INNATE AND ACQUIRED RESISTANCE TO AFRICAN TRYPANOSOMIASIS

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J. Parasitol., 87(1), 2001, p. 1–9q American Society of Parasitologists 2001

INNATE AND ACQUIRED RESISTANCE TO AFRICAN TRYPANOSOMIASIS

Samuel J. Black, J. Richard Seed*, and Noel B. Murphy†Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003

ABSTRACT: The review discusses the current field status of human and bovine trypanosomiases, and focuses on the molecularbasis of innate and acquired control of African trypanosomes in people, cattle, and Cape buffalo.

African trypanosomes are tsetse-transmitted protozoa that in-habit the extracellular compartment of their host’s blood, in theface of the humoral immune system, which they flout byswitching among antigenically distinct variant surface glyco-proteins (VSGs). Trypanosomes cause fatal sleeping sickness inpeople (Trypanosoma brucei rhodesiense, T. b. gambiense) anda fatal wasting disease, nagana, in domestic livestock (T. b.brucei, T. congolense, T. vivax). Nagana is endemic throughoutthe humid and semihumid zones of sub-Saharan Africa coin-cident with the distribution of tsetse, which infest an area em-bracing 36 countries and covering 1 3 107 km2. There are noebbs and flows of nagana, only an unrelenting presence againstwhich 30% of Africa’s cattle graze on the fringes of the tsetsehabitat, many sustained by chemotherapy and tsetse control pro-grams.

Unlike nagana, sleeping sickness is typically restricted to his-toric foci. However, when the tsetse population increases andhuman contact with the flies is frequent, as is presently the case,sleeping sickness can surge out of its historic foci and spreadthrough the rural population. About 60 million people are atrisk of sleeping sickness, but only 4 million are under surveil-lance.

Sleeping sickness has reached epidemic proportion in An-gola, the Democratic Republic of Congo, Uganda, and Sudan.Furthermore, its prevalence is high and increasing in Cameroon,Congo, Cote d’Ivoire, Central African Republic, Guinea,Mozambique, Tanzania, and Chad (http://www.who.int/emc/diseases/tryp/trypano.html). The number of cases is estimatedto be between 300,000 and one-half million, and allotted re-sources allow disease control in only a few locations (http://www.imc-la.com/sleeping.htm).

The upsurge in sleeping sickness is most likely linked topoverty and war. These have dismantled the infrastructure ofdisease surveillance and vector control in many regions of sub-Saharan Africa, and resulted in an increase in the exposure ofpeople to infected tsetse flies. Reversal of the current sleepingsickness epidemic will be facilitated by a return of civil order,the widespread application of tsetse traps and odor-baited tar-gets (Lancien, 1991; Vale, 1993; WHO, 1998), and rehabilita-tion of disease surveillance and treatment programs. In this re-gard, the World Health Organization is organizing and sup-porting an international control program to improve sleepingsickness diagnosis and treatment and to improve the local dis-ease control infrastructure. Although these are important andtimely goals, the labor-intensive strategies of vector control anddisease surveillance will remain vulnerable to civil unrest and

* Department of Epidemiology, University of North Carolina, ChapelHill, North Carolina 27599-7400.

† International Livestock Research Institute, P.O. Box 30709, Nairobi,Kenya.

poverty, and disease treatment will always be vulnerable to theacquisition of drug resistance in the target parasite populations.Indeed, resistance to all available trypanocidal drugs, includingthose that are effective against nagana, has already been re-ported (Chitambo and Arakawa, 1991; Ainanshe et al., 1992;Mohamed-Ahmed et al., 1992; Iten et al., 1997; Atouguia andCosta, 1999; Barrett and Fairlamb, 1999; Legros et al., 1999).The long-term control of sleeping sickness and nagana mayrequire the development of more sustainable control measures,e.g., protective vaccines and trypanosomiasis-resistant live-stock.

Although efficacious vaccines against animal and human try-panosomiases would revolutionize disease management, thesehave proven elusive. The bulk of immunological research intrypanosome-infected animals, which for the most part were ofthe susceptible phenotype, documents a depressing successionof waves of parasitemia, accompanied by antibodies againstVSGs and immune system activation leading to pathology rath-er than cure. Notwithstanding this common scenario, there arenumerous examples of control of trypanosomes by trypanoto-lerant breeds of livestock and by African wildlife. Furthermore,people and some old-world primates are resistant to infectionwith the trypanosome species that cause nagana, i.e., T. b. bru-cei, T. vivax, and T. congolense, and there is some evidence ofasymptomatic human sleeping sickness in the Luangwa valley,Zambia (Songa et al., 1991). Elucidation of the molecular basisfor control of trypanosomes in these hosts may suggest geneticor immunologic strategies to control trypanosomiasis in sus-ceptible mammals.

