Specific cellular and humoral immune response in Balb/c mice ...

Vaccine, Vol. 16, No. 6. pp. 608-612, 1998 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X/98 $19+0.00

PII: SO264-410X(97)00228-4

Specific cellular and humoral immune response in Balb/c mice immunised with an expression genomic library of TIypanosoma cruzi Esteban Alberti*$, Armando Acostat, Maria E. Sarmientot, Hidalgot, Teresita VidalJ-, Albert0 Fachado”, Luis Fonte”, Juan F. Infantej-, Carlos M. Finlay* and Gustav0 Sierra-j-

Carlos Luis Izquierdot,

An expression genomic library of Trypanosoma cruzi (T. cruzi) constructed using pcDNA3 plasmid was used for the immunisation (25 ug) of Balblc mice. Expression of T. cruzi antigens in the muscle of inoculated mice was detected by indirect immunofluorescence 7 days after immunisation. Specific IgG antibodies were significatively increased (P ~0.05) in animals that were reimmunised with 50 ,ug of the genomic library. An antigen specific lymphoproliferative response was detected in one animal of the group inoculated with one dose of the library 0 1998 Elsevier Science Ltd. All rights reserved Keywords: 7:

Chagas’

cruzi;genomic

disease,

expression

caused

by

library;

immunisation

the

parasite protozoan to affect 20 million people in Central and South America’,‘. The disease is transmitted by triatomine ‘kissing’ bugs associated with poor housing and living conditionsthrough blood transfusion4 or at birth from infected mothers to their babies4. Despite of improvements in vector control and in blood bank screening there is no fully effective treatment”. Thus, an effective vaccine against the disease is needed”-‘. The use of inactive parasites, fractionated parasite material and irradiated non infectious 7: cruzi trypomastigotes have been shown to induce partial protection in mice against a lethal 7: cruzi challenge”.“‘. One of the most promising new techniques in vaccine development is the immunisation with nucleic acids”. Immunisation with such vaccines has been reported to protect against influenza virus in mice”, against bovine herpes virus I (BHV-1) in cattleI and against Plasmodium yoelii14 and Leishmania major” in mice. Barry and collaborators reported the immunisation of mice with genomic libraries of Mycoplasma

TYypanosomacrazi, is estimated

*Department of Protozoology, Tropical Medicine Institute Pedro Kouri, Autopista Novia del Mediodia Km.6, PO Box 601, Marianao 13, Ciudad Habana, Cuba. tDepartment of Molecular Biology, Finlay Institute. Ave 27 No. 19805, La Coronela, La Lisa AP. 16017 Cod. 11600, Ciudad Habana, Cuba. SAuthor to whom all correspondence should be addressed. (Received 21 January 1997; revised version received 26 August 1997; accepted 9 September 1997)

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pulmonis’” resulting in specific humoral and cellular immune responses as well as protection after challenge. This latter approach could constitute a powerful methodological tool for the study of the immune response to different microorganisms, in particular, in cases as 7: cruzi where protective antigens have not been found”. The use of this technology could aid in the identification of genes codifying antigens associated with protection, thus paving the way for the development of new generation vaccines. The purpose of the present study was to investigate the degree of stimulation of the specific humoral and cellular immune responses in mice immuniscd with a genomic expression library of 7: cruzi in order to develop new methods of study of the immune response against this parasite.

MATERIALS

AND METHODS

Strains Epimastigotes of the Y(CT-IOC 106) strain of 7: cruzi kindly provided by Dr Maria Auxiliadora de Sousa (FIOCRUZ, Rio de Janeiro. Brazil) and HB 101 strain of Escherichia co/i (E. cofi) (Boehringer Mannheim Biochemica) were used. Animals Thirty five Balbic female mice (6-8 weeks old) were supplied by the National Centre for the Production of Laboratory Animals (CENPALAB, Havana, Cuba), and maintained in standard conditions, were used in the experiment.

