Immunosuppression, induced upon intradermal infections, causes failures of
vaccines against African trypanosomiases.
Henry Tabel1*, Guojian Wei1, Harold Bull2
1 Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan,
Canada, 2 Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada
Oct. 2, 2012
*e-mail address: henry.tabel@usask.ca
Abstract
African trypanosomes are hemoprotozoa that cause disease in humans and livestock. Each trypanosome
is covered by a single layer of about 107 identical molecules of surface glycoprotein. It is generally
believed that the almost unlimited capacity for antigenic variation of the surface glycoprotein is the
major impediment for developing vaccines against African trypanosomiases. We have discovered
contradictory evidence to this assumption. We found that, in contrast to infections of the blood,
infections of the skin with low numbers of trypanosomes are controlled by innate resistance and the
few killed trypanosomes induce enhanced susceptibility to reinfections. We argue that induction of
immunosuppression by trypanosomes infecting the skin is the major impediment for developing
effective vaccines. The role of suppressor macrophages and their L-arginine metabolism as well as the
role of NKT cells in the immunosuppression are discussed. We propose a novel strategy for vaccine
development to induce protective immunity to trypanosomal infections. The major thrust of this
strategy is to use an intradermal vaccination procedure that counteracts the induction of
immunosuppression and induces a Th1 imprint. We think this strategy may have relevance to the
development of vaccines against other hemoprotozoal infections.
Introduction
African trypanosomes are pathogens of humans and livestock. Various members of the genus
Trypanosoma are responsible for disease syndromes called African trypanosomiases [1]. Trypanosoma
brucei gambiense and T. b. rhodesiense cause sleeping sickness in humans, also called human African
trypanosomiasis (HAT), an emerging disease in East and Central Africa [2,3]. Infections in livestock
with T. congolense, T. vivax, or T. b. brucei cause anemia, hypocomplementemia, cachexia, and
susceptibility to secondary infections [1,4,5]. Various species of tsetse flies (Glossina spp.) can harbor
African trypanosomes and act as their intermediate hosts. Humans and animals become infected with
trypanosomes by bites of infected tsetse flies. A temporary local inflammation, the so-called chancre,
develops in the skin at the site of the bite [6]. The trypanosomes move from the skin into the blood via
the lymph system [7,8] (Fig. 1). African trypanosomes are single-cell, extracellular blood parasites.
Shared characteristics of the different species of African trypanosomes include the ability to produce
almost unlimited antigenic variation of their variant surface glycoprotein (VSG) [9,10,11] and to
induce a predominantly T cell-independent antibody response to the VSG [12,13,14,15,16], profound
immunosuppression [17], polyclonal B cell activation [18,19,20] and persistent hypocomplementemia
[5,21,22] in infected mammalian hosts. Infections of mammalian hosts lead to cycles of parasitemia
1
associated with expression of new VSGs [9,11].
Each new VSG initially elicits a strong immunoglobulin M (IgM) anti-VSG response [9,16,19], which
leads to phagocytosis of the trypanosomes, predominantly by macrophages of the liver [23,24,25,26].
Currently, there are no effective vaccines against African trypanosomiases, neither for humans nor for
livestock.
Figure 1. Mode of natural infections by African trypanosomes. Infected tsetse flies bite the host by
inserting the proboscis into the skin, inject saliva into the site and puncture a small blood vessel,
resulting into a small hemorrhage. The tsetse fly is sucking blood from the hemorrhage. During this
process trypanosomes are deposited into the skin. Trypanosomes enter the lymph system and then
reach the draining lymph node and the blood stream. Trypanosomes will circulate in the blood stream.
Whole trypanosomes or fractions thereof end up by antibody- and/or complement-mediated
phagocytosis in macrophages of liver and spleen.
Resistance to infections by African trypanosomes
In vitro, African trypanosomes can be killed by antibody/complement-mediated immune lysis
[23,24], antibody-mediated phagocytosis by macrophages [25,26,27] and nitric oxide (NO) produced
by macrophages [28,29]. Regarding infections in vivo, it is important to distinguish clearly between
resistance to intradermal infections by low numbers of parasites and resistance to blood stage
infections.
Primary intradermal infections by low numbers of parasites in the skin are controlled by innate
immunity mediated by induced nitric oxide [30]. At this stage, adaptive immune responses are not
protective but are immunosuppressive [30] (discussed below).
