An Alternative Splicing Variant of the Mixed-Lineage Leukemia 5 Protein Is a Cellular Adhesion Receptor for ScaA of Orientia tsutsugamushi.

Scrub typhus is a mite-borne disease caused by the obligately intracellular bacterium Orientia tsutsugamushi. We previously demonstrated that ScaA, an autotransporter membrane protein of O. tsutsugamushi, is commonly shared in various genotypes and involved in adherence to host cells. Here, we identified a mixed-lineage leukemia 5 (MLL5) mammalian trithorax group protein as a host receptor that interacts with ScaA. MLL5, identified by yeast two-hybrid screening, is an alternative splicing variant of MLL5 (vMLL5) which contains 13 exons with additional intron sequences encoding a tentative transmembrane domain. Indeed, vMLL5 is expressed on the plasma membrane as well as in intracellular compartments in eukaryotic cells and colocalized with adherent O. tsutsugamushi. In addition, ScaA-expressing Escherichia coli showed significantly increased adherence to vMLL5-overexpressing cells compared with vector control cells. We mapped the C-terminal region of the passenger domain of ScaA as a ligand for vMLL5 and determined that the Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain of MLL5 is an essential and sufficient motif for ScaA binding. We observed significant and specific inhibition of bacterial adhesion to host cells in competitive inhibition assays using the C-terminal fragment of ScaA or the SET domain of vMLL5. Moreover, immunization with the C-terminal fragment of ScaA provided neutralizing activity and protective immunity against lethal challenge with O. tsutsugamushi as efficiently as vaccination with the whole passenger domain of ScaA. These results indicate that vMLL5 is a novel cellular receptor for ScaA-mediated adhesion of O. tsutsugamushi and facilitates bacterial adhesion to host cells, thereby enhancing bacterial infection. IMPORTANCE O. tsutsugamushi is a mite-borne pathogen that causes scrub typhus. As an obligately intracellular pathogen, its adhesion to and invasion of host cells are critical steps for bacterial growth. However, the molecular basis of the bacterial ligand and host receptor interaction is poorly defined. Here, we identified a splicing variant of MLL5 (vMLL5) as a cellular adhesion receptor of ScaA, an outer membrane autotransporter protein of O. tsutsugamushi. We mapped the interacting domains in the bacterial ligand and host receptor and confirmed their functional interaction. In addition, immunization with the C-terminal region of ScaA, which involves an interaction with the SET domain of vMLL5, not only induces enhanced neutralizing antibodies but also provides protective immunity against lethal challenge with O. tsutsugamushi.

Epidemiological characteristics and spatiotemporal patterns of scrub typhus in Yunnan Province from 2006 to 2017.

Scrub typhus is an acute infectious disease in humans. A temporal, spatial and epidemiologic study was conducted to understand the characteristics of scrub typhus in Yunnan, to assist public health prevention and control measures. Based on the data on all cases reported in Yunnan during 2006-2017, we characterized the epidemiological features. Spatio-temporal patterns and Q-type cluster method were adopted to analyze the incidence of scrub typhus in Yunnan. In total, 27,838 scrub typhus cases were reported in Yunnan during 2006-2017. Of these, 49.53% (13,787) were male and 50.47% (14,051) were female (P > 0.05). Most patients were farmers (71.70%) (P < 0.05) and children aged 0-5 years (13.16%) (P < 0.01), which accounted for 84.86% of the total cases. An almost 20-fold increase in the number of patients was observed in 2017 (6,337 cases) compared to 2006 (307 cases). Baoshan and Lincang had the most cases accounting for 41.94%, while Diqing had the lowest incidence (only 3 cases). Sixteen municipalities infected were classified into three groups numbered in sequence. The incidence of scrub typhus in Yunnan is high and the annual incidence increased noticeably over time. Our results also indicate that surveillance and public education need to be focused on Baoshan, Lincang and Dehong.

The Effect of Long-lasting Permethrin Impregnated Socks on Tick Bite in Korea.