immune response

in mice immunised

Culture of the mi~roo~ar~isms T. cruzi. Epirnastigotes of the Y strain were harvested from 2-week olld cultures in Liver Infusion Tryptose (LIT) with 10% of fetal calf serum, as described by Camargo and colaborators’~. E. coli. Transformed and non transformed HB 101 strain cells were cultured in Luria Bertaini medium (LB) (1% Tryptone, 0.5% Yeast Extract and 0.5% NaCI) with and without a~mpicillin (50 jtg ml-‘) respectively. The cultures were incubated at 37°C with agitation, 200 rev. min ’ (Incubator Shaken Co., U.S.A.) Rest~ction and m~ification enzymes The enzymes BarnHI. EcoRV, Sau 3A, DNA Th and Alkaline Phosphatase (Boehringer Ligase Mannheim, Germany) were used according to the instructions of the manuf~~cturer. Soluble antigens of T.cru.d 5 x 10” epimastigotes of ‘I: cmzi were washed three times by centrifugation with phosphate buffered saline solution 0.1 M (PBS) pH 7.2 at 6OOgfor 10 min at 4°C. The pellet was resuspended in 2 ml of PBS and the cells were disrupted by sonication (Soniprep-120, MSE, U.K.) with an intensity of 18 Hz, three times (30 s each, with intervals of 60 s) on ice. The suspension obtained was centrifuged at I2OOOgfor 60 min at 4°C and the total protein content of the supernatant was determined” The resulting epimastigote antigenic solution was aliquoted and stored at -70°C until use. Construction of the I: cmlzi genomic library Genomic DNA of 7: cmzi epimastigotes was obtained as follows: 10” epimastigotes were resuspended in 1 ml of lysis buffer (100 mM Tris-HCL pH 8, 100 mM EDTA pH 8, 2% SDS, 150 mM NaCl and 200 pg ml- ’ of Proteinase K) and incubated for 2 h at 56°C. After extraction with one volume of phenol/ chloroform (l:l), the DNA was precipitated with two volumes of absolute ethanol. After centrifugation at 1OOOOgin a mini centrifuge (Costar, U.S.A.) for 15 min, the pellet was rinsed with 70% (v/v) ethanol, air-dried and then resuspended in 50 /ll of Tris-EDTA (TE) (Tris 10 mM, EDTA 1 mM pH 8). The integrity of the DNA was checked by agarose gel electrophoresis at 0.8% with ethidium bromide”‘. DNA concentration was estimated with the Gene Quant Apparatus (Pharmacia, Sweden). The genomic DNA of 7: cmzi was digested with the enzyme Sau 3A to a mean size of 2 Kb and ligated into the Barn HI site of dephosphorylated pcDNA3 plasmid (Invitrogen, U.S.A.), which carries the cytomegalovirus (CMV) major intermediate early promoter/enhancer region which drive the expression of inserted genes. E. coli Hb 101 was transformed with the library by electroporation”” (21 /IF, 2500 V, 4OOn) with the Gene Zapper 450/2500 Appal-atus (IBI, Kodak Company, U.S.A.). The library was amplified in 500 ml of LB with ampicillin and plasmid DNA was purified from the cultured cells by the alkaline lysis method”‘. The purified material was diluted in sterile NaCl 0.9% at a final concentration of 250 /lg ml-‘, aliquoted and stored at -20°C until used.

with a T. cruzi genomic

library: E. Alberti et al.

In order to check the presence of inserts of 7: cruzi DNA after the construction of the genomic library, plasmids obtained by the alkaline lysis method”’ from 20 colonies chosen at random among the E. coli transformants obtained in LB-agar 1% plates with ampicillin (50 /tg ml-‘), after the transformation with the library, were digested with the restriction enzyme EcoRV”‘. The presence of T. cmzi DNA inserts was determined by the demonstration, after agarose gel electrophoresis at 0.8% with ethidium bromide”‘, of single bands of higher sizes than linearized pcDNA3 vector or by the presence of more than one band which after summation of its sizes gave higher values than the size of the pcDNA3 plasmid. For the determination of the sizes of the DNA fragments was used the Molecular Weight Marker IV (Boehringer Mannheim, Germany) Immunisation protocol Animals were divided into 5 groups and inoculated as follows: 25 pg of the genomic library (II = 15) (Group L), 25 /lg of soluble antigens of 7: cruzi (n = 5) (Group T), 25 pg of plasmid pcDNA3 (n = 5) (Group P), 25 pg of genomic DNA of 7: cruzi (n = 5) (Group G) and an additional group of five animals that remained not immunised (Group C). Four weeks after the first inoculation, ten animals of the Group L were reimmunised with the genomic library; five of them with 25 ilg (Group L,) and the remaining animals with 50 fig (Group L,). All animals received the inoculations in a volume of 0.1 ml of sterile NaCl 0.9% by the intramuscular route in the internal region of the right thigh. Indirect immunofluorescence study One animal from groups L, T, P, G and C was sacrificed 7 days after the first immunisation and tissue sections of 4 pm of the inoculated muscle were cut by cryostat (1720 Leitz). All samples were incubated with a pool of high antibody titer human sera against 7: cruzi, obtained from patients of endemic areas (titers ranging from 1180 to f/1024 by Indirect Immuno~uorescence”) and with a pool of sera with negative results when tested by the same technique as negative control, diluted l/40 in PBS. After 30 min the slides were washed three times for 5 min with PBS and were incubated for 30 min with a goat anti-human immunoglobulin G fluorescein isothiocyanate conjugate (Sigma), diluted l/150. After three washes of 5 minutes in PBS the samples were mounted with a solution of glycerol PBS (9:l) and observed in a fluorescence microscope (Leizt, Germany) with a magnification of 25 x . ELBA Sera obtained at weekly intervals up to the fifth week after the first immunisation from the animals of all groups were studied by ELISA** for the determination of the specific IgG response to 7: cmzi antigens. Flat bottomed 96-well plates (Marxi-Sorp, Nunc, Denmark) were coated overnight with 100 ~1 per well of the solution of soluble antigens of 7: cruzi (5 pg ml-‘) in 0.05 M carbonate buffer pH 9.6. The plates were washed with 0.01 M phosphate buffer saline pH 7.2,

