Leishmania mexicana and Leishmania major: Attenuation of Wild-Type Parasites and Vaccination with the Attenuated Lines
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MAJOR ARTICLE
Leishmania mexicana and Leishmania major:
Attenuation of Wild-Type Parasites
and Vaccination with the Attenuated Lines
Hamid Daneshvar, Graham H. Coombs, Paul Hagan, and R. Stephen Phillips
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom
Downloaded from http://jid.oxfordjournals.org/ by guest on October 12, 2015
A method for attenuation of Leishmania species by culturing in vitro under gentamicin pressure has been
used successfully with Leishmania mexicana, L. major, L. infantum, and L. donovani. The attenuated lines
invaded but were unable to survive within bone marrow–derived macrophages in vitro, whereas wild-type
parasites survived and multiplied. The attenuated lines of L. mexicana and L. major both failed to induce
cutaneous lesions in the majority of BALB/c mice over a minimum 12-week observation period after sub-
cutaneous injection of stationary phase parasites. The attenuated line of L. mexicana retained its properties
in gentamicin-free medium over 40 subcultures. The attenuated lines of L. mexicana and L. major both induced
significant protection in mice against challenge with wild-type parasites.
Leishmania species are kinetoplastid protozoa and oblig- manicidal drugs are costly, frequently have unpleasant
atory intracellular parasites and are responsible for a wide side effects, and can be relatively ineffective against cu-
spectrum of diseases, ranging from local, self-healing skin taneous leishmaniasis. In addition, drug resistance ex-
ulcers (cutaneous leishmaniasis) to visceral leishmani- ists in various regions of endemicity [4, 5]. Persons who
asis, in which infection is located in the spleen and liv- have recovered from clinical leishmaniasis develop strong
er [1]. Visceral leishmaniasis is invariably fatal if not treat- immunity to reinfection; therefore, vaccination against
ed. The diseases affect ∼12 million people, with ∼1.5–2 leishmaniasis is feasible in principle [6]. Although there
million new cases occurring annually and 350 million have been a number of vaccine trials [7], there is cur-
rently no effective and safe vaccine against any form of
people at risk [2]. The World Health Organization
leishmaniasis. It has been argued that a successful leish-
(WHO) has identified leishmaniasis as a major target for
manial vaccine is most likely to be either a vaccine com-
eradication [3].
posed of attenuated promastigotes or a subunit vaccine
There has been much interest recently in attempts to
based on the antigens with demonstrable protective func-
vaccinate against Leishmania infection, because leish-
tion [8]. Several attenuated lines of Leishmania species
have been identified [9, 10], including L. mexicana de-
ficient in cysteine proteinase genes [11] and L. major
Received 30 September 2002; accepted 16 December 2002; electronically lacking the dihydrofolate reductase/thymidylate synthe-
published 23 April 2003.
tase gene [12]. Their potential as live vaccines has been
Financial support: Iranian Ministry of Health and Medical Education (scholarship
to H.D.); Institute of Biomedical and Life Sciences, University of Glasgow; United tested in laboratory animals. To date, the subunit vaccines
Nations Development Programme/World Bank/World Health Organization Special that have been tested, such as gp63 and gp46, have given
Programme for Research and Training in Tropical Disease (grant ID-A10764).
only partial protection, and their efficacy has depended
Reprints or correspondence: Prof. R. Stephen Phillips, Division of Infection and
Immunity, Institute of Biomedical and Life Sciences, Joseph Black Bldg., University often on the use of clinically unacceptable adjuvants [13].
of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom (S.Phillips@bio.gla Gentamicin, which has frequently been added to cul-
.ac.uk).
tures of promastigotes to prevent bacterial contami-
The Journal of Infectious Diseases 2003; 187:1662–8
2003 by the Infectious Diseases Society of America. All rights reserved. nation [14, 15], is an aminoglycoside that interacts with
0022-1899/2003/18710-0018$15.00 RNA [16]. The precise mechanism of bactericidal ac-
1662 • JID 2003:187 (15 May) • Daneshvar et al.tivity of aminoglycosides is not fully understood, but some fuged at 600 g for 10 min at 4C. The pellets were subsequently
hypotheses include disruption of ribosomal activity by breaking resuspended in complete DMEM supplemented with 20% (vol/
up polysomes, misreading of mRNA during protein synthesis, vol) HI-FCS, 100 U/mL penicillin G, 100 mg/mL streptomycin
and production of abnormal or nonfunctional proteins [17]. sulfate (Life Technologies), 50 mM 2–b-mercaptoethanol (Life
In the present study, attenuated lines of L. mexicana and Technologies), 2 mM l-glutamine, 1 mM pyruvate (Sigma),
other species were developed through exposure to gentamicin and 30% (vol/vol) L-929 cell-conditioned medium [21]. The
and evaluated for their ability to be vaccine candidates. cells were transferred to a 100-mm tissue culture dish (Greiner)
and were incubated at 37C in 5% CO2 95% air for 7 days.
