EXPERIMENTAL

PARASITOLOGY

38, 6-13

Plasmodium

( 1975)

bergh,ei: Immunogenic Enhancement Antigen by Adjuvant Addition’ ROBERT

Department

S.

of

DESOWITZ

Of Tropical Medicine University

and Medical Microbiology, School of Medicine, of Hawaii, Honolulu, Hawaii 96816

(Accepted

for publication

September

19, 1974)

DESOWITZ, R. S. 1975. Immunogenic enhancement of Plasmocliztm berg&i antigen by addition of various adjuvants. Experimental Parasitology 38, 6-13 Vaccines consisting of soluble Plasmodium berghei antigen in conjunction with a variety of adjuvants were injected into weanling white rats. Protective immunity, as evidenced by a lower mortality rate, reduced parasitemia and shortened course of infection, was induced by antigen in combination with the following adjuvants: Bordetella perk&s vaccine, levamisole, and polyinosinicsaponin, hexylamine, polycytidylic acid ( poly 1:C). Soluble antigen alone or combined with Freund’s complete adjuvant, bacterial cndotoxin, vitamin A, polyadenylic-polyuredelic acid (poly A:U) failed to induce any significant degree of protective immunity. INDEX DESCRIPTORS: Plasmodium berghei; Immunization; Rats; Adjuvants; Malaria; Vaccination.

It has been known for many years that bad antigens can be made immunogenic and good antigens can be persuaded to be even better by the addition of certain substances or combination of substances known as adjuvants. The best known ‘class of adjuvants is the oil and water mixtures such as Freund’s adjuvant, but as White (1967) has noted, the variety of adjuvants that have been successfully used reads like a “mediaeval alchemists’s shopping list.” It is also known that adjuvants have a selective activity in respect of enhancing different antigens. However, a systematic evaluation of adjuvants has not been heretofore applied to malaria immunization. Freund’s complete adjuvant (FCA) has been employed in association with nonliving antigens of avian (Freund et aL 1945; Coffin 1951), rodent (Zuckerman et ~2. 1967), and primate ( Freund et al. 1945; 1948; Targett and Fulton 1965;

Despite some 50 years of research on the problem, an effective practical means of artificial immunization against malaria by use of a nonliving vaccine is yet to be developed. One possible reason for this failure may be a relative lack of immunogenicity of the antigens employed for va’ccination. It has been shown that it requires a dose of approximately 1500 mg/kg of nonviable Plasmodium berghei antigen to induce partial immunity in the mouse (Jerusalem 1969) and 500 mg/kg in the weanling rat, an amount calculated by Jerusalem from the results reported by Zuckerman et al. ( 1967). If this were true, one possible means of overcoming this difficulty would be by adding adjuvants to the antigen. 1 This study was supported by the U. S. Amly Medical Research and Development Command Contract DADA 17-72-C-2112. This is contribution No. 1254 to the Army Research Programs on Malaria. 6 Copyright c 1975 by Academic Press, Inc. All rights oP reproduction in any form reserved.

P. bf3@&:

ANTIGEN

Brown et al. 1970; Schenkel et al. 1973) plasmodia. The results of all these experiments were variable but in genera1 would indicate that FCA can promote an adequate level of protective immunity. Desowitz (1967) reported that rats immunized with soluble P. berghei antigen bound to carboxymethylcellulose exhibited a parasitemia one-third as high as that of controls after challenge. Jacobs (1943) noted that bacterial endotoxin enhanced the immunogenicity of a vaccine of P. lophurae. The present communication reports the results of a program to screen effective adjuvants in a soluble P. berghei antigenweanling white rat experimental system. MATERIALS

