TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (2000) 94,328-332

HIV-Leishmania infanturn co-infection: humoral and cellular immune responses to the parasite after chemotherapy

Javier Moreno’, Carmen Caiiavate’, Cristina Chamizo’, Fernando Laguna* and Jorge Alvar’* ‘WHO Collaborating Centre for Leishmaniasis, Research Unit in Tropical Medicine and International Health (FIS-ISCIII), Servicio de Parasitologi’a, Centro National de Microbiologia, In&two de Salud Carlos III, Majadahonda (Madrid), Spain; ‘Departamento de Enfermedades Infecciosas, Centro de Investigaciones Clinicas, Institute de Salud Carlos III, Madrid, Spain

Abstract Specific serum antibodies, peripheral blood T-cell subsets, cellular response in vitro to soluble Leishmania antigens, phenotype of stimulated cells, and serum levels of tumour necrosis factor (TNF)-a and transforming growth factor (TGF)-Pl were studied in Spain in 17 patients co-infected with HIV and Leishmania infanturn who had been previously treated with pentavalent antimony. Both humoral and cellular responses to Leishmania sp. appeared diminished, 8 out of 17 patients were positive by indirect immunofluorescence, and immunoblotting detected heterogeneous antibody-binding pattern in 11 out of 13 subjects. A blastogenesis test was positive in 4 cases; 2 of them presented proliferation of CD4+ cells while CD8+ cells proliferated in the other 2 patients. Serum levels ofTNF-a were similar to those observed in patients infected with HIV only, while serum levels of TGF-0 1 were significantly lower in the co-infected patients. The inability of antibody response to control the parasite and the absence of specific T-cell immunity to Leishmania sp. would explain the high frequency of relapses reported in these patients. The decreased levels of TGF-fll could have an important role in the interaction between the 2 pathogens.

Keywords: Leishmaniu infanturn, HIV, co-infection, immune response, humoral immunity, cellular immunity, mmour necrosis factor, transforming growth factor, Spain

Introduction The spread of human immunodeficiency virus (HIV)

infection in leishmaniasis-endemic countries has re- sulted in a sharp increase in the reported numbers of cases of HIV-Leishmania co-infection in recent years. To date, over 1400 cases of co-infection have been reported in southern Europe (WHOAJNAIDS, 1998). It is currently estimated that 2570% of adult visceral leishmaniasis (VL) cases are related to HIV, and l-5-9% of AIDS cases suffer from newly acquired or re-activated VL (WHO, 1994). In these co-infected patients, VL presents with similar or more severe clinical symptoms than in immunocompetent patients and because of the former patients’ immunological condition may exhibit additional specific characteristics, e.g., parasitization of unusual locations such as skin (YEBRA et al., 1988), digestive tract (LAGUNA et al., 1994) or lung (BAl;nr~s et al.. 1995). Furthermore. uarasites isolated from these individuals present a high variability of zymodemes, some of which appear to be unique to HIV-infected individuals (JIM&%z et al., 1995). -

In immunocomuetent individuals, VL is associated with a clear humoral response, due to polyclonal B-cell activation, and a suppression of the cellular response to the parasite which is restored after successful chemo- therapy (CARVALHO et al., 198 1). The T-cell-mediated immune response is critical for cure and protection in secondary infections (MURRAY et al., 1989). Compared to immunocompetent patients with VL, co-infected patients present lower levels of specific antibodies (MARY et al., 1992) and a high rate of clinical relapses in spite of treatment (ALVAR et al., 1997). However, although numerous cases of co-infection have been reported, detailed investigations of the immune response of these patients are still lacking. A report of the cellular resnonse in 2 cases of AIDS-associated American tea-

I

mentary leishmaniasis caused by Leishmania braziliek is available (COUTINHO et al., 1996). Changes in blood subsets of HIV-L. donovani co-infected patients have also been reuorted (CENINI et al., 1994). and the cytokine profile of such patients has been recently

l Current address: Christ’s Colleae, Universim of Cambridge, -

- Cambridge CB2 3BU, UK. Address for correspondence: Dr Javier Moreno, WHO Colla- borating Centre for Leishmaniasis, Servicio de Parasitologia, Centro National de Microbiologia, Instimto de Salud Carlos III, 28220-Majadahonda (Madrid), Spain; phone +34 91 509 7901, fax +34 91 509 7034, e-mail fjmoreno@isciii.es

published (CACOPARDO et al., 1996; MEDRANO et al., 1998a).

