Seasonal influenza is still one of the major causes for mortality and morbidity worldwide (1
). Annual vaccinations are the most effective strategy to prevent and control influenza infections (2
). Most of the available influenza vaccines are administered via the intramuscular (i.m.) or subcutaneous route. These parenteral vaccines induce good systemic immune response but no mucosal immune response (3
), which limits their protective efficacy. In contrast, intranasal (i.n.) vaccines may induce both systemic and mucosal immune responses (6
). In addition, i.n. delivery of the vaccine does not require trained health care personnel for the administration of vaccine, is suitable for people with needle phobia, and circumvents the problem of needlestick injuries (10
A mucosal immune response is necessary for the protection of the upper respiratory tract, i.e., the port of entry for influenza virus (6
). The protection of the upper respiratory tract is mainly provided by sIgA (3
). Moreover, sIgA is known to induce cross-protection against variant viruses within the same subtype and also increase the protection during epidemics of heterologous viruses (6
). Furthermore, it is reported that the mucosal immune system develops early in life and is not affected by aging (19
). Therefore, a concomitant advantage of i.n. influenza immunization is that it can potentially provide effective immunity in all age groups and can be used for mass vaccination.
Currently, live attenuated influenza virus vaccines (LAIV) are marketed for i.n. administration. LAIV vaccines have shown to induce both systemic and mucosal immune responses. However, LAIV vaccine is licensed by the Food and Drug Administration only for persons aged 2–49 years but not for use in high-risk populations (elderly, children, and chronically ill patients) (21
). However, most of the marketed influenza vaccines are inactivated vaccines which can be administered safely via i.n. route to the whole population. A disadvantage of these vaccines is that they have shown to be poorly immunogenic when administered via this route (4
To increase the immunogenicity, inactivated influenza vaccines require adjuvants to potentiate the immune response when administered via the i.n. route. Several adjuvants are currently under development for i.n. immunizations like virus-like particles (23
), immunostimulating complexes (ISCOMS) (23
), lipids, nucleic acids (24
), and bacterial components (25
). However, the development of many of these adjuvants systems is hampered by safety and regulatory concerns (27
). For example, potent bacterial adjuvants like heat liable toxin of Escherichia coli
(LT) have shown severe side effects in humans (28
). Therefore, an adjuvant for i.n. inactivated influenza vaccine that is potent and safe for human use is still lacking.
The novel adjuvant Gram-positive enhancer matrix (GEM) particles are produced from the food-grade bacterium Lactococcus lactis
). L. lactis
is a nonpathogenic, noncolonizing Gram-positive bacterium. Moreover, L. lactis
is approved for human use by regulatory agencies and considered as a generally recognized as safe (GRAS) organism. The GEM particles are produced by heating the L. lactis
in acid, followed by washing with phosphate buffer (30
). The resulting particles are nonliving, deprived of intact surface proteins and intracellular content. The thick peptidoglycan cell wall, however, remains intact and provides the structural rigidity to constitute the bacterial-shaped peptidoglycan spheres of about 1 μm in size, referred to as GEM particles. The GEM particles have been studied for mucosal vaccination of malarial parasite antigen and pneumococcal antigens (31
). These studies demonstrated that antigens displayed on GEM particles induced higher immune response than antigen alone. Since GEM particles are a promising adjuvant for i.n. immunization, the aim of this study was to investigate the use of GEM particles as adjuvant for i.n. influenza vaccination.
In this study, we examined the immunogenicity in Balb/c mice of i.n. administered influenza subunit vaccine mixed with GEM particles as adjuvant. In earlier studies, the antigens (pneumococcal and malarial) were covalently bound to the GEM particles (29
). In contrast, in the present study, the particles were simply mixed with the antigens. The immune response was compared to i.m. and i.n. administered subunit influenza vaccine without the adjuvant.