Wortmannin br Results br Discussion For any new technology

Results
Discussion For any new technology a primary concern is safety and reactogenicity, especially for a vaccine that potentially contains powerful stimulators of the innate immune system. In these studies, we saw a correlation between dose and local reactions, but even at the highest dose of 5.9/100μg the vaccine had an acceptable reactogenicity profile, which was less than that reported for the licensed, Outer Membrane Vesicles (OMV) containing vaccine, 4CMenB (Toneatto et al., 2011). Importantly, as 1790GAHB comprises the outer membrane of Shigella, it contains appreciable levels of lipid A, which in the 1790-GMMA producing strain is genetically modified to be penta-acylated (Gerke et al., 2015) (320nmoles of lipid A per 100μg of protein, data not shown) and other potential activators of innate immune responses (e.g. lipidated proteins). In pre-clinical studies, rabbits receiving 5.9/100μg IM had an average temperature increase of 0.4°C compared to controls at 4h after injection (Gerke et al., 2015). In the clinical studies we saw no consistent temperature change as a function of dose and no significant pyrogenic response over the 4hour post vaccination. As the ratio of lipid A to protein is relatively constant and the structure of the outer membrane is similar in GMMA from related organisms (e.g. from other Shigella species), doses of up to at least 100μg of protein are likely to be acceptable even though the OAg content may vary considerably, depending on the average size of the OAg in each GMMA. These studies compared three routes of immunization (IN, ID and IM), which have different theoretical advantages: IN provides a needle free vaccination route and may elicit stronger mucosal immunity (Zaman et al., 2013), ID may provide dose sparing (Zehrung et al., 2013) and IM may provide strong systemic immunity and, as the standard vaccination route, may be easier to introduce into routine public health vaccination programs. All three routes gave strong IgG responses in rabbits when tested at the maximum human dose (Gerke et al., 2015), with the ID route giving significantly higher antibody responses than IM, and the IN route lower peak responses than the IM. These preclinical data Wortmannin with the clinical results, which show no detectable immune responses in most human subjects vaccinated via the ID or IN routes, even at the maximum doses tested. Therefore, data suggest that ID vaccination is unlikely to be a useful route for GMMA adsorbed on aluminium hydroxide. Alternatively, IN results seem to be consistent with human IN vaccination studies of S. flexneri 2a LPS adsorbed onto detergent extracted Neisseria meningitidis OMV where no or minimal circulating antibody was detected after two doses of 90μg of LPS, but significant IgG and serum IgA was measured following 360μg of LPS (Fries et al., 2001). While these subjects were given a different formulation, and a different serotype of Shigella, measured in a different way (μg of total LPS vs μg of OAg), never-the-less, these data suggest that the maximum IN dose in our study of 4.8/80μg is too low to definitively rule out the use of IN route with substantially larger doses. They also suggest that immunogenicity responses in rabbits are not predictive of human results. There is an interest in Shigella vaccines that induce strong mucosal immunity (i.e. sIgA responses) (Mani et al., 2016). Our limited results, from subjects immunized with 1790GAHB in cohort 3 of trial 2, did not show any significant IgA response at the tested doses (i.e., 0.59/10μg ID, 4.8/80μg IN and 0.29/5μg IM). We acknowledge that these results do not preclude the possibility that the use of an adjuvant, like double mutant LT, might induce a more robust mucosal response using the ID or IN routes. However, the key pathogenic step in shigellosis is the invasion of gut epithelial cells from the basal surface by bacteria that have crossed into the systemic circulation via M cells (Zychlinsky et al., 1994). Thus, although sIgA may play a role in reducing the bacterial inoculum, IgG is likely to play a key role in preventing disease. This central role for systemic IgG is consistent with the demonstrated field efficacy of an IM conjugate vaccine (Cohen et al., 1997) and the correlation between natural induced anti-OAg IgG levels and subsequent risk of infection (Cohen et al., 1991). Patients recovering from infection with Shigella have strain specific immunity against subsequent infection (Cohen et al., 1989). As strain specificity is determined by the anti-OAg response, we postulate that magnitude of anti-IgG OAg responses in convalescent patients is an indicator of the relevance of the IgG responses elicited in the studies with 1790GAHB. Importantly, in the study 1, subjects receiving ≥1.5/25μg dose IM had a reverse cumulative antibody distribution similar to that of convalescent patients, but with a higher median antibody level (305 vs 121 ELISA-units) and a relatively slow decay in antibody responses (median level of 241 at day 225, still exceeding the median level in convalescent patients).