The supernatant was refreshed with 20 mL of complete DMEM
MATERIALS AND METHODS on day 3. After removal of the nonadherent cells, the adherent
cells were collected, transferred into the individual wells of a
Mice. Female BALB/c mice were purchased from Harlan UK
chamber slide (Nunc/Invitrogen) at 2–3 ⫻ 10 6 cells/well, and
and were maintained at Glasgow University. The mice were
incubated overnight at 37C in 5% CO2 95% air. The next day,
∼12 weeks old at the start of each experiment.
the nonadherent cells were removed, and the adherent cells
Parasites. The 4 species of Leishmania used were L. mexi-
were exposed to stationary-phase promastigotes. The culture
cana (MNYC/BZ/62/M379), L. major MRHO/SU/59/P (referred
slides then were incubated in 5% CO2 95% air for 3 h at 32C
to as “LV39” in some publications; provided by Prof. E. Y. Liew,
for L. mexicana and L. major and at 37C for L. donovani and
Downloaded from http://jid.oxfordjournals.org/ by guest on October 12, 2015
Department of Immunology, Glasgow University), L. infantum
L. infantum. The free promastigotes then were removed by
JPCM5 (MCAN/ES/98/LIM-877), and L. donovani L82 (MHOM/
replacing the overlying medium with DMEM with 10% (vol/
ET/67/L82). These were cultivated in complete HOMEM (GIB-
vol) HI-FCS, and the cells were incubated for the appropriate
CO) [18] supplemented with 10% (vol/vol) heat-inactivated fetal
times (see Results) at 32C for L. mexicana and L. major and
calf serum (HI-FCS; Labtech International), as described else-
37C for L. donovani and L. infantum in 5% CO2 95% air. The
where [19]. The attenuated lines (designated “H line”) of L.
initial infection ratio of parasites to macrophages for L. major
mexicana, L. major, L. infantum, and L. donovani were generated
and L. mexicana, and for L. donovani and L. infantum were 1:
in the same medium supplemented with 10% (vol/vol) HI-FCS
1 and 2:1, respectively. At the end of the incubation period,
and gentamicin (Sigma) at 20 mg/mL. The process was initiated
with amastigotes of L. mexicana and L. major derived from cu- the macrophages were fixed and stained with Giemsa stain, and
taneous lesions of BALB/c mouse and amastigotes of L. donovani the infection status of the macrophages was determined by
and L. infantum derived from spleens of infected hamsters. The microscopy. The percentage of infected macrophages was de-
amastigotes were transferred into HOMEM supplemented with termined by examining at least 300 cells for each line.
10% (vol/vol) HI-FCS and were incubated at 25C in air, where- Stability of L. mexicana H line in medium free of genta-
upon they differentiated to promastigotes over 72 h. These were micin. To investigate the stability of attenuated lines in gen-
transferred into complete HOMEM with or without gentamicin tamicin-free medium in vitro, mid- to late-log–phase promas-
and were incubated again at 25C. Repeat subcultures were sub- tigotes of L. mexicana H line, after 58 passages in the presence
sequently made with mid- to late-log–phase promastigotes. Cul- of gentamicin, were subpassaged in medium with or without
tures of the parasites in the absence of gentamicin were main- gentamicin. The ability of promastigotes to infect macrophages
tained in parallel to those with the antibiotic, to confirm that and their survival within macrophages were subsequently ex-
attenuation was not simply the result of long-term cultivation. amined after 37 and 40 further passages in medium without
Attenuation (in terms of survival in macrophages, see below) gentamicin.
was apparent in L. mexicana, L. major, L. infantum, and L. don- Infection of mice with L. mexicana or L. major. Stationary-
ovani after 20, 11, 11, and 10 subpassages, respectively. Atten- phase promastigotes were harvested from culture (1–2 ⫻ 107
uation of L. mexicana and L. major by this process was carried cells/mL), and 200 mL of suspension (2.5 ⫻ 107 cells/mL of PBS)
out on 4 separate occasions and for the other 2 species, to date, was injected subcutaneously into the shaven rumps of BALB/c
on a single occasion. Similar attenuated lines were produced with mice. The lesion volume was measured weekly with a capillary
each repeat. micrometer (Royal) [22].
