Recombinant Glycoprotein Research Articles

Development of conditional cell lysis mutants of Saccharomyces cerevisiae as production hosts by modulating OCH1 and CHS3 expression.

The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants PMET3-OCH1 and PMET3-CHS3 and the double mutant PMET3-OCH1/PMET3-CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera β-glucosidase, with markedly reduced hypermannosylation, particularly in the PMET3-OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.

Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells

Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half‐life. In this study, a variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed‐batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high‐mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2‐F‐peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.

Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection

BackgroundPichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein.ResultsThe recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the α-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to ~ 46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O-mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb.ConclusionsrPstS-1 (98.9% sequence identity, O-mannosylated, and without tags) was produced and secreted by P. pastoris, demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.

Development of an antigen ELISA using monoclonal antibodies against recombinant VSG for the detection of active infections of Trypanosoma evansi in animals

Trypanosoma evansi, a haemo-flagellated protozoan parasite causes chronic wasting disease in a wide range of animals. For its diagnosis, blood smear examination is useful in clinical cases for direct identification of the parasite but in latent infection the carrier animals are difficult to screen out by conventional blood smear test. Harboring low level of parasites and showing no symptom, the carrier animals for surra can act as a source of infection. The level of parasitaemia fluctuates, especially during latent infection; moreover the antibodies which are not found early in the infection may persist even after recovery or chemotherapy. In the present study a double antibody sandwich ELISA exploring, monoclonal antibodies and hyperimmune serum, raised against recombinant variable surface glycoprotein has been developed to detect circulating trypanosome antigens. The developed antigen detection ELISA (Ag-ELISA) was evaluated using 652 blood samples collected from cattle, buffalo, equine and camel. The statistical analysis of the data showed diagnostic sensitivity and specificity at 97.4% and 96.4% respectively, with a positive-negative cut-off OD value >0.28. Furthermore, the detection limit of the assay was found to 7.15 trypanosomes per mL. The present finding revealed that the developed assay can be exploited as a potential diagnostic test in the detection of circulating trypanosome antigens and also can be used as a population screening test for multiple animal species for detection of active infection for further treatment and control of the disease.

Continuous translation of circularized mRNA improves recombinant protein titer

Recent success in demonstrating the translation of circular RNA open reading frames or circular mRNA, may offer a new avenue for improving recombinant protein production from cell and cell-free expression platforms. Initiation and termination are two rate limiting steps of translation. Circular RNA as a class of RNA is defined by covalent joining of terminal ends to give a closed loop structure. By encoding a gene lacking a stop codon on a circular RNA molecule an infinite open reading frame is generated permitting continuously translating circular mRNA (CTC mRNA). CTC mRNAs have shown promise in enhancing the production of multimeric polyproteins in bacterial cell-free expression systems. Problems arise when homogenous, functional post-translationally modified protein is required. To produce post-translationally modified, secreted protein from an CTC mRNA we investigated co-translational cleavage of nascent polypeptide chains by incorporating a 2 A “self-cleavage” peptide motif. Using a model recombinant human glycoprotein Erythropoietin (EPO) we demonstrate for the first time the ability to produce secreted protein from an infinite circular mRNA in live mammalian cells. Both cell-specific and volumetric productivity were improved by using circular mRNAs. This study pioneers the potential of recombinant protein production from CTC mRNA in mammalian cells.

Cloning, Expression and Purification of Recombinant Forms of Full Length and Extracellular Domain EBOV Glycoprotein within Mammalian Cell-Lines

