A self-associating hepatitis B surface antigen-derived peptide that is immunogenic in alum

A self-associating hepatitis B surface antigen-derived peptide that is immunogenic in alum V e n k a t a s a m y M a n i v e l * , A n u r a d h a T r i p a t h y t, H e m l a t a K u m a r * , S u b r a t K. P a n d a t a n d K a n u r y V.S. R a o *$

D u r g a p a l t,

Anil

We previously described an oligomeric synthetic peptide derived from the hepatit& B surface antigen that displayed a limited tendency to form self-associatin9 maeromolecular structures in solution. Here it is demonstrated that amino-terminal myristylation of this peptide results in near quantitative aggregation of the oligomeric peptide. The myristylated peptide is highly immunogenic when used in conjunction with alum as adjuvant in both the rabbit and rhesus monkey models. The antibody response 9enerated by peptide also cross-reacted with native antigen and was lono-lasting. Collectively the results described in this and previous reports offer an attractive new approach for generatin9 immunogenic peptide mimetics of conformational epitopes that may find application as vaccines. Keywords:Synthetic peptide; hepatitis B-surfaceantigen; self-associating;immunogenicity;alum

INTRODUCTION Early observations that synthetic antigens containing an immunodominant region of a protein can give rise to a specific immune response towards the intact native protein have paved the way for eventual development of peptide vaccines (see Ref. 1 for a review). However, the notoriously poor immunogenicity of synthetic peptides has severely impeded progress in this area 2 4. This is particularly true for vaccines intended for eventual human use when one has to contend with weak adjuvants such as alum 5, although efforts are under way to develop more potent, non-toxic formulations 6. Several strategies have been adopted to enhance the antibody response against weak synthetic immunogens. These include the use of liposomes 7, appropriate derivatization 8, synthesis of an anchored molecule presenting multiple copies of a single sequence9, generation of hybrid sequences with inbuilt adjuvanticity 1° and the use of interleukin-l-derived synthetic peptides as added coadjuvant al. While these approaches have shown varying degrees of promise, it would nevertheless be desirable to enhance the intrinsic immunogenicity of peptide immunogens. Our primary research interest has been to obtain synthetic peptides that can mimic conformational epitopes

*Virology Group, International Centre for Genetic Engineering and Biotechnology, Nil Campus, Shaheed Jeet Singh Marg, New Delhi 110 067, India. tDepartment of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India. tTo whom correspondence should be addressed. (Received 30 March 1992; revised 6 May 1992; accepted 12 May 1992) 0264-410)(/93/030366-o6 © 1993Butterworth-HeinemannLtd 366 Vaccine, Vol. 11, Issue 3, 1993

on native viral antigens. In this connection we have recently demonstrated that a cysteine-rich 24 amino acid synthetic peptide derived from the major protein of HBsAg spontaneously self-oligomerizes to reconstitute a conformational group-specific native epitope 12. This oligomeric peptide was immunogenic in animal models yielding an antibody response capable of recognizing a variety of HBsAg subtypes and could also immunoprecipitate the virions in vitro. It was proposed that this oligomeric peptide may represent a potential candidate peptide vaccine for hepatitis B. Subsequently we observed that, in solution, a fraction of this oligomeric peptide aggregates to form macromolecular structures of diverse size and shape 13. We were interested in determining whether it would be possible to significantly increase the proportion of the oligomeric peptide that forms aggregates. It was hypothesized that achieving such an objective would render the peptide more immunogenic, thus allowing its use in conjunction with relatively weaker adjuvants. In this report it is demonstrated that amino-terminal myristylation of the oligomeric peptide OS results in near quantitative macromolecular aggregation of the individual oligomeric units. Further, the myristylated peptide is highly immunogenic in both rabbits and rhesus monkeys when used in conjunction with alum as adjuvant. MATERIALS AND METHODS Materials

Plasma-derived HBsAg used in the ELISA experiments was the vaccine preparation (H-B-Vax) marketed by Merck, Sharp and Dohme (Westpoint, PA). This preparation contains purified HBsAg at a protein

A self-associating HBsAg peptide immunogen: V. Manivel et al.

concentration of 20 #g ml-1. Polyclonal horse antiHBsAg antiserum was purchased from Wellcome Laboratories (Beckenham, UK). All secondary antibodies conjugated to horseradish peroxidase (HRPO) and other reagents were purchased from Sigma Chemical Co. (St Louis, MO).