Interaction in vitro between stationary-phase promastigotes Immunization of mice with L. mexicana H line or L. major
and bone marrow–derived macrophages. Bone marrow–de- H line and challenge with homologous wild-type parasites.
rived macrophages from a culture of bone marrow cells were BALB/c mice were injected subcutaneously with 5 ⫻ 10 6 sta-
established as described elsewhere [20]. In brief, the femurs tionary-phase promastigotes of L. mexicana H line or L. ma-
and tibias of naive BALB/c mice were flushed out through the jor H line into their right shaven rumps, and another group
cut end with 5 mL of ice-cold Dulbecco’s modified Eagle me- was injected with PBS. After 12 weeks, the immunized mice
dium (DMEM; GIBCO). The cells were collected and centri- and control group mice were challenged subcutaneously with
Antibiotic Attenuation of Leishmania • JID 2003:187 (15 May) • 1663Downloaded from http://jid.oxfordjournals.org/ by guest on October 12, 2015
Figure 1. Percentage of infected bone marrow–derived macrophages after exposure to stationary-phase promastigotes of Leishmania mexicana
(A), L. major (B), L. donovani (C), and L. infantum (D). Data are mean SE (n p 3). H line, attenuated parasites; WT, wild type.
5 ⫻ 10 6 stationary-phase promastigotes of wild-type L. mexi- same. This prompted an analysis of the effect on the promas-
cana or L. major into the left side rump. The lesion volume tigotes of culturing with the aminoglycoside. After 20 passages
was measured weekly as described above. of L. mexicana in complete HOMEM supplemented with 20
Detection of L. mexicana parasites in infected mice. Skin mg/mL of gentamicin, the parasites showed reduced virulence
at the site of the infecting inoculum, draining lymph nodes, toward macrophages and mice. The attenuated parasites were
spleen, and liver were removed aseptically into RPMI medium designated as “H line.” Subsequently, attenuated lines of L.
(GIBCO). Subsequently, single-cell suspensions from all the major, L. donovani, and L. infantum were established by cul-
tissues apart from skin were prepared in complete HOMEM. turing in the presence of 20 mg/mL of gentamicin in complete
For each mouse, the liver, lymph nodes, and spleen were ho- HOMEM for 11, 10, and 11 passages, respectively. Comparison
mogenized separately. Bone marrow cells were collected from of these attenuated lines and wild-type parasites of each species
the femurs and tibias of the infected mice. Single-cell suspen- of Leishmania was made.
sions of epidermal cells were prepared from the skin at the site Infection of bone marrow–derived macrophages. The per-
of the infecting inoculum. The skin was cut into 0.75-cm strips, centage of macrophages infected with wild-type L. mexicana
and the strips were floated dermal side down on RPMI medium and L. mexicana H line at 9 h after infection was similar (53%)
supplemented with 1% trypsin for 90 min at 37C. The sheets and, in the case of the wild-type parasite, increased to 67% at
then were shaken to release the cells, and the cells were collect- 96 h after infection (figure 1A). The percentage of cells infect-
ed by sedimentation and were washed. The number of viable ed with L. mexicana H line decreased over time, from 53% at
parasites in these organs was assessed by limiting dilution, using 9 h to 0.4% at 96 h after infection. For L. mexicana H line,
serial doubling dilutions of the cell suspensions plated into flat- the number of amastigotes within the macrophage at 9 h was
bottomed 96-well microtiter plates [23]. The plates were in- 98 amastigotes/100 macrophages but declined to 2 amastigotes/
cubated at 25C for up to 14 days and were examined daily 100 macrophages at 96 h after infection (figure 2A). The initial
for the presence of promastigotes. level of infection of macrophages with wild-type L. major was
45%, which increased to 67% at 96 h after infection (figure
1B). In contrast to infection with wild-type L. major, the 40%
RESULTS
of macrophages infected with L. major H line at 9 h decreased
Attenuation of L. mexicana, L. major, L. donovani, and L. to 10% at 96 h after infection. The number of intracellular
infantum. Routine culturing of promastigotes of L. mexicana amastigotes of L. major H line within the macrophages after 9
in the presence of gentamicin resulted in the development of h was 98 amastigotes/100 macrophages, which decreased to 10
longer promastigotes, although the growth rate remained the amastigotes/100 macrophages at 96 h after infection (figure 2B).