Control of trypanosomes in resistant and susceptiblehosts

Infected mammals produce antibodies that react with the im-munodominant surface coat antigens of African trypanosomes,causing immune clearance of targeted parasites. The surfacecoat on each trypanosome is composed of about 10 millioncopies of a single species of VSG. African trypanosomes ex-press only 1 VSG gene at a time and evade immune control byswitching off the expressed VSG gene and replacing it with oneof their 1,000 or so genomic VSG genes, or their recombinants,a process that is sometimes associated with a change in theactive VSG gene expression site (Borst et al., 1998; Cross etal., 1998). The rate of VSG gene-switching varies from around1023 to around 1026 switches/trypanosome/generation depend-ing on the test organism (Turner and Barry, 1989). Consequent-ly, parasitemic waves are comprised of major and minor VSGtypes. Dominant VSG types in each wave stimulate the pro-duction of antibodies and are cleared, allowing growth-com-petent minority variants to seed the next wave.

The challenge posed to the immune system by VSG switch-

2 THE JOURNAL OF PARASITOLOGY, VOL. 87, NO. 1, FEBRUARY 2001

ing is further exacerbated by intraspecies variation among try-panosomes, which includes distinct genomic VSG gene reper-toires (Myler, 1993; Van Miervenne et al., 1995). Furthermore,additional diversity is evident in growth factor receptors. T.brucei can optimize its growth in a specific host by switchingamong alleles encoding their receptor for transferrin (Bitter etal., 1998). It is possible that the other trypanosome species aresimilarly equipped, and indeed that other important receptorsare polymorphic. The genes that encode the transferrin receptorare associated with the VSG gene expression site (Steverdinget al., 1995) and switching among alleles of the transferrin re-ceptor is achieved during VSG gene expression site switching(Bitter et al., 1998), although this system is somewhat leaky(Ansorge et al., 1999).

Trypanosome-infected susceptible hosts, which include peo-ple, most breeds of cattle, rabbits, and lab rodents, typically donot produce trypanodestructive antibodies other than those thatare VSG specific. In these hosts, parasitemia manifests as re-curring waves. Because a protective VSG-specific antibody re-sponse takes several days to develop and because the doublingtime of trypanosomes is about 7 hr, trypanosomiasis-susceptiblehosts typically develop high levels of parasitemia, severe try-panosomiasis-associated pathology, generalized immunodepres-sion, and, eventually, debilitating secondary infections. It is un-clear whether the high levels of trypanosome parasitemia thatdevelop in infected susceptible hosts result from inadequate ex-pression of innate protective mechanisms that limit the extentto which trypanosomes replicate during the developmentalphase of the acquired immune response, or an inability to re-spond to conserved protective antigens on trypanosomes, orinfection-induced generalized immune suppression, or a com-bination of these. It is clear, however, that both innate and ac-quired responses contribute to trypanosome control.

Innate resistance to trypanosomiasis: Human serumtrypanolytic factor

People are resistant to infection with T. b. brucei, but sus-ceptible to T. b. rhodesiense and T. b. gambiense. Early in the20th century, it was recognized that T. b. brucei is lysed byhuman serum, a trait that is presently used as a simple, althoughonly partly reliable, test to distinguish T. b. brucei from thehuman infective T. brucei subspecies (Rickman and Robson,1970; Robson and Rickman, 1978). The test can record falsenegatives, as human-infective T. brucei can give rise to humanserum-sensitive forms when freed from the selective pressureof human serum. T. b. brucei-lytic activity is also found inserum from some African ground-dwelling primates, which,like humans, are resistant to infections with T. b. brucei (Seedet al., 1990). Initial characterization of the trypanosome lyticfactor in human serum showed it to copurify with high-densitylipoprotein (HDL) and to be absent from patients with Tangierdisease, ‘‘an autosomal recessive disorder characterized by asevere deficiency of HDL’’ (Rifkin, 1978). Trypanocidal activ-ity of human serum requires Ca21 (D’Hondt et al., 1979) andis destroyed by phospholipase treatment, suggesting the in-volvement of HDL lipids (Rifkin, 1991a), a hypothesis that mayrequire modification in light of more recent data discussed be-low. Treatment of T. b. brucei with human trypanolytic factorresults in ‘‘irreversible acute damage to the normal permeability

properties of the trypanosome plasma membrane’’ (Rifkin,1984), and this cytopathic effect is inhibited by the inclusionof membrane stabilizers in the reaction buffer.

