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Immunization of guinea pigs against entamoebahistolytica using glucan as an adjuvant
Int. J. lmmunopharmac., Vol. 6, No. 5, pp. 483-491, 1984. Printed in Great Britain.
0192-0561f84 $3.00+ .00 (c~ 1984 International Society for Immunopharmaco|ogy
I M M U N I Z A T I O N OF G U I N E A PIGS A G A I N S T E N T A M O E B A H I S T O L Y T I C A U S I N G G L U C A N AS AN A D J U V A N T * ALPANA SHARMA, AFROZ UL HAQ, M. U. SIDDIQU1 and SOHAIL AHMAD Parasitic Immunology Laboratory, Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh 202001, India (Received 29 August 1983 and in final form 22 March 1984)
Abstract - - Beta 1- 3 polyglucose or glucan, an extract of cell wall of Saccharomyces cerevisiae, has been successfully employed in this laboratory as an effective immunopotentiator in experimental studies on amoebiasis. An antigen extract from Entamoeba histolytica was combined with/3, 1- 3 glucan for immunizing guinea pigs. In order to study the effectiveness of such vaccine preparations, several batches of guinea pigs were immunized with amoeba antigen alone, and in combination with various immunoadjuvants. Antigen inoculations were carried out via intraperitoneal route. Protective immune responses were obtained against amoeba antigen by using glucan as an adjuvant partner. The study showed that glucan can be safely used as an effective immune enhancer.
All parasite vaccines, purified or synthetic, are likely to prove weak immunogens needing some further immunopotentiation. Currently, the most effective way to develop marginal vaccines is to combine them with potent and safe adjuvants. For accomplishing an immunologic control of parasitic diseases, adjuvants with less toxicity must be found for clinical and veterinary applications. The objective of this paper is to briefly summarise the vaccination studies conducted against E. histolytica infection in a guinea pig model system. Immunizations were carried out with amoeba antigen in combination with/3, 1 - 3 glucan. These results were compared with similar immunizations carried out by using other immunoadjuvants. Particulate /3, 1 - 3 glucan was chosen because of its ability to act on several strategic parameters of humoral and cellular immunity (Di Luzio, Pisano & Saba, 1970; Browder, McNamee, Cohen, Taylor & Di Luzio, 1976; Di Luzio, 1976). It has been earlier shown to act as a potent activator of reticuloendothelial (RE) system (Di Luzio et al., 1970) and complement (Di Luzio, 1976). Glucan of fungal origin has been described in the literature as a promising candidate adjuvant for the prevention, or therapy, of experimental leprosy (Delville & Jacques, 1980), candidiasis (Williams, Cook, Hoffmann & Di
Luzio, 1978), leishmaniasis (Cook, Holbrook & Parker, 1980; Cook, Holbrook & Dougherty, 1982), sporotrichosis (Stevens, Stevens, Cook, Ichinose & Di Luzio, 1976), malaria (Gillet, Jacques & Herman, 1978), toxoplasmosis (Nguyen & Stadtsbaeder, 1980) and numerous other infectious diseases (Reynolds, Kastello, Harrington, Crabbs, Peters, Jemski, Scott & Di Luzio, 1980). Samples of/3, 1 - 3 glucan used in these investigations were kindly supplied by Professor P. J. Jacques (Centre Inter-universitaire de Recherches Multidisciplinaires en Affections Parasitaires et Mecanismes de Defense de l'Hote, Brussels, Belgium).
EXPERIMENTAL PROCEDURES
Antigen Axenic cultures of E. histolytica (Strain; NIH-200) were raised in Diamond's TP-S-1-monophasic medium (Diamond, 1968). Axenically cultivated amoebae were pooled and washed in normal saline. Amoebae were sedimented by centrifugation at 850 g for 5 min and antigen extraction accomplished according to the method of Kessel, Lewis, Pasquel & Turner (1965). Protein concentration was determined by the method
* Preliminary results of the above study were briefly published by us in IRCS Medical Sciences 8, 526 (1980). Further results of this study were presented in a symposium lecture given by one of us (SA) at lind InternationalCongress of Immunopharmacology held in 5 - 9 July, 1982 at Washington, DC. Abstract: International Journal of Immunopharmacology 4, 271 (1982). 483
484
ALPANA SHARMA,AFROZUL HAQ, M. U. SIDDIQUI and SOHAILAHMAD
of Lowry, Rosebrough, Farr & Randall (1951) at 700 nm, using Bausch & L o m b Spectronic 21 Spectrophotometer.
Immunization Eighty guinea pigs, each weighing 300 - 400 g, were equally divided into eight experimental groups. Each animal was immunized intraperitoneally with a total amount of 200/ag antigen protein given in three doses. The details o f the immunization schedule are shown in Table 1. Animals in groups I - III were inoculated with amoeba antigen given in combination with various adjuvants, while group IV animals received amoeba antigen alone. Animals in other control groups ( V - V I I ) were inoculated with fl, 1-3 glucan or F r e u n d ' s complete adjuvant (FCA, Difco), or aluminium hydroxide adjuvant (AIA, 2070 w/v). Group VIII animals received saline only. The glucan was administered at the rate of 40 m g / k g body weight. Immunization was completed over a period of four weeks including a week of rest (3rd week).
