JOURNAL OF PATHOLOGY, VOL. 164: 265-27 1 (1 99 1 )

HISTOPATHOLOGICAL STUDIES ON THE LOCAL REACTIONS INDUCED BY COMPLETE

FREUND'S ADJUVANT (CFA), BACTERIAL LIPOPOLYSACCHARIDE (LPS), AND SYNTHETIC

LIPOPEPTIDE (P3C) CONJUGATES FRANK WIEDEMANN*, RALF LINK*, KONRAD PUMPET, ULRICH JACOBSHAGENT, HANS E. SCHAEFER?,

KARL-HEINZ WIESMULLER~, ROLF-PETER HUMMEL~, GUNTHER J U N G ~ , WOLFGANG BESSLER* AND TRAUDEL BOLTZ*

*Institute of lmmunobiology and ?Department of Pathology, University of Freiburg, Stefan-Meier-Str. 8,0-7800 Freiburg. F.R.G.; $Institute of Organic Chemistry, University of Tubingen. Auf der Morgensteile 18. 0-7400 Tubingen,

F.R.G.

Received 25 October 1990 Accepted 10 January 1991

SUMMARY

The inflammatory reactions following subcutaneous application of adjuvants revealed characteristic pathological patterns. The injection of complete Freund's adjuvant (CFA) resulted in the formation of large lipid deposits encircled by an inflammatory reaction and concentrically arranged collagen bundles. Bacterial lipopolysaccharide (LPS) caused granulomatous aggregations of mononuclear cells with thrombotic vessel occlusions. Inoculation of the lipopeptide adjuvants induced accumulation of mononculear cells with only minimal fibrotic changes which were resolved after day 28. Lipopeptide conjugates based on the head group tripalmitoyl-S-glyceryl-cysteinyl-serin (P,CS) can thus be used as effective immunogens and adjuvants without long-term tissue damage.

KEY WORDS-Histopathoiogy, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (FA), lipopeptide (P,CS), lipopolysaccharide (LPS), synthetic vaccine, adjuvants.

INTRODUCTION

The rapid advances in recombinant DNA tech- nology and biochemistry have opened the way to antigenic determinants and vaccines via the syn- thesis of peptide segments." Because of the poor immunogenicity of these peptides, the search for appropriate carrier molecules and adjuvants in sub- unit vaccines is of A variety of irnrnunostimulatory reagents such as mineral oil, metabolizable lipid compounds, components of microorganisms, aluminium, and calcium salts enhance the immune response by different mechan- isms such as prolonged antigen presentation and

Addressee for correspondence: Professor Dr Giinther Jung, Institute of Organic Chemistry, University of Tiibingen, Auf der Morgenstelle 18, D-7400 Tiibingen, F.R.G.

0022-341 7/91/07026547 $05.00 0 199 1 by John Wiley & Sons, Ltd.

secretion of lymphokines.6 Bacterial cell wall com- ponents like lipopolysaccharide (LPS) or lipo- protein from Gram-negative bacteria have been demonstrated to be potent B-cell and macrophage stimulator^.^ The lipopeptide portion of the biologi- cally active N-terminal part of the lipoprotein of Escherichia coli, P,CS [N-palmitoyl-S-(2,3-bis(pal- mi toyloxy))-(2RS)-propyl)-(R)-cysteinyl-(S)- serine], can be coupled to synthetic peptides.' These low-molecular-weight conjugates with built-in adjuvanticity induced specific IgG and IgM responses in vivo and in vitro.'-" Moreover, a low- molecular-weight vaccine consisting of P,CS covalently coupled to an antigenic determinant of VPl, a structural protein of foot-and-mouth disease virus, induces protection against foot-and-mouth disease and serotype-specific virus neutralizing anti- bodies in guinea pigs after a single administration

266 F. WIEDEMANN E T A L .

without any additional adjuvant or carrier. I 2 , l 3

Furthermore, synthetic viral peptides covalently linked to P,CS can efficiently prime influenza-virus- specific cytotoxic T-lymphocytes in vivo.I4 The aim of the present study was to investigate the tissue reactions at the injection site induced by P,CS and P,CS covalently linked to a synthetic peptide derived from the amino acid sequence of the trans- membrane protein gp41 of HIV-1I5. The inflamma- tory changes after inoculation of P,CS were compared with those caused by complete Freund’s adjuvant, incomplete Freund’s adjuvant and LPS.

