The thermostability of the RTS,S/AS01 malaria vaccine can be increased by co-lyophilizing RTS,S and AS01.

BACKGROUND: Developing thermostable vaccines is a challenge for pharmaceutical companies due to the inherent instability of biological molecules in aqueous solution. The problem is even more stringent in regions subjected to high temperatures in which protective cold chain is difficult to maintain due to a lack of infrastructure. Here, a simple, cost-effective solution to increase the thermostability of the malaria candidate vaccine RTS,S/AS01 is described. This vaccine currently needs to be stored between 2 and 8  °C due to the sensitivity of liquid AS01 to higher temperatures. The strategy was to increase thermostability by co-lyophilizing the RTS,S antigen and AS01. METHODS: Co-lyophilization was achieved in a solution containing 5% sucrose, 10 mM potassium phosphate and 0.0312% polysorbate 80 at pH 6.1. The physicho-chemical characteristics and immunogenic properties of the resulting solid product, called CL-vac, fresh or stored at high temperature, were compared to those of the candidate RTS,S/AS01. RESULTS: CL-vac proved to be acceptable in terms of visual appearance and physico-chemical characteristics. The structural integrity of both RTS,S and AS01 within CL-vac and its equivalence to the RTS,S/AS01 candidate vaccine were shown. Further, the stability of CL-vac was demonstrated for storage periods including 1 year at 4  °C, 1 year at 30  °C, and up to 6 months at 37  °C. In addition, CL-vac could withstand a heat excursion consisting of 1 month at 45  °C after storage for 1 year at 30  °C. Equivalence and stability were demonstrated by the various analytical tools and the immunogenicity of the samples after storage was also demonstrated in mice. CONCLUSIONS: In conclusion, the co-lyophilization process appeared as a promising approach to increase RTS/AS01 vaccine thermostability.

Stability of an aluminum salt-adjuvanted protein D-conjugated pneumococcal vaccine after exposure to subzero temperatures.

Accidental exposure of a vaccine containing an aluminum-salt adjuvant to temperatures below 0°C in the cold chain can lead to freeze damage. Our study evaluated the potential for freeze damage in a licensed aluminum-salt-containing protein-D-conjugated pneumococcal vaccine (PHiD-CV; Synflorix, GSK) in conditions that included static storage, single subzero-temperature excursions, and simulated air-freight transportation. Several parameters were assessed including freezing at subzero temperatures, aluminum-salt-particle size, antigen integrity and immunogenicity in the mouse. The suitability of the WHO's shake test for identifying freeze-damaged vaccines was also assessed. During subzero-temperature excursions, the mean temperatures at which PHiD-CV froze (-16.7°C to -18.1°C) appeared unaffected by the type of vaccine container (two-dose or four-dose vial, or single-dose syringe), vaccine batch, rotational agitation, or the rate of temperature decline (-0.5 to -10°C/hour). At constant subzero temperature and in simulated air-freight transportation, the freezing of PHiD-CV appeared to be promoted by vibration. At -5°C, no PHiD-CV sample froze in static storage (>1 month), whereas when subjected to vibration, a minority of samples froze (7/21, 33%) within 18 hours. At -8°C with vibration, nearly all (5/6, 83%) samples froze. In these vibration regimes, the shake test identified most samples that froze (10/12, 93%) except two in the -5°C regime. Nevertheless, PHiD-CV-antigen integrity appeared unaffected by freezing up to -20°C or by vibration. And although aluminum-salt-particle size was increased only by freezing at -20°C, PHiD-CV immunogenicity appeared only marginally affected by freezing at -20°C. Therefore, our study supports the use of the shake test to exclude freeze-damaged PHiD-CV in the field.

Binding of mannose-binding lectin to fructosamines: a potential link between hyperglycaemia and complement activation in diabetes.