The aim of the present work is to provide information about the immunological status of co-infected patients compared to that of HIV-infected patients, and their ability to respond to Leishmania sp. after treatment. The presence of specific serum antibodies was determined by indirect immunofluorescent antibody test (IFAT), and their characteristics were determined by immunoblot. T- cell response was analysed through proliferation to Leishmania antigens and determination of T-cell subsets. Serum levels of tumour necrosis factor (TNF)-a and transforminn growth factor (TGF)-81 were also meas- ured and the& role in the interaction between the pathogens is discussed. Results obtained in this study may be of value in the treatment of co-infected patients.

Materials and Methods Subjects

The study included blood samples from 17 HIV- infected natients with a blood level of CD4+ cells <20O/n&1~ who were diagnosed with VL confirmed either by isolation of Leishmania sp. in bone-marrow aspirate or peripheral blood mononuclear cell (PBMC) culture or byxenodiagnosis and identified as L. infantum. The patients had been treated with pentavalent anti- mony at the conventional dose of 20 mg/kg daily for 28 days, in some cases with more than 1 course of treatment.

The data obtained were compared to those for a group of 15 HIV-infected patients of similar ages and risk groups, with <200 CD4+ cells/mm3, no clinical history of VL and without serum antibodies to L. infanturn.

Antibody levels Specific serum antibodies to Leishmania parasites were

determined bv IFAT using fixed L. infanturn as antigen. Antibody fixaiion was revealed with fhtorescein isothio- cyanate-conjugated sheep anti-human IgG (ICN, Aur- ora, OH, USA). A serum with a response at a dilution > 1:80 was considered positive.

Western Blot analysis L. infanturn soluble antigens (SLA) were electrophor-

etically separated in sodium dodecyl sulphate polyacry- lamide gel (SDS-PAGE) and proteins transferred on to nitrocellulose membrane (Schleicher and Schuell, Das- sel, Germany). The nitrocellulose was cut into strips which were incubated with the respective sera and with alkaline phosphatase-conjugated goat anti-human IgG

IMMUNE RESPONSES IN HILL. INFANTUM CO-INFECTION 329

(Bio Rad, Richmond, CA, USA). Enzymatic activity was revealed using 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Bio Rad, Richmond, CA, USA); a positive serum reflects one or more specific bands (MEDRANO et al., 1998b).

Mononuclear cells Heparinized peripheral blood was separated by cen-

trifugation (30 min at 800 g), using lymphocyte separa- tion medium at room temperature. The mononuclear cells were obtained and were stored frozen in liquid nitrogen until use.

Lymphocyte blastogenesis assay PBMCs were cultured in triplicate, in flat-bottom 96-

well microtitre plates, at a density of lo6 cells/ml in 0.1 muwell of RPM1 1640 (Gibco, Paisley, UK) sup- plemented with 100 IU/mL of penicillin, 100 pg/mL of streptomycin, 2 mM L-glutamine, 5 X 10m5 M 2-mercap- toethanol and 10% heat-inactivated fetal calf serum (Biological Industries, Israel), and incubated in a humi- dified atmosphere (37”C, 5% CO*) for 5 days with either 5 @mL phytohaemagglutinin (PHA), 10 pg/mL SLA or without antigen (blank) and pulsed during the last 20 h with 10 pi bromodeoxyuridine. Bromodeoxyuri- dine incorporation was determined using a specific ELISA system (Biotrak, Amersham, UK). The optimal concentration of antigens in proliferative assays was determined in previous experiments. The proliferative response is indicated as the stimulation index (SI) which represents the ratio of the mean absorbance measured at 450 nm after stimulation to the mean absorbance in the blank. A value of SI >2 was considered positive.

