PLOS NEGLECTED TROPICAL DISEASES

REVIEW

The neglected challenge: Vaccination against

rickettsiae
Anke Osterloh ID*
Research Center Borstel, Schleswig-Holstein, Germany

* aosterloh@fz-borstel.de

Abstract
Over the last decades, rickettsioses are emerging worldwide. These diseases are caused
by intracellular bacteria. Although rickettsioses can be treated with antibiotics, a vaccine
against rickettsiae is highly desired for several reasons. Rickettsioses are highly prevalent,
especially in poor countries, and there are indications of the development of antibiotic resis-
tance. In addition, some rickettsiae can persist and cause recurrent disease. The develop-
a1111111111 ment of a vaccine requires the understanding of the immune mechanisms that are involved
a1111111111
in protection as well as in immunopathology. Knowledge about these immune responses is
a1111111111
a1111111111 accumulating, and efforts have been undertaken to identify antigenic components of rickett-
a1111111111 siae that may be useful as a vaccine. This review provides an overview on current knowl-
edge of adaptive immunity against rickettsiae, which is essential for defense, rickettsial
antigens that have been identified so far, and on vaccination strategies that have been used
in animal models of rickettsial infections.
OPEN ACCESS

Citation: Osterloh A (2020) The neglected

challenge: Vaccination against rickettsiae. PLoS
Negl Trop Dis 14(10): e0008704. https://doi.org/ Introduction
10.1371/journal.pntd.0008704
Rickettsioses are emerging febrile infectious diseases that are caused by small obligate intracel-
Editor: Jeanne Salje, University of Oxford, UNITED
lular bacteria of the family of Rickettsiaceae that consists of 2 genuses, Rickettsia and Orientia.
KINGDOM
The genus Orientia contains at least 2 closely related members, Orientia tsutsugamushi and
Published: October 22, 2020 Orienta chuto [1] and maybe further, so far unnamed, novel Orientia species [2]. The genus
Copyright: © 2020 Anke Osterloh. This is an open Rickettsia is further divided into 3 major groups of pathogenic bacteria: the spotted fever
access article distributed under the terms of the group (SFG), the typhus group (TG), and the transitional group. The majority of rickettsiae,
Creative Commons Attribution License, which approximately 20 species, belongs to the SFG that also causes the majority of infections world-
permits unrestricted use, distribution, and
wide; prominent members being Rickettsia rickettsii; the causative agent of Rocky Mountain
reproduction in any medium, provided the original
author and source are credited. Spotted Fever (RMSF); Rickettsia conorii that causes Meditarranean Spotted Fever; Rickettsia
africae that is the etiologic agent of African Tick Bite Fever (ATBF); and Rickettsia parkeri.
Funding: Anke Osterloh is funded by the Deutsche
Rickettsia prowazekii and Rickettsia typhi are the 2 TG members and the causative agents of
Forschungsgemeinschaft (DFG, #OS 583/1-1) and
received the Memento Award 2020. The funders
epidemic and endemic typhus, while Rickettsia felis, Rickettsia akari, and Rickettsia australis
had no role in study design, data collection and build the transitional group of pathogenic rickettsiae.
analysis, decision to publish, or preparation of the Rickettsiae are zoonotic pathogens that are transmitted to humans by the bite of ticks or via
manuscript. the feces of infected lice and fleas, while Orientia species are transmitted by chiggers. Except
Competing interests: The authors have declared for R. prowazekii, which is transmitted from human to human via the body louse, rodents
that no competing interests exist. commonly serve as natural reservoirs for the bacteria [3]. However, transmission of Orientia

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 1 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

from rodents has not been formally proven. Some SFG rickettsiae (e.g., R. conorii) and O. tsu-
tsugamushi usually cause a characteristic eschar at the site of entry, while an eschar is not
observed in the infection with R. rickettsii and TG rickettsiae. Further symptoms of disease,
which are noticeable after approximately 7 to 14 days, are quite unspecific. With the onset of
disease, patients commonly present with high fever, and a high percentage shows a characteris-
tic spotted skin rash on days 3 to 5 of fever, which is caused by local bleedings and inflamma-
tory reactions. In the initial phase immediately after entering the body, rickettsiae first infect
phagocytic cells in the skin and then spread into endothelial cells (ECs) that build the inner
wall of the blood vessels [4,5], where they replicate free in the cytosol. SFG rickettsiae can
spread from cell to cell driven by actin-based motility without destroying the cell [6–8], while
TG rickettsiae are considered to accumulate in the cell until lysis [6]. In contrast, Orientia has
been shown to induce a kind of budding and leaves infected cells coated by cellular membrane
[9]. After release, the bacteria spread into adjacent cells and distribute in the body via the
blood stream. Rickettsiae can then enter nearly all tissues and organs and infect other cells,
especially monocytes/macrophages (MF) [4,5,10] that are also proposed to serve as a replica-
tive niche [11] and as a vehicle for the transport of the bacteria for dissemination through the
blood [12,13]. In vitro, rickettsiae can infect nonimmune cells including hepatocytes [14,15],
smooth muscle cells [16], neurons [17], and fibroblasts. The latter are also commonly used for
the in vitro culture of the bacteria for research and diagnostic purposes [18–21]. Whether neu-
rons or fibroblasts can get infected in vivo is not clear. Infection of these cells in patients has
not been observed.
Rickettsiae systemically spread in the body and can cause multi-organ failure with fatal out-
come. Complications that are often observed in severe cases of rickettsial infections are inter-
stitial pneumonia, meningoencephalitis or meningitis, myocarditis, nephritis, and liver
necrosis [22,23]. However, depending on the rickettsial species, the severity of disease and
lethality strongly varies. The highest lethality is observed for the infection with R. rickettsii,
nowadays 1%–7% [24] and 23% in the preantiobic era, and for the infection with R. prowazekii
(15% up to 30% under circumstances of poverty, starvation, and lack of nursing care
[23,25,26]). For that reason, these rickettsial species are classified as potential bioweapons.
With regard to the potential use of R. prowazekii and R. rickettsii as bioweapons, it is impor-
tant to mention that genetic manipulation of these agents to acquire antibiotic resistance is
possible. Meanwhile, genetic engineering has been shown for several rickettsial species [27–
32]. In addition, targeted gene knockout by homologous recombination was successful for R.
prowazekii [33] and for R. rickettsii employing a targetron plasmid vector [34]. Similar genetic
techniques may be used to introduce factors that enhance the pathogenicity of these potential
bioweapons.
Apart from the consideration of rickettsiae as potential bioweapons, rickettsioses are quite
common but still neglected and underdiagonized diseases that predominantly affect people in
poor countries where standards of hygiene are low. So far, the presence of O. chuto is consid-
ered to be restricted to the United Arab Emirates [1,3], while O. tsutsugamushi is common in
the Asia-Pacific region reaching from southern parts of eastern Russia, over Japan to China,
India, Pakistan, Indonesia, the north of Australia, and Afghanistan [35]. Scrub typhus is the
most common rickettsiosis in India [35]. R. typhi generally occurs worldwide and is also
endemic in Asia. Typhus, most likely the infection with R. typhi rather than R. prowazekii, is a
serious threat to public health in China, mainly northern China [36], where farmers and the
elderly are at enhanced risk [37]. Both O. tsutsugamushi and R. typhi have just recently been
recognized to be major causative agents of severe meningitis and meningoencephalitis with
high lethality rates [38]. Moreover, rickettsial infections generally occur worldwide with
increasing incidence and geographic expansion. Scrub typhus is reemerging in southern

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 2 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

China where an exponential increase in the incidence and geographic extension of the disease
is observed since 2006 [39]. Similar is true for South Korea where 70,914 cases of Scrub typhus
were reported by the Korea Center for Disease Control during 2001 to 2013, while no auto-
chthonic cases were recorded from 1951 to 1985 [40,41]. In addition, O. tsutsugamushi was
recently recognized in Chile [42], some countries of Africa, namely Kenia [43,44] and Senegal
[45], as well as in France where rodents were found to be positive for O. tsutsugamushi [45].
Similar is true for spotted fever rickettsioses (SFRs). A total of 495 cases of SFR were recorded
by the Centers for Disease Control and Prevention (CDC) in 2000, while around 5,500 cases
were reported in 2018. It is unclear, however, how many of these cases are RMSF caused by R.
rickettsii or other spotted fevers (https://www.cdc.gov/rmsf/stats/index.html). RMSF is also
reemerging after decades of quiescence in Mexico [46], Panama [47–49], Colombia [50,51],
and Brazil [52–54]. Also, steadily increasing case numbers and geographic expansion of R.
typhi infections are recognized in the United States of America. While 27 cases were reported
to the Texas State Health Department in 2003, 738 cases were confirmed in 2018 [55] (https://
www.dshs.texas.gov/IDCU/disease/murine_typhus/Statistics.aspx). In addition, sporadic cases
of epidemic typhus are reported from several states of eastern US in recent decades (https://
www.cdc.gov/typhus/epidemic/), which has been associated with contact to flying squirrels
that are considered a natural reservoir for R. prowazekii [56,57].
The spectrum of drugs for the therapy of rickettsial infections is currently still limited. Anti-
biotics that are active against rickettsiae are tetracyclines (e.g., doxycycline), macrolides (e.g.,
azithromycin) or chloramphenicol that inhibit the ribosomal biosynthesis of proteins, and rifa-
mycins (e.g., rifampin) that inhibit the bacterial RNA polymerase, and thus, RNA transcrip-
tion. Doxycycline is the antibiotic of choice for the treatment of all rickettsial infections.
The fact that rickettsiae respond to only few antibiotics bares 4 problems: (1) The unspecific
symptoms of rickettsial infections often lead to misdiagnosis and in the following to the treat-
ment with inappropriate antibiotics and to disease progression to a more severe outcome. (2)
Some people are doxycycline intolerant. In this case, alternatives are rare. Rifampin has been
successfully used for the treatment of ATBF in a patient with doxycycline intolerance [58].
This antibiotic, however, is not considered an acceptable and appropriate treatment for RMSF
[59], and it is questionable whether it is effective against other severe rickettioses such as epi-
demic typhus. (3) Apart from that, the risk of the development of resistance against these anti-
biotics is high. It has not only been shown that TG rickettsiae acquire resistance against
rifampin in laboratory experiments [60,61] but also resistance against this antibiotic occurs in
natural strains of SFG rickettsiae [62]. Similarly, resistance against doxycycline may be
acquired. In areas where O. tsutsugamushi is endemic resistance against doxycycline has been
reported [63–65]. True resistance of Orientia strains against doxycycline, however, is ques-
tioned because of the different methodology that was used in the few studies mentioned above
and because the supposedly resistant bacteria could also result from a lack of response to treat-
ment of the patient [66,67]. Standardized methods for the detection of resistance are still miss-
ing. (4) Finally, it is known that rickettsiae can persist despite the treatment with antibiotics.
Persistent infections after antibiotic treatment are observed in the infection with O. tsutsuga-
mushi [68], R. rickettsii [69,70], and R. prowazekii [71], and similar is assumed for R. typhi, the
second TG member [13]. R. prowazekii can reoccur years to decades after primary infection,
causing the so-called Brill Zinsser disease [72–75]. In addition, relapse of patients that had
been treated with antibiotics and recovered from O. tsutsugamushi infection is observed
months to years after primary infection [68]. Recurrence of other persisting rickettsial species
cannot be excluded, although it has not been described or recognized yet. In this context, it is
possible that treatment with wrong antibiotics or irresponsiveness of a patient to certain anti-
biotics may facilitate persistence.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 3 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

For these reasons, apart from the development of new drugs for therapeutic treatment of
the infection, vaccines that can prevent rickettsial infections are urgently needed and would be
beneficial in endemic areas as well as for travelers in these regions.
While innate immune responses are clearly important in early defense against rickettsial
infections [4,76,77], adaptive immunity is essential for protection. Vaccine development
requires the understanding of protective adaptive immune responses against rickettsiae, the
identification of immunogenic rickettsial antigens, and strategies to target and direct protec-
tive immune responses. This review provides a short overview on adaptive immunity against
rickettsial infections and mainly focuses on the efforts and progresses that have been made to
identify immunogenic targets and vaccine candidates.

Adaptive immunity against rickettsiae

Several studies in murine models of the infection with rickettsiae have clearly demonstrated
that adaptive immunity is essential for protection. This is clearly reflected by the fact that T
and B cell–deficient mice are highly susceptible to the infection with TG as well as SFG rickett-
siae [13,78,79]. The current knowledge on the role of B and T cells in defense against rickett-
siae is summarized in the following paragraphs and depicted in Fig 1.

Antibody-mediated immunity and B cell antigens

Antibodies produced by B cells can generally contribute to protection against invading patho-
gens by different mechanisms that depend on the constant fragment crystallizable (Fc) part:
(1) the opsonization of particles, which enhances the uptake and destruction by phagocytes;
(2) the activation of complement after binding to the pathogens surface, which can directly kill
the pathogen; and (3) in case of intracellular pathogens, the inhibition of cellular entry by
binding to surface molecules that are essential for cell surface receptor binding and uptake.
In rickettsial infections, it is assumed that antibodies play a minor role in defense in pri-
mary infection because specific high-affinity antibodies that are produced with the help of
CD4+ T cells are generated quite late in the infection. In humans, serum conversion in the
infection with R. typhi takes place around day 15 after the onset of symptoms [80] and even
later in the infection with R. conorii and R. africae (day 16 and day 25) [81]. In experimental
animal models, antibodies to rickettsiae appear after recovery of the animals. Therefore, it is
unlikely that they contribute to clearance in primary infection. Nonetheless, antibodies act
protectively and may help to prevent or ameliorate secondary infections. Passive immuniza-
tion of C3H SCID mice with polyclonal immune serum from R. conorii-infected mice pro-
tected the mice against a lethal challenge with R. conorii and even led to prolonged survival
and reduced bacterial load in already infected C3H SCID mice [79].
So far, it seems that there are only few dominant antigens that are recognized by potentially
protective antibodies in infected individuals. Members of the “surface cell antigen” (Sca) auto-
transporter family (namely Sca0 to 5) clearly represent immunodominant antigens that are
recognized by antibodies in the sera from experimentally infected animals as well as human
patients [82]. Among these, outer membrane protein A (OmpA/Sca0) that is expressed by SFG
but not by TG rickettsiae and OmpB (Sca5) that is expressed by all rickettsiae are the most
prominent antigens. Except for Sca4, which is found in the cytosol of the bacteria [83], these
high molecular weight proteins are expressed on the cell surface of rickettsiae where they are
easily accessible for antibodies. Especially, OmpA and OmpB are considered pathogenicity fac-
tors. Both proteins have been shown to be involved in the adherence of rickettsiae to host cells.
Escherichia coli bacteria expressing surface-exposed recombinant OmpB from Rickettsia
japonica or R. conorii acquired properties to adhere to and invade nonphagocytic Vero and

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 4 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

Fig 1. Immune response against rickettsiae. CD8+ cytotoxic T cells play the most important role against most
rickettsiae and can directly kill infected cells. Apart from the cytotoxic activity, CD8+ T cells also release IFNγ. The
cytotoxic activity of CD8+ T cells plays a dominant role in defense against SFG rickettsiae and Orienta, while the
release of IFNγ seems to be more important in long-term control of TG rickettsiae (A). IFNγ, which is released at high
amounts by CD4+ TH1 cells in addition to TNFα, acts against rickettsiae by activating antimicrobial mechanisms, e.g.,
NO production in MF and other infected cells (B). In the absence of IFNγ, CD4+ T cells develop into TH17 cells that
produce IL-17, IL-22, and TNFα. These cells can also protect against rickettsial infections by acting on MF via IL-17
and TNFα that induce the production of NO and the release of chemokines that attract neutrophils (PMNs). IL-22, in
addition to IL-17 and TNFα, also induces the production of NO, antimicrobial peptides, and other microbicidal
mechanisms in infected tissue cells. In this way, TH17 cells are capable to eliminate the bacteria. The combined release
of TNFα and IL-17, however, exerts pathological effects (C). The production of specific high-affinity antibodies by B
cells depends on T cell help. Specific antibodies are produced late in the infection with rickettsiae and are considered to
play a minor role in primary defense. Antibodies can contribute to defense most likely by opsonizing the bacteria for
the uptake and destruction by MF or the activation of complement to mediate direct bacterial killing (D). The most
promising way to achieve immunity against rickettsiae by a vaccine is the induction of specific cytotoxic CD8+ and/or
IFNγ-producing CD4+ TH1 cells, in best case in addition to antibody-producing B cells. IFNγ, interferon gamma; IL-
17, interleukin 17; IL-22, interleukin 22; iNOS, inducible nitric oxide synthase; MF, monocytes/macrophages; NO,
nitric oxide; PMN, polymorphonuclear neutrophils; ROS, reactive oxygen species; SFG, spotted fever group; TG,
typhus group; TNFα, tumor necrosis factor alpha.
https://doi.org/10.1371/journal.pntd.0008704.g001

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 5 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

