Abstract
Healing/protective responses in human visceral leishmaniasis (VL) are associated with stimulation/production of Th1 cytokines, such as interferon IFN-γ, and conversion in the leishmanin skin test (LST). Such responses were studied for 90 days in 44 adult healthy volunteers from VL non-endemic areas, with no past history of VL/cutaneous leishmaniasis (CL) and LST non-reactivity following injection with one of four doses of Alum-precipitated autoclaved Leishmania major (Alum/ALM) ± bacille Calmette–Guérin (BCG), a VL candidate vaccine. The vaccine was well tolerated with minimal localized side-effects and without an increase in antileishmanial antibodies or interleukin (IL)-5. Five volunteers (5/44; 11·4%) had significant IFN-γ production by peripheral blood mononuclear cells (PBMCs) in response to Leishmania antigens in their prevaccination samples (P = 0·001) but were LST non-reactive. On day 45, more than half the volunteers (26/44; 59·0%) had significantly high LST indurations (mean 9·2 ± 2·7 mm) and high IFN-γ levels (mean 1008 ± 395; median 1247 pg/ml). Five volunteers had significant L. donovani antigen-induced IFN-γ production (mean 873 ± 290; median 902; P = 0·001), but were non-reactive in LST. An additional five volunteers (5/44; 11·4%) had low IFN-γ levels (mean 110 ± 124 pg/ml; median 80) and were non-reactive in LST (induration = 00 mm). The remaining eight volunteers had low IFN-γ levels, but significant LST induration (mean 10 ± 2·9 mm; median 11). By day 90 the majority of volunteers (27/44; 61·4%) had significant LST induration (mean 10·8 ± 9·9 mm; P < 0·001), but low levels of L. donovani antigen-induced IFN-γ (mean 66·0 ± 62 pg/ml; P > 0.05). Eleven volunteers (11/44; 25%) had significantly high levels of IFN-γ and LST induration, while five volunteers had low levels of IFN-γ (<100 pg/ml) and no LST reactivity (00 mm). One volunteer was lost to follow-up. In conclusion, it is hypothesized that cellular immune responses to human VL are dichotomatous, and that IFN-γ production and the LST response are not in a causal relationship. Following vaccination and probably cure of VL infection, the IFN-γ response declines with time while the LST response persists. LST is a simple test that can be used to assess candidate vaccine efficacy.
Keywords: dichotomy, human visceral leishmaniasis, immune responses
Introduction
Visceral leishmaniasis (VL) is the most severe form of the human leishmaniases. In Sudan it is caused by Leishmania donovani [1–3]. The immune response to VL is characterized by polyclonal stimulation of B cells with production of large amounts of non-protective antileishmanial antibodies [4,5]. On the other hand, protective immune responses depend entirely on Th1 cells, which produce interferon (IFN)-γ and other Th1 cytokines [6,7]. The cellular immune responses usually follow subclinical infection, successful cure of VL or vaccination [8–14].
Surrogate markers of cellular immunity to human VL are not well characterized. Several studies showed that individuals who convert in the leishmanin skin test (LST) as a result of subclinical/clinical infections or vaccination are usually protected from infection/reinfection [12,15,16]. The LST is a delayed skin hypersensitivity reaction that can be induced in vivo by intradermal injection of dead L. major promastigotes [12,14,17–19]. For many years the LST has been used in epidemiological studies as a marker of exposure to Leishmania infections. It has also been used as an aide to diagnosis of clinical disease. The LST is invariably non-reactive in active VL and is reactive in about 50% of post-kala-azar dermal leishmaniasis patients and nearly 100% of active cutaneous leishmaniasis (CL) particularly at the advanced phases [3,6,13,20,21]. The test is usually positive a few weeks to a few months following successful treatment of VL [10]. The role of the LST as a correlate marker of immunity has been a subject of long debate. Most data point to the fact that it is the only available in vivo test that correlates, at least in part, with immunity to human VL/CL [10,12,15]. The other protective marker that can be evaluated in vitro is IFN-γ production by peripheral blood mononuclear cells (PBMCs). Recently, LST and IFN-γ production were successfully induced and studied in healthy volunteers using Alum-precipitated autoclaved Leishmania major + bacille Calmette–Guérin (BCG), a candidate vaccine for VL that was shown to be safe and immunogenic in humans [14]. This study was part of the special programme for research and training in tropical diseases (TDR)/World Health Organization (TDR/WHO) attempts to find an affordable vaccine for VL.