Background: Full length Ebolavirus glycoprotein (GP) intersperses the outer most lipid membrane to form spikes, where it mediates virus-host cell interaction. A secretory form of GP (sGP) is also produced by all 5 known Ebolavirus species. These attributes make GP an ideal target for research and development (R and D) of Ebolavirus and possibly pan-filovirus targeted Rapid Diagnostic Tests (RDTs), bio-therapeutics and vaccines. Prior cloning of recombinant Zaire ebolavirus (EBOV) GP has majorly used insect (baculovirus ) expression systems. We report the cloning, expression and purification of the full length and extracellular domain (ECD) forms of recombinant EBOV GP in mammalian cell-lines. Methods and results: 2034 and 1956 base-pair (bp) coding DNA sequences corresponding to the 669 and 643 amino acids (aa) residues of full length and ECD forms of EBOV GP were sub-cloned into the pTGE plasmids. Recombinant pTGE-plasmids were used to transfect 293-6E HEK mammalian cells grown in serum-free FreeStyleTM 293 Expression Medium. Cell lysates and or culture supernatants were used to obtain purified protein, followed by analysis on SDS-PAGE and Western blot. Purified full length GP was detected as membrane bound protein in cell lysates with estimated molecular weight of ~100 kDa (Cal.M.W.~71.67 kDa) on Western blot; and 0.02 mg GP (Concentration: 0.2 mg/mL, Purity: ~50%) derived. On the contrary, ECD GP was detected in supernatants of cell culture broth with estimated molecular weights of ~116 kDa based on SDS-PAGE and Western blot; and 1.6 mg (Concentration: 0.4 mg/ml, Purity: ~70%) of GP_ECD was obtained. Conclusion: Within mammalian cells, recombinant full length EBOV GP is predominantly expressed as transmembrane protein (tGP), while ECD GP is eluted into the culture medium. Both recombinant forms of GP are critical for the R and D of rapid diagnostic tests (RDTs).

Purification of Recombinant Glycoproteins from Pichia pastoris Culture Supernatants.

Pichia pastoris is a common host organism for the production of recombinant proteins. While unglycosylated recombinant proteins derived from this yeast can be purified efficiently by only a few conventional chromatography steps, the purification of glycosylated recombinant proteins is a very challenging process due to the intrinsic feature of the yeast of hypermannosylation. The resulting vast glycosylation pattern on the recombinant target protein masks its physicochemical properties hampering a conventional downstream process. Here, we describe a fast and efficient two-step chromatography strategy, where both steps are operated in flow-through mode, to purify recombinant glycoproteins from P. pastoris culture supernatants.

Towards Enhancing Therapeutic Glycoprotein Bioproduction: Interventions in the PI3K/AKT/mTOR Pathway.

Recombinant glycoproteins produced in mammalian cells are clinically indispensable drugs used to treat a broad spectrum of diseases. Their bio-manufacturing process is laborious, time consuming, and expensive. Investment in expediting the process and reducing its cost is the subject of continued research. The PI3K/Akt/mTOR signaling pathway is a key regulator of diverse physiological functions such as proliferation, global protein, and lipid synthesis as well as many metabolic pathways interacting to increase secretory capabilities. In this review we detail various strategies previously employed to increase glycoprotein production yields via either genetic or pharmacological over-activation of the PI3K/Akt/mTOR pathway, and we discuss their potential and limitations.Key words: mTORC1, CRISPR, specific productivity, translation.

Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites.

BackgroundN-linked glycans present in venoms, pollen and mites are recognized by IgE antibodies from >20% of allergic patients but have low or no allergenic activity.ObjectivesTo engineer recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from venoms, pollen and mites which can discriminate carbohydrate-specific IgE from allergenic, peptide-specific IgE.MethodsOne or two N-glycosylation sites were engineered into the N-terminus of the non-allergenic protein horse heart myoglobin (HHM) using synthetic gene technology. HHM 1 and HHM 2 containing one or two N-glycosylation sites were expressed in baculovirus-infected High-Five™ insect cells and a non-glycosylated version (HHM 0) was obtained by mutating the glycosylation motif. Recombinant HHM proteins were analyzed regarding fold and aggregation by circular dichroism and gel filtration, respectively. IgE reactivity was assessed by ELISA, immunoblotting and quantitative ImmunoCAP measurements. IgE inhibition assays were performed to study cross-reactivity with venom, plant and mite-derived carbohydrate IgE epitopes.ResultsHHM-glycovariants were expressed and purified from insect cells as monomeric and folded proteins. The HHM-glycovariants exhibited strictly carbohydrate-specific IgE reactivity, designed to quantify carbohydrate-specific IgE and resembled IgE epitopes of pollen, venom and mite-derived carbohydrates. IgE-reactivity and inhibition experiments established a hierarchy of plant glcyoallergens (nPhl p 4 > nCyn d 1 > nPla a 2 > nJug r 2 > nCup a 1 > nCry j 1) indicating a hitherto unknown heterogeneity of carbohydrate IgE epitopes in plants which were completely represented by HHM 2.ConclusionDefined recombinant HHM-glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites were engineered which made it possible to discriminate carbohydrate- from peptide-specific IgE reactivity.