Detection o f immune responses Antiamoebic antibodies were detected in sequential serum samples obtained from blood drawn at weekly intervals. Antibody detections were also carried out in samples obtained as pre and post immunized serum. Hemagglutinating antibody titers were determined by the modified technique of Krupp (1970). A 2.5°70 suspension of sheep red blood cells (SRBC) was tanned for 20 rain with tannic acid (1:100,000) in an ice bath at 4°C. The tanned cells were sensitized with antigen at pH 6.4 in a water bath at 37°C for 20 min. The test was performed in microtiter plates with U shaped bottoms. About fifteen serum samples obtained from confirmed human cases o f amoebic dysentery, or normal subjects, were used as reference sera for positive and negative controls, respectively. A serum was considered positive if its antibody titer was 1:64 or higher.
Skin reaction Following immunizations, each animal was skin
Table l. Immunization schedule Animal groups* I.
II.
III.
IV. V. VI. VII. VIII.
Weekly immunization doses (ml) I st 2nd 4th
Amoeba antigen + glucan Antigen Glucan
0.05 0.40
0.05 0.40
0.10 0.40
Amoeba antigen + FCA Antigen FCA
0.05 0.05
0.05 0.05
0.10 0. I 0
Amoeba antigen + AIA Antigen AIA
0.05 0.05
0.05 0.05
0.10 0.10
Amoeba antigen only Antigen
0.05
0.05
0.10
Glucan only Glucan
0.40
0.40
0.40
FCA only FCA
0.05
0.05
0.10
AIA only AIA
0.05
0.05
0.10
Saline only Saline
0.50
0.50
0.50
* Each animal group contained ten guinea pigs. 3rd week was treated as rest period, Glucan concentration 30 mg/ml. Amoeba antigen concentration 1 mg/ml. FCA: Freund's complete adjuvant. AIA: Aluminium hydroxide adjuvant (2o70 w/v). Total inoculum in each injection was made upto 0.5 ml with the addition of saline.
Negative
Negative
64_+37 (32- 128)
64_+26 (32 - 128)
128+52 ( 6 4 - 256)
1st
2nd
Negative
64_+37 (32 - 128)
128_+52 (63 -256)
256_+156 (128 - 512)
512_+209 (256- 1024)
* The given IHA titers are arithmetic mean of ten experiments _+ S.D. t Each group contained ten guinea pigs. The values given in parentheses indicate the range of IHA titers. FCA: Freund's complete adjuvant. A1A: Aluminium hydroxide adjuvant (2% w/v). An IHA titer less than 64 was taken as negative.
V, VI, VII and VIII Glucan, FCA, A1A and saline (controls)
IV (Antigen)
III (Antigen + AIA)
II (Antigen + FCA)
(Antigen + glucan)
I
Animal groupst
Negative
128_+74 (64 - 256)
256_+104 (128 -512)
512_+209 (256 - 1024)
4th
5th
6th
Negative
512_+209 (256 - 1024)
1024_+418 (512-2048)
Negative
512+_295 (256 - 1024)
1024_+591 (512- 2 0 4 8 )
2048_+1182 2048±836 (1024- 4096) (1024- 4096)
Negative
128_+52 (64 - 256)
512_+209 (256- 1024)
512_+295 (256 - 1024)
16384+_6688 16384_+6688 8192_+3344 (8192- 32768) (8192- 32768) (4096- 16384)
Weeks
2048+836 (1024- 4096)
3rd
Table 2. Reciprocal IHA titers in weekly sera samples* O
g
e<
>
r~
tm
> q~
O
486
ALPANA SHARMA, AFROZ UL HAQ, M. U. SIDDIQU! and SOHAIL AHMAD
sensitized by inoculating 12 /ag antigen protein intradermally. The reaction intensity was determined by measuring the diameter and thickness of erythema and induration at the skin test site at 48 h, following antigen inoculation. Skin tissue from the reaction site was excised for histological examination. Skin sections cut at 5 ta were stained with iron hematoxylin and eosin (H & E) and examined under microscope.
Challenging the animals All the test and control animals were subsequently challenged. The challenging dose containing 4 × 106 amoebae in 0.5 ml volume was inoculated intrahepatically, or intracecally. The amoebae used for challenging the immunized animals were of proven pathogenicity. They were isolated from a patient of amoebic liver abscess and maintained in laboratory cultures. The animals were sacrificed on post challenge day 14 for making gross observations of the viscera of the inoculated animals. The infected tissues from liver and cecum were later removed and fixed in formalin. The excised tissues from the two inoculated sites were sectioned at 4 - 5/a and fixed on the slides. The dehydrated sections were stained in H & E and made into permanent mounts. The stained slides were used for histopathological examination.