HeLa cells [84,85], which was dependent on the extracellular outer membrane-associated pas-
senger domain of the protein (OmpB36-1334) [85]. In addition, antibodies directed against
OmpA inhibited adherence of R. rickettsii to L929 cells in vitro [86].
It is therefore likely that antibodies against OmpA and/or OmpB can contribute to protec-
tion. Indeed, monoclonal antibodies directed against R. rickettsii (most likely recognizing
OmpA and OmpB) protected mice from a lethal short-term challenge with a large and toxic
dose of homologous bacteria [87–89] and prevented fever and rickettsemia in guinea pigs [89].
Furthermore, monoclonal antibodies that are directed against the extracellular passenger
domain of R. conorii OmpB are sufficient for protection of C3H/HeN mice against a lethal
challenge with R. conorii [90]. Finally, the application of polyclonal anti-R. conorii immune
serum as well as monoclonal anti-R. conorii OmpA or anti-OmpB antibodies protected even
immunodeficient C3H/HeN SCID mice against challenge with R. conorii [79].
The mechanism of protection by these antibodies is not clear, but there are hints that opso-
nization of the bacteria for the uptake by phagocytic cells rather than the inhibition of the
binding of the bacteria to nonphagocytic host cells may play a role in bacterial defense. It was
observed that the opsonization of R. conorii with either polyclonal or monoclonal antibodies
against OmpA and OmpB leads to enhanced engulfment of the bacteria by ECs (SVEC4–10)
and MF-like cells (J774A.1) in vitro [91]. In addition, bacterial growth in these cells was
reduced [91]. Also, the treatment of C3H/HeN SCID mice with polyclonal antiserum resulted
in enhanced killing of R. conorii by MF and the accumulation of rickettisal antigens in MF in
the spleen [79]. More recent in vitro investigations indicate that monoclonal antibodies recog-
nizing the extracellular passenger domain of OmpB also can induce complement-mediated
killing of the bacteria [90], which has not been described before.
For R. prowazekii, 4 B cell epitopes of OmpB (OmpB45-58, OmpB1239-1252, OmpB1259-1268,
and OmpB1287-1296) have been identified that are recognized by polyclonal antibodies from
rabbits immunized with purified OmpB [92]. Three of these are also recognized by antisera
from human patients (OmpB45-58, OmpB1239-1252, and OmpB1287-1296) [92]. In addition, differ-
ent Sca-derived peptides (Sca1753-665, Sca2496-509, Sca3314-327, Sca4263-276, and OmpB (Sca5651-
665)) from R. typhi that were fused to form multivalent antigens were found to induce antibody
response upon immunization of rabbits [93]. Whether these antibodies have protective prop-
erties, however, is unknown.
Apart from the high molecular weight OmpA and OmpB proteins, additional immunodo-
minant proteins are recognized by antibodies from infected individuals. One of these is the 60
heat shock protein GroEL. GroEL was found to be the most prominent antigen from R. conorii
that is recognized by antibodies in sera from immunized rabbits as well as from infected
patients [94]. Similar is true for GroEL from Rickettsia heilongjiangensis, Rickettsia helvetica,
and R. parkeri that is recognized by antibodies in the sera from patients as well as mice infected
with these pathogens [94–97]. GroEL acts as a chaperone that assists in the folding of proteins
in the cytosol of prokaryotice cells and is up-regulated under circumstances of stress. For R.
prowazekii, it was shown that GroEL is up-regulated in the early phase of infection [98] where
enhanced chaperone activity may be necessary. Despite its cytosolic chaperone function, how-
ever, GroEL appears in multiple isoforms in rickettsiae and has been shown to be surface
exposed in R. conorii [94] and R. heilongjiangensis [95]. A potentially protective function of
antibodies against GroEL, however, has not been investigated yet, but it is interesting that
GroEL is considered a promising vaccine candidate for other bacterial infections such as
Mycobacterium tuberculosis [99], Bacillus anthracis [100], and Helicobacter pylori [101,102].
Other immunodominant proteins that are recognized by antibodies in the infection with R.
heilongjiangensis are PrsA, RplY, RpsB, SurA, and YbgF [95] and Sta22, Sta47, Sta56, ScaA, and
ScaC in the infection with O. tsutsugamushi [103–107]. PrsA, a Parvulin-like peptidylprolyl

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 6 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

isomerase, presumably assists in the folding of periplasmatic and membrane proteins and is
likely expressed in the outer membrane of the bacteria. RplY and RpsB are ribosomal proteins
that are most likely expressed in the cytosol of the bacteria. The same is true for SurA, another
peptidylproly isomerase that acts as a chaperone. YbgF belongs to the Tol/Pal-system of bacteria
and is involved in the maintenance of the membrane integrity of the outer bacterial membrane
and can be surface exposed. ScaA, Sta56, and ScaC from O. tsutsugamushi are also proteins of
the outer membrane, while Sta22 and St47 are considered to locate in the cytosol, cytosolic
membrane, or periplasma. ScaA has been shown to be involved in adhesion of the bacteria to
nonphagocytic HeLa cells, which is blocked by anti-ScA antibodies but not by antibodies
against ScaB, ScaC, or ScaE [108]. Similarly, antibodies against recombinant Sta56 from the O.
tsutsugamushi Boryong strain that were produced in mice and rabbits inhibit adhesion and
infection of L929 cells by Orientia in vitro [109], and certain monoclonal Sta56-specific antibod-
ies were protective against challenge of mice with the homologous O. tsutsugamushi strain in
vivo [110]. The role and mode of action of antibodies against these antigens in defense against
rickettsiae in vivo, however, is still not clear and remains to be investigated.

T cell–mediated protection against rickettsiae

It is clearly undisputed that T cells rather than B cells play a critical role in protection against
rickettsial infections. The most important role in protection is ascribed to cytotoxic CD8+ T
cells that are capable of direct killing of infected cells. CD4+ T cells on the other hand can con-
tribute to protection by the release of effector molecules that can activate phagocytes and by
driving the activation of B cells to produce specific antibodies. CD8+ as well as CD4+ T cells
have been shown to be involved in protection against rickettsial infections in animal model
systems. In recent years, however, it is emerging that the importance of CD8+ and CD4+ T cell
populations in defense against SFG and TG rickettsiae may differ. The following paragraph
summarizes current knowledge on the role of CD8+ and CD4+ T cells in protection against
SFG, TG, and transitional rickettsiae and Orientia.

Role of CD4+ T cells and CD8+ T cells in defense against SFG and
transitional rickettsiae
In the experimental infection of C3H/HeN mice with R. conorii (SFG) and R. australis (transi-
tional group), a peak response of activated CD8+ T cells that release interferon gamma (IFNγ)
and exert enhanced cytotoxic function is observed at day 10 postinfection [111]. Experimental
animal models of the infection with these bacteria further indicate that CD8+ T cells are essen-
tial for defense against these pathogens. C3H/HeN mice that were depleted of CD8+ T cells
showed reduced survival, increased bacterial burden, and enhanced pathology in the infection
with a sublethal dose of R. conorii [111,112]. Furthermore, immune CD8+ T cells adoptively
transferred into C3H/HeN mice protected the animals against infection with a normally lethal
dose of R. conorii [112]. The importance of CD8+ T cells in defense against SFG rickettsiae is
further evidenced by the enhanced susceptibility of C57BL/6 MHCI-/- mice that lack CD8+ T
cells to a lethal outcome upon infection with R. australis compared to wild-type mice [111].
The cytotoxic activity of CD8+ T cells rather than the release of IFNγ seems to be the main
effector mechanism that acts against these bacteria. Firstly, the adoptive transfer of immune
CD8+ T cells from C57BL/6 IFNγ-/- mice into R. australis-infected C57BL/6 IFNγ-/- mice led
to reduced bacterial load and protection [111]. Secondly, C57BL/6 Perforin-/- mice where
CD8+ T cells lack the cytotoxic potential showed a higher susceptibility and lethality upon
infection with R. australis compared to wild-type as well as to C57BL/6 IFNγ-/- mice [111].
C57BL/6 Perforin-/- mice, however, were less susceptible to R. australis than C57BL/6 MHCI-/-

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 7 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

that do not possess CD8+ T cells at all [111]. Together, these findings indicate that the cyto-
toxic function of CD8+ T cells plays a dominant role in defense against these bacteria com-
pared to the release of IFNγ.
In contrast to the depletion of CD8+ T cells, the depletion of CD4+ T cells in C3H/HeN
mice by administration of neutralizing antibodies altered neither the course of disease in the
infection with a sublethal dose of R. conorii nor the bacterial load in different organs compared
to control mice that received control antibodies. Both groups cleared the infection with similar
kinetics [112]. Nonetheless, similar to the adoptive transfer of immune CD8+ T cells, the trans-
fer of immune but not naive CD4+ T cells into C3H/HeN mice was protective against a nor-
mally lethal infection with R. conorii [112]. The data demonstrate that CD4+ T cells can
contribute to defense against SFG rickettsiae, although CD8+ T cells obviously play a dominant
role.
The main effector molecules that are considered to be involved in CD4+ T cell–mediated
defense against intracellular pathogens are IFNγ and tumor necrosis factor alpha (TNFα).
Both cytokines can contribute to the killing and elimination of intracellular agents by activat-
ing the bactericidal function of phagocytic and responsive nonphagocytic cells, namely the
induction of the expression of inducible nitric oxide (NO) synthase (iNOS) and the production
of NO [113–116].
IFNγ and TNFα have also been involved in defense against R. conorii and R. australis. Both
cytokines induced bacterial killing in R. conorii-infected human cell lines in vitro, which was
dependent on the production of NO [117]. The neutralization of either IFNγ or TNFα leads to
reduced survival, overwhelming bacterial burden, and enhanced pathology in R. conorii-
infected C3H/HeN mice, which was associated with reduced NO production [118]. Further-
more, IFNγ-deficient C57BL/6 mice succumb to the infection with a normally sublethal dose
of R. conorii [118]. Finally, also IFNγ-/- C57BL/6 mice showed reduced survival in the infection
with R. australis [111].
Generally, C3H/HeN mice and C57BL/6 differ in their susceptibility to rickettsial infections
[76]. One reason for that may be the different ability of dendritic cells (DCs) to induce protec-
tive immune responses. R. conorii-infected DCs from C3H/HeN mice are less effective in the
in vitro induction of IFNγ production in CD4+ T cells than DCs from C57BL/6 mice, which
can be ascribed to lower major histocompatibility complex II (MHCII) expression and
reduced release of interleukin 12 (IL-12) [119]. In addition, higher frequencies of regulatory
CD4+FoxP3+ T cells are observed in C3H/HeN compared to C57BL/6 mice in the infection
with R. conorii [119]. In another study, it was shown that CD4+ T cells with an inducible regu-
latory phenotype (CD4+CD25+FoxP3-T-bet+CTLA4high) produced IFNγ and IL-10 in the
infection of C3H/HeN mice with a lethal dose of R. conorii and that these cells suppressed pro-
liferation and IL-2 release by CD4+ T cells in vitro [120]. These data suggest that immune sup-
pression by regulatory T cells may contribute to enhanced susceptibility.

Role of CD4+ T cells and CD8+ T cells in defense against TG rickettsiae

Corresponding to CD8+ T cells that develop in the infection with SFG rickettsiae, CD8+ T cells
from R. typhi-infected BALB/c and C57BL/6 mice produce increased levels of IFNγ and Gran-
zyme B upon in vitro restimulation with phorbol myristate acetate (PMA)/Ionomycin
[121,122], indicating enhanced cytotoxic activity. A peak response of activated CD8+ T cells in
the infection with R. typhi is observed at day 7 postinfection [121,122] and thus a little earlier
than in the infection with SFG rickettsiae. It has been previously shown that R. typhi persists in
these mouse strains [13] that are considered resistant to the infection as they do not develop
symptoms of disease. In concordance with the presence of persisting bacteria, levels of

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 8 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

activated CD8+ T cells do not decline to basal levels for a long period of time in BALB/c as well
as in C57BL/6 mice in the infection with R. typhi [121,122]. Moreover, enhanced amounts of
activated CD8+ T cells are detectable in R. typhi-infected BALB/c mice in periodic intervals
[122]. These findings indicate that activated CD8+ T cells are of importance for the control of
the persisting bacteria. In line with that, it was found that the depletion of CD8+ T cells in the
infection of C3H/HeN mice with R. typhi leads to enhanced bacterial burden and pathology
[123].
For a more detailed study of the role and function of CD8+ (and CD4+ T cells) in protection
against R. typhi, 2 animal models have been developed in recent years: immunodeficient
C57BL/6 RAG1-/- and BALB/c CB17 SCID mice, both of which lack adaptive immunity but
behave differently in the infection. C57BL/6 RAG1-/- mice can control the bacteria for approxi-
mately 3 months. Then, the bacteria suddenly start to grow more or less exclusively in the
brain. As a consequence of massive inflammation of the central nervous system, the animals
become ataxic and paralyzed and finally die [13]. The course of disease in BALB/c CB17 SCID
completely differs from that. These animals show high bacterial burden in all organs, develop
liver necrosis and splenomegaly, and die from high systemic inflammation within 3 weeks
[78].
In the C57BL/6 RAG1-/- model, the adoptive transfer of immune CD8+ T cells was 100%
protective against R. typhi even when transferred late in the infection shortly before the onset
of disease [121]. Similarly, the adoptive transfer of naive CD8+ into BALB/c CB17 SCID mice
prior to the infection with R. typhi was protective and none of the animals developed disease
or died [122]. CD8+ T cells, however, do not require cytotoxic activity to act against R. typhi.
BALB/c Perforin-/- mice where CD8+ T cells lack the cytotoxic function are not susceptible to
R. typhi [122]. Again, this is in contrast to the enhanced susceptibility of C57BL/6 Perforin-/-
mice to the infection with R. australis [111]. Moreover, the adoptive transfer of CD8+ Per-
forin-/- T cells still protected BALB/c CB17 SCID from R. typhi infection. As in the transfer of
immunocompetent CD8+ T cells, the mice did not even show symptoms of disease at any
point in time [122]. Thus, the cytotoxic activity of CD8+ T cells is not essential for protection
in the infection with R. typhi.
Further data show that the lack of the cytotoxic activity of CD8+ T cells can be compensated
by the release of IFNγ and vice versa. First of all, BALB/c IFNγ-/- mice are as resistant to R.
typhi as BALB/c Perforin-/- mice. Furthermore, the adoptive transfer of CD8+ IFNγ-/- T cells
into R. typhi-infected BALB/c CB17 SCID mice was as protective as the transfer of CD8+ Per-
forin-/- T cells [122]. In contrast to the infection with R. australis where the release of IFNγ by
CD8+ T cells was found to be not essential for protection [111], IFNγ was even more important
than the cytotoxic activity of CD8+ T cells in long-term control of R. typhi. Persisting bacteria
were not found in BALB/c CB17 SCID mice that received CD8+ Perforin-/- T cells but were
detectable by quantitative polymerase chain reaction (qPCR) in CD8+ IFNγ-/- T cell recipients,
predominantly in the brain [122]. In contrast to the infection with R. australis, these data sug-
gest that CD8+ T cells can protect against R. typhi either via cytotoxic activity or the release of
IFNγ.
Overall, CD8+ T cells clearly confer protection against the infection with R. typhi. This is
also demonstrated by the fact that C57BL/6 MHCII-/- mice that lack CD4+ T cells are resistant
against R. typhi and do not develop disease [121]. However, resistance against R. typhi was also
demonstrated for C57BL/6 MHCI-/- mice that lack CD8+ T cells [121]. This is in contrast to
the infection of the same mice with R. australis where the lack of CD8+ T cells results in
reduced survival [111]. These findings indicate that CD8+ T cells clearly play a dominant role
in protection against R. australis, while either CD8+ or CD4+ T cells are sufficient for defense
against R. typhi.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 9 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

That CD4+ T cells alone are sufficient to mediate protection against the infection with R.
typhi is further demonstrated by adoptive transfer of immune CD4+ T cells into susceptible T
and B cell–deficient C57BL6 RAG1-/- mice. Here, adoptively transferred immune CD4+ T cells
still protected the mice even when transferred late in the infection when the bacteria already
start to grow [121]. Furthermore, the adoptive transfer of naive CD4+ T cells, even at low
amounts (1 × 106), protected BALB/c CB17 SCID mice against challenge with R. typhi
[121,122]. In both systems, CD4+ T cells were capable to eliminate the bacteria below qPCR
detection limit, although CD8+ T cells were obviously more efficient and quicker in bacterial
clearance. CD4+ T cells from C57BL/6 as well as from BALB/c mice express huge amounts of
IFNγ and lower amounts of TNFα in the infection with R. typhi with a peak response around
day 7 postinfection, which is similar to the CD8+ T cell response [121,122]. Also, the CD4+ T
cell response does not return to basal levels [121,122], and CD4+ T cells are sporadically reacti-
vated in R. typhi-infected BALB/c mice as observed for CD8+ T cells [121,122]. IFNγ as well as
TNFα are activators of phagocytes such as MF and other cells and play an important role in
defense against R. conorii [118]. In line with that, immune CD4+ T cells act on MF in the
infection with R. typhi and activate the bactericidal activity of these cells via the release of IFNγ
and TNFα [121,122]. Similar to the killing of R. conorii, IFNγ and TNFα induce the produc-
tion of NO and bacterial killing of R. typhi in murine MF [122]. IFNγ was also shown to
inhibit the growth of R. prowazekii in murine and human fibroblasts [124]. Nonetheless, the
adoptive transfer of CD4+ T cells from BALB/c IFNγ-/- mice into R. typhi-infected BALB/c
CB17 SCID mice still protected a high percentage of the mice from a lethal outcome [122]. In
this setup, CD4+ T cells developed into TH17 cells that produced high amounts of IL-22 and
IL-17 in addition to lower amounts of TNFα [122]. Thus, TH1 as well as TH17 cells can be pro-
tective. For the latter, it has also been shown by neutralization experiments in vivo that the
combined release of IL-17 and TNFα has immunopathological effects, while the presence of
one or the other cytokine together with IL-22 is beneficial [122]. These data suggest that the
induction of specific CD4+ T cells, especially IFNγ-producing TH1 cells, could be sufficient for
protection against R. typhi.

