Influenza Vaccine Enhancement with Immunomodulating Peptide Thymosin Alpha 1
Clinical studies have shown that treatment with thymosin alpha 1 increases response to vaccination.
Abstract
Thymosin alpha 1 (trade name Zadaxin) is a 28 amino acid immune-stimulating peptide, which was originally isolated from thymic tissue, and is now produced by solid-phase peptide synthesis. Thymosin has been shown to activate toll-like receptor 9 (TLR9), a cell-surface receptor that plays a key role in triggering the body's innate and adaptive immune responses, allowing significant increases in antibody production and clearance of viral infections. In preclinical studies, treatment with thymosin led to increased influenza-specific cytotoxic T lymphocyte (CTL) responses following vaccination. In a Phase 2 clinical study, treatment with biweekly doses of thymosin after vaccination was shown to decrease the incidence of influenza in elderly subjects from 19% in patients receiving influenza vaccine alone to 6% in patients receiving vaccine in combination with thymosin alpha 1 (p = 0.002). Thymosin was also shown to increase the antibody response to influenza vaccines in a number of clinical studies. These data support the use of thymosin alpha 1 to increase response to vaccination.
As exemplified by the current outbreak of novel H1N1 Influenza A (swine flu), there is an urgent need for novel vaccine-enhancing compounds that could expand the available vaccine supply, as well as potentially broaden the response to a vaccine across multiple virus strains. A further and perhaps even more important need may exist in the treatment of historically difficult-to-treat populations with newly developed vaccines. These include the elderly and persons immune-suppressed by treatment with chemotherapy or other invasive procedures like dialysis.
SciClone Pharmaceuticals, Inc.
Thymosin alpha 1 (trade name Zadaxin) is a naturally-circulating 28 amino acid immune-stimulating peptide that has shown promise in enhancing the response to the influenza vaccine. Originally isolated from bovine thymus material, referred to as thymic fraction 5 (TF-5),1 thymosin alpha 1 is currently produced by solid-phase peptide synthesis and is commercially available in over 30 countries worldwide.
Over decades of research, thymosin alpha 1 has been shown to have a number of immunomodulating activities, centered primarily on augmentation of T-cell function. It has been shown to promote T-cell differentiation and maturation;2–7 increase natural killer (NK)-cell activity,8–10 production of interferon (IFN)-alpha, interleukin (IL)-2, and IL-3, expression of IL-2 receptor following activation by mitogens or antigens,8,11–17 production of migration inhibitory factor (MIF),18 and antibody response to T-cell–dependent antigens;19–21 and activate dendritic cell tryptophan catabolism.22,23 Thymosin alpha 1 also has been shown to antagonize dexamethasone-induced apoptosis in thymocytes in vitro.24,25 In vivo administration of thymosin alpha 1 to animals immunosuppressed by chemotherapy, tumor burden, or irradiation showed that thymosin alpha 1 protects against cytotoxic damage to bone marrow, tumor progression, and opportunistic infections, thereby increasing survival time and number of survivors.26–28 (see also 5,6,29,30) Many of the in vitro and in vivo effects of thymosin alpha 1 have been interpreted as influences on either differentiation of pluripotent stem cells to thymocytes or activation of thymocytes into activated T-cells. The pattern of enhanced cytokine production, i.e., IFN-alpha and IL-2, suggests that thymosin alpha 1 may influence progression to a T-helper 1 (Th1) type of immune response.31
On a subcellular level, thymosin alpha 1 has been shown to activate toll-like receptor 9 (TLR9), a receptor that plays a key role in triggering the body's innate and adaptive immune responses.22,32–34 TLR9 activation affects various intracellular cell-signaling pathways that have been implicated in thymosin alpha 1's effects. These include stimulation of the p38 MAPK and NFkappaB signaling pathways, which are important for the maturation of, and antigen presentation by, dendritic cells.35–38 Together with the macroscopic effects of thymosin alpha 1 described above, these effects on signaling pathways provide a rationale for why treatment with thymosin alpha 1 could lead to significant responses to vaccination.
