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Dengue fever, caused by dengue viruses (DENV 1-4) is a leading cause of illness and death in the tropics and subtropics. Therefore, an effective vaccine is urgently needed. Currently, the only available licensed dengue vaccine is a chimeric live attenuated vaccine that shows varying efficacy depending on serotype, age and baseline DENV serostatus. Accordingly, a dengue vaccine that is effective in seronegative adults, children of all ages and in immunocompromised individuals is still needed. We are currently researching the use of psoralen to develop an inactivated tetravalent dengue vaccine. Unlike traditional formalin inactivation, psoralen inactivates pathogens at the nucleic acid level, potentially preserving envelope protein epitopes important for protective anti-dengue immune responses. We prepared highly purified monovalent vaccine lots of formalin- and psoralen-inactivated DENV 1-4, using Capto DeVirS and Capto Core 700 resin based column chromatography. Tetravalent psoralen-inactivated vaccines (PsIV) and formalin-inactivated vaccines (FIV) were prepared by combining the four monovalent vaccines. Mice were immunized with either a low or high dose of PsIV or FIV to evaluate the immunogenicity of monovalent as well as tetravalent formulations of each inactivation method. In general, the monovalent and tetravalent PsIVs elicited equivalent or higher titers of neutralizing antibodies to DENV than the FIV dengue vaccines and this response was dose dependent. The immunogenicity of tetravalent dengue PsIVs and FIVs were also evaluated in nonhuman primates (NHPs). Consistent with what was observed in mice, significantly higher neutralizing antibody titers for each dengue serotype were observed in the NHPs vaccinated with the tetravalent dengue PsIV compared to those vaccinated with the tetravalent dengue FIV, indicative of the importance of envelope protein epitope preservation during psoralen inactivation of DENV.

The development of a safe and effective vaccine to protect against COVID-19 is a global priority due to the current high SARS-CoV-2 infection rate. Currently, there are over 160 SARS-CoV-2 vaccine candidates at the clinical or pre-clinical stages of development. Of these, there are only three whole-virus vaccine candidates produced using β-propiolactone or formalin inactivation. Here, we prepared a whole-virus SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) using a novel psoralen inactivation method and evaluated its immunogenicity in mice using two different adjuvants, alum and Advax-2. We compared the immunogenicity of SARS-CoV-2 PsIV against SARS-CoV-2 DNA vaccines expressing either full-length or truncated spike proteins. We also compared the psoralen-inactivated vaccine against a DNA prime, psoralen-inactivated vaccine boost regimen. After two doses, the psoralen-inactivated vaccine, when administered with alum or Advax-2 adjuvants, generated a dose-dependent neutralizing antibody responses in mice. Overall, the pattern of cytokine ELISPOT responses to antigen-stimulation observed in this study indicates that SARS-CoV-2 PsIV with the alum adjuvant promotes a Th2-type response, while SARS-CoV-2 PsIV with the Advax-2 adjuvant promotes a Th1-type response.

Phase 1 clinical trials with a DNA vaccine for dengue demonstrated that the vaccine is safe and well tolerated, however it produced less than optimal humoral immune responses. To determine if the immunogenicity of the tetravalent dengue DNA vaccine could be enhanced, we explored alternate, yet to be tested, methods of vaccine administration in non-human primates. Animals were vaccinated on days 0, 28 and 91 with either a low (1 mg) or high (5 mg) dose of vaccine by the intradermal or intramuscular route, using either needle-free injection or electroporation devices. Neutralizing antibody, IFN-γ T cell and memory B cell responses were compared to a high dose group vaccinated with a needle-free intramuscular injection delivery device similar to what had been used in previous preclinical and clinical studies. All previously untested vaccination methodologies elicited improved immune responses compared to the high dose needle-free intramuscular injection delivery group. The highest neutralizing antibody responses were observed in the group that was vaccinated with the high dose formulation via intradermal electroporation. The highest IFN-γ T cell responses were also observed in the high dose intradermal electroporation group and the CD8+ T cells were the dominant contributors for the IFNγ response. Memory B cells were detected for all four serotypes. More than a year after vaccination, groups were challenged with dengue-1 virus. Both the low and high dose intradermal electroporation groups had significantly fewer days of dengue-1 virus RNAemia compared to the control group. The results from this study demonstrate that using either an electroporation device and/or the intradermal route of delivery increases the immune response generated by this vaccine in non-human primates and should be explored in humans.