In this communication we hypothesize that protective Th1 immune responses to VL are dichotomatous with IFN-γ production, usually preceding conversion in the LST; IFN-γ production then wanes with time while LST conversion persists. We also introduce LST as a simple test that can be used to test the efficacy of VL candidate vaccines.
Materials and methods
Ethical considerations
The study protocol was reviewed and approved by the Ethical committees of the Institute of Endemic Diseases, University of Khartoum, the Federal Ministry of Health, Khartoum, Sudan and the Secretariat Committee of Research Involving Human Subjects (SCRIHS), TDR/WHO, Geneva. Volunteers were cared for by trained medical doctors during the course of the study.
Study volunteers
Following written informed consent, 44 adult healthy volunteers were allocated randomly to four alum-ALM (alum-precipitated autoclaved L. major) dose groups of 320 µg + BCG (group 1), 10·8 µg + BCG (group 2), 108 µg + BCG (group 3) and 320 µg without BCG (group 4). The volunteers were healthy, from VL/CL non-endemic areas, with no past history of VL/CL with a direct agglutination test (DAT) reciprocal titre of ≤ 400, and were LST non-reactive. Demographic, clinical and laboratory data were collected in a specially designed case report form (CRF). The vaccine was injected intradermally and the volunteers were followed closely for 90 days as follows: day 0 (injection day), days 3, 7, 14, 21, 45, 60 and 90 for local pain, nodule development, induration, ulceration, axillary and cervical lymphadenopathy, fever, rash, nausea and vomiting.
The LST was performed by intradermal injection of 0·1 ml of dead whole L. major promastigotes (Pasteur Institute of Iran, Tehran, Iran) on the volar aspect of the left arm. As control 0·1 ml of the vaccine diluent was injected on the volar aspect of the other arm. The test was read 72 h later using the ballpoint technique. A positive reading was taken as ≥ 5 mm. Serum was used for the DAT as described previously [17]. IFN-γ and interleukin (IL)-10 levels were measured from culture supernatants of isolated PBMCs using commercial kits (R&D GmbH, Weisbaden-Nordenstadt, Germany). Soluble Leishmania antigen at 10 µg/ml was used to stimulate PBMCs. Wells containing cell culture medium + PBMCs and cell culture medium + PBMCs + PHA at 12·5 µg/ml were used as negative and positive controls, respectively. Cultures were harvested after 24 h for phytohaemagglutinin (PHA) and 48 h for soluble Leishmania antigen. Anti-leishmanial, IgG1 and IgG4 antibodies were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Sigma, St Louis, Missouri, Diagnostic, USA) [14,19].
Results
Thirty-six males and eight females were enrolled into the study. There was no significant age difference between the volunteers in the different study groups. All volunteers were LST non-reactive and had DAT reciprocal titres of ≤ 400.
Vaccine tolerability
The vaccine was well tolerated by the volunteers with minimal: side-effects that were confined to the site of injection and necessitated no medical treatment.
LST, DAT and Leishmania-specific IgG1, IgG4 levels
All volunteers were LST non-reactive and had a DAT reciprocal titre of ≤ 400. Eighty per cent of the volunteers converted in LST (induration >5 mm) by day 45 and LST conversion persisted into day 90 (P < 0·03) (Table 1). In the vaccine group 4 (no BCG in the vaccine mixture) the leishmanin response was slow to develop and reached significant levels only by day 90 (P < 0·03) (Table 1).
Table 1.