T-cell subpopulation determination Percentages of CD4+ and CDS+ PBMCs and lym-

phoblast cells after culture in 24-well plates for 5 days in the presence of SLA (10 pghnL) were determined by flow cytometry (EPICS 75 1, Coulter) with specific anti- human CD8-FITC/CD4-RPE monoclonal antibodies (Labgen, Germany).

Cytokine determination Serum levels of TNF-a and TGF-6 were quantified

using commercially available ELISA kits (Predicta TNF- a Kit and Predicta TGF-fi 1 Kit, Genzyme, Cambridge, MA, USA). Each serum was assayed in duplicate following the procedures suggested by the manufacturer.

Results Anti-Leishmania antibodies were detected by IFAT in

serum of 8 of the 17 individuals tested while immuno- blotting showed that 11 out of 13 patients had specific antibodies (Table), confirming the latter technique as a more accurate tool to study leishmaniasis in HIV- Leishmania sp. co-infected patients. The antigen-binding patterns of the different patients were very heteroge- neous and the staining was weaker than in non-immu- nocompromised patients. Those subjects with a high titre of serum antibodies had a complex immunoblotting pattern and recognized a high number ofbands. The rest of the individuals, who presented low or undetectable antibody titres by IFAT, showed a partial or absent binding pattern (Fig. 1).

Proliferative responses to SLA were positive in 4 cases (Table). No correlation was found between this resoon- siveness and other factors such as antibody titre,* im- munoblotting pattern or percentages of T cells in peripheral blood. The phenotype of Leishmania-anti- gen-reactive blasts was determined by comparison of the CD4/CD8 ratio before and after SLA stimulation. Two cases showed a preferential response of CD4+ ceIIs: in Case 10 the CD4/CD8 ratio increased from 1.86 to 3.1 and in Case 14 from 0.1 to 0.46. In Cases 2 and 8 there was a preferential response of CD8+ cells, the CD4/CD8 ratio decreasing after antigen stimulation from 0.35 to 0.10 and from 0.34 to 0.2 1, respectively.

The percentages of peripheral blood CD4+ and CDS+ cells obtained in each co-infected case are shown in Figure 2. The mean percentage of CD4+ cells (18.0 f 2.5) was similar to that in non-Leishmania infected HIV individuals (20.4 & 1.5, P = 0.216) but the mean percentage of CD8+ cells (43.4 f 5.9) was greater than in the control group (31.1 k4.8, P = 0.060). A statistically significant difference was

Table. Characteristics of the study patients co-infected with HIV and L. infanturn

Antibody Immunoblot Stimulation Patient HIV risk group levels” pattemb index’

1 IVDU 1:640 Polyclonal 0.92 2 Homosexual 1:640 Polyclonal 2.03

: Homosexual 1:640 Polyclonal 1.11 IVDU 1:640 Polyclonal 1.02

5 Homosexual 1:640 Polyclonal 0.89

4 IVDU 1:320 Polyclonal 0.71 Homosexual 1:160 Oligoclonal 0.93

; IVDU 1:160 ND 3.12 IVDU Cl:40 Oligoclonal 0.94

10 Haemophilic Cl:40 Oligoclonal 2.20

:: IVDU Cl:40 Oligoclonal 1.23 IVDU Cl:40 Negative 0.91

13 Blood transfused Cl:40 Oligoclonal 1.59 14 Blood transfused <1:40 Negative 2.8gd 15 IVDU <1:40 1.02 16 Unknown <1:40 :: 1.46 17 Unknown <1:40 ND 1.29

“Serum antibody levels specific for L. infanturn were determined by immunofluorescent antibody test. Serum dilution 3 130 was considered positive. bImmunoblot pattern reflects the number of specific bands recognized by serum antibodies. Polyclonal, several bands (> IO); oligoclonal, few bands (< 10); negative, no bands. ‘Stimulation index represents the ratio of the mean lymphoproliferation with L. infanturn soluble antigens (SIA) to the mean lymphoproliferation in blank. Value > 2 was considered positive. Similar backgrounds were found for all the patients. din this assay SIA concentration was 20 pg/mL IVDU, intravenous drug user; ND, not done.

