Most infectious agents enter the body through mucosal surfaces such as the intestine or airways. Protective immune responses induced by such infections involve both cellular immune responses and systemic IgG, but at mucosal surfaces secretory IgA provides the most effective protection. Studies have indicated that IgA responses are dependent on immune responses in mucosal lymphoid tissues such as intestinal Peyer's patches and Nasal Associated Lymphoid Tissues (NALT) or tonsils [1
], where epithelial M cells acquire and transport antigens to underlying lymphoid tissue. Unfortunately, conventional vaccines rely instead on injected antigens, which induce IgG but not IgA. Live attenuated virus vaccines such as cold-adapted influenza (e.g., FluMist®
), or oral polio vaccine can provide better mucosal immunity, but these are a greater challenge to develop, and they require an expensive cold chain that complicates delivery in developing countries.
Vaccination at mucosal surfaces is a strategy that can help overcome the limitations of injected vaccines (needle disposal, trained medical staff required to administer the vaccine), but also to provide the benefit of mucosal IgA responses. Progress with this strategy has been made in animal studies using two distinct approaches that could be described as bioengineering versus immunological. In typical bioengineering approaches, vaccine antigens are encapsulated in polymer nanoparticles to package and protect the antigen (reviewed in [5
]); the particles are administered in an aerosol suspension for inhalation, or as a liquid suspension for intranasal instillation. Here, it is assumed that M cells will non-specifically acquire the encapsulated antigens from the lumen and initiate mucosal immune responses. However, antigen can also be acquired by dendritic cells in the mucosal epithelium [6
] and drain into other lymphoid tissues, so mucosal IgA responses are not always efficiently induced.
In contrast to bioengineering strategies, immunological approaches are based on targeting antigen delivery to M cells for specific uptake; direct targeting should provide greater control over the induced immune response than unregulated transport to draining lymph nodes. In animal models, targeting to M cells has been successful in inducing mucosal IgA responses. M cell targeting was achieved using a variety of ligands, including lectins or antibodies specific to a fucose moiety presented at the surface of mouse (but not human) M cells [8
], RGD peptides to bind exposed integrins [11
], and a Reovirus sigma protein specific for JAM-A [12
]. Challenges still remain, such as the identification of M cell target receptors that will reliably work in humans, and the identification of an effective mucosal adjuvant. Indeed, in the absence of an effective adjuvant, M cell targeting in mice has been found to be very effective in inducing immunological tolerance instead of immunity [12
We previously identified the tight junction protein Claudin 4 as a candidate M cell endocytosis receptor [15
]. Though Claudin 4 is normally found in tight junctions, it was also found redistributed into the cytoplasm of mouse and human M cells and appears to be part of the particle endocytosis machinery. To test the potential of Claudin 4 targeting, we developed a peptide derived from the c-terminal domain of the Clostridium perfringens
enterotoxin (CPE), which binds to the second external domain of Claudin 4 [18
]. Using fluorescently labeled microparticles and polymer nanoparticles displaying CPE or fusion proteins with CPE, we demonstrated that the CPE peptide retains Claudin 4 binding [20
] and mediates enhanced uptake by M cells in vivo [21
]. In addition, CD137 mutant mice that lack M cell function failed to take up Claudin 4-targeted particles, confirming the M cell-dependent uptake [23
]. Thus, using the CPE peptide, M cell targeting of mucosal vaccines might be possible in humans.
In the present study, we tested the mucosal immune response to engineered vaccine fusion proteins incorporating antigen and the CPE M cell targeting peptide. We report here that with an intranasal administration protocol, M cell targeted fusion proteins are effective in enhancing secretory IgA responses along with a systemic serum Th2-skewed IgG response.