Phenotypic Characterization of Peripheral T Cells and Their Dynamics in Scrub Typhus Patients

<div><h3>Background</h3><p>Scrub typhus, caused by <em>Orientia tsutsugamushi</em> infection, is one of the main causes of febrile illness in the Asia-Pacific region. Although cell-mediated immunity plays an important role in protection, little is known about the phenotypic changes and dynamics of leukocytes in scrub typhus patients.</p> <h3>Methodology/Principal Findings</h3><p>To reveal the underlying mechanisms of immunological pathogenesis, we extensively analyzed peripheral blood leukocytes, especially T cells, during acute and convalescent phases of infection in human patients and compared with healthy volunteers. We observed neutrophilia and CD4⁺ T lymphopenia in the acute phase of infection, followed by proliferation of CD8⁺ T cells during the convalescent phase. Massive T cell apoptosis was detected in the acute phase and preferential increase of CD8⁺ T cells with activated phenotypes was observed in both acute and convalescent phases, which might be associated or correlated with elevated serum IL-7 and IL-15. Interestingly, peripheral Treg cells were significantly down-regulated throughout the disease course.</p> <h3>Conclusions/Significance</h3><p>The remarkable decrease of CD4⁺ T cells, including Treg cells, during the acute phase of infection may contribute to the loss of immunological memory that are often observed in vaccine studies and recurrent human infection.</p> </div

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

Apoptosis and proliferation of T cells during <i>Orientia</i> infection.

<p>A and B. PBMCs were stained with antibodies against CD4, CD8, Annexin V, or Ki-67 and then analyzed by flow cytometry. The frequencies of Annexin V- (<i>A</i>) or Ki-67- (<i>B</i>) positive cells in CD4⁺ and CD8⁺ T cells were compared among healthy controls (HC, n = 6, open circle) and scrub typhus patients at acute phase (AP, n = 13–15, gray circle) or convalescent phase (CP, n = 6, black circle). Red bars indicate the mean value. C. The CD8/CD4 ratio of apoptotic or proliferating cells were compared among the patients and healthy volunteers. D. The levels of IL-7 and IL-15 (pg/ml) in the sera were measured and compared. Error bars indicate standard error from the mean value (C <i>and</i> D). <i>p</i> values were obtained using the Mann-Whitney <i>U</i> test. Statistically significant <i>p</i> values (<0.05) are shown in bold.</p

Profiles of type 1 and type 2 T cells in peripheral blood of scrub typhus patients.

<p>PBMCs were stained with antibodies against CXCR3, CCR4, CD4, and CD8 and then analyzed on a flow cytometer. A. The frequencies of Th1 (CD4⁺CXCR3⁺) or Th2 (CD4⁺CCR4⁺) within CD4⁺ T cells were compared among healthy controls (HC, n = 6, open circle) and the patients in acute phase (AP, n = 12, gray circle) or convalescent phase (CP, n = 5, black circle). B. The frequencies of Tc1 (CD8⁺CXCR3⁺) and Tc2 (CD8⁺CCR4⁺) within CD8⁺ T cells were compared among healthy controls and the patients as in <i>A</i>. Red bars indicate the mean value and <i>p</i> values were obtained using the Mann-Whitney <i>U</i> test. Statistically significant <i>p</i> values (<0.05) are shown in bold.</p

Profiles of CD4⁺Foxp3⁺ or CD4⁺CD25⁺⁺ regulatory T cells in scrub typhus patients.

<p>A. PBMCs were stained with antibodies against CD4 and CD25 or Foxp3 and then analyzed on a flow cytometer. Representative dot plots show the identification of CD25⁺⁺ T cells (upper panels) or Foxp3⁺ T cells (lower panels) within CD4⁺ T cells. Numbers in the plots indicate the frequencies (%) of the gated cells in total PBMCs. B. The frequency of CD4⁺CD25⁺⁺ or CD4⁺Foxp3⁺ T cells were compared between healthy controls (HC, n = 7, open circle) and scrub typhus patients at acute phase (AP, n = 10, gray circle) or convalescent phase (CP, n = 12, black circle). C. PBMCs were stained with antibodies against CD4 and Fas or CCR4, followed by intracellular staining of Foxp3 and/or CTLA-4 after fixation and permeablization. Mean fluorescent Intensities (MFI) representing CTLA-4, Fas, or CCR4 expression in CD4⁺Foxp3⁺ regulatory cells from healthy controls (HC, n = 7–8, open circle) or the patients (AP, n = 7–10, gray circle and CP, n = 9–12, black circle) were compared. Red bars indicate the mean value and <i>p</i> values were obtained using the Mann-Whitney <i>U</i> test or Wilcoxon signed-rank test. Statistically significant <i>p</i> values (<0.05) are shown in bold.</p

Profiles of peripheral blood leukocytes and T lymphocytes in scrub typhus patients.

<p>A. The frequencies (%) of neutrophils, lymphocytes, and monocytes in blood leukocytes from healthy controls (HC, n = 9, open circle) were determined using a hematology analyzer and compared to frequencies in scrub typhus patients during acute phase (AP, n = 17, gray circle) or convalescent phase (CP, n = 17, black circle) of infection. B. Peripheral blood mononuclear cells (PBMCs) from scrub typhus patients and healthy controls were stained with anti-CD3 and CD8 antibodies and analyzed by flow cytometry (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001789#pntd.0001789.s001" target="_blank">Figure S1</a>). The frequencies (% in lymphocytes) and absolute numbers (cells/mm²) of CD4⁺ or CD8⁺ T cells in PBMCs of the patients (AP, n = 15 and CP, n = 15) and healthy controls (HC, n = 9) were compared. The absolute number of each leukocyte subset was calculated based on the leukocyte differential counts and the frequency information obtained from flow cytometry. For example, CD4⁺ T cells count = (lymphocyte count) x (% of CD4+ T cells)/100. The data of each individual person are presented in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001789#pntd.0001789.s008" target="_blank">Table S3</a>. Red bars indicate the mean value and <i>p</i> values were obtained using the Mann-Whitney <i>U</i> test or Wilcoxon signed-rank test. Statistically significant <i>p</i> values (<0.05) are shown in bold.</p

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

Changes of effector and memory T cell subsets in scrub typhus patients.

<p>A. Representative contour plots show the frequencies of each subsets within CD4⁺ (upper panels) or CD8⁺ (lower panels) T cell populations. Numbers within the plots indicate the percentage of each subset. B. The frequencies of naïve (CCR7⁺CD45RA⁺), central memory (CM, CCR7⁺CD45RA⁻), and effector memory (EM, CCR7⁻CD45RA⁻), CD45RA⁺ EM (EM_CD45RA+, for CD8⁺ T cells), or CCR7⁻CD45RA⁺ (for CD4⁺ T cells) T-cell subsets in healthy controls (HC, n = 9) and scrub typhus patients at acute phase (AP, n = 15) or convalescent phase (CP, n = 17) were compared. Error bars indicate standard error of mean values. <i>p</i> values were obtained using the Mann-Whitney <i>U</i> test or Wilcoxon signed-rank test. *, <i>p</i><0.05; **, <i>p</i><0.01; and ***, <i>p</i><0.001 (compared with that of healthy control); †, <i>p</i><0.05 and ††, <i>p</i><0.01 (compared with that of the patients at acute phase).</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