Teik-Chye Chan

Scrub Typhus Vaccine Candidate Kp r56 Induces Humoral and Cellular Immune Responses in Cynomolgus Monkeys

ABSTRACT A truncated recombinant 56-kDa outer membrane protein of the Karp strain of Orientia tsutsugamushi (Kp r56) was evaluated in cynomolgus monkeys (Macaca fascicularis) for immunogenicity and safety as a vaccine candidate for the prevention of scrub typhus. This recombinant antigen induced strong humoral and cellular immune responses in two monkeys and was found to be well tolerated. Antigen-specific immunoglobulin M (IgM) and IgG were produced to almost maximal levels within 1 week of a single immunization. Peripheral blood mononuclear cells from vaccinated animals showed an induction of antigen-specific proliferation and gamma interferon production. The Kp r56 was not as efficient as infection with live organisms in preventing reinfection but was able to reduce the inflammation produced at the site of challenge. This report describes the results of the first systematic study of the immunogenicity of a recombinant scrub typhus vaccine candidate in a nonhuman primate model.

An Intradermal Inoculation Model of Scrub Typhus in Swiss CD-1 Mice Demonstrates More Rapid Dissemination of Virulent Strains of Orientia tsutsugamushi

Scrub typhus is an important endemic disease of the Asia-Pacific region caused by Orientia tsutsugamushi. To develop an effective vaccine to prevent scrub typhus infection, a better understanding of the initial host-pathogen interaction is needed. The objective of this study was to investigate early bacterial dissemination in a CD-1 Swiss outbred mouse model after intradermal injection of O. tsutsugamushi. Three human pathogenic strains of O. tsutsugamushi (Karp, Gilliam, and Woods) were chosen to investigate the early infection characteristics associated with bacterial virulence. Tissue biopsies of the intradermal injection site and draining lymph nodes were examined using histology and immunohistochemistry to characterize bacterial dissemination, and correlated with quantitative real-time PCR for O. tsutsugamushi in blood and tissue from major organs. Soluble adhesion molecules were measured to examine cellular activation in response to infection. No eschar formation was seen at the inoculation site and no clinical disease developed within the 7 day period of observation. However, O. tsutsugamushi was localized at the injection site and in the draining lymph nodes by day 7 post inoculation. Evidence of leukocyte and endothelial activation was present by day 7 with significantly raised levels of sL-selectin, sICAM-1 and sVCAM-1. Infection with the Karp strain was associated with earlier and higher bacterial loads and more extensive dissemination in various tissues than the less pathogenic Gilliam and Woods strains. The bacterial loads of O. tsutsugamushi were highest in the lungs and spleens of mice inoculated with Karp and Gilliam, but not Woods strains. Strains of higher virulence resulted in more rapid systemic infection and dissemination in this model. The CD-1 mouse intradermal inoculation model demonstrates features relevant to early scrub typhus infection in humans, including the development of regional lymphadenopathy, leukocyte activation and distant organ dissemination after low-dose intradermal injection with O. tsutsugamushi.

Development of a rapid method for determining the infectious dose (ID)50 of Orientia tsutsugamushi in a scrub typhus mouse model for the evaluation of vaccine candidates.

The infectious dose (ID) of an inoculum for which 50% of susceptible mice will become infected (ID(50)) with Orientia tsutsugamushi is usually determined by rechallenging mice that have already been challenged with O. tsutsugamushi to determine the lethal dose (LD)(50) titer of the inoculum. Those mice not killed by the initial challenge and which survived a rechallenge with 1000 LD(50) were considered immune and to have been initially infected with O. tsutsugamushi. Mice that succumbed to the rechallenge were considered not to have been initially infected. We have developed a more rapid method of determining the ID(50) of inocula for use in our vaccine studies based upon the observation that mice surviving initial challenge and that go on to survive rechallenge produced detectable IgG to O. tsutsugamushi antigens by enzyme-linked immunosorbent assay (ELISA). Mice that did not survive rechallenge, and therefore did not receive an initial infectious inoculum did not produce detectable IgG to O. tsutsugamushi antigens. Both original LD(50) and ID(50) titers determinations require observation of mice for 21 days post-challenge. Our new ID(50) determination does not require mice or the additional 21-day observation period for rechallenge and therefore is more rapid and cost-effective than the previous standard method of determining ID(50) titer necessary for the evaluation of vaccine candidates.

Short‐ and Long‐Term Immune Responses of CD‐1 Outbred Mice to the Scrub Typhus DNA Vaccine Candidate: p47Kp

Abstract: Orientia tsutsugamushi is an obligate intracellular bacterium that is the causative agent of scrub typhus. To develop an effective vaccine to prevent or ameliorate scrub typhus, knowledge of the protective immune response to O. tsutsugamushi needs to be ascertained. Our laboratory has demonstrated that the DNA vaccine vector pVR1012 carrying the O. tsutsugamushi Karp strain 47‐kDa protein gene (p47Kp) consistently provides outbred mice protection against homologous challenge.