BACKGROUND: Complement activation via the MBL pathway has been proposed to play a role in the pathogenesis of diabetic complications. As protein glycation is increased in diabetes, we tested the possibility that the glycation product fructoselysine is a ligand for MBL and that its interaction with this protein may initiate complement activation. METHODS: We investigated the binding of MBL to fructoselysine by chromatography of human serum on fructoselysine-Sepharose, followed by Western blot and mass spectrometry analysis. We also performed enzyme-linked immunosorbent assays using purified MBL and fructoselysine-derivatized (binding assay) or mannan-coated plates (inhibition assay). Complement activation was determined by the fixation of C3d following incubation of fructoselysine-derivatized plates with serum from subjects with different levels of MBL. RESULTS: MBL and its associated proteases were selectively purified from serum by chromatography on fructoselysine-Sepharose. Competition experiments indicated that MBL had a similar affinity for mannose, fructose and fructoselysine. MBL bound, in a highly cooperative manner, to fructoselysine-derivatized plates. This binding was associated with complement activation and was much lower with serum from subjects with low-MBL genotypes. CONCLUSIONS: MBL binding to fructoselysine and the ensuing complement activation may provide a physiopathological link between enhanced glycation and complement activation in diabetes. The cooperative character of this binding may explain the high sensitivity of diabetic complications to hyperglycaemia.

Many fructosamine 3-kinase homologues in bacteria are ribulosamine/erythrulosamine 3-kinases potentially involved in protein deglycation.

The purpose of this work was to identify the function of bacterial homologues of fructosamine 3-kinase (FN3K), a mammalian enzyme responsible for the removal of fructosamines from proteins. FN3K homologues were identified in approximately 200 (i.e. approximately 27%) of the sequenced bacterial genomes. In 11 of these genomes, from phylogenetically distant bacteria, the FN3K homologue was immediately preceded by a low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) homologue, which is therefore probably functionally related to the FN3K homologue. Five bacterial FN3K homologues (from Escherichia coli, Enterococcus faecium, Lactobacillus plantarum, Staphylococcus aureus and Thermus thermophilus) were overexpressed in E. coli, purified and their kinetic properties investigated. Four were ribulosamine/erythrulosamine 3-kinases acting best on free lysine and cadaverine derivatives, but not on ribulosamines bound to the alpha amino group of amino acids. They also phosphorylated protein-bound ribulosamines or erythrulosamines, but not protein-bound fructosamines, therefore having properties similar to those of mammalian FN3K-related protein. The E. coli FN3K homologue (YniA) was inactive on all tested substrates. The LMW-PTP of T. thermophilus, which forms an operon with an FN3K homologue, and an LMW-PTP of S. aureus (PtpA) were overexpressed in E. coli, purified and shown to dephosphorylate not only protein tyrosine phosphates, but protein ribulosamine 5-phosphates as well as free ribuloselysine 5-phosphate and erythruloselysine 4-phosphate. These LMW-PTPs were devoid of ribulosamine 3-phosphatase activity. It is concluded that most bacterial FN3K homologues are ribulosamine/erythrulosamine 3-kinases. They may serve, in conjunction with a phosphatase, to deglycate products of glycation formed from ribose 5-phosphate or erythrose 4-phosphate.

Identification of protein-ribulosamine-5-phosphatase as human low-molecular-mass protein tyrosine phosphatase-A.

Ribulosamines, which are substrates for the deglycating enzyme fructosamine-3-kinase-related protein, are presumably formed intracellularly through glycation of proteins with ribose 5-phosphate followed by dephosphorylation of resulting RN5Ps (ribulosamine 5-phosphates) by a putative RN5Pase (ribulosamine-5-phosphatase). Ribose 5-phosphate is known to be a potent glycating agent and we show in the present study that it reacts approximately 10 and 80-fold more rapidly with protein than ribose and glucose respectively. We also show that tissue extracts and, most particularly, erythrocyte extracts contain a protein-RN5Pase. We have purified this enzyme from human erythrocytes to near homogeneity and shown it to correspond to LMWPTP-A [low-molecular-mass ('weight') protein tyrosine phosphatase-A]. Human recombinant LMWPTP-A displayed an RN5Pase activity that was higher than its tyrosine phosphatase activity, indicating that this phosphatase may participate in protein deglycation, a new form of protein repair.

Identification of 3-deoxyglucosone dehydrogenase as aldehyde dehydrogenase 1A1 (retinaldehyde dehydrogenase 1).