Strategies for humanizing glycosylation pathways and producing recombinant glycoproteins in microbial expression systems.

Homogeneously glycosylated proteins are essential for analyzing the structure of N-glycans, studying their consequences inside cells, and developing therapeutic glycoproteins. However, the isolation of glycoproteins with homogeneous glycans from human is difficult since glycoforms slightly differ from each other with respect to molecular weight and charge. Microbial expression systems have numerous benefits in expression technology and have gained great attention, because they are more adaptable to the biotechnology industry. While selecting an expression host, the glycosylation pattern must be taken into account, because glycosylation strongly depends on cellular production system and selected production clone. This review discussed the technological developments in glycoengineering of microbial expression systems for humanizing the glycosylation profile and highlighted the expression potential of Leishmania expression system. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2752, 2019.

Targeted Diet Modification Reduces Multiple Sclerosis-like Disease in Adult Marmoset Monkeys from an Outbred Colony.

Experimental autoimmune encephalomyelitis (EAE) in common marmosets is a translationally relevant model of the chronic neurologic disease multiple sclerosis. Following the introduction of a new dietary supplement in our purpose-bred marmoset colony, the percentage of marmosets in which clinically evident EAE could be induced by sensitization against recombinant human myelin oligodendrocyte glycoprotein in IFA decreased from 100 to 65%. The reduced EAE susceptibility after the dietary change coincided with reduced Callitrichine herpesvirus 3 expression in the colony, an EBV-related γ1-herpesvirus associated with EAE. We then investigated, in a controlled study in marmoset twins, which disease-relevant parameters were affected by the dietary change. The selected twins had been raised on the new diet for at least 12 mo prior to the study. In twin siblings reverted to the original diet 8 wk prior to EAE induction, 100% disease prevalence (eight out of eight) was restored, whereas in siblings remaining on the new diet the EAE prevalence was 75% (six out of eight). Spinal cord demyelination, a classical hallmark of the disease, was significantly lower in new-diet monkeys than in monkeys reverted to the original diet. In new-diet monkeys, the proinflammatory T cell response to recombinant human myelin oligodendrocyte glycoprotein was significantly reduced, and RNA-sequencing revealed reduced apoptosis and enhanced myelination in the brain. Systematic typing of the marmoset gut microbiota using 16S rRNA sequencing demonstrated a unique, Bifidobacteria-dominated composition, which changed after disease induction. In conclusion, targeted dietary intervention exerts positive effects on EAE-related parameters in multiple compartments of the marmoset's gut-immune-CNS axis.

Bayesian assessment of two competitive enzyme-linked immunosorbent assays for the detection of bovine viral diarrhoea virus antibodies in bovine sera.

