Characterization and Stability of Trypanosoma cruzi 24-C4 (Tc24-C4), a Candidate Antigen for a Therapeutic Vaccine Against Chagas Disease.

Chagas disease due to chronic infection with Trypanosoma cruzi is a neglected cause of heart disease, affecting approximately 6-10 million individuals in Latin America and elsewhere. T. cruzi Tc24, a calcium-binding protein in the flagellar pocket of the parasite, is a candidate antigen for an injectable therapeutic vaccine as an alternative or a complement to chemotherapy. Previously, we reported that a genetically engineered construct from which all cysteine residues had been eliminated (Tc24-C4) yields a recombinant protein with reduced aggregation and improved analytical purity in comparison to the wild-type form, without compromising antigenicity and immunogenicity. We now report that the established process for producing Escherichia coli-expressed Tc24-C4 protein is robust and reproducibly yields protein lots with consistent analytical characteristics, freeze-thaw, accelerated, and long-term stability profiles. The data indicate that, like most proteins, Tc24-C4 should be stable at -80°C, but also at 4°C and room temperature for at least 30 days, and up to 7-15 days at 37°C. Thus, the production process for recombinant Tc24-C4 is suitable for Current Good Manufacturing Practice production and clinical testing, based on process robustness, analytical characteristics, and stability profile.

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. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1587-1594, 2018.

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

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.

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

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, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis.

The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 µM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a Th1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate.

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

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.

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.

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.

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.