AND METHODS

The selection of a suitable laboratory animal for testing the immunogenicity of P. berghei vaccines presents certain problems. The adult mouse in respect of P. berghei, does not seem to have a suffiand/or ciently adequate immunological physiological potential to respond to plasmodial vaccines that would normally be immunogenic. The weanling rat possesses a number of desirable ‘characteristics; not only is the course of infection in these animals sufficiently intense that mortality rates and parasitemia can be used as measures of antigen efficacity, but also their immune potential is sufficient to elicit an observable level of protection when appropriately stimulated. However, the well known ageresistance of the rat to P. beltghei limits their use to the sussceptible period, i.e., between approximately six and nine weeks of age (about 50-100 g in weight). Given this relatively short working time, only the primary response to a vaccine can generally be assayed. Thus, the selection of the weanling rat represents a compromise, but under the circumstances it is believed to be the best compromise possible. Inbred Wistar strain white rats weighing 50-55 g were used in these experiments. For screening purposes, groups of 10 ani-

7

ENHANCEMENT

mals were given a subcutaneous injection of the vaccine under trial and 10 animals set aside to serve as untreated controls. Ten days later all animals were given an intraperitoneal inoculation of 1 X lo6 P. berghei-infected rat erythrocytes (NYU-2 strain) and thin tail blood films taken daily or every other day thereafter. Parasitemia was assessed by obtaining the percentage of infected cells in 200 randomly observed erythrocytes in the Giemsa-stained thin ,blood film. When the screening trial indicated that a parti’cular antigen preparation possessed immunogenic activity, the experiment was repeated. Soluble P. berghei antigen was prepared by saponization of heavily infected rat blood followed by disintegration of the isolated parasites in a Hughes press. The method followed that described by Zuckerman et al. ( 1967). Each batch of antigen was standardized by Biuret analysis estimated to contain 1.5 g ,protein/lOO ml. The immunizing dose given was 0.1 ml of the soluble antigen alone or mixed with an equal volume of adjuvant dissolved, in the case of soluble adjuvants, in sterile physiological saline. The kinds and amounts of adjuvants given are shown in Table I. Precipitated antigen was prepared by adding, drop by drop, a 10% solution of precipitant (Table I) to 1 ml of plasmodial extract until what appeared to be maximum turbidity was observed. The precipitate was obtained by centrifugation. The supernatant was collected and more precipitant added to insure that the reaction had been completed. The precipitate was washed three times by centrifugation and finally resuspended in 1 ml lphysiological saline. Each rat received a 0.1 ml immunizing dose of the suspension. RESULTS

Evidence of immunogenic activity was considered to be present if one or more of the following criteria were met. As compared to the controls, the immunized

8

ROBERT

S, DES0 WIT2

group had (a) a lower mortality rate, (b) a lower average peak parasitemia in the survivors, (c) a shorter patent period, and (d) a longer survival time of the fatal infections. Table I, which summarizes the results of these experiments, shows that several adjuvant-antigen combinations and antigen preparations were immunogenic by these standards. Untreated Controls The mortality rate of the untreated control group was 62.870, the majority succumbing to a fulminating infection (average peak parasitemia, 65.8% ) within 19 days of challenge. The surviving control rats developed lower parasitemias (average peak 24.6% ) th an those of the fatal infections. Antigen Con&d.s A single immunizing dose of soluble antigen did not induce a protective immunity. Mortality rates and the course in infection in both the fatal infections and survivors was essentially the same as that of the untreated controls. Surfactants The two surfactants tested, saponin and hexylamine, induced a high order of protective immunity when used in combination with the soluble P. berghei antigen. The mortality rate was reduced from 62.8% in the controls to approximately 109) in the vaccinated groups. Moreover, the infections in the immunized survivor rats were generally of a relatively low parasitemia, the average peak being llojo, and in the case of the saponin: antigen-immunized group, it was of a transitory nature, the average patent period being three days shorter than that of the surviving controls. Saponin without antigen did not induce any protective effect.