Thymosin alpha 1 has an excellent safety profile. Adverse experiences have been infrequent and mild in over 70 studies conducted under US corporate investigational new drug applications (INDs), physician INDs, trials in foreign countries, and market experience to date in over 2,500 individuals who have received thymosin alpha 1.39,40 These studies have shown effectiveness in a variety of indications, including response to vaccination (described below) as well as various types of cancer (melanoma, non-small-cell lung cancer, glioma, head and neck, renal, breast, gastric, and myelodysplasia) and infectious disease (chronic hepatitis B and C, HIV, primary immune deficiency).
Thymosin Stimulation of Vaccine Responses
Immune senescence, a normal aging process, has been related to a gradual decline in thymus function and thymic peptide production. The lack of thymic factors may contribute to the decline in immune function, particularly the T-cell component. In the elderly, quantitative and qualitative analysis of a specific antibody response after vaccination has been shown to be compromised when compared with the response in young subjects.20,41 Impairment of cell-mediated immunity also has been demonstrated in hemodialysis patients.42–44
In two preclinical studies, administration of thymosin alpha 1 to mice was shown to increase antibody response to tetanus toxoid (TT) and influenza vaccination.19,21 In the first study, thymosin alpha 1 was administered at 0.5 ug/kg to old (23 months) and young (2–3 months) mice on the day of vaccination and for each of four additional days after vaccination. Old mice that were not treated with thymosin alpha 1 had a significantly lower antibody response than young mice. Thymosin alpha 1 treatment resulted in significantly greater (p < 0.05) anti-TT antibody in both young and old mice, and appeared to restore the antibody response in the old mice close to levels seen in the young animals. In the second study, mice were pretreated with thymosin alpha 1 (10 ug/day for five days), and virus-specific CTL response was determined after vaccination with the influenza vaccine (Figure 1). Thymosin treatment increased the vaccine response of old (24–26 months) mice to that seen in young (2–6 months) mice.
Figure 1. Thymosin alpha 1 treatment increases specific CTL response after influenza vaccination21
In vitro antibody synthesis by human peripheral blood lymphocytes (PBL) also has been shown to be augmented by treatment with thymosin alpha 1. In this study, Ershler and colleagues examined age-related changes in antibody response to influenza vaccine in humans, and tested the capacity of TF-5 or thymosin alpha 1 to enhance specific antibody synthesis in vitro.20 They found that the antibody response following influenza vaccination was lower in elderly subjects (>65 years) compared to young subjects (<30 years). PBLs were then isolated from immunized young and old subjects and were incubated in vitro with either TF-5 at doses of 50, 100, 200 ug/mL or thymosin alpha 1 at doses of 0.01, 0.1, or 1.0 ng/mL. In vitro augmentation of the antibody response by either TF-5 or thymosin alpha 1 was detected in PBLs from 16 of the 28 elderly volunteers, compared to seven of the 30 young volunteers. In addition, there were twice as many responders (six versus three) in the 1 ng/mL thymosin alpha 1 group compared to the 0.01 ng/mL group, indicating a possible dose response that correlates with the endogenous level of circulating thymosin alpha 1 in healthy individuals (about 1 ng/mL).
Because thymosin alpha 1 can enhance T-cell–dependent specific antibody production, the addition of thymosin alpha 1 to vaccination programs for immunocompromised individuals should be effective. Five clinical studies have been undertaken and have demonstrated effectiveness of thymosin alpha 1 in increasing vaccine response, both in elderly subjects or patients undergoing hemodialysis, and after either influenza or hepatitis vaccination, as shown in Tables 1 and 2, respectively.
Table 1. Response to influenza vaccine
In a pilot trial, the effect of thymosin alpha 1 on influenza vaccination was evaluated by Gravenstein, et al., at the University of Wisconsin and Cornell Medical Center.45 Nine elderly subjects (age range 65 to 99 years) who had been nonresponsive to influenza vaccination in the previous year were given biweekly thymosin alpha 1 injections for five weeks following a single injection of seasonal influenza vaccine. A total of 67% (6/9) responded with high levels of anti-influenza antibodies, compared to a historical rate of 10% after revaccination in elderly subjects.