At the blood stage of infection, antibodies are absolutely required for the control of parasitemia
[31,32,33,34]. Complement-mediated immune lysis of trypanosomes in vivo does not appear to be a
major protection mechanism providing long-term survival. Complement C5-deficient mice
(B10.D2/oSnJ) had survival times similar to those of control mice (B10.D2/nSnJ) when infected with
either T. b. rhodesiense [35] or T. congolense (Tabel & Wei, unpubl.). Considering the marked
hypocomplementemia in humans [21], livestock [5,22] and mice [36] infected with trypanosomes, the
benefit of immune lysis is presumably cancelled out by detrimental pathological effects [24]. Because
2
of antigenic variation, each wave of parasitemia represents a new antigenic variant and is controlled by
a variant-specific antibody response. This variant-specific immunity can be transferred by B cells but
not by T cells [32]. The anti-VSG antibody response is both T cell-independent and T cell-dependent
[12,16,37]. Although IgM antibodies to VSG are the major class of antibodies which do reduce
parasitemia [38,39] and mediate phagocytosis of trypanosomes by the macrophage system [26,39], they
do not enhance the survival time of infected mice [33,40]. There is evidence that IgG anti-parasite
antibodies are required for survival of mice infected with T. b. rhodesiense [40] or T. congolense [33]
and that anti-parasite-specific IgG2a and IgG3 antibodies, but not IgG1, are associated with protection
[33,41]. The latter observation would indicate that responses by Th1 cells are associated with
protection, which is confirmed by other findings [42,43]. Cumulative evidence suggests that for
maximum resistance against trypanosomes, parasite-specific Th1 cells activate macrophages via IFN-γ
to produce NO which is cytostatic for the trypanosomes [33,42,43,44]. The dilemma for the overall
outcome of the infection is the fact that parasite-specific Th1 cells also mediate immunopathology
[45,46,47,48,49] and NO is a major mediator of immunosuppression (see below).
Protective anti-parasite immune responses wane with progression of chronic infections [50,51].
Mice that have developed parasitemia will eventually die. Their survival time depends on the virulence
of the infecting parasite and the genetic resistance of the infected mice [52,53,54,55,56]. In some cases,
however, self-cure does occur [15,57].
Why are there no effective vaccines?
African trypanosomes have developed a highly sophisticated and complex system of antigenic
variation [9,10,11]. In the mammalian host, the whole parasite is covered with a coat of about 107
identical molecules of a glycoprotein, the variant surface glycoprotein (VSG). The VSG is anchored
into the cell membrane via a glycolipid, glycosylphosphatidylinositol (GPI) [58,59]. There is a widely
held belief that the almost unlimited capacity for antigenic variation of the surface glycoproteins by the
African trypanosomes is the major hurdle for producing a vaccine [17,60,61,62,63]. In view of our
recent experimental results on intradermal infections with low numbers of trypanosomes [30,64], we do
not share this belief.
Several comprehensive reviews on the immunobiology of infections with African trypanosomes
have been published [17,43,45,49,56,65,66,67,68]. Past research into the immunobiology has mostly
been based on the immune responses of mice infected intraperitoneally, a route of infection that leads
to development of parasitemia [17,33,43,56,65,69,70]. Although these studies have provided great
insight into the host-parasite relationship, they have neglected to investigate the very early
immunological events triggered by the infecting parasites. In nature, mammals become infected by skin
bites from trypanosome-infected tsetse flies [1]. Thus, we have developed a model for intradermal
infections of mice [30,64,71].
Intraperitoneal infections of mice with either T. brucei [16,17,31] or T. congolense [33,39] lead
to infections of the blood and definitely require antibodies to VSG for the control of parasitemia. In
contrast, we found that intradermal infections by low numbers (100-500) of African trypanosomes are
controlled by innate resistance involving induced nitric oxide (iNO) and TNF-α, but require neither
antibodies nor T cells for protection [30]. Relevant to these results, it was found that the average man
required a minimal dose of 300-450 metacyclic T. b. rhodesiense to be infected by the bite of a tsetse
fly [72]. We further provided evidence that primary intradermal infections were better controlled in
CD1d-/- or MHC class II-/- mice, indicating that the innate resistance to low numbers of trypanosomes
in primary intradermal infections is somewhat suppressed by CD1d-restricted natural killer T cells
3
(NKT cells) and MHC class II-restricted T cells [30]. Primary intradermal infections by 100-500
African trypanosomes which, in fact, are killed by innate resistance, not only failed to generate a longterm protective immunological state but resulted in enhanced susceptibility to intradermal challenges
[30]. Even more surprisingly, intradermal injection of mice with a trypanosomal lysate, i.e.,
trypanosomes killed by sonication, does not provide protection but makes such mice more susceptible
to an intradermal challenge [30]. The enhanced susceptibility to intradermal challenge is unrelated to
antigenic variation. We suggest that intradermal infections with low numbers of trypanosomes or
injections with mechanically killed trypanosomes prime the adaptive immune system to suppress
protective immunity to an intradermal challenge.
All previous attempts to produce vaccines against African trypanosomes were only partially
successful or failed entirely, because they did not deal with the stated problem of induction of
immunosuppression by infecting trypanosomes. A comprehensive review on previous vaccination
attempts has been published recently [73].
We propose that in any attempt to produce an effective vaccine, it would be crucial to address
the problem of induction of immunosuppression by the trypanosomes injected into the skin by infected
tsetse flies.
