Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA- or IgG-mediated protective mucosal immunity

Abstract

Local mucosal IgA antibodies play a central role in protection of the respiratory tract against influenza virus infection. Therefore, new-generation influenza vaccines should aim at stimulating not only systemic, but also local antibody responses. Previously, we demonstrated that the recombinant B subunit of the Escherichia coli heat-labile toxin (LTB) is a potent adjuvant towards nasally administered influenza subunit antigen. Here, we investigated the protection conferred by LTB-supplemented influenza subunit antigen given intranasally (i.n.) or intramuscularly (i.m.) to mice. Both i.n. and i.m. immunization with subunit antigen and LTB completely protected the animals against viral infection. Protection upon i.n. immunization was associated with the induction of antigen-specific serum IgG and mucosal IgA, whereas protection upon i.m. immunization correlated with strong serum and mucosal IgG, but not IgA responses. We conclude that LTB-supplemented influenza subunit antigen, given either i.n. or i.m, induces protective antibody-mediated mucosal immunity and thus represents a promising novel flu vaccine candidate.

Introduction

Annual influenza activity continues to be a considerable medical and economic burden. Influenza is a serious infectious disease with very extensive morbidity and significant often underestimated mortality, particularly among high-risk individuals including people with pulmonary or cardiac disease and the elderly [1], [2], [3], [4], [5]. Despite the fact that effective influenza vaccines are available, in many countries less than half of the people in high-risk groups are being vaccinated [2], [6], possibly due to a widespread belief that influenza is not a life-threatening disease and that vaccination is not particularly efficacious. However, a recent influenza outbreak in Hong Kong caused by an avian H5N1 virus [7] has demonstrated that a new influenza pandemic may occur, and this has resulted in a renewed awareness that vaccination against influenza is important.

Currently used influenza inactivated whole-virus and subunit vaccines are aimed at inducing serum antibody responses and are generally administered intramuscularly (i.m.). These vaccines are very successful in diminishing mortality among high-risk individuals [2], [5], [8]. Protection is mediated primarily by transudated plasma-derived neutralizing IgG antibodies, which inhibit infection of the lungs and the lower respiratory tract, and thus prevent viral pneumonia [2], [5], [8]. However, the upper respiratory tract is not adequately protected by plasma-derived antibodies, and therefore vaccinated individuals may still be infected and experience symptoms of flu [2], [5], [8]. It has been established that complete protection against influenza virus infection, as induced by natural infection, is mediated primarily by secretory IgA antibodies in the respiratory tract in addition to virus-neutralizing circulating IgG [9], [10], [11], [12]. Therefore, in order to achieve complete protection against influenza infection, new-generation influenza vaccines should aim at induction of a mucosal antibody response in the upper respiratory tract in addition to systemic IgG. In order to stimulate such a mucosal response, in general local immunization in conjunction with an appropriate mucosal adjuvant is required [13].

The Vibrio cholerae cholera toxin (CT) and closely related Escherichia coli heat-labile enterotoxin (LT) are among the most potent mucosal immunoadjuvants known to date [14], [15]. However, because of their intrinsic toxicity they can not be used as such for human vaccination purposes. Therefore, many attempts have been made to detoxify LT or CT with preservation of adjuvant activity, and several non-toxic variants of LT/CT have been studied in the context of experimental flu vaccines [16], [17], [18], [19], [20], [21], [22]. For example, Katz et al. observed that, upon oral immunization, both wild-type LT and an essentially non-toxic LT mutant, LT-R192G, strongly stimulate both humoral and cellular immune responses against an inactivated whole-virus influenza vaccine [16], [17]. Likewise, Tamura and coworkers have studied the adjuvant activity of the isolated B subunit of CT or LT in conjunction with influenza virus subunit antigen. In these studies, a trace of holotoxin was found to be essential for CTB or LTB to exhibit adjuvant activity, recombinant CTB or LTB alone being incapable of enhancing influenza-specific antibody responses [21], [22].

In contrast to Tamura and co-workers, we recently found that recombinant LTB by itself can act as a potent adjuvant towards intranasally (i.n.) administered influenza subunit antigen [23]. Moreover, in a subsequent study employing an LTB mutant deficient in G_M1-binding (LTB-G33D), we demonstrated that the adjuvant activity of LTB is directly related to its G_M1-binding capacity [24]. The observed adjuvanticity of recombinant LTB pertained to the induction of strong antibody responses in serum and high levels of IgA in respiratory and genitourinary tract secretions. In the present study, we investigated the protective capacity of these antibody responses in a murine challenge model. Remarkably, it is demonstrated that not only intranasal but also intramuscular immunization with LTB-supplemented influenza subunit antigen induces brisk serum, nasal, and bronchial antibody responses, and offers complete protection of both the lungs and the nasal cavity against viral infection.