Bacterial Adjuvants Pertussis vaccine proved to be an effective adjuvant. While the mortality rate of the pertussis adjuvanted P. berghei antigen was somewhat greater than the surfactant: antigen-immunized groups, the course of infection in the protected animals of both groups was similar, as characterized by a low average peak parasitemia ( 10.1% ) and a short patent period (12.2 days). Pertussis vaccine without the plasmodial antigen produced no demonstrable protection. Serratiu marcesem lipopolysaccharide endotoxin did not have a demonstrable immunoenhancing adjuvant effect. Synthetic Polynucleoticles When polyadenylic-polyuredelic acid ( poly A : U ) and polyinosinic-polycytidylic acid (poly I:C) were injected as a mixture with antigen, no protective immunity was induced. That there may be a strategic time sequence in administration is indicated by the group give poly 1:C two days before the injection of antigen. While the mortality rate in this group was only moderately reduced, the surviving rats seemed to have had a high level of induced protective immunity as reflected by an average peak parasitemia and a patent period approximately one half that of the surviving untreated control group. Phenylimidothiazole

(Levamisole)

Levamisole when given either 24 hr before or with the antigen had a definite immunoenhancing effect in that the mortality rate of these groups was approximately one-third that of the control groups. However, the degree of protective immunity induced by antigen with Levamisole adjuvant did not cause a reduction in parasitemia beyond that of the control group survivors nor was the patent period appreciably shorter. Levamisole alone or

Controls Soluble antigen alone Freunds complete adjuvant : antigen Saponin: antigen Saponin Hexylamine : antigen Pertussis vaccine: antigen Pertussis vaccine Serratia marcescens lipopolysaccharide: antigen Poly A: U : antigen Poly I : C : Antigen Poly I:C, Antigen given 2 days later Levamisole: antigen Levamisole, antigen given 24 hours later Antigen, Levamisole given 24 hours later Levamisole Vitamin A, antigen given 24 hours later Aluminum chloride precipatated antigen Ferric alum : precipitated antigen Aluminum ammonium sulfate (alum) precipitated antigen

Group

70.0 10.0 90.u 11.1 26.6 90.0

50.0 70.0 77.7 40.0

20 30 10 18 30 10 30 9 10 20 30 10 10 10 10 10 9 10

100 rg 100 rg ,500 pg 6.5 opu 6.5 opu

100 Pg 500 /hg 100 rg

100 rg 1 mg

1 mg

1 mg 1 mg

15 mg

50.0 50.0

20.0

40.0 26.6

53.3 77.8 60.0

62.8 57.5

150 40

Mortality (%I

Rats Vaccinated

No. in group

in Weanling

Amount adjuvant injected

Data on the Course of Infection

TABLE

I

23.8 25.1 9.5 22.7

52.5 f 49.4 f 28.2 f

21.9 29.2

20.0

30.5 20.9

49.8 f

46.6 f 47.7 f

35.3 f

27.7 f 31.6 f

48.4 f 54.1 f 43.9 f

25.4 22.6 20.7

25.5 10.1 8.9 17.8 22.9 15.7

53.4 14.0 60.5 17.1 23.7 61.0

f f f f f f

23.3 26.4

50.7 f 46.0 f

Average peak parasitemia of group (% f SD)

with a Nonliving

12.0 12.5

50.0 f

53.5 f

65.7 f

61.8 f

66.8 f 72.5 f

68.0 f

12.3

5.1

14.5

7.9

3.4 10.6

5.6

68.0 f 7.8 54.0 f 23.6

14.9 14.9 9.6

f 10.2 f 13.4 f 8.6 xt 1.4 f 6.0 f 13.3

63.5 f 63.4 f 58.5 f

68.3 37..5 61.6 64.0 59.0 64.0

65.8 f 65.0 f

Average peak parasitemia of fatal infections (% f SD)