Immunosuppression in humans and animals infected by African trypanosomes
Immunosuppression to heterologous antigens: Immunosuppression in mice infected by T.
brucei was discovered as depressed immune responses to heterologous antigens, measured by plaqueforming assays (PFC), i.e. lower numbers of B cells specific for sheep red blood cells (SRBC)
[14,19,55,74,75,76]. Humans [77], cattle [78,79,80,81] and mice [82] infected by African
trypanosomes also showed lower immune responses to vaccines. Another characteristic of T. brucei or
T. congolense infections of mice or cattle is the reduced proliferation of T cells in response to
stimulation by T cell mitogens, such as ConA or PHA [75,83,84,85,86,87,88,89,90,91]. These
observations would explain the enhanced susceptibility to secondary infections in T. b. gambiense
infections in humans [77] and T. congolense and T. vivax infections in cattle [4]. Infections of mice
with T. congolense or T. brucei lead to a disruption of the lymphoid architecture of the spleen, a
gradual disorganization of the white pulp with eventual lymphoid depletion [82,92].
Immunosuppression to trypanosomal antigens: The suppression of immune responses to the
infecting trypanosomes is not as well documented. Because intraperitoneal infections of mice always
lead to infections of the bloodstream, there is a technical problem of demonstrating parasite-specific
immunosuppression in such an experimental design. In a somewhat artificial experimental design, mice
were infected with live T. brucei together with complete Freund’s adjuvant into the skin.
Immunosuppression to trypanosomal antigens was demonstrated in these mice [50,93]. Lymph node
cells taken 1-2 weeks after infection showed a proliferative response to T. brucei antigens [93]. In
contrast, T cells taken from mice 3 weeks after infection no longer proliferated in response to T. brucei
antigens [50].
Sacks and Askonas [51] showed trypanosome-induced immunosuppression of anti-parasite
responses by infecting mice intraperitoneally with one clone of T. brucei and then immunizing these
mice with irradiated trypanosomes of a noncross-reacting clone. The suppression of antibody response
to the irradiated trypanosomes was virtually complete, resulting from acute infection by a highly
virulent clone. The authors also tested the anti-VSG antibodies to homologous variants in a chronic
infection after each of three waves of parasitemia. As the infections progressed, IgM and IgG anti-VSG
4
antibody responses declined. IgG antibodies declined more rapidly. After the third parasitemia, only
low levels of IgM anti-VSG antibodies were detectable.
Schleifer and Mansfield [94] infected mice with T. b. rhodesiense and prepared spleen cell
cultures from these mice. They measured the proliferative response to VSG. They found that the
proliferation index increased 3-fold, when they incorporated NG-monomethyl-L-arginine (NMMA), an
inhibitor of nitric oxide synthase. These results indicated an NO-mediated suppression of response of
VSG-specific T cells in the infected mice. Dagenais et al. [95] infected B10.BR mice intraperitoneally
with T. b. rhodesiense and produced T cell hybridomas from spleen cells collected 1 week after
infection. Of 11 hybridomas, which were specific for various VSG peptides, none of the hybridomas
recognized epitopes of the relatively conserved invariant sequences of the VSG C-terminal domain.
Although there is no proof, the results might indicate that T cells specific for the invariant VSG Cterminal domain become already suppressed or deleted within 1 week after the infection by T. b.
rhodesiense. Of course, for a vaccine to be protective, the host has to produce memory T cells specific
for common antigens of the parasite, such as the invariant domain of the VSG.
Mechanisms of immunosuppression
Historically, explanations for the mechanisms of immunosuppression had been conflicting, leaving
unresolved whether immunosuppression is mediated by suppressor macrophages [76,96] or by
suppressor T cells [97]. In 1984, Roelants and Pinder [17] carried out an extensive review and
concluded both types of cells are involved. Askonas’ lab has convincingly shown that macrophages
become immunosuppressive after antibody-mediated phagocytosis of T. brucei [65]. Suppressor
macrophages play a predominant role in the immunosuppression at the blood stage of infection by
African trypanosomes [76,83,87,88,89,91,98,99]. The induction of suppressor macrophages and their
effector mechanisms appear to be complex and are by no means sufficiently elucidated. Phagocytosis
of whole trypanosomes, membrane fractions or glycolipid fractions of the membrane made
macrophages immunosuppressive [65,100,101]. The major component of trypanosomes that activates
macrophages appears to be the GPI [102,103,104,105], the membrane anchor of the VSG, similar to the
activation of macrophages by the GPI of Plasmodium falciparum [103,104,106,107]. Such
macrophages are highly activated, producing prostaglandin E2, plasminogen activator, H2O2, O-2, IL-1,
but have lower expression of mannose receptor, FcR and CR3 [65]. They also produce enhanced
amounts of TNF-α, IL-6, IL-12, IL-10 and NO [28,108,109]. There appears to be a general
dysregulation of homeostasis of these macrophages, eventually leading to their apoptosis [110]. The
degree and pattern of activation depends on the genetics of the macrophage [28,108,109], the class of
antibody opsonizing the trypanosomes [28], the number of engulfed trypanosomes and the virulence of
the trypanosome [55,111].
