Oral adjuvants for viral vaccines in humans

Oral adjuvants for viral vaccines in humans Shigeaki lshizaka, Masahide Yoshikawa, Keiji Kitagami and Tadasu Tsujii The availability of various viral vaccines prompted extensive studies on oral adjuvants. In this study, parotid protein (parotin ), taurine and lithium were chosen as safe and potent oral adjuvants for rubella and influenza virus vaccines. The anti-rubella virus antibody titres in persons administered orally with parotin were significantly higher than those of persons with the non-adjuvanted vaccines. The combinations of oral adjuvants were used for vaccination with trivalent influenza vaccines. Each group, receiving taurine, taurine + lithium, taurine + parotin or taurine + parotin + lithium, was higher in the magnitude of increased influenza virus antibody titres than the non-adjuvant control group. Although taurine was shown to be an effective adjuvant in taurine responders but not in taurine non-responders, the combination of taurine, lithium and/or parotin gave higher increased rate of influenza antibody titres than with taurine alone. These results have shown to be a valuable approach to the clinical use of oral adjuvants.

Keywords:Oral adjuvants;viral vaccines;taurine; parotin Introduction The development of safe and potent adjuvants is expected to improve the effect of viral vaccines in human beings. Extensive research on the properties of many different oral adjuvants were previously performed by us for clinical applications 1-5. In this study, parotin, taurine and lithium were chosen as oral adjuvants for their safety in clinical use. Parotin (132 kDa), extracted from bovine parotid glands, exclusively influences differentiation and proliferation of cells from various mesenchymal tissues 6. Parotin induces polyclonal antibody production in mouse spleen cells 7 and human peripheral lymphocytesa, and is shown to be an effective adjuvant for hepatitis B virus surface (HBs) vaccine in mice 5. Furthermore, parotin itself possessed interleukin 1 (IL-.1)-like activity5. Recently, it was shown that taurine, a sulphur amino acid, augmented polyclonal antibody production and possessed adjuvant activity5. Taurine is also able to stimulate IL-1 secretion 5. Lithium enhanced the proliferation of T cell mitogen-stimulating lymphocytes9, and partially restored lipopolysaccharide (LPS) responsiveness in spleen cells of LPS-non-responder C3H/HeJ mice ~°. We investigated whether the combinations of these oral adjuvants could effectively stimulate viral vaccination responses.

tration of taurine, parotin and lithium in preliminary trials, the following administration method was chosen as the most effective one. Each group of ten volunteers was administered orally with 9g of taurine (Taisho Pharmaceutical Co., Tokyo), or 40mg of parotin (Teikoku Hormone Mfg. Co., Tokyo) or 400mg of lithium carbonate (Taisho Pharmaceutical Co.) on the same day and 1 day before influenza vaccine administration. Twenty healthy men with a mean age of 27.6 years (range 24-35 years) were injected subcutaneously with 0.5 ml of rubella virus vaccine (Foundation for Microbial Diseases, Osaka, Japan). Half of these persons were administered orally with 40 mg of parotin both on the preceding day and the day of vaccination.

In vitro culture The peripheral blood lymphocytes (1 x 10 6 ml-1) were cultured with parotin (500pgml-~), taurine (1, 5 or 10 mg ml - 1) or 100 #g ml - ~ of pokeweed mitogen (PWM) in RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo) containing 10% heat-inactivated human AB serum and kanamycin (60mgl-1) in each well of fiatbottomed microculture plates (Corning, NY, USA) at 37°C for 6 days. Each well contained 0.2ml of cell suspension, the adjuvants and culture medium.