RESULTS
Indirect hemagglutination (IHA) test The IHA titers of weekly serum samples were recorded. Table 2 shows the reciprocal IHA titers from various experimental groups. Erythrocytes sensitized with different antigen dilutions were first tested against reference serum of known antibody activity, and then against the test serum samples. Highest antibody titers in the experimental groups I - I V were recorded in the fourth week. The IHA titers from the animals immunized with amoeba antigen-glucan combination showed a typical antibody response, yielding a peak titer of 1:16,384. Skin test All the guinea pigs in groups I - IV developed delayed type skin hypersensitivity reactions. A positive skin reaction of a diameter greater than 5 mm was characterized by a zone of erythema and induration. These reactions were found maximum at around 48 h, subsiding thereafter. Figure 1 shows the appearance of a typical delayed type skin reaction at the site of antigen inoculation in antigen-glucan immunized
animals. A challenge dose containing 12 ~g antigen protein was found adequate to elicit such delayed type skin hypersensitivity reactions. The skin reactions appearing in the control animals were of very small size (diameter less than 5 ram) and were recorded as negative. Skin test results are shown in Table 3. Histological examination of the skin reaction site showed characteristic perivascular cuffing with prominent cell infiltration. The intercellular spaces were found filled with mononuclear cells, especially the lymphocytes and a few macrophages. A prominent intercellular cell infiltration can be seen in the photomicrograph from a skin reaction site (Fig. 2).
Lesion formation Experimental animals treated with glucan alone, or in combination with amoeba antigen did not show any skin lesions at the inoculation site. The animals receiving FCA or aluminium hydroxide adjuvant developed skin lesions at the site of inoculation.
Protection studies All glucan immunized animals throughout the entire post challenge period appeared completely normal, showing no sign of disease whatsoever. The gross examination of their viscera did not reveal any pathology. Liver and cecum of these animals were devoid of any macroscopic lesions and appeared completely normal. While in the unimmunized controls, similar organs not only showed lesions but also appeared inflammed and oedematous. The unprotected animals in various control groups developed frank diarrhoea, preceded with lassitude, listlessness and significant loss of appetite. Histopathological examination of the liver and cecum from control animals revealed ulcerative amoebic lesions accompanied with necrosis and considerable tissue damage. Tissue sections from liver showed a large number of small, locally decolourised raised areas. The hepatic cells gave a completely glassy appearance. Tissue histology from immunized animals did not reveal any such lesions, or other signs of an accompanying pathology. In fact, the animals immunopotentiated with glucan exhibited a qualitative difference in their protective response as revealed by a marked inflammatory reaction in the challenged tissues (Figs. 3a and b). The liver section showed prominent mononuclear cell infiltration around hepatic cells. Table 4 shows further results of this protection study. The results obtained in each test group were uniformly reproducible in repeated experiments.
Immunization of Guinea Pigs Against Entamoeba Histolytica Using Glucan as an Adjuvant
Fig. 1. Guinea pig showing positive delayed type skin hypersensitivity reaction.
Fig. 2. A skin section showing cellular infiltration at the reaction site.
487
488
ALPANA SHARMA, AFROZ UL HAQ, M. U. SIDDIQUI and SOHAIL AHMAD
Fig. 3. Photomicrographs showing tissue sections of protected animals immunized with a n t i g e n - g l u c a n combination. (a) Section of liver. (b) Section of cecum. Perivascular mononuclear cell infiltration areas are marked.
Immunization of Guinea Pigs Against Entarnoeba Histolytica Using Glucan as an Adjuvant
Table 3. Skin test results Total antigen inoculated/animal (ug)
Skin reaction diametert (mm)
Induration thicknesst (mm)
(Antigen + glucan)
200
15.50_+2.20
4.45_+1.85
I1 (Antigen + FCA)
200
14.00_+2.05
4.25_+1.60
111 (Antigen + AIA)
200
ll.50_+3.18
3.15_+0.62
IV (Antigen)
200
8.50_+1.18
2.75_+0.20
--
Negative
Negative
Animal groups* I
V, VI, VII and VIII Glucan, FCA, AIA and saline (controls)
* Each group contained ten guinea pigs. t Arithmetic mean of ten experiments -2_ S.D. FCA: Freund's complete adjuvant. AlA: Aluminium hydroxide adjuvant (2070 w/v). Skin reaction diameter and induration thickness of less than 5 and 2 mm respectively were taken as negative.
Table 4. Protection in experimental animals Animals showing disease after challenge
°7o protection
(Antigen + glucan)
0/10
100
II (Antigen + FCA)
3/10
70
III (Antigen + AIA)
6/10
40
IV (Antigen)
6/10
40
V (Glucan)
8/10
20
VI (FCA)
9/10
10
10/10
Zero
Animal groups* I
VII and VIII (AIA) and (saline) * Each group contained ten guinea pigs. FCA: Freund's complete adjuvant. A1A: Aluminium hydroxide adjuvant (207o w/v).
