Introduction
Worldwide, annual influenza epidemics are estimated to result in 250 000 to 500 000 deaths and up to 5 million cases of severe illness [
1]. Adults 65 years of age and older are particularly vulnerable to complications from influenza infection, and account for 90% of all influenza-associated deaths in the U.S. [
2]. Vaccination is the primary strategy to prevent and reduce morbidity and mortality associated with influenza [
3], and the World Health Organization (WHO) recommends annual vaccination for groups at high risk of complications, including the elderly, young children, pregnant women, health-care workers, and individuals with underlying medical conditions [
1]. However, it has proven difficult to stimulate potent immune responses in elderly individuals, and studies in mice, ferrets and humans show that antigen-specific immune responses decline with age [
4‐
9]. Accordingly, a quantitative review by Goodwin et al. shows that the clinical vaccine efficacy of injectable influenza vaccines was 17–53% in the elderly [
10] compared to 70–90% in younger adults [
11], and that following influenza vaccination, younger adults had up to 4 times higher antibody levels compared with older adults [
10].
Strategies to augment the immune response in older adults include using alternative routes of vaccine delivery, a higher dose of antigen, or addition of an adjuvant. The Fluzone
® High-Dose influenza vaccine (Sanofi Pasteur Inc.) was designed for people 65 years and older, and contains four times the amount of hemagglutinin (HA) contained in standard-dose vaccines [
12,
13]. The adjuvanted injectable influenza vaccine Fluad
® was developed (by Novartis) for older adults. Both Fluzone
® High-Dose and Fluad
® have demonstrated enhanced humoral immune responses compared to standard-dose non-adjuvanted vaccines [
12‐
15]. Nevertheless, immunization advisory committees such as the Advisory Committee on Immunization Practices (ACIP) in the U.S., and the National Advisory Committee on Immunization (NACI) in Canada, consider that there is currently insufficient evidence to make a recommendation for their routine use in the elderly population. There is currently only one intranasal influenza vaccine on the western market, which is a live attenuated influenza vaccine (LAIV) called Fluenz
®/Flumist
® (MedImmune, AstraZeneca). However, LAIV is not licensed for adults >18 years in Europe, and not recommended for individuals ≥50 years in the U.S. It has been speculated that LAIV’s limited efficacy in adults and elderly could be due to its inability to infect individuals with pre-existing immunity [
16‐
18]. Following vaccination with LAIV, viral shedding can occur and there is a possibility that the vaccine strain is transmitted [
19], which could be problematic if LAIV recipients come into contact with severely immunocompromised persons.
Here we sought to evaluate a vaccine based on split influenza antigen together with the lipid-based adjuvant Endocine™ in aged mice. Endocine™ is a mucosal adjuvant that has been shown to be safe and well tolerated in both pre-clinical and clinical studies [
20‐
24]. We have previously demonstrated that vaccines formulated with Endocine™ enhance both humoral and cell-mediated immune responses in mice after intranasal vaccination [
20], and a study in ferrets demonstrated that Endocine™ induces high HI and virus neutralization titers, and fully protects ferrets from virus replication in the lungs [
22]. Furthermore, a recent study by Falkeborn et al. showed that an Endocine™-adjuvanted influenza vaccine evoked serum IgG and virus neutralization titers to comparable levels as cholera toxin (CT) in mice, and induced significantly higher serum and mucosal influenza-specific IgA titers than an alum-adjuvanted vaccine administered parenterally [
25]. In the current study we tested if Endocine™ could enhance systemic and mucosal humoral immune responses to influenza antigen in aged mice.
Materials and methods
Mice
Female BALB/c mice were purchased from Charles River, Germany and used for vaccinations at 2 or 20 months of age. All animal experiments were approved by the regional animal experimental ethics committee in Stockholm (North), Sweden. The study was performed in accordance with institutional guidelines at Adlego Biomedical AB, Stockholm, Sweden.
Antigen and adjuvant
All mice were vaccinated with A/California/07/2009(H1N1)pdm split influenza antigen from season 2012/2013 (kindly provided by Mitsubishi Tanabe Pharma Corporation, Kanagawa, Japan) with or without the adjuvant Endocine™ (Eurocine Vaccines AB, Stockholm, Sweden), except the control groups which received saline. The adjuvant Endocine™ consists of the lipids mono-olein and oleic acid [
21,
22,
24].
Vaccination and sampling
Mice (n = 8-11 per group) received 3 μg HA +/- 2% Endocine™ intranasally in 5 μL/nostril, or 50 μL of 3 μg HA subcutaneously in one hind leg. During vaccination, the mice were anaesthetized with isoflurane (IsoFlo® vet, Orion Pharma Animal Health, Sollentuna, Sweden). The mice were immunized three times at three-week intervals (day 0, 21 and 42). Blood samples and nasal lavages were collected one day before each immunization and three weeks after the last immunization at termination. The samples were stored at -20 °C until analysis. After sacrifice, the lungs were removed, put in PBS and frozen at -70 °C.
Determination of influenza-specific antibodies by ELISA
All serum samples were analyzed individually for influenza specific IgG, IgA, and subclass IgG (IgG1, IgG2a) with an enzyme-linked immunosorbent assay (ELISA). The samples were tested against the trivalent split vaccine Inflexal from season 2012/2013 (Cruzell, Madrid, Spain), consisting of A/California/07/2009 (H1N1), A/Victoria/361/2001 (H3N2) and B/Wisconsin/1/2010 influenza strains. Serological responses were measured as previously described [
20] with the exception that the plates were coated with Inflexal at a concentration of 1.5 μg HA/mL. Nasal lavage was also analyzed for nasal IgA against Inflexal. These samples were incubated overnight in +4 °C on the plate and then analyzed as previously described [
20]. The lungs were homogenized, flushed with PBS and the solution was collected and centrifuged to remove tissue and cell debris. To analyze total IgA, plates were coated with 1 μg/mL of Goat-anti mouse IgA (MyBiosource.com). The samples were then incubated overnight in +4 °C on the plate. To detect total IgA, Mouse Immunoglobulins AP (DAKO) diluted 1:3,000 and p-nitrophenyl phosphate (pNPP) (Sigma-Aldrich) were used. To analyze influenza specific IgA and IgG in lung homogenate, plates were coated with Inflexal and analyzed as previously described [
20].
Hemagglutination inhibition (HI)
Samples were pooled in each group from each time point except serum samples from day 63 which were analysed individually. Sera were tested in hemagglutination inhibition (HI) as previously described [
26] against pH1N1 A/California/07/09 virus at Viroclinics Biosciences B.V.
Statistical analysis
Statistical analysis was performed using GraphPad Prism (La Jolla, CA, US). Analysis of immunological parameters was performed using Kruskal-Wallis one-way ANOVA. When significant, Mann-Whitney U-test was applied for comparison between two groups. A p-value of <0.05 was considered statistically significant.
Acknowledgments
The authors thank Professor Marie Larsson, Linköping University, Sweden, who provided writing assistance. The authors also thank Mitsubishi Tanabe Pharma Corporation, Kanagawa, Japan, for providing influenza split antigens.