P. berghei Antigen

f f f f f f

9.7 f

35.0 f

21.6 f

39.0 f

26.4 f 23.0 f

28.0 f

7.7 f 21.0 f

28.7 f 21.5 f 21.7 f

18.6 11.4 50.0 11.8 10.1 34

24.6 f 28.8 f

7.5

6.5

13.0

13.4

7.4 6.2

12.2

3.7 9.6

22.5 2.1 7.8

5.0 5.6 0 6.3 4.0 0

12.5 16.3

Average peak parasitemia of survivors (% f SD)

in Conjunction

f f f f f f

2.7 1.4 4.0

0 2.6 0 1.5 2.7 0

3.7 3.1

13.4 f

19.0 f

15.0 f

19.2 f

16.8 f 15.0 f

16.0 f

1.5

1.4

1.7

2.5

1.1 1.4

2.8

8.9 f 3.5 14.7 f 2.1

17.4 f 16.0 f 16.2 f

15.0 12.9 15.0 16.3 12.2 16.0

16.0 f 16.4 f

20.7 f

19.1 f

18.2 f

18.8 f

18.2 f 22.0 f

22.0 f

4.8

2.1

2.9

3.7

0.45 2.8

0

1.0 2.8

20.5 f 20.0 f

2.0 1.5 3.3 0 2.0 1.9 2.0 1.0 3.0

f f f f f f

2.7 3.2

19.2 f 19.0 f 18.6 f

15.3 14.6 20.8 17.0 16.0 20.8

19.2 f 18.4 f

Average survival time : fatal infections (Days f SD)

of Adjuvants

Average patency of survivors (Days rt SD)

with a Variety

ii

!2 $ z

10

ROBERT

S. DESOWITZ

given 24 hr after antigen did not show an appreciable adjuvant effect. Other Adjuvan,ts Neither vitamin A nor Freund’s complete adjuvant possessed any immunoenhancing properties. Precipitated

Antigen

Suspensions of antigen that had been precipitated by ferric alum and aluminum The ‘chloride were not immunogenic. screening trial of aluminum alum-precipitated antigen indicates that this preparation may induce a protective immunity. The mortality rate in this group was somewhat lower than that of the untreated controls (40% as compared to 62.8% ) and the surviving va’ccinated animals had a relatively transient course of infection characterized by a low-peak parasitemia. DISCUSSION

The objective of this study was to determine whether a feebly immunogenic plasmodial antigen could be made to induce a protective immunity when combined with active adjuvants. The results indicate that within the limitations of the certain experimental system employed, adjuvants, such as surfactants, B. pertussis vaccine and levamisole, when given in conjunction with soluble P. berghei antigen appreciably decrease mortality, lower the parasitemia and attenuate the course of infection. While the principle of selective adjuvation is believed to have been demonstrated it should be kept in mind that the present investigation was essentially of a screening nature. Further systematisc study is required to determine the most efficacious adjuvant : antigen systems and strategic immunizing schedule. The mechanism(s) by which different adjuvants exert their immunoenhancing effect is still not well understood (Munder and Modolell 1973). The difficulty in at-

tempting to relate the mode of adjuvant action to immunity is further ‘compounded by the many uncertainties still surrounding the mechanisms responsible for functional immunity in malaria. The results obtained in this present study are similar to that of Johnson et al. (1963) who demonstrated the ineffectiveness of Freund’s complete adjuvant and the effectiveness of saponin and syntheti’c surfactants as adjuvants of soluble antigens in inducing protective immunity in mice to Trypanosoma congolense. Gall (1967) has hypothesized that the active surfactant adjuvants, c.g., saponin, and various synthetic nonionic, anionic and cationic agents act as linking molecules between tissue cells and the antigen, “their highly charged polar groups reacting with the protein antigens and their nonpolar halves with the lipid elements of the cell membrane.” He also has suggested (Gall 1966) that these adjuvants damage the membranes of lymphoid cells. Dresser (1968) extended this hypothesis by further suggesting that the damage stimulates division of immunologically competent cells. Munder and Modolell (1973) present evidence that these adjuvants act by inducing profound changes in the phospholipid metabolism of macrophages. However, if there is a common modality in the mechanism of surfactant adjuvation there may also exist some specific antigen-adjuvant relationship. For example, vitamin A, which is another putative membrane-affecting adjuvant (Dresser 1968), was not immunoenhancing for P. berghei antigen, whereas saponin and the amine surfactant hexylamine was. Bordetella pertussis vaccine, an effective adjuvant for a wide variety of antigens, is also thought to act by inducing an accelerated and prolonged multiplication of antibody-forming cells (Finger et al. 1967; Rowley et al. 1968). Since it has been shown that IgG-producing cells are more sensitive to the adjuvant activity of B. pertussis than IgM producers (Dresser et

