Nematospiroides dubius: Influence of adjuvants on immunity in mice vaccinated with antigens isolated by affinity chromatography from adult worms

Nematospiroides dubius: Influence of adjuvants on immunity in mice vaccinated with antigens isolated by affinity chromatography from adult worms

EXPERIMENTAL PARASITOLOGY Nematospiroides Mice Vaccinated 68, 67-73 (1989) Influence of Adjuvants on Immunity in with Antigens Isolated by Affin...

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Mice Vaccinated


67-73 (1989)

Influence of Adjuvants on Immunity in with Antigens Isolated by Affinity Chromatography from Adult Worms dubius:




Department of Parasitology, University of Queensland, St. Lucia, Australia 4067, and TSIRO, Animal Science, Long Pocket Laboratories, Indooroopilly, Australia 4068

Division of

MONROY, F. G., ADAMS, J. H., DOSSON, C., AND EAST, I. J. 1989. Nematospiroides dubius: Influence of adjuvants on immunity in mice vaccinated with antigens isolated by aflinity chromatography from adult worms. Experimental Parasitology 68, 67-73. Five adjuvants were examined for their ability to potentiate the immune response of mice to soluble antigens from adult Nematospiroides dub&s prepared by affiity chromatography against antibodies from repeatedly infected mice as ligands (IMIgAg). Immunized mice were better protected against N. dubius by IMIgAg injected intraperitoneally with either pertussigen (75%) or aluminium hydroxide (Alum) (67%) as adjuvants than with Freund’s complete (54%) or incomplete adjuvants (31%). Protection was correlated with elevated specific antibody values and with cellular responses. Quil A was toxic to recipient mice at the concentration used. Alum may be a more practical adjuvant than pertussigen, which may activate protective immunity only in specific recipient genotypes and oil-based adjuvants which appear to be less efficient, to vaccinate mice with soluble parasite antigens. o 1989 Academic

Press, Inc.

DESCRIPTORS AND ABBREVIATIONS: Nematospiroides dubius; Nematode; Adjuvan&; Vaccines; Immunity; Parasite antigens; Adult worm homogenate (AWH); Immunoglobulins (Igs); Affinity chromatography using Igs from nomal and immune mouse serum (NMS, IMS) as ligands to prepare antigens (NMIgAg, IMIgAg); Phosphate-buffered saline (PBS); Intraperitoneal (ip); Enzyme-linked immunosorbent assay (ELISA); sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE); Freund’s complete and incomplete adjuvants (FCA, FIA); Aluminium hydroxide (Alum); White blood cell counts (WBC); Packed red cells (PVC); Mesenteric lymph node (MLN); Delayed-type hypersensitivity (DTH). INDEX

varied results (Van Zandt 1971; Cypess 1970; Goven and De Buysscher 1980; Monroy et al. 1985, Monroy and Dobson 1986). In one other case, however, genetically susceptible C57BL/6 mice were protected to an impressive high degree by vaccines made with soluble extracts of adult N. dubius and pertussigen as the adjuvant (Mitchell and Munoz 1983). Here three categories of adjuvants (antigen depot, bacterial, and surface-active agents) were assessed for their capacity to enhance protective immunity in mice against N. dubius induced by soluble parasite antigens (extracted from whole adult worms by affinity chromatography using Igs from IMS); FCA improved the efficacy


Adjuvants potentiate the protective efficacy of vaccines; some preferentially activate cell-mediated and others humoral immunity. It is the propensity of individual adjuvants to amplify and to sustain specific elements of protective immunity which illustrates their importance in prophylaxis. Attempts to vaccinate mice against Nematospiroides dubius have commonly employed the oil-based adjuvant FCA with ’ To whom correspondence should be addressed. ’ Present address: Laboratory of Parasitic Diseases, National Institutes of Health, Building 5, Room 112, Bethesda, MD 20892, U.S.A. 67

OO14-4894l89 $3.00 Copyright 0 1989 by Academic Press. Inc. AU rights of reproduction in any fom reserved.