BACKGROUND: The risk of tick-borne diseases is decreased by increasing awareness and knowledge through prevention education. The aim of the present study was to evaluate the effect of long-lasting permethrin impregnated (LLPI) socks for tick bites. METHODS: A randomized open label study was conducted to determine the effectiveness of LLPI socks for prevention of tick bites among 367 adults living in a rural area. Participants completed questionnaires at the start of follow-up (July 2014) and at the end of follow-up (December 2014), and tick bites were reported. RESULTS: A total of 332 subjects completed the follow-up survey. The tick bite rate of the two groups was not significantly different (3.6% vs. 3.1%). But the tick bite rate of lower extremities of subjects wearing LLPI socks was significantly lower compared to that of subjects wearing general socks. CONCLUSION: The tick bite rate was not different between the two groups, but the tick bite rate of lower extremities of LLPI was significantly lower than general groups. Further study is needed to investigate the effect of LLPI clothes with larger populations.

Epidemiological Characteristics of Rodents and Chiggers with Orientia Tsutsugamushi in the Republic of Korea.

A survey of rodents and chiggers associated with Orientia tsutsugamushi was conducted in a rural region of the Republic of Korea (Korea) between 2014 and 2018. Overall Apodemus agrarius 15.2% had the highest seropisitive for O. tsutsugamushi, followed by Myodes regulus 11.4%. Monthly risk factors using logistic regression analysis were not associated with O. tsutsugamushi infections in rodents. The overall prevalence rate of O. tsutsugamushi among chiggers was 0.3%. The chigger (Leptotrombidium scutellare) and monthly (October) risk factors were associated with O. tsutsugamushi human infections (P<0.05). Orientia tsutsugamushi infections are endemic in rodents in Korea and people, for example, soldiers who are active outdoors, must employ preventive measures, especially during October (P<0.05). When there are many reports of O. tsutsugamushi infections in Korea. The Boryong strain 85.7% (2/14) was the most common strain detected in chiggers, followed by the Shimokoshi 7.1% (1/14) and Karp 7.1% strains.

Scrub Typhus.

Scrub typhus, also known as tsutsugamushi disease, is caused by Orientia sp. and approximately 1 million new cases are reported annually. This article discusses the importance of scrub typhus and its clinical presentation, diagnosis, treatment, and prevention.

Extracellular vesicle-associated antigens as a new vaccine platform against scrub typhus.

Scrub typhus is an acute vector-borne disease caused by infection with the intracellular gram-negative bacterium Orientia tsutsugamushi (Ot). The rapid production of an efficient vaccine against Ot using novel strategies is required because of the global increase in mortality caused by these infections; however, no commercial vaccine is currently available. Ot induces T-cell-mediated immunogenic responses upon infection; therefore, a new rapidly producible vaccine that maximizes T-cell responses against Ot is required. In this study, we sought to develop a model vaccine platform for T-cell-mediated Ot infection using T-cell-immunity associated Salmonella-derived extracellular vesicles (EVs). For this purpose, we optimized DNA sequences encoding the full-length Ot proteins, TSA56, ScaA, ScaC, ScaD, and ScaE, and their expression in Salmonella. The sequences were incorporated into a new platform vector, pKST, which ectopically and concurrently produces Ot proteins and EVs. Expression analysis using pKST-antigen plasmids showed that TSA56 and ScaC produced antigen-associated EVs and showed strong T-cell immunogenic responses. We found that mice vaccinated with EVs derived from TSA56-expressing cells were protected from Salmonella-induced mortality. Therefore, our findings showed that Salmonella EV-associated antigen is a model platform for T-cell immune response infections. Our system could help prepare EV-antigen vaccines against scrub typhus in an easy and rapid manner.

Immunization with a recombinant antigen composed of conserved blocks from TSA56 provides broad genotype protection against scrub typhus.

Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Despite the wide range of approaches explored during the last seventy years, an effective prophylactic vaccine is not yet available. Here, we developed a novel recombinant antigen derived from conserved regions of 56 kDa type-specific antigen (TSA56), a major outer membrane protein responsible for genetic heterogeneity and antigenicity, and evaluated it as a protective vaccine antigen. Our findings demonstrate that immunization with conserved blocks of TSA56 (cTSA56) not only provides protective immunity against lethal challenges with the homologous genotype, but also confers significantly better protection against heterologous genotypes than TSA56. Adoptive transfer of CD4+ or CD8+ T cells from immunized mice provided significantly enhanced protection against lethal challenge, whereas immune B cells failed to do so, indicating that cellular immunity against the conserved epitopes plays a protective role. Moreover, immunization with a 10-mer peptide mixture, screened from CD8+ T cell epitopes within the conserved region of TSA56, provided enhanced protection against lethal challenge with O. tsutsugamushi. Therefore, this novel recombinant antigen is a promising candidate for scrub typhus vaccine against a wide range of O. tsutsugamushi genotypes.