Infections due to bovine viral diarrhoea virus (BVDV) are endemic in most cattleproducing countries throughout the world and bovine viral diarrhoea is considered a transboundary disease. The key elements of a BVDV control programme are vaccination, biosecurity, elimination of persistently infected (PI) animals and surveillance. The aim of this study was to assess the sensitivity (Se) and the specificity (Sp) of two commercial competitive enzyme-linked immunosorbent assays (ELISAs) based on selected immune-dominant BVDV proteins: the non-structural protein NS3 (p80) and the recombinant envelope glycoprotein E0(Erns). Both tests were used on individual serum samples from randomly sampled young bovines in southern Belgium in order to detect specific BVDV antibodies. The Se and Sp were assessed using a Bayesian approach and were estimated, respectively, at 97.2% (with 95% credibility interval [Cr I]: 95.1-99.8) and 98.7%(95% Cr I: 96.6-99.9) for the first test and 95.8% (95% Cr I: 91.1-99.7) and 96.1%(95% Cr I: 95.1-97.7) for the second test. The results obtained with the two tests were not significantly different. In addition, using both ELISAs, the current BVDV exposure among young bovines in southern Belgium was estimated at 23.3% (95%Cr I: 20.6-26.2). Combining virological testing of all newborns to detect PI animals with regular serological testing of young stock using ELISAs is recommended in the surveillance of BVDV.

Bayesian assessment of two competitive enzyme-linked immunosorbent assays for the detection of bovine viral diarrhoea virus antibodies in bovine sera

Infections due to bovine viral diarrhoea virus (BVDV) are endemic in most cattleproducing countries throughout the world and bovine viral diarrhoea is considered a transboundary disease. The key elements of a BVDV control programme are vaccination, biosecurity, elimination of persistently infected (PI) animals and surveillance. The aim of this study was to assess the sensitivity (Se) and the specificity (Sp) of two commercial competitive enzyme-linked immunosorbent assays (ELISAs) based on selected immune-dominant BVDV proteins: the non-structural protein NS3 (p80) and the recombinant envelope glycoprotein E0(Erns). Both tests were used on individual serum samples from randomly sampled young bovines in southern Belgium in order to detect specific BVDV antibodies. The Se and Sp were assessed using a Bayesian approach and were estimated, respectively, at 97.2% (with 95% credibility interval [Cr I]: 95.1–99.8) and 98.7%(95% Cr I: 96.6–99.9) for the first test and 95.8% (95% Cr I: 91.1–99.7) and 96.1%(95% Cr I: 95.1–97.7) for the second test. The results obtained with the two tests were not significantly different. In addition, using both ELISAs, the current BVDV exposure among young bovines in southern Belgium was estimated at 23.3% (95%Cr I: 20.6–26.2). Combining virological testing of all newborns to detect PI animals with regular serological testing of young stock using ELISAs is recommended in the surveillance of BVDV.

Improved yield of rhEPO in CHO cells with synthetic 5' UTR.

The impact of local structure on mRNA translation is not well-defined pertaining to the 5' UTR. Reports suggest structural remodelling of the 5' UTR can significantly influence mRNA translation both in cis and trans however a new layer of complexity has been applied to this model with the now known reversible post-transcriptional chemical modification of RNA. N6-methyladenosine (m6A) is the most abundant internal base modification in mammalian mRNA. It has been reported that mRNAs harbouring m6A motifs in their 5' UTR have improved translation efficiency. The present study evaluated the addition of putative m6A motifs to the 5' UTR of a model recombinant human therapeutic glycoprotein, Erythropoietin (EPO), in a direct comparison with an A to T mutant and a no adenosine control. The m6A construct yielded significantly improved EPO titer in transient batch culture over no adenosine and m6T controls by 2.84 and 2.61-fold respectively. This study highlights that refinement of transgene RNA elements can yield significant improvements to protein titer.

Neonatal alloimmune thrombocytopenia due to a new alloantigen Bl(a) defined by an Asp458Gly substitution in GPIIIa.