of these vaccines when injected ip (Monroy and Dobson 1987). MATERIALSAND METHODS Parasite and host. Nematospiroides dubius larvae were cultured from eggs voided by infections maintained in female Quackenbush mice for 53 generations. Female, 6-week-old BCFl mice (CBA male x C57BLI 6 female) were obtained from the central animal breeding house, University of Queensland, and were infected, killed, and necropsied and the worms were recovered as previously described (Monroy et al. 1985). Antigens. N. dubius AWH was prepared from worms that were recovered 20 days after infection. Worms were washed several times in 0.15 M saline, resuspended in 3 ml phosphate-buffered saline (PBS) (0.01 M, 0.15 M NaCl, 10 mM EDTA, pH 7.2), and homogenized in a glass tissue grinder in an ice bath. The AWH was clarified by centrifugation at lO,OOOg,4 C for 45 min, sterilized by passage through a 0.45 pm pore size membrane, assayed for protein content by Bio-Rad (Richmond, CA, U.S.A.) protein assay, and stored at - 70 C. Sera. IMS were raised in BCFl mice given seven infections with 100 L3 N. dubius on Days 0, 7,28, 35, 49, 56, and 63, and were treated with 0.1 ml 10% levamisole HCl on Days 21, 42, and 70. Uninfected BCFI mice provided NMS. Igs were extracted from NMS and IMS to prepare normal (NMIg) and immune (IMIg) aftinity chromatography columns (Monroy and Dobson 1987). Aflnity chromatography. Five milliliters of N. dubius AWH (5 mg/ml) were filtered through three columns in sequence. The first included CNBrSepharose 4B blocked by glycine-HCl, pH 8.0, as a guard column; the second included normal, and the third contained immune mouse Igs coupled to CNBrSepharose 4B (Monroy and Dobson 1987). Unbound AWH was collected, pooled, concentrated to 5 ml, and recycled five times through regenerated columns until depleted of reactive components. Bound proteins were eluted from each column after each of the five cycles with 0.2 M glycineHC1, pH 2.5, into Tris to bring the solution to pH 8.0 and monitored at 280 nm. Antigens (NMIgAg, IMIgAg) eluted from each column were pooled, concentrated, assayed for protein content, and stored at -70 C. Serology. A double-antibody ELISA identified mutine anti-N. dubius antibody isotypes. Unlabeled goat antibodies (specific for mouse IgA, IgM, IgGl, IgG2a, IgGZb, IgG3, IgE; Nordic Immunological Reagents) were added in appropriate dilution (Fig. 5) to AWH murine antibody complexes fixed on microtiter plates and bound anti-isotype antibodies were detected with HRP-conjugated rabbit anti-goat Igs diluted 1:2000 and enzyme substrate 4-chloro-1-naphtol(25 mg dissolved

in 10 ml methanol in 100 ml 0.05 M acetate buffer, pH 5.0, and 10 11130% H,O,). Hematology. Total WBC were done in a Coulter counter (Coulter Electronics, Ltd., England); 20 pl heparinized blood was discharged into 10 ml isotonic saline solution and three drops of Zap-oglobin were added immediately before the counts were made (total leucocytes/n& x 1000). Eosinophil leucocytes were counted in heparinized blood samples mixed 10: 190 p,l in Discombe’s fluid (Discombe 1946), reacted for a few minutes at 4 C, and counted (mm3 X 100) in a hemocytometer. The hematocrit (PCV) was determined using a microhematocrit system (Gelman Hawksley Ltd., England). Electrophoresis. Affinity-purified antigens were analyzed either directly by SDS-PAGE or were transferred to nitrocelhrlose membranes and reacted with either NMS or IMS (Monroy and Dobson 1987). Experimental Design. N. dubius IMIgAg, at 150 &ml, was mixed with adjuvants to give a vaccine dose of 15 t&O.1 ml per mouse. Five groups of eight, 6-week-old female BCFl were vaccinated ip twice at intervals of 8 days with 15 ug, IMIgAg emulsified 1:l with FCA and FIA. Commercial Alum gel was used at a ratio of 3:l IMIgAg. Alum (CSL, Australia; 6.3 mg/ ml) was mixed with N. dubius AWH and bovine serum albumin (BSA) and gave optimum flocculation at this ratio. Lyophilized pertussigen, 400 ng, was reconstituted and mixed with IMIgAg (Mitchell and Munoz 1983). Quil A (Superfos a/s, Denmark), as 1.5% lyophilized dry matter, was resuspended in PBS and mixed with antigen at 100 pg/mouse. Four groups of eight mice were injected with FCA, IFA, Alum, and pertussigen adjuvant-saline mixtures; another was kept as primary infection controls; and one group of mice was not infected (see Table 1). Mice were infected with 100 L3 N. dubius 10 days after the second injection and were killed 28 days later when their worm burdens, serum ELISA antibody levels, PCV, WBC, and splenic coefficients (mg spleen/g body weight; Baker 1955) were measured. None of the adjuvant control and infected-vaccinated mice showed gross peritoneal reactions at necropsy. Differences between groups with metric mean values were analyzed by paired Student’s t-test. A value of P < 0.05 was taken as significant.