Nitric oxide: In 1992, Sternberg et al. discovered that NO produced by macrophages is a mediator
of immunosuppression in T. brucei infection of mice [89]. NO is a major mediator of
immunosuppression in mice infected intraperitoneally [69,98,112,113,114], but only during the early
phase of infection of the blood [69,112]. The phagocytosis of trypanosomal antigen does not result in
the production of a great amount of NO in macrophages. It is the stimulation of such macrophages by
IFN-γ that, in synergy with TNF-α, induces the synthesis of high amounts of NO [28,69,94,112,114].
In turn, the highly activated macrophages stimulate the interacting T cells to unusually high synthesis
of IFN-γ [69,90,91,94,112,115,116].
Parasite-specific Th1 cells potentially induce protective immune responses by eliciting an IFN-γmediated NO synthesis in antigen-presenting macrophages, leading to killing of trypanosomes. Under
5
heavy trypanosome load, the macrophages can turn into “suppressor macrophages” preventing, by a
NO-mediated mechanism, the proliferation of Th1 cells [94,113]. The production of cytokines by the
incapacitated Th1 cells apparently continues [113]. In T. brucei-infected mice [86,87,117] and T.
congolense-infected cattle [88] the IL-2 receptor of T cells is down-regulated. There is evidence that
NO [84] as well as IFN-γ [118] mediate the down-regulation of the IL-2 receptor.
NO is not the only mediator of immunosuppression. Prostaglandin E2 produced by the suppressor
macrophages also contributes to the immunosuppression [65,87,94], possibly by reducing the
production of IL-2 [87].
M1 versus M2 macrophages: The diverse biological activity of macrophages is mediated by
phenotypically distinct subpopulations of cells that develop in response to inflammatory mediators in
their microenvironment. Two major populations have been characterized: classically activated M1
macrophages and alternatively activated M2 macrophages [45,119]. The M1 type develops upon
activation by IFN-α/β, IFN-γ and/or TNF-α. The M2 type develops after activation by IL-10, IL-4,
and/or IL-13 [120]. Activation of the inducible nitric oxide synthase (iNOS or NOS2) has been
regarded as one of the most specific marker for M1 macrophages and activation of arginase 1 (Arg1)
the most specific marker of M2 macrophages121. Both types of macrophages have been associated with
immunosuppression. The L-arginine metabolism in macrophages controls T-lymphocyte function
[121,122]. Both the arginase pathway and the iNOS pathway use L-arginine as their substrate. Both
pathways compete for the available L-arginine and cross-regulate each other [121]. In tumors, M1
macrophages produce high amounts of NO which has been found to interfere with IL-2R signaling in T
cells [120,121]. The NO-producing suppressor macrophages observed in C57BL/6 mice infected
intraperitoneally by T. brucei appear to be of M1 type. The suppressive effect of M2 macrophages in
Leishmania major skin infections of BALB/c mice has been associated with local depletion of Larginine by arginase, impaired proliferation of T cells in the skin lesion and impaired production of
IFN-γ [122]. In mammary carcinoma 4T1 of BALB/c mice, suppressor macrophages are polarized by
interleukin 13 (IL-13) towards the M2 type with increased metabolism of L-arginine by enhanced
activity of arginase [123]. Enhanced arginase activity can lead to exhaustion of L-arginine. T cells
stimulated and cultured in the absence of L-arginine, present a sustained down-regulation of CD3zeta
preventing the normal expression of the T cell receptor (TCR) [124].
Despite the distinct expression of iNOS and Arg1 in M1 and M2 macrophages, respectively, some
macrophages have been shown to express both iNOS and Arg1 [120]. Thus, macrophages of mixed
characteristic do exist. In tumor bearing mice, Foxp3+CD4+ Tregs can be induced by a pathway
requiring IL-10 and IFN-γ [125]. This is reminiscent of macrophage-mediated immunosuppression in
highly susceptible BALB/c mice intraperitoneally infected by T. congolense , which is mediated by IL10 and IFN-γ but not by NO [91].
BALB/c mice are highly susceptible to T. congolense [56] and also more susceptible to T. brucei
[126] than relatively resistant C57BL/6 mice. In mice intraperitoneally infected with T. brucei, arginase
mRNA is expressed higher in peritoneal macrophages of infected BALB/c than in those of infected
C57BL/6 mice. In co-cultivation with macrophages, T. brucei directly induces increased arginase 1 and
arginase 2 mRNA levels in macrophages as well as increases macrophage arginase activity, with higher
levels in BALB/c than in C57BL/6 mice [127]. The mechanism is unknown. Under the same
conditions, neither iNOS expression nor NO production is stimulated. Host arginase appears to be a
marker of susceptibility/resistance to trypanosome infections [127]. From two days on after infection,
arginase activity is increasingly up-regulated in peritoneal macrophages of Swiss mice subcutaneously
infected with T. brucei. Under the same conditions, increasing iNOS activity is delayed by a couple of
days [44].
