**5. Metallochelation liposomes for construction of experimental recombinant vaccines**

Only few papers report the implementation of the metallochelating lipids in the attachment of the recombinant proteins or synthetic peptides with His-Tag anchor (short peptide consisting of 4 to 6 molecules of histidine). Both reversible character and high affinity of the metallochelating bonds are very useful for the preparation of various self-assembling supramolecular structures useful for the construction of experimental vaccines (Chikh et al., 2002; Malliaros et al., 2004; Masek et al., 2011a; Masek et al., 2011b). As an example of synthetic liposome-based recombinant vaccine we can use metallochelating liposomes and recombinant antigen rOspc-6HisTag derived from the pathogen Borrelia burgdorferi.

### **5.1 rOspC antigen** *Borrelia* **as an example for construction of metallochelation liposome-based vaccines**

Lyme disease or Lyme borreliosis is an infectious disease caused by spirochetes of the *Borrelia burgdorferi* sensu lato complex vectored by ticks of the genus *Ixodes*. At least three species are pathogenic for humans, *B. burgdorferi* sensu stricto, *B. afzelii*, and *B. garinii*. The initial stage of Lyme disease is commonly associated with skin rash occurring within few weeks after the tick bites. Later the infection can spread to bloodstream and insult of joints, heart, and nervous system. Although not common, some patients experience late stage symptoms like arthritis, nervous system complications, or acrodermatitis chronica atrophicans. Here, slower response to antibiotics therapy, sometimes taking weeks or months to recover, or eventually, incomplete resolution is observed. In a few cases, antibiotic-refractory complications persist for months to years after antibiotic therapy, most likely due to infection-induced autoimmunity. Therefore, alternative approaches such as preventive immunisation are needed, mainly in the endemic areas (Krupka M, 2007; Tilly et al., 2008).

Protective immune response to *Borrelia* involves non-specific activity of complement, phagocytic cells and *Borrelia*-specific Th1-dependent response leading to production of complement-activating antibodies, in mouse presented mostly by IgG2a (IgG2b). During natural infection, nevertheless, *Borrelia* and tick saliva modulate the immune response toward non-protective Th2 type response, associated with production of neutralizing, poorly opsonizing *Borrelia*-specific antibodies (Vesely et al., 2009). *Borrelia* outer surface proteins OspA and OspC are among the most promising antigens for elicitation of opsonizing antibodies. The applicability of OspA antigen is limited because *Borrelia* expresses it mainly in the tick and the antibodies thus should act outside of the vaccinee's organism (Pal et al., 2000). Therefore continuously high level of OspA-specific antibodies is required to prevent *Borrelia* transfer. In contrast, OspC is expressed during the transfer and the initial stage of infection. In this case the vaccine-induced immune memory has enough time to initiate the production of opsonizing antibodies preventing *Borrelia* spreading (Tilly et al., 2006).

OspC antigen can be used here as an example of reverse vaccinology approach. Full length recombinant OspC is difficult to prepare in high yield and purity. Production of Osp-s for vaccination purposes is hindered by low yield of fully processed lipidized Osp antigens or low immunogenicity of their non-lipidized versions. In our experiments, removing of N' terminal lipidation signal was associated with an increase of the recombinant protein yield and purity but, as demonstrated also for other *Borrelia* lipoproteins, a decrease in immunogenicity (Erdile & Guy, 1997; Gilmore et al., 2003; Lovrich et al., 2005; Weis et al., 1994). Induction of OspC-specific opsonizing antibodies to non – lipidised OspC could be

**5. Metallochelation liposomes for construction of experimental recombinant** 

Only few papers report the implementation of the metallochelating lipids in the attachment of the recombinant proteins or synthetic peptides with His-Tag anchor (short peptide consisting of 4 to 6 molecules of histidine). Both reversible character and high affinity of the metallochelating bonds are very useful for the preparation of various self-assembling supramolecular structures useful for the construction of experimental vaccines (Chikh et al., 2002; Malliaros et al., 2004; Masek et al., 2011a; Masek et al., 2011b). As an example of synthetic liposome-based recombinant vaccine we can use metallochelating liposomes and recombinant antigen rOspc-6HisTag derived from the pathogen Borrelia burgdorferi.