330 JAVIER MORENO ETAL.

kDa N P 1 2 3 4 5 6 7 91011121314

213 123 85

50

33

28

8

Fig. 1. Reactivity of antibodies to L. infanturn soluble antigens in sera of HIV-L. infanturn co-infected patients and controls by irnmunoblot. Lane N, a healthy donor; lane I’, a positive immunocompetent visceral leishmaniasis patient; the rest of the lane numbers correspond to the cases studied (see the Table).

HIV-L. infantum HIV-L. infanturn

Fig. 2. Percentages of CD4+ and CD8+ cells in peripheral blood mononuclear cells of HIV-L. infanturn co-infected pa- tients and HIV+ controls. Bars represent mean percentage for each group.

observed in the CD4/CD8 mean ratio: 0.94 in HIV+ patients compared to 055 in co-infected patients, P = 0.0345. The lower CD4/CD8 ratio observed in HIV-L. infanturn co-infected individuals is due to the higher percentage of CD8+ cells observed.

Mean TNF-a levels in the serum of co-infected pa- tients (33.8 f 8.9 pg/mL) were no different from the levels in the HIV-infected patients (26.5 & 4.8 pg/mL, P = 0.226). In contrast, serum levels of TGF-pl were significantly lower in the group of co-infected patients (18.7 f 2.8 ng/mL) compared to the control group (57.0 Z!Z 9.0 ng/mL, P = 0.0008) (Fig. 3).

Discussion Successful chemotherapy of VL in non-immunocom-

promised patients reduces the antigenic load through parasite destruction, resulting in a decrease of serum levels of specific and non-specific antibody and restora- tion of specific T-cell mediated immunitv (HALDAR et al., 1983). The present study documents the lack of a nrotective snecific immune resnonse in HIV-L. infun- &n co-infected patients despiie treatment with peLta- valent antimonial compounds. This condition would explain the low rate of clinical and parasitological response (h&DRANO et al., 1992) as well as high

60

@ 50 5 40 0 HIV-Leishmania

2 30 HIV

* 20

10

0 TNF-a TGF+l WmL) (ng/mL)

Fig. 3. TNF-a and TGF-p serum levels in HIV- L. infanturn co- infected patients and HIV+ controls. Bars represent mean values f SD.

frequencv of relapses observed in these natients (ALVAR etai., 19g7). *

Five of the 17 cases included in our study showed high titres of anti-Leishmaniu antibodv (1:640). The rest of the cases presented low or absent s’p&ific antibody levels, in part due to the IFAT technique which has been reported to have a limited value in diagnosis of leishma- niasis in HIV+ patients (MEDRANO et al., 1998b). The use of a more sensitive technique, immunoblotting, revealed more positive cases, 11 out of 13, and confirm& the usefulness of this techniaue for the diaznosis of VL in HIV+ subjects (KUBAR et ai, 1998). Imm<noblot analy- sis also confirmed the partial or weak humoral response of co-infected patients when compared to non-immuno- compromised VL patients, an aspect previously reported by MARY et al. (1992).

The absence of a cellular response to Leishmania antigens in 13 out of 17 co-infected patients might explain the high frequency of treatment failures. Com- parable data were found by COUTINHO et al. (1996) in 2 cases ofAIDS plus American tegumentary leishmaniasis, 1 of which showed a lymphoproliferative response after treatment but not the other case. The later relapse suffered by the 4 patients who showed responsiveness to SLA during our study supports the loss of specific cell- mediated immunity along the process. It is probable that this cellular response is lost because of the preferential death of memory cells upon activation in HIV+ patients (JANOSSY et al., 1993).

