Whole inactvated influenza virus as an adjuvant for influenza peptide antigens

Influenza peptide antigens have the potential to induce cross-protective influenza-specific T cells. However, short peptide antigens are poorly immunogenic and therefore need to be formulated with a potent adjuvant. In this study, it was investigated whether whole inactivated influenza virus (WIV) can act as an adjuvant for influenza peptide antigens. The immunogenicity of WIV mixed with the HLA-A2.1-restricted influenza peptide GILGFVFTL (GIL) was assessed in HLA-A2.1 transgenic mice by quantification of peptide-specific IFN-γ+ CD8+ T cells after vaccination. Subsequently, a design of experiments (DoE) approach was utilized to study the synergistic effect between WIV adjuvant and peptide antigen at different doses. Moreover, the roles of WIV fusogenicity, peptide-WIV association and co-localization on the adjuvanticity of WIV were investigated. To assess whether WIV could also act as an adjuvant for other peptides, a peptide pool with three wild type (WT) influenza peptides was adjuvanted with WIV. In addition, three chemically enhanced peptide ligands (CPLs) derived from the WT peptides, which possessed a higher binding affinity to the MHC molecules, were adjuvanted with WIV and screened for their immunogenicity compared to the WT peptides. WIV was found to be a potent adjuvant for the GIL peptide. The DoE study revealed that WIV was able to act as an adjuvant at even low concentrations. Co-localization of the peptide antigen and WIV adjuvant was important for the induction of a peptide-specific immune response, whereas peptide-WIV association and WIV fusogenicity were not. WIV was also able to act as an adjuvant for both WT and CPL peptide pools. This study shows the potential of WIV as an adjuvant for influenza peptides. The simple formulation process and the existing safety track record of WIV make this an attractive adjuvant, which could also be used for non-influenza antigens. Whole inactivated influenza vaccine as a peptide adjuvant 77 introduCtion Seasonal influenza vaccines mediate their protective effect mainly through the induction of virusspecific neutralizing antibodies. These antibodies are directed against the influenza surface proteins hemagglutinin (HA) and neuraminidase (NA). However, these proteins can undergo changes due to antigenic shifts and drifts. These antigenic changes impair the neutralizing ability of antibodies induced by vaccines, rendering these vaccines ineffective. Therefore, additional immune responses such as cellular responses against influenza need to be induced to increase vaccine effectiveness [1]. Cellular immune responses represent a potential alternative to antibody-mediated immune responses. Recently, Sridhar et al. found that cellular immune responses correlated with reduced morbidity in patients infected with pandemic influenza [2]. Similarly, Wang et al. showed that patients with early influenza-specific CD8+ T cell responses recovered faster from severe H7N9-induced disease [3]. These studies confirmed that cellular responses against influenza can indeed be effective. Cellular immune responses such as cytotoxic T cells (CTLs) can effectively clear virus-infected host cells, thereby inhibiting viral replication and spread. Unlike most vaccine-induced antibodies, these CTLs recognize epitopes located on internal influenza proteins, which are conserved in many influenza strains. Owing to the conserved nature of these epitopes, cellular responses directed against these epitopes are potentially cross-reactive. Short linear peptides representing these epitopes are therefore attractive antigens for the development of cross-reactive influenza vaccines. Peptide antigens as such however suffer from low immunogenicity, due to inefficient delivery to antigen presenting cells (APCs) and the absence of pathogen-associated molecular patterns (PAMPs) or adjuvants to activate the APCs. Delivery of the peptide antigen to the cytoplasm of APCs is considered to be crucial for proper processing and subsequent presentation on MHC-I molecules, while activation of the APCs is important for licensing of naïve effector and memory CD8+ T cells [4]. Formulation of peptide antigens with an appropriate adjuvant (which can be a delivery system or an immunopotentiator [5]) is thus crucial to induce a cellular immune response against the peptide antigen. Water-in-oil emulsions such as Incomplete Freund’s Adjuvant (IFA) are commonly used and effective adjuvants for peptides, but are associated with severe adverse events such as lesion formation at the site of injection [6]. Thus, alternative adjuvants for peptide antigens are highly sought after. Particulate adjuvant systems such as liposomes or virosomes formulated with influenza peptides derived from internal proteins have proven to be effective for the induction of peptide-specific CTLs [7, 8], especially in combination with Toll-like receptor (TLR) agonists [9, 10]. However, the formulation of peptides into these delivery systems can be complicated and may result in low encapsulation rates. Adjuvants that can be directly admixed with peptide antigens would therefore be preferable. Whole inactivated influenza virus (WIV) possesses an innate adjuvant capability in the form of viral single-stranded RNA (ssRNA). Previously it was shown that influenza ssRNA is a potent TLR7 agonist