| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Immunology, University of Glasgow, Glasgow, United Kingdom; and
Molecular Biology and Immunology Division, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 x 104 clones each. Mice
immunized with one of the sublibraries (SL1A) developed an even
stronger protective effect than that induced by L1. SL1A was further
divided into 20 sublibraries (SL2) of 103 clones each.
One of the SL2 libraries (SL2G) induced a strong protective effect
against L. major infection. In direct comparative
studies, the protective effect of the sublibraries was in the order of
SL2G > SL1A > L1. Lymphoid cells from mice vaccinated with
SL2G produced more IFN-
and NO, compared with cells from control
mice injected with EV. Serum from the vaccinated mice also contained
more parasite-specific IgG2a Ab, compared with controls. Therefore,
these data demonstrate that ELI is feasible against this complex
intracellular parasitic infection, by preferentially inducing the
development of Th1 responses. Furthermore, by sequential division of
the libraries, this approach may be used to enrich and identify
protective genes for effective gene vaccination against other parasitic
infections. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A number of candidate vaccine molecules against experimental murine leishmaniasis have been identified. These include: gp63 (3), gp46 (4), p-4, p-8 (5), thiol-specific antioxidant protein (6), promastigote surface Ag-2 (7), LeIF (8, 9), and lipophosphoglycan associated proteins (10). However, all of these molecules demonstrated only partial protection; some only increase recovery from disease in resistant hosts and often require clinically unacceptable adjuvants.
Genetic vaccination has recently provided a promising new approach to vaccination, and protective responses with DNA vaccines against several pathogens have been demonstrated (reviewed in Refs. 11, 12). We have shown earlier that the highly susceptible BALB/c mice became more resistant to L. major infection following i.m. immunization with a eukaryotic expression plasmid containing cDNA encoding gp63 (13). Subsequently, it was shown that immunization with plasmid expressing the leishmania LACK gene (encoding gp24) induced a strong protective response against L. major infection (14). These data suggest the feasibility of DNA vaccination against leishmaniasis.
It is likely that effective vaccination against a complex parasitic infection such as leishmaniasis would require a multivalent vaccine containing a number of candidate genes. Identification of the protective genes may be greatly facilitated by the use of genomic expression library immunization (ELI)4. Mice immunized i.m. with a partial genomic library of Mycoplasma pulmonis developed significant protective responses against a challenge infection (15). Theoretically, once a protective pool of genomic library of a pathogen is identified, it can be subdivided into smaller pools, eventually leading to the identification of a small number of protective genes.
We have tested this hypothesis using the murine cutaneous leishmaniasis model of L. major in BALB/c mice. We report here that mice immunized with a genomic expression library of the parasite developed significant protective immunity against a challenge infection. The strength of the protective effect increased with the sequentially selected sublibraries. Protection was associated with the preferential induction of a specific Th1 cell response. Therefore, these results demonstrate, for the first time, the feasibility of genomic ELI against a eukaryotic parasite. Importantly, they show the potential application of genomic ELI in the identification and characterization of protective genes against clinical parasitic infections.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Promastigotes of L. major (LV39) were passaged and maintained in vitro, as previously described (16). All animals used were 6- to 8-wk-old female BALB/c mice obtained from Harlan Olac (Bicester, U.K.).
Genomic library construction and purification
Genomic DNA (gDNA) of L. major was isolated from
stationary-phase promastigotes as previously described
(17). gDNA was digested with MbolI to a median
size of 0.6 kb and cloned into BeglII and BamHI
site of the expression vectors, CMV-GH-F1, CMV-GH-F2, and CMV-GH-F3, in
three separate reading frames (kind gifts of Dr. S. A. Johston,
University of Texas, Dallas, TX). Escherichia coli (DH5
;
Life Technologies/BRL, Paisley, U.K.) were transformed and plated onto
Luria broth (LB) plates containing ampicillin (100 µg/ml; Sigma,
Dorset, U.K.). Several separate stocks consisting of
105 transformants for each expression vector were
suspended in 1.5 ml LB, containing 50% (v/v) glycerol and stored at
-70°C. These stocks were designated L. major library 1,
2, and 3 (L1-L3) for each of the transformants cloned into the
expression vectors CMV-GH-F1, CMV-GH-F2, and CMV-GH-F3, respectively.