One of the metabolic fates of 3-deoxyglucosone, a product of protein deglycation and a potent glycating agent, is to be oxidized to 2-keto-3-deoxygluconate, but the enzyme that catalyzes this reaction is presently unknown. Starting from human erythrocytes, which are known to convert 3-deoxyglucosone to 2-keto-3-deoxygluconate, we have purified to near homogeneity a NAD-dependent dehydrogenase that catalyzes this last reaction at neutral pH. Sequencing of a 55 kDa band co-eluting with the enzymatic activity in the last step indicated that it corresponded to aldehyde dehydrogenase 1A1 (ALDH1A1), an enzyme known to catalyze the oxidation of retinaldehyde to retinoic acid. Overexpression of human ALDH1A1 in HEK cells led to a more than 20-fold increase in 3-deoxyglucosone dehydrogenase activity. In mouse tissues 3-deoxyglucosone dehydrogenase activity was highest in liver, intermediate in lung and testis, and negligible or undetectable in other tissues, in agreement with the tissue distribution of ALDH1A1 mRNA. 3-deoxyglucosone dehydrogenase activity was undetectable in tissues from ALDH1A1(-/-) mice. ALDH1A1 appears therefore to be the major if not the only enzyme responsible for the oxidation of 3-deoxyglucosone to 2-keto-3-deoxygluconate. The urinary excretion of 2-keto-3-deoxygluconate amounted to 16.7 micromol/g creatinine in humans, indicating that 3-deoxyglucosone may be quantitatively a more important substrate than retinaldehyde for ALDH1A1.

Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair.

Fructosamine-3-kinase (FN3K) is a recently described protein-repair enzyme responsible for the removal of fructosamines, which are the products of a spontaneous reaction of glucose with amines. We show here that, compared with glucose, glucose 6-phosphate (Glu-6-P) reacted 3-6-fold more rapidly with proteins and 8-fold more rapidly with N-alpha-t-Boc-lysine, being therefore a more significant intracellular glycating agent than glucose in skeletal muscle and heart. Fructosamine 6-phosphates, which result from the reaction of amines with Glu-6-P, were not substrates for FN3K. However, a phosphatase that dephosphorylates protein-bound fructosamine 6-phosphates was found to be present in rat tissues. This enzyme was purified to near homogeneity from skeletal muscle and was identified as magnesium-dependent phosphatase-1 (MDP-1), an enzyme of the haloacid dehalogenase family with a putative protein-tyrosine phosphatase function. Human recombinant MDP-1 acted on protein-bound fructosamine 6-phosphates with a catalytic efficiency >10-fold higher than those observed with its next best substrates (arabinose 5-phosphate and free fructoselysine 6-phosphate) and >100-fold higher than with protein-phosphotyrosine. It had no detectable activity on fructosamine 3-phosphates. MDP-1 dephosphorylated up to approximately 75% of the fructosamine 6-phosphates that are present on lysozyme after incubation of this protein with Glu-6-P. Furthermore, lysozyme glycated with Glu-6-P was converted by MDP-1 to a substrate for FN3K. We conclude that MDP-1 may act physiologically in conjunction with FN3K to free proteins from the glycation products derived from Glu-6-P.

Plant ribulosamine/erythrulosamine 3-kinase, a putative protein-repair enzyme.

FN3K (fructosamine 3-kinase) is a mammalian enzyme that catalyses the phosphorylation of fructosamines, which thereby becomes unstable and detaches from proteins. The homologous mammalian enzyme, FN3K-RP (FN3K-related protein), does not phosphorylate fructosamines but ribulosamines, which are probably formed through a spontaneous reaction of amines with ribose 5-phosphate, an intermediate of the pentose-phosphate pathway and the Calvin cycle. We show in the present study that spinach leaf extracts display a substantial ribulosamine kinase activity (approx. 700 times higher than the specific activity of FN3K in erythrocytes). The ribulosamine kinase was purified approx. 400 times and shown to phosphorylate ribulose-epsilon-lysine, protein-bound ribulosamines and also, with higher affinity, erythrulose-epsilon-lysine and protein-bound erythrulosamines. Evidence is presented for the fact that the third carbon of the sugar portion is phosphorylated by this enzyme and that this leads to the formation of unstable compounds decomposing with half-lives of approx. 30 min at 37 degrees C (ribulosamine 3-phosphates) and 5 min at 30 degrees C (erythrulosamine 3-phosphates). This decomposition results in the formation of a 2-oxo-3-deoxyaldose and inorganic phosphate, with regeneration of the free amino group. The Arabidopsis thaliana homologue of FN3K/FN3K-RP was overexpressed in Escherichia coli and shown to have properties similar to those of the enzyme purified from spinach leaves. These results indicate that the plant FN3K/FN3K-RP homologue, which appears to be targeted to the chloroplast in many species, is a ribulosamine/erythrulosamine 3-kinase. This enzyme may participate in a protein deglycation process removing Amadori products derived from ribose 5-phosphate and erythrose 4-phosphate, two Calvin cycle intermediates that are potent glycating agents.