HIV and L. infanturn infections induce similar altera- tions of the immune system that appear enhanced in co-infected patients, although the effects of the viral infection predominate when these alterations are not synergistic. These changes are related to the progression of HIV infection, being more evident in subjects with AIDS and L. do&vanzYinfection than in subiects with AIDS-related comnlex and L. donovani infection KEN- INI et al., 1994). in our study, HIV-L. infant&n co- infected patients did not have changed percentages of CD4-t cells when compared to non-L. infanturn HIV+ patients, although the percentages prior to chemo- therapy were not analysed. The higher percentage of CD8+ cells observed in co-infected patients might be due to the specific role of these cells in the response to the parasite after treaUnent (COUTINHO et al., 1996). Char- acterization of blast cells after stimulation showed that both CD4+ and CD8f cells are able to respond to SLA. In experimental VL it has been shown that the acquisi- tion ofresistance to L. donovanirequires both CD4+ and CD8f cells, but CD8+ cells alone contribute to the host defence in secondary infections (MURRAY et al., 1992). We have observed in the group of patients with lympho- proliferative responses to SLA that the ability of CD4+ or CD8t cells to proliferate did not induce acquired resistance since all of them suffered new relapses, as

IMMUNE RESPONSES IN HIV-L. INFANTUM CO-INFECTION 331

already commented. Although more data are needed to obtain conclusions, it is probable that the phenotype of proliferating cells depends on the CD4+ or CD8+ phenotype of memory cells that have still not been lost CJ~N0ssy et al., 1993).

It is clear that immunosuppression induced by HIV infection permits the re-activation of latent-Leishmania sp. infection and facilitates the development of VJ2 from new infections. Furthermore, since both HIV and L&h- mania parasites can invade and replicate within macro- phages, it is possible that the interaction between these hathogens could lead to exacerbation of the infections. Thus. TREMBLAY et al. (19961 have DrODOSed that Leis&ania SD. infection cab indbce HIV re&ication in macrophagei, a proposal that is supported bithe finding that Leishmaniu sp. infection in vitro of macrophage lines infected with HIV increases TNF-a-mediated virus replication (BERNIER et al., 1995). On the other hand, WOLDAY et ~2. (1998) have shown that live and killed HIV-l increases the growth of L. donovani in monocyte- derived cells.

Nevertheless, studies carried out in co-infected pa- tients have shown discrepant results about the increased replication of HIV induced by Leishmaniu sp. infection. MEDRANO et ~2. (1998a) did not observe differences between the number of HIV-l RNA copies before the onset of VL and at the time of diagnosis, while PREISER et al. (1996) renorted an increased number of HIV-l RNA dopies’in &-infected patients when compared with non- Leishmuniu HIV+ patients. More significant is the important increase of HIV replication observed in the co- infected patients by CACOPARDO et al. (1996) and MEDRANO et al. (1998a) after successful VL treatment, while BERHE et aZ. (1999) have reported the marked increase of HIV viral load in co-infected natients who failed anti-leishmanial chemotherapy. Tl& increase of virus replication after VL treatment in co-infected pa- tients did not correlate with TNF-a serum levels (ME- DRANO et al., 1998a). The determination of TNF-a serum levels in our study did not show differences between the co-infected and control groups, results that do not seem to agree with a TNF-a-mediated induction of HIV expression in vivo. Nevertheless, a specific role for TNF-a cannot be discarded, since POLI et al. (1990) have shown an autocrine mechanism of mF-a: mediated HIV induction in oromonocvtic cell lines.