Role of CD4+ T cells and CD8+ T cells in defense against Orientia

T cells also play a dominant role against Orientia that resembles Rickettsia species with regard
to lifestyle and targeting of ECs. It was first observed that BALB/c mice that recovered from an
infection with the Gilliam strain of O. tsutsugamushi resist a normally lethal infection with the
more virulent Karp strain of O. tsutsugamushi [125]. It was then further demonstrated that the
adoptive transfer of nonadherent spleen from mice that recovered from the O. tsutsugamushi
Gilliam infection conferred protection against lethal challenge with O. tsutsugamushi Karp
[126]. The protective effect was clearly dependent on T cells, because mice that received
immune spleen cells depleted of T cells were not protected anymore, while the depletion of B
cells had no effect [126].
In more recent studies, it becomes clear that CD8+ T cells play a more critical role than
CD4+ T cells in defense against Orientia. The infection of humans with Orientia leads to a loss
rather than an increase in peripheral CD4+ T cells including regulatory T cells (Tregs) in the
acute phase of infection, while activated CD8+ T cells increase [127]. Similarly, intravenous or
subcutaneous infection of C57BL/6 mice as well as subcutaneous infection of BALB/c mice
with O. tsutsugamushi Karp leads to a much stronger increase of CD8+ T cells than CD4+ T
cells within the first 14 days after infection [128,129]. Furthermore, the CD8+ T cell response
lasts for a long period of time (at least 135 days) in C57BL/6 mice [128]. In the subcutaneous
infection model as well as in the intravenous infection, the bacteria spread to nearly all organs

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 10 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

with highest bacterial loads in the lung [129,130], which is accompanied by an increase of
CD8+ T cell infiltrates within the third weak after infection [128]. The prominent role of CD8+
T cells in defense was further demonstrated by the study of mice that were either depleted of
CD8+ T cells or CD8+ T cell deficient as well as by the adoptive transfer of CD8+ versus CD4+
T cells. Depletion of CD8+ T cells in O. tsutsugamushi-infected BALB/c mice results in uncon-
trolled bacterial growth and death of the animals [128]. The same is true for the infection of
CD8+ T cell–deficient C57BL/6 mice, either infected intravenously or via the skin. These mice
show increasing bacterial loads in lung, kidney, liver, and spleen; more severe lesions in the
organs; and die through a normally sublethal infection with O. tsutsugamushi [128,129]. The
adoptive transfer of CD8+ T cells from immune BALB/c mice that recovered from the suble-
thal skin infection protected animals that were challenged with the homologous strain via the
normally lethal intraperitoneal route [128]. The same observations were made when immune
CD8+ T cells were transferred into C57BL/6 mice that were intravenously infected with a lethal
dose of Orientia [129]. The long-lasting CD8+ T cell response seems to be associated with the
persistence of the bacteria that has been described for humans [68,131] as well as for mice
[132,133], because the depletion of CD8+ T cells in O. tsutsugamushi-infected C57BL/6 at day
84 postinfection leads to reactivation of the bacteria [128]. The protective effect of CD8+ T
cells seems to rely on the cytoxic activity of these cells rather than cytokine production. This is
evidenced by the observation that Perforin-/- C57BL/6 mice die through the infection during
the first 14 days, similar to CD8+ T cell–deficient mice, and show enhanced bacterial burden
in several organs [128]. These studies demonstrate that CD8+ T cells seem to be indispensable
for protection against Orientia.
Nonetheless, CD4+ T cells also contribute to protection. While the transfer of immune
CD8+ T cells is 100% protective in the intravenous C57BL/6 infection model, the transfer of
CD8+ T cell–depleted immune spleen cells still protects approximately 50% of the animals. In
addition, the onset of disease in these mice is delayed [129]. Although B cells were present in
the cell preparation used for transfer, it is likely that this protective effect can be largely
ascribed to CD4+ T cells, most probable TH1 cells that produce IFNγ and TNFα. These cyto-
kines are induced in CD4+ T cells by Orientia-infected DCs in vitro [134] and are considered
to contribute to protection against Orientia by the mechanisms mentioned earlier. In line with
that, the adoptive transfer of an IFNγ-producing T cell line generated from immune BALB/c
mice after sublethal infection with O. tsutsugamushi Gilliam conferred protection against lethal
intraperitoneal challenge with the homologous strain [135]. Furthermore, in O. tsutsugamu-
shi-infected BALB/c and C57BL/6 mice, the bacteria were predominantly found in MF, and
inflammatory iNOS-expressing MF infiltrates were detectable in the organs [128,130]. The
mice produced enhanced levels of IFNγ that clearly contributed to the inhibition of bacterial
growth in an iNOS-dependent fashion [128].
However, CD4+ as well as CD8+ T cells may also contribute to pathology. Xu and colleagues
observed that the expression of IFNγ and Granzyme B as well as of TNFα and monocyte che-
moattractant protein-1 (MCP-1) was enhanced in CD8+ T cell- and MHCI-deficient Orientia-
infected C57BL/6 mice [129]. In addition, these mice showed more severe liver and kidney
damage. Similarly, O. tsutsugamushi-infected BALB/c mice depleted of CD8+ T cells showed
enhanced serum levels of IFNγ and stronger MF responses in liver and lung with an increase
of these cells as well as an increase in iNOS expression [128]. These findings indicate that the
absence of CD8+ T cells probably leads to enhanced activation of CD4+ T cells and cytotoxic
natural killer (NK) cells with enhanced IFNγ production as a compensatory mechanism. Such
enhanced inflammatory response can result in more severe pathology as observed in the infec-
tion with R. typhi upon the adoptive transfer of immune CD4+ T cells into C57BL/6 RAG1-/-
mice where CD4+ T cells, when transferred late in the infection, promote MF-mediated

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 11 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

inflammation in the brain [121]. In this context, it is also interesting that elevated levels of
IFNγ and TNFα are found in the peritoneal lavage of experimentally O. tsutsugamushi-infected
C3H/HeN mice and BALB/c mice with higher levels in susceptible C3H/HeN mice compared
to resistant BALB/c mice [136,137]. The infection of humans with O. tsutsugamushi is also
associated with elevated serum levels of these cytokines in addition to other inflammatory
cytokines, several chemokines, as well as of Granzymes A and B as indicators of the activation
of cytotoxic CD8+ T and NK cells [138–142]. Although important for protection, a contribu-
tion of these mediators to pathology cannot be excluded.
Last but not least, human and mouse may differ in their immune response and susceptibil-
ity to the infection with Orientia and other pathogens. In contrast to mice, the longevity of
immunity against Orientia in humans seems to be limited. CD4+ and CD8+ T cells that specif-
ically react to membrane proteins of the bacteria decline in infected humans from 1 year after
infection [143], which is different from the long-lasting T cell response in C57BL/6 mice
[128], and it was suggested that this may be due to a lack of memory response. To achieve a
better understanding of human immune response, Jiang and colleagues just recently tested a
humanized mouse, the DRAGA mouse which is based on an immunodeficient mouse that
was reconstituted with human hemotopoietic stem cells in the infection with O. tsutsugamu-
shi. Footpad inoculation of O. tsutsugamushi Karp into these mice leads to the dissemination
of the bacteria into various organs with highest bacterial loads in the lung as observed in
infected BALB/c and C57BL/6 mice [128–130]. The humanized DRAGA mice develop
splenomegaly and liver necrosis, and the infection is lethal in a dose-dependent manner,
whereas C3H/HeJ or BALB/c mice that are infected via the same route survive the infection
with the same dose [130,144]. A strong TH1 response with the production of high amounts of
IFNγ, TNFα, IL-12, and IL-2 as well as an increase of activated human CD4+ and CD8+ T
cells was observed in DRAGA mice. In addition, regulatory T cells and the production of IL-
10 were significantly enhanced [144], which is also observed in the initial phase of the infec-
tion in C57BL/6 mice [129]. Overall, the infection and disease of these humanized mice
largely resembles the infection of normal mice and humans. The expansion of CD8+ T cells,
however, seems to be much more pronounced in normal mice as well as in humans compared
to the DRAGA mouse.

T cell–mediated cross-protection and T cell antigens

Animals as well as humans show cross-protective immune response against different rickettsial
species. The experimental infection of guinea pigs with R. typhi renders the animals resistant
to R. prowazekii and vice versa, and similar is true for humans [145]. Antigen preparations
from R. rickettsii, R. sibirica, and R. australis but not R. akari produce reactivation of immune
spleen cells from R. conorii-infected C3H/HeJ mice, while spleen cells from R. akari-immu-
nized mice react to R. conorii and other SFG rickettsiae [146]. Furthermore, immunization of
C3H/HeN mice with a sublethal dose of R. conorii and R. australis protects the animals against
a lethal challenge with the heterologous pathogen [147]. These data demonstrate cross-immu-
nity between SFG and transitional rickettsiae. Even cross-protection between SFG and TG
rickettsiae has been described. C3H/HeN mice immunized with sublethal doses of R. conorii
or R. typhi were protected against lethal challenge with one or the other rickettsial species
[148]. Therefore, the identification of immunodominant T cell antigens that are present in a
broader range of rickettsiae may lead to the identification of vaccine candidates that can confer
protection against a broader range of rickettsial species.
The following paragraph summarizes the current knowledge on the efficiency of different
immunization strategies and rickettsial T cell antigens identified so far.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 12 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

Immunization and rickettsial T cell antigens

Vaccination with inactivated rickettsiae as whole cell antigen (WCA)
One of the first vaccines against rickettsial infection was developed by R. Spencer and R.
Parker in 1924. The vaccine was produced by triturating R. rickettsii-infected ticks, which were
produced in the laboratory by feeding them on infected guinea pigs [149]. The bacteria were
then inactivated in phenol and formalin. Another early vaccine was produced in embryonated
chicken eggs (Cox vaccine). Application of the inactivated R. rickettsii Cox-type vaccine prior
to the onset of disease after naturally acquired infection reduces the severity of illness in man
[150]. Both vaccines induce the production of antibodies [151]. When given 3 to 6 months
before exposure to live bacteria, both vaccines, however, only lead to milder illness but do not
prevent the disease [151]. Another vaccine against R. rickettsii was produced in embryonal
chicken fibroblasts followed by formalin inactivation by the US military in the 1970s
[152,153]. This formalin-killed vaccine applied 2 times completely protected cynomolgus
monkeys (Macaca fasciularis) that were infected subcutaneaously 2 months after the last
immunization with virulent R. rickettsii [154]. It also protected rhesus monkeys [155] and led
to milder disease in human volunteers [156].
In the 1920s, R. L. Weigl produced a vaccine against epidemic typhus by intrarectal injec-
tion of R. prowazekii into lice that were fed on humans. The bacteria were then isolated by trit-
uration of the intestinal canals of infected lice and inactivated in phenol. Two or 3 injections of
this material protected guinea pigs [157]. This vaccine was also used during World War II to
protect German soldiers from the disease [158]. An interesting note here is that the Weigl lab
produced vaccine lots with different potency for protection of which the stronger ones were
given to resistance fighters and the weaker ones to the German Army. In addition, Weigl
smuggled the vaccine into ghettos, which was done under huge risk as the German forces
monitored his work. At the same time, the US military also produced a vaccine against R. pro-
wazekii and grew the bacteria in chicken egg yolk sacs. The formalin-inactivated bacteria were
then used for the vaccination of US soldiers during World War II and led to a milder form of
disease and protection against severe disease [159]. Further vaccines against epidemic typhus
were developed by R. Castaneda and H. Zinsser who isolated R. prowazekii either from the
lungs of intranasally infected rabbits (Castaneda vaccine) [159] or the tunica vaginalis and
peritoneum of infected rats (Zinsser-Castaneda vaccine) followed by inactivation of the bacte-
ria in formalin [160]. This vaccine protected guinea pigs when applied subcutaneously or
intraperitoneally [161]. F. Veintemillas found that at least 3 doses of the Zinsser-Castaneda
vaccine are needed to protect guinea pigs [162].
Similar attempts were made for the vaccination against O. tsutsugamushi with either forma-
lin-fixed homogenized lungs from infected cotton rats [163,164] or formalin-killed purified O.
tsutsugamushi [165]. Both vaccination strategies, however, were not effective in humans [165]
and led to only limited protection against the homologous strain in mice [166,167]. In contrast
to that, it was shown in a more recent study that immunization of C3H/HeN mice with forma-
lin-killed O. tsutsugamushi protected the animals against challenge with the homologous strain
and induced immunity that lasted longer than 8 months [168].
Overall, complete protection against R. prowazekii, R. rickettsii, or O. tsutsugamushi
employing formalin-inactivated bacteria as a vaccine was generally not achieved in humans so
far, which maybe ascribed to alterations in the antigenic determinants due to the fixation
method.
Alternative inactivation methods to avoid possible losses in antigenicity are killing of the
bacteria by heat (56˚C) or irradiation. Vaccination with irradiated O. tsutsugamushi
completely protected mice against challenge with the homologous strain [169–171], and in a

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 13 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

very recent study, the vaccinating potential of heat-killed bacteria was analyzed in a canine
model of RMSF. Dogs were immunized twice with heat-inactived R. rickettsii grown either in
embryonated eggs or in Vero cells and then challenged with live R. rickettsii intravenously.
This vaccine protected the dogs from severe RMSF and reduced tissue lesions [172].
Together, these findings indicate that the method of inactivation for vaccine preparation
plays a critical role for the protective potential.
Vaccination with live avirulent or attenuated rickettsiae. Apart from the immunization
with intact but inactivated rickettsiae, other approaches employed live bacteria for immuniza-
tion. As early as 1936, H. Zinsser immunized guinea pigs with a mixture of live R. prowazekii
and serum from convalescent guinea pigs or immunized horses [173] potentially containing
opsonizing or neutralizing antibodies. In this way, he achieved immunity in the treated ani-
mals against challenge with R. prowazekii. One month after immunization, the animals were
still immune against the bacteria. Another example is the immunization of humans with a
low-virulence strain of O. tsutsugamushi, which induced solid protection [174]. Similarly, the
infection with O. tsutsugamushi followed by early antibiotic treatment resulted in protection
against the homologous strain [175,176].
A safer possibility of immunization may be the use of avirulent or attentuated rickettsiae. A
human isolate of R. prowazekii that was obtained during the second World War and passaged
several times in embryonated chicken eggs turned out to be of low virulence. Vaccination with
this strain (Madrid E) has been tested in prisoner volunteers in the Mississippi State Prison
and was found to protect humans against the infection with a virulent R. prowazekii strain and
to confer long-term immunity. The vast majority of vaccinated people was still protected
against the infection with virulent R. prowazekii up to approximately 5 years [177]. The same
avirulent strain R. prowazekii was also used in field trials in South America and Burundi [178].
The use of avirulent R. prowazekii, however, bares the risk of reversion to the pathogenic form.
Virulence of R. prowazekii Madrid E is steadily increasing after passages in mice and guinea
pigs [179], and reversion to the virulent form of R. prowazekii (Evir) is likely also the reason
for the fact that 14% of the people vaccinated with R. prowazekii Madrid E showed mild illness
around 9 to 14 days postimmunization [178]. The loss of virulence of the R. prowazekii Madrid
E strain is a result of a point mutation in the gene encoding for the S-adenosylmethionine-
dependent methyltransferase (RP028/RP027) leading to the absence of this enzyme [180],
which results in the hypomethylation of surface proteins. Therefore, OmpB of the attenuated
Madrid E strain of R. prowazekii is hypomethylated compared to the same protein from viru-
lent Evir and naturally occuring R. prowazekii [181]. The virulent reisolate Evir shows a rever-
sion of this mutation and expresses this enzyme again [182].
A more promising and safer way may be the generation of stably attenuated rickettsial
strains that are suitable for vaccination by introducing mutations into virulence genes or by
deletion of such genes. Although systems for the targeted introduction or deletion of genes
in the rickettsial genome are still limited, some have been described. Phopholipase D, which
is involved in phagosomal escape of the bacteria, is considered a virulence factor for R. pro-
wazekii [183], and site-directed knockout of the gene (pld) encoding for this enzyme by
transformation and homologous recombination resulted in an attenuated strain of R. prowa-
zekii. Immunization with these bacteria induced protective immunity in guinea pigs against
challenge with virulent R. prowazekii [33]. Other target proteins might be surface proteins
that are involved in bacterial adhesion and invasion such as OmpA (only SFG rickettsiae)
and OmpB. It has been shown, however, that a targeted knockout of OmpA does not disturb
infectivity of R. rickettsii in the infection of guinea pigs [34]. Here, a LtrA group II intron ret-
rohoming system has been used to insert intronic RNA at the OmpA target site in the rickett-
sial genome.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 14 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

The use of attenuated mutant or knockout strains for vaccination is promising, and meth-
ods for genetic engineering are evolving. Yet, rickettsial virulence factors that are essential for
infectivity and pathogenicity need to be identified.

Immunization with antigen-presenting cells (APCs) and recombinant

antigens
The safest and most uncomplicated way to achieve immunity against rickettsial infections
would be the immunization with recombinant protein antigens or APCs that are capable to
induce protective adaptive immune responses. Achievements on this topic of research in recent
years as well as different vaccination strategies are summarized below. An overview on rickett-
sial antigens described in the literature and mentioned in the following chapters is given in Fig
2 (TG antigens), Fig 3 and Fig 4 (SFG antigens), and Fig 5 (Orientia antigens). The supposed
function and localization of these proteins within the bacteria is provided in Figs 6 and 7.
Immunization with transfected antigen-expressing APCs to activate CD8+ T cells.
Because CD8+ T cells play a dominant role in defense against intracellular rickettsiae, the
induction of specific CD8+ T cells is a promising way to achieve immunity against rickettsial
infections. To induce CD8+ T cell responses, antigens have to be directed into the MHC class I
presentation pathway, which makes this approach experimentally difficult. Another problem
is that rickettsial CD8+ T cell antigens and epitopes are still unknown.
Bioinformatic prediction tools (S1 Fig) can be of help for the identification of immunogenic
proteins and epitopes. Employing such bioinformatic algorithms, Gazi and colleagues and
Caro-Gomez and colleagues identified for the first time CD8+ T cell antigens of R. prowazekii.