MATERIALS AND METHODS

Reagents and adjuvant preparations P,CS was synthesized as described elsewhere.n

The peptide segment HIV-1(586606) (R-ILA- VERYLKDQQLLGIWG-Aba-S; Aba = a-amino- butyric acid as a substitute for cysteine) was obtained by using the Fmoc/tBu approach for the solid phase method of fully automated stepwise pep- tide synthesis. In the last steps, the adjuvant and membrane anchor group P,CS was attached to the peptide using methods described elsewhere. l o

Complete Freund’s adjuvant (CFA) and incomplete Freund’s adjuvant (IFA) were purchased from Difco (Detroit, U.S.A.); lecithin (1,2-dipal- mitoyl-sn-glycero-3-phosphocholine) from Sigma (Taufkirchen, F.R.G.). CFA and IFA were emulsi- fied with an equal volume of sterile, pyrogen-free saline or lipopeptide solution by sonication. P,CS and P,CS-HIV-1 peptide conjugate were dissolved in saline with an equal amount of lecithin by soni- cation. Lipopolysaccharide (LPS) from S. abortus egui was kindly provided by Dr Kleine, Freiburg.

Animals Six- to eight-week-old female BALB/c mice were

provided by Professor Rossbach, Institute for Bio- medical Research, Fiillinsdorf, Switzerland. The animals were divided into nine groups and injected with 5Opl subcutaneously in the ventral aspect of the flank (group 1: FCA; group 2: FCA+50pg HIV- 1 peptide; group 3: FCA + 50 pg P,CS-HIV-1 peptide conjugate; group 4: FIA; group 5: 50pg P,CS; group 6 : 50pg P,CS-HIV-1 peptide conju- gate; group 7: LPS; group 8: saline; group 9: 50 pg lecithin).

Histology Four to eight animals of each group were killed

under ether anaesthesia at intervals of 2,5,7,14,28,

and 84 days after injection of the adjuvant prep- arations. The injection sites were excised, fixed in buffered formalin, and processed by standard methods for the histological studies. Paraffin sec- tions were stained with haematoxylin and eosin and Elastic van Gieson stain. In addition, selected sections were stained for chloroacetate esterase.

Immunization and assay of antigen-specijic antibody Groups of four mice were immunized intraperi-

toneally with 0.2 ml of the indicated antigen emulsi- fied in sterile saline with an equal amount of lecithin by sonication. Preliminary studies demonstrated that injection of the lipopeptide conjugate via the intraperitoneal route as well as via the subcutaneous route resulted in comparable antibody levels. The mice were bled at day 14 and the sera stored at -20°C. Antibody levels were measured by enzyme- linked immunoassay (ELISA). Briefly, 96-well polystyrene microtitre plates (Dynatech, M 129B, Denkendorf, F.R.G.) were coated with sonicated P,CS-HIV- l(586-606) conjugate in phosphate- buffered saline (PBS) at a concentration of 1 pg/well and air-dried at 37°C overnight. The wells were blocked with I per cent BSA in PBS, washed, and incubated overnight with the serum samples in serial dilutions. The plates were washed and 5Opl of affinity-purified horseradish peroxidase-conjugated rabbit anti-mouse IgG (Dianova, Hamburg, F.R.G.) was added for 2 h. After washing, 50p1 of substrate solution (5 mg 2,2’-azino-bis(3- ethyl-benzthiazoline-6-sulphonate) (Sigma Chemie, Taufkirchen, F.R.G.) and 0.03 per cent H,O, in 10 ml of 0.1 M citrate buffer (pH 4.2) was added for 15 min. The optical densities were analysed at 405nm using an automated ELISA reader (SLT EAR 400, Austria).