489
490
ALPANA SHARMA, AFROZ UL HAQ, M. U. SIDDIQUI and SOHAILAHMAD DISCUSSION
Results of this study provide ample p r o o f that guinea pigs are able to generate a protective immune response, following amoeba-glucan vaccination. The protective response comprised of a strong cell mediated immunity along with raised levels of circulating antibodies. Such highly protective responses were obtainable only in those animals which were immunized with glucan as an adjuvant partner. Animals receiving other adjuvants, or immunized with amoeba antigen alone, did not appear to elicit a protective response of a similar order. The action of glucan as an effective adjuvant is well documented in the literature. It has been shown to enhance the proliferation and stimulation activity, mainly of the reticuloendothelial cells (Wooles & Di Luzio, 1963; Wooles & Di Luzio, 1964; Morrow & Di Luzio, 1965; Di Luzio et al., 1970; Di Luzio & Morrow, 1971; Di Luzio, 1976). Use of glucan as a powerful adjuvant has met with considerable success in certain earlier studies. For example, it has been shown to enhance the immunity against rodent malaria (Holbrook, Cook & Parker, 1981). These authors have further shown the usefulness of glucan as an adjuvant in their studies on murine babesiosis (Benach, Habicht, H o l b r o o k & Cook, 1982). More recently, glucan has been shown to exert a protective effect in hamsters against visceral leishmaniasis (Cook et al., 1982). On the basis of information so far available, it seems like glucan is an ideally suited adjuvant for use with parasite antigens. The optimum dose recommended for administering glucan as an adjuvant is around
40 m g / k g body weight. Patchen & Mac Vittie (1982) in their extensive studies have described that glucan administration up to 100 m g / k g acts as a stimulator of RE system in a dose dependent manner. The F C A treated animals were found more predisposed to skin lesions, while glucan treated animals did not show any such tendency for lesion formation. There are some earlier reports to show that glucan administration promotes tumor regression, besides its immunoamplifying effect. Some workers have shown that glucan possesses a definite antitumor activity as revealed by their investigations on certain areas of host defense (Mansell, Ichinose, Reed, Krementz, M c N a m e e & Di Luzio, 1975; Mansell, Di Luzio, McNamee, Rowden & Proctor, 1976; Cook, Taylor, Cohen, H o f f m a n n , Rodrique, Malshet & Di Luzio, 1977; H a m u r o , Hadding & Bitter-Suermann, 1978). In our opinion, glucan adequately fulfills the necessary prerequisites for its use as a safe adjuvant partner with amoeba and, perhaps other parasite antigens. At the moment glucan appears an extremely good candidate adjuvant for giving further trials to such an amoeba vaccine in non human primates, for which efforts are presently underway.
Acknowledgements - - Financial support for this work was
provided through a project grant of the Department of Science & Technology (DST), Govt. of India, New Delhi. One of the authors (AS) also received a fellowship from the Lady Tata Memorial Trust, Bombay.
REFERENCES
BENACH, J. L., HABICHT, G. S., HOLBROOK, T. W. & CooK, J. A. (1982). Glucan as an adjuvant for a murine Babesia microti immunization trial. Infect. Immun., 45, 947- 951.
BROWDER, W., MCNAMEE, R., COHEN, C., TAYLOR, D. & D1 LUZlO, N. R. (1976). Induction of immunity to leukemia by the conjoint use of glucan and glutaraldehyde treated tumor cells. J. Reticuloendothel. Soc., 20, 55a. COOK, J~ A., HOLBROOK,T. W. & PARKER, B. W. (1980). Visceral lieshmaniasis in mice: protective effect of glucan. J. Reticuloendothel. Soc., 27, 567-573. COOK, J, A., HOLBROOK,T. W. & DOUGHERTY,W. J. (1982). Protective effect of glucan against visceral leishmaniasis in hamsters. Infect. hnmun., 37, 1261 - 1269. COOK, J. A., TAYLOR,D., COHEN,C., HOFFMANN,E. O., RODRIQUE,J., MALCHET,V. & DI LUZIO,N. R. (1977). Evaluation of effector cells mediating the antitumor action of glucan. J. Reticuloendothel. Sue., 22, 21 -34. DELVILLE, J. & JACQUES, P. J. (1980). Therapeutic and prophylactic effects of yeast glucan and related polysaccharidic immunomodulators in experimental leprosy. Int. J. Immunopharmac., 2, 183. DIAMOND, L. S. (1968). Techniques of Axenic cultivation of Entamoeba histolytica. Schaudinn, 1903 and E. histolytica - - like amebae. J. Parasit., 54, 1047- 1056. DI Luz~o, N. R., PISANO, J. C. & SAaA, T. M. (1970). Evaluation of the mechanism of glucan-induced stimulation of the reticuloendothelial system. J. Reticuloendothel. Soc., 7, 731- 742. DI Luzlo, N. R. (1976). Pharmacology of the reticuloendothelial system - - Accent on glucan. In The Reticuloendothelial System in Health and Disease: Function and Characteristics (Eds Reichard, S. M. and Escobar, M. R.) pp. 412 - 421, Plenum Press, New York.