Escherichia coli NeuNAc (N-acetylneuraminic acid) synthase catalyses the condensation of PEP (phosphoenolpyruvate) and ManNAc (N-acetylmannosamine) to form NeuNAc and is encoded by the neuB gene. Campylobacter jejuni has three neuB genes, one of which is very similar to the E. coli neuB gene. We have characterized the C. jejuni neuraminic acid synthase with respect to acylamino sugar specificity and stereochemistry of the PEP condensation. We determined the specificity of C. jejuni NeuNAc synthase for N-acetylmannosamine, N-butanoylmannosamine, N-propionoylmannosamine and N-pentanoylmannosamine. We find that, although this enzyme exhibits similar Km values for N-acylmannosamine molecules with different N-acyl groups, the kcat/Km values decreased with increasing chain length. NeuNAc synthase is a member of a PEP-utilizing family of enzymes that form oxo acids from PEP and a monosaccharide. This family includes KDO 8-P (2-keto-3-deoxy-D-manno-octulosonate 8-phosphate) synthase and DAH 7-P (2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate) synthase. Both enzymes catalyse the condensation of the re face of the aldehyde group of the monosaccharide with the si face of the PEP molecule. The C. jejuni NeuNAc synthase catalysed the condensation of Z- and E-[3-2H]PEP with ManNAc, yielding (3S)-3-deutero-NeuNAc and (3R)-3-deutero-NeuNAc respectively. The condensation of Z-[3-F]PEP and ManNAc yielded (3S)-3-fluoro-NeuNAc. Results of our studies suggest that the C. jejuni NeuNAc synthase, similar to KDO 8-P synthase and DAH 7-P synthase, catalyses the condensation of the si face of PEP with the aldehyde sugar. The present study is the first stereochemical analysis of the reaction catalysed by a bacterial NeuNAc synthase.

Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but not D-ribose 5-phosphate (R5P) nor the four carbon analogue D-erythrose 4-phosphate (E4P). However, E. coli KDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate. The mechanism of this abortive utilization of PEP was investigated using [2,3-(13)C(2)]-PEP and [3-F]-PEP, and the reaction products were determined by (13)C, (31)P, and (19)F NMR to be pyruvate, phosphate, and 2-phosphoglyceric acid (2-PGA). The formation of pyruvate and 2-PGA suggests that the reaction catalyzed by KDO8-P synthase may be initiated via a nucleophilic attack to PEP by a water molecule. In experiments in which the homologous enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7-P) synthase was incubated with D,L-glyceraldehyde 3-phosphate (G3P) and [2,3-(13)C(2)]-PEP, pyruvate and phosphate were the predominant species formed, suggesting that the reaction catalyzed by DAH7-P synthase starts with a nucleophilic attack by water onto PEP as observed in E. coli KDO8-P synthase.

[figure: see text] Neisseria gonorrhoeae 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase catalyzes an aldol-type condensation between D-erythrose 4-phosphate and phosphoenolpyruvate (PEP) to form 3-deoxy-D-arabino-heptulosonate 7-phosphate and not 3-deoxy-D-riboheptulosonate 7-phosphate. Similar to the Escherichia coli enzyme, N. gonorrhoeae DAH 7-P synthase condenses D-arabinose 5-phosphate with PEP to give 3-deoxy-D-manno-octulosonate 8-phosphate. Therefore, the stereochemistry of the reaction catalyzed by N. gonorrhoeae DAH 7-P synthase at C1 of the phosphorylated monosaccharide is the same as that for the E. coli enzyme, namely, re face attack.

Purpose. Vitamin A (retinol) and its metabolites comprise the natural retinoids. While the biological action of these molecules are thought to be primarily mediated by ca. 55 kDa nuclear retinoic acid receptors, a number of structurally similar 15-20 kDa proteins are involved in the transport, and possibly metabolism, of these compounds. The milk protein β-lactoglobulin B (β-LG) is an 18 kDa protein which binds retinol and may be involved in oral delivery of retinol to neonates. β-LG also binds drugs and other natural products and is of potential interest as a protective delivery vehicle.

The active site residues of the proposed metal binding site of DAH 7-P synthase (phe) were probed by site-directed mutagenesis of C61 to glycine and serine, H64 to glycine, and with the double mutant C61H/H64C. While C61S and C61H/H64C were inactive, both C61G and H64G were active. All mutants, regardless of enzymatic activity, bound one equivalent of Fe2+ per monomeric unit. Even though C61 and H64 were shown not to be metal ligands for the DAH 7-P synthase (phe), they may provide some of the backbone interactions/secondary structural elements necessary to properly form the metal binding pocket.