Table 2. Response to hepatitis B vaccine
This pilot trial was followed by a double-blind, randomized, placebo-controlled study conducted by the same researchers, at the Wisconsin Veterans' Administration Medical Center, Madison.46 Ninety male veterans over 64 years of age (range 65 to 99 years) were randomized to receive either thymosin alpha 1 or placebo biweekly for four weeks following injection with the trivalent vaccine. Effective immunization was defined as a four-fold or greater rise in antibody titer over a period of three to six weeks as measured by ELISA. About 69% (31/45) of thymosin-treated subjects were effectively immunized, compared to 52% (21/40) with placebo (p = 0.023). The differences seen were greater in subjects older than 77 years; the relationship between antibody levels and age also was significant (p < 0.039). As seen in the mouse studies, the antibody levels seen after treatment with thymosin alpha 1 were comparable to those seen in younger subjects.
In a study conducted at George Washington University, 330 elderly subjects were vaccinated with the trivalent seasonal influenza vaccine (B/Ann Arbor, A/H3N2 Leningrad, A/H1N1 Taiwan).47 Biweekly doses of thymosin alpha 1 were given for either two weeks (120 subjects) or four weeks (100 subjects) after vaccination; placebo was given for two weeks (110 subjects). In the subjects treated with thymosin for four weeks, greater antibody levels were seen compared to placebo or two weeks of thymosin (p = 0.015). In addition to the improved antibody response, the subjects treated with thymosin alpha 1 for four weeks also had a significantly decreased incidence of influenza, from 19% in patients receiving influenza vaccine with placebo to 5.5% in patients receiving the vaccine in combination with thymosin alpha 1 (p = 0.002) (Figure 2).
Figure 2. Thymosin alpha 1 treatment decreases influenza incidence47
In a study with 97 patients immunocompromised by chronic renal failure and undergoing hemodialysis, vaccination with monovalent A/Taiwan/1/86 (H1N1) vaccine followed by biweekly injections of thymosin for four weeks also led to improved response.48 Four weeks after vaccination, 71% (34/48) of subjects treated with thymosin alpha 1 had a four-fold or higher titer of specific antibody, compared to 43% (21/49) for those treated with placebo after vaccination (p < 0.002). This effect was long-lasting: eight weeks after vaccination the thymosin-treated subjects still had a rate of 65% response, compared to only 24% in the placebo-treated patients (p < 0.001).
Finally, the effect of thymosin on increasing response to vaccination was also seen with hepatitis B vaccine. In a study with 23 hemodialysis patients, nonresponders to a previous course of Heptavax vaccination were retreated with three vaccine injections one-month apart, with five biweekly injections of thymosin given after each vaccination. When response was measured three months later, 64% (7/11) of thymosin-treated patients had clinically significant anti-HBsAg titers, compared to 17% (2/12) of placebo-treated patients. The effect of thymosin was also long-lasting: measured after 12 months, 45% of the thymosin-treated patients still had clinically significant titers, whereas none of the placebo-treated patients retained theirs (p < 0.002).
Summary
Clinical studies have shown that treatment with thymosin alpha 1 increases response to vaccination, providing up to six times the typical response seen. In these studies, thymosin alpha 1 was provided as a dose of about 1.6 mg/kg (0.9 mg/m2), injected twice-weekly after vaccination. Future studies will evaluate higher doses, which could allow for a shorter course of administration.
Thymosin alpha 1 is one of only a few immunomodulating compounds that have been approved for human use and does not appear to induce any of the side effects and toxicities commonly associated with agents in this class, such as interferon and interleukin-2. Thymosin has shown great utility in subjects older than 80 years of age, and was even used safely in patients as old as 101 years. Taken together, these data support the use of treatment with thymosin alpha 1 to increase response to vaccination.
Cynthia W. Tuthill is senior vice president and chief scientific officer at SciClone Pharmaceuticals, Inc., Foster City, CA, 650.358.3450, ctuthill@sciclone.comStefan Gravenstein is professor of medicine, Brown University and clinical director, quality improvement, at Quality Partners, Providence, RI, and William B. Ershler is deputy clinical director, clinical research branch, intramural research program, National Institute of Aging, National Institutes of Health, Baltimore, MD, and director at the Institute for Advanced Studies in Geriatric Medicine, Gaithersburg, MD.