Fructosamine 3-kinase-related protein and deglycation in human erythrocytes.

Fructosamine 3-kinase (FN3K), an enzyme initially identified in erythrocytes, catalyses the phosphorylation of fructosamines on their third carbon, leading to their destabilization and their removal from protein. We show that human erythrocytes also contain FN3K-related protein (FN3K-RP), an enzyme that phosphorylates psicosamines and ribulosamines, but not fructosamines, on the third carbon of their sugar moiety. Protein-bound psicosamine 3-phosphates and ribulosamine 3-phosphates are unstable, decomposing at pH 7.1 and 37 degrees C with half-lives of 8.8 h and 25 min respectively, as compared with 7 h for fructosamine 3-phosphates. NMR analysis indicated that 1-deoxy-1-morpholinopsicose (DMP, a substrate for FN3K and FN3K-RP), like 1-deoxy-1-morpholinofructose (DMF, a substrate of FN3K), penetrated erythrocytes and was converted into the corresponding 3-phospho-derivative. Incubation of erythrocytes with 50 mM allose, 200 mM glucose or 10 mM ribose for 24 h resulted in the accumulation of glycated haemoglobin, and this accumulation was approx. 1.9-2.6-fold higher if DMP, a competitive inhibitor of both FN3K and FN3K-RP, was present in the incubation medium. Incubation with 50 mM allose or 200 mM glucose also caused the accumulation of ketoamine 3-phosphates, which was inhibited by DMP. By contrast, DMF, a specific inhibitor of FN3K, only affected the glucose-dependent accumulation of glycated haemoglobin and ketoamine 3-phosphates. These data indicate that FN3K-RP can phosphorylate intracellular, protein-bound psicosamines and ribulosamines, thus leading to deglycation.

Fructosamine 3-kinase is involved in an intracellular deglycation pathway in human erythrocytes.

Fructosamine 3-kinase, which phosphorylates low-molecular-mass and protein-bound fructosamines on the third carbon of their deoxyfructose moiety, is quite active in erythrocytes, and was proposed to initiate a process removing fructosamine residues from proteins. In the present study, we show that incubation of human erythrocytes with 200 mM glucose not only caused the progressive formation of glycated haemoglobin, but also increased the level of an anionic form of haemoglobin containing alkali-labile phosphate, to approx. 5% of total haemoglobin. 1-Deoxy-1-morpholinofructose (DMF), a substrate and competitive inhibitor of fructosamine 3-kinase, doubled the rate of accumulation of glycated haemoglobin, but markedly decreased the amount of haemoglobin containing alkali-labile phosphate. The latter corresponds therefore to haemoglobin bound to a fructosamine 3-phosphate group (FN3P-Hb). Returning erythrocytes incubated with 200 mM glucose and DMF to a low-glucose medium devoid of DMF caused a decrease in the amount of glycated haemoglobin, a transient increase in FN3P-Hb and a net decrease in the sum (glycated haemoglobin+FN3P-Hb). These effects were prevented by DMF, indicating that fructosamine 3-kinase is involved in the removal of fructosamine residues. The second step of this 'deglycation' process is most likely a spontaneous decomposition of the fructosamine 3-phosphate residues to a free amine, 3-deoxyglucosone and P(i). This is consistent with the findings that 2-oxo-3-deoxygluconate, the product of 3-deoxyglucosone oxidation, is formed in erythrocytes incubated for 2 days with 200 mM glucose in a sufficient amount to account for the removal of fructosamine residues from proteins, and that DMF appears to inhibit the formation of 2-oxo-3-deoxygluconate from elevated glucose concentrations.