Vaccine 29 (2011) 9385–9390 Contents lists available at SciVerse ScienceDirect Vaccine jou rn al h om epa ge: www.elsevier.com/locate/vaccine Evaluating functional antibodies in rhesus monkeys immunized with hepatitis B virus surface antigen vaccine with novel adjuvant formulations Daniel C. Freed, Victoria M. Towne, Danilo R. Casimiro, Qinjian Zhao 1 , Tong-Ming Fu ∗ Department of Vaccine Basic Research, Department of Vaccine Processing Research and Development, Merck Research Laboratories, West Point, PA, USA a r t i c l e i n f o Article history: Received 25 April 2011 Received in revised form 6 September 2011 Accepted 29 September 2011 Available online 11 October 2011 Keywords: HBV HBsAg Adjuvant ISS1018 ISCOMATRIX TM adjuvant RFHBs1 Inhibition titers Functional Abs a b s t r a c t Effective and safe novel adjuvants are of great interest to the vaccine research community. In this study, we describe our evaluation of adjuvant formulations containing a TLR9 agonist adjuvant (ISS1018) or ISCOMATRIX TM adjuvant for a two-dose regimen of hepatitis B virus surface antigen virus-like parti- cle vaccine in mice and rhesus macaques. Our results show a 10–20 fold improvement in Ab binding titers determined in an antigen-sandwich assay for adjuvant formulations with ISCOMATRIX TM adju- vant, in comparison to routine aluminum formulation. Furthermore, we optimized a competition assay to evaluate a functional component of immune sera, using a conformation-dependent and protective mAb, RFHBs1, as the probe. Although good correlation was observed between Ab binding titers from the antigen-sandwich assay and functional titers from the in-solution competition against RFHBs1, the latter assessment provided a much more stringent ranking of adjuvant formulations than the former. These results indicate the importance of evaluating functional Abs when assessing and comparing novel adju- vant formulations, as it provides another angle to investigate the effects of change in adjuvant composition on antigenic integrity of the testing vaccines. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Adjuvant is an important component of an effective vaccine. The concept of adjuvant and the use of aluminum salt as a vaccine adju- vant for human use were introduced in the 1930s. Despite highly active research on novel adjuvants in the last two decades, there is only one new adjuvant approved for human use in the United States. A recombinant human papilloma virus vaccine with AS04, an adjuvant formulation of aluminum hydroxide and monophos- phoryl lipid A (MPL), was approved in 2009 after an extensive safety and efficacy trial [1]. There are challenges and experimental hurdles in adjuvant research in preclinical stages [2,3]. Firstly, one of the critical concerns for new adjuvants is the safety and tolerability in humans. However, there is no reliable animal model or biomarkers to assess this attribute. In addition, the incidence for such adverse effects is so low that the convincing link can Abbreviations: HBsAg, hepatitis B virus surface antigen; pHBsAg, plasma-derived HBsAg; HBV, hepatitis B virus; VLP, virus-like particle; HPV, human papilloma virus; MPL, monophosphoryl lipid A; AAHS, amorphous aluminum hydroxylphosphate sulfate; GMT, geometric mean titers; TLR9, Toll-like receptor 9. ∗ Corresponding author. Tel.: +1 215 652 8270; fax: +1 215 993 3489. E-mail addresses: zhaoqinjian@xmu.edu.cn (Q. Zhao), tong-ming fu@merck.com (T.-M. Fu). 1 Present address: Xiamen University, School of Public Health, Xiamen, Fujian 361005, PR China. only be established through large scale clinical trials. This concern is illustrated in a recent phase III trial where a subject devel- oped Wegener’s granulomatosis, a rare form of vasculitis, after receiving two doses of HBsAg vaccine with a TLR9 agonist, ISS1018 (http://investors.dynavax.com/releasedetail.cfm?ReleaseID=305191). Secondly, the mechanisms of action underlying a successful adju- vant are not fully understood [4]. Many novel adjuvants have been assessed for their ability to activate immature dendritic cells in cultures, or to elicit T cell responses or “proper” cytokine profiles in animals. Although these could constitute or reflect complex host immune responses to a successful vaccination, their values in assessing a novel adjuvant have yet to be validated. The most reliable markers to assess novel adjuvants should be antigen-specific immune responses, preferably those defined as correlates for protection in efficacy trials. Similarly, antigens that have not been fully validated in clinical studies are of limited value in assessing novel adjuvants in preclinical studies. Because of these challenges, many investigations on novel adju- vants were conducted using HBsAg vaccine as a model antigen. It seems to be an attractive option since an antigen-sandwich binding assay is widely used for evaluating the Abs in clinical trials, and a correlate for protection of 10 mIU/mL is accepted based on some early publications [5–7]. However, a functional serological assay, such as the one reported with a murine mAb RFHBs1 [8], has not been utilized for evaluation of Abs responses to HBsAg vaccine in the context of adjuvants. To study the impact of 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.09.122