The high level of virus- replicatioh observed after treatment could be related to the decreased level of TGF-fil observed by us in these patients. TGF-fll is a potent inhibitor of diverse aspects of cellular and humor- al immunity. It is overexpressed in HIV infection and contributes to immune defects (KEKOW et al., 1990), but this cytokine has also been demonstrated tb be a sup- Dressor of HIV exnression in both chronicallv infected promonocytic celis as well as in primary &onocyte- derived macrophages (POLI et al., 199 1). TGF-Pl is also produced by murine macrophages exposed to L. bruzi- liensis and causes susceptibility to disease (BARRAL et al., 1993). These opposed effects could support the lack of interaction in t&se macrophages co-in&ted with both uathoeens. while the decrease of TGF-I31 after theranv could?nduce viral replication in these cells. Therefore, more research is needed before synergism between HIV and Leishmuniu parasites can be definitively established, at least in monocytesimacrophages. It is also possible that VL treatment induces the T cell activation, which is known to produce viral replication in these T cells. Reduced levels of TGF-Bl cornoared to non-VL HIV+ patients could also heip T-ceil activation and viral replication in co-infected patients, since increased titres of TGF-pl correlate closely with defective responses of CD4+ lymphocytes (KEKOW et al., 1990). The question that deserves more research is whether chemotherapy against Leishmuniu infection could induce a decrease of TGF-61 serum levels that could allow increased viral replication in macrophages or activated T cells.

We can conclude that HIV-L. injuntum co-infected patients are able to generate a T-cell response against the parasite after treatment; nevertheless, this response is lost with progression of viral infection, which allows new relapses. Since HIV-Leishmuniu sp. co-infection could induce both an uncontrollable spread of the parasite and increased virus replication, it is important that the factors influencing the co-infection are understood, so that effective treatments against leishmaniasis in co-infected patients can be designed, avoiding an increase in HIV replication.

Acknowledgements The authors thank Professor D. McMahon-Pratt for useful

comments. This study was supported by a WHO/CTD Tech- nical Agreement (L3/181/42) and a Fondo de Investigaciones Sanitarias grant (9510008-02). J. M. was supported by an ‘Instituto de Salud Carlos III’ fellowship. C. C. was supported bv a FPI fellowshiu from the Ministerio de Educaci6n v Cultura. Spain. J. A. was supported by grants from the @ondo de Investigaciones Sanitarias (B.A.E. num. 99/5038) and Christ’s College, University of Cambridge, UK.

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Received 3 August 1999; revised 2SJanua y 2000; accepted forpublication 26Januaty 2000

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The Tropical Health and Education Trust (THET)

Since THET was established in 1988, it has actively supported training for health care in tropical countries, where resources are limited. THET works with those who are responsible for such training so that their goals can be reached, as they prepare their staff and students for the tasks that they will be called on to do, relevant to the needs of their local community.

THET achieves this strategy through collaborative links between a hospital or medical/nursing school in the United Kingdom and its counterpart overseas. These institutional links are made up of individual links, each of which is carefully planned over a number of years. Precise goals are set so that progress, which is closely monitored, can be used as a basis for additional funding. Links cover many disciplines from nursing, clinical medicine, community health, laboratory and library services, and management, to fieldwork by students.

We respond to requests to establish these links and, while we do not prescribe what the pattern of a link should be, we are only prepared to seek funding for those that are relevant and needed, and that directly or indirectly contribute to the health care of poor people. THET does not fund individuals, except when newly acquired personal skills contribute to an institution and the service that it provides.

Trustees: Professor Eldryd Parry (chairman), Professor Roy Anderson, FRS, Professor Brian Greenwood, FRS, Miss Thelma Holland, Mrs Helen Parry, Mr John Rennie FRCS, Mr Richard Southwell, QC and Professor David Warrell.

For further information please contact THET, Euston House, 24 Eversholt Street, London NW1 IAD, UK; phone +44 (0)20 7611 8705/6, fax +44 (0)20 7611 0683/4, e-mail vpthetl@aol.com