Rapid detection and identification of biological molecules is of utmost importance in a number of applications including clinical diagnosis and detection of foodborne pathogens as well as environmental contaminants. Mass spectrometry affords a high degree of accuracy (specificity) and sensitivity without a need for extensive sample purification. Atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry has become a useful technique in a number of important applications, including identification of proteins, and structural analysis of oligosaccharides, phosphopeptides, and lipids. Sample preparation methods for AP-MALDI mass spectrometry are relatively simple, rapid and can be easily automated for high throughput sample analysis. AP-MALDI MS/MS analysis has been successfully utilized to identify the presence of different types of bioagents such as Escherichia coli (E. coli), spores of several Bacilli, Saccharomyces cerevisiae (yeast), ovalbumin, and Clostridium botulinum Neurotoxin Type A in environmental samples. AP-MALDI imaging mass spectrometry is an emerging technique for direct analysis of biological tissue sections that can be useful for profiling spatial distribution of drugs, peptides and proteins in tissue sections of plants, animals, and humans.

An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 degrees C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with K(m)=3.6 microM for phosphoenolpyruvate and 3.8 microM for D-arabinose 5-phosphate and kcat=5.9 s(-1) at 37 degrees C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

The five-carbon phosphorylated monosaccharide analogues, D-arabinose 5-phosphate, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate, were separately condensed with (Z)- and (E)-[3-(2)H]-phosphoenolpyruvate (PEP) in the presence of Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (phe) to give in the case of (Z)-[3-(2)H]-PEP (3S)-[3-(2)H]-3-deoxy-D-manno-octulosonate 8-phosphate, (3S)-[3-(2)H]-3-deoxy-D-altro-octulosonate 8-phosphate, and (3S)-[3-(2)H]-3,5-dideoxy-D-altro-octulosonate 8-phosphate, respectively, whereas incubation with (E)-[3-(2)H]-PEP gives the corresponding (3R)-monosaccharides. These results are in complete agreement with the observed facial selectivity of DAH 7-P synthase for its normal substrates D-erythrose 4-phosphate and PEP and provide direct evidence that DAH 7-P synthase (phe) catalyzes the si face addition of the C3 of PEP to the re face of C1 of the phosphorylated monosaccharides tested. Products formed by DAH 7-P synthase (phe)-catalyzed condensation of (Z)- and (E)-[3-F]-PEP with E 4-P were completely characterized by (1)H and (19)F NMR analysis for the first time. Results of our studies suggest that disappearence of the double bond between C2 and C3 of PEP and formation of a bond between C3 of PEP and C1 of the phosphorylated monosaccharide tested occur in concert during the DAH 7-P synthase-catalyzed condensation reaction.

The five-carbon phosphorylated monosaccharide analogues, D-arabinose 5-phosphate, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate, were separately condensed with (Z)- and (E)-[3-(2)H]-phosphoenolpyruvate (PEP) in the presence of Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (phe) to give in the case of (Z)-[3-(2)H]-PEP (3S)-[3-(2)H]-3-deoxy-D-manno-octulosonate 8-phosphate, (3S)-[3-(2)H]-3-deoxy-D-altro-octulosonate 8-phosphate, and (3S)-[3-(2)H]-3,5-dideoxy-D-altro-octulosonate 8-phosphate, respectively, whereas incubation with (E)-[3-(2)H]-PEP gives the corresponding (3R)-monosaccharides. These results are in complete agreement with the observed facial selectivity of DAH 7-P synthase for its normal substrates D-erythrose 4-phosphate and PEP and provide direct evidence that DAH 7-P synthase (phe) catalyzes the si face addition of the C3 of PEP to the re face of C1 of the phosphorylated monosaccharides tested. Products formed by DAH 7-P synthase (phe)-catalyzed condensation of (Z)- and (E)-[3-F]-PEP with E 4-P were completely characterized by (1)H and (19)F NMR analysis for the first time. Results of our studies suggest that disappearence of the double bond between C2 and C3 of PEP and formation of a bond between C3 of PEP and C1 of the phosphorylated monosaccharide tested occur in concert during the DAH 7-P synthase-catalyzed condensation reaction.