Subsequent work (Hajduk et al., 1989) has shown that the T.b. brucei-lytic activity of human serum HDL is due to a veryhigh density subfraction (VHDL) containing apolipoproteins A-I, A-II, C-I, C-II, and C-III. Furthermore, its trypanocidal ac-tivity is inhibited by monoclonal antibodies specific for apo A-I and apo A-II, suggesting that these components may be in-volved in the interaction of the lipoprotein with trypanosomes,or in the killing mechanism, or both. The role of human apoA-I in the lytic event is ambiguous. Gillett and Owen (1992a)showed it to be capable, when used alone, of causing lysis ofT. b. brucei, whereas earlier studies from Rifkin (1991b)showed that not to be the case. The latter’s conclusions aresupported by reconstitution experiments from Tytler et al.(1995), showing that apo A-1, apo L-III, and apo L-I in humanVHDL contribute to lysis of T. b. brucei, but individually arenot cytotoxic. Additional studies (Smith et al., 1995) haveshown that trypanolytic VHDL also contains human haptoglo-bin-related protein (hpr) and paraoxonase–arylesterase and isendocytosed by trypanosomes, possibly together with boundhemoglobin, which endows the particle with peroxidase activ-ity. An antibody against the hpr inhibited the T. b. brucei lyticactivity of human serum, as did addition of the H2O2-catabolicenzyme catalase to the incubation medium (Smith et al., 1995).These observations led to the hypothesis that endocytosed hu-man VHDL reacts with endogenous H2O2 to cause lipid per-oxidation of the trypanosomal lysosomal membrane, resultingin release of its enzymes and autolysis. Smith and Hajduk(1995) went on to show that haptoglobin in human serum in-hibits endocytosis of the lytic VHDL by trypanosomes and dif-fers in concentration in different human sera, accounting fordifferences in their T. brucei-lytic activity. Ortiz-Ordonez andSeed (1995) showed that the endocytosed lytic factor could berecovered from lysed trypanosomes and retained its trypanol-ytic activity.

The idea that endocytosis of a single species of VHDL isresponsible for the T. b. brucei-lytic activity of human serumwas shaken by observations of Lorenz et al. (1994) that showedlysis to be mediated by particles of 214–440 kDa and of.1,000-kDa, of which both species contained apo A-I. Thesedata support earlier studies of Barth (1989) that indicate thelytic factor to be a .1,000-kDa complex of 4 peptides. Raperet al. (1996) verified the presence of 2 distinct lytic factors inhuman serum and confirmed that the trypanocidal activity ofisolated human VHDL was inhibited by serum haptoglobin, but,importantly, that addition of haptoglobin to intact human serumhad no effect on its trypanolytic activity. This led the investi-gators to conclude that the .1,000-kDa fraction is mainly re-sponsible for the T. b. brucei-lytic activity of unfractionatednormal human serum. In search of the relation between the 2trypanolytic particles, Raper et al. (1999) developed improvedpurification protocols and undertook biochemical comparisons.Their analysis of the trypanocidal VHDL particle showed it tocontain apo A-I and hpr, and to have trace amounts of paraox-onase, apo A-II, and haptoglobin, but to have no detectablehemoglobin. The test organisms were grown in vivo and mayhave contained hemoglobin in their endosomal system. Hence,the investigation raises questions regarding, but does not ex-

BLACK ET AL.—HOST CONTROL OF AFRICAN TRYPANOSOMES 3

clude, a role for hemoglobin in the mechanism of T. b. bruceilysis by human VHDL. Characterization of the .1,000-kDa try-panocidal protein complex showed it to contain mainly IgM,apo A-I, and hpr, but less than 1% (and possibly no) lipid (Tom-linson et al., 1997; Muranjan et al., 1998; Raper et al., 1999).The mechanism of trypanosome lysis by this particle and itsrelation to VHDL-mediated lysis are not resolved. However, thepresence of apo A-I and hpr in both particles may account fortheir similar activities.

In addition to their resistance to infection with T. b. brucei,humans do not develop infections with T. congolense and T.vivax. Whereas there is little or no published information onhow humans resist these parasites, ongoing experiments by Or-inda, Murphy, and Black (data not shown) indicate that humanserum lyses T. congolense and T. vivax, as well as T. b. brucei,as does material that copurifies with serum VHDL. Inclusionof 10 mM ammonium chloride in the incubation buffer reducedthe end-trypanocidal titer of human serum against T. b. bruceiand T. congolense by 4-fold, but did not eliminate the activityor affect the kinetics of trypanosome killing at high serum con-centration. These data show that processing in an acid com-partment enhances, but may not be essential for, the expressionof the T. congolense- and T. b. brucei-trypanolytic activity ofunfractionated human serum. Addition of 1 mg of bovine cat-alase per milliliter of reaction buffer delayed killing of T. b.brucei at low serum concentration, but had little or no effecton their lysis at high concentration of human serum, and hadno protective effect for T. congolense at any serum concentra-tion. The extracellular destruction of H2O2 by catalase accel-erates the downloading of, but does not eliminate, intracellularH2O2. A lack of effect of the agent, as was seen with T. con-golense at all serum concentrations, and with T. brucei at highserum concentration only, does not necessarily exclude a rolefor H2O2 in the lysis process.

