Phytol-based novel adjuvants in vaccine formulation: 1. assessment of safety and efficacy during stimulation of humoral and cell-mediated immune responses.

BACKGROUND: Vaccine efficacy depends significantly on the use of appropriate adjuvant(s) in the formulation. Phytol, a dietary diterpene alcohol, is similar in structure to naturally occurring isoprenoid adjuvants; but little is known of its adjuvanticity. In this report, we describe the relative safety and efficacy of phytol and its hydrogenated derivative PHIS-01 compared to commercial adjuvants. METHODS: We tested adjuvant properties using a formulation consisting of either a hapten, phthalate-conjugated to a protein, keyhole limpet hemocyanin (KLH), or ovalbumin (OVA) emulsified with the test adjuvants in mice without any surfactant. Humoral immunity was assessed in terms of titer, specificity, and isotypic profiles. The effect on cell-mediated immunity was studied by assaying the induction of either OVA- or B-lymphoma-specific cytotoxic T-lymphocyte (CTL) activity. RESULTS AND DISCUSSION: The phytol compounds, particularly PHIS-01, elicit increased titers of all major IgG subclasses, especially IgG2a. Unlike commercial adjuvants, both phytol compounds are capable of inducing specific cytotoxic effector T cell responses specific to both OVA and B-lymphoma tested. Phytols as adjuvants are also distinctive in that they provoke no adverse anti-DNA autoimmune response. Intraperitoneally administered phytol is comparable to complete Freund's adjuvant in toxicity in doses over 40 ug/mouse, but PHIS-01 has no such toxicity. CONCLUSION: These results and our ongoing studies on antibacterial immunity show that phytol and PHIS-01 are novel and effective adjuvants with little toxicity.

Phytol-based novel adjuvants in vaccine formulation: 2. Assessment of efficacy in the induction of protective immune responses to lethal bacterial infections in mice.

BACKGROUND: Adjuvants are known to significantly enhance vaccine efficacy. However, commercial adjuvants often have limited use because of toxicity in humans. The objective of this study was to determine the comparative effectiveness of a diterpene alcohol, phytol and its hydrogenated derivative PHIS-01, relative to incomplete Freund's adjuvant (IFA), a commonly used adjuvant in augmenting protective immunity in mice against E. coli and S. aureus, and in terms of inflammatory cytokines. METHODS: Vaccines, consisting of heat-attenuated E. coli or S. aureus and either of the two phytol-based adjuvants or IFA, were tested in female BALB/c mice. The vaccines were administered intraperitoneally at 10-day intervals. The efficacy of the phytol and PHIS-01, as compared to IFA, was assessed by ELISA in terms of anti-bacterial antibody and inflammatory cytokines. We also examined the ability of the vaccines to induce specific protective immunity by challenging mice with different doses of live bacteria. RESULTS AND DISCUSSION: IFA, phytol, and PHIS-01 were equally efficient in evoking anti-E. coli antibody response and in providing protective immunity against live E. coli challenges. In contrast, the antibody response to S. aureus was significant when PHIS-01 was used as the adjuvant. However, in terms of the ability to induce protective immunity, phytol was most effective against S. aureus. Moreover, during challenges with live E. coli and S. aureus immune mice produced much less IL-6, the mediators of fatal septic shock syndromes. CONCLUSION: Our results show that vaccine formulations containing phytol and PHIS-01 as adjuvants confer a robust and protective immunity against both Gram-negative and Gram-positive bacteria without inducing adverse inflammatory cytokine due to IL-6.