Immunization of Guinea Pigs Against Entamoeba Histolytica Using Glucan as an Adjuvant
491
DI Luzlo, N. R. & MORROW, S. H. (1971). Comparative behaviour of soluble and particulate antigens and inert colloid in reticuloendothelial-stimulated or depressed mice. J. Reticuloendothel. Soc., 9, 273- 287. GILLET, J., JACQUES, P. J. t~: HERMAN, F. (1978). Particulate /3, 1-3 glucan and causal prophylaxis of mouse malaria (Plasmodium berghei ). In Advances in Experimental Medicine and Biology (Eds Escobar, M. R. and Friedman, H.) pp. 307-313, Plenum Press, New York. HAMURO, J., HADDING, U. & BI'ITER-SUERMANN,D. (1978). Solid phase activation of alternative pathway of complement by/3, 1-3 glucan and its possible role for tumor regressing activity. Immunology, 34, 695-705. HOLBROOK, T. W., COOK, J. A. & PARKER, B. W. (1981). Glucan-enhanced immunogenicity of killed erythrocytic stages of Plasmodium berghei. Infect. Immun., 32, 542 - 546. KESSEL, J. F., LEWIS, W. P., PASQUEL,C. M. & TURNER, J. A. (1965). Indirect hemagglutination and complement fixation tests in amebiasis. Am. J. trop. Med. Hyg., 14, 540- 550. KRUPP, I. M. (1970). Modification of the indirect hemagglutination test for amebiasis. J. clin. Path., 22, 530- 533. LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L. & RANDALL,R. J. (1951). Protein measurement with the Folin-phenol reagent. J. biol. Chem., 193, 265-275. MANSELL, P. W. A., DI LUZlO, N. R., MCNAMEE, R., ROWDEN, C. & PROCTOR, J. W. (1976). Recognition factors and nonspecific macrophage activation in the treatment of neoplastic disease. Ann. N.Y. Acad. Sci., 277, 20-44. MANSELL, P. W. A., ICHINOSE, H., REED, R. J., KREMENTZ,E. T., MCNAMEE,R. & DI LuzIo, N. R. (1975). Macrophagemediated destruction of human malignant cells in vivo. J. natn. Cancer Inst., 54, 571 -580. MORROW, S. H. & DI LuzIo, N. R. (1965). The fate of foreign red cells in mice with altered reticuloendothelial function. Proc. Soc. exp. Biol. Med., 119, 647- 652. NGUYEN,B. T. • STADTSBAEDER,S. 0980). Comparative biological and antitoxoplasmic effects of particulate and watersoluble polysaccharides, in vitro. In Advances in Experimental Medicine and Biology (Eds Escobar, M. R. and Friedman, H.) pp. 255-268, Plenum Press, New York. PATCHEN, M. L. & MAC VITTIE, T. J. (1982). In Advances in Experimental Medicine and Biology (Eds Norman, S. J. and Sorkin, E.) pp. 267-272, Plenum Press, New York. REYNOLDS, J. A., KASTELLO, M. D., HARRINGTON, D. C., CRABBS, C. L., PETERS, C. J., JEMSKI, J. V., SCOTT, G. H. & DI LUZIO, N. R. (1980). Glucan-induced enhancement of host resistance to selected infectious diseases. Infect. Immun., 30, 51-57. STEVENS,M., STEVENS,P., COOK,J. A., ICHINOSE.H. & DI LUZIO, N. R. (1976). Protective effect of the stimulant glucan on sporotrichosis infections of mice. J. Reticuloendothel. Soc., 20, 66a. WILLIAMS,D. L., COOK, J. A., HOFFMANN,E. O. t~ DI LUZlO, N. R. (1978). Protective effect of glucan in experimentally induced candidiasis. J, Reticuloendothel. Soc., 23, 4 7 9 - 490. WOOLES,W. R. t~ DI LUZIO, N. R. (1963). Reticuloendothelial function and the immune response. Science, 142, 1078 - 1080. WOOLES, W. R. & DI LUZIO, N. R. (1964). The phagocytic and proliferative response of the reticuloendothelial system following glucan administration. J. Reticuloendothel. Soc., 1, 160- 169.
0192-0561f84 $3.00+ .00 (c~ 1984 International Society for Immunopharmaco|ogy
I M M U N I Z A T I O N OF G U I N E A PIGS A G A I N S T E N T A M O E B A H I S T O L Y T I C A U S I N G G L U C A N AS AN A D J U V A N T * ALPANA SHARMA, AFROZ UL HAQ, M. U. SIDDIQU1 and SOHAIL AHMAD Parasitic Immunology Laboratory, Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh 202001, India (Received 29 August 1983 and in final form 22 March 1984)
Abstract - - Beta 1- 3 polyglucose or glucan, an extract of cell wall of Saccharomyces cerevisiae, has been successfully employed in this laboratory as an effective immunopotentiator in experimental studies on amoebiasis. An antigen extract from Entamoeba histolytica was combined with/3, 1- 3 glucan for immunizing guinea pigs. In order to study the effectiveness of such vaccine preparations, several batches of guinea pigs were immunized with amoeba antigen alone, and in combination with various immunoadjuvants. Antigen inoculations were carried out via intraperitoneal route. Protective immune responses were obtained against amoeba antigen by using glucan as an adjuvant partner. The study showed that glucan can be safely used as an effective immune enhancer.
