The Position of His-Tag in Recombinant OspC and Application of Various Adjuvants Affects the Intensity and Quality of Specific Antibody Response after Immunization of Experimental Mice.

Lyme disease, Borrelia burgdorferi-caused infection, if not recognized and appropriately treated by antibiotics, may lead to chronic complications, thus stressing the need for protective vaccine development. The immune protection is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies, associated with the Th1 immune response. Surface antigen OspC is involved in Borrelia spreading through the host body. Previously we reported that recombinant histidine tagged (His-tag) OspC (rOspC) could be attached onto liposome surfaces by metallochelation. Here we report that levels of OspC-specific antibodies vary substantially depending upon whether rOspC possesses an N' or C' terminal His-tag. This is the case in mice immunized: (a) with rOspC proteoliposomes containing adjuvants MPLA or non-pyrogenic MDP analogue MT06; (b) with free rOspC and Montanide PET GEL A; (c) with free rOspC and alum; or (d) with adjuvant-free rOspC. Stronger responses are noted with all N'-terminal His-tag rOspC formulations. OspC-specific Th1-type antibodies predominate post-immunization with rOspC proteoliposomes formulated with MPLA or MT06 adjuvants. Further analyses confirmed that the structural features of soluble N' and C' terminal His-tag rOspC and respective rOspC proteoliposomes are similar including their thermal stabilities at physiological temperatures. On the other hand, a change in the position of the rOspC His-tag from N' to C' terminal appears to affect substantially the immunogenicity of rOspC arguably due to steric hindrance of OspC epitopes by the C' terminal His-tag itself and not due to differences in overall conformations induced by changes in the His-tag position in rOspC variants

Synthetic molecular adjuvants MT06 and MPLA.

<p>Left panel shows MT06, a non-pyrogenic lipophilic derivative of muramyl dipeptide (MDP) analogue—Nor-AbuMDP. The right panel shows monophosphoryl lipid A, a derivative of lipopolysaccharide from <i>S</i>. <i>minnesota</i>.</p

Doses and vaccine formulations.

<p>Doses and vaccine formulations.</p

Characterization of metallochelating N' and C´ terminal His-tag rOspC proteoliposomes by immuno EM and DLS.

<p>The EM picture of <b>A)</b> plain nanoliposome, <b>B</b>) metallochelation nanoliposome with surface bound N´ terminal His-tag rOspC proteins. Metallochelation liposomes were incubated with N' and C' terminal His-tag rOspC proteins followed by incubation with pooled sera from rOspC-immunized mice (1:50 dilution) followed by addition of protein A-labeled 10-nm colloidal gold particles. After 12-h, the proteoliposomes were negatively stained by ammonium molybdate and observed using Philips Morgagni transmission EM. Black dots represent immunogold particles on rOspC proteins bound to the liposome surface (black arrows). rOspC protein molecules (white dots) forms chains on proteoliposome surfaces (white arrows). <b>C)</b> The increase of hydrodynamic radius after binding of rOspC proteins was measured by DLS. <b>D)</b> Tabular data characterizing the size of plane and rOspC proteoliposomes in detail.</p

SDS-PAGE and MS analyses of recombinant N' and C' terminal His-tag rOspC.

<p><b>A)</b> Recombinant N' and C' terminal His-tag rOspC proteins were separated using SDS-PAGE and stained by CBB G-250. The differences in mobility of both rOspC variants are caused by the length of labeling tags and spacers and correspond to theoretical prediction based on cDNA sequences: 23.96 kDa for C' and 27.12 for N' terminal His-tag rOspC. <b>B, C</b>) MALDI-TOF peptide mass fingerprinting of rOspC proteins. All spectra were acquired using CHCA matrix on Microflex LRF20 MALDI-TOF mass spectrometer. Panel <b>B</b>) refers to C' terminal His-tag rOspC and panel <b>C</b>) N' terminal His-tag rOspC.</p

Determination of thermal stability of His-tag rOspC proteins by DLS, DSC and circular dichroism.

<p><b>A, B</b>) rOspC ellipticity induced by temperature changes monitored by circular dichroism. CD spectra were obtained at room temperature (22°C) using a Chirascan CD spectrometer. Data were collected from 185 to 260 nm at 100 nm/min, 1 s response time, and 2 nm bandwidth using a 0.1 cm quartz cuvette. Thermal unfolding of rOspC proteins was followed by monitoring the ellipticity at 195 and 222 nm over the temperature range of 20 to 80°C, with a resolution of 0.1°C, at a heating rate of 1°C/min. <b>C</b>) Determination of thermal stability by measurement transition midpoints of rOspC proteins using DSC. Scans ran from 10 to 85°C at the scan rate of 1°C per minute. <b>D</b>) Thermal stability of rOspC determined by DLS. The hydrodynamic diameter of the proteins was monitored over the temperature range of 25–55°C. The lines are created from hydrodynamic radius measurements at the temperature gradient of 1°C/ min. <b>E</b>) Transition temperatures (°C) of of N' and C' terminal His-tag rOspC calculated from DLS, DSC, and FTIR measurements.</p

CD spectra of rOspC proteins.

<p>Secondary structure of N' and C' terminal His-tag rOspC proteins determined by CD and FTIR. <b>A</b>) CD and FTIR spectra were obtained at room temperature (22°C) using a Chirascan CD spectrometer and Tensor 27 FTIR, respectively. <b>B)</b> Comparison of secondary structures of rOspC proteins obtained by calculations based on FTIR and CD measurements.</p