C. Patrick McAtee

Identification of differentially regulated proteins in metronidozole resistant Helicobacter pylori by proteome techniques

Resistance to metronidazole (MTZ) is common among Helicobacter pylori strains in many societies, and results from loss of function mutations in genes for one or more cellular nitroreductases. When functional, these enzymes convert MTZ from a harmless prodrug to mutagenic and bacteriocidal products (probably hydroxylamine‐type compounds), and in the process may generate active reactive oxygen metabolites. Here we examine the protein profiles of a derivative of strain 26695 that is resistant to moderate levels of MTZ because of mutation in rdxA (HP0954), the gene for the most important of these nitroreductases. The strain was grown with and without 18 μg/mL of MTZ to assess whether sublethal exposure triggers an adaptive response. Bacterial lysates were subjected to two‐dimensional (2‐D) electrophoresis and protein bands were identified by mass spectrometry and sequence analysis. Several proteins were decreased at least two‐fold during growth with MTZ, yet the levels of various isoforms of alkylhydroperoxide reductase (AHP) (encoded by ahpC HP1563) were increased. AHP is an essential enzyme, and had been linked to resistance to oxygen toxicity in various prokaryotic and eukaryotic systems; we propose that the ability of an rdxA mutant strain to increase AHP abundance during exposure to MTZ is critically important in the realization of the resistance phenotype. More generally, these results highlight the potential of proteome analysis to tracing out how pathogenic bacteria cope with the challenges imposed on them by therapy or host responses to infection.

Characterization of a Helicobacter pylori vaccine candidate by proteome techniques

In a previous two-dimensional (2D) gel electrophoretic study of protein antigens of the gastric pathogen, Helicobacter pylori recognized by human sera, one of the highly and consistently reactive antigens, a protein with Mr of approximately 30,000 (Spot 15) seemed to be of special interest because of low yields on N-terminal protein sequencing. This suggested possible N-terminal modification, as the N-terminal sequence analysis of this 30,000 protein (Spot 15) did not provide a definitive match within the H. pylori genomic database. This protein was isolated by 2D polyacrylamide gel electrophoresis, evaluated by liquid chromatography-mass spectrometry, and found to consist of two related species of approximately 28,100 and 26,500. In parallel, the proteins within this spot were digested in situ with the endoprotease Lys-C. Analysis of the Lys-C digest by matrix-assisted laser desorption time-of-flight mass spectrometry, peptide mapping, and sequence analysis was conducted. Comparison of the mass and sequence of the Lys-C peptides with those derived from a H. pylori genomic library identified an open reading frame of approximately 300 base pairs as the source of the Spot 15 protein. This corresponded to HP0175 in the recently reported H. pylori genome sequence, an open reading frame with some homology to Campylobacter jejeuni cell binding protein 2. Mass spectral and sequence analysis indicated that Spot 15 was a processed product generated by proteolytic cleavage at both the carboxy and amino termini of the 34 open reading frame precursor.

Isolation of Monoclonal Antibody Charge Variants by Displacement Chromatography

This unit discusses the important parameters in designing and optimizing a separation of monoclonal antibody (mAb) charge variants from process streams by ion‐exchange displacement chromatography, including sample preparation and selection of matrix, column, and appropriate buffer. A protocol is provided for determination of optimal column binding and displacement conditions, including cleaning and regeneration of the displacement columns. Curr. Protoc. Protein Sci. 69:8.10.1‐8.10.13.

Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human chagas disease

ABSTRACT A therapeutic vaccine for human Chagas disease is under development by the Sabin Vaccine Institute Product Development Partnership. The aim of the vaccine is to significantly reduce the parasite burden of Trypanosoma cruzi in humans, either as a standalone product or in combination with conventional chemotherapy. Vaccination of mice with Tc24 formulated with monophosphoryl-lipid A (MPLA) adjuvant results in a Th1 skewed immune response with elevated IgG2a and IFNγ levels and a statistically significant decrease in parasitemia following T. cruzi challenge. Tc24 was therefore selected for scale-up and further evaluation. During scale up and downstream process development, significant protein aggregation was observed due to intermolecular disulfide bond formation. To prevent protein aggregation, cysteine codons were replaced with serine codons which resulted in the production of a non-aggregated and soluble recombinant protein, Tc24-C4. No changes to the secondary structure of the modified molecule were detected by circular dichroism. Immunization of mice with wild-type Tc24 or Tc24-C4, formulated with E6020 (TLR4 agonist analog to MPLA) emulsified in a squalene-oil-in-water emulsion, resulted in IgG2a and antigen specific IFNγ production levels from splenocytes that were not significantly different, indicating that eliminating putative intermolecular disulfide bonds had no significant impact on the immunogenicity of the molecule. In addition, vaccination with either formulated wild type Tc24 or Tc24-C4 antigen also significantly increased survival and reduced cardiac parasite burden in mice. Investigations are now underway to examine the efficacy of Tc24-C4 formulated with other adjuvants to reduce parasite burden and increase survival in pre-clinical studies.

Expression and purification of an engineered, yeast-expressed Leishmania donovani nucleoside hydrolase with immunogenic properties.