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH7P) synthase (phenylalanine repressible) catalyze similar aldol-type condensations between phosphoenolpyruvate (PEP) and the monosaccharides d-arabinose 5-phosphate (A5P) and d-erythrose 4-phosphate (E4P), respectively (Fig. ​(Fig.1).1). Both enzymes from Escherichia coli are tetrameric, and although their quaternary structures are dissimilar, the monomeric subunits that comprise each enzyme are nearly superimposable (9). As one might expect from this degree of subunit similarity, the facial selectivities of both enzymes from E. coli have been shown to be re-face with respect to the phosphorylated monosaccharide and si-face with respect to PEP, resulting in an (R) configuration at the C-4 position of each product (2, 4, 7, 8). Reports from this laboratory have shown that E. coli DAH7P synthase (encoded by aroG) is capable of utilizing arabinose 5-phosphate, ribose 5-phosphate, and 2-deoxyribose 5-phosphate to synthesize the corresponding eight carbon monosaccharides (14), while E. coli KDO8P synthase is unable to utilize E4P as a substrate (D. L. Howe, G. Y. Sheflyan, and R. W. Woodard, unpublished data). The facial selectivity of DAH7P synthase with regard to both PEP and the alternate substrates listed above is the same as with the natural substrates (16a). FIG. 1 The reactions catalyzed by E. coli KDO8P and DAH7P synthases with their respective substrates and the proposed reaction for the N. gonorrhoeae KDO8P synthase-catalyzed condensation of E4P and PEP. Open arrow, location of newly formed C-C bond. It has recently been reported not only that KDO8P synthase from Neisseria gonorrhoeae utilizes E4P as an alternate substrate but also that the facial selectivity of the enzyme with respect to this monosaccharide is si-face, resulting in an (S) configuration at the C-4 position of the product (16). This suggests that the enzyme from N. gonorrhoeae is distinct from all other reported KDO8P synthases, in terms of both its substrate ambiguity and its stereoselectivity. However, these unusual results were obtained using enzyme isolated directly from N. gonorrhoeae that was neither rigorously purified nor thoroughly biochemically characterized (16). The product of the enzymatic reaction, C7-X (original authors' designation), was neither isolated nor purified, and its stereochemistry was assigned solely on the intensity and rapid production of the chromophore produced in the classic Aminoff assay (periodate-thiobarbituric acid assay) for 3-deoxy-monosaccharides (1, 10). This assay is known to be sensitive to the stereochemistry of the hydroxyl groups of the monosaccharide (11, 18) but is also known to give false-positive results with compounds such as shikimic acid, guanosine, and adenosine, as well as with DNA (6). Since studies in our laboratory are aimed at the elucidation of the similarities and differences between KDO8P and DAH7P synthase, we were intrigued by the reported use of E4P by a KDO8P synthase and decided to further investigate this unusual finding. An open reading frame corresponding to the KDO8P synthase gene (kdsA) was retrieved via a TBLASTN search of the N. gonorrhoeae FA 1090 databank at the University of Oklahoma Advanced Center for Genome Technology (http://www.genome.ou.edu/gono_blast.html) using a fragment of E. coli KDO8P synthase. Standard PCR methodologies were employed to amplify this gene sequence from N. gonorrhoeae DNA. The amplified product was isolated, restricted, and ligated into the expression vector pT7-7 (17). The ligation mixture was used to transform competent E. coli XL1-Blue cells (Stratagene). Plasmids isolated from several transformants were subjected to DNA sequencing, and one plasmid with the expected sequence was isolated (pT7-7-NGKDO). In our sequence, position 119 is threonine; this residue is absolutely conserved in all known KDO8P synthase sequences (M. R. Birck and R. W. Woodard, submitted for publication). KDO8P synthase from N. gonorrhoeae was overexpressed in E. coli BL21(DE3) cells harboring pT7-7-NGKDO and purified, as previously reported, using anion-exchange chromatography (14; W. P. Taylor and R. W. Woodard, submitted for publication). The resultant protein was determined to be >95% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The apparent molecular mass was determined by size-exclusion chromatography and was found to be 88.2 kDa. This value is similar to those reported for the KDO8P synthases from E. coli, Aquifex aeolicus, and Salmonella enterica serovar Typhimurium (3, 12–14; Taylor and Woodard, submitted). The N. gonorrhoeae KDO8P synthase-catalyzed condensation of A5P