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Immune response in mice immunised with a T. crud genomic library: E. Alberti et al. 0.05% tween-20 (PBS-Tween) and btocked with 3% skim milk in PBS for 1 h at 37°C. The plates were incubated 2 h at room temperature with 100 611of the serum samples, diluted 11200 in PBS 1% skim milk. in duplicate. After three washes, the plates were incubated for I h at room temperature with a rabbit anti-mouse immunoglobulin G peroxidase conjugate (Sigma) at l/l000 dilution. Colour development was carried out with OPD (Sigma) and the optical density at 492 nm (OD4y2) were obtained using an ELISA Reader (Flow Laboratories). All the serum samples of each weekly extraction were studied in the same ELISA plate and each experiment was done twice. Cell proliferation assay Two animals from groups L, T, P, G and C (at that time there were not animals available of the groups Ll and L2) were sacrificed 12 weeks after the first inoculation, by cervical dislocation. Spleens were removed in sterile conditions and single cefi suspensions were prepared in RPM1 1640 (G&co) supplemented with 50 /lg ml of Gentamicin and 10% of heat inactivated foetal calf serum. Proliferative responses to soluble 7: cmzi antigens (0.1 ;lg mll’) were assayed in triplicate cultures of 100 ~1 containing 1 x 10” spleen cells. The cultures were established in 96-well round-bottomed microtiter plates (Nunc, Denmark) at 37°C in 5% CO, for 6 days. Six hours before the end of the incubation period, 1 &i of [‘HI thymidine (specific activity: 5.0 Ci mmoll’) (Amersham, U.K.) was added to each well. The cells were harvested on fiberglass paper using a cell harvester (Titertek, Denmark) and radioactivity was measured in a liquid scintillation counter (LKB, Sweden). The Stimulation Index (SI) was determined as follows: SI = counts mini’ of the stimulated cultures/counts mini of unstimulated cultures. Values of SI 2 2 were considered positive. Statistical processing For the analysis of the specific IgG response all groups were compared using the one way ANOVA

test. Tukey’s test was used to determine significant

the pairs with

differences.

RESULTS After the restriction analysis of the plasmids obtained from E. cofz’ transft~rmed with the library was demonstrated the presence of Tcruzi DNA inserts in the 70% of the plasmids analysed (data non shown), thus demonstrating that a high proportion of the plasmids in the inoculum had 7: crzlzi genetic material. Strong fluorescent staining (Figure la) was observed in the muscle of the animals immunised with the genomic library after incubation with the high titer pool of sera. In contrast, the other control groups gave negative results (Figure Ih). All the samples gave negative results after incubation with the pool of negative sera. With reference to the determination of specific IgG antibodies by ELISA, there were not statistical differences among the groups studied in the first 4 weeks after the first immunisation (data non shown). in the fifth week’s determination, specific antibody levels showed a significant (PcO.05) increase in the group reimmunised with 50 ,ug of the genomic library (Group L?) and in the group that receive soluble antigens of 7: cn4zi (Group T) compared with Groups C, G, P, L and Ll (Fignrr 2). responses were Positive lymphoprolife~tive obtained in the two animals of the group immuniscd with 7: mm antigens (Group T) (Figure 3). One of the animals of the group immunised with the genomic library (Group L) gave a positive lymphoproliferative response (SI = 2.2) (Figure 3). The SI of the animals belonging to the non immunised group (Group C) and to the groups immunised with plasmid (Group P) and with genomic DNA (Group G) were below 2 (Figure 3).

DISCUSSION expression of 7: cn4zi antigens in the muscle of animals inoculated with the genomic expression library

The

Figure 1 (a) Indirect immunofluorescence. Strong immunofluorescent signal obtained from the muscle of an animal immunised with the genomic expression library of T. cruzi (group t), 7 days after the immunisation. (b) Absence of fluorescence in the muscle of an animal immunised with pcDNA3 plasmid (group P), 7 days after immunisation. Similar results were obtained from animals of the groups C, T, G and C.

