Photocage-Selective Capture and Light-Controlled Release of Target Proteins.

Photochemical transformations enable exquisite spatiotemporal control over biochemical processes; however, methods for reliable manipulations of biomolecules tagged with biocompatible photo-sensitive reporters are lacking. Here we created a high-affinity binder specific to a photolytically removable caging group. We utilized chemical modification or genetically encoded incorporation of noncanonical amino acids to produce proteins with photocaged cysteine or selenocysteine residues, which were used for raising a high-affinity monoclonal antibody against a small photoremovable tag, 4,5-dimethoxy-2-nitrobenzyl (DMNB) group. Employing the produced photocage-selective binder, we demonstrate selective detection and immunoprecipitation of a variety of DMNB-caged target proteins in complex biological mixtures. This combined orthogonal strategy permits photocage-selective capture and light-controlled traceless release of target proteins for a myriad of applications in nanoscale assays.

Synthesis of human parainfluenza virus 4 nucleocapsid-like particles in yeast and their use for detection of virus-specific antibodies in human serum.

The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system, to explore its structural and antigenic properties and to evaluate its applicability in serology. The use of an optimized gene encoding HPIV4 N protein amino acid (aa) sequence GenBank AGU90031.1 allowed high yield of recombinant N protein forming nucleocapsid-like particles (NLPs) in yeast. A substitution L332D disrupted self-assembly of NLPs, confirming the role of this position in the N proteins of Paramyxovirinae. Three monoclonal antibodies (MAbs) were generated against the NLP-forming HPIV4 N protein. They recognised HPIV4-infected cells, demonstrating the antigenic similarity between the recombinant and virus-derived N proteins. HPIV4 N protein was used as a coating antigen in an indirect IgG ELISA with serum specimens of 154 patients with respiratory tract infection. The same serum specimens were tested with previously generated N protein of a closely related HPIV2, another representative of genus Rubulavirus. Competitive ELISA was developed using related yeast-produced viral antigens to deplete the cross-reactive serum antibodies. In the ELISA either without or with competition using heterologous HPIV (2 or 4) N or mumps virus N proteins, the seroprevalence of HPIV4 N-specific IgG was, respectively, 46.8, 39.6 and 40.3% and the seroprevalence of HPIV2 N-specific IgG-47.4, 39.0 and 37.7%. In conclusion, yeast-produced HPIV4 N protein shares structural and antigenic properties of the native virus nucleocapsids. Yeast-produced HPIV4 and HPIV2 NLPs are prospective tools in serology.

Yeast-generated virus-like particles as antigens for detection of human bocavirus 1-4 specific antibodies in human serum.

Human bocaviruses (HBoV) are non-enveloped, single-stranded DNA viruses, classified into the genus Bocavirus in the family Parvoviridae. Self-assembled virus-like particles (VLPs) composed of the major capsid protein VP2 of HBoV1-4 and mosaic VLPs composed of both VP2 and VP1 capsid proteins of HBoV1 were generated in yeast Saccharomyces cerevisiae and used to detect HBoV-specific IgG in human serum. Recombinant HBoV VLPs were similar to native HBoV particles in size and morphology. The prevalence of HBoV infection in a group of Lithuanian patients with clinical symptoms of respiratory tract infection was studied using purified yeast-generated VLPs as antigens in a competitive enzyme immunoassay (EIA). After depletion of cross-reactive antibodies, the seroprevalence of HBoV1 was 44.2 % and the seroprevalence of HBoV2-4 was 35.7 %. Mosaic VLPs consisting of HBoV1 VP1 and VP2 proteins showed a stronger reactivity with HBoV1 IgG-positive human serum specimens, and two equivocal serum specimens were reinterpreted as positive. Thus, mosaic VLPs offer a more sensitive tool for HBoV1 serology than currently available serodiagnostics tests based on VP2 VLPs. In conclusion, yeast S. cerevisiae represents an efficient expression system for generating recombinant HBoV1-4 VLPs of diagnostic relevance.

Synthesis of human parainfluenza virus 2 nucleocapsid protein in yeast as nucleocapsid-like particles and investigation of its antigenic structure.