ABSTRACT Leishmania donovani is the major cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality second only to malaria. No human vaccine is currently available. A 36 kDa L. donovani nucleoside hydrolase (LdNH36) surface protein has been previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris and its purification at the 20 L scale to establish suitability for future pilot scale manufacturing. To improve efficiency of process development and ensure reproducibility, 4 N-linked glycosylation sites shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues. The mutant LdNH36 (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modeling, and the mutated amino acids are located outside the major immunogenic domain. Immunogenic properties of the LdNH36-dg2 recombinant protein were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)). Mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive to both LdNH36-dg2 and LdNH36 wild-type. While the point mutations did affect the hydrolase activity of the enzyme, the IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of the wild-type LdNH36. The results indicate that LdNH36-dg2 as expressed in and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing in support of future first-in-humans phase 1 clinical trials.

Displacement Chromatography of Proteins

This unit discusses the important parameters in designing and optimizing a separation by ion‐exchange displacement chromatography, including preparing the sample and choosing a matrix, column, and buffer. Protocols are provided for testing a column, determining binding and elution conditions, displacing the sample, and cleaning, regenerating, and storing of displacement columns. Curr. Protoc. Protein Sci. 59:8.9.1‐8.9.14.

Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate

Abstract From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218–amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast–Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials.

Fitness, nutrition and the molecular basis of chronic disease

The onset of chronic disease is often the prelude to the subsequent physiological and mental twilight in the aging population of modern society. While rates of obesity, specific types of cancer and cardiovascular disorders seem to be on the rise in this group, many new therapies have addressed diseases that have been largely untreatable in the past. Alzheimer’s disease has also recently come to the forefront of ongoing maladies most typically associated with an aging population. Ironically, though, many people seem to be living longer than expected. Recent biochemical, nutritional and genomic approaches have been able to elucidate some of the complex mechanisms, which lead to chronic diseases associated with an aging population such as Alzheimer’s, metabolic syndrome, tumor metastasis and cardiovascular disease. These diseases and their sequalae seem to be related in many respects, with the common culprit being the inflammatory environment created by the presence of excess fat – particularly within the vascular network. Although a substantial effort has been focused on the development of new-line therapeutics to address these issues, nutrition and overall fitness and their effects on stalling or potentially reversing the advent of these diseases has not been fully embraced in the research arena. This review discusses the role of the inflammatory environment in the development of chronic diseases in the aging population and also proposes a common pathology. The benefits that improvements and dedication in nutrition and fitness approaches may offer at the molecular level are also discussed.

3M-052 as an adjuvant for a PLGA microparticle-based Leishmania donovani recombinant protein vaccine

It is believed that an effective vaccine against leishmaniasis will require a T helper type 1 (TH 1) immune response. In this study, we investigated the adjuvanticity of the Toll-like receptor (TLR) 7/8 agonist 3M-052 in combination with the Leishmania donovani 36-kDa nucleoside hydrolase recombinant protein antigen (NH36). NH36 and 3M-052 were encapsulated in separate batches of poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs). The loading efficiency for NH36 was 83% and for 3M-052 was above 95%. In vitro stimulation of bone marrow-derived dendritic cells, measured by IL-12 secretion, demonstrated that 3M-052 (free or MP-formulated) had a concentration-dependent immunostimulatory effect with an optimum concentration of 2 µg/mL. In immunogenicity studies in BALB/c mice, MP-formulated NH36 and 3M-052 elicited the highest serum titers of TH 1-associated IgG2a and IgG2b antibodies and the highest frequency of IFNγ-producing splenocytes. No dose dependency was observed among MP/NH36/3M-052 groups over a dose range of 4-60 µg 3M-052 per injection. The ability of MP-formulated NH36 and 3M-052 to elicit a TH 1-biased immune response indicates the potential for PLGA MP-formulated 3M-052 to be used as an adjuvant for leishmaniasis vaccines.

Deamidation and isoaspartate formation during in Vitro aging of a recombinant hepatitis e vaccine candidate

Publisher Summary Deamidation reduce the biological activity of several recombinant biopharmaceuticals. Formation of isoaspartate via the deamidation of asparaginyl residues or isomerization of aspartyl residues constitutes a major source of instability in proteins and peptides. Isoaspartate arises through an intramolecular rearrangement that produces a succinimide (cyclic imide) intermediate. Spontaneous hydrolysis of the imide occurs with a half-life of several hours and generates an aspartyl residue linked to its C-flanking neighbor through the aspartate β-carbonyl. Isoaspartate-bearing proteins and peptides are specific substrates for a widely distributed protein methyltransferase, PIMT that uses S-adenosyl-L-methionine (Adomet) as its methyl donor. The predicted amino acid sequence of the r62-kDa vaccine candidate contained several potential deamidation “hotspots” in which an asparagine residue was flanked on its carboxyl side by a threonine, glycine or serine residue, or in an example, the putative site(s) were flanked by a threonine or a serine on the amino terminal side. By methylation of isoaspartate residues generated through in vitro aging, it has been determined that three tryptic peptides contained notable isoaspartate accumulation. The presence of isoaspartate in these peptides has been confirmed by sequence analysis and mass spectrometry.