A checkerboard titration of unfractionated human sera andtrypanosome isolates indicated that individual serum samplesoften killed some isolates of T. congolense and T. brucei betterthan other isolates on the basis of end trypanolytic titers. Fur-thermore, the spectrum of isolates that were killed by any serumvaried, so that trypanosomes that were poorly killed by 1 hu-man serum could be effectively killed by another, which in turnmay have had little activity against other isolates. Seed et al.(1993) made similar observations while evaluating the impactof injecting different human sera into mice that were infectedwith different clones of T. b. rhodesiense. These data suggestthat interactions among polymorphic serum and parasite com-ponents affect the trypanolytic process. Complete characteriza-tion of the trypanolytic particles and resolution of their killingmechanism(s) may suggest ways to engineer this very effectiveinnate protective mechanism into cattle.

Comparative analyses of human serum-sensitive and humanserum-resistant T. brucei indicate that both bind human VHDL,but the lytic factor is endocytosed by the former only (Hagerand Hajduk, 1997). Furthermore, most isolates of T. b. rhode-siense preferentially express a human serum resistance-associ-ated (SRA) gene (De Greef et al., 1989; Xong et al., 1998;Milner and Hajduk, 1999), which upon transfection into T. b.brucei confers resistance to lysis by human serum (Xong et al.,1998). It is noteworthy that T. b. brucei that were transfectedwith the SRA gene resisted lysis by undiluted human serum (E.

Pays, pers. comm.). These data suggest that the SRA gene en-ables T. b. brucei to resist all of the trypanolytic factors inhuman serum. Elucidation of the mechanism through which ex-pression of the SRA gene results in acquisition of human serumresistance by T. b. brucei may lead to the development of block-ing drugs that will render SRA-positive T. b. rhodesiense sus-ceptible to lysis by human serum and thus alleviate acute hu-man trypanosomiasis. However, this may be an overly optimis-tic outlook. There are SRA-negative T. b. rhodesiense, and T.b. gambiense, which also infect people, do not express SRA.Consequently there are SRA-independent mechanisms that en-dow upon human-infective T. brucei the capacity to resist lysisby human serum.

Trypanosomiasis-resistant cattle

Trypanosomiasis resistance is found in individual cattle ofmany different breeds (Akol et al., 1986; Roelants et al., 1987;Mattioli and Wilson, 1996; Dolan, 1998), but is enriched insome breeds, notably the N’dama (Murray et al., 1990; Palingand Dwinger, 1993). Trypanotolerant cattle can survive in areasof low tsetse and trypanosome challenge without chemotherapyand can self-cure. Immunity in cattle that have undergone self-cure after trypanosome infection is directed only against theinfecting parasites and their progeny. The immune animals re-main susceptible to infection with trypanosomes that differ inVSG gene repertoires from the priming organisms (Akol andMurray, 1983; Wellde et al., 1989; Paling et al., 1991a,b; Dwin-ger et al., 1992). The kinetics and specificity of anti-trypano-some immunity in N’dama indicate that it is mediated predom-inantly by anti-VSG antibodies and not by antibodies directedagainst invariant antigens.

During the early stages of trypanosome infection, levels ofparasitemia are similar in N’dama and Boran cattle, but thetrypanotolerant N’dama cattle develop less severe anemia thanthe more susceptible Boran cattle. The extent to which this re-flects differences in immune responses is unclear. The infectedN’dama develop higher-titered IgG1 responses against trypano-some antigens than their Boran counterparts (Authie et al.,1993a,b; Taylor et al., 1996), including a response against theT. congolense cysteine protease, congopain, and it is possiblethat antibodies against these non-VSG components affect try-panosome-induced pathology (Authie, 1994).

