of heterologous strains in mouse models thus far.</p></div

Adhesion function of ScaA.

<p>(A) HeLa cells were incubated with fluorescent microbeads coated with GST or GST-ScaA (ScaA) for 1 h, washed extensively, and fixed. Cell-bound microbeads (green) were visualized by fluorescence microscopy after staining of cell nuclei (blue). Scale bars, 10 μm. (B) Relative binding of the microbeads coated with GST (dotted line) or GST-ScaA (thick line) to HeLa cells was quantified directly using fluorescence-activated cell sorter (FACS) analysis. The gray histogram represents unbound cells (cells not incubated with microbeads). (C) Immunofluorescence microscopy using an anti-ScaA antibody revealed the presence of ScaA on the surface of the recombinant <i>E</i>. <i>coli</i> (lower panels). Preimmune serum did not detect the recombinant protein (upper panels). Scale bars, 5 μm. (D) <i>E</i>. <i>coli</i> transformed with the pET28a vector or with pScaA was induced with IPTG and incubated with HeLa cells. After being washed to remove adherent bacteria, the cells were fixed, permeabilized, and stained with an anti-<i>E</i>. <i>coli</i> antibody (green) and ToPro-3 for nuclear staining (blue). Scale bars, 10 μm. (E) CFU-based quantification of adherent <i>E</i>. <i>coli</i> transformed with the vector or pScaA was performed. The results are presented as percentages of adherent bacteria relative to the total bacterial input. Data are representative of three independent assays for each of the host cells. **, <i>p</i> < 0.01. (F) Inclusion of anti-ScaA serum in the medium (α-ScaA) significantly inhibited adhesion of <i>E</i>. <i>coli</i> expressing ScaA into host cells. After addition of anti-ScaA or preimmune serum into infection media, CFU-based quantification of adherent <i>E</i>. <i>coli</i> transformed with the vector or pScaA was performed. **, <i>p</i> < 0.01.</p

Similarity plots of a set of <i>tsa56</i> and <i>scaA</i> sequences from the indicated strains compared to sequences from the Boryong strain.

<p>Each plotted point is the percent identity within a sliding window of 100 bp or 100 amino acids wide centered on the position plotted, with a step size between points of 10 bp or amino acids. Diagrams above the graphs show the relative sizes of TSA56 and ScaA proteins and their sequence motiffs. Yellow box: signal peptide, gray box: antigenic domain, green box: variable domain, blue box: transmembrane domain, pink box: repeated sequences, brown box: autotransporter domain.</p

Protective role of anti-ScaA immunity.

<p>(A) Anti-ScaA antibody inhibited <i>O</i>. <i>tsutsugamushi</i> infection into host cells. HeLa cells were infected with the pathogen in the presence of the indicated anti-Sca antibodies or nonimmune serum. At 4 h after infection, bacterial infection was examined using confocal microscopy after differential immunoflourescent staining (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003585#sec005" target="_blank">materials and methods</a>). (B) The <i>O</i>. <i>tsutsugamushi</i> per host cell ratio was determined from three independent experiments in (A). **, <i>p</i> < 0.01. (C) Survival curves of immunized mice following lethal challenge with <i>O</i>. <i>tsutsugamushi</i>. Mice (<i>n</i> = 5/group) were immunized with the indicated antigen from the Boryong strain and challenged intraperitoneally with 100 x LD₅₀ of <i>O</i>. <i>tsutsugamushi</i> Boryong strain. Their survival was monitored until all the surviving mice recovered from the disease. This graph is a representative survival curve of two experiments. **, <i>p</i> < 0.01 when compared with non-immunized group (PBS).</p