Synthesis of myristylated peptide OS The 124-147 sequence of HBsAg (subtype adw) was synthesized by the solid-phase method as previously described12. After completion of synthesis the terminal protecting group was removed and the amino-terminus myristylated in the presence of a fourfold molar excess of myristic acid and diisopropylcarbodimide. The myristylated peptide was then cleaved from the solid support and side-chains deprotected simultaneously with hydrogen fluoride (using the Multiple Peptide System, San Diego, CA).

Sucrose density gradient centrifugation of peptide OS-Myr The procedure used was identical to that previously described x3. Briefly, 100/~g of peptide in 100/A of distilled water was overlaid onto a 10-30% linear sucrose gradient and centrifuged in an SW 50.1 rotor at 50000 rev min -1 for 18 h at 5°C. At the end of the run fractions of ~ 400/A were collected and the percentage of sucrose in each fraction determined by refractometry. Individual fractions were dialysed exhaustively against distilled water and 100 #1 aliquots lyophilized for estimation of peptide present by the ninhydrin procedure ~4. The remainder was diluted twofold in 2 x phosphate-buffered saline (PBS, pH 7.4) and 100 #1 portions coated in duplicate onto wells of an ELISA plate to determine antigen distribution.

Adsorption of peptide onto alum To 400 #1 of potassium aluminium sulphate (0.2 M) was added 400 #1 of a 1 mg ml- 1 solution of peptide in distilled water and the mixture vortexed briefly (10 s). To this was added 180 ~1 of a 1 M sodium bicarbonate solution in a dropwise manner after which it was incubated at 22°C for 15 min and then centrifuged at 500 rev min - ~ for 15 min. The supernatant was discarded and the alum gel washed twice with 1 ml of PBS and then finally resuspended in 1.2 ml PBS. In preliminary experiments it was established that under these conditions ~60% of added peptide is adsorbed. Thus the above formulation represents a final peptide concentration of 200 #g ml- 1. In the experiment with monkeys the final volume of reconstitution was adjusted to give the desired dose of immunogen in a 1 ml volume.

Enzyme-linked immunosorbent assay (ELISA) Wells were coated with 1 #g antigen in 100/A PBS (at 37°C for 3 h). Subsequently they were blocked with 100 /A of 5% (w/v) solution of non-fat dried milk powder at 37°C for 2 h and washed. Amounts of 100 ~tl of appropriate dilutions of antiserum were added and incubated at 4°C overnight. After washing, wells were incubated with 100 /~1 of HRPO-labelled secondary antibody at 37°C for 1.5 h. For monkey sera, the secondary antibody employed was HRPO-labelled goat anti-human IgG. The chromogen used was O-phenyl-

enediamine (OPD) and absorbance was measured at 490 nm. In vitro proliferation of T lymphocytes from immunized

monkeys Lymphocytes were isolated from the peripheral blood mononuclear cells (PBMC) as previously described 15. Cells were then resuspended in 2 ml of bovine serum. Of this, 1.4 ml of cell suspension was passed over a nylon wool column (Leukopak, Fenwal Laboratories, Morton Grove, IL) to obtain purified T cells. T cells were plated at 1 x 105 cells/well along with an equal number of non-T-cell PBMCs which had been previously treated with mitomycin C for 45 min at 37°C. Cultures were performed in 96-well flat-bottomed microtitre plates either with or without peptide at 37°C in a humidified atmosphere containing 5% CO2 for 6 days. At the end of this period, cells were pulsed with 0.5 #Ci of [3H]thymidine (New England Nuclear, Boston, MA) and harvested 20 h later. Incorporated radioactivity was determined by scintillation counting and the stimulation index determined 16. RESULTS