Identification of B- and T-cell epitopes on HtrA protein of Orientia tsutsugamushi.

Orientia tsutsugamushi, a cause of scrub typhus is emerging as an important pathogen in several parts of the tropics. The control of this infection relies on rapid diagnosis, specific treatment, and prevention through vector control. Development of a vaccine for human use would be very important as a public health measure. Antibody and T-cell response have been found to be important in the protection against scrub typhus. This study was undertaken to predict the peptide vaccine that elicits both B- and T-cell immunity. The outer-membrane protein, 47-kDa high-temperature requirement A was used as the target protein for the identification of protective antigen(s). Using BepiPred2 program, the potential B-cell epitope PNSSWGRYGLKMGLR with high conservation among O. tsutsugamushi and the maximum surface exposed residues was identified. Using IEDB, NetMHCpan, and NetCTL programs, T-cell epitopes MLNELTPEL and VTNGIISSK were identified. These peptides were found to have promiscuous class-I major histocompatibility complex (MHC) binding affinity to MHC supertypes and high proteasomal cleavage, transporter associated with antigen processing prediction, and antigenicity scores. In the I-TASSER generated model, the C-score was -0.69 and the estimated TM-score was 0.63 ± 0.14. The location of the epitope in the 3D model was external. Therefore, an antibody to this outer-membrane protein epitope could opsonize the bacterium for clearance by the reticuloendothelial system. The T-cell epitopes would generate T-helper function. The B-cell epitope(s) identified could be evaluated as antigen(s) in immunodiagnostic assays. This cocktail of three peptides would elicit both B- and T-cell immune response with a suitable adjuvant and serve as a vaccine candidate.

Spatiotemporal Patterns and Risk Factors for Scrub Typhus From 2007 to 2017 in Southern China.

BACKGROUND: Substantial outbreaks of scrub typhus, coupled with the discovery of this vector-borne disease in new areas, suggest that the disease remains remarkably neglected. The objectives of this study were to map the contemporary and potential transmission risk zones of the disease and to provide novel insights into the health burden imposed by scrub typhus in southern China. METHODS: Based on the assembled data sets of annual scrub typhus cases and maps of environmental and socioeconomic correlates, a boosted regression tree modeling procedure was used to identify the environmental niche of scrub typhus and to predict the potential infection zones of the disease. Additionally, we estimated the population living in the potential scrub typhus infection areas in southern China. RESULTS: Spatiotemporal patterns of the annual scrub typhus cases in southern China between 2007 and 2017 reveal a tremendous, wide spread of scrub typhus. Temperature, relative humidity, elevation, and the normalized difference vegetation index are the main factors that influence the spread of scrub typhus. In southern China, the predicted highest transmission risk areas of scrub typhus are mainly concentrated in several regions, such as Yunnan, Guangxi, Guangdong, Hainan, and Fujian. We estimated that 162 684 million people inhabit the potential infection risk zones in southern China. CONCLUSIONS: Our results provide a better understanding of the environmental and socioeconomic factors driving scrub typhus spread, and estimate the potential infection risk zones beyond the disease's current, limited geographical extent, which enhances our capacity to target biosurveillance and help public health authorities develop disease control strategies.

[Epidemiology of scrub typhus and influencing factors in Yunnan province, 2006-2013].

Objective: To understand the epidemiological characteristics of scrub typhus and influencing factors in Yunnan province, and provide further information for the prevention and control of scrub typhus. Methods: Based on the incidence data of scrub typhus reported in Yunnan from 2006 to 2013, the epidemiological characteristics of scrub typhus were analyzed and related environmental factors were identified with panel negative binomial regression model. Results: A total of 8 980 scrub typhus cases were reported during 2006-2013 in Yunnan. The average annual incidence was 2.46/100 000, with an uptrend observed. Natural focus expansion was found, affecting 71.3% of the counties in 2013. The epidemic mainly occurred in summer and autumn with the incidence peak during July-October. The annual incidence was higher in females than in males. More cases occurred in children and farmers, the proportions of cases in farmers and pre-school aged children showed an obvious increase. Panel negative binomial regression model indicated that the transmission risk of scrub typhus was positive associated with monthly temperature and monthly relative humidity. Furthermore, an "U" pattern between the risk and the increased coverage of cropland and grassland as well as an "inverted-U" pattern between the risk and increased coverage of shrub were observed. Conclusion: It is necessary to strengthen the scrub typhus surveillance in warm and moist areas as well as the areas with high coverage of cropland and grassland in Yunnan, and the health education in children and farmers who are at high risk.