References
1. Goldstein AL, Low TL, McAdoo M, McClure J, Thurman GB, Rossio J, et al. Thymosin alpha1: isolation and sequence analysis of an immunologically active thymic polypeptide. Proc Natl Acad Sci USA. 1977;74(2):725–9.
2. Ho AD, Ma DD, Price G, Hunstein W, Hoffbrand AV. Biochemical and immunological differentiation of human thymocytes induced by thymic hormones. Immunol. 1983;50(3):471–6.
3. Ho AD, Stehle B, Dietz G, Hunstein W, Hoffbrand AV. Terminal differentiation of cord blood lymphocytes induced by thymosin fraction 5 and thymosin alpha 1. Scand J Immunol. 1985;21(3):221–5.
4. Knutsen AP, Freeman JJ, Mueller KR, Roodman ST, Bouhasin JD. Thymosin-alpha1 stimulates maturation of CD34+ stem cells into CD3+4+ cells in an in vitro thymic epithelia organ coculture model. Int J Immunopharmacol. 1999;21(1):15–26.
5. Ohta Y, Sueki K, Yoneyama Y, Tezuka E, Yagi Y. Immunomodulating activity of thymosin fraction 5 and thymosin alpha 1 in immunosuppressed mice. Cancer Immunol Immunotherapy 1983;15:108–13.
6. Ohta Y, Tezuka E, Tamura S, Yagi Y. Thymosin alpha 1 exerts protective effect against the 5-FU induced bone marrow toxicity. Int J Immunopharmacol. 1985;7:761–68.
7. Ahmed A, Wong D. T-lymphocyte maturation: cell surface markers and immune functions induced by T-lymphocyte cell-free products and thymosin polypeptides. Annals New York Academy Sci. 1979;332:81–94.
8. Serrate S, Schulof R, Leondaridis L, Goldstein AL, Sztein MB. Modulation of human natural killer cell cytotoxic activity, lymphokine production, and interleukin 2 receptor expression by thymic hormones. J Immunol. 1987;139:2338–343.
9. Di Francesco P, Pica F, Gaziano R, Favalli C, Garaci E. In vivo recovery of natural killer cell activity by the association of thymosin a 1 and cytokines during cocaine administation. Med Sci Res. 1994;22:41-42.
10. Favalli C, Mastino A, Jezzi T, Grelli S, Goldstein AL, Garaci E. Synergistic effect of thymosin alpha 1 and alpha beta-interferon on NK activity in tumor-bearing mice. Int J Immunopharmacol. 1989;11(5):443–50.
11. Sztein M, Serrate S, Goldstein A. Modulation of interleukin-2 receptor expression on normal human lymphocytes by thymic hormones. Proc Natl Acad of Sci USA. 1986;83:6107–111.
12. Sztein M, Serrate S. Characterization of the immunoregulatory properties of thymosin alpha 1 on interleukin-2 production and interleukin-2 receptor expression in normal human lymphocytes. Int J Immunopharmacol. 1989;11:789–800.
13. Svedersky L, Hui A, May L, McKay P, Stebbing N. Induction and augmentation of mitogen-induced immune interferon production in human peripheral blood lymphocytes by Na-desacetylthymosin alpha 1. Eur J Immunol. 1982;12:244–47.
14. Hsia J, Sarin N, Oliver JH, Goldstein AL. Aspirin and thymosin increase interleukin-2 and interferon-gamma production by human peripheral blood lymphocytes. Immunopharmacol. 1989;17(3):167–173.
15. Leichtling KD, Serrate SA, Sztein MB. Thymosin alpha 1 modulates the expression of high affinity interleukin-2 receptors on normal human lymphocytes. Int J Immunopharmacol. 1990;12:19–29.
16. Mutchnick MG, Appelman HD, Chung HT, Aragona E, Gupta TP, Cummings GD, et al. Thymosin treatment of chronic hepatitis B: a placebo-controlled pilot trial. Hepatol. 1991;14:409–15.
17. Cursaro C, Margotti M, Favarelli L, Ferri E, Gramenzi A, Di Giammarino L, et al. Thymosin alpha 1 plus interferon alpha enhance the immune and antiviral response of patients with hepatitis C virus infection. Hepatol. 1998;28(4):361A.
