Nonclinical safety assessments of a novel synthetic toll-like receptor 4 agonist and saponin based adjuvant.

A full nonclinical safety package was performed to support the clinical use of SPA14, a novel liposome-based vaccine adjuvant containing the synthetic toll-like receptor 4 agonist E6020 and saponin QS21. E6020 and QS21 were tested negative for their potential genotoxic effects in Ames, micronucleus, or mouse-lymphoma TK (thymidine kinase) assay. To evaluate the potential local and systemic effects of SPA14, two toxicity studies were performed in rabbits. In the first dose range finding toxicity study, rabbits received two intramuscular injections of SPA14 at increasing doses of E6020 combined with two antigens, a control (saline), the two antigens alone, or the antigens adjuvanted with a liposome-based adjuvant AS01B. No systemic toxicity was detected, supporting the dose of 5 µg of E6020 for the subsequent pivotal study. In the second repeated dose toxicity study, rabbits received four intramuscular injections of SPA14 alone, a control (saline), SPA14 combined with two antigens, the two antigens alone, or the antigens combined with AF03 adjuvant, which is a squalene-based emulsion. SPA14 alone or in combination with the antigens was well tolerated and did not cause any systemic toxicity. Finally, two safety pharmacology studies were conducted to assess potential cardiovascular and respiratory effects of E6020 and SPA14 in conscious telemetered cynomolgus monkeys and beagle dogs, respectively. One subcutaneous injection of E6020 in monkeys and one intramuscular injection of SPA14 in dogs had no consequences on respiratory and cardiovascular functions. Altogether these results support the clinical development of SPA14.

Lessons from NK Cell Deficiencies in the Mouse.

Since their discovery in the late 1970s, in vivo studies on mouse natural killer (NK) cell almost entirely relied on the use of depleting antibodies and were associated with significant limitations. More recently, large-scale gene-expression analyses allowed the identification of NKp46 as one of the best markers of NK cells across mammalian species. Since then, NKp46 has been shown to be expressed on other subsets of innate lymphoid cells (ILCs) such as the closely related ILC1 and the mucosa-associated NCR(+) ILC3. Based on this marker, several mouse models specifically targeting NKp46-expressing cell have recently been produced. Here, we review recent advances in the generation of models of deficiency in NKp46-expressing cells and their use to address the role of NK cells in immunity, notably on the regulation of adaptive immune responses.

Design and preclinical characterization of a novel vaccine adjuvant formulation consisting of a synthetic TLR4 agonist in a thermoreversible squalene emulsion.

We describe the development, analytical characterization, stability and preclinical efficacy of AF04, a combination adjuvant comprising the synthetic toll-like receptor 4 (TLR4) agonist, E6020, formulated in AF03, a thermoreversible squalene emulsion. By using AF04 with the recombinant major outer membrane protein of Chlamydia trachomatis (Ct-MOMP) and with the recombinant surface glycoprotein gB from human cytomegalovirus (CMV-gB) as model antigens, we show that AF03 and E6020 can synergize to augment specific antibody and Th-1 cellular immune responses in mice. In terms of formulation, we observe that the method used to incorporate E6020 into AF03 affects its partition between the oil and water phases of the emulsion which in turn has a significant impact on the tolerability (IV pyrogenicity test in rabbits) of this novel adjuvant combination.

Modern Vaccine Adjuvant/Formulation--Session 9: Adjuvants.

The Session 9 of the Modern Vaccine Adjuvant/Formulation meeting pointed out the permanent need for vaccine improvement and for adjuvant development. Indeed, the increasing use of recombinant subunit vaccines for both parenteral and mucosal vaccination necessitates the development of improved adjuvants. This session dealt with strategies for the development of new vaccine adjuvants with respect to the availability of new molecules targeting specifically the receptors of the systemic or mucosal immune system.

AF03, an alternative squalene emulsion-based vaccine adjuvant prepared by a phase inversion temperature method.

AF03 is a squalene-based emulsion adjuvant that is present in the adjuvanted pandemic influenza vaccine, Humenza™. In this report, we describe the design and development of this novel adjuvant formulation from the selection of the oil and surfactant system used in the adjuvant composition to the phase inversion temperature emulsification process that afforded AF03 as a long-term stable and well calibrated oil-in-water emulsion. The emulsion was characterized by its particle sizes, surface and interfacial tensions, viscosity, and long-term stability.

Characterization of surfactants in an oil-in-water emulsion-based vaccine adjuvant using MS and HPLC-MS: structural analysis and quantification.

Mass spectrometry (MS) and high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) techniques were developed to characterize two surfactants, cetheareth-12 and sorbitan oleate, used to manufacture AF03, an emulsified oil-in-water (O/W) adjuvant. MS was first used to characterize the chemical structure and determine the composition of the two surfactants. The two surfactants appeared as complex products, in particular with respect to the nature of the fatty alcohols and the distribution of the number of ethylene oxides in cetheareth-12, and with respect to the different sorbitan-bound fatty acids (oleic, linoleic and palmitic acids) in sorbitan oleate. Subsequently, once the ions of interest were determined and selected, HPLC-MS was developed and optimized to quantify and to "quality control" the two surfactants as raw materials and as ingredients in the final O/W emulsion bulk and filled products.

Improvement of immunogenicity of meningococcal lipooligosaccharide by coformulation with lipidated transferrin-binding protein B in liposomes: implications for vaccine development.

Among various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group B Neisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluated in vitro using the Limulus Amebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, an in vivo study demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.