Subjects and methods Subjects and vaccination Fifty healthy men with a mean age of 29.2 years (range 23-38 years) were injected subcutaneously with the trivalent influenza virus vaccine (Takeda Pharmaceutical Co., Osaka, Japan) containing 200 CCA of A/Kumamoto/ 37/79 (H1N1), 350 CCA of A/Ishikawa/7/82 (H3N2) and 150CCA of B/Singapore/222/79 twice at 1-month intervals. After testing suitable doses and timing of adminisThird Department of Internal Medicine, Nara Medical University, 840 Shijo-Cho, Kashihara, Nara 634, Japan. (Received 17 October 1989; revised 22 January 1990; accepted 23 January 1990) 0264410X/90/040337-05 © 1990Butterworth-HeinemannLtd

Cell preparation Human lymphocytes were isolated from peripheral blood by centrifugation over Ficoll-Isopaque as described previously 8. Monocyte-depleted lymphocytes were prepared by incubation of lymphocytes for 1 h in a plastic petri dish to remove adherent cells. The non-adherent cells were treated with mouse IgG2b monoclonal antibody Leu-M3 (Becton Dickinson Monoclonal Center Inc., CA, USA) at 1:100/106 cells for 60min in ice. After washing twice in balanced salt solution, the cells were incubated with low-toxicity rabbit complement diluted 1:10 (Cederlane Laboratories Limited, Ontario, Canada) for 60min at 37°C.

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Oral adjuvants: S. Ishizaka et al.

T cell-depleted lymphocytes were obtained by erythrocyte-rosette assay and treatment with OKT3 monoclonal antibody and complement as previously described x~.

Antibody measurement Anti-influenza antibodies in the sera from the vaccine recipients were measured by the haemagglutinin inhibition test ~2. IgG anti-rubella virus antibodies in sera were determined by an ELISA procedure. In brief, formalininactivated, zonal centrifugation-purified rubella virions of M-33 strain grown in Vero cells served as an antigen. A volume of 10#gml -~ rubella virus antigens in 0.05M carbonate buffer (pH 9.6) were added to each well of flat-bottomed ELISA plates (Dynatech, Alexandrin, VA, USA) and absorbed to a solid-phase by incubation in the plates overnight at 4°C. After washing three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20, the test sera diluted from 1:2 to 1:200 in PBS containing 0.1% bovine serum albumin (BSA) and 0.05% Tween 20 are dispensed into each well. After a 1-h incubation, the plates were washed three times, and 100/zl of rabbit anti-human immunoglobulin G'conjugated to horse-radish peroxidase (diluted 1:2000) is added to each well. The plates were again incubated at 37°C for 1 h, washed, and developed with 0.4 mg ml- ~ of o-phenylene diamine and 0.4/zl of 30% H20 2 in 0.01 M citrate buffer (pH 5.0) at 37°C for 30min. The absorbance was read at 492nm in a microELISA auto reader MR 580 (Dynatech Laboratories, VA, USA). The number of IgM plaque-forming cells (PFC) was determined by the protein A plaque assay .3 as previously described ~4. In brief, 25/A of cell suspension of protein A-coupled sheep red blood cells (SRBC), 25/A of a 1/50 dilution of anti-human IgM (DAKOPATTS, Copenhagen, Denmark) and 25 F1 of SRBC absorbed guineapig complement (diluted 1/6) were added to 0.5% agar (0.2 ml) containing 0.005% DEAE dextran. The mixture was placed on petri dishes, covered with a coverglass and incubated at 37°C for 4 h.

Statistical analysis Wilcoxon's rank sum test was used to compare the results of the adjuvant group with those of the non-adjuvant group. Statistical significance of the data in Figure 2 was determined by the Student's t test.

parotin. The antibody titres, for rubella virus in men given rubella vaccines adjuvanted with parotin, were significantly higher than those given the non-adjuvanted rubella vaccines (Figure I). Four subjects already had anti-rubella virus antibody prior to vaccination. The mean antibody levels. (=A) of the vaccinees given the non-adjuvanted vaccines 1 day before, 1 month and 2 months after the vaccination were 0.391, 0.442 and 0.557, respectively. Those of parotin-administered vaccinees 1 day before, 1 month and 2 months after the vaccination were 0.370, 0.499 and 0.76, respectively.