A review of the global epidemiology of scrub typhus.

Scrub typhus is a serious public health problem in the Asia-Pacific area. It threatens one billion people globally, and causes illness in one million people each year. Caused by Orientia tsutsugamushi, scrub typhus can result in severe multiorgan failure with a case fatality rate up to 70% without appropriate treatment. The antigenic heterogeneity of O. tsutsugamushi precludes generic immunity and allows reinfection. As a neglected disease, there is still a large gap in our knowledge of the disease, as evidenced by the sporadic epidemiologic data and other related public health information regarding scrub typhus in its endemic areas. Our objective is to provide a systematic analysis of current epidemiology, prevention and control of scrub typhus in its long-standing endemic areas and recently recognized foci of infection.

Differences in agricultural activities related to incidence of scrub typhus between Korea and Japan.

OBJECTIVES: The purpose of this study was to establish a basis for improving or strengthening the preventive strategy against scrub typhus in Korea by comparing and analyzing the difference of prevention behaviors contributing to the occurrence of scrub typhus in Japan and Korea. METHODS: The survey was carried out in Jeollabuk-do, which is a high risk and high incidence area, and Fukuoka Prefecture, which is a high risk and low incidence area. The study included 406 Korean farmers and 216 Japanese farmers. Data were collected through face-to-face surveys by interviewers who had completed standardized education. RESULTS: Korean farmers have a higher percentage of agricultural working posture that involved contact with weeds than Japanese farmers (p<0.05). The frequency and proportion of weeding were lower in Korean farmers than in Japanese farmers (p<0.05). The level of knowledge about scrub typhus was significantly higher among Korean farmers than among Japanese farmers (p<0.05). Mostly, the behavior of agriculture work was more appropriate for Japanese farmers than for Korean farmers (p<0.05). The total average level of agricultural work was lower in Korea than in Japan, lower in men than women, and lower in part-time farmers than full-time farmers (p<0.05). CONCLUSIONS: This study suggests that it is reasonable to develop and provide a program that can improve the level of preventive behavior taking into consideration the characteristics of the subject in order to reduce the incidence of diseases in high-risk areas for scrub typhus.

A Recombinant 47-kDa Outer Membrane Protein Induces an Immune Response against Orientia tsutsugamushi Strain Boryong.

We investigated the 47-kDa outer membrane protein (OMP), which is a periplasmic serine protease and an antigenic major surface protein of Orientia tsutsugamushi, as a vaccine candidate. We developed a conventional subunit vaccine expressing recombinant 47-kDa OMP (rec47) and a DNA vaccine (p47). In mouse immunization experiments, intranasal immunization with rec47 alone or with rec47 plus heat-labile enterotoxin B subunit from Escherichia coli or plus cholera toxin (CT) as adjuvants induced a higher amount of rec47-specific antibodies than intramuscular immunization with p47 alone or with p47 plus pBOOST2-samIRF7/3 (pB) as adjuvant. Moreover, the combination of rec47 and CT induced a strong cellular immune response to 47-kDa OMP, as demonstrated by a spleen cell proliferation assay, and also induced Th1- and Th2-type cytokine production, as demonstrated by a cytokine enzyme-linked immunosorbent assay. Intranasal immunization with rec47 plus CT was the most effective method for the induction of humoral and cell-mediated immune responses. Furthermore, relatively strong protection against homologous O. tsutsugamushi strain Boryong challenge was observed in mice immunized with rec47 plus CT. Therefore, 47-kDa OMP is an attractive candidate for developing a prophylactic vaccine against scrub typhus by O. tsutsugamushi infection.

Generation of protective immunity against Orientia tsutsugamushi infection by immunization with a zinc oxide nanoparticle combined with ScaA antigen.