IMIgAg showed at least 11 polypeptides between MW 20,000 and 100,000 which stained with Coomassie blue after reducing SDS-PAGE in a 10% gel. Most bands were located between MW 40,000 to 90,000 and a MW 78,000 component was strongly



AGAINST N. dubius

TABLE I Number of Nematospiroides dubius and the Packed Cell Volume from Female BCFl Mice Vaccinated with IMIgAg Emulsified in Different Adjuvants Infected 28 Days with 100 L3 Mice n FCA/IMIgAg FCA IFA/IMIgAg IFA Alum/lMIgAg Alum Pertussigen/IMIgAg Pertussigen Control Mean control Noninfected

8 8 7 I 8 8 8 8 9 40 9

Worm recovery x


Protection %

25 55 38 58 18 52 13 48 62 55 -

4 4 6 6 3 6 3 6 7 2 -

54* 0.3 31* 67* 5 15* 12 -




42.2 39.1 39.0 42.4 46.6 48.6 46.8 48.5 42.0 42.4

0.8 0.8 2.4 1.4 2.4 1.6* 1.6” 1.3* 0.4



* Significant P < 0.05.

stained (Fig. 1A). Seventeen bands were showed polypeptides at MW 200,000, stained from IMIgAg silver stain under re- 78,000,74,000,60,000, and 24,000 which reducing conditions in a 12% gel, and compo- acted with IMS (Fig. 1B). nents at MW 42,000 and 52,000 stained IMIgAg was mixed with different adjustrongly (Fig. 1C). Immunoblots of IMIgAg vants to formulate vaccines which were assayed in mice challenged with N. dubius. Fewer worms were recovered from mice vaccinated with IMIgAg emulsified with IFA (31%), FCA (55%), Alum (67%), and 180 pertussigen (75%) (r = 2.69, 4.09, 5.30, I -180 5.91; P < 0.05, 0.01) compared with the 116 mean of the adjuvant and no treatment con116 (? trols (see Table 1). The mice which were p injected with vaccines that included Quil A ,-84 2 _ either with or without IMIgAg died within 3, 24 hr. Similar numbers of worms were recovered from all the adjuvant saline treated -4% =and no-treatment groups of mice; these data were pooled to give a mean control value (Table 1). Parasite epg were the same 12-28 days after infection for the different adjuvant control and primary infection mice and data 26were pooled. Epg of vaccinated mice given IMIgAg with different adjuvants were conA 6 sistently lower than those for the controls; FIG. 1. SDS-PAGE and Western blot of affinityfewest eggs were voided by mice treated puritied Nematospiroides dubius IMIgAg in a 10% gel: and IMIgAg(A) Coomassie blue-stained IMIgAg; (B) IMIgAg re- with IMIgAg-Alum pertussigen (t = 2.76, 2.61; P < 0.02) comacted with IMS 1:200 dilution; (C) silver-stained IMIgAg in a 12.5% gel. pared with others immunized with IMIgAg-



FCA and IMIgAg-FIA (t = 1.75, 1.42; NS) (Fig. 2). Hematocrits were significantly elevated 28 days after infection in the blood of mice treated with Alum, pertussigen, and IMIgAg-pertussigen compared with controls (t = 3.06, 3.35 and 2.21; P < 0.01 and 0.05; Table 1). Only mice vaccinated with IMIgAg-pertussigen showed a significant leucocytosis (c = 2.83; P < 0.02). All adjuvant control mice had higher leucocyte counts than mice vaccinated with IMIgAg in different adjuvants, except for mice injected with Alum preparations (Fig. 3). All groups of infected mice developed splenomegaly compared with noninfected mice; this was most apparent in mice injected with FCA and IFA either with or without IMIgAg which showed the same degree of splenomegaly as did infected control mice (Fig. 4). ELISA showed that IMIgAg plus either Alum or pertussigen preferentially stimulated IgGl; whereas FIA, FCA, and pertussigen induced more IgG2b anti-N. dubius class antibodies. IgE antibodies were consistently elevated in infected mice vacci-

nated with IMIgAg in pertussigen as an adjuvant and to a lesser extent with Alum (Fig. 5). DISCUSSION

The mode of action and efficacy of adjuvants relative to the reactions they induce has been explained as selective augmentation of the immune response (Bomford 1980). Monroy and Dobson (1987) vaccinated and protected mice to 38% with IMIgAg alone and to 57% with IMIgAg in FCA, which agrees closely with the level of protection (50%) achieved with FCA here and we were encouraged that protective immunity was increased when IMIgAg was administered with pertussigen (75%) and Alum (67%) as adjuvants. The effectiveness of humoral immunity against parasites depends on the antigen epitopes which bind antibody isotypes capable of mediating protective reactions. IgE and IgGl are the only murine antibodies which bind to mast cells by Fc receptors and can elicit anaphylactic reactions (Bloch 1967). Anti-N. dubius ELISA revealed that

FIG. 2. Fecal parasite epg from female BCFl mice vaccinated with IMIgAg in different adjuvants 20 days after infection with 100 L3 N. dubius. The shaded area is the control k SE.