1664 • JID 2003:187 (15 May) • Daneshvar et al.Downloaded from http://jid.oxfordjournals.org/ by guest on October 12, 2015
Figure 2. No. of parasites within infected bone marrow–derived macrophages after exposure to stationary-phase promastigotes of Leishmania
mexicana (A), L. major (B), L. donovani (C), and L. infantum (D). Data are mean SE (n p 3). H line, attenuated parasites; WT, wild type.
Similar trends were found for both L. donovani (figures 1C and was completely healed by 12 weeks after infection. At 12 weeks,
2C) and L. infantum (figures 1D and 2D). parasites were found at the site of injection of the attenuated
Stability of L. mexicana H line in gentamicin-free me- parasites, and their dissemination was limited to the draining
dium. The stability of the gentamicin-attenuated lines was popliteal lymph node, whereas wild-type parasites could be
assessed with L. mexicana H line. L. mexicana H line promas- found in the spleen, liver, lung, draining popliteal lymph node,
tigotes were transferred into complete HOMEM with or with- and the skin where the parasites were injected. In a further
out gentamicin. After 40 further passages, stationary-phase pro- experiment, 2 groups (5 BALB/c mice/group) were injected with
mastigotes were prepared and exposed to macrophages. The L. major H line (12 passages) or wild-type L. major (14 pas-
percentage of macrophages infected with L. mexicana H line sages). Wild-type L. major induced progressive nonhealing le-
grown in medium with or without gentamicin decreased over sions over 12 weeks, whereas no lesions developed with L. major
time to 2% or 4% at 96 h after infection. In the same assay, H line over the same time period.
the percentage of macrophages infected with wild-type L. mexi- Vaccine potential of L. mexicana H line and L. major H
cana cultured for the same number of passages increased to line. Experiments were carried out to determine whether
90% at 96 h after infection. Thus, the attenuated L. mexicana pretreatment of mice with the attenuated lines of L. mexicana
H line remained attenuated, despite being cultured in the ab- or L. major could protect susceptible BALB/c mice from in-
sence of gentamicin. fection with wild-type L. mexicana or wild-type L. major. All
Infectivity of the attenuated lines of L. mexicana and L. nonvaccinated mice infected with wild-type L. mexicana de-
major to mice. The in vitro results suggested that L. mexicana veloped progressive nonhealing lesions, and the mean lesion
H line and L. major H line are unlikely to survive within murine size at 14 weeks after infection was 12600 mm3. In contrast,
macrophages in significant numbers beyond 96 h. Subcuta- the lesion developed slowly in vaccinated mice, and the mean
neous inoculation of wild-type L. mexicana or L. major into lesion size at 18 weeks after infection was only ∼500 mm3 and
BALB/c mice results in progressive lesions. To examine the thereafter declined. Another important effect of vaccination
infectivity of the attenuated line, groups of 5 BALB/c mice were with the attenuated line was that the wild-type challenge par-
injected subcutaneously with 5 ⫻ 10 6 stationary-phase L. mexi- asites were confined to the site of injection, at least up to 12
cana H line (25 passages) or wild-type L. mexicana (22 passages) weeks after challenge. Wild-type parasites given alone dissemi-
promastigotes. In contrast to the progressive nonhealing lesion nated to the internal organs, as noted above. The ability of L.
after infection with wild-type parasites, 4 mice infected with L. major H line to protect susceptible BALB/c mice against in-
mexicana H line developed no lesions during the period of fection with wild-type L. major also was examined. All non-
study (12 weeks), and 1 mouse developed a small lesion that vaccinated mice developed progressive nonhealing lesions up
Antibiotic Attenuation of Leishmania • JID 2003:187 (15 May) • 1665to 12 weeks after infection. The mean lesion size in nonvac-
cinated mice was 12000 mm3 at 12 weeks after infection. In
contrast to lesions in nonvaccinated mice, lesions developed
slowly in vaccinated mice, and the mean lesion size was !1000
mm3 at 12 weeks (figure 3B).