Neonatal alloimmune thrombocytopenia (NAIT) commonly arises due to antibodies against a small number of well-defined human platelet antigens (HPAs). A minority of NAIT cases occur due to maternal immunization against low-frequency polymorphisms in platelet glycoprotein that result in new immunogenic epitopes. Antibodies to these novel epitopes can be detected by the incubation of maternal serum with paternal platelets and is usually performed after initial investigation using HPA-typed panel platelets has failed to provide evidence of NAIT. The propositus and the parents from a case of suspected neonatal alloimmune thrombocytopenia (NAIT) were investigated using serologic and molecular techniques to detect and identify relevant platelet-specific antibodies and for HPA typing. Calculations of molecular dynamics were undertaken to explore potential variations in the molecular structure. Maternal antibodies were detected that were reactive only in crossmatch with paternal platelets using the platelet immunofluorescence test (PIFT) and a GPIIb/IIIa monoclonal antibody immobilization of platelet antigen (MAIPA) assay. In the propositus and father, a novel mutation c.1373 A > G was found in exon 10 of ITGB3 resulting in the substitution of an aspartic acid for a glycine (p.Asp458Gly). Recombinant GPIIIa glycoprotein mutated to contain the novel mutation and expressed in HEK293 cells with GPIIb was also specifically recognized by maternal antibodies. Calculations of molecular dynamics identified that the mutation was in a structurally constrained site. This case describes a low-frequency platelet antigen (Asp458Gly) that defines a further alloantigenic target in NAIT. The case emphasizes the role of the platelet crossmatch as the single most useful tool to establish evidence of immunization of low-frequency platelet glycoprotein polymorphisms. A crossmatch should always be performed where there is strong clinical evidence of NAIT but initial laboratory investigations are not confirmatory.

The metabolic switch can be activated in a recombinant strain of Streptomyces lividans by a low oxygen transfer rate in shake flasks

BackgroundIn Streptomyces, understanding the switch from primary to secondary metabolism is important for maximizing the production of secondary metabolites such as antibiotics, as well as for optimizing recombinant glycoprotein production. Differences in Streptomyces lividans bacterial aggregation as well as recombinant glycoprotein production and O-mannosylation have been reported due to modifications in the shake flask design. We hypothetized that such differences are related to the metabolic switch that occurs under oxygen-limiting conditions in the cultures.ResultsShake flask design was found to affect undecylprodigiosin (RED, a marker of secondary metabolism) production; the RED yield was 12 and 385 times greater in conventional normal Erlenmeyer flasks (NF) than in baffled flasks (BF) and coiled flasks (CF), respectively. In addition, oxygen transfer rates (OTR) and carbon dioxide transfer rates were almost 15 times greater in cultures in CF and BF as compared with those in NF. Based on these data, we obtained respiration quotients (RQ) consistent with aerobic metabolism for CF and BF, but an RQ suggestive of anaerobic metabolism for NF.ConclusionAlthough the metabolic switch is usually related to limitations in phosphate and nitrogen in Streptomyces sp., our results reveal that it can also be activated by low OTR, dramatically affecting recombinant glycoprotein production and O-mannosylation and increasing RED synthesis in the process.

Vaccine nanoparticles displaying recombinant Ebola virus glycoprotein for induction of potent antibody and polyfunctional T cell responses

The recent outbreaks of Ebolavirus (EBOV) in West Africa underscore the urgent need to develop an effective EBOV vaccine. Here, we report the development of synthetic nanoparticles as a safe and highly immunogenic platform for vaccination against EBOV. We show that a large recombinant EBOV antigen (rGP) can be incorporated in a configurational manner into lipid-based nanoparticles, termed interbilayer-crosslinked multilamellar vesicles (ICMVs). The epitopes and quaternary structure of rGP were properly maintained on the surfaces of ICMVs formed either with or without nickel nitrilotriacetic acid (NTA)-functionalized lipids. When administered in mice, rGP-ICMVs without NTA-lipids efficiently generated germinal center B cells and polyfunctional T cells while eliciting robust neutralizing antibody responses. This study suggests the potential of vaccine nanoparticles as a delivery platform for configurational, multivalent display of large subunit antigens and induction of neutralizing antibody and T cell responses.

Comprehensive Glycoproteomic Analysis of Chinese Hamster Ovary Cells.