N-terminal myristylation yields near quantitative aggregation of the HBsAg-derived peptide In a prior report we demonstrated that a cysteine-rich 24 amino acid synthetic peptide derived from the major protein of hepatitis B surface antigen spontaneously self-assembles via intermolecular disulphide bridging 12. This oligomeric peptide (peptide OS) was shown to represent a conformational, group-specific or "a' determinant of HBsAg. Although peptide OS was immunogenic with the Freund's adjuvant formulation 12, it was subsequently found to be non-immunogenic in rabbits when used in conjunction with alum as adjuvant (data not shown). To overcome this problem we exploited our more recent observation that, in solution, a small proportion of peptide OS forms macromolecular aggregates 13. Preliminary results indicated that these aggregates represented non-covalent association of individual oligomeric units of peptide 13. To enhance the relative extent of selfassociation a myristylated derivative of peptide OS (Peptide OS-Myr) was prepared where the myristyl group was added onto the amino terminus (see Materials and methods). An Ellman analysis 17 of the product was negative, indicating that all the cysteines were engaged in disulphide bonds. A titration of polyclonal anti-OS serum 12 against either the myristylated (peptide OSMyr) or non-myristylated (peptide OS) forms gave identical profiles (Figure la). Thus amino-terminal myristylation does not impair the ability of the OS sequence to form the previously described disulphidedependent conformational epitope 12. Interestingly, when polyclonal anti-HBsAg antiserum was employed, peptide OS-Myr showed a distinctly higher reactivity as opposed to the non-myristylated peptide OS (Figure lb). From Figure la it does not appear that the differential reactivities of peptide OS and OS-Myr to anti-HBsAg are due to differences in the ability to coat onto wells of the ELISA plate. Myristylation of the peptide OS therefore yields a closer approximation of the native HBsAg epitope.

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A self-associating HBsAg peptide immunogen: V. Manivel 2.0

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Figure 1 Polyclonal anti-OS and anti-HBsAg antisera recognize peptide OS-Myr in an ELISA assay. Wells coated with 1 #g of either peptide OS ( O ) or OS-Myr ( Q ) were incubated with either polyclonal rabbit anti-OS antiserum ((a), Ref. 12) or polyclonal horse anti-HBsAg antiserum (b) at the indicated dilutions. Values are the absorbance obtained at 490 nm after subtracting that obtained for an irrelevant peptide representing the sequence 167-181 of human interleukin-1 which was used as control. Data are the mean of triplicate determinations

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Sucrose density gradient centrifugation of peptide OS-Myr. For experimental details refer to Materials and methods. (a) Reactivity obtained for each fraction in an ELISA assay with either polyclonal anti-OS ( © ) or polyclonal anti-HBsAg ( O ) antiserum. (b) Peptide distribution obtained among the various fractions by the ninhydrin method (Materials and methods, Ref. 14)

To examine its self-associating properties, peptide OS-Myr was centrifuged through a 10-30% sucrose gradient and the resulting fractions analysed for peptide present (see Materials and methods). These results are shown in Figure 2a. Remarkably, almost all of the peptide OS-Myr appeared to form high-density aggregates. While there was some accumulation of peptide around 14°/'o sucrose, the majority had traversed the entire gradient

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aggregated peptide which is normally observed in the fraction below 10% sucrose 13. This is in sharp contrast to the profile obtained for non-myristylated peptide OS where the majority exists in non-aggregated form with only a fraction entering the gradient 13. Thus aminoterminal myristylation has a dramatic positive influence on the ability of peptide OS to form high-density aggregates. The individual fractions obtained from the above experiment were also analysed for antigenicity with either polyclonal anti-OS or polyclonal anti-HBsAg antisera (Figure 2b). Interestingly, while anti-OS antibodies preferentially recognize that component of peptide present in the lighter fractions, the anti-HBsAg antisera recognize the high-density component. These results along with that shown in Figure lb lend further support to our earlier contention 13 that the self-aggregated components of peptide OS represent the more 'native' mimetic of the cognate HBsAg epitope. Examination of peptide OS-Myr by transmission electron microscopy ~3 showed a predominance of fairly uniform spherical aggregates of diameters ranging from 100 to 120 nm (Figure 3). However, a few structurally ill-defined aggregates were also visible (see Figure 3).

Alum-adsorbed peptide OS-Myr is immunogenic in rabbits To examine the immunogenicity of peptide OS-Myr in an adjuvant system relevant for human use, an alum-adsorbed preparation of peptide was injected into three New Zealand white rabbits (see Materials and methods). Sera collected from individual rabbits after a single boost were analysed for anti-OS and anti-HBsAg antibodies. All three rabbits gave an anti-OS response which also cross-reacted with HBsAg (Figure 4). Further, in all three rabbits, anti-OS and anti-HBsAg titres were comparable (Figure 4a and b) indicating that the majority of antibodies obtained in response to peptide also recognize the native antigen.

A self-associating HBsAg peptide immunogen: V. Manivel et al.