Table 1

Effects of monocytes and T cells on polyclonal IgM antibody

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Time after immunizotion (months) Rgute 2 Stimulating effects of taurine, lithium and par.tin on anti-influenza virus antibody production. Fifty healthy male subjects were injected subcutaneously with the trivalent influenza virus vaccine containing 200CCA of A/Kumamoto(H1N1), 350CCA of A/Ishikawa(H3N2) and 150CCA of B/Singapore twice at one month intervals. Each of ten vaccinees group was administered orally with 9g of taurine, or 40rng of par.tin or 400rag of lithium carbonate in the same day and 1 day before influenza vaccine administration. Each point indicates the mean of haemagglutination-inhibition (HI) antibody titres against N B a n g k o k (a), A/Kumamoto (b) and B/Singapore (c) influenza virus before and after vaccination. + , p<0.05; + + , p<0.01; + + + , p<0.001, x , Control; (3, taurine; O , taurine+lithium; A . taurine+parotin; A , taurine+lithium+parotin

Capacity o f taurine and lithium to enhance anti-influenza virus antibody responses An increase in an anti-influenza virus antibody of each group who received taurine, taurine + lithium, taurine + par,tin or taurine+lithium + p a r . t i n as oral adjuvants was significantly higher than that of the non-adjuvant control group (Figure 2). Furthermore, each group given a combination of taurine and lithium and/or par.tin had relatively higher HI titres compared with the vaccinees who received taurine alone. In vitro polyclonal antibody production induced

by taurine in peripheral blood lymphocytes from taurine-administered vaccinees Peripheral blood lymphocytes from ten taurineadministered vaccinees were cultured with taurine for 6 days. Subjects (five of ten) with an increase in polyclonal antibody responses above 2 for stimulation index (SI) were considered to be responders. Responders in taurineadministered vaccinees were 50% (five of ten). As shown in Figure 3, taurine exerted adjuvant effects on antiinfluenza virus antibody responses in taurine responders but not in taurine non-responders. D i s c u s s i o n

Previously it was demonstrated that par.tin induced relative T cell- and macrophage-independent polyclonal antibody production in mice 5. Parotin also augmented T cell- and monocyte-independent responses in human peripheral lymphocytes (Table I). An active glycopeptide (FrAA-1) was isolated from the subunit (45kDa) of

parotin by tryptic digestion 16. FrAA-1 (9,1 kDa), consisted of 58 amino acids containing carbohydrates, and was also a polyclonal B cell activator for mouse and human lymphOcytes s. The adjuvant activities of parotin fail to be inactivated even if parotin is orally administered because the active fragment FrAA-1 of parotin is resistant to trypsin digestion 16. It is suggested that the carbohydrate residue in FrAA-1 plays an important role in the protein's ability to resist protein digestion. Furthermore, there is evidence to suggest that parotin itself possesses IL-l-like activity 5. The mechanism by which parotin acts as an adjuvant for anti-rubella virus antibody responses may depend on polyclonal B cell activation and IL-l-like activity by parotin. It is well documented that taurine is one of the most abundant free amino acids present in mammalian tissues and has never been found in amino acid sequences of a protein. Taurine is considered to act as a neurotransmitter 17'~s, a membrane stabilizer ~9, the regulator of cardial contractility 2° and a detoxifier 2L22 (conjugation with toxic substances such as bile acids and retinoids). Recent experiments in our own laboratory have demonstrated that taurine can significantly augment anti-HBs antibody responses in mice 5. The magnitude of adjuvant effects observed in taurine-administered persons is related to that of in vitro polyclonal antibody responses to taurine (Figure 3), Such observations clearly tempt one to consider that polyclonal antibody responses induced by taurine in lymphocytes from vaccinees should be tested before the administration of taurine as an adjuvant. It is well known that lithium has been used clinically for the treatment of various diseases such as manic-