and PEP was monitored by observing the disappearance of the α,β-unsaturated carbonyl absorbance (λ = 232 nm, ɛ = 2,840 M−1 cm−1) of PEP (14). All reactions were performed in 100 mM Tris-acetate, pH 7.4, at 37°C. The kinetic parameters were determined from progress curves of substrate consumption, as previously described (Taylor and Woodard, submitted), and are as follows: for PEP, Km = 3.1 ± 0.2 μM; for A5P, Km = 8.5 ± 0.1 μM; for both PEP and A5P, kcat = 1.0 ± 0.1 s−1. The Michaelis constants for both PEP and A5P are very similar to those reported for both E. coli and S. enterica serovar Typhimurium KDO8P synthase, although the value for kcat was reduced approximately three- to sixfold (12; Taylor and Woodard, submitted). The effect of divalent metal ions on enzymatic activity was investigated by quantitating the amount of KDO8P produced using the periodate-thiobarbituric acid assay (1, 10, 14). Inclusion of Mn2+ or Zn2+ (1 mM) in the assay mixture had no effect on activity. Preincubation of the enzyme with a 10 mM concentration of either metal prior to reaction led to a small decrease in enzyme activity. Prolonged treatment of purified enzyme with 5 mM EDTA resulted in no loss of enzymatic activity. Purified KDO8P synthase (12 μM) was incubated with 24.3 mM A5P, 23.7 mM PEP, 0.5 mM MnCl2, and 1 mM dithiothreitol in 100 mM Tris-acetate buffer (pH 7.5) in a final volume of 2.0 ml. Even though it is shown in the present study that N. gonorrhoeae KDO8P synthase does not require any metal ion for activity, MnCl2 was included in the reaction mixture in order to mimic the reaction conditions reported by Subramaniam et al. (16). Tris-acetate was substituted for phosphate buffer in the present study since inorganic phosphate has been shown to inhibit E. coli KDO8P synthase (5). Incubation of KDO8P synthase from N. gonorrhoeae with E4P and PEP in 75 mM phosphate buffer (pH 7.0), however, gave no indication of deoxymonosaccharide formation as determined by periodate-thiobarbituric acid analysis. The reaction mixture was incubated at 37°C for 5 h, and then the reaction was quenched by the addition of 10% trichloroacetic acid (2.0 ml). The 3-deoxy-α-keto sugar acid component of the reaction mixture was purified by anion-exchange chromatography as previously described (14). The 500 MHz 1H-nuclear magnetic resonance (NMR) spectrum of the product isolated from the above reaction is shown in Fig. ​Fig.22 and is in complete agreement with that reported previously for 3-deoxy-d-manno-octulosonic acid 8-phosphate obtained from the E. coli KDO8P synthase-catalyzed condensation of A5P and PEP (Fig. ​(Fig.2)2) (2). In a parallel experiment, 33 mM E4P was substituted for A5P and the mixture was subjected to the reaction conditions and purification protocol described above. Periodate-thiobarbituric acid analysis of the quenched enzymatic reaction mixture before chromatography as well as analysis of all column fractions after purification yielded no indication of a keto-deoxy-monosaccharide acid product. 1H-NMR analysis of the column fractions where acidic phosphorylated monosaccharides normally elute (fractions 30 to 40) gave no signals representative of the condensation product (14). Starting materials were recovered in fractions 23 to 27 (E4P) and fractions 35 to 39 (PEP). Thus, we found that KDO8P synthase from N. gonorrhoeae exhibits the same reaction stereochemistry and substrate selectivity observed for E. coli KDO8P synthase (2, 4). FIG. 2 500-MHz 1H-NMR spectra of the product derived from the condensation of A5P and PEP as catalyzed by E. coli KDO8P synthase (X, peak from buffer) (a) and N. gonorrhoeae KDO8P synthase (b). While the present report demonstrates that E4P is not a substrate for N. gonorrhoeae KDO8P synthase, the question remains as to the actual identity of the C7-X compound identified by Subramanian et al. (16). Based on the reported rate of oxidation as well as the fact that C7-X is not a substrate for dehydroquinate synthase, it is tempting to suggest that this compound is indeed 3-deoxy-d-ribo-heptulosonate 7-phosphate. It is known that 2-keto-3-deoxy-6-phosphogluconate aldolase catalyzes the aldol-like condensation of E4P and pyruvic acid to form 3-deoxy-d-ribo-heptulosonate 7-phosphate (15). Alternatively, a minor DAH7P synthase contaminant could prove to be the source of C7-X formation, although this would also require addition to the si-face of E4P, in contrast to the stereochemistry of all other known DAH7P synthase-catalyzed reactions. Efforts are underway in our laboratory to clone, overexpress, and purify N. gonorrhoeae DAH7P synthase in order to address this latter possibility.