Fig 2. TG antigens. The figure summarizes the antigens identified from TG rickettsiae [92,93,190,191,197,198]. APCs, antigen-presenting cells; MAP, multiple antigen
peptide; TG, Typhus group.
https://doi.org/10.1371/journal.pntd.0008704.g002

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 15 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

Fig 3. SFG antigens. The figure summarizes the antigens identified from SFG rickettsiae (R. rickettsii) [172,195,199,201–205,207,208]. enc., encoding; IFNγ, interferon
gamma; IgG1, immunoglobulin G1; IgG2a, immunoglobulin G2a; IL-12, interleukin 12; rec., recombinant; SFG, spotted fever group; TNFα, tumor necrosis factor alpha.
https://doi.org/10.1371/journal.pntd.0008704.g003

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 16 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

Fig 4. SFG antigens. The figure summerized the antigens identified from SFG rickettsiae (R. conorii and R. heilongjiangensis) [79,94,95,192,194,196,200,201,206]. DCs,
dendritic cells; IFNγ, interferon gamma; SFG, spotted fever group; TNFα, tumor necrosis factor alpha.
https://doi.org/10.1371/journal.pntd.0008704.g004

They analyzed 834 proteins from R. prowazekii for 9mer peptides that can be presented in
MHC class I H-2KK molecules using NetMHCpan, IEBD-Ann, and SYFPEITHI [184–187].
The proteins identified with these methods were further analyzed with RANKPEP, an algo-
rithm that evaluates both MHC class I binding affinity and proteasome processing [188], Vax-
ign and Vaxitope [189]. Using these bioinformatic approaches, they identified 5 proteins from
R. prowazekii (RP403, RP598, RP739, RP778, and RP884) that may be recognized by CD8+ T
cells. They further expressed these proteins in SVEC4-10 ECs. These cells derive from C3H/
HeJ mice and express the costimulatory molecules CD137L and CD80, facilitating T cell acti-
vation. In this way, MHCI presentation was achieved, and the cells were further used as APCs
for the immunization of C3H/HeN mice followed by a lethal challenge with R. typhi. Immuni-
zation of the mice prior to the infection with R. typhi led to increased production of IFNγ and
Granzyme B by CD8+ T cells and protected the mice from lethal outcome [190,191]. Further-
more, immunization of C3H/HeN mice with SVEC4-10 cells expressing a mixture of these
antigens even led to partial protection against a lethal challenge with R. conorii [191]. Thus,
these findings not only demonstrate that these antigens are recognized by CD8+ T cells but
also that immunization with these proteins can confer cross-protection between the 2 TG rick-
ettsiae as well as SFG rickettsiae, at least in part.
Immunization with antigen-pulsed APCs. Another approach for the use of APCs for
immunization is to feed the cells with protein antigen. Going this way, one would expect that

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 17 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

Fig 5. Orientia antigens. The figure summarizes the antigens identified from Orientia [103–109,209–215]. DCs, dendritic cells; IFNγ, interferon gamma; IL-2, interleukin
2.
https://doi.org/10.1371/journal.pntd.0008704.g005

the proteins are taken up by the APCs to be processed and presented in the context of MHCII
leading predominantly to the induction of CD4+ T cells.
Meng and colleagues pulsed DCs with different overlapping recombinant OmpB fragments
(OmpB371-702, OmpB689-1033, OmpB991-1363, and OmpB1346-1643) from R. heilongjiangensis and
subsequently adoptively transferred the DCs into C3H/HeN mice. Mice that were immunized
in this way with proteins OmpB689-1033, OmpB 991–1363, or OmpB1346-1643 were protected
against subsequent infection with R. heilongjiangensis and showed reduced bacterial load,
while protein OmpB371-702 did not have this effect [192]. In addition, OmpB689-1033, OmpB991-
+
1363, or OmpB1346-1643 but not OmpB371-702 induced IFNγ and TNFα expression in CD4 as
+
well as CD8 T cells upon restimulation of T cells from immunized mice with DCs pulsed with
the respective OmpB antigen [192], indicating that immunization with DCs pulsed with these
antigens leads to the generation of a CD4+ TH1 and probably cytotoxic CD8+ T cell responses.
The authors of this study further show that incubation of DCs with all 4 recombinant OmpB
fragments or WCA leads to comparable up-regulation of the costimulatory molecules CD40
and CD86 as well as to the up-regulation of MHC class II [192], indicating a stimulatory capac-
ity of the recombinant OmpB fragments and of WCA. Therefore, the differences in the protec-
tive capacity and the induction of cytokine production by T cells cannot be explained by
differences in the stimulatory capacity of the proteins on the APCs. Important to mention here
is that antibodies were generally not generated in mice immunized with pulsed DCs, whether
OmpB protein fragments or WCA were used. Thus, antibodies obviously do not play a role in
protection in this experimental setup, while an induction of antigen-specific CD4+ as well as of
CD8+ T cells can be achieved.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 18 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

Fig 6. Description of protein function and predicted and/or experimentally evidenced subcellular location. The predicted function and predicted and/or
experimentally evidenced location of the immunogenic rickettsial antigens is depicted [86,94,95,204,216–221]. C, cytoplasm; Exp., experimentally evidenced location; IM,
inner membrane; ns, non-secreted; OM, outer membrane; P, periplasm; SFG, spotted fever group; Sp., species; TG, typhus group; TSA, Type Surface Antigen.
https://doi.org/10.1371/journal.pntd.0008704.g006

Vaccination with recombinant proteins and peptides. The experimental vaccination

strategies mentioned so far are time consuming and expensive and not suitable for large-scale
vaccine production. Therefore, it is still an effort to identify antigenic proteins and peptides

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 19 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

Fig 7. Overview on the antigens identified from Orientia, TG, and SFG rickettsiae and localization of these proteins. The picture
provides an overview on the antigens identified from Orientia, TG, and SFG rickettsiae and their subcellular localization. PM, periplasm;
SFG, spotted fever group; TG, typhus group.
https://doi.org/10.1371/journal.pntd.0008704.g007

that can directly be used for the induction of protective immunity. Some candidates are
described below.
OmpA/OmpB. OmpA and OmpB are considered immunodominant antigens that are
recognized by T and B cells from experimentally infected animals as well as from humans suf-
fering from rickettsioses. For example, different OmpB fragments from R. rickettsii were
expressed in THP-1 MF, and T cells from patients that were positively tested for the presence
of R. rickettsii, R. typhi, or R. felis by PCR/restriction fragment length polymorphism (RFLP)
reacted to these cells with enhanced IL-2 and IFNγ production and induced IL-12p70, IL-6,
and TNFα in the antigen-expressing THP-1 MF [193]. This response is likely to be ascribed
mainly to CD8+ T cells because expression of the OmpB fragments in the cells results in
MHCI presentation. These data also suggest cross-reaction of T cells to conserved epitopes of
OmpB from SFG and TG rickettsiae, which makes this protein a promising vaccine candidate
for the vaccination against a broad range of rickettsiae.
Regarding OmpA and OmpB, different vaccination strategies have been applied that are
described below.
E. coli expressing recombinant OmpA from R. conorii. There is 1 study where E. coli-
expressing recombinant OmpA from R. conorii, instead of purified protein, was used for
immunization. Immunization of guinea pigs with lysates of these OmpA-expressing E. coli
protected the animals against R. conorii and partially against R. rickettsii infection [194]. The
underlying mechanisms of protection, however, were not further analyzed.
Immunization with recombinant OmpA and OmpB. In most studies, either purified
natural rickettsial proteins, recombinant proteins, protein fragments, or peptides were used
for immunization. For example, immunization of guinea pigs with baculovirus-expressed
purified recombinant R. rickettsii OmpA was protective against the infection with R. rickettsii
[195] as was the immunization of guinea pigs with truncated OmpA from R. heilongjiangensis
against homologous bacteria and against R. rickettsii [196]. Guinea pigs and mice were also

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 20 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

immunized with purified native OmpB from R. typhi, which was protective against the infec-
tion with this agent [197,198]. For R. prowazekii, it has been shown that rabbits immunized
with recombinant OmpB develop antibodies against this protein, and these were used to iden-
tify specific OmpB B cell epitopes that were also recognized by antibodies from human patients
[92].
In a recent study, Wang and colleagues identified 5 CD4+ T cell epitopes from the OmpB
protein of R. rickettsii (OmpB152-166 (QNVVVQFNNGAAIDN), OmpB399-413 (NTDFGNLAA
QIKVPN), OmpB563-577 (TIDLQANGGTIKLTS), OmpB698-712 (TNPLAEINFGSKGVN), and
OmpB1411-1425 (NLMIGAAIGITKTDI)) and 1 peptide from the YbgF protein of R. rickettsii
(YbgF57–71 (LQHKIDLLTQNSNIS)). Immunization of C3H/HeN mice with these peptides
either alone, pooled, or expressed as a recombinant fusion protein resulted in enhanced
expression of IFNγ and TNFα by CD4+ T cells as well as increased immunoglobulin G1 (IgG1)
and IgG2a production in the infection with R. rickettsii. Furthermore, immunization with the
pooled peptides led to reduced bacterial burden [199].
Also, CD8+ T cell epitopes have been identified in the OmpB protein from SFG rickettsiae.
Five synthetic peptides of the OmpB protein from R. conorii (OmpB708-716 (SKGVNVDTV),
OmpB789-797 (ANSTLQIGG), OmpB812-820 (IVEFVNTGP), OmpB735-743 (ANVGSFVFN), and
OmpB749-757 (IVSGTVGGQ) induced IFNγ expression by CD8+ T cells from R. conorii-
infected C3H/HeN mice upon restimulation in vitro [200]. CD8+ T cells that were reactive to
OmpB708-716, OmpB789-797, and OmpB812-820 additionally showed enhanced proliferation and
cytotoxic activity against R. conorii-infected SVEC4-10 cells, which were not observed with
OmpB735-743 and OmpB749-757 [200]. Whether immunization with these peptides leads to pro-
tective immunity against the infection with R. conorii or other SFG rickettsiae where these pep-
tides are conserved remains to be investigated. If these peptides can induce protective
immunity, it is unlikely that they can mediate cross-protection against TG rickettsiae because
the mentioned antigenic OmpB peptides are not expressed by R. prowazekii and R. typhi
except for OmpB749-757.
Cross-protection between SFG rickettsiae has been demonstrated for the vaccination with
recombinant OmpA and OmpB fragments. Immunization with R. rickettsii OmpA and OmB
fragments (Fig 3) can effectively induce cross-protection against R. conorii. Effectiveness and
cross-protection employing these proteins, however, differs depending on the species the pro-
teins derive from. Another study shows that vaccination of C3H/HeN mice with recombinant
OmpB from R. conorii, though inducing high titers of antibodies recognizing the protein, was
not protective against R. rickettsii, whereas the immunization with the corresponding OmpB
from R. rickettsii prevented a lethal outcome of the infection with R. conorii [201]. Thus, the
antigenic potential of nearly identical proteins from different rickettsial species may differ.
Heterologous prime/boost vaccination. Protective immunization may not only require
repeated vaccination with recombinant antigens but different methods of application. A prom-
ising approach is heterologous prime-boost vaccination, in which the same antigen is applied
by different methods. Few studies describe such attempts employing either bacteria that
express recombinant antigen or antigen-encoding DNA for primary vaccination followed by
boost immunization with the respective recombinant protein antigen.
Crocquet-Valdes and colleagues showed that immunization of C3H/HeN mice with Myco-
bacterium vaccae that express the DNA encoding for either R. rickettsii OmpA980-1301 or
OmpA755-1301 followed by boost immunization with recombinant OmpA755-1301 or OmpA703-
1288 fragments leads to partial protection against a lethal outcome of R. conorii infection [202].
The same was observed when DNA encoding for OmpA980-1301 or OmpA755-1301 was used for
primary immunization and boost immunization with the before-mentioned recombinant pro-
teins [202]. The authors further observed that lymphocytes from mice immunized with the

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 21 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

mentioned DNAs followed by boost immunization with OmpA980-1301 or OmpA755-1301 pro-

tein produced enhanced levels of IFNγ upon restimulation with R. conorii WCA in vitro, dem-
onstrating the recognition of T lymphocyte epitopes within these fragments. In another work
from the same group, mice were repeatedly immunized with DNA encoding for OmpA703-1288
or OmpA1644-6710 followed by boost vaccination with recombinant OmpA703-1288 or
OmpA1644-6710 protein or with DNA encoding for OmpB451-846 or OmpB754-1308 followed by
boost immunization with recombinant OmpB451-846 or OmpB754-1308 proteins with a similar
outcome [203]. T lymphocytes from immunized mice in this way produced IFNγ upon restim-
ulation with OmpB fragments OmpA703-1288 and OmpA1644-2213 or OmpB451-846 and
OmpB754-1308, respectively, and 100% of the mice were protected against lethal challenge with
R. conorii upon immunization with mixed plasmid DNA encoding for all 4 protein fragments
[203].
Although OmpA and OmpB may be promising candidates for vaccination, it is not clear
whether immunization against these proteins can indeed confer protection in species other
than mice. Very recently, it has been shown that only the immunization with R. rickettsii WCA
but not with a combination of recombinant Adr2 and a recombinant OmpB fragment pro-
tected dogs against RMSF [172].
Adr1, Adr2, TolC, OmpW, Porin 4, and YbgF. Little is known about the experimental
immunization of animals with other potentially antigenic rickettsial proteins. Knowledge
about these antigens is therefore combined in this section. Gong and colleagues showed that
immunization of C3H/HeN mice with recombinant Adr1, TolC, OmpW, or Porin 4 from R.
rickettsii results in reduced bacterial load after challenge with homologous bacteria [204], and
immunization of C3H/HeN mice with recombinant R. rickettsii YbgF protein leads to
enhanced proliferation and IFNγ production by CD8+ and CD4+ T cells from R. rickettsii-
infected mice upon in vitro restimulation with YbgF [205]. Similiarly, Qi and colleagues found
that YbgF is recognized by CD4+ T cells from R. heilongjiangensis-infected C3H/HeN mice. In
addition, antibodies are generated against this protein, and the immunization with YbgF leads
to reduced bacterial burden upon challenge with R. heilongjiangensis [206].
Apart from Adr1, Adr2 also has immunogenic potential. Recombinant Adr2 from R. rick-
ettsii was used for the immunization of C3H/HeN mice and found to be protective against the
infection [207]. CD4+ and CD8+ from R. rickettsii-infected animals produced higher levels of
IFNγ and showed increased antibody production upon prior immunization recombinant with
Adr2 [207], both of which may contribute to protection. Similarly, vaccination of C3H/HeN
mice with a combination of R. rickettsii Adr2 and a fragment of OmpB leads to enhanced IFNγ
production by CD4+ T cells and TNFα release by CD8+ T cells, increased IgG2a and IgG1 gen-
eration, and enhanced protection against R. rickettsii [208].
Adr1, Adr2, TolC, OmpW, Porin 4, and YbgF therefore might represent promising vaccine
candidates apart from OmpA and OmpB, although the immunogenicity of these proteins and
the immune reactions that are induced have to be further investigated.
O. tsutsugamushi Sta22, Sta47, Sta56, ScaA, ScaC, ScaD, and ScaE. O. tsutsugamushi
phylogenetically differs from other rickettsiae and represents a unique genus of Rickettsiceae.
For O. tsutsugamushi, other immunogenic proteins have been described, namely Sta22, Sta47,
and Sta56. O. tsutsugamushi-infected mice develop antibodies and specific CD4+ T cells
against Sta22 [103]. The same is true for O. tsutsugamushi-infected humans. Humans addition-
ally develop Sta56-specific antibodies and CD4+ T cells [104–106] as well as antibodies recog-
nizing Sta47 [209]. Further investigations focused on the Sta47 and Sta56 proteins. The
immunization of mice with DNA encoding for Sta56 was partially protective against the infec-
tion with the homologous O. tsutsugamushi strain [210], and mice vaccinated with recombi-
nant Sta56 protein were found to be completely protected against challenge with the

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 22 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

homologous O. tsutsugamushi strain [109,211,212]. In these animals, enhanced levels of anti-

bodies were observed, and lymphocytes showed increased proliferation, IFNγ, and IL-2 release
upon restimulation in vitro with homologous O. tsutsugamushi WCA [109,211]. Similarly, vac-
cination of mice with a fusion protein of Sta46 and Sta56 was partially protective against chal-
lenge with homologous O. tsutsugamushi strain [213]. However, only weak protection was
achieved in primates (Macaca fascicularis) upon immunization with a recombinant fragment
of Sta56 (AA 80–456). All of these animals developed fever and rickettsiemia [214].
Other immunodominant antigens are ScaA, C, D, and E from Orientia. O. tsutsugamushi
patients develop antibodies against these surface proteins with a stronger response to ScaA
and C compared to Sca E and D that are differentially expressed by different O. tsutsugamushi
strains [107]. In the literature, there is 1 description in which C57BL/6 mice were immunized
with purified recombinant ScaA, ScaC, or Sta56 that derived from the O. tsutsugamushi Bor-
yong strain. Immunization with ScaA but not ScaC or Sta56 led to protection and enhanced
survival of the animals upon infection with the homologous O. tsutsugamushi strain. In addi-
tion, immunization with recombinant ScaA from the strain Boryong also led to enhanced sur-
vival upon infection with the Karp strain as well as in the infection with the Kato strain,
although to weaker extent [108]. In another study from the same group, immunization was
performed with antigens coupled to nanoparticles. Ha and colleagues coupled ScaA from O.
tsutsugamushi to zinc oxide nanoparticles. These particles are taken up by DCs in vitro and
induce protective immunity in vivo in mice. Animals vaccinated with the ScaA-coupled nano-
particles were protected against lethal challenge with O. tsutsugamushi and developed antibod-
ies against ScaA as well as IFNγ-producing CD4+ TH1 and CD8+ T cells similar to the
vaccination of mice with ScaA plus adjuvants [215].

Concluding remarks
In recent years, few but promising vaccination strategies against rickettsial infections in experi-
mental animal models have been described. OmpA and OmpB are the most prominent anti-
gens that may serve as vaccine candidates, although it is not yet clear whether immunization
with these proteins can indeed confer protection. The observations made in immunized and
experimentally infected animals, however, are encouraging that the development of a vaccine
is possible. Apart from these proteins, only very few other rickettsial antigens have been
described. Further research should focus on the idenfication of new rickettsial antigens and
the analysis of their immunogenic potential. This research is essential for the development of a
protective vaccine that can serve as a prophylaxis against rickettsial infections in endemic
areas that are predominantly found in poor countries, as well as for travelers of these regions.