Leishmanin skin indurations (mm) and IFN-γ (pg/ml) levels at screening and during the post-vaccination follow-up visits.
| Vaccination groups | ||||
|---|---|---|---|---|
| Screening and follow-up days | Group 1 | Group 2 | Group 3 | Group 4 |
| Day 0 | ||||
| ″LST mm | 00 | 00 | 00 | 00 |
| ″IFN-γ picrogram/ml | ||||
| Day 45 | ||||
| ″LST mm | 9·0 ± 3·5/9* | 6·6 ± 4·1/6** | 9·6 ± 4·2/10* | 4·5 ± 4·8/5 |
| ″IFN-γ picrogram/ml | 692 ± 663/1209 | 690 ± 478/670 | 930 ± 506/1263 | 547 ± 456/410 |
| Day 90 | ||||
| ″LST mm | 9·3 ± 3·7* | 8·6 ± 2·2/9* | 10·4 ± 3·2/10* | 5·8 ± 4·8/6** |
| ″IFN-γ picrogram/ml | 501 ± 472/193 | 236 ± 419/48 | 348 ± 479/119 | 117 ± 208/31 |
Continuous variables were expressed as mean ± s.d./median.
P < 0·01;
P < 0·03.
There was no significant rise in the antileishmanial antibodies (IgG1 and IgG4) as measured by the DAT and ELISA in all study groups in all follow-up visits.
IFN-γ production and lack of concordance between IFN-γ levels and LST reactivity
Significant spontaneous IFN-γ production by PBMCs (>1000 pg/ml) was seen in two volunteers in prevaccination samples (P = 0·001), with no significant spontaneous production at days 45 and 90. Significant L. donovani antigen-induced IFN-γ production in the prevaccination sample was seen in five volunteers (mean level 1189 ± 23 pg/ml; P = 0·001), while their levels were 731 ± 668 and 816 ± 514 at days 45 and 90, respectively. On the other hand, their mean LST induration increased significantly from 00 mm at screening to 9·0 ± 5·2 mm and 11·0 ± 2·9 mm on days 45 and 90, respectively (P = 0·008) (Table 2).
Table 2.
Discordant IFN-γ levels and LST indurations in prevaccination samples.
| Group 1 | Group 2 | Group 3 | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 804 | 814 | 833 | 841 | 819 | ||||||
| Vaccination groups Volunteers’ ID Follow days | LST | IFN-γ | LST | IFN-γ | LST | IFN-γ | LST | IFN-γ | LST | IFN-γ |
| Screening | 00 | 1199 | 00 | 1200 | 00 | 1191 | 00 | 1208 | 00 | 1151 |
| Day 45 | 09 | ″00 | 13 | 1259 | 11 | ″00 | 00 | 1172 | 12 | 1224 |
| Day 90 | 07 | 693 | 12 | 1203 | 14 | 139 | 11 | 1229 | Lost to follow-up | |
On day 45, more than half the volunteers (26/44; 59·0%) had significantly high LST indurations (mean 9·2 ± 2·7 mm) and IFN-γ levels (mean 1008 ± 395; median 1247 pg/ml). Five volunteers had significant L. donovani antigen-induced IFN-γ production (mean 873 ± 290; median 902; P < 0·001), but were non-reactive in LST. By day 90, four of these volunteers had converted in LST (mean induration of 6·0 ± 4·0 mm) and their IFN-γ levels had dropped to screening levels. An additional five volunteers (5/44; 11·4%) had low IFN-γ levels (mean 110 ± 124 pg/ml; median 80) and were non-reactive in LST (induration = 00 mm). The remaining eight volunteers had low IFN-γ levels, but significant LST induration (mean 10 ± 2·9 mm; median 11) (Table 3).
Table 3.