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3 Polyclonal antibody production and adjuvant activity induced by taurine, a, Human peripheral lymphocytes were cultured with 1, 5 or 10 mg m1-1 of taurine for 6 days. The number of IgM ?FC was measured by protein A plaque assay. All values represent arithmetic means_.+s.d, of IgM PFC per 10= viable cells ill triplicate cultures. Sludent's t test: +, p<0.05; -t-t-,j p<0.01; t I I ,, p<0.001 b (A/Bangkok), c (A/Kukamoto) and d (B/Singapore). Administration of taurine and influenza vaccines was performed as described in Subjects and methods. All points represent geometric means ___s.d. of HI titres in triplicate experiments. The dotted line indicates values of vaccine alone. The different symbols represent ten different individuals. Results are compared with each control value before immunization. Student's t test: ++,, p<0.01; I I I, ,p<0.001 Figure

depressive illness 23, hyperthyroidism24, granulocytopoiesis 2s'26 and neutropenia 27'2s. Lithium has been shown to enhance mitogen-stimulated polyclonal antibody production I°'29. Cyclic AMP plays an important role in the modulation of the immune responses. Lithium also produces greater stimulation of RNA synthesis in murine splenic B cells 1°, but induces neither polyclonal antibody production, nor IL-1 or IL-2 secretion s. These results indicate that lithium, a potent inhibitor of adenylate cyclase, may exert a direct or indirect effect on chromosomes leading to the induction of new RNA synthesis through the modification of cyclic AMPdependent events in lymphocytes. The combination of taurine, lithium and parotin for oral adjuvants showed higher increased rate of antibody titres than taurine alone (Figure 2). Parotin, taurine and lithium as unique and safe oral adjuvants may successfully improve clinical efficacy of vaccines for viral peptides, hepatitis C type virus or HIV in the near future.

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Acknowledgement This work was supported in part by a Grant in-Aid for Scientific Research (No. 61480181)from the Ministry of Education, Science and Culture, Japan.

References 1 2

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Ishizaka, S., Hasuma, T., Otani, S. and Morisawa, S. Lymphocyte activation by purified carrageenan. J. Immunol. 1980,125, 2232-2235 Sugawara, I. and Ishizaka, S. Polysaccharides with sulfate groups are human T-cell mitogens and routine polyclonal B-cell activators (PBAs). I. Fucoidan and heparin. Ceil. Immunol. 1982, 74, 162-171

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13

14 15 16

Ishizaka, S., Sugawara, I., Hasuma, T., Morisawa, S. and M611er, G. Immune responses to xanthan gum. I. The characteristics of lymphocyte activation by xanthan gum. Eur. J. Immunol. 1983, 13, 225-231 Ishizaka, S., Kikuchi, E., Higashino, T., Kinoshita, K. and Tsujii, T. Effects of acetate on the immune system of mice. Int. J. Immunopharmac. 1990, 12, 135-143 Kuriyama, S., Tsujii, T., Ishizaka, S., Kikuchi, E., Kinoshita, K., Nishimura, K. et al. Enhancing effects of oral adjuvants on anti-FIBs responses induced by hepatitis B vaccine. Clin. Exp. Immunol. 1988, 72, 383-389 Ito, Y. Parotin: A salivary gland hormone. Ann. NY Acad. Sci. i980, 85, 228-312 Ishizaka, S. and Morisawa, S. B cell activating properties of parotid protein. Microbiol. Immunol. 1979, 23, 481-485 Ishizaka, S. and Sugawara, I. Polyclonal antibody production induced by parotid protein and its active glycopeptide in mouse and human lymphocytes. Immunopharmacology 1983, 6, 133-142 Hart, D.A. Modulation of concanavalin A stimulation of hamster lymphoid cells by lithium chloride. Cell. Immunol. 1979, 43,113-122 Ishizaka, S. and M611er, G. Lithium chloride induces partial responsiveness to LPS in nonresponder B cells. Nature 1982, 239, 363-385 Sugawara, I. and Ishizaka, S. Carrageenan, highly sulfated polysaccharides and macrophage-toxin agents: newly found human T lymphocyte mitogens. Immunobiology 1982, 163, 527-538 Kendal, A. and Dowdle, W.R. Influenza virus. In: Manual of Clinical Laboratory Immunology. 3rd Edn (Eds Rose, N.R., Friedman, H. and Fahey, J.L) American Society for Microbiology, Washington, DC 1986, pp. 515-520 Gronowicz, E., Coutinho, A. and Melchers, F. A plaque assay for all cells secreting Ig of a given type or class. Eur. J. Immunol. 1976, 6, 588-590 Ishizaka, S. Modification of lipopolysaccharide non-responder cells in low cell density culture. Immunol. Lett. 1983, 6, 343-350 Janossy, G. and Graves, M. Functional analysis of murine and human B lymphocyte subsets. Transplant. Rev. 1975, 24, 177-236 Aomuma, S., Kohama, Y., Komiyama, Y., Fujimoto, Y. and Nomura, S. Amino acid sequence of an active fragment, FrAA-1 of salivary gland hormone. Chem. Pharm. Bull. 1980, 28, 417-423