P. berghei:

ANTIGEN

ENHANCEMENT

11

al. 1970; Finger et al. 1968), it is probable that a higher than usual level of IgG antibody occurs in the primary response after immunization with this type of adjuvantantigen ‘combination. There is convincing eviden’ce that specific IgG antibody plays an important role in protective immunity to malaria (Diggs and Osler 1969; Stechschulte et al. 1969), and the protection afforded by immunization with B. pertussis: P. berghei antigen shown in this study, may be due to an augmented concentration of this immunoglobulin. The mode of bacterial endotoxin adjuvation may differ from that of B. pertussis in that it is variously thought to stimulate immunocompetent cells of both thymus and bone marrow origins (Nakano et al. 1973) and to accelerate the aging of macrophages (Winchurch and Braun 1969). In this present study, Serratiu marcescens endotoxin had little or no adjuvant activity; however, Martin et al. (1967) and MacGregor et al. (1969) reported that Escherichiu coli endotoxin alone had a moderately suppressive effect on the course of P. berghei infections of rats and mice and SalmoneZla typhosa endotoxin had a similar effelct on the course of P. Zophurae in ,chickens (Barrett et al. 1971). It has been similarly assayed for a nonspecific effect against trypanosomes with variable results. It induced a degree of resistance in rats against Typarzosomu equiperdum (Foris et al. 1970) but increased the susceptibility of mice to T. cruzi ( Kierszenbaum and Saavedra 1972). Immunization with poly 1% and antigen did not cause a marked reduction in the mortality rate of the group as compared to the controls. However, the animals that were protected appeared to have had a high level of immunity; the average peak parasitemia was less than one third that of the control survivors, and the course of infections was shortened by an average of seven days. Poly I: C alone, when injected

induced P. berghei, as evidenced by a lengthened prepatent period and a prolonged course of infection (Jahiel et al. 1969). The authors attributed the protection afforded by the polynucleotide to its interferon-inducing ability. However, the synthetic ~polynucleotides appear to have a diverse immunological effect when used as an adjuvant to specific antigens. Under these #conditions, they have been shown to amplify the proliferation of T cells (Cone and Johnson 1972), and enhance the production of IgG and IgM antibodies ( Schmidtke and Johnson 1971) . Recently, Schenkel et al. (1973) reported that poly A:U with P. know?& antigen was not immunogenic for rhesus monkeys. Phenylimidothiazole (levamisole) is anthelminthic drug that has excited current interest in its immunologic activity, particularly for its apparent #potent ability to restore activity in deficient or depressed states. Renoux and Renoux (1971) have shown that levamisole does not affect the course of primary invasion of bacteria, but rather increases resistance to challenge in hosts previously sensitized to the antigen. It is therefore of interest that in these present experiments, levamisole was an effective adjuvant in enhancing the primary response when given with or before the P. berghei antigen. There is evidence the levamisole acts by increasing phagocytic activity of macrophages (Hoebeke and Franchi 1973). The effect on humoral immunity is uncertain. An increase of antibody titer has been observed in some studies (Brugmans et al. 1973; Kulkarni et al. 1973) but not in others (Renoux and Renoux 1973). It will be of interest to elucidate the mechanism( s ) by which levamisole and other active adjuvants exert their immunoenhancing effect against rodent malaria. Of even greater importance, however, is the determination of whether the observations made for the rodent ma-

into mice, has been shown to induce a modest protective effect against sporozoite-

laria system can be extended to the primate malarias.