Discordant IFN-γ levels and LST indurations on day 45 of follow-up.
| Screening | D45 | D90 | ||||
|---|---|---|---|---|---|---|
| Vol. ID. | LST | IFN-γ | LST | IFN-γ | LST | IFN-γ |
| Volunteers with high IFN-γ and non-reactive LST (n = 5) | ||||||
| ″Group 1 | ||||||
| ″821 | 00 | 26 | 00 | 902 | 08 | ″44 |
| ″841 | 00 | 1208 | 00 | 1172 | 11 | 1229 |
| ″Group 2 | ||||||
| ″810 | 00 | ″16 | 00 | 831 | 05 | ″34 |
| ″822 | 00 | ″03 | 00 | 421 | 00 | ″12 |
| ″842 | 00 | ″61 | 00 | 1051 | 06 | ″31 |
| Volunteers with low IFN-γ high LST (n = 8) | ||||||
| ″Group 1 | ||||||
| ″804 | 00 | 1199 | 09 | ″00 | 07 | 693 |
| ″828 | 00 | 478 | 12 | ″00 | 12 | 1221 |
| ″823 | 00 | 06 | 07 | ″00 | 08 | 193 |
| ″833 | 00 | 1191 | 11 | 00 | 14 | 139 |
| ″Group 2 | ||||||
| ″825 | 00 | 03 | 11 | 195 | 11 | ″05 |
| ″836 | 00 | ″24 | 12 | 102 | 09 | ″06 |
| ″Group 3 | ||||||
| ″806 | 00 | 144 | 14 | ″00 | 15 | 146 |
| ″Group 4 | ||||||
| ″805 | 00 | 147 | 07 | ″00 | 07 | ″10 |
By day 90 the majority of volunteers (27/44; 61·4%) had significant LST induration (mean 10·8 ± 9·9 mm; P < 0·001), but low levels of L. donovani antigen-induced IFN-γ (mean 66·0 ± 62 pg/ml; P > 0·05). Eleven volunteers (11/44; 25%) had significantly high levels of IFN-γ and LST induration, while five volunteers had low levels of IFN-γ (<100 pg/ml) and no LST reactivity (00). One volunteer was lost to follow-up (Table 4).
Table 4.
Discordant IFN-γ levels and LST indurations on day 90 of follow-up.
| Screening | D45 | D90 | ||||
|---|---|---|---|---|---|---|
| Vol. ID. | LST | IFN-γ | LST | IFN-γ | LST | IFN-γ |
| Volunteers with significant LST induration and low IFN-γ (n = 27) | ||||||
| ″Group 1 (n = 5) | ||||||
| ″807 | 00 | 63 | 12 | 1237 | 12 | 129 |
| ″811 | 00 | ″38 | 08 | 1296 | 10 | 165 |
| ″818 | 00 | ″18 | 09 | 1209 | 08 | 33 |
| ″823 | 00 | ″06 | 07 | ″00 | 08 | 193 |
| ″833 | 00 | 1191 | 11 | ″00 | 14 | 139 |
| ″Group 2 (n = 9) | ||||||
| ″801 | 00 | 100 | 06 | 1088 | 05 | 58 |
| ″812 | 00 | 195 | 06 | 670 | 06 | 29 |
| ″815 | 00 | ″63 | 06 | 320 | 07 | 131 |
| ″820 | 00 | ″29 | 05 | 283 | 10 | 48 |
| ″821 | 00 | ″26 | 00 | 902 | 08 | 44 |
| ″825 | 00 | ″03 | 11 | 195 | 11 | 05 |
| ″829 | 00 | 135 | 09 | 1302 | 07 | 04 |
| ″836 | 00 | ″24 | 12 | 102 | 09 | 06 |
| ″838 | 00 | ″40 | 07 | 1310 | 10 | 143 |
| ″Group 3 (n = 7) | ||||||
| ″806 | 00 | 144 | 14 | ″00 | 15 | 146 |
| ″824 | 00 | 160 | 09 | 1263 | 11 | 93 |
| ″827 | 00 | ″11 | 13 | 1301 | 10 | 171 |
| ″830 | 00 | ″35 | 05 | 248 | 10 | 42 |
| ″835 | 00 | 148 | 14 | 1272 | ||
| ″840 | 00 | ″07 | 00 | 286 | 12 | 00 |
| ″843 | 00 | 129 | 09 | 1290 | 07 | 93 |
| ″Group 4 (n = 6) | ||||||
| ″805 | 00 | 147 | 07 | ″00 | 07 | 10 |
| ″803 | 00 | ″09 | 08 | 397 | 08 | 16 |
| ″810 | 00 | ″16 | 00 | 831 | 05 | 34 |
| ″817 | 00 | 501 | 10 | 1258 | 13 | 266 |
| ″837 | 00 | 137 | 00 | ″00 | 06 | 09 |
| ″842 | 00 | 61 | 00 | 1051 | 06 | 31 |
IL-5 production
The mean IL-5 levels on days 45 and 90 were not significantly different from the screening levels (P > 0·05).