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-~0 In parallel with the above experiment two additional rabbits were also immunized with an identical dose of an alum-adsorbed preparation of non-myristylated peptide. However, anti-peptide or anti-HBsAg antibody could not be detected at dilutions as low as 1:100 (data not shown). These results demonstrate that amino-terminal myristylation does indeed enhance the immunogenicity of peptide OS, presumably by ensuring near quantitative macromolecular clustering of individual oligomeric peptide units.

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4 Alum-adsorbed peptide OS-Myr is immunogenic in rabbits. Three female New Zealand white rabbits (6-8 weeks old) were immunized intramuscularly with 200 pg each of alum-adsorbed peptide OS-Myr. After 4 weeks (day 28) these rabbits were again boosted with an identical dose. Blood was collected from the retro-orbital plexus 2 weeks after the first (1 g) and 1 week after the second (2 g) immunizations and sera titrated against (a) peptide or (b) native HBsAg. Titres were calculated as that dilution of serum that gave an absorbance 2.1 times that of the corresponding pre-immune serum at a dilution of either 1 : 100 (for 1g) or 1 : 1000 (for 2°). Titres of individual rabbits are presented as the mean of three independent determinations. Deviation from the mean was < 1 5 % in all cases Rgure

lmmunogenicity of alum-adsorbed peptide OS-Myr in rhesus monkeys Next, a comparative evaluation was performed of the immunogenicity of varying doses of peptide OS-Myr along with that of a commercially available vaccine preparation in rhesus monkeys. For this experiment four rhesus monkeys (5-7 kg in weight) were taken. One received the vaccine at the recommended dose for humans (20 #g) while the three remaining monkeys received either 50, 250 or 500 #g doses of an alum-adsorbed preparation of peptide OS-Myr. A primary immunization was followed by two booster immunizations 4 and 15 weeks later and the IgG anti-peptide antibody titre followed at 2 week intervals. A weak primary response was obtained in all four monkeys (Figure 5). However, after the first boost, antibody titres increased significantly with near identical levels in monkeys that received either the vaccine or a 50/~g dose of peptide. Higher doses of peptide yielded even higher levels of antibody, reaching a titre of about 1 : 100000 with a 500 #g dose of peptide (Figure 5). After reaching their peak levels, the anti-OS titres slowly declined from the eighth week in all four monkeys, though for the monkey immunized with 500/~g peptide titres were still about 50% of the peak value at the time

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Figure5 Alum-adsorbed peptide elicits an antibody response in rhesus monkeys. Four monkeys, either male or female (weight 5-7 kg), were used for this experiment. Three were immunized with either 50 (monkey 745), 250 (monkey 1442) or 500 (monkey 1109) #g of peptide OS-Myr that had been adsorbed onto alum. The fourth monkey received a commercially available hepatitis B vaccine (20 #g/dose, Engerix B, SmithKline Biologicals, Rixensart, Belgium). Each monkey received three doses of immunogen at 0, 1 and 3 months (indicated by arrow). Blood was collected at the indicated times and sera assayed for anti-OS-Myr titre by ELISA (Materials and methods, see also legend to Figure 4). ( © ) Monkey no. 1757; ( 0 ) monkey no. 745; ( A ) monkey no. 1442; ( A ) monkey no. 1109

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A self-associating

HBsAg peptide

immunogen:

V. Manivel

et al.

of the third immunization (Figure 5). A third immunization at 15 weeks resulted in a boosted response in all four monkeys with monkeys 1757 and 745 again giving comparable anti-OS titres. The persistence of circulating anti-OS antibodies was followed up over a subsequent 22 week period at the end of which significant levels of antibodies were still detectable (Figure 5). Sera obtained at 17 weeks (2 weeks after the third immunization) of the experiment were also screened for their ability to cross-react with HBsAg and the results are presented in Table 1. As might be expected, the vaccine-immunized monkey (monkey 1757) had an anti-HBsAg titre that was significantly greater than that obtained for the peptide-immunized monkeys. Obviously, immunization with HBsAg elicits antibodies to epitopes in addition to that present in the 124-147 sequence. In the case of monkeys immunized with peptide OS-Myr, anti-HBsAg titres ranged from about 40% (monkeys 1442 and 1109) to 60% (monkey 745) of that obtained as anti-OS-Myr titre (Table I). Thus a significant proportion of antibodies obtained in response to peptide OS-Myr in rhesus monkeys is also capable of recognizing native HBsAg. It is, however, not clear at present why the anti-OS-Myr and anti-HBsAg titres in peptideimmunized monkeys do not correlate more closely, as observed in rabbits (see Figure 4). Peptide OS-Myr stimulates T cells from monkeys immunized with either HBsAg or homologous peptide