RESULTS

Group 1: injection of CFA The inoculation of CFA resulted in the formation

of a large lipid deposition in the subcutaneous tissue surrounded by an inflammatory reaction. At day 2, the infiltrating wall consisted of dense layers of polymorphonuclear leucocytes and macrophages around necrotic areas. In the 7-day specimens, the majority of the cells surrounding the lipid vacuole (Fig. 1) were macrophages and lymphocytes fol- lowed by a zone with fibrin exudate and few poly- morphs. In the external layer fibroblasts increased

LOCAL REACTIONS INDUCED BY ADJUVANTS 267

Fig. I-Day 7 after CFA injection. Infiltrating wall consisting of fibrin exudate and inflammatory cells around necrotic areas. Elastic van Gieson (EVG)

in number. Formation ofcollagen and vascular pro- liferation could be demonstrated (Fig. 2). By 3 months, lipid vacuoles were still present, sur- rounded by macrophages, lymphocytes, and fibro- blasts, and encircled by concentrically arranged compact collagen bundles (Fig. 3). Multiple granulomas were scattered throughout the densely infiltrated subcutaneous tissue.

Groups 2 and 3: injection of HIV-I (586-606) peptide solution or P,CS-HIV-I (586406) peptide conjugate solution each emulsijied in CFA

Application of CFA in combination with HIV- l(586606) peptide or CFA together with P,CS- HIV- I (58G606) peptide conjugate revealed similar histopathological patterns to those following injec- tion of CFA alone. By day 28, the lipid depot was surrounded by inflammatory cells and dense collagen fibres (Fig. 4).

Group 4: injection o f IFA The lipid vacuoles were similar to those seen after

application of CFA but the surrounding inflamma- tory reaction with neutrophil granuloctyes was less

Fig. 2-Day 7 after CFA injection. The fibrin exudate zone is surrounded by a layer of macrophages, lymphocytes, and poly- morphonuclear leucocytes, and encircled by concentric collagen bundles. EVG

pronounced. By day 7, collagen formation was detectable. At day 28, the wall consisted of several layers of macrophages, neutrophils, fibrocytes and collagen.

Groups 5 and 6: injection of P,CS or P,CS-HIV-I peptide conjugate

At day 2 following inoculation of P,CS, accumu- lation of polymorphonuclear leucocytes with patchy fibrinoid necroses could be detected in the slightly oedematous connective tissue (Fig. 5). By day 7, thelesion appeared to besmallerand flattened. The dominant cell types were macrophages and epitheloid cells surrounded by fibroblasts. In some sections giant cells could be demonstrated (Fig. 6). In contrast to the fibrotic changes following CFA, only slight collagen formation could be observed. At day 28, a small residual infiltrate with lympho- cytes, foamy cells, and occasional giant cells remained at the injection site (Fig. 7). By day 84, the infiltration was completely resolved. Similar histopathological changes could be detected after injection of P,CS-HIV- I(586-606) peptide conjugate.

268 F. WIEDEMANN E T A L.

Flg. 3-Day 28 after CFA injection. Granuloma formation in the subcutaneous tissue. Vessels and nerves are surrounded by

Fig, &Day 7 after injection of the peptideconjugate P,CS-HIV- l(586606) emulsified in CFA. Similar infiltration to that

concentric collagen bundles. EVG following the injection of CFA alone. EVG

Group 7: injection of LPS During the first week (days 2 and 5 ) after injection

of LPS the subcutaneous tissue was densely infil- trated with neutrophils and granulomatous aggre- gations of mononuclear cells (Fig. 8). In the hyperaemic blood vessels, thrombi could be detected (Fig. 9), probably induced by platelet adhesion to the vessel walls. By day 28, the inflammatory reaction was resolved.

Groups 8 and 9: injection of saline or lecithin

had no histologically visible lesions at any time. Control animals (injected with saline or lecithin)

Serological response to the antigens Immunization with a single dose of the lipopep-

tide conjugate resulted in a specific IgG response (Fig. 10) at day 14 post-immunization. Higher anti- body titres could be observed following application of P,CS-HIV-1(586-606) conjugate in CFA. Inoculation of the non-conjugated HIV- 1 peptide alone did not induce a significant IgG response. Antibodies against the P,CS moiety could not be detected (data not shown).