**5.1 rOspC antigen** *Borrelia* **as an example for construction of metallochelation** 

Lyme disease or Lyme borreliosis is an infectious disease caused by spirochetes of the *Borrelia burgdorferi* sensu lato complex vectored by ticks of the genus *Ixodes*. At least three species are pathogenic for humans, *B. burgdorferi* sensu stricto, *B. afzelii*, and *B. garinii*. The initial stage of Lyme disease is commonly associated with skin rash occurring within few weeks after the tick bites. Later the infection can spread to bloodstream and insult of joints, heart, and nervous system. Although not common, some patients experience late stage symptoms like arthritis, nervous system complications, or acrodermatitis chronica atrophicans. Here, slower response to antibiotics therapy, sometimes taking weeks or months to recover, or eventually, incomplete resolution is observed. In a few cases, antibiotic-refractory complications persist for months to years after antibiotic therapy, most likely due to infection-induced autoimmunity. Therefore, alternative approaches such as preventive immunisation are needed, mainly in the endemic

Protective immune response to *Borrelia* involves non-specific activity of complement, phagocytic cells and *Borrelia*-specific Th1-dependent response leading to production of complement-activating antibodies, in mouse presented mostly by IgG2a (IgG2b). During natural infection, nevertheless, *Borrelia* and tick saliva modulate the immune response toward non-protective Th2 type response, associated with production of neutralizing, poorly opsonizing *Borrelia*-specific antibodies (Vesely et al., 2009). *Borrelia* outer surface proteins OspA and OspC are among the most promising antigens for elicitation of opsonizing antibodies. The applicability of OspA antigen is limited because *Borrelia* expresses it mainly in the tick and the antibodies thus should act outside of the vaccinee's organism (Pal et al., 2000). Therefore continuously high level of OspA-specific antibodies is required to prevent *Borrelia* transfer. In contrast, OspC is expressed during the transfer and the initial stage of infection. In this case the vaccine-induced immune memory has enough time to initiate the production of

OspC antigen can be used here as an example of reverse vaccinology approach. Full length recombinant OspC is difficult to prepare in high yield and purity. Production of Osp-s for vaccination purposes is hindered by low yield of fully processed lipidized Osp antigens or low immunogenicity of their non-lipidized versions. In our experiments, removing of N' terminal lipidation signal was associated with an increase of the recombinant protein yield and purity but, as demonstrated also for other *Borrelia* lipoproteins, a decrease in immunogenicity (Erdile & Guy, 1997; Gilmore et al., 2003; Lovrich et al., 2005; Weis et al., 1994). Induction of OspC-specific opsonizing antibodies to non – lipidised OspC could be

opsonizing antibodies preventing *Borrelia* spreading (Tilly et al., 2006).

**vaccines** 

**liposome-based vaccines** 

areas (Krupka M, 2007; Tilly et al., 2008).

enhanced by appropriate adjuvants and carriers like such as various modification of liposomes. It was reported that immunisation of mice with non-lipidated OspC in strong adjuvants (Complete Freund's Adjuvant, TiterMax, or Alum) could induce intense OspCspecific antibody responses (Earnhart et al., 2007; Earnhart and Marconi, 2007; Gilmore et al., 1996; Gilmore and Mbow, 1999; Ikushima et al., 2000). Here we demonstrated that similarly strong response could be elicited by immunisation of experimental mice with metallochelating nanoliposomes with entrapped lipophilic derivatives of norAbu-MDP as a potent adjuvant molecule.

A) Scheme of metallochelating liposome with His-Tag recombinant protein antigen bound to the surface. Lipophilic nor-AbuMDP analogue as adjuvant is exposed on the surface of proteoliposome; B) Upper photograph (confocal microscop) shows human dendritic cell with phagocytosed prototypical vaccination nanoparticles cumulated in giant lysozomes (green – HLA-DR marked with antiHLA-DR antibodies; red – Lyssamine-rhodamine labelled vaccination particles). Lower photograph (Hofman modulation contrast and fluorescence microscopy) shows dendritic cells with accumulated vaccination particles inside (red fluorescence)

Fig. 12. Schematic drawing of prototypical recombinant protein vaccination nanoparticle and interaction with human dendritic cells.

### **5.2 Structure of OspC metallochelating proteoliposome**

Protein-His-Tag/metallochelating lipid complex is anchored in phospholipid bilayer by its lipid moiety. Egg yolk or soya phosphatidyl choline contain large portion of unsaturated fatty acids, therefore DOGS-NTA lipid is freely miscible with these lipids and phase separation do not occur in liposomal bilayer. In other words, contribution of lipids to the formation of various protein structures on the liposomal surface is negligible.

A) Structure of plain metallochelating liposome (TEM) B) Structure of metallochelating liposome with OspC bound onto the surface (white arrows – OspC); black arrow – OspC marked by 10nm immunogold particles; small red arrow – bead chain of OspC molecules; C) Schematic presentation of bead chain model of proteoliposome D) Size distribution and hydrodynamic diameter of OspC, plain metallochelating liposomes and OspC proteoliposomes analysed by DLS. The size distribution of parent monodispersed metallochelating liposomes (dashed line) was compared with that of OspC proteoliposomes (full line). As a reference, size distribution of OspC (dotted line) is shown. Numbers in brackets represent mean hydrodynamic diameters of particles

Fig. 13. Characterisation of OspC preoteoliposomes.

