**5.3 Immunization experiments**

New synthetic nonpyrogenic lipophilic analogues of NorAbu-MDP modified at a peptide part by two different hydrophobic ligands (Fig. 15) and for the first time these defined synthetic molecules were used in combination with metallochelating liposomes to construct an experimental recombinant vaccine. The important finding was that both MT06 and MT05 adjuvants exerted a high adjuvant effect comparable or better than MDP but proved itself as nonpyrogenic (rabbit pyrogenicity test) and safe. While alum induced stronger antibody response in IgG1 subtype, both MT06 derivatives and liposomal-MDP induced stronger immune response in both IgG2 subtypes. Interestingly, in comparison with MT06, the analogue MT05 induced stronger response in IgG1, IgG3 and IgM isotypes, respectively. This interesting finding pointed on the effect of lipophilic residues, which could not be supposed as the only accessory part of the molecule, but can significantly affect the quality of immune response. The position (peptide or sugar part) and the character (hydrophobicity and bulkiness) of the lipophilic function can affect the interaction with appropriate receptors as well as the metabolic degradation of the molecule. This aspect has not been described in the literature yet and is of interest for our understanding of the mechanism of action.

OspC itself did not elicit detectable OspC-specific antibodies of IgG, IgM, and IgA isotypes (Ig\*). Similarly, when OspC bound onto the surface of liposomes was used for immunisation, only negligible increase of OspC-specific Ig\* was detected after the second immunisation. In contrast, immunisation with OspC plus adjuvants (FCA, AlOH, MDP, MT05, and MT06) elicited strong OspC-specific antibody responses with ELISA titres of the same magnitude. The contributions of particular IgG isotypes for the immune response showed differences among various adjuvants (Fig. 15) (FCA was no tested for the IgG isotype response).

Mice (5 per group) were immunised by i.d. application of various liposome - adjuvant formulations of rOspC. Pooled sera from each group were used for ELISA analysis of specific antibodies titers. Naive sera were obtained before immunisation. ELISA plates were coated with 100 µl of rOspC (1 µg/ml), incubated with anti-mouse IgG1 (A), anti-mouse IgG2a (B), anti-mouse IgG2b (C), anti-mouse IgG3 (D), or anti-mouse IgM (E) and after addition of OPD plus H2O2, the absorbance was read at 490 nm on ELISA reader. The results are expressed as the end point titers +/- SD. Mean values are expressed in the table under each graph. Formulae of tested compounds in this experiment (F).

Although AlOH adjuvant induced strong OspC-specific antibody responses in total immunoglobulin level (Ig\*) and IgG1 isotype (Fig. 15A), the response in complementactivating IgG isotypes (IgG2a and IgG2b; (Fig. 15B,C) was only modest. In comparison with AlOH, the synthetic adjuvant MT06 when combined with liposomes-bounded OspC induced strong OspC-specific response in isotypes IgG2a and at lesser extent in IgG2b (Fig. 15B). Application of another synthetic adjuvant MT05 was associated with dominancy of IgG3 and IgM (Fig. 15D,E). Furthermore, we compare responses to synthetic norAbu-MDP adjuvants with the response to liposomes-bounded OspC plus MDP, which elicits strongest OspC-specific antibodies in IgG2b and in lesser extent in IgG2a isotype (Fig. 15B,C).

Non-bound OspC was separated from the proteoliposomes by gel permeation chromatography using Superose 6 column. OspC was detected in various fractions from GPC by immunoblot. OspC GPC elution profile is correlated with immunoblot assay.

New synthetic nonpyrogenic lipophilic analogues of NorAbu-MDP modified at a peptide part by two different hydrophobic ligands (Fig. 15) and for the first time these defined synthetic molecules were used in combination with metallochelating liposomes to construct an experimental recombinant vaccine. The important finding was that both MT06 and MT05 adjuvants exerted a high adjuvant effect comparable or better than MDP but proved itself as nonpyrogenic (rabbit pyrogenicity test) and safe. While alum induced stronger antibody response in IgG1 subtype, both MT06 derivatives and liposomal-MDP induced stronger immune response in both IgG2 subtypes. Interestingly, in comparison with MT06, the analogue MT05 induced stronger response in IgG1, IgG3 and IgM isotypes, respectively. This interesting finding pointed on the effect of lipophilic residues, which could not be supposed as the only accessory part of the molecule, but can significantly affect the quality of immune response. The position (peptide or sugar part) and the character (hydrophobicity and bulkiness) of the lipophilic function can affect the interaction with appropriate receptors as well as the metabolic degradation of the molecule. This aspect has not been described in the literature yet and is of interest for our

OspC itself did not elicit detectable OspC-specific antibodies of IgG, IgM, and IgA isotypes (Ig\*). Similarly, when OspC bound onto the surface of liposomes was used for immunisation, only negligible increase of OspC-specific Ig\* was detected after the second immunisation. In contrast, immunisation with OspC plus adjuvants (FCA, AlOH, MDP, MT05, and MT06) elicited strong OspC-specific antibody responses with ELISA titres of the same magnitude. The contributions of particular IgG isotypes for the immune response showed differences among various adjuvants (Fig. 15) (FCA was no tested for the IgG

Mice (5 per group) were immunised by i.d. application of various liposome - adjuvant formulations of rOspC. Pooled sera from each group were used for ELISA analysis of specific antibodies titers. Naive sera were obtained before immunisation. ELISA plates were coated with 100 µl of rOspC (1 µg/ml), incubated with anti-mouse IgG1 (A), anti-mouse IgG2a (B), anti-mouse IgG2b (C), anti-mouse IgG3 (D), or anti-mouse IgM (E) and after addition of OPD plus H2O2, the absorbance was read at 490 nm on ELISA reader. The results are expressed as the end point titers +/- SD. Mean values are expressed in the table

Although AlOH adjuvant induced strong OspC-specific antibody responses in total immunoglobulin level (Ig\*) and IgG1 isotype (Fig. 15A), the response in complementactivating IgG isotypes (IgG2a and IgG2b; (Fig. 15B,C) was only modest. In comparison with AlOH, the synthetic adjuvant MT06 when combined with liposomes-bounded OspC induced strong OspC-specific response in isotypes IgG2a and at lesser extent in IgG2b (Fig. 15B). Application of another synthetic adjuvant MT05 was associated with dominancy of IgG3 and IgM (Fig. 15D,E). Furthermore, we compare responses to synthetic norAbu-MDP adjuvants with the response to liposomes-bounded OspC plus MDP, which elicits strongest OspC-specific antibodies in IgG2b and in lesser extent in

under each graph. Formulae of tested compounds in this experiment (F).

**5.3 Immunization experiments** 

understanding of the mechanism of action.

isotype response).

IgG2a isotype (Fig. 15B,C).

Fig. 15. ELISA analyses of specific antibody titers in sera of immunised mice.