Stocks were thawed, amplified overnight in 1.5 liters of LB with
ampicillin (100 µg/ml; Sigma), and plasmid DNA was purified from
pools of transformants for each library using Qiagen (Sussex, U.K.)
kit, according to the manufacturer’s specifications. Subsequent
partitioning of the L. major L1 into smaller groups of
clones (sublibraries) was performed by thawing a stock vial, plating
the transformants overnight on LB plates containing ampicillin, and
resuspending the desired number of transformants in 1.5 ml LB,
containing 50% (v/v) glycerol, followed by storage at -70°C.
Subsequent plasmid DNA isolation was performed as described above. If
one of these sublibraries was protective following vaccination of mice,
this stock was again plated on LB plates containing ampicillin, and the
same procedure as described above followed.
Immunization and challenge infection
Mice were injected i.m. at two sites in the thigh skeletal muscle with 100 µg of plasmid DNA suspended in 100 µl of saline. In some experiments, mice were injected in their hind footpads with 100 µg of plasmid DNA suspended in 50 µl of saline. Mice were boosted one to three times at 2-wk intervals. Then, 2 wk after the final boost, groups of mice were either sacrificed for immunological assays or infected in one hind footpad with 105 stationary-phase promastigotes of L. major. Lesion development was measured at regular intervals with a constant-pressure dial caliper (Kroeplin, Munich, Germany) and expressed as the footpad thickness increase of the infected hind foot, compared with the uninfected hind foot. At the end of experiments, mice were sacrificed and infected footpads removed. The number of parasites present in infected footpads were quantified using a limiting dilution method previously described (18). Draining lymph nodes and spleens were harvested for in vitro proliferation and cytokine production.
Cell proliferation and cytokine production
Single cell suspensions were obtained by gentle homogenization.
Cells were washed twice with RPMI 1640 (Life Technologies/BRL) and
resuspended in RPMI at 5 x 106 cells/ml
supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml
penicillin, 100 µg/ml streptomycin (all from Life Technologies/BRL),
and 50 µM 2-ME (Sigma). Cells were stimulated with either 2 µg/ml
anti-CD3 Ab (PharMingen, Kent, U.K.) or L. major Ags
(18) for up to 96 h. Proliferation assays were
performed in triplicates in 96-well culture plates (Nunc, Roskilde,
Denmark), with addition of 1 µCi of
[3H]thymidine (Amersham Life Sciences, Bucks,
U.K.) in 25 µl during the final 16 h of culture before
harvesting onto a glass-fiber filter (Packard, Wallac, Milton Keynes,
U.K.) using a Micromate 196 Harvester (Packard).
[3H]thymidine incorporation was measured using
a Matrix 96 direct 
counter (Packard). In some experiments,
duplicate cultures were performed for up to 96 h and supernatants
collected and stored at -70°C until cytokines in the culture
supernatant were determined by ELISA using paired Ab (PharMingen), as
previously described (19). The lower limit of
detection of the assays was as follows: murine IFN-, 10 pg/ml;
murine IL-4, 40 pg/ml.
Measurement of delayed-type hypersensitivity (DTH), NO, and Ab
Groups of mice were injected in the right hind footpad with 50 µl of PBS containing killed L. major Ags (equivalent to 106 promastigotes). Footpad swelling was measured at regular intervals for up to 72 h with a constant-pressure dial caliper (Kroeplin). Ab against Leishmania was measured by an ELISA method using 96-well plates coated with soluble L. major Ags, as previously described (20). Rabbit anti-mouse IgG1 and IgG2a Abs were obtained from PharMingen. Nitrite was measured by the Greiss method (21).
Statistical analysis.
Statistical significance (p < 0.05) of differences between treatment groups was determined by a one-way ANOVA and the Student’s t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To capture the majority of the parasite Ags, gDNA of the entire
L. major promastigote was digested and cloned into three
different frames of the expression vector, CMV-GH-F1, CMV-GH-F2, and
CMV-GH-F3 (15), and three expression libraries (L1, L2,
and L3) were prepared, each containing 105
clones. Groups of BALB/c mice were immunized i.m. in the thigh with 100
µg plasmid DNA of L1, L2, L3, or a combination of the three (mixed).