The ability of trypanosomiasis-resistant cattle to sustainhealth and to develop serodeme-specific immunity under lowchallenge with T. brucei, T. congolense, and T. vivax attests tothe husbandry skills of the African pastoralists who have man-aged indigenous herds of cattle whether they were domesticatedin Africa or later introduced from other regions (Bradley et al.,1996; Hanotte et al., 2000). Because resistance is reported toall the currently available drugs against trypanosomes, ‘‘cattlewith reduced susceptibility to trypanosomosis (trypanosomia-sis) and a lower requirement for drug therapy or prophylaxismay have a major contribution to make to future sustainablelivestock production in the tsetse infected areas of East Africa’’(Dolan, 1998). It would be useful if the trypanosomiasis-resis-tant phenotype could be crossed, or engineered, into improvedcattle. However, this will be a difficult task. The mechanismsunderlying the sustained health of the trypanotolerant cattle un-der low to moderate challenge with tsetse-transmitted trypano-


somes are complex and unresolved (Kamanga-Sollo et al.,1991; Williams et al., 1996; Taylor, 1998), may differ amongbreeds of trypanotolerant cattle, and may involve several loci.

Studies are underway at the International Livestock ResearchInstitute, Nairobi, Kenya to map the genetic basis of the try-panotolerant phenotype of N’dama cattle by segregation anal-ysis. However, relevant loci have not yet been identified andlimitations imposed by numbers of progeny may preclude finemapping of the trait. Similar studies in mice (Kemp et al., 1997;Iraqi et al., 2000) suggest the involvement of ‘‘a chromosomeregion of large effect, possibly comprising more than 1 resis-tance locus, on chromosome 17, and of further loci on chro-mosomes 1 and 5’’. Further resolution of the genetic basis forthe limited trypanotolerance seen in mice may provide insightsinto possible resistance genes in cattle. However, it is also pos-sible that the different host species have distinct resistancemechanisms.

Trypanosomiasis-resistant African wildlife

Nature’s experiments have been going on for several millionyears among tsetse flies, trypanosomes, and African wildlife.Several sub-Saharan mammals, including Cape buffalo (Syn-cerus caffer), have been selected through evolution to be highlyresistant to trypanosomiasis (Ashcroft, 1959; Ashcroft et al.,1959; Giegy et al., 1971; Rurangirwa et al., 1986; Mulla andRickman, 1988; Reduth et al., 1994). For example, Cape buf-falo that are bred in captivity from trypanosomiasis-free parentsshow no signs of disease upon infection with T. brucei (Reduthet al., 1994), T. vivax (Dwinger et al., 1986), T. congolense(Grootenhuis et al., 1990; Olubayo et al., 1990), or mixtures ofthese (Wang et al., 1999). Control of trypanosome infectionsby wildlife species typically involves early control of parasit-emia. When infection is initiated by intravenous injection witha cloned trypanosome variant type, Cape buffalo develop a sin-gle wave of patent parasitemia, after which the infection be-comes cryptic and can be detected only by injection of bloodinto mice or by tsetse pickup.

Cape buffalo that have cleared a trypanosome infection havea heightened ability to control subsequent infections with un-related trypanosomes. For example, a Cape buffalo, 5641, thathad been exposed to T. brucei GUTat 3.1 and T. bruceiGUTR22 developed only cryptic parasitemia upon infectionwith unrelated T. brucei ILTat 1.4 (Reduth et al., 1994). Noparasites were detected in the blood before infection with T.brucei ILTat 1.4 and, thereafter, the infection was characterizedby the presence of 1 to 10 mouse-infective T. brucei ILTat or-ganisms per milliliter of blood. The immediate suppression ofT. brucei ILTat parasitemia that was established in the blood ofCape buffalo 5641 was not mediated by antibodies against ILTat1.4 VSG, or the VSGs of several other ILTat variants (Reduthet al., 1994). Similar analyses in Cape buffalos 7810, 7813, and7752 showed that they effectively controlled T. brucei A4 andT. congolense IL 1180 after a single wave of parasitemia (Wanget al., 1999), and did not develop a parasitemic wave uponsubsequent infection with unrelated T. brucei GUTat 3.1, T.congolense IL 2642, T. congolense IL 3000, and T. congolenseIL 3338 (N. Murphy, Q. Wang, and S. Black, unpubl. obs.).The studies suggest that infected Cape buffalo develop broad-acting anti-trypanosome immunity that severely restrains try-

panosome population expansion irrespective of VSG gene rep-ertoires.

