5). Only the two subjects who received 1010 BMB72 had IgA responses against listeriolysin (data not shown). Responses to influenza nucleoprotein were not detected in these assays. These results were interpreted to represent low level mucosal immune response against the listerial vector only. Serological immune responses XL765 in vivo were modest at best, with isolated individuals having four-fold or greater titer increases in ELISAs directed against listeriolysin or sonicated listerial antigen (denoted in Table 2 as one or two positive assays). No individual seroconverted to the recombinant nucleoprotein antigen.

Virtually all individuals had relatively high titers directed against recombinant nucleoproteins at baseline, which did not change over time (i.e. ≥1:640). Sera from other species (mouse and rabbit) studied similarly in ELISAs did not have similarly high baseline values, so these were interpreted to ATM/ATR assay represent bona fide pre-existing immune responses to this influenza protein, rather than inadequate blocking or another technical problem with the assay. A high baseline is not unexpected, as most

subjects had evidence of cellular immunity to influenza A, and it is expected that most healthy young adults would have been exposed to influenza. Grouped by vaccine given, there was no statistically significant increase in IgG mean titers (GMT; pre-immune to peak value) directed against listerial sonicate, listeriolysin or nucleoprotein, as exemplified in Figure 6b (for listeriolysin). Baseline listeriolysin titers were high, which is not unexpected. Antibodies to streptolysins

present in commensal and pathogenic streptococci cross-react with listeriolysin (34). Our volunteers were required to have previously received penicillin Erythromycin or ampicillin, commonly administered to treat Group A streptococcal pharyngitis. Overall, mean serum IgA titers did increase modestly when considered as a group for both vaccine organisms (Fig. 6a). All subjects had positive control responses to the lectin PHA (usually TNTC), and all but one to the CEF control pool (subject No. 11 had both robust PHA responses and responses to sonicated listerial antigen, but no apparent response to CEF or influenza nucleoprotein peptides). Most subjects (17/22) had convincing baseline immune responses to at least one of the Influenza A nucleoprotein peptide test pools (tens to many hundreds of spots per million PBMC, see exemplary data in Fig. 7a). About two-thirds of the subjects (14/22) had some baseline responses to the listeriolysin peptide pools, with mean baseline value 21 (range 0–205) SFC/106 PBMC, comparable to others’ published work (35). ELISpot data were analyzed by individual and as a group by vaccine administered, irrespective of dose, as responses overall did not appear dose-related. Values were analyzed as pre-immune vs.