The aim of this study was to investigate the suitability of yeast Saccharomyces cerevisiae expression system for the production of human parainfluenza virus type 2 (HPIV2) nucleocapsid (N) protein in the form of nucleocapsid-like particles (NLPs) and to characterize its antigenic structure. The gene encoding HPIV2 N amino acid (aa) sequence RefSeq NP_598401.1 was cloned into the galactose-inducible S. cerevisiae expression vector and its high-level expression was achieved. However, this recombinant HPIV2 N protein did not form NLPs. The PCR mutagenesis was carried out to change the encoded aa residues to the ones conserved across HPIV2 isolates. Synthesis of the modified proteins in yeast demonstrated that the single aa substitution NP_598401.1:p.D331V was sufficient for the self-assembly of NLPs. The significance of certain aa residues in this position was confirmed by analysing HPIV2 N protein structure models. To characterize the antigenic structure of NLP-forming HPIV2 N protein, a panel of monoclonal antibodies (MAbs) was generated. The majority of the MAbs raised against the recombinant NLPs recognized HPIV2-infected cells suggesting the antigenic similarity between the recombinant and virus-derived HPIV2 N protein. Fine epitope mapping revealed the C-terminal part (aa 386-504) as the main antigenic region of the HPIV2 N protein. In conclusion, the current study provides new data on the impact of HPIV2 N protein sequence variants on the NLP self-assembly and demonstrates an efficient production of recombinant HPIV2 N protein in the form of NLPs.

Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes.

Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes.

Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies.

BACKGROUND: Porcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production. RESULTS: In this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test. CONCLUSIONS: We have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2-suspected samples.

Generation of recombinant porcine parvovirus virus-like particles in Saccharomyces cerevisiae and development of virus-specific monoclonal antibodies.

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.

Development and characterization of new monoclonal antibodies against human recombinant CA XII.

Carbonic anhydrases (CAs) are enzymes that catalyse the reversible hydration of CO2 to bicarbonate. CA XII is considered a potential biomarker of tumor cells and a promising target for specific therapies. The aim of the current study was to develop new monoclonal antibodies (MAbs) against human recombinant CA XII and evaluate their diagnostic potential. An extracellular catalytic domain of human CA XII was expressed in E. coli and used as an immunogen. Seven stable hybridoma cell lines producing high-affinity IgG antibodies against human CA XII were generated. The majority of MAbs were highly specific to CA XII and did not cross-react with human recombinant CA I, CA II, CA VII, and CA XIII. In order to demonstrate the diagnostic value of the MAbs, they were employed for the immunohistochemistry analysis of CA XII expression in tissues. Two MAbs (15A4 and 4A6) demonstrated a strong and specific immunostaining of CA XII in human tissue specimens. Flow cytometry analysis of 5 human tumor cell lines with the MAb 15A4 revealed its immunoreactivity with cellular CA XII. In conclusion, the MAbs raised against recombinant catalytic domain of CA XII recognize cellular CA XII and represent a promising diagnostic tool for the immunodetection of CA XII-expressing cells.

Immunogenic properties of amyloid beta oligomers.

BACKGROUND: The central molecule in the pathogenesis of Alzheimer's disease (AD) is believed to be a small-sized polypeptide - beta amyloid (Aß) which has an ability to assemble spontaneously into oligomers. Various studies concerning therapeutic and prophylactic approaches for AD are based on the immunotherapy using antibodies against Aß. It has been suggested that either active immunization with Aß or passive immunization with anti-Aß antibodies might help to prevent or reduce the symptoms of the disease. However, knowledge on the mechanisms of Aß-induced immune response is rather limited. Previous research on Aß1-42 oligomers in rat brain cultures showed that the neurotoxicity of these oligomers considerably depends on their size. In the current study, we evaluated the dependence of immunogenicity of Aß1-42 oligomers on the size of oligomeric particles and identified the immunodominant epitopes of the oligomers. RESULTS: Mice were immunized with various Aß1-42 oligomers. The analysis of serum antibodies revealed that small Aß1-42 oligomers (1-2 nm in size) are highly immunogenic. They induced predominantly IgG2b and IgG2a responses. In contrast, larger Aß1-42 oligomers and monomers induced weaker IgG response in immunized mice. The monoclonal antibody against 1-2 nm Aß1-42 oligomers was generated and used for antigenic characterization of Aß1-42 oligomers. Epitope mapping of both monoclonal and polyclonal antibodies demonstrated that the main immunodominant region of the 1-2 nm Aß1-42 oligomers is located at the amino-terminus (N-terminus) of the peptide, between amino acids 1 and 19. CONCLUSIONS: Small Aß1-42 oligomers of size 1-2 nm induce the strongest immune response in mice. The N-terminus of Aß1-42 oligomers represents an immunodominant region which indicates its surface localization and accessibility to the B cells. The results of the current study may be important for further development of Aß-based vaccination and immunotherapy strategies.