18. Thurman GB, Seals C, Low TLK, Goldstein AL. Restorative effects of thymosin polypeptides on purified protein derivative-dependent migration inhibition factor production by the peripheral blood lymphocytes of adult thymectomized guinea pigs. J Biological Response Modifiers. 1984;3(2):160–173.
19. Ershler W, Hebert J, Blow A, Granter S, Lynch J. Effect of thymosin alpha one on specific antibody response and susceptibility to infection in young and aged mice. Int J Immunopharmacol. 1985;7:465–71.
20. Ershler W, Moore A, Socinski M. Influenza and aging: age-related changes and the effects of thymosin on the antibody response to influenza vaccine. J Clin Immunol. 1984;4:445–54.
21. Effros RB, Casillas A, Walford RL. The effect of thymosin alpha 1 on immunity to influenza in aged mice. In: Aging: Immunology and Infectious Disease: Mary Ann Liebert, Inc; 1988. p. 31–40.
22. Romani L, Bistoni F, Perruccio K, Montagnoli C, Gaziano R, Bozza S, et al. Thymosin alpha1 activates dendritic cell tryptophan catabolism and establishes a regulatory environment for balance of inflammation and tolerance. Blood. 2006;108(7):2265–74.
23. Shrivastava P, Singh SM, Singh N. Effect of thymosin alpha 1 on the antitumor activity of tumor-associated macrophage-derived dendritic cells. J Biomed Sci. 2004;11(5):623–30.
24. Osheroff PL. The effect of thymosin on glucocorticoid recepters in lymphoid cells. Cellular Immunol. 1981;60:376–85.
25. Baumann CA, Badamchian M, Goldstein AL. Thymosin alpha 1 antagonizes dexamethasone and CD3-induced apoptosis of CD4+CD8+ thymocytes through the activation of cAMP and protein kinase C dependent second messenger pathways. Mech Age Develop. 1997;94:85–101.
26. D'Agostini C, Palamara AT, Favalli C, Sivilia M, Febbraro G, Bue C, et al. Efficacy of combination therapy with amantadine, thymosin alpha 1 and alpha/beta interferon in mice infected with influenza A virus. Int J Immunopharmacol. 1996;18(2):95–102.
27. Rasi G, Silecchia G, Sinibaldi-Vallebona P, Spaziani E, Pierimarchi P, Sivilia M, et al. Anti-tumor effect of combined treatment with thymosin alpha 1 and interleukin-2 after 5-fluorouracil in liver metastases from colorectal cancer in rats. Int J Cancer. 1994;57:701–5.
28. Pica F, Fraschetti M, Matteucci C, Tuthill C, Rasi G. High doses of thymosin alpha 1 enhance the anti-tumor efficacy of combination chemo-immunotherapy for murine B16 melanoma. Anticancer Research. 1998;18:3571–78.
29. Hadden JW, Saha A, Sosa M, Hadden EM. Immunotherapy with natural interleukins and/or thymosin alpha 1 potently augments T-lymphocyte responses of hydrocortisone-treated aged mice. Int J Immunopharmacol. 1995;17(10):821–28.
30. Shrivastava P, Singh SM, Singh N. Antitumor activation of peritoneal macrophages by thymosin alpha-1. Cancer Invest. 2005;23(4):316–22.
31. Sjogren MH. Thymalfasin: an immune system enhancer for the treatment of liver disease. J Gastroenterol Hepatol. 2004;19(12):S69–72.
32. Bozza S, Gaziano R, Bonifazi P, Zelante T, Pitzurra L, Mongagnoli C, et al. Thymosin alpha 1 activates the TLR9/MyD88/IRF7-dependent murine cytomegalovirus sensing for induction of anti-viral responses in vivo. Int Immunol. 2007;19:1261–70.
33. Giuliani C, Napolitano G, Mastino A, Di Vincenzo S, D'Agostini C, Grelli S, et al. Thymosin-alpha1 regulates MHC class I expression in FRTL-5 cells at transcriptional level. Eur J Immunol. 2000;30(3):778–86.
34. Romani L, Bistoni F, Gaziano R, Bozza S, Montagnoli C, Perruccio K, et al. Thymosin alpha 1 activates dendritic cells for antifungal Th1 resistance through toll-like receptor signaling. Blood. 2004;103(11):4232–9.