Our investigations show that the initial control of trypano-some parasitemia in Cape buffalo is due to the production ofVSG-specific antibodies and the coincident generation of try-panocidal H2O2 that is produced during catabolism of endoge-nous purine by serum xanthine oxidase (Muranjan et al., 1997;Wang et al., 1999). The capacity to accumulate H2O2 in Capebuffalo serum during purine catabolism to a level that is harm-ful to trypanosomes results from an infection-associated declinein blood catalase, an enzyme that destroys H2O2 (Wang et al.,1999). The types and concentrations of purines that are avail-able in infected Cape buffalo plasma have not been studied.However, it has been shown that endogenous purine-catabolicenzymes in Cape buffalo serum, together with trypanosome-associated guanine deaminase (Fish et al., 1982), allow catab-olism of adenosine, guanosine, inosine, guanine, hypoxanthine,and xanthine to uric acid, yielding trypanocidal H2O2 (Wang etal., 2000). In addition, Cape buffalo serum contains purines thatare associated with macromolecules and are made available tothe catabolic enzymes only in the presence of trypanosomes(Wang et al., 1999), thus maximizing the exposure of trypano-somes to H2O2 and minimizing colateral damage to the host.

Several features of bloodstream-stage trypanosomes ensure ahigh sensitivity to H2O2: The organisms obtain all of their en-ergy through glycolysis and their ATP store is sufficient to sup-port only a few seconds of normal activity (Bienen et al., 1991;Muranjan et al., 1997). Thus, they require continuous energymetabolism to meet their energy needs. The parasites lack cat-alase and rely on coupled redox reactions fueled by the reduc-ing power of NADPH to inactivate H2O2 (Fairlamb and Cerami,1992; Flohe et al., 1999). However, the utilization of NADPHdeflects glucose 6-phosphate from the glycolytic to the pentosephosphate pathway by unleashing glucose 6-phosphate dehy-drogenase. Bloodstream-stage T. brucei suppress expression ofgenes encoding ribulose-5-phosphate 39-epimerase and trans-ketolase (Cronin et al., 1989), and thus terminate their NADPH-yielding pentose phosphate pathway at ribulose 5-phosphate in-stead of recycling this product back into the glycolytic pathway.Consequently, the glucose metabolic pathways in bloodstream-stage T. brucei appear to be configured to yield ATP orNADPH, but not both at the same time. This may account forthe almost instantaneous depletion of the ATP content of blood-stream-stage trypanosomes in the presence of xanthine and xan-thine oxidase (Muranjan et al., 1997). The coadaptations ofCape buffalo and African trypanosomes that lead to efficientproduction of H2O2 on one hand and high sensitivity to H2O2

on the other are consistent with an ancient relationship thatensured survival of both the parasite and its host.

A xanthine oxidase-dependent plasma oxidative defense mayalso be expressed in giraffe and eland, which have similar levelsof serum xanthine oxidase to Cape buffalo (Black et al., 1999).However, it is certainly not common to all sub-Saharan mam-mals, many of which lack plasma xanthine oxidase activity(Black et al., 1999). Furthermore, the plasma oxidative defenseis short-lived even in Cape buffalo. Cape buffalo blood catalasereturns to preinfection levels by about a month after infection,thus precluding the accumulation of a trypanocidal concentra-tion of H2O2 in the plasma whether it be generated directly bycatabolism of purine in the plasma or released by cells under-


going a respiratory burst. In addition, the VSG- and species-unrestricted trypanocidal activity of undiluted Cape buffalo se-rum declines within a few days after remission of the sole par-asitemic wave, most likely because the level of purine substratedeclines (Wang et al., 1999). Whereas the plasma oxidative re-sponse may account for the transitory purging of trypanosomesfrom the Cape buffalo bloodstream, it does not result in sterileimmunity. Parasites reoccupy the Cape buffalo bloodstreamwithin 2 wk after remission of the parasitemic wave and aresustained at a level of only a few organisms per milliliter ofblood. The stable suppression of trypanosome parasitemia thatresults in the prolonged cryptic phase of infection, together withthe enhanced control of heterologous trypanosomes, extend be-yond the period of depressed catalase activity. We thereforehypothesize that the Cape buffalo develop trypanosome growth-inhibitory humoral factors in addition to H2O2.

Our ongoing studies support this contention and suggest arole for antibodies. Immunoglobulin of the IgG class that wasisolated from Cape buffalo blood 6 mo after their infectionsuppressed growth of bloodstream-form T. b. brucei and T. con-golense in axenic cultures, whereas IgG from preinfection se-rum or from infected cattle did not affect growth of the parasites(data not shown). The immune Cape buffalo IgG did not reactwith the VSG of the test parasites, but did stain the flagellarpocket of the organisms. These data are consistent with the ideathat antibodies against trypanosome flagellar pocket compo-nents, which include growth factor receptors as discussed be-low, facilitate the long-term suppression of trypanosome para-sitemia in infected Cape buffalo.