Control mice were injected with the empty vector (EV) or PBS. The
vaccination was repeated three times at 2-wk intervals and challenged
in the footpad with 105 stationary phase L.
major promastigotes 2 wk after the last immunization. Lesion
development was measured at regular intervals. Mice vaccinated with L1
developed smaller lesions than the controls injected with EV (Fig. 1
A). Mice immunized with L2,
L3, or a mixture of the three libraries developed lesions
indistinguishable from mice injected with EV (Fig. 1, B–D).
No difference in the lesion development was detected between mice
vaccinated with EV or PBS alone (data not shown), showing that the
plasmid alone containing bacterial DNA did not induce a significant
degree of protection. Additional experiments showed that similar
results were obtained when mice were given two instead of three
immunizations (data not shown).
|
To enrich the protective genes, L1 was partitioned into five
sublibraries (SL1A-SL1E) containing 2 x
104 clones each. Groups of mice were then
vaccinated with the purified plasmids from each of the sibling
libraries, and one of the sibling libraries (SL1A) showed a further
decrease in footpad lesion development following a challenge infection,
compared with mice vaccinated with L1 (Fig. 2A). By day 50 postinfection,
the lesions in the group injected with EV began to ulcerate, and the
experiments were terminated as required by the guidelines of the animal
experimentation of the Home Office, U.K. At this stage, mice vaccinated
with L1 or SL1A had a mean footpad parasite load 1.5 and 2.5
log10 lower than that of the control group
immunized with EV, respectively (Fig. 2B).
|
103 clones. Groups of mice
were then vaccinated with purified plasmid from each of the SL2 groups
and their ability to induce protective immunity examined. Control mice
were vaccinated with SL1A or EV. Of the 20 groups of mice vaccinated
with the SL2 libraries, 4 had lesions similar to those of control
EV-injected mice, 15 had lesions not significantly different from those
vaccinated with SL1A, and 1 (SL2G) showed a further decrease in lesion
development compared with the SL1A-vaccinated mice (Fig. 2
C). Mice vaccinated with SL1A or SL2G also contained 3.5
and 6 log10 lower parasite loads compared with
mice vaccinated with EV, respectively (Fig. 2D). The
protective effect is systemic and not localized, since mice immunized
and boosted in the right foot and challenged in the right or the
contralateral left footpad exhibited similar levels of protection (data
not shown). Furthermore, similar results were obtained whether SL2G was
delivered i.m. in the thigh or injected s.c. in the footpad (data not
shown). Immunological responses induced by expression library immunization
Anti-CD3 activation.
Mice vaccinated with SL1A or SL2G were sacrificed 2 wk after the third
immunization, and draining lymph node cells were collected and cultured
with immobilized anti-CD3 Ab (polyclonal T cell activation) in
vitro. Cells from mice immunized with SL2G produced significantly
stronger proliferation and secreted more IFN- than cells from mice
immunized with SL1A, which, in turn, showed a stronger proliferative
response and produced more IFN- than cells from the control mice
injected with EV (Fig. 3, A
and B). In contrast, cells from all three groups of mice
produced low and indistinguishable levels of IL-4. No proliferative
response or cytokine production was detected when cells were cultured
with leishmanial Ags, presumably the level of specific T cell response
induced by the libraries before a challenge infection was too low to be
detected by the in vitro assays.
|
, IL-4, and nitrite
concentrations. Cells from mice immunized with EV produced minimum
amounts of the cytokines or NO. In contrast, cells from mice immunized
with SL1A produced significant amounts of IFN- and NO, which were
markedly enhanced in the cultures of cells from mice immunized with
SL2G (Fig. 4, A and
B). All cultures produced modest amounts of IL-4. Similar
results were obtained with spleen cells (data not shown). Thus, mice
immunized with plasmid DNA of SL2G developed significant Th1 responses
and produced substantial amounts of NO.
|
A).
Similarly, cells from mice immunized with SL2G produced significantly
stronger specific T cell proliferation, compared with cells from mice
injected with EV (Fig. 5B).
|
). In contrast,
specific IgG1 Ab was not detectable before infection and was
indistinguishable between the two groups of mice following infection.
