Safety and immunogenicity of adjuvanted and unadjuvanted subunit influenza vaccines administered intranasally to healthy adults.

Antigen-specific mucosal immunity is thought to be important for protection against influenza virus infection. Currently licensed parenteral influenza vaccines stimulate the production of serum antibodies, but are poor inducers of mucosal immunity. The adjuvant MF59 has been shown to enhance the humoral immune response to parenteral influenza vaccine in humans and the mucosal immune response to intranasally-administered influenza vaccine in mice. We conducted an open-label safety study followed by an observer-blind, randomized trial comparing the immune response to intranasally-administered subunit influenza vaccine adjuvanted with MF59, unadjuvanted subunit influenza vaccine, and placebo. Adverse reactions did not occur significantly more frequently in vaccinees than placebo recipients. Of 31 subjects receiving 2 doses of MF59-adjuvanted influenza vaccine, 19 (61%), 8 (26%), and 11 (35%) developed a mucosal IgA response to influenza A/H1N1, A/H3N2, and B, respectively. The percentage of subjects with a serum antibody response was slightly lower. The immune responses to adjuvanted vaccine were not significantly different from those to unadjuvanted vaccine. Both vaccines gave more frequent responses than seen in placebo recipients, indicating the potential of intranasal inactivated vaccines to stimulate local IgA responses.

Isoaspartate in peptides and proteins: formation, significance, and analysis.

Formation of isoaspartyl peptide bonds (isoAsp) is one of the most common forms of non-enzymatic degradation of peptides and proteins under mild conditions. IsoAsp arises when certain Asn-Xaa and Asp-Xaa sites undergo a spontaneous intramolecular rearrangement to form a succinimide which subsequently hydrolyzes to generate a mixture of isoAsp-Xaa and Asp-Xaa linkages in a ratio of approximately 2:1. This pathway is responsible for the much greater susceptibility of asparagine, compared with glutamine, to deamidation at neutral and alkaline pH. Rearrangement occurs most readily at Asn-Gly, Asn-Ser, and Asp-Gly sequences where the local polypeptide chain flexibility is high. Formation of isoAsp can decrease the biological activity of a protein pharmaceutical, alter its susceptibility to proteolytic degradation, and elicit autoimmunity. The enzyme protein L-isoaspartyl methyltransferase can be used to measure isoAsp sites in the low pmol range with or without the use of radioisotopes.

Spontaneous alterations in the covalent structure of synapsin I during in vitro aging.

Synapsin I purified from bovine brain was incubated for 30 days at pH 7.4 and 37 degrees C. Samples were taken at various times and assayed for isoaspartate content using protein-L-isoaspartyl methyltransferase. During the first 22 days, synapsin accumulated isoaspartyl sites at a rate of > or = 6 sites per day per 100 molecules of synapsin. Concomitant with isoaspartate formation, synapsin underwent two other types of modification: a substantial degree of spontaneous intermolecular cross-linking via the formation of disulfide bonds, and a second, less pronounced, irreversible aggregation. The irreversible aggregation apparently results from covalent cross-linking of a non-disulfide nature or possibly a strong hydrophobic interaction. Isoaspartate accumulated in both aggregated and non-aggregated forms of synapsin during in vitro aging. These findings demonstrate that synapsin is capable of significant spontaneous covalent alteration under physiological conditions. These modifications may play a role in the function of, or limit the lifetime of, synapsin in vivo.

Major degradation products of basic fibroblast growth factor: detection of succinimide and iso-aspartate in place of aspartate.

The degradation products of basic fibroblast growth factor (bFGF) were isolated by ion exchange HPLC (HP-IEC) and characterized. The predominant product at pH 5 was a succinimide in place of aspartate15 as determined by LC/MS, N-terminal sequencing, and susceptibility to degradation at pH > 6.5. The rate of appearance of the succinimidyl-bFGF at 22 degrees C was comparable to that reported for small peptides, consistent with a high flexibility predicted for asp15-gly. Tryptic mapping together with [3H]-methylation indicated that iso-aspartate was formed at the position of asp15. Size exclusion HPLC indicated the presence of intact and truncated dimers and trimers which associated through disulfide linkages. Two truncated monomer forms were found that co-eluted by HP-IEC; the cleavages were determined to be at asp28-pro and asp15-gly using LC/MS and N-terminal sequencing. These degradation products which occurred at sites that are away from receptor or heparin binding domains of bFGF remained bioactive in a cell proliferation assay.

Deamidation and isoaspartate formation during in vitro aging of recombinant tissue plasminogen activator.

When incubated at pH 7.3, 37 degrees C, human recombinant tissue plasminogen activator accumulated 0.77 mol of isoaspartate per mol of plasminogen activator over a 14-day period. Isoaspartate was detected by enzymatic transfer of 3H-labeled methyl groups from S-adenosyl-L-methionine in a reaction catalyzed by protein L-isoaspartyl methyltransferase. Analysis of tryptic peptides derived from aged plasminogen activator revealed that the two major sites of isoaspartate accumulation resulted from deamidation of Asn58 in the sequence -FNGG- and Asn177 in the sequence -GNSD-. Significant levels of isoaspartate also accumulated via deamidation of Asn37 in the sequence -CNSG-. All three sites occur in sequences predicted from studies with synthetic peptide to be unstable. All three sites appear to be on the surface of the protein, and all three occur in regions of the protein predicted to have higher than average chain mobility. These findings add support to the idea that sequence and flexibility play major roles in determining susceptibility to deamidation and peptide bond isomerization at Asn and Asp sites under mild conditions. These studies also illustrate the utility of enzymatic methylation for characterizing sites of deamidation in a large protein that contains numerous disulfide bonds and several sites of glycosylation.