PMCA-replicated PrPD in urine of vCJD patients maintains infectivity and strain characteristics of brain PrPD: Transmission study.

The presence of abnormal, disease-related prion protein (PrPD) has recently been demonstrated by protein misfolding cyclic amplification (PMCA) in urine of patients affected with variant Creutzfeldt-Jakob disease (vCJD), a prion disease typically acquired from consumption of prion contaminated bovine meat. The complexity and multistage process of urine excretion along with the obligatory use of PMCA raise the issue of whether strain characteristics of the PrPD present in vCJD brains, such as infectivity and phenotype determination, are maintained in urine excreted PrPD and following amplification by PMCA. We inoculated transgenic mice expressing normal human PrP with amplified urine and brain homogenate achieving the same 100% attack rate, similar incubation periods (in both cases extremely long) and histopathological features as for type and severity of the lesions. Furthermore, PrPD characteristics analyzed by immunoblot and conformational stability immunoassay were indistinguishable. Inoculation of raw vCJD urine caused no disease, confirming the extremely low concentration of PrPD in vCJD urine. These findings show that strain characteristics of vCJD brain PrPD, including infectivity, are preserved in PrPD present in urine and are faithfully amplified by means of PMCA; moreover, they suggest that the PrPD urine test might allow for the diagnosis and identification of disease subtype also in sporadic CJD.

Co-occurrence of chronic traumatic encephalopathy and prion disease.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease associated with repetitive traumatic brain injury (TBI). CTE is generally found in athletes participating in contact sports and military personnel exposed to explosive blasts but can also affect civilians. Clinically and pathologically, CTE overlaps with post-traumatic stress disorder (PTSD), a term mostly used in a clinical context. The histopathology of CTE is defined by the deposition of hyperphosphorylated tau protein in neurons and astrocytes preferentially with perivascular distribution and at the depths of the cortical sulci. In addition to hyperphosphorylated tau, other pathologic proteins are deposited in CTE, including amyloid ß (Aß), transactive response (TAR) DNA-binding protein 43 kDa (TDP-43) and α-synuclein. However, the coexistence of prion disease in CTE has not been observed. We report three cases of histopathologically validated CTE with co-existing sporadic prion disease. Two were identified in a cohort of 55 pathologically verified cases of CTE submitted to the CTE Center of Boston University. One was identified among brain tissues submitted to the National Prion Disease Pathology Surveillance Center of Case Western Reserve University. The histopathological phenotype and properties of the abnormal, disease-related prion protein (PrPD) of the three CTE cases were examined using lesion profile, immunohistochemistry, electrophoresis and conformational tests. Subjects with sporadic Creutzfeldt-Jakob disease (sCJD) matched for age, PrP genotype and PrPD type were used as controls. The histopathology phenotype and PrPD properties of the three CTE subjects showed no significant differences from their respective sCJD controls suggesting that recurring neurotrauma or coexisting CTE pathology did not detectably impact the prion disease phenotype and PrPD conformational characteristics. Based on the reported incidence of sporadic prion disease, the detection of two cases with sCJD in the CTE Center series of 55 CTE cases by chance alone would be highly unlikely (p = 8.93*10- 6). Nevertheless, examination of a larger cohort of CTE is required to conclusively determine whether the risk of CJD is significantly increased in patients with CTE.

Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.

Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.

Escherichia coli-expressed near full length HIV-1 envelope glycoprotein is a highly sensitive and specific diagnostic antigen.

BACKGROUND: The Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r)-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies. METHODS: A deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni(II)-affinity chromatography. Biotinylated and europium(III) chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n=131), that included an in-house panel and four commercially procured panels. RESULTS: In-frame deletion of three hydrophobic regions, spanning amino acid residues 1-43, 519-538 and 676-706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C, and human T cell leukemia viruses. CONCLUSIONS: This study has demonstrated the expression of r-gp160 variant in E. coli, by deletion of hydrophobic regions, and its purification in reasonable yields. This underscores the potential for cost saving in antigen production. Evaluation of this antigen in a double antigen sandwich two-step assay showed it to be a highly sensitive and specific HIV-1 diagnostic reagent. The amenability of this assay to the one-step format suggests its potential utility in developing a rapid point-of-care HIV-1 diagnostic test.

Optimization of conditions for secretion of dengue virus type 2 envelope domain III using Pichia pastoris.

We have developed a recombinant clone of the methylotrophic yeast Pichia pastoris capable of secreting dengue virus type 2 envelope domain III (sEDIII-2). We explored various induction parameters including media composition, temperature, pH, and methanol concentration, to optimize conditions for sEDIII-2 expression in shake flask culture. Induction at 20°C in the presence of 2% (v/v) methanol in a medium buffered to pH 5.8 resulted in highest secretion of sEDIII-2. This yield could be further enhanced up to 70% by repeated induction of the same initial biomass. Using a fed-batch cultivation strategy, we observed that shake-flask yields can be scaled up ∼8-fold in a bioreactor. We obtained ∼94% purity with >70% recovery after purification. This study, which demonstrates for the first time the feasibility of secreting envelope domain III using the P. pastoris host, will be relevant to sub-unit approaches to dengue vaccine development.

Expression, purification and characterization of in vivo biotinylated dengue virus envelope domain III based tetravalent antigen.

Dengue is a rapidly spreading mosquito-borne viral disease prevalent in over a hundred countries around the world. A definitive identification of dengue infection depends on reliable dengue diagnostic tests. This study describes the design, expression and purification of an in vivo biotinylated chimeric dengue antigen to exploit the high affinity of biotin-streptavidin interaction to detect anti-dengue antibodies. This chimeric antigen incorporates the envelope domain III (EDIII) of the four dengue virus serotypes. A biotin acceptor peptide was fused with the chimeric dengue antigen for in vivo biotinylation in Escherichia coli through simultaneous co-expression of the biotin ligase, BirA. Despite the localization of the chimeric dengue antigen to the insoluble fraction of induced E. coli cells, it was found to be biotinylated in vivo. It was purified to near homogeneity using affinity chromatography with final yields of 20mg protein of approximately 95% purity, from 1L of induced E. coli shake flask culture, and the efficiency of biotinylation was estimated to be approximately 85%. Mouse antibodies specific to recombinant EDIII of each of the four dengue serotypes, captured on microtiter wells sensitized with anti-mouse immunoglobulin antibodies, were recognized specifically and with high efficiency by the biotinylated antigen in conjunction with streptavidin-enzyme conjugate. An evaluation of the biotinylated antigen against a panel of pre-characterized dengue-positive and dengue-negative human sera (n=164), in an antibody capture ELISA format, showed that it manifested 100% specificity, but also suggested that additional epitopes may need to be included in its design to enhance sensitivity.

Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen.

BACKGROUND: Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. RESULTS: Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. CONCLUSION: In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.