Supporting information
S1 Fig. A selection of bioinformatic software tools for the prediction of cellular protein
location, immunogenicity, MHC processing, and B cell epitopes. The table provides a selec-
tion of bioinformatic software tools for the prediction of cellular protein location, immunoge-
nicity, processing for MHCI, or MHCII presentation and B cell epitopes.
(PDF)

Acknowledgments
I thank Prof. Bernhard Fleischer and Prof. Christoph Hoelscher for carefully reading the man-
uscript and discussions.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 23 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

References
1. Izzard L, Fuller A, Blacksell SD, Paris DH, Richards AL, Aukkanit N, et al. Isolation of a novel Orientia
species (O. chuto sp. nov.) from a patient infected in Dubai. J Clin Microbiol 2010; 48(12):4404–9.
https://doi.org/10.1128/JCM.01526-10 PMID: 20926708; PubMed Central PMCID: PMC3008486.
2. Weitzel T, Martinez-Valdebenito C, Acosta-Jamett G, Jiang J, Richards AL, Abarca K. Scrub Typhus
in Continental Chile, 2016-2018(1). Emerg Infect Dis 2019; 25(6):1214–7. https://doi.org/10.3201/
eid2506.181860 PMID: 30835200; PubMed Central PMCID: PMC6537721.
3. Prevention CDCA. CDC Yellow Book 2020: Health information for international travel. 2020:326–328.
4. Mansueto P, Vitale G, Cascio A, Seidita A, Pepe I, Carroccio A, et al. New insight into immunity and
immunopathology of Rickettsial diseases. Clin Dev Immunol 2012; 2012:967852. Epub 2011/09/14.
https://doi.org/10.1155/2012/967852 PMID: 21912565; PubMed Central PMCID: PMC3170826.
5. Sahni SK, Rydkina E. Host-cell interactions with pathogenic Rickettsia species. Future Microbiol 2009;
4(3):323–39. Epub 2009/03/31. https://doi.org/10.2217/fmb.09.6 PMID: 19327117; PubMed Central
PMCID: PMC2775711.
6. Hackstadt T. The biology of rickettsiae. Infect Agents Dis 1996; 5(3):127–43. Epub 1996/06/01. PMID:
8805076.
7. Heinzen RA. Rickettsial actin-based motility: behavior and involvement of cytoskeletal regulators. Ann
N Y Acad Sci 2003; 990:535–47. Epub 2003/07/16. https://doi.org/10.1111/j.1749-6632.2003.
tb07424.x PMID: 12860687.
8. Weddle E, Agaisse H. Principles of intracellular bacterial pathogen spread from cell to cell. PLoS
Pathog 2018; 14(12):e1007380. https://doi.org/10.1371/journal.ppat.1007380 PMID: 30543716;
PubMed Central PMCID: PMC6292572.
9. Kim MJ, Kim MK, Kang JS. Involvement of lipid rafts in the budding-like exit of Orientia tsutsugamushi.
Microb Pathog 2013; 63:37–43. https://doi.org/10.1016/j.micpath.2013.06.002 PMID: 23791848.
10. Radulovic S, Price PW, Beier MS, Gaywee J, Macaluso JA, Azad A. Rickettsia-macrophage interac-
tions: host cell responses to Rickettsia akari and Rickettsia typhi. Infect Immun 2002; 70(5):2576–82.
Epub 2002/04/16. https://doi.org/10.1128/iai.70.5.2576-2582.2002 PMID: 11953398; PubMed Central
PMCID: PMC127898.
11. Curto P, Riley SP, Simoes I, Martinez JJ. Macrophages Infected by a Pathogen and a Non-pathogen
Spotted Fever Group Rickettsia Reveal Differential Reprogramming Signatures Early in Infection.
Front Cell Infect Microbiol 2019; 9:97. https://doi.org/10.3389/fcimb.2019.00097 PMID: 31024862;
PubMed Central PMCID: PMC6467950.
12. Drevets DA, Leenen PJ, Greenfield RA. Invasion of the central nervous system by intracellular bacte-
ria. Clin Microbiol Rev 2004; 17(2):323–47. https://doi.org/10.1128/cmr.17.2.323-347.2004 PMID:
15084504; PubMed Central PMCID: PMC387409.
13. Osterloh A, Papp S, Moderzynski K, Kuehl S, Richardt U, Fleischer B. Persisting Rickettsia typhi
Causes Fatal Central Nervous System Inflammation. Infect Immun 2016; 84(5):1615–32. https://doi.
org/10.1128/IAI.00034-16 PMID: 26975992.
14. Walker DH, Popov VL, Wen J, Feng HM. Rickettsia conorii infection of C3H/HeN mice. A model of
endothelial-target rickettsiosis. Lab Investig 1994; 70(3):358–68. Epub 1994/03/01. PMID: 7511715.
15. Pongponratn E, Maneerat Y, Chaisri U, Wilairatana P, Punpoowong B, Viriyavejakul P, et al. Electron-
microscopic examination of Rickettsia tsutsugamushi-infected human liver. Tropical Med Int Health
1998; 3(3):242–8. Epub 1998/05/21. https://doi.org/10.1046/j.1365-3156.1998.00231.x PMID:
9593364.
16. Walker DH, Harrison A, Henderson F, Murphy FA. Identification of Rickettsia rickettsii in a guinea pig
model by immunofluorescent and electron microscopic techniques. Am J Pathol 1977; 86(2):343–58.
Epub 1977/02/01. PMID: 402079; PubMed Central PMCID: PMC2032096.
17. Joshi SG, Kovacs AD. Rickettsia rickettsii infection causes apoptotic death of cultured cerebellar gran-
ule neurons. J Med Microbiol 2007; 56(Pt 1):138–41. https://doi.org/10.1099/jmm.0.46826-0 PMID:
17172530.
18. McDade JE, Stakebake JR, Gerone PJ. Plaque assay system for several species of Rickettsia. J Bac-
teriol 1969; 99(3):910–2. Epub 1969/09/01. https://doi.org/10.1128/JB.99.3.910-912.1969 PMID:
4984178; PubMed Central PMCID: PMC250118.
19. Wike DA, Tallent G, Peacock MG, Ormsbee RA. Studies of the rickettsial plaque assay technique.
Infect Immun 1972; 5(5):715–22. Epub 1972/05/01. https://doi.org/10.1128/IAI.5.5.715-722.1972
PMID: 4629250; PubMed Central PMCID: PMC422430.
20. Hanson B. Improved plaque assay for Rickettsia tsutsugamushi. Am J Trop Med Hyg. 1987; 36
(3):631–8. Epub 1987/05/01. https://doi.org/10.4269/ajtmh.1987.36.631 PMID: 3107412.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 24 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

21. Policastro PF, Peacock MG, Hackstadt T. Improved plaque assays for Rickettsia prowazekii in Vero
76 cells. J Clin Microbiol 1996; 34(8):1944–8. Epub 1996/08/01. https://doi.org/10.1128/JCM.34.8.
1944-1948.1996 PMID: 8818887; PubMed Central PMCID: PMC229159.
22. Rathi N, Rathi A. Rickettsial infections: Indian perspective. Indian Pediatr 2010; 47(2):157–64. Epub
2010/03/17. https://doi.org/10.1007/s13312-010-0024-3 PMID: 20228429.
23. Kuloglu F. Rickettsial infections. Disease and Molecular Medicine. 2013; 1 (2):39–45.
24. Regan JJ, Traeger MS, Humpherys D, Mahoney DL, Martinez M, Emerson GL, et al. Risk factors for
fatal outcome from rocky mountain spotted Fever in a highly endemic area-Arizona, 2002–2011. Clin
Infect Dis 2015; 60(11):1659–66. https://doi.org/10.1093/cid/civ116 PMID: 25697742; PubMed Cen-
tral PMCID: PMC4706357.
25. Raoult D, Woodward T, Dumler JS. The history of epidemic typhus. Infect Dis Clin N Am 2004; 18
(1):127–40. Epub 2004/04/15. https://doi.org/10.1016/S0891-5520(03)00093-X PMID: 15081509.
26. Dill T, Dobler G, Saathoff E, Clowes P, Kroidl I, Ntinginya E, et al. High seroprevalence for typhus
group rickettsiae, southwestern Tanzania. Emerg Infect Dis 2013; 19(2):317–20. https://doi.org/10.
3201/eid1902.120601 PMID: 23347529; PubMed Central PMCID: PMC3559041.
27. Qin A, Tucker AM, Hines A, Wood DO. Transposon mutagenesis of the obligate intracellular pathogen
Rickettsia prowazekii. Appl Environ Microbiol 2004; 70(5):2816–22. Epub 2004/05/07. https://doi.org/
10.1128/aem.70.5.2816-2822.2004 PMID: 15128537; PubMed Central PMCID: PMC404435.
28. Liu ZM, Tucker AM, Driskell LO, Wood DO. Mariner-based transposon mutagenesis of Rickettsia pro-
wazekii. Appl Environ Microbiol 2007; 73(20):6644–9. https://doi.org/10.1128/AEM.01727-07 PMID:
17720821; PubMed Central PMCID: PMC2075046.
29. Clark TR, Lackey AM, Kleba B, Driskell LO, Lutter EI, Martens C, et al. Transformation frequency of a
mariner-based transposon in Rickettsia rickettsii. J Bacteriol 2011; 193(18):4993–5. Epub 2011/07/19.
https://doi.org/10.1128/JB.05279-11 PMID: 21764933; PubMed Central PMCID: PMC3165637.
30. Clark TR, Ellison DW, Kleba B, Hackstadt T. Complementation of Rickettsia rickettsii RelA/SpoT
restores a nonlytic plaque phenotype. Infect Immun 2011; 79(4):1631–7. Epub 2011/02/09. https://doi.
org/10.1128/IAI.00048-11 PMID: 21300770; PubMed Central PMCID: PMC3067566.
31. Baldridge GD, Burkhardt N, Herron MJ, Kurtti TJ, Munderloh UG. Analysis of fluorescent protein
expression in transformants of Rickettsia monacensis, an obligate intracellular tick symbiont. Appl
Environ Microbiol 2005; 71(4):2095–105. https://doi.org/10.1128/AEM.71.4.2095-2105.2005 PMID:
15812043; PubMed Central PMCID: PMC1082560.
32. Hauptmann M, Burkhardt N, Munderloh U, Kuehl S, Richardt U, Krasemann S, et al. GFPuv-express-
ing recombinant Rickettsia typhi: a useful tool for the study of pathogenesis and CD8+ T cell immunol-
ogy in Rickettsia typhi infection. Infect Immun 2017. https://doi.org/10.1128/IAI.00156-17 PMID:
28289147.
33. Driskell LO, Yu XJ, Zhang L, Liu Y, Popov VL, Walker DH, et al. Directed mutagenesis of the Rickettsia
prowazekii pld gene encoding phospholipase D. Infect Immun 2009; 77(8):3244–8. Epub 2009/06/10.
https://doi.org/10.1128/IAI.00395-09 PMID: 19506016; PubMed Central PMCID: PMC2715659.
34. Noriea NF, Clark TR, Hackstadt T. Targeted knockout of the Rickettsia rickettsii OmpA surface antigen
does not diminish virulence in a mammalian model system. MBio. 2015; 6(2). https://doi.org/10.1128/
mBio.00323-15 PMID: 25827414; PubMed Central PMCID: PMC4453529.
35. Chakraborty S, Sarma N. Scrub Typhus: An Emerging Threat. Indian J Dermatol 2017; 62(5):478–85.
https://doi.org/10.4103/ijd.IJD_388_17 PMID: 28979009; PubMed Central PMCID: PMC5618834.
36. Gao Y, Yan D, Liu K, Sun J, Niu Y, Liu X, et al. Epidemiological characteristics and spatiotemporal pat-
terns of typhus group rickettsiosis at the county level in China, 2005–2017. Int J Infect Dis 2020;
91:60–7. https://doi.org/10.1016/j.ijid.2019.11.018 PMID: 31760046.
37. Chen R, Kou Z, Xu L, Cao J, Liu Z, Wen X, et al. Analysis of epidemiological characteristics of four nat-
ural-focal diseases in Shandong Province, China in 2009–2017: A descriptive analysis. PLoS One
2019; 14(8):e0221677. https://doi.org/10.1371/journal.pone.0221677 PMID: 31454372; PubMed Cen-
tral PMCID: PMC6711524.
38. Dittrich S, Rattanavong S, Lee SJ, Panyanivong P, Craig SB, Tulsiani SM, et al. Orientia, rickettsia,
and leptospira pathogens as causes of CNS infections in Laos: a prospective study. Lancet Glob
Health 2015; 3(2):e104–12. https://doi.org/10.1016/S2214-109X(14)70289-X PMID: 25617190.
39. Ren J, Sun J, Wang Z, Ling F, Shi X, Zhang R, et al. Re-emergence of scrub typhus in Zhejiang Prov-
ince, southern China: A 45-year population-based surveillance study. Travel Med Infect Dis 2019.
https://doi.org/10.1016/j.tmaid.2019.05.013 PMID: 31125615.
40. Lee HW, Cho PY, Moon SU, Na BK, Kang YJ, Sohn Y, et al. Current situation of scrub typhus in South
Korea from 2001–2013. Parasit Vectors 2015; 8:238. https://doi.org/10.1186/s13071-015-0858-6
PMID: 25928653; PubMed Central PMCID: PMC4416255.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 25 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

41. Burchard G, Fleischer B. Tsutsugamushi-Fieber breitet sich weltweit aus—Bisherige Angaben zu

Endemiegebieten müssen revidiert werden. Flugmedizin-Tropenmedizin-Reisemedizin—Georg
Thieme Verlag KG Stuttgart. 2016; 23(06):267. https://doi.org/10.1055/s-0042-120836
42. Balcells ME, Rabagliati R, Garcia P, Poggi H, Oddo D, Concha M, et al. Endemic scrub typhus-like ill-
ness, Chile Emerg Infect Dis 2011; 17(9):1659–63. https://doi.org/10.3201/eid1709.100960 PMID:
21888791; PubMed Central PMCID: PMC3322051.
43. Thiga JW, Mutai BK, Eyako WK, Ng’ang’a Z, Jiang J, Richards AL, et al. High seroprevalence of anti-
bodies against spotted fever and scrub typhus bacteria in patients with febrile Illness, Kenya Emerg
Infect Dis 2015; 21(4):688–91. https://doi.org/10.3201/eid2104.141387 PMID: 25811219; PubMed
Central PMCID: PMC4378494.
44. Maina AN, Farris CM, Odhiambo A, Jiang J, Laktabai J, Armstrong J, et al. Q Fever, Scrub Typhus,
and Rickettsial Diseases in Children, Kenya, 2011–2012. Emerg Infect Dis 2016; 22(5):883–6. https://
doi.org/10.3201/eid2205.150953 PMID: 27088502; PubMed Central PMCID: PMC4861507.
45. Cosson JF, Galan M, Bard E, Razzauti M, Bernard M, Morand S, et al. Detection of Orientia sp. DNA
in rodents from Asia, West Africa and Europe. Parasit Vectors 2015; 8:172. https://doi.org/10.1186/
s13071-015-0784-7 PMID: 25884521; PubMed Central PMCID: PMC4374543.
46. Alvarez-Hernandez G, Roldan JFG, Milan NSH, Lash RR, Behravesh CB, Paddock CD. Rocky Moun-
tain spotted fever in Mexico: past, present, and future. Lancet Infect Dis 2017; 17(6):e189–e96. https://
doi.org/10.1016/S1473-3099(17)30173-1 PMID: 28365226.
47. Estripeaut D, Aramburu MG, Saez-Llorens X, Thompson HA, Dasch GA, Paddock CD, et al. Rocky
Mountain spotted fever, Panama Emerg Infect Dis 2007; 13(11):1763–5. Epub 2008/01/26. https://doi.
org/10.3201/eid1311.070931 PMID: 18217566; PubMed Central PMCID: PMC3375809.
48. Tribaldos M, Zaldivar Y, Bermudez S, Samudio F, Mendoza Y, Martinez AA, et al. Rocky Mountain
spotted fever in Panama: a cluster description. J Infect Dev Ctries 2011; 5(10):737–41. Epub 2011/10/
15. https://doi.org/10.3855/jidc.2189 PMID: 21997944.
49. Martinez-Caballero A, Moreno B, Gonzalez C, Martinez G, Adames M, Pachar JV, et al. Descriptions
of two new cases of Rocky Mountain spotted fever in Panama, and coincident infection with Rickettsia
rickettsii in Rhipicephalus sanguineus s.l. in an urban locality of Panama City, Panama. Epidemiol
Infect 2018; 146(7):875–8. https://doi.org/10.1017/S0950268818000730 PMID: 29619916.
50. Hidalgo M, Orejuela L, Fuya P, Carrillo P, Hernandez J, Parra E, et al. Rocky Mountain spotted fever,
Colombia Emerg Infect Dis 2007; 13(7):1058–60. Epub 2008/01/25. https://doi.org/10.3201/eid1307.
060537 PMID: 18214179; PubMed Central PMCID: PMC2878212.
51. Hidalgo M, Miranda J, Heredia D, Zambrano P, Vesga JF, Lizarazo D, et al. Outbreak of Rocky Moun-
tain spotted fever in Cordoba, Colombia Mem I Oswaldo Cruz 2011; 106(1):117–8. Epub 2011/02/23.
https://doi.org/10.1590/s0074-02762011000100019 PMID: 21340366.
52. del Sa DelFiol F, Junqueira FM, da Rocha MC, de Toledo MI, Filho SB. [Rocky Mountain spotted fever
in Brazil]. Rev Panam Salud Publica 2010; 27(6):461–6. https://doi.org/10.1590/s1020-
49892010000600008 PMID: 20721447.
53. de Oliveira SV, Guimaraes JN, Reckziegel GC, Neves BM, Araujo-Vilges KM, Fonseca LX, et al. An
update on the epidemiological situation of spotted fever in Brazil. J Venom Anim Toxins incl Trop Dis
2016; 22(1):22. https://doi.org/10.1186/s40409-016-0077-4 PMID: 27555867; PubMed Central
PMCID: PMC4994305.
54. Faccini-Martinez AA, Munoz-Leal S, Acosta ICL, de Oliveira SV, de Lima Dure AI, Cerutti CJ, et al.
Confirming Rickettsia rickettsii as the etiological agent of lethal spotted fever group rickettsiosis in
human patients from Espirito Santo state, Brazil Ticks Tick Borne Dis 2018;9(3):496–9. https://doi.org/
10.1016/j.ttbdis.2018.01.005 PMID: 29371125.
55. Murray KO, Evert N, Mayes B, Fonken E, Erickson T, Garcia MN, et al. Typhus Group Rickettsiosis,
Texas, USA, 2003–2013. Emerg Infect Dis 2017; 23(4):645–8. https://doi.org/10.3201/eid2304.
160958 PMID: 28322701; PubMed Central PMCID: PMC5367421.
56. Reynolds MG, Krebs JS, Comer JA, Sumner JW, Rushton TC, Lopez CE, et al. Flying squirrel-associ-
ated typhus, United States Emerg Infect Dis 2003; 9(10):1341–3. Epub 2003/11/12. https://doi.org/10.
3201/eid0910.030278 PMID: 14609478; PubMed Central PMCID: PMC3033063.
57. Chapman AS, Swerdlow DL, Dato VM, Anderson AD, Moodie CE, Marriott C, et al. Cluster of sylvatic
epidemic typhus cases associated with flying squirrels, 2004–2006. Emerg Infect Dis 2009; 15
(7):1005–11. Epub 2009/07/25. https://doi.org/10.3201/eid1507.081305 PMID: 19624912; PubMed
Central PMCID: PMC2744229.
58. Strand A, Paddock CD, Rinehart AR, Condit ME, Marus JR, Gillani S, et al. African Tick Bite Fever
Treated Successfully With Rifampin in a Patient With Doxycycline Intolerance. Clin Infect Dis 2017; 65
(9):1582–4. https://doi.org/10.1093/cid/cix363 PMID: 28505276; PubMed Central PMCID:
PMC5850440.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 26 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