6
Immunity to infections is mediated by memory T cells and B cells, which are generated from naïve
precursor cells after exposure to the microbial antigen. Upon interaction of naïve T cells with the
antigen-presenting cell, naïve T cells rapidly proliferate and differentiate into effector T cells. This
phase of proliferation lasts about 1 week and is followed by a contraction phase of about 14 days
during which about 90 % of the effector T cells die whereas the remaining cells differentiate into
memory T cells [128,129]. We conclude that, in African trypanosomiasis, there is a lack of expansion
of trypanosome-specific Th1 cells and little or no development of Th1 memory cells specific for
variant and common parasite antigens.
We contend that, at the intradermal stage of infection, the immunosuppression is predominantly
controlled by suppressor T cells [30] and possibly a mixed M1/M2 macrophages environment
[44,120,127], whereas at the early parasitemic stage of infected C57BL/6 mice, this process is
predominantly controlled by M1 suppressor macrophages [69,94,112].
Immunobiology of intradermal infections
Knowledge of the immunological events in primary intradermal infections by African
trypanosomes is very limited. Nevertheless our recent investigation [30] provides some unexpected
findings and allows some fundamental conclusions.
Resistance to low numbers of trypanosomes in primary intradermal infections: We have shown that,
upon intradermal infection, low numbers (100-500) of trypanosomes are killed by innate immunity.
Contrary to blood stage infections, intradermal infections do not require B cells or T cells to induce
resistance. The innate resistance is mediated by induced NO and TNF-α [30].
What processes might lead to innate resistance to intradermal infections by low numbers of
trypanosomes? T. brucei [130] and T. congolense [131] isolated from infected blood can have
cleavage products of complement component C3 on their surface. T. brucei [132,133] as well as T.
congolense [134] can form filopodia (slender membrane protrusions). Immune complexes of anti-VSG
antibody and complement have been shown to be shed via filopodia [134], which, in turn, can be taken
up by macrophages [27]. Thus, it is conceivable that trypanosomes with iC3b on their surface will
temporarily attach to macrophages via CR3 (CD11b/CD18) [26]. It might be possible that, even in the
absence of anti-VSG antibodies, a certain threshold number of iC3b-CR3 interactions might induce
formation of filopodia. We speculate that these filopodia might separate from trypanosomes and then
are engulfed by macrophages, without harming the trypanosomes involved. This process might activate
these macrophages to synthesize TNF-α and induce them to synthesize and secrete NO. Macrophagederived TNF-α might, in an autocrine fashion, enhance synthesis of NO by the macrophages [135]. We
suggest that all trypanosomes that subsequently attach to these activated macrophages are killed by
NO-mediated products, such as nitrosylated proteins [136,137] (Fig. 2). Potential enhancement of the
innate resistance by CD1d-restricted NKT cells [71] appears to be suppressed by other T cells
[30,49,71].
7
Figure 2. Proposed mechanism of resistance to low numbers of trypanosomes in primary intradermal
infections. We anticipate that trypanosomes release a yet unknown substance that induces Arg 1 and Arg 2 in
macrophages that are in close vicinity to these trypanosomes [129], skewing such macrophages towards M2
type. We contend that filopodia are engulfed by macrophages via iC3b-CR3 interaction and that the iNOS
pathway gets stimulated by the GPI contained in the filopodia, skewing such macrophages predominantly to a
M1 type (see text). The activated macrophages then kill surrounding trypanosomes via NO mediated process,
predominantly via S-nitrosylated proteins [138].
Immunosuppression in primary intradermal infections: Highly susceptible BALB/c mice infected
intradermally with 104 T. congolense develop parasitemia and die within 10 days. Treatment with an
optimal dose of anti-CD25 antibodies of such infected BALB/c mice prevents the development of
parasitemia and the mice remain entirely healthy [71]. There is evidence that induction of
CD4+CD25highFoxp3+ T cells (Treg) down-regulate potential protective immune responses, since the
spleens of the mice treated with anti-CD25 antibodies had a 100% reduction of CD4+CD25high T cells
and a 75% reduction of CD4+CD25+Foxp3+ T Cells [71].
Intradermal infections of relatively resistant C57BL/6 mice with 104 T. congolense lead to
development of parasitemia and disease [30]. CD1d-/- C57BL/6 mice infected with the same dose are
completely protected and MHC class-II-/- C57BL/6 mice are mostly protected (8/10) [30]. These results
indicate that in wild-type C57BL/6 mice intradermally infected with 104 T. congolense, the innate
immunity is down-regulated by CD1d-restricted NKT cells139 and also by MHC class-II-restricted T
cells.