T cells purified from the peripheral blood of monkeys described in Figure 5 were challenged in vitro with peptide OS-Myr (Materials and methods). These results are given in Table 2. As is evident, lymphocyte proliferation was Table 1

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Table 2 Purified T lymphocytes from immunized responsive to in vitro challenge with peptide OS-Myr

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Blood was drawn from monkeys (figure 5) at 2 weeks after the second immunization (day 44). From this T cells were purified and cultured either in the presence or absence of peptide OS-Myr (12 pglwell) as described in Materials and methods. Proliferative responses were assessed by incorporation of [BH]thymidine. Data are expressed as the stimulation index (SI) which was calculated as the ratio of counts obtained in the presence of added peptide to that obtained in the absence of peptide OS-Myr. Values are the mean of quadruplicate determinations ( * s.e.)

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obtained regardless of whether the monkey was immunized with HBsAg or peptide OS-Myr. This suggests that immunization with HBsAg results in the recruitment of a T-helper cell population specific for an epitope within the sequence between residues 124 and 147 of the major protein. In addition to an in vitro challenge with peptide OS-Myr, monkey T lymphocytes were also challenged with purified HBsAg. However, the stimulation index obtained was low ( < 3.0) in all four monkeys (data not shown). The reason that peptide OS-Myr evokes a stronger lymphocyte proliferative response than purified HBsAg is not clear at the present time. However, it is known that native HBsAg is very poor at stimulating proliferation of lymphocytes’*. DISCUSSION Early enthusiasm over the possibility of developing peptide vaccines against a variety of pathogens has now given way to the realization that some inherent limitations need to be overcome if they are to become even viable. Of these, the two principal ones relate to the inability of linear synthetic peptides to mimic native conformational epitopes accurately and to poor immunogenicity. In order to address these problems we selected the major protein of the hepatitis B surface antigen as a model. In initial experiments it was demonstrated that a sequence representing the cysteine-rich 124- 147 sequence of HBsAg spontaneously self-oligomerizes in a disulphidedependent manner l2 . We had speculated that the odd number of cysteine residues and appropriate distribution of putative p-turn sequences may have been responsible for inter- and intramolecular bridging to reconstitute the native ‘a’ epitope l2 . Based on the above hypotheses we recently designed and synthesized a chimeric peptide that included the sequence from gp120 and gp41, the end part of HIV-l. This peptide was also found to spontaneously self-oligomerize on cleavage from the solid support to regenerate a native conformational epitope”. Thus an approach involving synthesis of self-oligomerizing peptide sequences to mimic native conformational epitopes may have broader applicability. Having a peptide mimetic of a conformational group-specific determinant of HBsAg”, the next objective was to obtain a form of peptide OS that was immunogenic in alum, one of the few adjuvants approved for human use to date. For this use was made of our earlier observations that, in solution, a small fraction of peptide OS tends to form macromolecular aggregates13. It was thought that by increasing the proportion of peptide OS that forms macromolecular aggregates it may be possible to enhance its immunogenicity. It was consequently decided to investigate whether myristylation of peptide OS might achieve this objective. It was anticipated that addition of myristic acid would result in micelle-like interactions due to the hydrophobic fatty acid tail and the relatively polar peptide head, thus promoting aggregation. The results described in the report demonstrate that the amino-terminal myristylation of peptide does indeed yield near quantitative aggregation, resulting predominantly in spherical particles of diameter lOO- 120 nm. Presumably because of its macromolecular nature, peptide OS-Myr is also highly immunogenic when used in conjunction with alum as adjuvant. At comparable doses (50 and 20 ,ug) peptide OS-Myr and vaccine give

A self-associating HBsAg peptide i m m u n o g e n : V. M a n i v e l et al.

comparable levels of OS epitope-specific titres in these monkeys. Higher doses of peptide result in antibody levels that are further elevated. The long-term stability of the antibody response to peptide was similar to that obtained against vaccine and circulating antibody levels would be detected at least 22 weeks after the second boost. Finally, immunization of monkeys with peptide primes Th cells for an HBsAg-relevant T epitope. It is not known at present whether anti-OS-Myr antibodies can neutralize HBV infection and lack of an adequate in vitro system has hampered our progress on this aspect. Protection studies performed in chimpanzees will be needed to answer this question. Nevertheless the cumulative results described in this and previous reports 12'13'19 indicate that our approach involving generation of self-assembling peptide sequences that can regenerate native conformational epitopes followed by myristylation to obtain enhanced macromolecular aggregation of enhanced immunogenicity provides a promising new strategy for the development of peptide vaccines. However, it remains to be seen if myristylation aids macromolecular aggregation of self-oligomerized peptides other than the one described here. This aspect is currently under investigation.