DISCUSSION
An effective vaccine would be a very useful tool in the control
of leishmaniasis. In some areas, persons have been deliberately
infected with virulent L. major at a site where any resulting scar
would not normally be seen [24]. In time, these persons develop
solid immunity to natural challenge but, in many cases, at the
expense of an unpleasant clinical episode. These observations
prompted the search for live vaccines composed of attenuated
or avirulent parasites [25], and preclinical data, to date, suggest
Downloaded from http://jid.oxfordjournals.org/ by guest on October 12, 2015
that this approach holds the most promise for an antileishmania
vaccine [13]. These findings are consistent with reports that at-
tenuated forms of other pathogens, such as intracellular micro-
organisms and helminths, can serve as highly effective vaccines
[26]. Avirulent or attenuated Leishmania parasites have been pro-
duced by g-irradiation [27], long-term culture [9], selection for
Figure 3. Lesion size after infection with Leishmania mexicana (A;
temperature sensitivity [28], and chemical mutagenesis and more
n p 14) or L. major (B; n p 5) in BALB/c mice vaccinated with 5 ⫻ 106
recently by disruption of virulence genes. For example, L. major stationary-phase promastigotes of L. mexicana or L. major. Lesion size was
was attenuated by targeted deletion of the metabolic gene dihy- measured weekly. Data are mean SE.
drofolate reductase/thymidylate synthetase [12]. L. mexicana has
been attenuated by removal of cysteine proteinase genes [22],
and L. donovani was attenuated by inactivation of the biopterin culture media to prevent bacterial contamination [14, 15], at-
transporter BT1 by gene disruption [29]. tenuates Leishmania organisms is unknown. Disruption of ribo-
In the present study, we report a novel method of attenuat- somal activity by breaking up of polysomes or inducing mis-
ing Leishmania species, initially working with wild-type L. mex- reading of mRNA during protein synthesis, resulting in in-
icana, by repeated in vitro subculture of promastigotes in the complete protein synthesis, are likely mechanisms [17]. The
presence of gentamicin. Subsequently, the method was shown reproducibility of the attenuation process is encouraging, al-
to be effective with 3 other species of Leishmania, namely L. though our studies have yet to pinpoint the precise point during
major, L. donovani, and L. infantum, and presumably might be the subculturing process in the presence of gentamicin when
applicable not only to other species of Leishmania but also detectable attenuation occurs.
possibly to other microorganisms. Attenuation in L. mexicana The stability of the attenuated cell lines in the absence of
and L. major was evaluated by 2 criteria: first, that promastigotes gentamicin is of major importance if these lines are to be con-
of the attenuated lines could enter but not survive in bone sidered as vaccine candidates. This was confirmed through cul-
marrow–derived macrophages, and second, that mice inocu- turing promastigotes of L. mexicana H line in antibiotic-free
lated subcutaneously with attenuated parasites did not develop medium for an extended period (40 passages) with the attenu-
skin lesions. For the visceral species, attenuation has been de- ation remaining stable. The attenuated line of L. mexicana re-
termined to date simply on the basis of failure of amastigotes mained stable in antibiotic-free medium through a test period
to survive in bone marrow–derived macrophages. In all cases, of 40 passages. The other lines have yet to be put through such
parasites were grown in parallel in culture medium with or rigorous testing, but, on the basis of these results, there is no
without gentamicin; therefore, the attenuation cannot be as- reason to suggest that the gentamicin-induced attenuation is
cribed simply to long-term culture. Wild-type parasites cultured unstable.
in the absence of gentamicin retained their infectivity to macro- The vaccine efficiency of L. mexicana H line (which appears
phages and to mice and failed to protect mice against subse- to be associated with a CD4⫹ Th1 response; author’s unpub-
quent challenge with further wild-type parasites. The mecha- lished data) was greater than that exhibited by L. major H line
nism whereby gentamicin, which was routinely added to the but was significant for both. The cross-species value of the
1666 • JID 2003:187 (15 May) • Daneshvar et al.method clearly provides promise that the approach to atten- CTD/LEISH/98.9, UNAIDS/98.23. Geneva, World Health Organiza-
tion, 1998.
uation for vaccine purposes also will be valuable with other
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