Identifying a stable bulk dmLT adjuvant formulation at a clinically relevant concentration.

Double mutant heat-labile toxin (dmLT) is a novel vaccine adjuvant under development with several different vaccine candidates. Studies using existing dmLT adjuvant stocks require significant dilution to achieve a clinically relevant dose. This dilution leads to wastage of the adjuvant. This manuscript describes a limited formulation study to improve the stability of bulk dmLT at a more clinically relevant concentration (20 µg/mL) with minimal changes to the existing bulk dmLT formulation. In vitro methods were used to evaluate dmLT stability after lyophilization and short-term accelerated stability studies. The addition of the excipient polysorbate 80 (PS80) at 0.05 % to the existing dmLT formulation was identified as the lead modification that provided improved stability of the lyophilized dmLT at 20 µg/mL through 4 weeks at 40 °C.

Structure-based analysis of the beta8 interactive sequence of human alphaB crystallin.

The functional importance of the beta8 sequence ((131)LTITSSLS(138)), which is on the surface of the alpha crystallin core domain of human alphaB crystallin, was evaluated using site-directed mutagenesis. Ultraviolet circular dichroism determined that mutating the surface-exposed, nonconserved residues, Leu-131, Thr-132, Thr-134, Ser-135, Ser-136, and Ser-138 individually or in combination (alphaAbeta8 and CEbeta8), had no measurable effect on secondary and tertiary structure. Size exclusion chromatography determined the size of the complexes formed by the beta8 mutants to be 6-8 subunits larger than wt alphaB crystallin. In chaperone assays, the protective effect of the L131S, T132A, and S135C mutants of the beta8 sequence was similar to wt alphaB crystallin when beta(L) crystallin and alcohol dehydrogenase were the chaperone substrates and decreased to 66% when citrate synthase was the chaperone substrate. In contrast, the chaperone activity for all three substrates was dramatically reduced for the T134K, S138A, S136H, and CEbeta8 mutants. The prominent location of Thr-134, Ser-136, and Ser-138 on the exposed surface of the alpha crystallin core domain could account for the effect on complex assembly and chaperone activity. Modulation of chaperone activity by the exposed residues of the beta8 sequence in the alpha crystallin core domain was independent of complex size. The results established the beta3-beta8-beta9 surface of the alpha crystallin core domain as an interface for complex assembly and chaperone activity.

The function of the beta3 interactive domain in the small heat shock protein and molecular chaperone, human alphaB crystallin.

Knowledge of the interactive domains on the surface of small heat shock proteins (sHSPs) is necessary for understanding the assembly of complexes and the activity as molecular chaperones. The primary sequences of 26 sHSP molecular chaperones were aligned and compared. In the interactive beta3 sequence, 73DRFSVNLDVKHFS85 of human alphaB crystallin, Ser-76, Asn-78, Lys-82, and His-83 were identified as nonconserved residues on the exposed surface of the alpha crystallin core domain. Site-directed mutagenesis produced the mutant alphaB crystallins: S76E, N78G, K82Q, and H83F. Domain swapping with homologous beta3 sequences, 32EKFEVGLDVQFFT44 from Caenorhabditis elegans sHSP12.2 or 69DKFVIFLDVKHFS81 from alphaA crystallin, resulted in the mutant alphaB crystallins, CE1 and alphaA1, respectively. Decreased chaperone activity was observed with the point mutants N78G, K82Q, and H83F and with the mutant, CE1, in aggregation assays using betaL crystallin, alcohol dehydrogenase (ADH), or citrate synthase (CS). The S76E mutant had minimal effect on chaperone activity, and domain swapping with alphaA crystallin had no effect on chaperone activity. The mutations that resulted in altered chaperone activity, produced minimal modification to the secondary, tertiary, and quaternary structure of human alphaB crystallin as determined by ultraviolet circular dichroism spectroscopy, chymotrypsin proteolysis, and size exclusion chromatography. Chaperone activity was influenced by the amount of unfolding of the target proteins and independent of complex size. The results characterized the importance of the exposed side chains of Glu-78, Lys-82, and His-83 in the interactive beta3 sequence of the alpha crystallin core domain in alphaB crystallin for chaperone function.

Interactive domains for chaperone activity in the small heat shock protein, human alphaB crystallin.

Protein pin arrays identified seven interactive sequences for chaperone activity in human alphaB crystallin using natural lens proteins, beta(H) crystallin and gammaD crystallin, and in vitro chaperone target proteins, alcohol dehydrogenase and citrate synthase. The N-terminal domain contained two interactive sequences, (9)WIRRPFFPFHSP(20) and (43)SLSPFYLRPPSFLRAP(58). The alpha crystallin core domain contained four interactive sequences, (75)FSVNLDVK(82) (beta3), (113)FISREFHR(120), (131)LTITSSLS(138) (beta8), and (141)GVLTVNGP(148) (beta9). The C-terminal domain contained one interactive sequence, (157)RTIPITRE(164), that included the highly conserved I-X-I/V motif. Two interactive sequences, (73)DRFSVNLDVKHFS(85) and (131)LTITSSLSDGV(141), belonging to the alpha crystallin core domain were synthesized as peptides and assayed for chaperone activity in vitro. Both synthesized peptides inhibited the thermal aggregation of beta(H) crystallin, alcohol dehydrogenase, and citrate synthase in vitro. Five of the seven chaperone sequences identified by the pin arrays overlapped with sequences identified previously as sequences for subunit-subunit interactions in human alphaB crystallin. The results suggested that interactive sequences in human alphaB crystallin have dual roles in subunit-subunit assembly and chaperone activity.