The ocular conjunctiva and conjunctiva-associated lymphoid tissue as a mucosal immunization route: humoral and cellular immune responses against Salmonella typhimurium Bacterial Ghosts

Abstract

Purpose: In this work we explored the efficacies of Salmonella typhimurium (ST) Bacterial Ghosts (BGs), using the conjunctival route of immunization, to induce mucosal and systemic immunity against ST.

Methods: BALB/c and C57BL/6 mice were immunized via the conjunctiva with ST BGs in three different doses (50, 25 and 12.5 μg/mouse). Three immunizations were performed at 2 weeks interval and the evaluation of local and systemic immune response was done 2 and 8 weeks after the last immunization. This research was conducted in compliance with the “ARVO Statement of the Use of Animals in Ophthalmic and Visual Research” and the “Declaration of Helsinki”.

Results: ST BGs were well tolerated; non-toxic and successfully uptaken by the mouse conjunctival cells. We have found ST BG-specific IgG and IgA in tears and sera of both mice strains, as well as, IgG positive ST BG-specific cells. It was shown that the amount of ST BG-specific antibodies in mice' sera strongly correlated with the presence of ST BG-specific B cells in draining lymph nodes. 8 weeks after the last immunization, the levels of anti-ST BG IgG and IgA in the sera of BALB/c mice were higher than in C57BL/6. BALB/c mice exhibited also more prominent anti-ST BG IgA concentration in mice’ tears. The strongest IgG and IgA response was obtained with the highest dose used for immunization (50 μg/mouse). The conjunctival application of ST BGs in both mouse strains induced skewing of ST BG-specific immune responses toward a Th1/Th17 profile, as determined by the stimulation of IFNγ and IL-17A secretion and/or the concurrent pronounced reduction of IL-4 secretion.

Conclusions: Conjunctival immunization induced a strong ST BG-specific local and systemic immune response, and a Th1-biased response that is critical for control of Salmonella species. ST BGs, non-living shells of Gram negative ST bacteria, possess excellent safety properties and could be used as a candidate vaccine for an ocular delivery.

Keywords: 474 conjunctiva • 555 immunomodulation/immunoregulation