The Chinese hamster ovary (CHO) cell line is a major expression system for the production of therapeutic proteins, the majority of which are glycoproteins, such as antibodies and erythropoietin (EPO). The characterization glycosylation profile of therapeutic proteins produced from engineered CHO cells and therapeutic functions, as well as side effects, are critical to understand the important roles of glycosylation. In this study, a large scale glycoproteomic workflow was established and applied to CHO-K1 cells expressing EPO. The workflow includes enrichment of intact glycopeptides from CHO-K1 cell lysate and medium using hydrophilic enrichment, fractionation of the obtained intact glycopeptides (IGPs) by basic reversed phase liquid chromatography (bRPLC), analyzing the glycopeptides using LC-MS/MS, and annotating the results by GPQuest 2.0. A total of 10 338 N-linked glycosite-containing IGPs were identified, representing 1162 unique glycosites in 530 glycoproteins, including 71 unique atypical N-linked IGPs on 18 atypical N-glycosylation sequons with an overrepresentation of the N-X-C motifs. Moreover, we compared the glycoproteins from CHO cell lysate with those from medium using the in-depth N-linked glycoproteome data. The obtained large scale glycoproteomic data from intact N-linked glycopeptides in this study is complementary to the genomic, proteomic, and N-linked glycomic data previously reported for CHO cells. Our method has the potential to monitor the production of recombinant therapeutic glycoproteins.

Immunogenicity of the Multi-Epitopic Recombinant Glycoproteins of Newcastle Disease Virus: Implications for the Serodiagnosis Applications.

BackgroundNewcastle disease virus (NDV) is a dangerous viral disease, infecting a broad range of birds, and has a fatal effect on the poultry industries. The attachment and consequently fusion of the virus to the host cell membrane is directed by the two superficial glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) which is considered as the important targets for the poultry immune response.ObjectivesThe principal goal of this investigation was to realize the potential efficacy of the E. coli expression system for the production of the multi-epitopic HN, and F proteins with respect to the ability for the stimulation of the immune system and production of the cross-reactive antibodies in mice.Materials and MethodsThe recombinant HN and F (rHN, rF) have accumulated almost 40% of the total bacterial proteins. The presence of rHN and rF proteins recognized by the Western blotting with specific anti-HN, anti-F, anti-Newcastle B1, and anti-poly 6x His-tag antibodies. Furthermore, both rHN and rF have shown the specific reactivity against the Newcastle B1 antiserum as a standard strain.ResultsThe ELISA analysis showed that the higher dilutions of the antibody against Newcastle B1 could react with the as least quantity as 100 ng of the purified rHN, and rF. Cross-reactivity analysis of the sera from the mice immunized with Newcastle B1 in two time points indicated that the raise of anti-Newcastle B1, anti-HN and anti-F antibodies peaked at 28 days post immunization (dpi). Moreover, temporal variation in IgG titration between both time points was significant at 5% probability level.ConclusionThe results provided valuable information about the cross-reactivity patterns and biological activity of the multi-epitopic proteins compared to the NDV standard strain which was determined by the Western blotting and ELISA.

Immunoprotective response induced by recombinant glycoprotein D in the BALB/c respiratory mouse model of Equid alphaherpesvirus 1 infection

Equid alphaherpesvirus 1 (EHV-1) infection causes abortion, respiratory disease, perinatal deaths and neurological disorders in horses. The natural infection and available vaccines provide only partial and short-lived protection against reinfections. In the present study, we analyzed the ability of purified baculovirus-expressed glycoprotein D (gD) administered by different routes to induce protective immunity in BALB/c mice after challenge with the EHV-1 AR8 strain. Clinical signs varied among the different groups of mice immunized by parenteral routes, and, although gD induced a specific serum IgG response, it did not prevent the virus from reaching the lungs. Intranasally immunized mice showed no clinical signs, and virus isolation from lungs, histological lesions and antigen detection by immunohistochemistry were negative. In addition, by this route, gD did not stimulate the production of serum IgG and IgA. However, a specific IgA response in the respiratory tract was confirmed in intranasally immunized mice. Thus, we conclude that the mucosal immune response could reduce the initial viral attachment and prevent the virus from reaching the lungs. Our findings provide additional data to further study new immunization strategies in the natural host.