CD1d is an MHC class-I-like molecule that presents glycolipid antigens to a subset of T cells
called natural killer T cells (NKT cells [138,139]. There are two subpopulations of NKT cells that vary
in the programming of the T cell receptor (TCR): invariant NKT cells (iNKT), type I and variant NKT
cells, type II. Both types of NKT cells recognize, with their TCR, lipids presented by CD1d expressed
on the surface of antigen-presenting cells (APC) [140]. Type I NKT cells, upon interacting with APC,
predominantly produce IFN-γ and activate the iNOS pathway in the APC, whereas type II NKT cells
produce IL-13 and activate the Arg 1 pathway in APC [140,141]. There is evidence that there is crossregulation between the two types of NKT cells [140,141].
Since the innate resistant to intradermal infections by low numbers of trypanosomes is mediated
by the iNOS pathway [30] whereas a subpopulation of NKT cells down-regulate this resistance [30],
we speculate that the suppressive NKT cells might be type II NKT cells [140,141] which would
predominantly produce IL-13 and activate the Arg 1 pathway in macrophages that have engulfed
trypanosome GPI (Fig. 3).
8
Figure 3. Minimal model: immunosuppression at primary intradermal infections by low numbers of
trypanosomes. Macrophages that have engulfed filopodia of trypanosomes [27] or whole killed trypanosomes
will process trypanosome antigens and present them at their cell surface. GPI of the membrane VSG (mVSG)
will be presented via CD1d to NKT cells [30,139]. We argue that the NKT cells are predominantly type II NKT
cells that release IL-13 (see text) which, in turn, skew the macrophages toward M2 type. Thus the antigenpresenting macrophages will predominantly be a mixed M1/M2 type (see text). MHC class II will present
peptides of variant and invariant domains of soluble VSG (sVSG) as well as other invariant peptides to MHC
class II-restricted T cells. The microenvironment will skew the naïve MHC class II-restricted T cells towards
Tregs [30,71], presumably via TGF-β produced by macrophages. The Tregs, in turn, activate the ARG 1
pathway of macrophages by production of IL-10. We propose that many of the naïve trypanosome-specific T
cells that develop into Th1 effector cells are deleted by apoptosis, due to peroxynitrite (ONOO-) produced by
macrophages under conditions of shortage of L-arginine supply [121] or functionally impaired, such as by downregulation of CD3zeta [124].
We have to keep in mind that our intradermal infections were performed by syringe and needle.
In natural infections, the tsetse fly injects the trypanosomes together with fly saliva. The saliva of tsetse
flies [142] as well of mosquitoes [143] induce Th2 responses. The tsetse fly saliva will likely alter the
microenvironment of the infection site of the skin. Thus, IL-4 produced by Th2 cells will skew APCs
towards activating the Arg 1 pathway and, like the suppressor T cells, interfere with the innate
resistance.
TNF-α- and iNOS-producing dendritic cells (Tip-DCs) are a major population of cells in
the liver of C57BL/6 mice infected with T. b. brucei [144]. The most abundant APC in the skin are
Langerhans cells [145]. Presently, we have no information to what degree Langerhans cells or other
dendritic cells might be involved in the resistance or suppression of resistance to intradermal infections
by trypanosomes.
If our hypothesis is correct that the initial activation of macrophages occurs by phagocytosis of
trypanosome filopodia via iC3b/CR3 interaction, there will be a predominant induction of the iNOS
9
pathway by trypanosome GPI. There are, however, reports that iC3b/CR3 interactions lead to the
sequential production of TGF-β and IL-10 and down-regulation of IL-12 by the APC, exerting an
immunosuppressive effect on Th1 cells [146,147,148]. Considering the profound complement
activation in humans [21], cattle [5,149], sheep [22] and mice [36] infected with African trypanosomes,
there might be another, yet to be identified, circuit of immunosuppression mediated by interactions
with complement components C3b and/or iC3b [150].
Immunosuppression in secondary intradermal infections: Primary intradermal infections with
low numbers of T. brucei or T. congolense, associated with innate resistance, fail to induce long-term
immunity. Instead, primary intradermal infections with low numbers of trypanosomes result in
enhanced susceptibility to intradermal challenges [30]. Even more surprisingly, intradermal injection
with a single dose of killed T. brucei or T. congolense can induce a suppressive immune response as
demonstrated by enhanced susceptibility to intradermal challenge by T. brucei or T. congolense [30].
Moreover, we found that intradermal injection with a lysate of T. congolense equivalent to 106
trypanosomes induces a significantly higher susceptibility to challenge than a lysate equivalent to 103
trypanosomes [30]. Thus, there is a dose-dependent effect.