DISCUSSION

Following injection of the potent mineral oil adjuvants CFA and IFA, the histopathological pic- ture was characterized by long-term retention of the oil in the tissue surrounded by an inflammatory reaction and pronounced fibrosis.16 Abscess forma- tion and other severe side effects such as fever, tumours, and autoimmune disease preclude the use of these adjuvants in humans.17

Bacteria like mycobacteria, B. pertussis, and anaerobic coryneforms as well as bacterial cell wall components such as lipopolysaccharides and lipo- proteins from Gram-negative bacteria have been shown to augment the humoral and cellular immune response. The wide spectrum of the biological activities of LPS can be attributed to the lipid A portion including polyclonal B-cell activation, pyrogenicity, adherence of platelets to vascular endothelium, and the release of a variety of biologi- cally active factors. Because of their undesirable side effects, the use of LPS and lipid A as adjuvants is limited.

Another class of adjuvants comprises synthetic analogues of bacterial cell wall fractions such as

LOCAL REACTIONS INDUCED BY ADJUVANTS 269

Fig. S-Day 2 after application of lipopeptide adjuvant P,CS. Accumulation of polymorphonuclear leucocytes with patchy fibrinoid necroses. EVG

Fig. &Day 7 after injection of P,CS. Giant cells arc present in the inflammatory reaction

Fig. 8- Day 5 after injection of lipopolysaccharide LPS. Densely infiltrated subcutaneous tissue with accumulationsofneutrophils and mononuclear cells

Fig. 7-Day 28 after injection of P,CS. Residual infiltrate with lymphocytes and foamy cells. EVG

270 F. WIEDEMANN ET AL.

Fig. 9-Day 5 after injection of LPS. Thrombotic occlusion of a venule. EVG

SERUM DILUTION

Fig. IO-IgG antibody responses of BALB/c mice at day 14 after immunization. (0-0) HIV-1(58&606) peptide, 0.1 pmol; (M-M) peptide conjugate P,CS-HIV-1(586-606), 0.1 pmol; (A-A) peptide conjugate P,CS-HIV-1(58&606), 0.1 pmol emulsified in CFA

muramyl peptides" and lipopeptide analogue^.^-^ After subcutaneous injection the lipopeptide ana- logue P,CS induced a pronounced inflammatory reaction with an accumulation of lymphocytes,

neutrophils, macrophages, giant cells, and epitheloid cells. The resolution of these granulomas occurred after several days and was nearly complete after 28 days. Similar histopathological patterns could be seen after injection of P,CS-HIV-1 peptide conju- gate emulsion. There are several mechanisms by which P,CS may exert its adjuvant activity. B cells are activated to proliferation and immunoglobulin ~ec re t ion .~ ,~ The stimulation of macrophages leads to the production of IL1, prostaglandin^,'^ and TNF.*' Furthermore, the release of lymphokines and enzymes may result in the rapid resolution of the granulomas, collagen resorption, and the lack of appreciable fibrosis.2i

To enhance the immunogenicity of synthetic anti- gens, the development of new adjuvant and carrier systems is of critical importance. The effectiveness of most adjuvant materials is associated with the accumulation and stimulation of lymphocytes, macrophages, and other immunocompetent cells. The release of lymphokines may participate in immun~potentiation.~ However, granuloma forma- tion and the development of abscesses and keloids, which has been described in humans following immunization with a mineral oil adjuvant vaccine,** emphasizes the value of adjuvants which do not produce unacceptable inflammatory reactions. The present investigation shows the induction of specific IgG antibodies against the synthetic P,CS-HIV- l(586-606) conjugate after a single dose without any additional adjuvant or carrier. These results demon- strate that a completely synthetic lipopeptide immu- nogen with built-in adjuvanticity could be used for effective immunization. Owing to the high chemical and heat stability, refrigeration for storage is not necessary.23 Furthermore, these compounds can be synthesized within a short time in any amount and of reproducible quality. The lipopeptide adju- vants induce local granuloma formation correlated with effective antigen presentation and immuno- potentiation but no long-term irritancy and tissue damage.

ACKNOWLEDGEMENTS

We wish to thank Professor Rossbach for the generous gift of the BALB/c mice. Thanks are due to Ms P. Koch and the staff of the Department of Pathology for their expert technical assistance. This study was supported by the Erwin-Riesch-Stiftung and the Bundesministerium fur Forschung und Tecnologie FK 03 18768.

L O C A L REACTIONS I N D U C E D BY A D J U V A N T S 27 1

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