Murine cytokine responses following multiple oral immunizations using lipid‐formulated mycobacterial antigens

Abstract

Oral vaccination of mice with live Mycobacterium bovis BCG in lipid-formulation induces a gamma-interferon response that can be measured systemically, and confers protection against aerosolized mycobacterial challenge. Here, we have investigated cytokine responses following the vaccination, drawing comparisons between mice that received single or multiple oral immunizations and between mice receiving formulations containing live BCG or non-replicating mycobacterial antigens. Single oral immunization with lipid-formulated live BCG invoked secreted and cellular IFN-gamma responses in mice 8 weeks post-vaccination, the magnitudes of which were significantly elevated in mice receiving multiple immunizations over the 8-week period. Single oral immunization with live BCG also invoked an interleukin-2 response (but not TNF-alpha or IL-4), although the magnitude was not elevated by multiple immunizations. Multiple immunizations with lipid-formulated soluble or particulate non-replicating mycobacterial antigens failed to invoke cytokine responses, except for a low-level IFN-gamma response in mice multiple immunized with lipid-formulated heat-killed BCG. These results are discussed in contrast to the known patterns of cytokine induction following parenteral-route immunization with live or non-replicating mycobacterial antigens and with practical reference to the development of oral-delivery vaccines against tuberculosis.

Murine immune responses to oral BCG immunization in the presence or absence of prior BCG sensitization

“…In previous studies, murine IFN‐γ responses have been measured 2 months after oral vaccination using BCG in a lipid matrix 12 , 16 , 23 . Here, we first conducted a kinetic study of induced IFN‐γ responses, and determined that such responses were maximal at 5 months after vaccination (Figure 1); thus more detailed study of the influence of prior BCG sensitization on subsequent oral vaccine efficacy was conducted, using this time period as the optimal interval between oral vaccination and readout.…”

“…Cell‐free supernatants were harvested from cultured cells and assessed by capture enzyme‐linked immunosorbent assays, using murine cytokine reagents as described previously; 23 data were expressed as secreted ng or pg of cytokine per ml of cell culture supernatant. For ELISPOT assay, splenocytes were co‐cultured in the presence or absence of PPD‐B for 18 h, before IFN‐γ + cell spots were identified using anti‐murine IFN‐γ reagents, as described previously; 23 data were expressed as the frequency of IFN‐γ‐secreting cells per 10 6 splenocytes. All immunological and bacteriological data were analysed statistically by one‐way analysis of variance of log 10 ‐transformed data, using Tukey's post hoc tests to identify between‐group differences.…”

“…In previous studies, murine IFN-g responses have been measured 2 months after oral vaccination using BCG in a lipid matrix. 12,16,23 Here, we first conducted a kinetic study of induced IFN-g responses, and determined that such responses were maximal at 5 months after vaccination ( Figure 1); thus more detailed study of the influence of prior BCG sensitization on subsequent oral vaccine efficacy was conducted, using this time period as the optimal interval between oral vaccination and readout. Five months after oral vaccination, Purified Protein Derivative (PPD-B; Prionics Inc., Schlieren, Switzerland)-stimulated cellular and secretory IFN-g responses and interleukin (IL)-2 production were significantly elevated in mice that received the oral vaccine only or the injected vaccine only, and also in mice that received the oral vaccine as a sequel to a subcutaneous (s.c.) BCG immunization ( Table 1).…”

Strategies in Oral Immunization

“…These include oral vaccination with live Mycobacterium bovis BCG in lipid formulation to protect against tuberculosis (Cross et al 2007), Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae antigens ), adjuvanted influenza subunit vaccine (Amorij et al 2007), Lactococcus lactis as a live vector or virus-like particles expressing human papillomavirus type 16 L1 Thones and Muller 2007), and entericcoated capsules containing adenoviral vectors expressing HIV antigens and peptides (Mercier et al 2007). Recent experimental studies have investigated the feasibility of PEGylated PLGA-based nanoparticles to target antigens directly to M cells in the gut following oral administration (Garinot et al 2007).…”

Oral bacillus Calmette-Guérin vaccine against tuberculosis: why not?

“…A single oral immunisation with lipid-formulated live BCG invoked secreted and cellular IFN-γ responses in mice eight weeks post-vaccination, the magnitudes of which were significantly elevated in mice receiving multiple immunisations over the eight-week period. Interestingly, the magnitude of IFN-γ responses in mice was amplified by repeated oral immunisations of live BCG, whereas the magnitude of IL-2 production did not increase with multiple immunisations ( Cross et al 2008 ).…”