59. Biggs HM, Behravesh CB, Bradley KK, Dahlgren FS, Drexler NA, Dumler JS, et al. Diagnosis and
Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted
Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis—United States. MMWR Morb Mortal
Wkly Rep. 2016; 65(2):1–44. https://doi.org/10.15585/mmwr.rr6502a1 PMID: 27172113.
60. Rachek LI, Tucker AM, Winkler HH, Wood DO. Transformation of Rickettsia prowazekii to rifampin
resistance. J Bacteriol 1998; 180(8):2118–24. Epub 1998/04/29. https://doi.org/10.1128/JB.180.8.
2118-2124.1998 PMID: 9555894; PubMed Central PMCID: PMC107138.
61. Troyer JM, Radulovic S, Andersson SG, Azad AF. Detection of point mutations in rpoB gene of rifam-
pin-resistant Rickettsia typhi. Antimicrob Agents Chemother 1998; 42(7):1845–6. Epub 1998/07/14.
https://doi.org/10.1128/AAC.42.7.1845 PMID: 9661032; PubMed Central PMCID: PMC105694.
62. Drancourt M, Raoult D. Characterization of mutations in the rpoB gene in naturally rifampin-resistant
Rickettsia species. Antimicrob Agents Chemother 1999; 43(10):2400–3. Epub 1999/10/03. https://doi.
org/10.1128/AAC.43.10.2400 PMID: 10508014; PubMed Central PMCID: PMC89490.
63. Watt G, Chouriyagune C, Ruangweerayud R, Watcharapichat P, Phulsuksombati D, Jongsakul K,
et al. Scrub typhus infections poorly responsive to antibiotics in northern Thailand. Lancet 1996; 348
(9020):86–9. Epub 1996/07/13. https://doi.org/10.1016/s0140-6736(96)02501-9 PMID: 8676722.
64. Watt G, Kantipong P, Jongsakul K, Watcharapichat P, Phulsuksombati D, Strickman D. Doxycycline
and rifampicin for mild scrub-typhus infections in northern Thailand: a randomised trial. Lancet 2000;
356(9235):1057–61. https://doi.org/10.1016/S0140-6736(00)02728-8 PMID: 11009140.
65. Rajapakse S, Rodrigo C, Fernando SD. Drug treatment of scrub typhus. Trop Dr 2011; 41(1):1–4.
https://doi.org/10.1258/td.2010.100311 PMID: 21172901.
66. Kelly DJ, Fuerst PA, Richards AL. The Historical Case for and the Future Study of Antibiotic-Resistant
Scrub Typhus. Trop Med Infect Dis. 2017; 2(4). https://doi.org/10.3390/tropicalmed2040063 PMID:
30270920; PubMed Central PMCID: PMC6082054.
67. Wangrangsimakul T, Phuklia W, Newton PN, Richards AL, Day NPJ. Scrub Typhus and the Miscon-
ception of Doxycycline Resistance. Clin Infect Dis 2020; 70(11):2444–9. https://doi.org/10.1093/cid/
ciz972 PMID: 31570937; PubMed Central PMCID: PMC7245148.
68. Chung MH, Lee JS, Baek JH, Kim M, Kang JS. Persistence of Orientia tsutsugamushi in humans. J
Korean Med Sci 2012; 27(3):231–5. Epub 2012/03/02. https://doi.org/10.3346/jkms.2012.27.3.231
PMID: 22379331; PubMed Central PMCID: PMC3286767.
69. Parker RT, Menon PG, Merideth AM, Snyder MJ, Woodward TE. Persistence of Rickettsia rickettsii in
a patient recovered from Rocky Mountain spotted fever. J Immunol 1954; 73(6):383–6. Epub 1954/12/
01. PMID: 13212060.
70. Hove MG, Walker DH. Persistence of rickettsiae in the partially viable gangrenous margins of ampu-
tated extremities 5 to 7 weeks after onset of Rocky Mountain spotted fever. Arch Pathol Lab Med
1995; 119(5):429–31. Epub 1995/05/01. PMID: 7748070.
71. Bechah Y, Paddock CD, Capo C, Mege JL, Raoult D. Adipose tissue serves as a reservoir for recru-
descent Rickettsia prowazekii infection in a mouse model. PLoS One 2010; 5(1):e8547. Epub 2010/
01/06. https://doi.org/10.1371/journal.pone.0008547 PMID: 20049326; PubMed Central PMCID:
PMC2797295.
72. Stein A, Purgus R, Olmer M, Raoult D. Brill-Zinsser disease in France. Lancet 1999; 353(9168):1936.
https://doi.org/10.1016/S0140-6736(99)01995-9 PMID: 10371575.
73. Turcinov D, Kuzman I, Herendic B. Failure of azithromycin in treatment of Brill-Zinsser disease. Antimi-
crob Agents Chemother 2000; 44(6):1737–8. Epub 2000/05/19. https://doi.org/10.1128/aac.44.6.
1737-1738.2000 PMID: 10817744; PubMed Central PMCID: PMC89948.
74. Lutwick LI. Brill-Zinsser disease. Lancet 2001; 357(9263):1198–200. https://doi.org/10.1016/s0140-
6736(00)04339-7 PMID: 11323068.
75. Faucher JF, Socolovschi C, Aubry C, Chirouze C, Hustache-Mathieu L, Raoult D, et al. Brill-Zinsser
disease in Moroccan man, France, 2011. Emerg Infect Dis 2012; 18(1):171–2. https://doi.org/10.3201/
eid1801.111057 PMID: 22261378; PubMed Central PMCID: PMC3310116.
76. Osterloh A. Immune response against rickettsiae: lessons from murine infection models. Med Micro-
biol Immunol 2017. https://doi.org/10.1007/s00430-017-0514-1 PMID: 28770333.
77. Sahni A, Fang R, Sahni SK, Walker DH. Pathogenesis of Rickettsial Diseases: Pathogenic and
Immune Mechanisms of an Endotheliotropic Infection. Ann Rev Pathol 2019; 14:127–52. https://doi.
org/10.1146/annurev-pathmechdis-012418-012800 PMID: 30148688; PubMed Central PMCID:
PMC6505701.
78. Papp S, Moderzynski K, Rauch J, Heine L, Kuehl S, Richardt U, et al. Liver Necrosis and Lethal Sys-
temic Inflammation in a Murine Model of Rickettsia typhi Infection: Role of Neutrophils, Macrophages

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 27 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

and NK Cells. PLoS Negl Trop Dis 2016; 10(8):e0004935. https://doi.org/10.1371/journal.pntd.

0004935 PMID: 27548618.
79. Feng HM, Whitworth T, Olano JP, Popov VL, Walker DH. Fc-dependent polyclonal antibodies and
antibodies to outer membrane proteins A and B, but not to lipopolysaccharide, protect SCID mice
against fatal Rickettsia conorii infection. Infect Immun 2004; 72(4):2222–8. Epub 2004/03/25. https://
doi.org/10.1128/iai.72.4.2222-2228.2004 PMID: 15039346; PubMed Central PMCID: PMC375156.
80. Dumler JS, Taylor JP, Walker DH. Clinical and laboratory features of murine typhus in south Texas,
1980 through 1987. JAMA 1991; 266(10):1365–70. Epub 1991/09/11. PMID: 1880866.
81. Fournier PE, Jensenius M, Laferl H, Vene S, Raoult D. Kinetics of antibody responses in Rickettsia
africae and Rickettsia conorii infections. Clin Diagn Lab Immunol 2002; 9(2):324–8. Epub 2002/03/05.
https://doi.org/10.1128/cdli.9.2.324-328.2002 PMID: 11874871; PubMed Central PMCID:
PMC119950.
82. Teysseire N, Raoult D. Comparison of Western immunoblotting and microimmunofluorescence for
diagnosis of Mediterranean spotted fever. J Clin Microbiol 1992; 30(2):455–60. Epub 1992/02/01.
https://doi.org/10.1128/JCM.30.2.455-460.1992 PMID: 1537916; PubMed Central PMCID:
PMC265077.
83. Uchiyama T. Intracytoplasmic localization of antigenic heat-stable 120- to 130-kilodalton proteins
(PS120) common to spotted fever group rickettsiae demonstrated by immunoelectron microscopy.
Microbiol Immunol 1997; 41(10):815–8. Epub 1997/01/01. https://doi.org/10.1111/j.1348-0421.1997.
tb01933.x PMID: 9403508.
84. Uchiyama T. Adherence to and invasion of Vero cells by recombinant Escherichia coli expressing the
outer membrane protein rOmpB of Rickettsia japonica. Ann N Y Acad Sci 2003; 990:585–90. Epub
2003/07/16. https://doi.org/10.1111/j.1749-6632.2003.tb07431.x PMID: 12860694.
85. Chan YG, Cardwell MM, Hermanas TM, Uchiyama T, Martinez JJ. Rickettsial outer-membrane protein
B (rOmpB) mediates bacterial invasion through Ku70 in an actin, c-Cbl, clathrin and caveolin 2-depen-
dent manner. Cell Microbiol 2009; 11(4):629–44. Epub 2009/01/13. https://doi.org/10.1111/j.1462-
5822.2008.01279.x PMID: 19134120; PubMed Central PMCID: PMC2773465.
86. Li H, Walker DH. rOmpA is a critical protein for the adhesion of Rickettsia rickettsii to host cells. Microb
Pathog 1998; 24(5):289–98. Epub 1998/06/13. https://doi.org/10.1006/mpat.1997.0197 PMID:
9600861.
87. Anacker RL, List RH, Mann RE, Hayes SF, Thomas LA. Characterization of monoclonal antibodies
protecting mice against Rickettsia rickettsii. J Infect Dis 1985; 151(6):1052–60. Epub 1985/06/01.
https://doi.org/10.1093/infdis/151.6.1052 PMID: 3923129.
88. Anacker RL, McDonald GA, List RH, Mann RE. Neutralizing activity of monoclonal antibodies to heat-
sensitive and heat-resistant epitopes of Rickettsia rickettsii surface proteins. Infect Immun 1987; 55
(3):825–7. Epub 1987/03/01. https://doi.org/10.1128/IAI.55.3.825-827.1987 PMID: 2434430; PubMed
Central PMCID: PMC260417.
89. Lange JV, Walker DH. Production and characterization of monoclonal antibodies to Rickettsia rickett-
sii. Infect Immun 1984; 46(2):289–94. Epub 1984/11/01. https://doi.org/10.1128/IAI.46.2.289-294.
1984 PMID: 6209219; PubMed Central PMCID: PMC261528.
90. Chan YG, Riley SP, Chen E, Martinez JJ. Molecular basis of immunity to rickettsial infection conferred
through outer membrane protein B. Infect Immun 2011; 79(6):2303–13. Epub 2011/03/30. https://doi.
org/10.1128/IAI.01324-10 PMID: 21444665; PubMed Central PMCID: PMC3125829.
91. Feng HM, Whitworth T, Popov V, Walker DH. Effect of antibody on the rickettsia-host cell interaction.
Infect Immun 2004; 72(6):3524–30. Epub 2004/05/25. https://doi.org/10.1128/IAI.72.6.3524-3530.
2004 PMID: 15155660; PubMed Central PMCID: PMC415703.
92. Ching WM, Wang H, Jan B, Dasch GA. Identification and characterization of epitopes on the 120-kilo-
dalton surface protein antigen of Rickettsia prowazekii with synthetic peptides. Infect Immun 1996; 64
(4):1413–9. Epub 1996/04/01. https://doi.org/10.1128/IAI.64.4.1413-1419.1996 PMID: 8606109;
PubMed Central PMCID: PMC173934.
93. Sears KT, Ceraul SM, Gillespie JJ, Allen ED Jr., Popov VL, Ammerman NC, et al. Surface proteome
analysis and characterization of surface cell antigen (Sca) or autotransporter family of Rickettsia typhi.
PLoS Pathog 2012; 8(8):e1002856. Epub 2012/08/23. https://doi.org/10.1371/journal.ppat.1002856
PMID: 22912578; PubMed Central PMCID: PMC3415449.
94. Renesto P, Azza S, Dolla A, Fourquet P, Vestris G, Gorvel JP, et al. Proteome analysis of Rickettsia
conorii by two-dimensional gel electrophoresis coupled with mass spectrometry. FEMS Microbiol Lett
2005; 245(2):231–8. Epub 2005/04/20. https://doi.org/10.1016/j.femsle.2005.03.004 PMID:
15837377.
95. Qi Y, Xiong X, Wang X, Duan C, Jia Y, Jiao J, et al. Proteome analysis and serological characterization
of surface-exposed proteins of Rickettsia heilongjiangensis. PLoS One 2013; 8(7):e70440. Epub

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 28 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

2013/07/31. https://doi.org/10.1371/journal.pone.0070440 PMID: 23894656; PubMed Central PMCID:

PMC3720918.
96. Hajem N, Weintraub A, Nimtz M, Romling U, Pahlson C. A study of the antigenicity of Rickettsia helve-
tica proteins using two-dimensional gel electrophoresis. APMIS 2009; 117(4):253–62. Epub 2009/04/
03. https://doi.org/10.1111/j.1600-0463.2009.02435.x PMID: 19338513.
97. Pornwiroon W, Bourchookarn A, Paddock CD, Macaluso KR. Immunoproteomic profiling of Rickettsia
parkeri and Rickettsia amblyommii. Ticks Tick Borne Dis 2015; 6(6):829–35. https://doi.org/10.1016/j.
ttbdis.2015.07.012 PMID: 26234571; PubMed Central PMCID: PMC4575651.
98. Gaywee J, Radulovic S, Higgins JA, Azad AF. Transcriptional analysis of Rickettsia prowazekii inva-
sion gene homolog (invA) during host cell infection. Infect Immun 2002; 70(11):6346–54. Epub 2002/
10/16. https://doi.org/10.1128/iai.70.11.6346-6354.2002 PMID: 12379714; PubMed Central PMCID:
PMC130406.
99. Mustafa AS. Development of new vaccines and diagnostic reagents against tuberculosis. Mol Immu-
nol 2002; 39(1–2):113–9. https://doi.org/10.1016/s0161-5890(02)00048-2 PMID: 12213334.
100. Sinha K, Bhatnagar R. GroEL provides protection against Bacillus anthracis infection in BALB/c mice.
Mol Immunol 2010; 48(1–3):264–71. https://doi.org/10.1016/j.molimm.2010.08.001 PMID: 20832865.
101. Yamaguchi H, Osaki T, Taguchi H, Sato N, Toyoda A, Takahashi M, et al. Effect of bacterial flora on
postimmunization gastritis following oral vaccination of mice with Helicobacter pylori heat shock pro-
tein 60. Clin Diagn Lab Immunol 2003; 10(5):808–12. https://doi.org/10.1128/cdli.10.5.808-812.2003
PMID: 12965909; PubMed Central PMCID: PMC193875.
102. Nilsson CL, Larsson T, Gustafsson E, Karlsson KA, Davidsson P. Identification of protein vaccine can-
didates from Helicobacter pylori using a preparative two-dimensional electrophoretic procedure and
mass spectrometry. Anal Chem 2000; 72(9):2148–53. https://doi.org/10.1021/ac9912754 PMID:
10815978.
103. Hickman CJ, Stover CK, Joseph SW, Oaks EV. Molecular cloning and sequence analysis of a Rickett-
sia tsutsugamushi 22 kDa antigen containing B- and T-cell epitopes. Microb Pathog 1991; 11(1):19–
31. Epub 1991/07/01. https://doi.org/10.1016/0882-4010(91)90090-w PMID: 1724548.
104. Chen WJ, Niu DS, Zhang XY, Chen ML, Cui H, Wei WJ, et al. Recombinant 56-kilodalton major outer
membrane protein antigen of Orientia tsutsugamushi Shanxi and its antigenicity. Infect Immun 2003;
71(8):4772–9. https://doi.org/10.1128/iai.71.8.4772-4779.2003 PMID: 12874360; PubMed Central
PMCID: PMC166048.
105. Seong SY, Park SG, Huh MS, Jang WJ, Kim HR, Han TH, et al. Mapping of antigenic determinant
regions of the Bor56 protein of Orientia tsutsugamushi. Infect Immun 1997; 65(12):5250–6. Epub
1997/12/11. https://doi.org/10.1128/IAI.65.12.5250-5256.1997 PMID: 9393823; PubMed Central
PMCID: PMC175756.
106. Ramaiah A, Koralur MC, Dasch GA. Complexity of type-specific 56 kDa antigen CD4 T-cell epitopes of
Orientia tsutsugamushi strains causing scrub typhus in India. PLoS One 2018; 13(4):e0196240.
https://doi.org/10.1371/journal.pone.0196240 PMID: 29698425; PubMed Central PMCID:
PMC5919512.
107. Ha NY, Kim Y, Choi JH, Choi MS, Kim IS, Kim YS, et al. Detection of antibodies against Orientia tsu-
tsugamushi Sca proteins in scrub typhus patients and genetic variation of sca genes of different
strains. Clin Vaccine Immunol 2012; 19(9):1442–51. https://doi.org/10.1128/CVI.00285-12 PMID:
22787193; PubMed Central PMCID: PMC3428396.
108. Ha NY, Sharma P, Kim G, Kim Y, Min CK, Choi MS, et al. Immunization with an autotransporter protein
of Orientia tsutsugamushi provides protective immunity against scrub typhus. PLoS Negl Trop Dis
2015; 9(3):e0003585. https://doi.org/10.1371/journal.pntd.0003585 PMID: 25768004; PubMed Cen-
tral PMCID: PMC4359152.
109. Seong SY, Kim HR, Huh MS, Park SG, Kang JS, Han TH, et al. Induction of neutralizing antibody in
mice by immunization with recombinant 56 kDa protein of Orientia tsutsugamushi. Vaccine 1997; 15
(16):1741–7. Epub 1997/11/19. https://doi.org/10.1016/s0264-410x(97)00112-6 PMID: 9364677.
110. Seong SY, Kim MK, Lee SM, Odgerel Z, Choi MS, Han TH, et al. Neutralization epitopes on the anti-
genic domain II of the Orientia tsutsugamushi 56-kDa protein revealed by monoclonal antibodies. Vac-
cine 2000; 19(1):2–9. https://doi.org/10.1016/s0264-410x(00)00167-5 PMID: 10924780.
111. Walker DH, Olano JP, Feng HM. Critical role of cytotoxic T lymphocytes in immune clearance of rick-
ettsial infection. Infect Immun 2001; 69(3):1841–6. https://doi.org/10.1128/IAI.69.3.1841-1846.2001
PMID: 11179362; PubMed Central PMCID: PMC98091.
112. Feng H, Popov VL, Yuoh G, Walker DH. Role of T lymphocyte subsets in immunity to spotted fever
group Rickettsiae. J Immunol 1997; 158(11):5314–20. PMID: 9164951.
113. Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem 1994; 269(19):13725–8.
PMID: 7514592.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 29 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

