CELLULAR

IMMUNOLOGY

Distinctive

21, 2‘+3-249 (1976)

Adjuvanticity of Synthetic Analogs of Mycobacterial Water-Soluble Components F.

AUDIEERT

AND

L.

CHBDID

1

ImmunothP’rapie Expirimenfale,

Instituf Pasteur, Groupe de Recherche No. 31 du C.N.R.S,. Paris, France AND

P.

LEFRANCIER

In&&t

AND

Choay,

Received

Paris,

October

J.

CHOAY

France 8, 1975

Synthetic N-acetyl-muramyl+alanyl-n-isoglutamine represents the minimal structure capable of duplicating the activity of mycobacteria in Freund’s complete adjuvant. In contrast to mycobacterial adjuvant preparations, that function only in the form of water-in-oil emulsions, this compound and a second synthetic analog (N-acetylmuramyl-L-alanyl-n-isoglutamic acid) augment the humoral immune responses of mice equally as well as aqueous solutions. Whereas N-acetyl-muramyl-L-alanyl-uisoglutamic acid administered to guinea pigs in water-in-oil emulsion has no effect, N-acetyl-muramyl-L-alanyl-D-isoglutamine induces delayed hypersensitivity to ovalbumin and azobenezene-arsonate N-acetyl-L-tyrosine and increases the level of antibody against ovalbumin. Under these conditions, challenge with the synthetic adjuvants themselves evokes no skin responses. Moreover, Freund’s complete adjuvant sensitizes guinea pigs to tuberculin and to native mycobacterial water-soluble adjuvant but not to the synthetic analogs.

INTRODUCTION The chemistry of adjuvant fractions of mycobacteria has been investigated for many years, but it is only recently that the relevant structures have been identified. It has been established that water-soluble fractions can substitute for mycobacterial cells in Freund’s complete adjuvant (FCA) (l-4). One such component, referred to as water-soluble adjuvant (WSA) was extracted by Adam et al. from Mycobacterium smegmatis and proved to be an arabinogalactan linked to a peptidoglycan (1). Subsequently, neutral sugars were removed from the peptidoglycan moiety (5) and monomeric peptidoglycans extracted from M. smeg~mztis and EschericClia COG also turned out to be active adjuvants (6, 7). Most recently, it was demonstrated that synthetic N-acetyl-muramyl-L-alanyl-D-isoglutamine had the minimal structure required for adjuvant activity (8, 9). In the latter studies, the adjuvant activity was demonstrated by immunization of guinea pigs with 1 Address reprint request to : Dr. L. Chtiid, 28 rue du Dr. Roux, F 5015 Paris, France.

Institut Pasteur ImmunothCrapie Experimentale,

243 Copyright @ 1976 by Academic Press, All rights of reproduction in any form

244

AUDIBERT

ET

AL.

ovalbumin in a water-in-oil (Bay01 F, Arlacel) emulsion : Precipitating antibodies were increased and delayed hypersensitivity was induced. Although WSA administered with antigen in saline has also been shown to increase the immune response both in Z&O (10) and irt vitro (11)) this effect was slight compared to that obtained with the same material administered in Freund’s incomplete adjuvant (FIA). Concurrently, we found however that acylated derivatives of WSA, even when given in saline, markedly enhanced the humoral antibody response to soluble antigens (unpublished observation). This development led us to test N-acetylmuramyl-r.-ananyl-n-isoglutamine (Mur-NAc-L-Ala-n-iso-Gln) and a synthetic analog having no amide function, N-acetyl-muramyl-L-alanyl-n-glutamic acid ( Mur-NAc-L-Ala-D-Glu) administered with antigen to ascertain their adjuvant capabilities in the absence of water-in-oil emulsion. In these experiments lipopolysaccharide (LPS) was administered to controls since endotoxins given with a saline solution of antigen constitute strong adjuvants of the antibody response (12, 13). In the work reported here we have also investigated the antigenic and adjuvant activity of these compounds administered to guinea pigs in FIA alone and with antigens such as ovalbumin and azobenzene-arsonate N-acetyl+tyrosine ( ABA-tyrosine) . Studies with ABA-tyrosine were particularly revealing inasmuch as this antigen is not immunogenic in FIA but does induce a pure state of delayed hypersensitivity when given in FCA (14, 15). MATERIALS

AND

METHODS

Animals. Two-month-old Swiss common stock mice (C.N.R.S. Orleans Center), Bouscat rabbits (4-6 weeks old) and male Hartley guinea pigs (Pasteur Institute) weighing 350 g were used. Preparations. WSA was extracted from M. smegmatis cells according to the procedure described by Adam et al. (16). The synthetic analog, Mur-NAc-L-AlaD-Glu, was prepared by Lefrancier and Choay (unpublished data). Lipopolysaccharide, prepared by the phenol-water procedure from S. enteritidis, FIA and FCA were purchased from Difco Laboratories. Antigens. These were highly purified preparations of egg albumin and bovine serum albumin (BSA) (Miles Laboratories). Azobenzene-arsonate N-acetyl L-tyrosine (ABA-tyrosine) and azobenzene arsonate of BSA (ABA-BSA) were prepared according to procedures previously described (15). Purified and old tuberculin (Pasteur Institute) extracted from Mycobacteriurn bovis were also used. Antibody estimation. Anti-ovalbumin and anti-BSA were determined by passive hemagglutination with formalinized antigen-coated sheep red blood cells (17). Anti-ovalbumin was also evaluated by quantitative precipitation, by means of Folin’s method and anti-BSA by the antigen-binding Farr technique (IS). RESULTS Adjuvant

Activity

of Synthetic

Analog Administered

with BSA in Saline

In these experiments, controls were immunized with 0.5 mg of BSA and a recall of 0.1 mg of BSA 30 days later. The experimental groups (eight mice each) received with the first injection of BSA, either LPS (30-100 ,ug) or Mur-NAc-LAla-o-iso-Gln (30-300 pg) and were then bled at various intervals.

SYNTHETIC

ADJUVANTS,

EFFECTIVE

TABLE

IN

24.5

SALINE

1

Comparison of Mur-NAc-L-Ala-D-iso-Gin with LPS for Enhancement of the Immune Response of Mice to BSA in Saline Immunization

Primary

response

_

Day 14

Day 28 ---

Antigen (controls) Antigen + LPS, 30 fig Antigen + LPS, 100 pg Antigen + Mur-NAc-L-AlaD-iso-Gin, 30 pg Antigen + Mur-NAc-L-AlaD-iso-Gin, 300 pg

_

Secondary response ______--.

---.

Day 34 -.--_

_

Day 36 _~.. ABC

PAa ABC.

PA

ABC

PA

ABC

Distinctive adjuvanticity of synthetic analogs of mycobacterial water-soluble components.

CELLULAR IMMUNOLOGY Distinctive 21, 2‘+3-249 (1976) Adjuvanticity of Synthetic Analogs of Mycobacterial Water-Soluble Components F. AUDIEERT AND...
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