This is consistent with the results obtained above that SL2G
preferentially induced a Th1 response.
|
To determine whether genomic ELI induced long-term pathology, groups of mice were immunized and boosted i.m. with 100 µg of L1, EV, or PBS and were observed over a 2-yr period. All of the mice lived normally and showed no sign of ill health. Histological examinations of tissue sections at the sites of vaccination over this period showed no sign of pathology, compared with control mice injected with PBS (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and
IgG2a production and NO synthesis. We have demonstrated earlier that vaccination with plasmid encoding gp63 also preferentially induced a Th1 response (13). Others have shown that immunization with leishmanial DNA encoding LACK induced a protective Th1 response (14), whereas injection with the LACK protein promoted the disease-exacerbating Th2 response against L. major infection (23). Thus, DNA vaccination holds considerable promise for vaccination against diseases in which Th1 responses are desirable. However, the route of vaccination appears to be of critical importance. In early experiments, we compared s.c. and i.m. immunization with that of subdermal immunization using the gene gun method (24). We found that mice immunized and boosted with 100 µg of L1 injected s.c. or i.m. consistently showed significantly improved resistance against L. major infection, whereas, mice immunized and boosted with 20 ng of L1 delivered by the gene gun did not (data not shown). This is consistent with an earlier report that DNA delivered by the gene gun preferentially induced Th2 responses (25). It is noteworthy that i.m. immunization induced systemic immunity because vaccination was equally protective whether the challenge infection was administered in the same footpad as the DNA injection or at the contralateral foot. While there is no significant difference whether two or three immunizations were administered, immunization with one injection alone was insufficient to induce a detectable degree of protection (data not shown). The increased protective effect and Th1 responses when vaccinated with the successive smaller libraries could represent the enrichment of the protective genes/gene fragments. It is of interest to note that while some of the sibling libraries were not protective, none of them was diseases exacerbative. Within the limitation of the assay used, this argues against the presence of a dominant disease-promoting gene in the system used here.
The fact that there was no significant difference in disease development in mice injected with EV or PBS also argues against a protective effect of the bacterial CpG motif in the expression plasmid in our system. However, it should be noted that the effect of CpG tends to be short-lived and requires the simultaneous administration of CpG with the immunizing Ags (26). In our system, mice were challenged 14 days after the last boosting, by which time any effect of CpG would have waned. In addition, of the three expression libraries (L1-L3), which were cloned into three different frames of the expression vector, only L1 showed any detectable protective effect. This result also supports the leishmanial-specific nature of the immunization and argues against the notion that the protective effect was mediated by nonspecific sequences of leishmanial oligonucleotides. The use of a genomic library, rather than a cDNA, library has the advantage of avoiding the selection of stage-specific genes or genes with a high level of expression. Thus, the protective genes enriched, and ultimately identified, could be effective against the promastigote and the amastigote stages of the parasite. Furthermore, the use of the same strong promoter (CMV) would ensure the equal expression of the cloned genes, whatever the levels of their expression in the parasite.
The genome size of Leishmania spp. has been estimated to be
5 x 107 bp with 25% of the gDNA made up
of various noncoding forms of repetitive sequences (27).
However, due to the absence of introns in the parasite genome, the
construction of the initial library L1 is likely to represent most of
the coding sequences of L. major. In addition, it is likely
that the protective clones identified in this study represent
protective epitopes rather than full-length proteins, as the 600-bp
sequences cloned into the expression vectors would more often represent
gene fragments rather than full-length genes of L. major.
While sterile immunity has not been achieved so far, the incremental
protective effect with successive subdivision of the libraries into
smaller pools of clones is encouraging. The degree of protection
achieved so far, determined by lesion development and parasite load, is
at least comparable to those attained with single protein Ags
(3, 4, 5, 6, 7, 8, 9), the whole soluble leishmanial Ags (data not
shown), or gp63 DNA. Thus, the method described here has the potential
of enriching and identifying novel protective genes for effective
vaccination against leishmaniasis. A full characterization of the
protective immune responses will be reported with the identification of
the protective genes.
Finally, the vaccination schedule appears not to induce any sign of clinical abnormality or local pathology over a 24-mo period studied. This, together with the potential of identifying and characterizing a small number of protective genes, places the process of sequential selection of protective libraries described in this report in a promising position in developing an effective DNA vaccine against leishmaniasis. The general applicability of this approach to other complex parasitic infections remains to be explored.
|
Footnotes |
|---|
2 D.P. and D.X. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. F. W. Liew, Department of Immunology, University of Glasgow, Western Infirmary, Glasgow G11 6NT, U.K. E-mail address:
4 Abbreviations used in this paper: ELI, expression library immunization; gDNA, genomic DNA; EV, empty vector; DTH, delayed-type hypersensitivity; L, library; SL, sublibrary.