114. Kamijo R, Harada H, Matsuyama T, Bosland M, Gerecitano J, Shapiro D, et al. Requirement for tran-
scription factor IRF-1 in NO synthase induction in macrophages. Science 1994; 263(5153):1612–5.
https://doi.org/10.1126/science.7510419 PMID: 7510419.
115. Koide N, Mu MM, Hassan F, Islam S, Tumurkhuu G, Dagvadorj J, et al. Lipopolysaccharide enhances
interferon-gamma-induced nitric oxide (NO) production in murine vascular endothelial cells via aug-
mentation of interferon regulatory factor-1 activation. J Endotox Res 2007; 13(3):167–75. https://doi.
org/10.1177/0968051907080894 PMID: 17621559.
116. Chan ED, Riches DW. Potential role of the JNK/SAPK signal transduction pathway in the induction of
iNOS by TNF-alpha. Biochem Biophys Res Commun 1998; 253(3):790–6. https://doi.org/10.1006/
bbrc.1998.9857 PMID: 9918806.
117. Feng HM, Walker DH. Mechanisms of intracellular killing of Rickettsia conorii in infected human endo-
thelial cells, hepatocytes, and macrophages. Infect Immun 2000; 68(12):6729–36. Epub 2000/11/18.
https://doi.org/10.1128/iai.68.12.6729-6736.2000 PMID: 11083788; PubMed Central PMCID:
PMC97773.
118. Feng HM, Popov VL, Walker DH. Depletion of gamma interferon and tumor necrosis factor alpha in
mice with Rickettsia conorii-infected endothelium: impairment of rickettsicidal nitric oxide production
resulting in fatal, overwhelming rickettsial disease. Infect Immun 1994; 62(5):1952–60. Epub 1994/05/
01. https://doi.org/10.1128/IAI.62.5.1952-1960.1994 PMID: 8168962; PubMed Central PMCID:
PMC186451.
119. Fang R, Ismail N, Soong L, Popov VL, Whitworth T, Bouyer DH, et al. Differential interaction of den-
dritic cells with Rickettsia conorii: impact on host susceptibility to murine spotted fever rickettsiosis.
Infect Immun 2007; 75(6):3112–23. Epub 2007/04/04. https://doi.org/10.1128/IAI.00007-07 PMID:
17403875; PubMed Central PMCID: PMC1932850.
120. Fang R, Ismail N, Shelite T, Walker DH. CD4+ CD25+ Foxp3- T-regulatory cells produce both gamma
interferon and interleukin-10 during acute severe murine spotted fever rickettsiosis. Infect Immun
2009; 77(9):3838–49. Epub 2009/07/01. https://doi.org/10.1128/IAI.00349-09 PMID: 19564386;
PubMed Central PMCID: PMC2738046.
121. Moderzynski K, Papp S, Rauch J, Heine L, Kuehl S, Richardt U, et al. CD4+ T Cells Are as Protective
as CD8+ T Cells against Rickettsia typhi Infection by Activating Macrophage Bactericidal Activity.
PLoS Negl Trop Dis 2016; 10(11):e0005089. https://doi.org/10.1371/journal.pntd.0005089 PMID:
27875529.
122. Moderzynski K, Heine L, Rauch J, Papp S, Kuehl S, Richardt U et al. Cytotoxic effector functions of T
cells are not required for protective immunity against fatal Rickettsia typhi infection in a murine model
of infection: Role of TH1 and TH17 cytokines in protection and pathology. PLoS Negl Trop Dis. 2017;
11 (2):e0005404. https://doi.org/10.1371/journal.pntd.0005404 PMID: 28222146
123. Walker DH, Popov VL, Feng HM. Establishment of a novel endothelial target mouse model of a typhus
group rickettsiosis: evidence for critical roles for gamma interferon and CD8 T lymphocytes. Lab Inves-
tig 2000; 80(9):1361–72. Epub 2000/09/27. https://doi.org/10.1038/labinvest.3780144 PMID:
11005205.
124. Turco J, Winkler HH. Gamma-interferon-induced inhibition of the growth of Rickettsia prowazekii in
fibroblasts cannot be explained by the degradation of tryptophan or other amino acids. Infect Immun
1986; 53(1):38–46. Epub 1986/07/01. https://doi.org/10.1128/IAI.53.1.38-46.1986 PMID: 3087883;
PubMed Central PMCID: PMC260072.
125. Catanzaro PJ, Shirai A, Hilderbrandt PK, Osterman JV. Host defenses in experimental scrub typhus:
histopathological correlates. Infect Immun 1976; 13(3):861–75. Epub 1976/03/01. https://doi.org/10.
1128/IAI.13.3.861-875.1976 PMID: 1270135; PubMed Central PMCID: PMC420689.
126. Shirai A, Catanzaro PJ, Phillips SM, Osterman JV. Host defenses in experimental scrub typhus: role of
cellular immunity in heterologous protection. Infect Immun 1976; 14(1):39–46. Epub 1976/07/01.
https://doi.org/10.1128/IAI.14.1.39-46.1976 PMID: 820646; PubMed Central PMCID: PMC420841.
127. Cho BA, Ko Y, Kim YS, Kim S, Choi MS, Kim IS, et al. Phenotypic characterization of peripheral T cells
and their dynamics in scrub typhus patients. PLoS Negl Trop Dis 2012; 6(8):e1789. https://doi.org/10.
1371/journal.pntd.0001789 PMID: 22905277; PubMed Central PMCID: PMC3419201.
128. Hauptmann M, Kolbaum J, Lilla S, Wozniak D, Gharaibeh M, Fleischer B, et al. Protective and Patho-
genic Roles of CD8+ T Lymphocytes in Murine Orientia tsutsugamushi Infection. PLoS Negl Trop Dis
2016; 10(9):e0004991. https://doi.org/10.1371/journal.pntd.0004991 PMID: 27606708; PubMed Cen-
tral PMCID: PMC5015871.
129. Xu G, Mendell NL, Liang Y, Shelite TR, Goez-Rivillas Y, Soong L, et al. CD8+ T cells provide immune
protection against murine disseminated endotheliotropic Orientia tsutsugamushi infection. PLoS Negl
Trop Dis 2017; 11(7):e0005763. https://doi.org/10.1371/journal.pntd.0005763 PMID: 28723951;
PubMed Central PMCID: PMC5536391.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 30 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

130. Keller CA, Hauptmann M, Kolbaum J, Gharaibeh M, Neumann M, Glatzel M, et al. Dissemination of
Orientia tsutsugamushi and inflammatory responses in a murine model of scrub typhus. PLoS Negl
Trop Dis 2014; 8(8):e3064. https://doi.org/10.1371/journal.pntd.0003064 PMID: 25122501; PubMed
Central PMCID: PMC4133189.
131. Smadel JE, Ley HL Jr., Diercks RH, Cameron JA. Persistence of Rickettsia tsutsugamushi in tissues
of patients recovered from scrub typhus. Am J Hyg 1952; 56(3):294–302. Epub 1952/11/01. https://
doi.org/10.1093/oxfordjournals.aje.a119553 PMID: 12996497.
132. Fox JP. The long persistence of Rickettsia orientalis in the blood and tissues of infected animals. Fed
Proc 1948; 7(1 Pt 1):305. Epub 1948/03/01. PMID: 18915957.
133. Shirai A, Chan TC, Gan E, Huxsoll DL. Persistence and reactivation of Rickettsia tsutsugamushi infec-
tions in laboratory mice. Jpn J Med Sci Biol 1979; 32(3):179–84. Epub 1979/06/01. https://doi.org/10.
7883/yoken1952.32.179 PMID: 120458.
134. Chu H, Park SM, Cheon IS, Park MY, Shim BS, Gil BC, et al. Orientia tsutsugamushi infection induces
CD4+ T cell activation via human dendritic cell activity. J Microbiol Biotechnol 2013; 23(8):1159–66.
https://doi.org/10.4014/jmb.1303.03019 PMID: 23727805.
135. Kodama K, Kawamura S, Yasukawa M, Kobayashi Y. Establishment and characterization of a T-cell
line specific for Rickettsia tsutsugamushi. Infect Immun 1987; 55(10):2490–5. Epub 1987/10/01.
https://doi.org/10.1128/IAI.55.10.2490-2495.1987 PMID: 2443453; PubMed Central PMCID:
PMC260735.
136. Koh YS, Yun JH, Seong SY, Choi MS, Kim IS. Chemokine and cytokine production during Orientia tsu-
tsugamushi infection in mice. Microb Pathog 2004; 36(1):51–7. https://doi.org/10.1016/j.micpath.
2003.08.006 PMID: 14643640.
137. Yun JH, Koh YS, Lee KH, Hyun JW, Choi YJ, Jang WJ, et al. Chemokine and cytokine production in
susceptible C3H/HeN mice and resistant BALB/c mice during Orientia tsutsugamushi infection. Micro-
biol Immunol 2005; 49(6):551–7. https://doi.org/10.1111/j.1348-0421.2005.tb03761.x PMID:
15965303.
138. Iwasaki H, Takada N, Nakamura T, Ueda T. Increased levels of macrophage colony-stimulating factor,
gamma interferon, and tumor necrosis factor alpha in sera of patients with Orientia tsutsugamushi
infection. J Clin Microbiol 1997; 35(12):3320–2. https://doi.org/10.1128/JCM.35.12.3320-3322.1997
PMID: 9399546; PubMed Central PMCID: PMC230174.
139. Chierakul W, de Fost M, Suputtamongkol Y, Limpaiboon R, Dondorp A, White NJ, et al. Differential
expression of interferon-gamma and interferon-gamma-inducing cytokines in Thai patients with scrub
typhus or leptospirosis. Clin Immunol 2004; 113(2):140–4. https://doi.org/10.1016/j.clim.2004.08.006
PMID: 15451469.
140. de Fost M, Chierakul W, Pimda K, Dondorp AM, White NJ, Van der Poll T. Activation of cytotoxic lym-
phocytes in patients with scrub typhus. Am J Trop Med Hyg. 2005; 72(4):465–7. PMID: 15827287.
141. Kramme S, An le V, Khoa ND, Trin le V, Tannich E, Rybniker J, et al. Orientia tsutsugamushi bacter-
emia and cytokine levels in Vietnamese scrub typhus patients. J Clin Microbiol 2009; 47(3):586–9.
Epub 2009/01/16. https://doi.org/10.1128/JCM.00997-08 PMID: 19144812; PubMed Central PMCID:
PMC2650899.
142. Chung DR, Lee YS, Lee SS. Kinetics of inflammatory cytokines in patients with scrub typhus receiving
doxycycline treatment. J Infect 2008; 56(1):44–50. https://doi.org/10.1016/j.jinf.2007.09.009 PMID:
17976731.
143. Ha NY, Kim Y, Min CK, Kim HI, Yen NTH, Choi MS, et al. Longevity of antibody and T-cell responses
against outer membrane antigens of Orientia tsutsugamushi in scrub typhus patients. Emerg Microbes
Infect 2017; 6(12):e116. https://doi.org/10.1038/emi.2017.106 PMID: 29259327; PubMed Central
PMCID: PMC5750460.
144. Jiang L, Morris EK, Aguilera-Olvera R, Zhang Z, Chan TC, Shashikumar S, et al. Dissemination of
Orientia tsutsugamushi, a Causative Agent of Scrub Typhus, and Immunological Responses in the
Humanized DRAGA Mouse. Frontiers Immunol 2018; 9:816. https://doi.org/10.3389/fimmu.2018.
00816 PMID: 29760694; PubMed Central PMCID: PMC5936984.
145. Woodward TE. Murine and epidemic typhus rickettsiae: how close is their relationship? The Yale J
Biol Med 1982; 55(3–4):335–41. PMID: 6817526; PubMed Central PMCID: PMC2596437.
146. Jerrells TR, Jarboe DL, Eisemann CS. Cross-reactive lymphocyte responses and protective immunity
against other spotted fever group rickettsiae in mice immunized with Rickettsia conorii. Infect Immun
1986; 51(3):832–7. Epub 1986/03/01. https://doi.org/10.1128/IAI.51.3.832-837.1986 PMID: 3949382;
PubMed Central PMCID: PMC260973.
147. Feng HM, Walker DH. Cross-protection between distantly related spotted fever group rickettsiae. Vac-
cine 2003; 21(25–26):3901–5. Epub 2003/08/19. https://doi.org/10.1016/s0264-410x(03)00301-3
PMID: 12922124.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 31 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

148. Valbuena G, Jordan JM, Walker DH. T cells mediate cross-protective immunity between spotted fever
group rickettsiae and typhus group rickettsiae. J Infect Dis 2004; 190(7):1221–7. Epub 2004/09/04.
https://doi.org/10.1086/423819 PMID: 15346331.
149. Spencer RR, Parker RR. Rocky Mountain spotted fever: vaccination of monkeys and man. Public
Health Rep. 1925; 40:2159–2167. PMID: 19315003
150. Ecke RS, Gilliam AG, et al. The effect of Cox-type vaccine on louse-borne typhus fever; an account of
61 cases of naturally occurring typhus fever in patients who had previously received one or more injec-
tions of Cox-type vaccine. Am J Trop Med Hyg. 1945; 25:447–62. PMID: 21010811.
151. DuPont HL, Hornick RB, Dawkins AT, Heiner GG, Fabrikant IB, Wisseman CL Jr., et al. Rocky Moun-
tain spotted fever: a comparative study of the active immunity induced by inactivated and viable patho-
genic Rickettsia rickettsii. J Infect Dis 1973; 128(3):340–4. Epub 1973/09/01. https://doi.org/10.1093/
infdis/128.3.340 PMID: 4199563.
152. Kenyon RH, Pedersen CE Jr. Preparation of Rocky Mountain spotted fever vaccine suitable for human
immunization. J Clin Microbiol 1975; 1(6):500–3. Epub 1975/06/01. https://doi.org/10.1128/JCM.1.6.
500-503.1975 PMID: 809483; PubMed Central PMCID: PMC275168.
153. Kenyon RH, Sammons LS, Pedersen CE Jr. Comparison of three rocky mountain spotted fever vac-
cines. J Clin Microbiol 1975; 2(4):300–4. Epub 1975/10/01. PMID: 810494; PubMed Central PMCID:
PMC362799.
154. Gonder JC, Kenyon RH, Pedersen CE Jr. Evaluation of a killed Rocky Mountain spotted fever vaccine
in cynomolgus monkeys. J Clin Microbiol 1979; 10(5):719–23. Epub 1979/11/01. https://doi.org/10.
1128/JCM.10.5.719-723.1979 PMID: 120877; PubMed Central PMCID: PMC273254.
155. Maugh TH, 2nd. Rickettsiae: a new vaccine for Rocky Mountain spotted fever. Science 1978; 201
(4356):604. Epub 1978/08/18. https://doi.org/10.1126/science.97783 PMID: 97783.
156. Clements ML, Wisseman CL Jr., Woodward TE, Fiset P, Dumler JS, McNamee W, et al. Reactogeni-
city, immunogenicity, and efficacy of a chick embryo cell-derived vaccine for Rocky Mountain spotted
fever. J Infect Dis 1983; 148(5):922–30. Epub 1983/11/01. https://doi.org/10.1093/infdis/148.5.922
PMID: 6415182.
157. Weigl RL. Die Methoden der aktiven Fleckfieberimmunisierung. Bull Int Acad Polonaise Sci et lettres
(d Méd). 1930: 25.
158. Weigl R. Immunization against typhus fever in Poland during World War II. Tex Rep Biol Med 1947; 5
(2):177–9. PMID: 20255936.
159. Walker DH. The realities of biodefense vaccines against Rickettsia. Vaccine 2009; 27 Suppl 4:D52–5.
https://doi.org/10.1016/j.vaccine.2009.07.045 PMID: 19837287; PubMed Central PMCID:
PMC2909128.
160. Zinsser H, Castaneda MR. Studies on Typhus Fever: Vii. Active Immunization against Mexican
Typhus Fever with Dead Virus. J Exp Med 1931; 53(4):493–7. Epub 1931/03/31. https://doi.org/10.
1084/jem.53.4.493 PMID: 19869859; PubMed Central PMCID: PMC2131980.
161. Zinsser H, Castaneda MR. Studies on Typhus Fever: X. Further Experiments on Active Immunization
against Typhus Fever with Killed Rickettsia. J Exp Med 1933; 57(3):381–90. Epub 1933/02/28. https://
doi.org/10.1084/jem.57.3.381 PMID: 19870137; PubMed Central PMCID: PMC2132240.
162. Veintemillas F. Vaccination against typhus fever with the Zinsser-Castaneda Vaccine, in: The Journal
of Immunology. J Immunol. 1939; 36 (5):339–348.
163. Buckland FE, Dudgeon A. Scrubtyphus vaccine; large-scale production. Lancet 1945; 2:734–7. Epub
1945/01/01. PMID: 21003850.
164. Card WI, Walker JM. Scrub-typhus vaccine; field trial in South-east Asia. Lancet 1947; 1(6450):481–3.
https://doi.org/10.1016/s0140-6736(47)91989-2 PMID: 20294827.
165. Berge TO, Gauld RL, Kitaoka M. A field trial of a vaccine prepared from the Volner strain of Rickettsia
tsutsugamushi. Am J Hyg 1949; 50(3):337–42. Epub 1949/11/01. https://doi.org/10.1093/
oxfordjournals.aje.a119366 PMID: 15391985.
166. Bailey CA, Diercks FH, Proffitt JE. Preparation of a serological antigen and a vaccine for experimental
tsutsugamushi disease. J Immunol 1948; 60(3):431–41. PMID: 18890201.
167. Rights FL, Smadel JE. Studies on scrib typhus; tsutsugamushi disease; heterogenicity of strains of R.
tsutsugamushi as demonstrated by cross-vaccination studies. J Exp Med 1948; 87(4):339–51. https://
doi.org/10.1084/jem.87.4.339 PMID: 18904219; PubMed Central PMCID: PMC2135778.
168. Choi Y, Kim KS, Kim TY, Cheong HS, Ahn BY. Long-term egg-yolk adaptation of the Orientia tsutsuga-
mushi for preparation of a formalinized immunogen. Vaccine 2006; 24(9):1438–45. Epub 2005/11/22.
https://doi.org/10.1016/j.vaccine.2005.07.113 PMID: 16297509.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 32 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