Establishment of Orientia tsutsugamushi Lc-1 (Rickettsiales: Rickettsiaceae) Infection in ICR Outbred Mice (Rodentia: Muridae) by Needle Challenge

ABSTRACT Orientia tsutsugamushi is a pathogen transmitted by Leptotrombidium that causes scrub typhus. To develop an infection mouse model, a mite-derived isolate of O. tsutsugamushi was established from a laboratory-maintained colony of Leptotrombidium chiangraiensis (O. tsutsugamushi Lc-1). This Lc-1 isolate was initially presented to ICR (CD-1) mice by feeding an infected Lc chigger on the ear of a mouse. Once the Lc-1 was adapted to the ICR mice, quantitative real-time polymerase chain reaction was used to investigate O. tsutsugamushi genomic equivalent copies in tissues and sera. Furthermore, times to onset of the signs of infection are reported in this study. This study provides information useful for future research on this host-pathogen interaction and the associated vaccine efficacy trials.

Development of an Orientia tsutsugamushi Lc-1 Murine Intraperitoneal Challenge Model for Scrub Typhus: Determination of Murine Lethal Dose (MuLD50), Tissue Bacterial Loads, and Clinical Outcomes

Currently, no vaccine has been developed to protect humans from naturally acquired heterologous Orientia tsutsugamushi infections. To enhance the validity of vaccine candidates, we are developing a murine chigger challenge model with the O. tsutsugamushi Lc-1-infected Leptotrombidium chiangraiensis Line-1. To this end, an intraperitoneal (i.p.) murine challenge model using an O. tsutsugamushi Lc-1 isolate was developed for eventual validation of the chigger challenge model. We have determined that the murine lethal dose that kills 50% of the challenged mice (MuLD50) of a liver/spleen homogenate developed from O. tsutsugamushi Lc-1-infected ICR Swiss mice to be 10(-6.9). Employing different inoculum doses of this homogenate, the bacterial load using quantitative real-time PCR (qPCR) was determined to range from 60 to 1.6 × 10(5) genome equivalent copies (GEC)/μL of liver and 33.4 to 2.2 × 10(5) GEC/μL of spleen tissue. The clinical outcomes relative to homogenate dose levels followed a dose-dependent pattern. The successful development and characterization of the O. tsutsugamushi Lc-1 i.p. challenge model will assist in the development and validation of a mouse chigger challenge scrub typhus model.

Dissemination of Orientia tsutsugamushi, a Causative Agent of Scrub Typhus, and Immunological Responses in the Humanized DRAGA Mouse

Scrub typhus is caused by Orientia tsutsugamushi, an obligated intracellular bacterium that affects over one million people per year. Several mouse models have been used to study its pathogenesis, disease immunology, and for testing vaccine candidates. However, due to the intrinsic differences between the immune systems in mouse and human, these mouse models could not faithfully mimic the pathology and immunological responses developed by human patients, limiting their value in both basic and translational studies. In this study, we have tested for the first time, a new humanized mouse model through footpad inoculation of O. tsutsugamushi in DRAGA (HLA-A2.HLA-DR4.Rag1KO.IL2RγcKO.NOD) mice with their human immune system reconstituted by infusion of HLA-matched human hematopoietic stem cells from umbilical cord blood. Upon infection, Orientia disseminated into various organs of DRAGA mice resulted in lethality in a dose-dependent manner, while all C3H/HeJ mice infected by the same route survived. Tissue-specific lesions associated with inflammation and/or necroses were observed in multiple organs of infected DRAGA mice. Consistent with the intracellular nature of Orientia, strong Th1, but subdued Th2 responses were elicited as reflected by the human cytokine profiles in sera from infected mice. Interestingly, the percentage of both activated and regulatory (CD4+FOXP3+) human T cells were elevated in spleen tissues of infected mice. After immunization with irradiated whole cell Orientia, humanized DRAGA mice showed a significant activation of human T cells as evidenced by increased number of human CD4+ and CD8+ T cells. Specific human IgM and IgG antibodies were developed after repetitive immunization. The humanized DRAGA mouse model represents a new pre-clinical model for studying Orientia-human interactions and also for testing vaccines and novel therapeutics for scrub typhus.

Comparative pan-genomic analyses of Orientia tsutsugamushi reveal an exceptional model of bacterial evolution driving genomic diversity

Orientia tsutsugamushi, formerly Rickettsia tsutsugamushi, is an obligate intracellular pathogen that causes scrub typhus, an underdiagnosed acute febrile disease with high morbidity. Scrub typhus is transmitted by the larval stage (chigger) of Leptotrombidium mites and is irregularly distributed across endemic regions of Asia, Australia and islands of the western Pacific Ocean. Previous work to understand population genetics in O. tsutsugamushi has been based on sub-genomic sampling methods and whole-genome characterization of two genomes. In this study, we compared 40 genomes from geographically dispersed areas and confirmed patterns of extensive homologous recombination likely driven by transposons, conjugative elements and repetitive sequences. High rates of lateral gene transfer (LGT) among O. tsutsugamushi genomes appear to have effectively eliminated a detectable clonal frame, but not our ability to infer evolutionary relationships and phylogeographical clustering. Pan-genomic comparisons using 31 082 high-quality bacterial genomes from 253 species suggests that genomic duplication in O. tsutsugamushi is almost unparalleled. Unlike other highly recombinant species where the uptake of exogenous DNA largely drives genomic diversity, the pan-genome of O. tsutsugamushi is driven by duplication and divergence. Extensive gene innovation by duplication is most commonly attributed to plants and animals and, in contrast with LGT, is thought to be only a minor evolutionary mechanism for bacteria. The near unprecedented evolutionary characteristics of O. tsutsugamushi, coupled with extensive intra-specific LGT, expand our present understanding of rapid bacterial evolutionary adaptive mechanisms.