All parasite vaccines, purified or synthetic, are likely to prove weak immunogens needing some further immunopotentiation. Currently, the most effective way to develop marginal vaccines is to combine them with potent and safe adjuvants. For accomplishing an immunologic control of parasitic diseases, adjuvants with less toxicity must be found for clinical and veterinary applications. The objective of this paper is to briefly summarise the vaccination studies conducted against E. histolytica infection in a guinea pig model system. Immunizations were carried out with amoeba antigen in combination with/3, 1 - 3 glucan. These results were compared with similar immunizations carried out by using other immunoadjuvants. Particulate /3, 1 - 3 glucan was chosen because of its ability to act on several strategic parameters of humoral and cellular immunity (Di Luzio, Pisano & Saba, 1970; Browder, McNamee, Cohen, Taylor & Di Luzio, 1976; Di Luzio, 1976). It has been earlier shown to act as a potent activator of reticuloendothelial (RE) system (Di Luzio et al., 1970) and complement (Di Luzio, 1976). Glucan of fungal origin has been described in the literature as a promising candidate adjuvant for the prevention, or therapy, of experimental leprosy (Delville & Jacques, 1980), candidiasis (Williams, Cook, Hoffmann & Di
Luzio, 1978), leishmaniasis (Cook, Holbrook & Parker, 1980; Cook, Holbrook & Dougherty, 1982), sporotrichosis (Stevens, Stevens, Cook, Ichinose & Di Luzio, 1976), malaria (Gillet, Jacques & Herman, 1978), toxoplasmosis (Nguyen & Stadtsbaeder, 1980) and numerous other infectious diseases (Reynolds, Kastello, Harrington, Crabbs, Peters, Jemski, Scott & Di Luzio, 1980). Samples of/3, 1 - 3 glucan used in these investigations were kindly supplied by Professor P. J. Jacques (Centre Inter-universitaire de Recherches Multidisciplinaires en Affections Parasitaires et Mecanismes de Defense de l'Hote, Brussels, Belgium).
EXPERIMENTAL PROCEDURES
Antigen Axenic cultures of E. histolytica (Strain; NIH-200) were raised in Diamond's TP-S-1-monophasic medium (Diamond, 1968). Axenically cultivated amoebae were pooled and washed in normal saline. Amoebae were sedimented by centrifugation at 850 g for 5 min and antigen extraction accomplished according to the method of Kessel, Lewis, Pasquel & Turner (1965). Protein concentration was determined by the method
* Preliminary results of the above study were briefly published by us in IRCS Medical Sciences 8, 526 (1980). Further results of this study were presented in a symposium lecture given by one of us (SA) at lind InternationalCongress of Immunopharmacology held in 5 - 9 July, 1982 at Washington, DC. Abstract: International Journal of Immunopharmacology 4, 271 (1982). 483
484
ALPANA SHARMA,AFROZUL HAQ, M. U. SIDDIQUI and SOHAILAHMAD
of Lowry, Rosebrough, Farr & Randall (1951) at 700 nm, using Bausch & L o m b Spectronic 21 Spectrophotometer.
Immunization Eighty guinea pigs, each weighing 300 - 400 g, were equally divided into eight experimental groups. Each animal was immunized intraperitoneally with a total amount of 200/ag antigen protein given in three doses. The details o f the immunization schedule are shown in Table 1. Animals in groups I - III were inoculated with amoeba antigen given in combination with various adjuvants, while group IV animals received amoeba antigen alone. Animals in other control groups ( V - V I I ) were inoculated with fl, 1-3 glucan or F r e u n d ' s complete adjuvant (FCA, Difco), or aluminium hydroxide adjuvant (AIA, 2070 w/v). Group VIII animals received saline only. The glucan was administered at the rate of 40 m g / k g body weight. Immunization was completed over a period of four weeks including a week of rest (3rd week).
Detection o f immune responses Antiamoebic antibodies were detected in sequential serum samples obtained from blood drawn at weekly intervals. Antibody detections were also carried out in samples obtained as pre and post immunized serum. Hemagglutinating antibody titers were determined by the modified technique of Krupp (1970). A 2.5°70 suspension of sheep red blood cells (SRBC) was tanned for 20 rain with tannic acid (1:100,000) in an ice bath at 4°C. The tanned cells were sensitized with antigen at pH 6.4 in a water bath at 37°C for 20 min. The test was performed in microtiter plates with U shaped bottoms. About fifteen serum samples obtained from confirmed human cases o f amoebic dysentery, or normal subjects, were used as reference sera for positive and negative controls, respectively. A serum was considered positive if its antibody titer was 1:64 or higher.
Skin reaction Following immunizations, each animal was skin
Table l. Immunization schedule Animal groups* I.
II.
III.
IV. V. VI. VII. VIII.
Weekly immunization doses (ml) I st 2nd 4th
Amoeba antigen + glucan Antigen Glucan
0.05 0.40
0.05 0.40
0.10 0.40
Amoeba antigen + FCA Antigen FCA
0.05 0.05
0.05 0.05
0.10 0. I 0
Amoeba antigen + AIA Antigen AIA
0.05 0.05
0.05 0.05
0.10 0.10
Amoeba antigen only Antigen
0.05
0.05
0.10
Glucan only Glucan
0.40
0.40
0.40
FCA only FCA
0.05
0.05
0.10
AIA only AIA
0.05
0.05
0.10
Saline only Saline
0.50
0.50
0.50
* Each animal group contained ten guinea pigs. 3rd week was treated as rest period, Glucan concentration 30 mg/ml. Amoeba antigen concentration 1 mg/ml. FCA: Freund's complete adjuvant. AIA: Aluminium hydroxide adjuvant (2o70 w/v). Total inoculum in each injection was made upto 0.5 ml with the addition of saline.