35. Yao Q, Doan LX, Zhang R, Bharadwaj U, Li M, Chen C. Thymosin-alpha1 modulates dendritic cell differentiation and functional maturation from human peripheral blood CD14+ monocytes. Immunol Lett. 2007;110(2):110–20.
36. Yao W, Zhu Q, Yuan Y, Qiao M, Zhang Y, Zhai Z. Thymosin alpha 1 improves severe acute pancreatitis in rats via regulation of peripheral T cell number and cytokine serum level. J Gastroenterol Hepatol. 2007;22(11):1866–71.
37. Iijima N, Yanagawa Y, Onoe K. Role of early- or late-phase activation of p38 mitogen-activated protein kinase induced by tumour necrosis factor-alpha or 2,4-dinitrochlorobenzene during maturation of murine dendritic cells. Immunol 2003;110(3):322–8.
38. Arrighi JF, Rebsamen M, Rousset F, Kindler V, Hauser C. A critical role for p38 mitogen-activated protein kinase in the maturation of human blood-derived dendritic cells induced by lipopolysaccharide, TNF-alpha, and contact sensitizers. J Immunol. 2001;166(6):3837–45.
39. Goldstein A, Goldstein A. From lab to bedside: emerging clinical applications of thymosin alpha 1. Expert Opinion Biological Therapy 2009;9:593-6-8.
40. Tuthill C. Issues in pharmaceutical development of thymosin alpha 1 from preclinical studies through marketing. Annals New York Acad Sci. 2007;1112:351–56.
41. Ershler W, Moore A, Hacker M, Ninomiya J, Naylor P, Goldstein A. Specific antibody synthesis in vitro. II. Age-associated thymosin enhancement of antitetanus antibody synthesis. Immunopharmacology. 1984;8:69–77.
42. Crosnier J, Jungers P, Courouc AM, Laplanche A, Benhamou E, Degos F, et al. Randomised placebo-controlled trial of hepatitis B surface antigen vaccine in French haemodialysis units: I, Medical staff. Lancet 1981;1(8218):455–9.
43. Grob P, Binswanger U, Zaruba K, Joller-Jemelka H, Schmid M, Hacki W, et al. Immunogenicity of a hepatitis B subunit vaccine in hemodialysis and in renal transplant recipients. Antiviral Research 1983;3:43–52.
44. Stevens CE, Alter HJ, Taylor PE, Zang EA, Harley EJ, Szmuness W. Hepatitis B vaccine in patients receiving hemodialysis. Immunogenicity and efficacy. N Engl J Med. 1984;311(8):496–501.
45. Gravenstein S, Ershler WB, Drumaskin S, Schwab R, Weksler ME. Anti-influenza antibody response: augmentation in elderly "non-responders" by thymosin alpha 1. Gerontologist. 1986;26:150A.
46. Gravenstein S, Duthie EH, Miller BA, Roecker E, Drinka P, Prathipati K, et al. Augmentation of influenza antibody response in elderly men by thymosin alpha one. A double-blind placebo-controlled clinical study. J Ame Geriatric Soc. 1989;37:1–8.
47. McConnell L, Gravenstein S, Roecker E, Spencer S, Simon G, Ershler W. Augmentation of influenza antibody levels and reduction in attack rates in elderly subjects by thymosin alpha 1. Gerontologist. 1989;29:188A.
48. Shen SY, Corteza QB, Josselson J, Gravenstein S, Ershler WB, Sadler JH, et al. Age-dependent enhancement of influenza vaccine responses by thymosin in chronic hemodialysis patients. In: Goldstein AL, editor. Biomedical Advances in Aging. New York: Plenum Press; 1990. pp. 523–30.
49. Shen S, Josselson J, McRoy C, Sadler J, Chretien P. Effect of thymosin alpha 1 on Heptavax-B vaccination among hemodialysis patients. Kidney Int. 1987:31:217.
50. Shen S, Josselson J, McRoy C, Sadler J, Chretien P. Effects of thymosin alpha 1 on peripheral T-cell and Heptavax-B vaccination in previously non-responsive hemodialysis patients. Hepatol. 1987;7(5):1120.