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Immune response

in mice immunised

0.35

n S.D 1

qM-n

~~

0 05 0 L

Ll

G

L2

T

C

P

Groups Figure 2 Specific IgG antibody response to soluble antigens of T. cruzi 5 weeks after the first immunisation. ELBA (mean OD@’ value; S.D.). (L) Group immLlnised with one dose (25 1’9) of the T. cruzi expression library (0.167; 0.0187); (Ll) group reimmunised (25 j(g) with the T. cruzi genomic library (0.178; 0.0099); (L2) group reinmunised (50 L(g) with the T. cruzi genomic library (0.218; 0.0135)*; (T) Group immunised (25 L(g) with soluble T. cruzi antigens (0.284; 0.0564)*; (I’) group immunised (25 ,(g) with the plasmid pcDNA3 (0.177; 0.0152); (G) group immunised (251(g) with genomic DNA of T.cruzi (0.189; 0.0286); (C) group not immunised (0.159; 0.0212;~. (*Significant statistical difference compared with groups L, Ll P, G and C.; PcO.05). N = 5 in each group.

suggests two non mutually exclusive possibilities: the processing of 7: cntzi RNA into mature messenger RNA (mRNA) by the splicing machinery of the muscular cells in a similar way as done by the parasite and/or the expression of 7: cruzi gene without introns. In this sense, the use of monoclonal antibodies or specific antiserum directed to antigens codified by genes with introns in the study by immunofluorescence of the muscle of animals immunised with the genomic library could demonstrated the expression of this kind of genes. Using an alternative approach, Northern blot studies of muscle of immunised animals could clarify this issue. The confirmation of the expression of genes with introns by the muscular cells could have a great impact in the development of DNA based vaccines against parasitic diseases because up to now the vaccination is done with cDNA”.‘~. The use of genomic DNA could

= ‘” E ‘E) .E .-6 z z ‘S m

10 a 6 4 2 -r C

P

G

-F L

T

Groups

Figure 3 Lymphoproliferative response against soluble T. cruzi antigens. (C) Group not immunised; (G) group immunised (25 L(g) with T. cruzi genomic DNA; (P) group immunised (25 j(g) with the plasmid pcDNA3; (L) group immunised with one dose (25 A(g) of the T. cruzi expression library; (T) group immunised (25 Irg) with soluble T. cruzi antigens. Each bar represents the SI value of one animal. SI values ~2 were considered positive.


library: E. Alberti et al.

simplify the technology of production of nucleic acid vaccines against parasites. The presence of autoantibodies in the sera of patients of Chagas’ disease has a key role in the immunopathogeny of the disease’,‘“. Thus, this fact should be considered in the interpretation of indirect immunofluorescence studies done with sera of Chagas’ patients as a source of primary antibodies. The fact that positive results were obtained with the muscular tissue of animals immunised with the genomic library, and not with the muscle of the control animals (Groups G, P and C) excludes the possibility of recognition of normal constituents of muscle by the pool of positive sera used in this study. It seems that the antigens expressed by muscle cells contained epitopes corresponding to ‘natural’ antigens because they were recognised by sera of patients with Chagas’ disease who had been exposed to the wild type microorganism. In fact, one of the postulated advantages of the immunisation with nucleic acids is the capability of the muscle cells to express the parasitic antigens in a native conformation”. The significant increase in the OD”’ values in the group reimmunised with 50 /lg of the genomic library (Group L2) with respect to Groups L, Ll, G and C argues in favour of the presence of specific IgG antibodies produced after the administration of the booster in the animals of this group, which was not evident with the administration of one dose of the library or a booster of 25 pg. In further experiments we have demonstrated the recognition of proteins of 7: cruzi in Western blot studies by the sera of the animals immunised with the genomic library (Alberti et al. manuscript in preparation). The positive lymphoproliferative response obtained in one of the animals immunised with one dose of the genomic library (Group L) suggests a specific stimulatory effect of the administration of the library. The different immune response observed in the two animals of this group should be interpreted with caution because many factors could influence the results obtained with this technique in animals immunised with genomic libraries, among them, the most important are the level of expression of the different genes administered and the degree of overlapping between the genes administered to each animal and the antigens of 7: cruzi present in the sonicate used for the stimulation of lymphocytes. It should be noted that a genomic library was used for the immunisation of the so each animal could have received an animals, inoculum with different genetic composition with regard to 7: cmzi DNA. In conclusion, our results demonstrated the feasibility of immunisation with a genomic library of 7: cruzi. In challenge experiments in course, using higher numbers of animals, we are studying the protective capability induced by the administration of the genomic library.

ACKNOWLEDGEMENTS We acknowledge that E. Alberti and A. Acosta made equal contributions to this work. The authors are grateful to Dr Juraj Ivanyi, Head of the TB and Related Infections Unit, MRC, London, for the critical


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Immune response in mice immunised


review of the manuscript and to Dr Xiaojiu Zhu for help in the initial phase of the work.

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