Head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: Effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model

Abstract

In order to study the influence of antigen composition, spatial organization of antigen and the route of administration, four cell culture-derived, inactivated, nonadjuvanted influenza vaccine formulations, i.e. whole inactivated virus (WIV), split, subunit and virosome vaccines were prepared from a single antigen batch. We directly compared the immunogenicity and efficacy of these vaccine formulations after intramuscular (i.m.) or intranasal (i.n.) administration in mice. Prime and boost vaccination were followed by a potentially lethal homologous aerosol challenge. For all vaccines, the i.m. route induced higher serum humoral immune responses as compared to the i.n. route and protected all mice against challenge at a dose of 5 μg. Upon i.n. immunization only WIV and split vaccines induced detectable IgG titers and partial protection against challenge but only very low HI titers were induced in almost all mice. WIV induced mainly IgG2a/c titers via both routes, whereas split vaccine induced exclusively IgG1 titers via both routes. Subunit and virosome vaccines induced exclusively IgG1 via the i.m. route. Mucosal sIgA levels were only detected after i.n. vaccination with WIV. Furthermore, vaccines containing all viral components (WIV and split vaccine) induced higher serum HI titers and serum antibody titers than subunit and virosome vaccines. The differences in magnitude and quality of immune responses of split and WIV, having the same composition, are likely related to their distinct spatial organization. In conclusion, the direct comparison between WIV, split, subunit and virosomes, shows that the differences in immune responses between these well known influenza vaccines can be explained by both the composition and particulate structure of these vaccine formulations.

Introduction

Since the outbreaks of highly pathogenic avian influenza (H5N1) in poultry in 1996 in Southeast Asia, resulting in lethal human infections [1], the threat of a new influenza pandemic has become real. This has prompted efforts to produce more effective influenza vaccines. In addition to adjuvant research, needle-free immunization routes are being explored [2].

There are several types of nonadjuvanted, inactivated influenza vaccines, like whole inactivated virus (WIV), split, subunit and virosome vaccines. Split and subunit influenza vaccines are widely used for seasonal intramuscular (i.m.) vaccination, generally well tolerated and considered to induce similar immune responses [3], [4], although some studies show that split is more immunogenic than subunit vaccine [5], [6]. Nonadjuvanted virosomes demonstrate comparable tolerability and immunogenicity to subunit and split vaccines in humans [7]. WIV induces strong immune responses after i.m. immunization and is superior to split and subunit vaccines in naïve human populations [8], [9], [10]. However, whole inactivated influenza virus (WIV), used as a vaccine until the 1980s, was withdrawn form the market because side effects like fever and headache were more frequently observed than with split and subunit vaccines [10]. These adverse reactions were attributed to the presence of impurities derived from production in eggs and have mainly been observed in influenza B strains [11]. However, it has been suggested that WIV may work best in case of a pandemic [12].

Currently, most influenza vaccines are still produced in eggs. This has major disadvantages like the prompt need for eggs, especially in a pandemic situation, limited production capacity and varying vaccine yields. Furthermore, these vaccines have induced allergic reactions to egg-derived protein impurities. Several companies aim to overcome these disadvantages of egg-derived vaccine production by using mammalian cell lines for vaccine production, and some publication have shown comparable immunogenicity of MDCK-derived and egg-derived split vaccines [13] as well as subunit vaccines [14].

When compared to vaccines administered via i.m. injection, intranasal (i.n.) vaccination offers several advantages [15] such as simple, needle-free administration and less adverse reactions. Furthermore, i.n. vaccines can induce mucosal immune responses which may play an important role in the first line of defense against pathogens transmitted via the airways like influenza virus [16]. Regarding i.n. immunization, the immunogenic properties of nonadjuvanted, inactivated influenza vaccines are less clear and only a live attenuated influenza vaccine is currently licensed in the US and Russia and seems to be equally or more effective than current i.m. vaccines [17], [18]. From the nonadjuvanted inactivated influenza vaccines, WIV seems the most immunogenic one [19], [20].

All inactivated influenza vaccines are dosed on the amount of hemagglutinin (HA), but the total antigen composition and spatial organization of the vaccine components is different for WIV, split, subunit and virosome vaccines, as illustrated in Fig. 1. WIV and split vaccine contain all viral components, including all viral proteins and the viral genomic single-stranded RNA (ssRNA), which is complexed with viral nucleoprotein (NP) and viral polymerase proteins into ribonucleoprotein particles (RNPs). Subunit and virosomes on the other hand, contain mainly HA and small amounts of neuraminidase, another envelope glycoprotein. When comparing the spatial organization, WIV and virosomes have their antigens organized in vesicles resembling the size of the original virus (100–300 nm), whereas subunit vaccine does not contain this particulate structure and split vaccines consist of a mixture of solubilized membrane proteins and viral internal components (Fig. 1).

Although several reports have been published on the immunogenicity of various influenza vaccines via i.m. and/or i.n. vaccination in humans and mice (e.g. [3], [4], [9], [21], [22], [23], [24]), a direct comparison of these vaccine formulations is missing.

When studying the influences of antigen composition, spatial organization of vaccine components and route of administration on the magnitude and quality of induced immune responses, the antigen source, and the fact that human subjects have varying histories of influenza infections may bias the results. Therefore, and because there is a trend towards replacing egg-based vaccines by cell culture-derived vaccines, we made a head-to-head comparison of four types of nonadjuvanted, cell culture-derived, inactivated influenza vaccines, prepared from the same antigen batch in unprimed mice.