Negative
Negative
64_+37 (32- 128)
64_+26 (32 - 128)
128+52 ( 6 4 - 256)
1st
2nd
Negative
64_+37 (32 - 128)
128_+52 (63 -256)
256_+156 (128 - 512)
512_+209 (256- 1024)
* The given IHA titers are arithmetic mean of ten experiments _+ S.D. t Each group contained ten guinea pigs. The values given in parentheses indicate the range of IHA titers. FCA: Freund's complete adjuvant. A1A: Aluminium hydroxide adjuvant (2% w/v). An IHA titer less than 64 was taken as negative.
V, VI, VII and VIII Glucan, FCA, A1A and saline (controls)
IV (Antigen)
III (Antigen + AIA)
II (Antigen + FCA)
(Antigen + glucan)
I
Animal groupst
Negative
128_+74 (64 - 256)
256_+104 (128 -512)
512_+209 (256 - 1024)
4th
5th
6th
Negative
512_+209 (256 - 1024)
1024_+418 (512-2048)
Negative
512+_295 (256 - 1024)
1024_+591 (512- 2 0 4 8 )
2048_+1182 2048±836 (1024- 4096) (1024- 4096)
Negative
128_+52 (64 - 256)
512_+209 (256- 1024)
512_+295 (256 - 1024)
16384+_6688 16384_+6688 8192_+3344 (8192- 32768) (8192- 32768) (4096- 16384)
Weeks
2048+836 (1024- 4096)
3rd
Table 2. Reciprocal IHA titers in weekly sera samples* O
g
e<
>
r~
tm
> q~
O
486
ALPANA SHARMA, AFROZ UL HAQ, M. U. SIDDIQU! and SOHAIL AHMAD
sensitized by inoculating 12 /ag antigen protein intradermally. The reaction intensity was determined by measuring the diameter and thickness of erythema and induration at the skin test site at 48 h, following antigen inoculation. Skin tissue from the reaction site was excised for histological examination. Skin sections cut at 5 ta were stained with iron hematoxylin and eosin (H & E) and examined under microscope.
Challenging the animals All the test and control animals were subsequently challenged. The challenging dose containing 4 × 106 amoebae in 0.5 ml volume was inoculated intrahepatically, or intracecally. The amoebae used for challenging the immunized animals were of proven pathogenicity. They were isolated from a patient of amoebic liver abscess and maintained in laboratory cultures. The animals were sacrificed on post challenge day 14 for making gross observations of the viscera of the inoculated animals. The infected tissues from liver and cecum were later removed and fixed in formalin. The excised tissues from the two inoculated sites were sectioned at 4 - 5/a and fixed on the slides. The dehydrated sections were stained in H & E and made into permanent mounts. The stained slides were used for histopathological examination.
RESULTS
Indirect hemagglutination (IHA) test The IHA titers of weekly serum samples were recorded. Table 2 shows the reciprocal IHA titers from various experimental groups. Erythrocytes sensitized with different antigen dilutions were first tested against reference serum of known antibody activity, and then against the test serum samples. Highest antibody titers in the experimental groups I - I V were recorded in the fourth week. The IHA titers from the animals immunized with amoeba antigen-glucan combination showed a typical antibody response, yielding a peak titer of 1:16,384. Skin test All the guinea pigs in groups I - IV developed delayed type skin hypersensitivity reactions. A positive skin reaction of a diameter greater than 5 mm was characterized by a zone of erythema and induration. These reactions were found maximum at around 48 h, subsiding thereafter. Figure 1 shows the appearance of a typical delayed type skin reaction at the site of antigen inoculation in antigen-glucan immunized
animals. A challenge dose containing 12 ~g antigen protein was found adequate to elicit such delayed type skin hypersensitivity reactions. The skin reactions appearing in the control animals were of very small size (diameter less than 5 ram) and were recorded as negative. Skin test results are shown in Table 3. Histological examination of the skin reaction site showed characteristic perivascular cuffing with prominent cell infiltration. The intercellular spaces were found filled with mononuclear cells, especially the lymphocytes and a few macrophages. A prominent intercellular cell infiltration can be seen in the photomicrograph from a skin reaction site (Fig. 2).
Lesion formation Experimental animals treated with glucan alone, or in combination with amoeba antigen did not show any skin lesions at the inoculation site. The animals receiving FCA or aluminium hydroxide adjuvant developed skin lesions at the site of inoculation.
Protection studies All glucan immunized animals throughout the entire post challenge period appeared completely normal, showing no sign of disease whatsoever. The gross examination of their viscera did not reveal any pathology. Liver and cecum of these animals were devoid of any macroscopic lesions and appeared completely normal. While in the unimmunized controls, similar organs not only showed lesions but also appeared inflammed and oedematous. The unprotected animals in various control groups developed frank diarrhoea, preceded with lassitude, listlessness and significant loss of appetite. Histopathological examination of the liver and cecum from control animals revealed ulcerative amoebic lesions accompanied with necrosis and considerable tissue damage. Tissue sections from liver showed a large number of small, locally decolourised raised areas. The hepatic cells gave a completely glassy appearance. Tissue histology from immunized animals did not reveal any such lesions, or other signs of an accompanying pathology. In fact, the animals immunopotentiated with glucan exhibited a qualitative difference in their protective response as revealed by a marked inflammatory reaction in the challenged tissues (Figs. 3a and b). The liver section showed prominent mononuclear cell infiltration around hepatic cells. Table 4 shows further results of this protection study. The results obtained in each test group were uniformly reproducible in repeated experiments.