N. dubius




cmirol mlwed

FIG. 3. Total blood leucocytes/mm3 in female BCFl mice vaccinated with IMIgAg in diierent adjuvants 28 days after infection with 100 L3 N. dubius. The shaded area is the control + SE.

Alum, pertussigen, and FIA stimulated predominantly IgGl antibodies; whereas FIA and FCA induced IgG2a and IgG2b; and pertussigen stimulated IgE, IgG2b, and IgGl antibodies. The most effective class of antibody to passively transfer resistance in mice against N. dubius is IgGl and the most protective immunity has been attributed to this antibody class (Pritchard et al. 1983; Williams and Behnke 1983; Monroy and Dobson 1987).

7 kj . a

1 l.FCA Z.FCA/Ag


3 9lFA 4JFAlAg


5 aALuM &ALUM&g






FIG. 4. Splenic coefficients for female BCFl mice vaccinated with N. dubius IMIgAg in different adjuvants. The shaded area is the control + SE.

Mitchell and Munoz (1983) protected susceptible C57BL/6 mice to 95% against N. dubius with a pertussigen-based vaccine. Here we increased PCV and leucocytosis and reduced worm numbers by 75% with IMIgAg and pertussigen as adjuvant. Such cell responses occur because pertussigen selectively induces activity in reticuloendothelial tissues (Munoz et al. 1981). The difference in effect can be attributed either to the mouse strain or to the route of vaccination. Here BCFl (hybrids of highly susceptible CBA males x C57BL/6 females) mice were immunized ip with IMIgAgpertussigen, whereas Mitchell and Munoz (1983) gave AWH, ip, and pertussigen intravenously. Quil A is a pure and negatively charged surface-active saponin which at 100 t&mouse ip exceeded the toxic limits for these mice. Morein et al. (1987) reported that 10-50 kg Quil A is toxic in 20-g mice. It is likely that IMIgAg-Quil A could have disrupted local lymphoid tissue and mucous membranes. Similar concentrations of Quil A delivered subcutaneously are not toxic (Opdebeeck, personal communication). N. dubius infections are long lived in mice and are associated with pronounced












12345676 lFCA 2.FCA /Ag


12345678 3. IFA 4. IFA/Ag



FIG. 5. Immunoglobulin isotype ELISA on serum from female BCFl mice vaccinated with N. IMIgAg in different adjuvants. Dilutions for each isotype are marked in the top right comer.


hematological changes (Baker 1962), increased levels of circulating parasitespecific antibody (Dobson 1982), and marked splenomegaly and splenic lymphocytosis (Baker 1955). Vaccines which contained FCA and FIA induced significant splenomegaly whereas those with Alum or pertussigen did not. Moreover, IMIgAg administered with Alum and with pertussigen improved protective immunity against N. dubius in mice. Alum induces strong humoral rather than cell-mediated immunity and pertussigen enhances cell-dependent antibody responses (Allison and Byars 1986). The low splenic coefficient in mice treated with Alum and pertussigen-based vaccines suggests that the MLN regulated the flow of immunological reagents from surviving worms without an exaggerated involvement of the spleen in these reactions.

It is possible therefore that immunogens delivered ip with either Alum or pertussigen were restricted in their action to the intestine and adjacent MLN and therefore were more protective because they did not activate peripheral immunomodulatory cellular events which may enhance the survival of the parasite. Pertussigen will potentiate specific DTH (Sewell et al. 1983) and may reinforce the migration of DTH-mediator cells from spleen to the blood because it either inhibits the induction or interferes with the function of splenic T-suppressor cells (Tamura er al. 1985). One disadvantage of pertussigen as an adjuvant is that it may only be active in specific recipient genotypes (Mitchell and Munoz 1983). Thus adjuvants like Alum and pertussigen may either selectively induce immune responses against particular immunogens to


AGAINST N. dubius

enhance protective immunity or selectively inhibit the interaction of immunosuppressors from N. dubius which depress protective effector reactions. There are advantages, therefore, in selecting specific adjuvants like Alum which enhanced protective antibody responses to improve immunity against gastrointestinal parasites, particul&rly where the vaccine contains soluble protein antigens.