We presently have no information on the immune response that leads to enhanced susceptibility
at the time of challenge. In this respect, it is worth commenting on an unexpected observation. We have
characterized a protein of T. congolense as a homologue of the major surface protease, gp63 of
Leishmania major [151], which we called Tco-MSP-D [64]. We have cloned and purified a protein
encoding the amino-terminal domain of Tco-MSP-D. We detect Tco-MSP-D in the serum of T.
congolense-infected mice. Mice immunized with the amino-terminal domain of Tco-MSP-D generate a
persisting IgG1 antibody response. Surprisingly, such immunized mice show an enhanced
susceptibility to an intradermal challenge with T. congolense, suggesting that antibodies to Tco-MSP-D
might enhance a yet unknown virulence mechanism of Tco-MSP-D. We speculate that Tco-MSP-D
might, like gp63 [151], be an enzyme that affects the complement cascade, cleaving C3b to iC3b and
thus preventing the amplification of the terminal complement pathway that is required for complementmediated lysis of target cells. We presently have, however, not investigated whether antibodies to TcoMSP-D are a) induced at primary intradermal infections with low numbers of T. congolense or b) might
be present at the time of challenge.
What strategy should be pursued to produce an effective vaccine?
As discussed above, we suggest a trypanosome-specific Th1 imprint is required for resistance against
trypanosome infections (Fig. 4). We propose that inhibiting the arginase pathway [44] and adequately
supplying L-arginine [121], combined with intradermal immunization with low numbers of
trypanosomes, will ameliorate or abolish the immunosuppressive environment, lead to induction of a
trypanosome-specific Th1 imprint and, in turn, enhance innate resistance.
Intradermal injections of a lysate of T. congolense, clone TC13 equivalent to 1000 parasites or
injections of a lysate of 100 T. brucei, strain Whatat 1.1 induced enhanced susceptibility to intradermal
challenge with T. congolense, clone TC14 or T. brucei, strain 10-26, respectively [30]. These results
clearly point to important conclusions: 1) Antigenic variation does not come into play in resistance to
intradermal infections by low number of parasites. 2) For a vaccine to be successful, the induction of
immunosuppression has to be prevented.
Route of immunization: Since in natural infections, the host is first exposed to trypanosome
antigens in the skin, immunizations should be carried out intradermally.
10
Interference with immunosuppressive environment. The strategy should be to suppress the
arginase pathway and prevent a shortage of L-arginine, and to enhance the iNOS pathway in such a
way that it does not inhibit, but enhances the induction of Th1 cells.
a) Incorporation of anti-trypanosomal drugs: Difluoromethyl ornithine (DFMO), an inhibitor
of the ornithine decarboxylase, a key enzyme in the arginase pathway, is now used in the treatment of
human sleeping sickness [60]. It might be the drug of first choice to be incorporated at optimal
concentration in the vaccine. Other potential candidates might be suramin, also being used for
treatment of sleeping sickness [60] and berenil, effective for treating cattle infected with T. congolense
[152].
b) Arginase inhibitors: Nω-hydroxy-nor-L-arginine (norNOHA) is an inhibitor of arginase [127].
When macrophages of BALB/c mice, which had been infected with T. b. brucei, were co-cultured with
T. b. brucei, the incorporation of norNOHA increased NO synthesis of the macrophages and
cytotoxicity for co-cultured trypanosomes [127]. Thus, incorporation of arginase inhibitors in the
vaccine could prevent depletion of L-arginine by arginase and enhance the iNOS pathway [121,127].
c) L-Arginine. Addition of L-arginine is expected to enhance this effect [121,122,127].
Incorporation of appropriate concentrations of anti-trypanosomal drugs or arginase inhibitors and
L-arginine should not pose a logistical problem.
The next proposals are less practical. Nevertheless, we propose that incorporation of the following
antibodies into a vaccine might be of benefit in counteracting the immunosuppressive effects on
inducing a protective Th1 response.
d) Anti-cytokine/cytokine receptor antibodies: The incorporation of anti-IL-13, anti-IL-10R,
anti-IL4Rα and/or anti-TGF-β antibodies would, in different ways, skew the microenvironment
towards a Th1 response. By blocking IL-13, IL-10R and/or IL4Rα, the induction of the arginase
pathway in antigen-presenting cells would be inhibited [121,140]. Neutralizing TGF-β would inhibit
the induction of Tregs.
e) Anti-iC3b antibodies, immunoconglutinin: As discussed above, complement factor iC3b has
been found to mediate inhibition of induction of Th1 cell responses [146,147,148]. Rabbits infected
with T. b. brucei have been found to have high titers of immunoconglutinin [153]. Antibodies to iC3b
(immunoconglutinin) [154], that block the function of iC3b, might reduce the inhibition of induction of
Th1 cell responses. Such antibodies, however, are not readily available.
Dose: The emphasis has to be on a low dose of antigen. One has to prevent hyperactivation of
macrophages. Our quantitative data on injections of trypanosome lysates indicate that fewer
trypanosomes induce less immunosuppression [30]. In experimental Leishmania major infections,
susceptible BALB/c mice infected with a small number of parasites could be made resistant to a larger,
normally pathogenic, challenge [155]. Bretscher et al. [156] convincingly argue that immunizations
with low doses of non-pathogenic antigens or pathogens lead to a Th1 imprint, rather than a Th2
imprint. Since we aim at inducing a Th1 imprint to obtain a protective immune response against
African trypanosomes, more antigen is not better, less is best.