BACKGROUND: Zinc oxide nanoparticle (ZNP) has been applied in various biomedical fields. Here, we investigated the usage of ZNP as an antigen carrier for vaccine development by combining a high affinity peptide to ZNP. RESULTS: A novel zinc oxide-binding peptide (ZBP), FPYPGGDA, with high affinity to ZNP (K a  = 2.26 × 106 M-1) was isolated from a random peptide library and fused with a bacterial antigen, ScaA of Orientia tsutsugamushi, the causative agent of scrub typhus. The ZNP/ZBP-ScaA complex was efficiently phagocytosed by a dendritic cell line, DC2.4, in vitro and significantly enhanced anti-ScaA antibody responses in vivo compared to control groups. In addition, immunization with the ZNP/ZBP-ScaA complex promoted the generation of IFN-γ-secreting T cells in an antigen-dependent manner. Finally, we observed that ZNP/ZBP-ScaA immunization provided protective immunity against lethal challenge of O. tsutsugamushi, indicating that ZNP can be used as a potent adjuvant when complexed with ZBP-conjugated antigen. CONCLUSIONS: ZNPs possess good adjuvant potential as a vaccine carrier when combined with an antigen having a high affinity to ZNP. When complexed with ZBP-ScaA antigen, ZNPs could induce strong antibody responses as well as protective immunity against lethal challenges of O. tsutsugamushi. Therefore, application of ZNPs combined with a specific soluble antigen could be a promising strategy as a novel vaccine carrier system.

An outbreak of scrub typhus in military personnel despite protocols for antibiotic prophylaxis: doxycycline resistance excluded by a quantitative PCR-based susceptibility assay.

Scrub typhus is caused by the obligate intracellular bacterium Orientia tsutsugamushi and is endemic to many countries in the Asia-Pacific region, including tropical Australia. We describe a recent large outbreak amongst military personnel in north Queensland. A total of 45 clinical cases were identified (36% of all potentially exposed individuals). This occurred despite existing military protocols stipulating the provision of doxycycline prophylaxis. Doxycycline resistance in O. tsutsugamushi has been described in South-East Asia, but not Australia. In one case, O. tsutsugamushi was cultured from eschar tissue and blood. Using quantitative real-time PCR to determine susceptibility to doxycycline for the outbreak strain, a minimum inhibitory concentration (MIC) of ≤0.04 µg/mL was found, indicating susceptibility to this agent. It seems most probable that failure to adhere to adequate prophylaxis over the duration of the military exercise accounted for the large number of cases encountered rather than doxycycline resistance.

Scrub typhus in Bhutan: a synthesis of data from 2009 to 2014.

Scrub typhus is an acute, febrile illness, caused by the bacterium Orientia tsutsugamushi, that affects millions annually in the endemic Asia-Pacific region. In untreated cases, the case-fatality rates range from 6% to 35%. In Bhutan, there was a probable outbreak in Gedu in 2009, which resulted in heightened awareness of the disease. Nevertheless, information on scrub typhus in Bhutan is limited and scattered and the epidemiology has yet to be established. To report the current picture of scrub typhus in Bhutan, this review gathered data from scholarly databases, surveillance reports, the Annual health bulletin, research publications and laboratory test reports from hospitals. The weight of evidence indicates an increasing burden of scrub typhus since the Gedu incident, coupled with increased awareness and testing. Another outbreak in a rural primary school in 2014 resulted in two deaths. More hospitals now have testing facilities and laboratory-confirmed cases have been increasing since 2009, with seasonal trends. This review highlights the need for in-depth surveillance and reporting, increased awareness among health-care workers, and initiation of prevention and control programmes in the country.

Immunization with an autotransporter protein of Orientia tsutsugamushi provides protective immunity against scrub typhus.

BACKGROUND: Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi 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 O. tsutsugamushi. METHODOLOGY/PRINCIPAL FINDINGS: We examined the potential role of ScaA protein, an autotransporter protein of O. tsutsugamushi, in bacterial pathogenesis and evaluated the protective attributes of ScaA immunization in lethal O. tsutsugamushi 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 O. tsutsugamushi. CONCLUSIONS/SIGNIFICANCE: 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.

A nonhuman primate scrub typhus model: protective immune responses induced by pKarp47 DNA vaccination in cynomolgus macaques.