tion of immunity in mice to Nematospiroides dubius by use of a double-emulsion adjuvant. Journal of Parasitology 66(2), 346-347. MITCHELL, G. F., AND MUNOZ, J. J. 1983. Vaccination of genetically susceptible mice against chronic infection with Nematospiroides dubius using pertussigen as adjuvant. Australian Journal of Experimental Biology and Medical Science 61, 425-434. MONROY, F. G., BRINDLEY, P. J., AND DOBSON, C. 1985. Vaccination against Nematospiroides dubius in mice using adult worm extracts as antigens. Australian Journal of Experimental Biology and Medical Science 63, 423430. MONROY, F. G., AND DOSSON, C. 1986. Protective anACKNOWLEDGMENTS tigens in fractions of adult Nematospiroides dubius. Australian Journal of Experimental Biology and This work was supported by a CSIRO/University of Medical Science 62, 523-530. Queensland collaborative research fund and the AusMONROY, F. G., AND DOSSON, C. 1987. Mice vaccitralian research grant scheme. nated against Nematospiroides dubius with antigens isolated by affinity chromatography from adult REFERENCES worms. Immunology and Cell Biology 65, 223-230. MOREIN, B., L~VGREN, K., HOGLUND, S., AND ALLISON, A. C., AND BYARS, N. E. 1986. An adjuvant SUNDQUIST, B. 1987. The ISCOM: An immunostimformulation that selectively elicits the formation of ulating complex. Immunology Today 8, 333-338. antibodies of protective isotypes and of cellMUNOZ, J. J., ARAI, H., BERGMAN, R. K., AND SAmediated immunity. Journal of Immunological DOWSKY, P. L. 1981. Biological activities of crystalMethods 95, 157-168. line pertussigen from Bordetella pertussis. Infection BAKER, N. F. 1955. The pathogenesis of trichoand Immunity 33, 820-829. strongylid parasites: Some effects of NematospiroiD. I., WILLIAMS, D. J. L., BEHNKE, des dubius on the erythrocyte patterns and spleens PRITCHARD, J. M., AND LEE, D. G. 1983. The role of IgGl hyof mice. Experimental Parasitology 4, 526-541. pergammaglobulinaemia in immunity to the gastroBAKER, N. F. 1962. The nature and etiology of the intestinal nematode Nematospiroides dubius: The leukocytic response of Webster mice infected with immunochemical purification, antigen-specificity in Nematospiroides dubius. Journal of Parasitology in vivo anti-parasite effect of IgGl from immune se48,438+41. rum. Immunology 49, 353-365. BLOCH, K. J. 1%7. The anaphylactic antibodies of SEWELL, W., MUNOZ, J. J., AND VADAS, M. A. 1983. mammals including man. Progress in Allergy 10,84Enhancement of the intensity, persistence and pas150. sive transfer of delayed-type hypersensitivity leBOMFORD, R. 1980. The comparative reactivity of adsions by pertussigen in mice. Journal of Experimenjuvants for humoral and cell-mediated immunity. I. talMedicine 157,2087-2096. Effect on the antibody response to bovine serum TAMURA, S., TANAKA, H., TAKAYAMA, R., SATO, H., albumin and sheep red blood cells of Freund’s inSATO, Y., AND UCHIDA, N. 1985. Break of unrecomplete and complete adjuvants, alhydrogel, Cosponsiveness of delayed-type hypersensitivity to rynebacterium parvum, Bordetella pertussis, musheep red blood cells by pertussigen toxin. Cellular ramyl dipeptide and saponin. Clinical and ExperiImmunology 92, 376-390. mental Immunology 39,426-434. VAN ZANDT, P. D. 1971. Immunity relationships in CYPESS, R. 1970. Artificial production of acquired imwhite mice infected with Nematospiroides dubius. munity in mice by footpad injections of crude larval III. Artificial immunization with antigen prepared extract of Nematospiroides dubius. Journal of Parfrom adults. Journal of the Elisha Mitchell Scientific asitology 56, 320. Sociery 87, 74-76. DISCOMBE, G. 1946. Criteria of eosinophilia. Lancer 1, WILLIAMS, D. J., AND BEHNKE, J. M. 1983. Host pro195-l%. tective antibodies and serum immunoglobulin isoD~BSON, C. 1982. Passive transfer of immunity with types in mice chronically infected or repeatedly imserum in mice infected with Nematospiroides dumunized with the nematode parasite Nematospiroibius: Influence of quality and quantity of immune des dubius. immunology 48, 37-47. serum. International Journal for Parasitology 12, 207-213. Received 20 May 1988; accepted with revision 13 Sep GOVEN, A. J., AND DE BUYSSCHER,V. 1980. Productember 1988