Acquired resistance to trypanosomiasis: T. brucei growthfactor receptors, endocytosis, and inhibitory antibodies

There are reports of surface-exposed trypanosome plasmamembrane antigens in addition to VSG (Coppens et al., 1988;Olenick et al., 1988; Ziegelbauer et al., 1992; Borst and Fair-lamb, 1998; Pays and Nolan, 1998). An important group ofthese is located in an invagination of the trypanosome surfacecalled the flagellar pocket. The trypanosome subpellicular mi-crotubule network is interrupted at the flagellar pocket, whichis the sole site of endocytosis and exocytosis in trypanosomes(Balber, 1990). Non-VSG surface components of the flagellarpocket include a variety of enzymes (Steiger et al., 1980; Walterand Opperdoes, 1982; Tosomba et al., 1996) as well as recep-tors for required host macromolecules, namely, transferrin(Salmon et al., 1994; Steverding et al., 1995), low-density li-poprotein (LDL) (Coppens et al., 1995), HDL (Gillett andOwen, 1992b), and T. b. brucei lytic VHDL present in humanserum (Hager et al., 1994). Antibodies against these flagellarpocket-associated antigens may affect trypanosome replicationand mediate acquired immunity, particularly because the organ-isms require lipoproteins (LDL or HDL) (Gillett and Owen,1987; Coppens et al., 1988; Black and Vandeweerd, 1989; Mor-gan et al., 1993, 1996) and transferrin (Black and Vandeweerd,1989; Schell et al., 1991; Steverding, 1998) for sustained rep-lication. In this regard, antibodies against the T. brucei trans-ferrin receptor have been shown to inhibit growth of blood-stream-stage trypanosomes (Salmon et al., 1994), but onlywhen the receptor and transferrin interaction is of low affinity(Borst et al., 1996). In addition, antibodies against the T. brucei

LDL receptor have been shown to both prevent uptake andprocessing of LDL and to inhibit T. brucei growth in vitro(Coppens et al., 1988), although this effect is not evident on allclones of trypanosomes, as discussed below. The LDL receptoris conserved throughout the Kinetoplastida, has at least 1 sur-face-accessible epitope that distinguishes it from the mamma-lian LDL receptor, and is immunogenic (Bastin et al., 1996),suggesting that it might be a useful target for immunoprophy-laxis against both animal and human trypanosomiasis.

T. brucei lipid acquisition and immune control

There is some confusion over how trypanosomes interactwith lipoproteins. One set of experiments show that blood-stream-stage T. brucei meet their lipid requirements mainly byreceptor-mediated endocytosis of LDL (Coppens et al., 1995),which would account for the capacity of T. brucei LDL recep-tor-specific antibodies to prevent trypanosome replication inmedium supplemented with intact serum and, hence, containingboth HDL and LDL (Coppens et al., 1988). The endocytosedLDL is processed in an acid compartment, allowing receptorrecycling, possibly by acid-induced ligand–receptor dissocia-tion (Coppens et al., 1993). The protein component of endo-cytosed LDL is proteolysed and a portion remains associatedwith trypanosomes, whereas the remainder is released into theextracellular medium (Coppens et al., 1993).

However, another set of experiments suggests a more com-plex lipid acquisition process. Bloodstream-stage S427 and ILTatT. brucei isolates were found to grow equally well in axenicculture when provided with bovine, rabbit, or rat LDL, or HDL,as their sole lipid source (Black and Vandeweerd, 1989). Theseparasites acquired lipids from bovine, rabbit, and rat LDL, andHDL, with similar efficiency, took up radiolabeled lipids fromthe particles without accumulation of radiolabeled protein com-ponents (Vandeweerd and Black, 1989, 1990), and did not con-tain material in their endosomal system that reacted with poly-clonal or monoclonal antibodies specific for bovine LDL andHDL apolipoproteins, even after propagation in the presence ofbovine LDL and HDL for a month (P. Webster and S. Black,unpubl. obs.). The S427 and ILTat T. brucei isolates took upradiolabeled cholesterol from rat, rabbit, and bovine HDL andLDL by contact-mediated desorption and diffusion processes,which also occur between lipoproteins and all other cell types,between adjacent cells, and between adjacent lipoproteins. Theparasites acquired lipoprotein-associated cholesteryl ester andphosphatidylcholine via different lipid class-specific uptakepathways that could be distinguished by inhibitors (Vandeweerdand Black, 1989, 1990; Vandeweerd, 1990). In addition, theuptake by the cultured trypanosomes of cholesterol, cholesterylester, and phosphatidylcholine from rat, rabbit, and bovine LDLcould also be inhibited by the inclusion of either unlabeled LDLor HDL, from any of the species, in the incubation medium(Vandeweerd and Black, 1989). Similar results were obtainedwith T. b. brucei ILTat 1.4, T. b. brucei GUTat 3.1, and T. b.rhodesiense 1292 that had not been selected by growth in vitro,although uptake of cholesteryl ester relative to phosphatidyl-choline was much reduced in these in vivo-derived parasites(Vandeweerd, 1990), possibly due to lower expression of a cho-lesteryl ester transport protein. The observations suggest eitherthat trypanosomes acquire most of their lipids from LDL and