Immunization of Guinea Pigs Against Entamoeba Histolytica Using Glucan as an Adjuvant
Fig. 1. Guinea pig showing positive delayed type skin hypersensitivity reaction.
Fig. 2. A skin section showing cellular infiltration at the reaction site.
487
488
ALPANA SHARMA, AFROZ UL HAQ, M. U. SIDDIQUI and SOHAIL AHMAD
Fig. 3. Photomicrographs showing tissue sections of protected animals immunized with a n t i g e n - g l u c a n combination. (a) Section of liver. (b) Section of cecum. Perivascular mononuclear cell infiltration areas are marked.
Immunization of Guinea Pigs Against Entarnoeba Histolytica Using Glucan as an Adjuvant
Table 3. Skin test results Total antigen inoculated/animal (ug)
Skin reaction diametert (mm)
Induration thicknesst (mm)
(Antigen + glucan)
200
15.50_+2.20
4.45_+1.85
I1 (Antigen + FCA)
200
14.00_+2.05
4.25_+1.60
111 (Antigen + AIA)
200
ll.50_+3.18
3.15_+0.62
IV (Antigen)
200
8.50_+1.18
2.75_+0.20
--
Negative
Negative
Animal groups* I
V, VI, VII and VIII Glucan, FCA, AIA and saline (controls)
* Each group contained ten guinea pigs. t Arithmetic mean of ten experiments -2_ S.D. FCA: Freund's complete adjuvant. AlA: Aluminium hydroxide adjuvant (2070 w/v). Skin reaction diameter and induration thickness of less than 5 and 2 mm respectively were taken as negative.
Table 4. Protection in experimental animals Animals showing disease after challenge
°7o protection
(Antigen + glucan)
0/10
100
II (Antigen + FCA)
3/10
70
III (Antigen + AIA)
6/10
40
IV (Antigen)
6/10
40
V (Glucan)
8/10
20
VI (FCA)
9/10
10
10/10
Zero
Animal groups* I
VII and VIII (AIA) and (saline) * Each group contained ten guinea pigs. FCA: Freund's complete adjuvant. A1A: Aluminium hydroxide adjuvant (207o w/v).
489
490
ALPANA SHARMA, AFROZ UL HAQ, M. U. SIDDIQUI and SOHAILAHMAD DISCUSSION
Results of this study provide ample p r o o f that guinea pigs are able to generate a protective immune response, following amoeba-glucan vaccination. The protective response comprised of a strong cell mediated immunity along with raised levels of circulating antibodies. Such highly protective responses were obtainable only in those animals which were immunized with glucan as an adjuvant partner. Animals receiving other adjuvants, or immunized with amoeba antigen alone, did not appear to elicit a protective response of a similar order. The action of glucan as an effective adjuvant is well documented in the literature. It has been shown to enhance the proliferation and stimulation activity, mainly of the reticuloendothelial cells (Wooles & Di Luzio, 1963; Wooles & Di Luzio, 1964; Morrow & Di Luzio, 1965; Di Luzio et al., 1970; Di Luzio & Morrow, 1971; Di Luzio, 1976). Use of glucan as a powerful adjuvant has met with considerable success in certain earlier studies. For example, it has been shown to enhance the immunity against rodent malaria (Holbrook, Cook & Parker, 1981). These authors have further shown the usefulness of glucan as an adjuvant in their studies on murine babesiosis (Benach, Habicht, H o l b r o o k & Cook, 1982). More recently, glucan has been shown to exert a protective effect in hamsters against visceral leishmaniasis (Cook et al., 1982). On the basis of information so far available, it seems like glucan is an ideally suited adjuvant for use with parasite antigens. The optimum dose recommended for administering glucan as an adjuvant is around
40 m g / k g body weight. Patchen & Mac Vittie (1982) in their extensive studies have described that glucan administration up to 100 m g / k g acts as a stimulator of RE system in a dose dependent manner. The F C A treated animals were found more predisposed to skin lesions, while glucan treated animals did not show any such tendency for lesion formation. There are some earlier reports to show that glucan administration promotes tumor regression, besides its immunoamplifying effect. Some workers have shown that glucan possesses a definite antitumor activity as revealed by their investigations on certain areas of host defense (Mansell, Ichinose, Reed, Krementz, M c N a m e e & Di Luzio, 1975; Mansell, Di Luzio, McNamee, Rowden & Proctor, 1976; Cook, Taylor, Cohen, H o f f m a n n , Rodrique, Malshet & Di Luzio, 1977; H a m u r o , Hadding & Bitter-Suermann, 1978). In our opinion, glucan adequately fulfills the necessary prerequisites for its use as a safe adjuvant partner with amoeba and, perhaps other parasite antigens. At the moment glucan appears an extremely good candidate adjuvant for giving further trials to such an amoeba vaccine in non human primates, for which efforts are presently underway.
Acknowledgements - - Financial support for this work was
provided through a project grant of the Department of Science & Technology (DST), Govt. of India, New Delhi. One of the authors (AS) also received a fellowship from the Lady Tata Memorial Trust, Bombay.
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Immunization of Guinea Pigs Against Entamoeba Histolytica Using Glucan as an Adjuvant
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