The particle size of AnExILs is amenable for uptake by antigen presenting cells (Foged et al. 2005; Tabata et al. 1996; Xiang et al. 2006; Yoshida and Babensee 2006) . The amount of pIVEX2.2EM-LacZ-6HIS encapsulated in liposomes (20 nM) was sufficient to initiate in vitro protein expression in liposomal compartments. b-galactosidase was successfully produced and quantified in liposomes. Enzymatic activity was used as a fast and sensitive method for evaluating biological activity and quantification of expressed b-galactosidase in AnExILs and showed that the produced enzyme has a correct folding and is active in cellfree protein synthesis. Western blot analysis showed that antigenicity of the expressed b-galactosidase was preserved. These results indicate that cell-free protein synthesis can efficiently produce antigens avoiding complexity and maintenance of cell viability associated with recombinant and in vivo systems (Jewett et al. 2008 ). Furthermore, it was demonstrated that the use of AnExILs can circumvent current problems of low immunogenicity with non-adjuvanted DNA vaccines (Liu and Ulmer 2005; Rosenberg et al. 2003; Trimble et al. 2009 ). As opposed to i.m. vaccination with liposomal DNA vaccine, i.m. immunization with AnExILs resulted in strong humoral immune responses. This result is in agreement with other studies showing that DNA vaccination is incapable of inducing strong antibody responses most probably because of poor transfection of DNA in endogenous cells, which leads to low doses of antigens produced (Abdulhaqq and Weiner 2008; Lu 2008 ). Moreover, AnExILs were able to induce significantly higher antibody responses as compared to conventional liposomes encapsulating antigen with or without pDNA. These results demonstrate strong immunostimulating effect of AnExILs as alternative for DNA and protein vaccines. Factors that might contribute to the adjuvant effect of AnExILs include efficient delivery of antigen to APCs, possible adjuvant activities of pDNA (due to CpG methylation pattern) (Klinman et al. 2009 ) and the presence of pathogen-associated molecular patterns (PAMPs) in the bacterial S30 extract which can activate the innate immune system via pattern recognition receptors. AnExIL-ON with surfaced-linked b-galactosidase was tested in mice as an alternative to AnExIL-IN formulation, in which antigen is entrapped inside vesicles. From the results of the vaccination studies with AnExIL-ON, it can be concluded that antigen presentation on the surface of the liposomes could significantly enhance systemic immune responses as compared to the other tested formulations and even a single immunization was sufficient to strongly stimulate an immune response superior to those achieved after i.m. immunization with conventional liposomal vaccines. Several studies have previously shown that surfacelinked liposomal antigens could enhance presentation of antigens to APCs and induce stronger immune responses Matsui et al. 2010; Ohno et al. 2009; Takagi et al. 2009; Taneichi et al. 2006b ). The impact of the AnExILs and other control vaccines on the antibody subtype profile was also investigated. In most of infectious diseases protection against viral or bacterial infections are mainly mediated by neutralizing immunoglobulins that bind to the viral or bacterial antigens (Gissmann 2009; Ho et al. 2002; Montefiori et al. 2007; Montefiori and Mascola 2009; Sattentau 2008; Smith 2009 ). In mice IgG1 and IgG2a antibodies are known to contribute to virus neutralization. IgG2a has been reported to contribute in complement activation and antibody-dependent cell-mediated immunity and is more effective than IgG1 in protection against viral infections by preventing virus replication (Coutelier et al. 1987; Hocart et al. 1989 ). On the other hand, the absolute concentration of IgG is important for reducing the viral shedding (Hocart et al. 1989; Hovden et al. 2005) . Therefore, induction of a combined IgG2a/IgG1 (T-helper 1 /T-helper 2 ) response may improve vaccine efficacy especially against viral infections. In this study, i.m. immunizations with all vaccines resulted in a mixed Th 1 /Th 2 immune response and i.m. administered AnExILs were able to markedly enhance both the IgG1 and the IgG2a response after i.m. vaccination as compared to protein and DNA liposomal formulations (Fig. 4a, b) , which may be advantageous for protection against viral or intracellular bacterial infections. The IgG2a/IgG1 ratio of AnExIL-IN was the highest among those of other vaccines after prime vaccination (Fig. 4c) and that of AnExIL-ON was substantially increased after booster immunization (Fig. 4d) . These data suggest that the quality of the immune response to b-galactosidase vaccine is substantially affected by the characteristic of AnExIL formulations.