Choice of trypanosomal material: For mice to be immunized against T. brucei or T. congolense,
an optimal dose might be a lysate equivalent to 100-500 organisms of the respective parasites.
Final comment: Our proposal to use a vaccination procedure that enhances Th1 cell differentiation
appears to run counter to the observation that Th1 cell/IFN-γ-induced NO mediates profound
immunopathology and immunosuppression in African trypanosomiasis [49]. NO, however, is a doubleedged sword. Our reasoning is based on the observation that high concentrations of NO are
immunosuppressive whereas low concentrations of NO enhance Th1 cell differentiation [157].
11
Figure 4. Minimal model of envisioned protective immune response to vaccination. We propose
that intradermal immunization with a low dose of antigens of the whole parasite is necessary but not
sufficient. We suggest that the immunization has to be accompanied by a treatment that inhibits the
arginase pathway of antigen-presenting cells. We anticipate that such vaccination procedure modestly
enhances the iNOS pathway of antigen-presenting cells to induce a Th1 imprint, i.e., memory Th1 cells
specific for relevant trypanosomal antigens
Does the proposed strategy have relevance to development of vaccines against other infections?
Malaria. Malaria is associated with immunosuppression [158,159]. Presently, there are still no
effective vaccines against malaria, in spite of vast scientific efforts. Guilbride et al. [160,161] published
an extensive investigation into immunization studies against malaria, covering a period from 1965 to
2010. The authors came to the conclusion that Plasmodium sporozoites induce a skin-stage–initiated
immunosuppression inhibiting vaccine function. Intradermal vaccination against rabies has been shown
to be as safe and immunogenic as intramuscular vaccination [162]. Thus, there does not appear to be an
intrinsic property of the skin that necessarily leads to immunosuppression when a pathogen enters. We
propose that pathogenic Plasmodia and Trypanosoma share parameters that initiate immunosuppression
upon intradermal infections, but that these parameters are absent in rabies virus. Thus, as in African
trypanosomiases, the challenge is to overcome the early induced immunosuppression to obtain an early
Th1 imprint that leads to a protective immune memory.
Leishmaniases. Visceral leishmaniasis caused by L. donovanni is associated with antigenspecific immunosuppression during acute disease [163]. Excessive increase of arginase is observed in
macrophages of non-healing lesions of cutaneous leishmaniasis caused by L. major and has been found
to be the cause for local suppression of Th1 cell responses. The local depletion of L-arginine impairs
the T cells within the skin lesion to proliferate and to produce IFN-γ [122]. As in intradermal
infections with African trypanosomes, intradermal injections of killed L. major enhance susceptibility
to secondary infections [164]. Again, we see a great similarity between African trypanosomiases and
leishmaniases as to the early induction of suppression of Th1 responses, preventing the development of
12
a sound Th1 memory. We contend that a vaccine strategy similar to the one we have proposed for
African trypanosomiases might be effective.
Summary
There is the general belief that success in producing vaccines against African trypanosomiases is
unlikely because of the almost unlimited capacity for antigenic variation of African trypanosomes. This
conclusion, however, is based on the immunological knowledge of the blood stage of infection by
African trypanosomes. We recently found that intradermal infections with low numbers of
trypanosomes is controlled by innate immunity and that this innate immunity is, in fact, compromised
by suppressive adaptive immune responses. On the basis of these findings, we propose a vaccine
strategy that aims at a) preventing the early induction of suppression of Th1 responses by inhibiting the
arginase pathway, but enhancing the iNOS pathway of antigen-presenting cells and b) using the
optimally lowest dose of antigens of the whole parasite to induce a Th1 imprint (Fig. 4). We suggest
that similar approaches might be successful in producing vaccines against other pathogenic protozoa
that manipulate the immune system.
Key Papers in the Field
• Wei G, Bull H, Zhou X, Tabel H (2011) Intradermal infections of mice by low
numbers of African trypanosomes are controlled by innate resistance but enhance
susceptibility to reinfection. J Infect Dis 203: 418-429
• Roelants GE, Pinder M (1984) Immunobiology of African trypanosomiasis.
Contemp Top Immunobiol 12: 225-274
• Duleu S, Vincendeau P, Courtois P, Semballa S, Lagroye I, et al. (2004) Mouse
strain susceptibility to trypanosome infection: an arginase-dependent effect. J
Immunol 172: 6298-6303
• Bronte V, Zanovello P (2005) Regulation of immune responses by L-arginine
metabolism. Nat Rev Immunol 5: 641-654
• Guilbride DL, Gawlinski P, Guilbride PD (2010) Why functional pre-erythrocytic
and bloodstage malaria vaccines fail: a meta-analysis of fully protective
immunizations and novel immunological model. PLoS One 5: e10685
Acknowledgments
The authors are grateful to Juliane Deubner who drew the diagrams of the presented figures. We thank
John Allen and Vikram Misra for reading the first draft of the manuscript and for providing
constructive criticism.
13
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