12

ROBERT S. DESOWITZ ACKNOWLEDGMENTS

The skillful technical assistance Kramer is gratefully acknowledged.

of Mr. Kenton

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ANTIGEN

KIERSZENBAUM, F., AND SAAVEDRA, L. E. 1972. The effects of bacterial endotoxin on the infection of mice with Trypunosoma cruzi. Journal of Protozoology 19, 655-657. KULKARNI, V. B., MULBAGAL, A. N., PARANJAPE, V. L., KHOT, J. B., AND MANDA, A. V. 1973. Immunostimulating effect of tetramisole on antibody formation against Newcastle disease virus in chicks. Indian Veterinary Journal 50, 225-227. MACGREGOR, R. R., SHEAGREN, J. N., AND WOLFF, S. M. 1969. Endotoxin-induced modification of Plasmodium berghei infection in mice. Journal of Immunology 102, 131-138. MARTIN, L. K., EINHEBER, A., SADUN, E. H., AND WREN, L. E. 1967. Effect of bacterial endotoxin on the course of Plasmodium berghei infection. Experimental Parasitology 20, 186199. MUNDER, P. G., AND MODOLELL, M. 1973. Adjuvant induced formation of lysophosphatides and their role in the immune reslponse. International Anchives of Allergy 45, 133-135. NAKANO, M., UCHIYAMA, T., AND SAITO, K. 1973. Adjuvant effect of endotoxin; antibody response to sheep erythrocytes in mice after transfer of syngeneic lymphoid cells treated with bacterial lipopolysaccharide in vitro. JournaZ of Immunology 110, 408-413. RENOUX, G., AND RENOUX, M. 1971. Immunostimulant effect of an imidothiazole in the immunization of mice infected with Brucellu abortus. Comptes Rendus de I’Academie des Science 272, 349-355. RENOUX, G., AND RENOUX, M. 1973. Stimulation of anti-Brucella vaccination in mice by tetramisalt. Infection sole, a phenyl-imidothiazole and Immunity 8, 544-552. ROWLEY, D. A., FITCH, F. W., MOSIER, D. E., SOLLIDAY, S., COPPLESON, L. W., AND BROWN, B. W. 1968. The rate of division of antibody-

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forming cells during the early primary imJournal of Experimental mune response. Medicine 127, 983-1002. SCHENKEL, R. H., SIMPSON, 6. L., AND SILVERMAN, P. H. 1973. Vaccination of Rhesus monkeys (Macaca mulutta) against Plasmodium knowlesi by the use of nonviable antigen. Bulletin Would Health Organization 48,597-604. SCHMIDTKE, J. R., AND JOHNSON, A. G. 1971. Regulation of the immune system by synthetic polynucleotides. I. Characteristics of adjuvant action on antibody synthesis. Journal of Immunology 106, 1191-1200. STECHSCHULTE, D. U., BRIGGS, N. T., AND WELLDE, B. T. 1969. Characterization of protective antibodies produced in Plasmodium berghei infected rats. Military Medicine 134, 11401152. TARGETT, G. A. T., AND FULTON, J. D. 1965. Immunization of Rhesus monkeys against Plusmodium knowlesi malaria. Experimental Parasitology 17, 180-193. WHITE, R. G. 1967. Concepts relating to the mode of action of adjuvants. In “International Symposium on Adjuvants of Immunity,” pp. 3-12. Symposium series in immunological standardization. S. Karger AG, Basel, Switzerland. WINCHURCH, R., AND BRAUN, W. 1969. Antibody formation: Premature production by endotoxin or synthetic nucleotides in newborn mice. Nature (London) 223, 843-844. ZUCKERMAN, A., HAMBURGER, J., AND SPIRA, D. 1967. Active immunization of rats with a cell-free extract of the erythrocytic parasites of Plasmodium berghei. Experimental Parasitology 21, 84-97. ZUCKERILIAN, A., SPIRA, D., AND HAMBURGER, J. 1967. A procedure for the harvesting of mammalian plasmodia. Bulletin of the World Health Organization 37, 431-436.

Plasmodium berghei: immunologic enhancement of antigen by adjuvant addition.

EXPERIMENTAL PARASITOLOGY 38, 6-13 Plasmodium ( 1975) bergh,ei: Immunogenic Enhancement Antigen by Adjuvant Addition’ ROBERT Department S. of...
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