169. Eisenberg GH Jr., Osterman JV. Gamma-irradiated scrub typhus immunogens: broad-spectrum
immunity with combinations of rickettsial strains. Infect Immun 1979; 26(1):131–6. Epub 1979/10/01.
https://doi.org/10.1128/IAI.26.1.131-136.1979 PMID: 115796; PubMed Central PMCID: PMC414584.
170. Eisenberg GH Jr., Osterman JV. Gamma-irradiated scrub typhus immunogens: development and
duration of immunity. Infect Immun 1978; 22(1):80–6. Epub 1978/10/01. https://doi.org/10.1128/IAI.
22.1.80-86.1978 PMID: 103828; PubMed Central PMCID: PMC422119.
171. Eisenberg GH Jr., Osterman JV. Experimental scrub typhus immunogens: gamma-irradiated and for-
malinized rickettsiae. Infect Immun 1977; 15(1):124–31. Epub 1977/01/01. https://doi.org/10.1128/IAI.
15.1.124-131.1977 PMID: 401770; PubMed Central PMCID: PMC421337.
172. Alhassan A, Liu H, McGill J, Cerezo A, Jakkula L, Nair ADS, et al. Rickettsia rickettsii Whole-Cell Anti-
gens Offer Protection against Rocky Mountain Spotted Fever in the Canine Host. Infect Immun. 2019;
87(2). https://doi.org/10.1128/IAI.00628-18 PMID: 30396898; PubMed Central PMCID:
PMC6346123.
173. Zinsser H, Macchiavello A. Further Studies on Typhus Fever: On Homologous Active Immunization
against the European Strain of Typhus Fever. J Exp Med 1936; 64(5):673–87. Epub 1936/10/31.
https://doi.org/10.1084/jem.64.5.673 PMID: 19870560; PubMed Central PMCID: PMC2133449.
174. Kawamura R, Kasahar S, Toyama T, Nishinarita F, Tsubaki S. On the prevention of tsutsugamushi.
Results of preventive inoculations for people in the endemic region, and laboratory tests with the Pes-
cadores strain. Trop Dis Bull. 1940; 37:269–270.
175. Kekcheyeva N. A living chemo-vaccine prepared from rickettsia tsutsugamushi. Acta Med Biol (Nii-
gata). 1967; 15:113–6. Epub 1967/12/01. PMID: 4968946.
176. Kekcheyeva N. Preventive immunization against tsutsugamushi fever. J Hyg Epidemiol Microbiol
Immunol 1968; 12(1):14–7. PMID: 5752387.
177. Fox JP, Jordan ME, Gelfand HM. Immunization of man against epidemic typhus by infection with aviru-
lent Rickettsia prowazeki strain E. IV. Persistence of immunity and a note as to differing complement-
fixation antigen requirements in post-infection and post-vaccination sera. J Immunol 1957; 79(4):348–
54. Epub 1957/10/01. PMID: 13481387.
178. Wisseman CL Jr. Concepts of louse-borne typhus control in developing countries: the use of the living
attenuated E strain typhus vaccine in epidemic and endemic situations. Adv Exp Med Biol 1972; 31
(0):97–130. Epub 1972/01/01. https://doi.org/10.1007/978-1-4684-3225-1_9 PMID: 4211883.
179. Balayeva NM, Nikolskaya VN. Enhanced virulence of the vaccine strain E of Rickettsia prowazeki on
passaging in white mice and guinea pigs. Acta Virol. 1972; 16:80–82. PMID: 4400680
180. Liu Y, Wu B, Weinstock G, Walker DH, Yu XJ. Inactivation of SAM-methyltransferase is the mecha-
nism of attenuation of a historic louse borne typhus vaccine strain. PLoS One 2014; 9(11):e113285.
https://doi.org/10.1371/journal.pone.0113285 PMID: 25412248; PubMed Central PMCID:
PMC4239044.
181. Ching WM, Wang H, Davis J, Dasch GA. Amino acid analysis and multiple methylation of lysine resi-
dues in the surface protein antigen of Rickettsia prowazekii. Angeletti RH, editor Techniques in protein
chemistry IV Academic Press, Inc; San Diego. 1993:307–14.
182. Zhang JZ, Hao JF, Walker DH, Yu XJ. A mutation inactivating the methyltransferase gene in avirulent
Madrid E strain of Rickettsia prowazekii reverted to wild type in the virulent revertant strain Evir. Vac-
cine 2006; 24(13):2317–23. Epub 2005/12/21. https://doi.org/10.1016/j.vaccine.2005.11.044 PMID:
16364512.
183. Whitworth T, Popov VL, Yu XJ, Walker DH, Bouyer DH. Expression of the Rickettsia prowazekii pld or
tlyC gene in Salmonella enterica serovar Typhimurium mediates phagosomal escape. Infect Immun
2005; 73(10):6668–73. Epub 2005/09/24. https://doi.org/10.1128/IAI.73.10.6668-6673.2005 PMID:
16177343; PubMed Central PMCID: PMC1230948.
184. Nielsen M, Lundegaard C, Worning P, Lauemoller SL, Lamberth K, Buus S, et al. Reliable prediction
of T-cell epitopes using neural networks with novel sequence representations. Protein Sci 2003; 12
(5):1007–17. https://doi.org/10.1110/ps.0239403 PMID: 12717023; PubMed Central PMCID:
PMC2323871.
185. Nielsen M, Lundegaard C, Worning P, Hvid CS, Lamberth K, Buus S, et al. Improved prediction of
MHC class I and class II epitopes using a novel Gibbs sampling approach. Bioinformatics 2004; 20
(9):1388–97. https://doi.org/10.1093/bioinformatics/bth100 PMID: 14962912.
186. Lin HH, Ray S, Tongchusak S, Reinherz EL, Brusic V. Evaluation of MHC class I peptide binding pre-
diction servers: applications for vaccine research. BMC Immunol 2008; 9:8. https://doi.org/10.1186/
1471-2172-9-8 PMID: 18366636; PubMed Central PMCID: PMC2323361.
187. Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: database for
MHC ligands and peptide motifs. Immunogenetics 1999; 50(3–4):213–9. https://doi.org/10.1007/
s002510050595 PMID: 10602881.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 33 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

188. Reche PA, Reinherz EL. Prediction of peptide-MHC binding using profiles. Methods Mol Biol 2007;
409:185–200. https://doi.org/10.1007/978-1-60327-118-9_13 PMID: 18450001.
189. He Y, Racz R, Sayers S, Lin Y, Todd T, Hur J, et al. Updates on the web-based VIOLIN vaccine data-
base and analysis system. Nucleic Acids Res 2014; 42(Database issue):D1124–32. https://doi.org/10.
1093/nar/gkt1133 PMID: 24259431; PubMed Central PMCID: PMC3964998.
190. Gazi M, Caro-Gomez E, Goez Y, Cespedes MA, Hidalgo M, Correa P, et al. Discovery of a protective
Rickettsia prowazekii antigen recognized by CD8+ T cells, RP884, using an in vivo screening platform.
PLoS One 2013; 8(10):e76253. https://doi.org/10.1371/journal.pone.0076253 PMID: 24146844;
PubMed Central PMCID: PMC3797808.
191. Caro-Gomez E, Gazi M, Goez Y, Valbuena G. Discovery of novel cross-protective Rickettsia prowaze-
kii T-cell antigens using a combined reverse vaccinology and in vivo screening approach. Vaccine
2014; 32(39):4968–76. https://doi.org/10.1016/j.vaccine.2014.06.089 PMID: 25010827; PubMed Cen-
tral PMCID: PMC4145598.
192. Meng Y, Xiong X, Qi Y, Duan C, Gong W, Jiao J, et al. Protective immunity against Rickettsia heilong-
jiangensis in a C3H/HeN mouse model mediated by outer membrane protein B-pulsed dendritic cells.
Sci China Life Sci 2015; 58(3):287–96. https://doi.org/10.1007/s11427-014-4720-4 PMID: 25270001.
193. Dzul-Rosado K, Balam-Romero J, Valencia-Pacheco G, Lugo-Caballero C, Arias-Leon J, Peniche-
Lara G, et al. Immunogenicity of OmpA and OmpB antigens from Rickettsia rickettsii on mononuclear
cells from Rickettsia positive Mexican patients. J Vector Borne Dis 2017; 54(4):317–27. https://doi.org/
10.4103/0972-9062.225836 PMID: 29460861.
194. Vishwanath S, McDonald GA, Watkins NG. A recombinant Rickettsia conorii vaccine protects guinea
pigs from experimental boutonneuse fever and Rocky Mountain spotted fever. Infect Immun 1990; 58
(3):646–53. Epub 1990/03/01. https://doi.org/10.1128/IAI.58.3.646-653.1990 PMID: 2106490;
PubMed Central PMCID: PMC258514.
195. Sumner JW, Sims KG, Jones DC, Anderson BE. Protection of guinea-pigs from experimental Rocky
Mountain spotted fever by immunization with baculovirus-expressed Rickettsia rickettsii rOmpA pro-
tein. Vaccine 1995; 13(1):29–35. Epub 1995/01/01. https://doi.org/10.1016/0264-410x(95)80007-z
PMID: 7762273.
196. Jiao Y, Wen B, Chen M, Niu D, Zhang J, Qiu L. Analysis of immunoprotectivity of the recombinant
OmpA of Rickettsia heilongjiangensis. Ann N Y Acad Sci 2005; 1063:261–5. Epub 2006/02/17. https://
doi.org/10.1196/annals.1355.042 PMID: 16481525.
197. Bourgeois AL, Dasch GA. The species-specific surface protein antigen of Rickettsia typhi: immunoge-
nicity and protective efficacy in guinea pigs. in: Burgdorfer W and Anacker R L (ed), Rickettsiae and
rickettsial diseases Academic Press, New York, NY. 1981:71–80.
198. Dasch G, Bourgeois AL, Rollwagen FM. The surface protein antigen of Rickettsia typhi: in vitro and in
vivo immunogenicity and protective capacity in mice. Raoult D, Brouqui P eds Rickettsiae and Rickett-
sial Diseases at the Turn of the Third Millenium Paris: Elsevier. 1999:116–122.
199. Wang P, Xiong X, Jiao J, Yang X, Jiang Y, Wen B, et al. Th1 epitope peptides induce protective immu-
nity against Rickettsia rickettsii infection in C3H/HeN mice. Vaccine 2017; 35(51):7204–12. https://doi.
org/10.1016/j.vaccine.2017.09.068 PMID: 29032899.
200. Li Z, Diaz-Montero CM, Valbuena G, Yu XJ, Olano JP, Feng HM, et al. Identification of CD8 T-lympho-
cyte epitopes in OmpB of Rickettsia conorii. Infect Immun 2003; 71(7):3920–6. Epub 2003/06/24.
https://doi.org/10.1128/iai.71.7.3920-3926.2003 PMID: 12819078; PubMed Central PMCID:
PMC161984.
201. Riley SP, Cardwell MM, Chan YG, Pruneau L, Del Piero F, Martinez JJ. Failure of a heterologous
recombinant Sca5/OmpB protein-based vaccine to elicit effective protective immunity against Rickett-
sia rickettsii infections in C3H/HeN mice. Pathog Dis. 2015; 73(9):ftv101. https://doi.org/10.1093/
femspd/ftv101 PMID: 26519448; PubMed Central PMCID: PMC4732028.
202. Crocquet-Valdes PA, Diaz-Montero CM, Feng HM, Li H, Barrett AD, Walker DH. Immunization with a
portion of rickettsial outer membrane protein A stimulates protective immunity against spotted fever
rickettsiosis. Vaccine 2001; 20(5–6):979–88. Epub 2001/12/12. https://doi.org/10.1016/s0264-410x
(01)00377-2 PMID: 11738766.
203. Diaz-Montero CM, Feng HM, Crocquet-Valdes PA, Walker DH. Identification of protective components
of two major outer membrane proteins of spotted fever group Rickettsiae. Am J Trop Med Hyg. 2001;
65(4):371–8. Epub 2001/11/06. https://doi.org/10.4269/ajtmh.2001.65.371 PMID: 11693887.
204. Gong W, Xiong X, Qi Y, Jiao J, Duan C, Wen B. Identification of novel surface-exposed proteins of
Rickettsia rickettsii by affinity purification and proteomics. PLoS One 2014; 9(6):e100253. https://doi.
org/10.1371/journal.pone.0100253 PMID: 24950252; PubMed Central PMCID: PMC4065002.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 34 / 35

PLOS NEGLECTED TROPICAL DISEASES Vaccination against rickettsiae

205. Gong W, Qi Y, Xiong X, Jiao J, Duan C, Wen B. Rickettsia rickettsii outer membrane protein YbgF
induces protective immunity in C3H/HeN mice. Hum Vac Immunother 2015; 11(3):642–9. https://doi.
org/10.1080/21645515.2015.1011572 PMID: 25714655; PubMed Central PMCID: PMC4514262.
206. Qi Y, Xiong X, Duan C, Jiao J, Gong W, Wen B. Recombinant protein YbgF induces protective immu-
nity against Rickettsia heilongjiangensis infection in C3H/HeN mice. Vaccine 2013; 31(48):5643–50.
https://doi.org/10.1016/j.vaccine.2013.09.064 PMID: 24113261.
207. Gong W, Xiong X, Qi Y, Jiao J, Duan C, Wen B. Surface protein Adr2 of Rickettsia rickettsii induced
protective immunity against Rocky Mountain spotted fever in C3H/HeN mice. Vaccine 2014; 32
(18):2027–33. https://doi.org/10.1016/j.vaccine.2014.02.057 PMID: 24582636.
208. Gong W, Wang P, Xiong X, Jiao J, Yang X, Wen B. Enhanced protection against Rickettsia rickettsii
infection in C3H/HeN mice by immunization with a combination of a recombinant adhesin rAdr2 and a
protein fragment rOmpB-4 derived from outer membrane protein B. Vaccine 2015; 33(8):985–92.
https://doi.org/10.1016/j.vaccine.2015.01.017 PMID: 25597943.
209. Chen HW, Zhang Z, Huber E, Mutumanje E, Chao CC, Ching WM. Kinetics and magnitude of antibody
responses against the conserved 47-kilodalton antigen and the variable 56-kilodalton antigen in scrub
typhus patients. Clin Vaccine Immunol 2011; 18(6):1021–7. https://doi.org/10.1128/CVI.00017-11
PMID: 21508168; PubMed Central PMCID: PMC3122618.
210. Ni YS, Chan TC, Chao CC, Richards AL, Dasch GA, Ching WM. Protection against scrub typhus by a
plasmid vaccine encoding the 56-KD outer membrane protein antigen gene. Am J Trop Med Hyg
2005; 73(5):936–41. PMID: 16282307.
211. Seong SY, Huh MS, Jang WJ, Park SG, Kim JG, Woo SG, et al. Induction of homologous immune
response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused
with the maltose-binding protein MBP-Bor56. Infect Immun 1997; 65(4):1541–5. Epub 1997/04/01.
https://doi.org/10.1128/IAI.65.4.1541-1545.1997 PMID: 9119501; PubMed Central PMCID:
PMC175167.
212. Choi S, Jeong HJ, Ju YR, Gill B, Hwang KJ, Lee J. Protective immunity of 56-kDa type-specific antigen
of Orientia tsutsugamushi causing scrub typhus. J Microbiol Biotechnol 2014; 24(12):1728–35. https://
doi.org/10.4014/jmb.1407.07048 PMID: 25112312.
213. Yu Y, Wen B, Wen B, Niu D, Chen M, Qiu L. Induction of protective immunity against scrub typhus with
a 56-kilodalton recombinant antigen fused with a 47-kilodalton antigen of Orientia tsutsugamushi
Karp. Am J Trop Med Hyg. 2005; 72(4):458–64. PMID: 15827286.
214. Chattopadhyay S, Jiang J, Chan TC, Manetz TS, Chao CC, Ching WM, et al. Scrub typhus vaccine
candidate Kp r56 induces humoral and cellular immune responses in cynomolgus monkeys. Infect
Immun 2005; 73(8):5039–47. https://doi.org/10.1128/IAI.73.8.5039-5047.2005 PMID: 16041019.
215. Ha NY, Shin HM, Sharma P, Cho HA, Min CK, Kim HI, et al. Generation of protective immunity against
Orientia tsutsugamushi infection by immunization with a zinc oxide nanoparticle combined with ScaA
antigen. J Nanobiotechnol 2016; 14(1):76. https://doi.org/10.1186/s12951-016-0229-2 PMID:
27887623; PubMed Central PMCID: PMC5124320.
216. Garza DA, Riley SP, Martinez JJ. Expression of Rickettsia Adr2 protein in E. coli is sufficient to pro-
mote resistance to complement-mediated killing, but not adherence to mammalian cells. PLoS One.
2017; 12(6):e0179544. https://doi.org/10.1371/journal.pone.0179544 PMID: 28662039; PubMed Cen-
tral PMCID: PMC5491016.
217. Park H, Lee JH, Gouin E, Cossart P, Izard T. The rickettsia surface cell antigen 4 applies mimicry to
bind to and activate vinculin. J Biol Chem 2011; 286(40):35096–103. Epub 2011/08/16. https://doi.org/
10.1074/jbc.M111.263855 PMID: 21841197; PubMed Central PMCID: PMC3186400.
218. Riley SP, Goh KC, Hermanas TM, Cardwell MM, Chan YG, Martinez JJ. The Rickettsia conorii auto-
transporter protein Sca1 promotes adherence to nonphagocytic mammalian cells. Infect Immun 2010;
78(5):1895–904. Epub 2010/02/24. https://doi.org/10.1128/IAI.01165-09 PMID: 20176791; PubMed
Central PMCID: PMC2863548.
219. Fish AI, Riley SP, Singh B, Riesbeck K, Martinez JJ. The Rickettsia conorii Adr1 Interacts with the C-
Terminus of Human Vitronectin in a Salt-Sensitive Manner. Front Cell Infect Microbiol 2017; 7:61.
https://doi.org/10.3389/fcimb.2017.00061 PMID: 28299286; PubMed Central PMCID: PMC5331051.
220. Riley SP, Patterson JL, Nava S, Martinez JJ. Pathogenic Rickettsia species acquire vitronectin from
human serum to promote resistance to complement-mediated killing. Cell Microbiol 2014; 16(6):849–
61. https://doi.org/10.1111/cmi.12243 PMID: 24286496; PubMed Central PMCID: PMC4028375.
221. Kleba B, Clark TR, Lutter EI, Ellison DW, Hackstadt T. Disruption of the Rickettsia rickettsii Sca2 auto-
transporter inhibits actin-based motility. Infect Immun 2010; 78(5):2240–7. Epub 2010/03/03. https://
doi.org/10.1128/IAI.00100-10 PMID: 20194597; PubMed Central PMCID: PMC2863521.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0008704 October 22, 2020 35 / 35