Discussion
Successful recovery from VL due to L. donovani is usually followed by development of a lifelong cell-mediated response to the disease. This response can be demonstrated in vivo as a positive LST. The exact role of LST as a surrogate marker of immunity has not been widely acknowledged, but evidence has been accumulating over the past few years to this effect. The LST measures delayed-type hypersensitivity (probably measures protection) in the skin, which is the primary portal of entry for the Leishmania parasite. The possible correlation of LST with immunity to VL has been documented previously in large vaccine efficacy studies [12,15,22].
The data provided by this study showed that IFN-γ production and the LST reactivity which follow vaccination do not develop concomitantly. IFN-γ production seems to precede LST conversion and tends to wane with time, unlike the LST induration. On day 45 more than half the volunteers had both high LST induration and IFN-γ levels. A third of the volunteers (13/44) showed discordant IFN-γ and LST results: eight volunteers showed low IFN-γ levels and high LST induration, while the remaining five had significant IFN-γ levels and non-reactive LST. By day 90, the IFN-γ levels had dropped to low levels while the LST induration persisted in 60% of volunteers. A quarter of volunteers had significantly high IFN-γ and LST induration. The rest of the volunteers had low IFN-γ levels and were LST non-reactive. This discordance between IFN-γ production and LST conversion could probably provide an explanation for the fact that some individuals in VL endemic areas in Sudan, although LST non-reactive, do not develop VL. These individuals most probably produce protective levels of IFN-γ induced by natural infection. It is likely that these individuals were exposed to low levels of the parasite antigen through bites of infected sand flies which were just enough to induce cells capable of producing IFN-γ, but not enough to induce LST skin reactivity.
Although the LST induration was not significantly different between the groups (Table 1), the mean LST induration was low in group 2 (10·8 µg) compared to groups 1 and 3 (108 µg and 320 µg). The adjuvant effect of the BCG in LST conversion was shown by the relatively low induration observed in group 4.
It is likely that there are two facets of protective immunity to leishmaniasis. The first one is mediated by IFN-γ production through less differentiated T cells or other cells such as natural killer (NK) cells. The other (LST skin conversion) is carried out by more specialized and differentiated T cells capable of induction of a varied inflammatory reaction through chemotactic mediators, leading to development of induration at the site of antigen injection. The first reaction is quicker to combat parasite at the port of entry (skin). If this concept is proved to be correct, then we need to re-evaluate our field tools for testing the immunogenicity/efficacy of candidate vaccines for VL. In the previous efficacy trials some volunteers did not convert in the LST, but also did not develop VL [12]. This could have been due to lack of exposure or, alternatively, to the IFN-γ mediated protection without the LST response.
We conclude that the cellular immune response to human leishmaniasis is dichotomatous composed of IFN-γ production that declines with time and which may or may not be accompanied by LST conversion that persists. IFN-γ production and LST skin conversion probably measure two different facets of protective cellular immunity to leishmaniasis. This phenomenon is apparent in vaccine-induced immune responses and the variation seen in individuals may be due to their genetic diversity. Further studies are required to establish the mechanisms of protection and their surrogate markers. LST conversion is a simple in vivo test that can be used to test VL candidate vaccines without the need for expensive and time-consuming field studies.
Acknowledgments
This work received financial support from the UNDP/World Bank/WHO/TDR (Project ID980791).
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