We developed an intradermal (ID) challenge cynomolgus macaque (Macaca fascicularis) model of scrub typhus, the leading cause of treatable undifferentiated febrile illness in tropical Asia, caused by the obligate intracellular bacterium, Orientia tsutsugamushi. A well-characterized animal model is required for the development of clinically relevant diagnostic assays and evaluation of therapeutic agents and candidate vaccines. We investigated scrub typhus disease pathophysiology and evaluated two O. tsutsugamushi 47-kDa, Ag-based candidate vaccines, a DNA plasmid vaccine (pKarp47), and a virus-vectored vaccine (Kp47/47-Venezuelan equine encephalitis virus replicon particle) for safety, immunogenicity, and efficacy against homologous ID challenge with O. tsutsugamushi Karp. Control cynomolgus macaques developed fever, classic eschars, lymphadenopathy, bacteremia, altered liver function, increased WBC counts, pathogen-specific Ab (IgM and IgG), and cell-mediated immune responses. Vaccinated macaques receiving the DNA plasmid pKarp47 vaccine had significantly increased O. tsutsugamushi-specific, IFN-γ-producing PBMCs (p = 0.04), reduced eschar frequency and bacteremia duration (p ≤ 0.01), delayed bacteremia onset (p < 0.05), reduced circulating bacterial biomass (p = 0.01), and greater reduction of liver transaminase levels (p < 0.03) than controls. This study demonstrates a vaccine-induced immune response capable of conferring sterile immunity against high-dose homologous ID challenge of O. tsutsugamushi in a nonhuman primate model, and it provides insight into cell-mediated immune control of O. tsutsugamushi and dissemination dynamics, highlights the importance of bacteremia indices for evaluation of both natural and vaccine-induced immune responses, and importantly, to our knowledge, has determined the first phenotypic correlates of immune protection in scrub typhus. We conclude that this model is suitable for detailed investigations into vaccine-induced immune responses and correlates of immunity for scrub typhus.

Protective immunity of 56-kDa type-specific antigen of Orientia tsutsugamushi causing scrub typhus.

Scrub typhus, caused by infection with Orientia tsutsugamushi, is a mite-borne zoonotic disease endemic to the Asian-Pacific region. In Korea, the incidence of this disease has increased with climate changes, and over 10,000 cases of infection were reported in 2013. Although this infection is treatable with antibiotics such as doxycycline and azithromycin, an effective prophylactic vaccine against O. tsutsugamushi would be more desirable for preventing scrub typhus in endemic areas. In this study, we investigated the 56-kDa type-specific antigen (TSA56), which is a major outer membrane protein of O. tsutsugamushi, as a vaccine candidate. Intranasal immunization of recombinant TSA56 (rec56) induced a higher level of TSA56- specific IgG than that induced by intramuscular immunization of tsa56-expressing DNA (p56). Both types of immunization induced a cell-mediated immune response to TSA56, as demonstrated by the splenic cell proliferation assay. Mice immunized with p56, followed by rec56 plus heat-labile enterotoxin B subunit from E. coli, had a stronger protection from a homologous challenge with the O. tsutsugamushi Boryong strain than with other combinations. Our preliminary results suggest that an effective human vaccine for scrub typhus can include either recombinant TSA56 protein or tsa56-expressing DNA, and provide the basis for further studies to optimize vaccine performance using additional antigens or different adjuvants.

Diversity of the 47-kD HtrA nucleic acid and translated amino acid sequences from 17 recent human isolates of Orientia.

Orientia tsutsugamushi, the etiologic agent of potentially fatal scrub typhus, is characterized by a high antigenic diversity, which complicates the development of a broadly protective vaccine. Efficacy studies in murine and nonhuman primate models demonstrated the DNA vaccine candidate pKarp47, based upon the O. tsutsugamushi Karp 47-kD HtrA protein gene, to be a successful immunoprophylactic against scrub typhus. To characterize 47-kD HtrA protein diversity among human isolates of Orientia, we sequenced the full open reading frame (ORF) of the 47-kD HtrA gene and analyzed the translated amino acid sequences of 17 patient isolates from Thailand (n=13), Laos (n=2), Australia (n=1), and the United Arab Emirates (UAE) (n=1) and 9 reference strains: Karp (New Guinea), Kato (Japan), Ikeda (Japan), Gilliam (Burma), Boryong (Korea), TA763, TH1811 and TH1817 (Thailand), and MAK243 (China). The percentage identity (similarity) of translated amino acid sequences between 16 new isolates and 9 reference strains of O. tsutsugamushi ranged from 96.4% to 100% (97.4% to 100%). However, inclusion of the recently identified Orientia chuto sp. nov. reduced identity (similarity) values to 82.2% to 83.3% (90.4% to 91.4%). These results demonstrate the diversity of Orientia 47-kD HtrA among isolates encountered by humans and therefore provide support for the necessity of developing a broadly protective scrub typhus vaccine that takes this diversity into account.