This is advantageous for study of protein-protein interaction of proteins anchored on the surface of liposomes. Moreover, anchoring of proteins via metallochelating bond produces highly oriented binding of proteins because the proteins are attached to liposomes exclusively by His-tagged end of the polypeptide chain. If some interaction between liposomal surface bound proteins exists, we can observe formation of various structures which are conditioned by the character and number of protein-protein interactions. Another important feature of metallochelating proteoliposomes is relatively high surface concentration of proteins. This concentration could be set by changing the DOGS-NTA/phosphatidyl choline ratio in the lipid mixture used for preparation of liposomes.

The ultrastructure of the rOspC-His-tag proteoliposomes was revealed by TEM (Fig. 7). rOspC-His tag as an example of the preoteoliposomal structure "Bead chain" model. Binding of individual molecules of OspC protein onto the liposomal surface is clearly visible

A) Structure of plain metallochelating liposome (TEM) B) Structure of metallochelating liposome with

immunogold particles; small red arrow – bead chain of OspC molecules; C) Schematic presentation of bead chain model of proteoliposome D) Size distribution and hydrodynamic diameter of OspC, plain metallochelating liposomes and OspC proteoliposomes analysed by DLS. The size distribution of parent

proteoliposomes (full line). As a reference, size distribution of OspC (dotted line) is shown. Numbers in

This is advantageous for study of protein-protein interaction of proteins anchored on the surface of liposomes. Moreover, anchoring of proteins via metallochelating bond produces highly oriented binding of proteins because the proteins are attached to liposomes exclusively by His-tagged end of the polypeptide chain. If some interaction between liposomal surface bound proteins exists, we can observe formation of various structures which are conditioned by the character and number of protein-protein interactions. Another important feature of metallochelating proteoliposomes is relatively high surface concentration of proteins. This concentration could be set by changing the DOGS-NTA/phosphatidyl choline ratio in the lipid mixture used for preparation of liposomes. The ultrastructure of the rOspC-His-tag proteoliposomes was revealed by TEM (Fig. 7). rOspC-His tag as an example of the preoteoliposomal structure "Bead chain" model. Binding of individual molecules of OspC protein onto the liposomal surface is clearly visible

OspC bound onto the surface (white arrows – OspC); black arrow – OspC marked by 10nm

monodispersed metallochelating liposomes (dashed line) was compared with that of OspC

brackets represent mean hydrodynamic diameters of particles Fig. 13. Characterisation of OspC preoteoliposomes.

on the rim of the liposome and immunogold staining confirmed the identity of OspC molecules as well as preservation of the epitopes recognised by polyclonal antibodies (Fig. 7b). In the case of recombinant OspC, TEM micrograph (Fig. 7b) showed that the individual molecules of OspC antigen were bound onto the surface of the liposome and some organisation in beads-like structures was revealed. This observation testifies against the simplification of the proteoliposomal structures and hence against accepting the simple schematic concept based on the random distribution on the liposomal surface.

Binding of OspC onto the surface of metallochelating liposomes was proved by an increase of hydrodynamic diameter as assayed by DLS. The increase of the size of proteoliposomes is well distinguished from plain liposomes, even if the increase of the size is only 5.5 nm (Fig.. 13D). This precise measurement was allowed by a preparation of parent monodispersed metallochelating liposomes and pointed to the importance of using monodispersive liposomal preparation for such a study (Fig. 8). In the case of homogenous coating of liposomes by OspC, the increase in the size should be of about 7.4 nm, theoretically. A lower increase of the size (5.5 nm) indicates only partial coating of the liposomal surface by the protein and this is in a good accordance with the structure revealed by TEM (Fig. 7b).

Binding of OspC onto metallochelating liposomes was confirmed also by GPC used as an independent method (Fig. 9) The liposomal fraction was separated from free protein and OspC was assayed by SDS PAGE followed by immunoblot (Fig. 9C). The vast majority of OspC was shown to be bound onto liposomes and was only slightly ripped from their surface by shearing forces taking place during penetrating through GPC column. The tailing character of the OspC elution profile is supportive to this explanation. Stability of the metallochelating bond in model biological fluid was studied by incubation in undiluted human serum. In spite of the presence of serum, it was estimated that more than 60% of OspC was still associated with liposomes. Based on this data, the half life of OspC proteoliposomes in serum was estimated to be at least 1 hour.

Fig. 14. Stability analysis of OspC proteoliposomes by GPC.

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.
