Conformational stability and antibody response to the 18kda heat- shock protein formulated into different vehicles

Abstract

Protein stability is one of the most important obstacles for successful formulation in the development of new-generation vaccines. Here, the 18kDa heat-shock protein (18kDa-hsp) was chemically modified though conjugation with bovine serum albumin or by esterification with N-hydroxysuccinimide ester of palmitic acid. The biologically active conformation of the protein was preserved after chemical modification. The immune responses to the recombinant 18kDa-hsp from Mycobacterium leprae were studied in different presentations: free, copolymerized with bovine serum albumin in aggregates (18kDa-hsp-BSA), and either surface linked to liposomes or entrapped into liposomes. Measuring the antibody production of immunized genetically selected mice has compared the adjuvant effects of liposomes and proteic copolymer. Among the two liposome preparations, the strongest response was obtained with the surface-exposed antigen-liposomes. The copolymer 18kDa-hsp-BSA conferred a high titer of antibody in injected mice, and persisted 70 d after immunization. This approach should prove very useful for designing more effective vaccines by using 18kDa-hsp as carrier protein.

A S52P mutation in the ‘α‐crystallin domain’ of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function

“…However, the functional behaviour of wildtype HSP18 (HSP18S 52 ) is still not understood properly. Despite having less idea about the functionality of wild-type HSP18, this sHSP has been used for the development of second-generation vaccines for leprosy as a carrier protein [29]. Very few attempts have been FEBS Journal 280 (2013) 5994-6009 ª 2013 FEBS made to understand the structure of this protein.…”

“…Mitra et al [30] identified tryptic digested fragments in M. leprae HSP18, which appeared to be a potent stimulator of CD4 + T-cell responses in normal and leprotic patients. Costa et al [29] showed that the native conformation of wild-type HSP18 was preserved after hydrophobic modification by acylation. We also made an attempt to understand the structure of wild-type and mutant HSP18 and theoretically demonstrated that mutant HSP18 variant (HSP18P 52 ) has a different secondary structure compared to that of the wild-type HSP18 ( HSP18S 52 ) [31].…”

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems

“…We developed the large-scale production of 18 kDa-hsp at low cost in order to be able to introduce this T cell stimulation into our vaccines (7,8). Our main goal was to co-encapsulate the 18 kDa-hsp together with poor antigens within safe and pluripotent supports, such as liposomes (9,10) or biodegradable microspheres (11). These supports can also be excellent vehicles with adjuvant and controlled release capacity.…”

“…To make this association with the liposome membrane possible, the 18 kDa-hsp was modified by acylation of the lysine residues to increase its hydrophobicity (9). The N-acyl-18 kDa-hsp circular dichroism spectra were obtained using a JASCO spectropolarimeter model J720.…”

“…After 15 min at room temperature, the reaction was stopped with H 2 SO 4 . Absorbance was read automatically at 450 nm (9).…”