HDL without endocytosis of the intact particles, or that theendocytic compartment they enter is a sorting zone from whichsome lipids are imported into the organisms while processedapolipoproteins and other lipids are rapidly exported back intothe extracellular space. The studies do not exclude a role forreceptor-mediated binding of LDL and HDL in lipid acquisitionby trypanosomes, although they suggest that the receptor maybe unable to discriminate between HDL and LDL and amonglipoproteins that are isolated from rabbit, rat, and bovine serum.

The T. brucei studied by Black and Vandeweerd (1989) donot appear to be affected by exposure to T. brucei LDL recep-tor-specific antibodies in vitro. The parasites can be propagatedequally well in culture medium supplemented with 10 to 50%vol. intact or heat-inactivated T. brucei LDL receptor-specificor nonimmune rabbit serum in place of fetal bovine serum(FBS) (S. Black, unpubl. obs. obtained using sera that weregenerously supplied by Dr. Coppens). Furthermore, the para-sites were observed to replicate to a similar extent in serum-free medium supplemented with lipoprotein-depleted FBS, alimiting amount of FBS-LDL, and with or without a high con-centration of IgG specific for the LDL receptor of T. brucei andgenerously provided by Dr. Coppens (S. Black, unpubl. obs.).The latter result might reflect either heterogeneity of protectiveepitopes on T. brucei LDL receptors, or a lack of requirementfor these LDL receptors in crucial interactions between LDLand T. brucei in axenic cultures. With respect to the formerpossibility, other investigators have shown that immunizationof mice with purified T. brucei LDL receptor from 1 strain wasnot sufficient to confer protection against another strain of theparasites, although the immunization did delay the developmentof parasitemia for a short period (Bastin et al., 1996; P. Bastin,pers. comm.). The impact of LDL receptor immunization oncontrol of a homologous challenge was not evaluated.

Polyvalent antibody responses against flagellar pocketpolypeptides

The restricted impact of antibodies against transferrin andLDL receptors on T. brucei growth does not exclude a role forantibodies against trypanosome receptors in trypanosomegrowth control. A combination of antibodies against several re-ceptors may be required to affect growth of the parasites. Im-munization with a crude subcellular fraction of T. brucei thatmay include flagellar pocket antigens can protect rats (Powell,1978), rabbits (Powell, 1993), sheep (Powell and Mathaba,1978), and cattle (Mkunza et al., 1995) against infection. Sim-ilar experiments in BALB/c mice did not result in protection(C. Powell and S. Black, unpubl. obs.). However, the mouse isa poor model for vaccine studies against African trypanosomes,as this species typically develops high parasitemia and becomesrapidly immunosuppressed. Indeed, immunization of mice withother preparations of T. brucei flagellar pocket antigens yieldedonly limited protection (Olenick et al., 1988), whereas immu-nization with clathrin-coated vesicles that were presumed tocontain endocytosed receptors actually enhanced virulence,leading to speculation that some T. brucei receptor-specific an-tibodies made by mice may block the capacity of T. brucei tointeract with growth-inhibitory ligands, or simulate growth fac-tors, or interact with their target receptor proximal to a protec-tive epitope and sterically hinder the binding of a protective

antibody, or any combination of these (Shapiro, 1994). If so, itmay prove essential to target particular epitopes on T. bruceireceptors to consistently achieve protection. Difficulties in ob-taining adequate amounts and diversity of trypanosome recep-tors for immunization studies have limited exploitation of thisapproach. However, the discovery by Nolan et al. (1999) thatmany, if not all, trypanosome flagellar pocket receptors bear N-linked glycans containing linear poly-N-acetyllactosamine pro-vides a means for their isolation and thus, their immunologicalevaluation.

ACKNOWLEDGMENTS

We thank Derek Nolan, Etienne Pays, and Philippe Bastin for theirwillingness to share unpublished data and for useful discussion. Re-search on the Cape buffalo was supported by NIH R01 AI 35646 anda grant from USAID to expedite collaboration between the InternationalLivestock Research Institute and the University of Massachusetts atAmherst.

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INNATE AND ACQUIRED RESISTANCE TO AFRICAN TRYPANOSOMIASIS