Received for publication March 2, 1999. Accepted for publication May 19, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-activated human monocytes: involvement of the nitric oxide synthase pathway. J. Biol. Chem. 269:9811.This article has been cited by other articles:
|
|
 |
|
  A. M. Talaat and K. Stemke-Hale Expression Library Immunization: a Road Map for Discovery of Vaccines against Infectious Diseases Infect. Immun., November 1, 2005; 73(11): 7089 - 7098. [Full Text] [PDF]
|
|
|
|
|
|
T. Scorza, K. Grubb, P. Smooker, A. Rainczuk, D. Proll, and T. W. Spithill Induction of Strain-Transcending Immunity against Plasmodium chabaudi adami Malaria with a Multiepitope DNA Vaccine Infect. Immun., May 1, 2005; 73(5): 2974 - 2985. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Liu, B. Hu, D. Xu, and F. Y. Liew CD4+CD25+ Regulatory T Cells Cure Murine Colitis: The Role of IL-10, TGF-{beta}, and CTLA4 J. Immunol., November 15, 2003; 171(10): 5012 - 5017. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Fachado, A. Rodriguez, J. Molina, J. C. Silverio, A. P. M. P. Marino, L. M. O. Pinto, S. O. Angel, J. F. Infante, Y. Traub-Cseko, R. R. Amendoeira, et al. Long-Term Protective Immune Response Elicited by Vaccination with an Expression Genomic Library of Toxoplasma gondii Infect. Immun., September 1, 2003; 71(9): 5407 - 5411. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Rainczuk, T. Scorza, P. M. Smooker, and T. W. Spithill Induction of Specific T-Cell Responses, Opsonizing Antibodies, and Protection against Plasmodium chabaudi adami Infection in Mice Vaccinated with Genomic Expression Libraries Expressed in Targeted and Secretory DNA Vectors Infect. Immun., August 1, 2003; 71(8): 4506 - 4515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Xu, H. Liu, M. Komai-Koma, C. Campbell, C. McSharry, J. Alexander, and F. Y. Liew CD4+CD25+ Regulatory T Cells Suppress Differentiation and Functions of Th1 and Th2 Cells, Leishmania major Infection, and Colitis in Mice J. Immunol., January 1, 2003; 170(1): 394 - 399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Garg and R. L. Tarleton Genetic Immunization Elicits Antigen-Specific Protective Immune Responses and Decreases Disease Severity in Trypanosoma cruzi Infection Infect. Immun., October 1, 2002; 70(10): 5547 - 5555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. N. Coler, Y. A. W. Skeiky, K. Bernards, K. Greeson, D. Carter, C. D. Cornellison, F. Modabber, A. Campos-Neto, and S. G. Reed Immunization with a Polyprotein Vaccine Consisting of the T-Cell Antigens Thiol-Specific Antioxidant, Leishmania major Stress-Inducible Protein 1, and Leishmania Elongation Initiation Factor Protects against Leishmaniasis Infect. Immun., August 1, 2002; 70(8): 4215 - 4225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Campos-Neto, J. R. Webb, K. Greeson, R. N. Coler, Y. A. W. Skeiky, and S. G. Reed Vaccination with Plasmid DNA Encoding TSA/LmSTI1 Leishmanial Fusion Proteins Confers Protection against Leishmania major Infection in Susceptible BALB/c Mice Infect. Immun., June 1, 2002; 70(6): 2828 - 2836. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. C. Melby, J. Yang, W. Zhao, L. E. Perez, and J. Cheng Leishmania donovani p36(LACK) DNA Vaccine Is Highly Immunogenic but Not Protective against Experimental Visceral Leishmaniasis Infect. Immun., August 1, 2001; 69(8): 4719 - 4725. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. C. Melby, G. B. Ogden, H. A. Flores, W. Zhao, C. Geldmacher, N. M. Biediger, S. K. Ahuja, J. Uranga, and M. Melendez Identification of Vaccine Candidates for Experimental Visceral Leishmaniasis by Immunization with Sequential Fractions of a cDNA Expression Library Infect. Immun., October 1, 2000; 68(10): 5595 - 5602. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