Oral adjuvants: S. Ishizaka et al.

17 Gruener, R. and Bryant, H.J. Excitability modulation by taurine: action in axon membrane permeabilities. J. Pharmacol. Exp. Ther. 1975, 194, 514-521 18 Kuriyama, K. Taurine as a neuromodulator. Fed. Proc. 1980, 39, 2680-2684 19 Huxtable, R. and Bressler, R. Effect of taurine on a muscle intracellular membrane. Biochim. Biophys. Acta 1973, 323, 573-583 20 Read, W.O. and Welty, J.D. Effect of taurine on epinephrine and digoxin induced irregularities of the dog heart. J. Pharmacol. Exp. Ther. 1963, 139, 283-289 21 Portman, O.W. and Mann, G.V. The disposition of taurine-S35 and taurocholate-S= in the rat: dietary influences. J. Biol. Chem. 1955, 213, 733-743 22 Share, K.L., Sietsema, W.K. and DeLuca, H.F. The biological activity of retinotaurine. J. Nutr. 1982, 112, 1626-1630 23 Cade, J.F.J. Lithium in Medical Practice (Eds Johnson, F.N. and Johnson, S.) MTP Press, Lancaster, 1978, pp. 5-6 24 Temple, R., Berman, M., Carlson, H., Robbins, J. and Wolff, J. The use of lithium in Graves' disease. Mayo C/in. Proc. 1972, 47, 872-878

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Tisman, G., Herbert, V. and Rosenblatt, S. Evidence that lithium induces human granulocyte proliferation: elevated serum vitamin BI= binding capacity in vivo and granulocyte colony proliferation in vitro. Br. J. Haematol. 1973, 24, 767-771 26 Turner, A.R. and Allalunis, M.J. Oral lithium carbonate increases colony stimulating activity production from human mononuclear cells. In: Advances in Experimental Medicine and Biology (Eds Rossof, A.H. and Robinson, W.A.) Plenum Press, New York, 1980, 127, pp. 127-136 27 Barrett, A.J., Griscelli, C., Buriot, D. and Faille, A. Lithium therapy in congenital neutropenia. Lancet 1977, li, 1357-1358 28 DeAlarcon, P.A., Goldberg, J., Nelson, D.A. and Stockman, J.A. Lithium therapy in childhood neutropenia. J. Pediatr. 1983, 102, 149-152 29 Weetman, A.P., McGegor, A.M., Lazarus, J.H., Smith, B.R. and Hall, R. The enhancement of immunoglobulin synthesis by human lymphocytes with lithium. C/in. Immuno/. Immunopathol. 1982, 22, 400-407

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