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REFERENCES 1 Arnon, R. Synthetic peptides as the basis for vaccine design. Mol. Immunol. 1991, 28, 209-215 2 Morein, B. and Simons, K. Subunit vaccines against enveloped viruses: virosomes, micelles and other protein complexes. Vaccine 1985, 3, 83-93 3 Boudrealt, A. and Thibodeau, L. Mouse response to influenza immunosomes. Vaccine 1985, 3, 231-234 4 Goodman-Snitkoff, G., Eisele, L.E., Heimer, E.P., Felix, A.M., Anderson, T.T., Fuerst, T.R. and Manino, R.J. Defining minimal requirements for antibody production to peptide antigens. Vaccine 1990, 8, 257-262 5 Bomford, R. Aluminium salts: perspectives in their use as adjuvants. In: Immunological Adjuvants and Vaccines (Eds Gregordias, G., Allison, A.C. and Poste, G.) Plenum Press, New York, 1989, pp. 35-41

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Allison, A.C. Antigens and adjuvants for a new generation of vaccines. In: Immunological Adjuvants and Vaccines (Eds Gregordias, G., Allison, A.C. and Poste, G.) Plenum Press, New York, 1989, pp. 1-12 Van Rooijen, N. and Su, D. Immunoadjuvant action of liposomes: Mechanisms. In: Immunological Adjuvants and Vaccines (Eds Gregordias, G., Allison, A.C. and Poste, G.) Plenum Press, New York, 1989, pp. 95-106 Krug, M., Folkers, G., Haas, B., Hess, G., Weismuller, K.-H., Freund, S. and Jung, G. Molecular dynamics of the co-helical epitope of a novel synthetic lipopeptide Foot-and-Mouth disease virus vaccine. Biopolymers 1989, 28, 499-512 Tam, J.P. Synthetic peptide vaccine design: Synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl Acad. Sci. USA 1988, 85, 5409-5413 Rao, K.V.S. and Nayak, A.R. Enhanced immunogenicity of a sequence derived from hepatitis B virus surface antigen in a composite peptide that includes the immunostimulatory region from human interleukin-l. Proc. NaU Acad. Sci. USA 1990, 87, 5519-5522 Manivel, V. and Rao, K.V.S. Interleukin-1 derived synthetic peptide as an added co-adjuvant in vaccine formulations. Vaccine 1991, 9, 395-397 Manivel, V., Ramesh, R., Panda, S.K. and Rao, K.V.S. A synthetic peptide spontaneously self-assembles to reconstruct a groupspecific, conformational determinant of hepatitis B surface antigen. J. Immunol. 1992, 148, 4006-4011 Rao, K.V.S., Panda, S.K. and Manivel, V. Macromolecular selfassociation of a synthetic peptide derived from the hepatitis B surface antigen: Construction of a quaternary epitope. Vaccine (in press) Allen, G. Methods for the detection of peptides. In: Sequence of Proteins and Peptides Elsevier Science Publishers, Amsterdam, 1986, pp. 155-160 Boyum, A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Invest. 1968, 21, 77-89 Cells, E., Daweir, O. and Otvos, LR. Recognition of hepatitis B surface antigen by human T lymphocytes. J. Immunol. 1986, 148, 1898-1815 EIIman, G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82, 70-77 Celis, E., Kato, I., Miller, R.W. and Chang, T. Regulation of the human immune response to HBsAg: Effect of antibodies and antigen conformation in the stimulation of helper T cells by HBsAg. Hepatology 1985, 5, 744-751 Tripathy, S.P., Kumar, A., Manivel, V., Panda, S.K. and Rao, K.V.S. Design and synthesis of a self-assembling peptide derived from the envelope proteins of human immunodeficiency virus type 1: An approach to heterovalent immunogens. J. Immunol. 1992, 148, 4012-4020

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