The use of recombinant pseudotype virus-like particles harbouring inserted target antigen to generate antibodies against cellular marker p16INK4A.

Protein engineering provides an opportunity to generate new immunogens with desired features. Previously, we have demonstrated that hamster polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) are highly immunogenic and can be employed for the insertion of foreign epitopes at certain surface-exposed positions. In the current study, we have designed pseudotype VLPs consisting of an intact VP1 protein and VP2 protein fused with the target antigen--cellular marker p16(INK4A)--at its N terminus. Both proteins coexpressed in yeast were self-assembled to pseudotype VLPs harbouring the inserted antigen on the surface. The pseudotype VLPs were used for generation of antibodies against p16(INK4A) that represents a potential biomarker for cells transformed by high-risk human papillomavirus (HPV). The pseudotype VLPs induced in immunized mice a strong immune response against the target antigen. The antisera raised against pseudotype VLPs showed specific immunostaining of p16(INK4A) protein in malignant cervical tissue. Spleen cells of the immunized mice were used to generate monoclonal antibodies against p16(INK4A) protein. The specificity of antibodies was proven by the immunostaining of HPV-transformed cells. In conclusion, the current study demonstrates the potential of pseudotype VLPs with inserted target antigen as a new type of immunogens to generate antibodies of high diagnostic value.

Genetic and biochemical diversity of Gardnerella vaginalis strains isolated from women with bacterial vaginosis.

Gardnerella vaginalis is considered a substantial player in the progression of bacterial vaginosis (BV). We analysed 17 G. vaginalis strains isolated from the genital tract of women diagnosed with BV to establish a potential link between genotypes/biotypes and the expression of virulence factors, vaginolysin (VLY) and sialidase, which are assumed to play a substantial role in the pathogenesis of BV. Amplified ribosomal DNA restriction analysis revealed two G. vaginalis genotypes. Gardnerella vaginalis isolates of genotype 2 appeared more complex than genotype 1 and were subdivided into three subtypes. Biochemical typing allowed us to distinguish four different biotypes. A great diversity of the level of VLY production among the isolates of G. vaginalis may be related to a different cytotoxicity level of the strains. We did not find any correlation between VLY production level and G. vaginalis genotype/biotype. In contrast, a link between G. vaginalis genotype and sialidase production was established. Our findings on the diversity of VLY expression level in different clinical isolates and linking sialidase activity with the genotype of G. vaginalis could help to evaluate the pathogenic potential of different G. vaginalis strains.

Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis.

BACKGROUND: Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance. RESULTS: Single-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G4S)4 were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells. CONCLUSIONS: Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis.

Generation of recombinant metapneumovirus nucleocapsid protein as nucleocapsid-like particles and development of virus-specific monoclonal antibodies.

Human metapneumovirus (hMPV) is a member of the Pneumovirinea subfamily within the Paramyxoviridea family. Since its discovery in 2001, hMPV has been isolated in several continents, which suggests its prevalence worldwide. hMPV resembles human respiratory syncytial virus with regard to disease symptoms and its ability to infect and cause disease in young infants as well as individuals of all ages. The aim of the current study was to construct an efficient high-level yeast expression system for the generation of hMPV nucleocapsid (N) protein and to develop monoclonal antibodies (MAbs) suitable for hMPV detection. The genome of hMPV was isolated from oral fluid of an infected patient by using specific primers and reverse transcriptase polymerase chain reaction (RT-PCR). DNA sequence corresponding to the N protein gene was inserted into yeast expression vector under inducible GAL7 promoter. SDS-PAGE analysis of crude lysates of yeast Saccharomyces cerevisiae harbouring recombinant plasmid revealed the presence of a protein band of approximately 43 kDa corresponding to the molecular weight of hMPV N protein. Electron microscopy analysis of purified N protein revealed nucleocapsid-like structures with typical herring-bone morphology: rods of 20 nm diameter with repeated serration along the edges and central core of 5 nm. Recombinant hMPV N protein was reactive with human serum specimens collected from patients with confirmed hMPV infection. After immunization of mice with recombinant hMPV N protein, a panel of MAbs was generated. The specificity of newly generated MAbs was proven by immunofluorescence analysis of hMPV-infected cells. Epitope mapping using truncated variants of hMPV N revealed localization of linear MAb epitopes at the N-terminus of hMPV N protein, between amino acid residues 1 and 90. The MAbs directed against conformational epitopes did not recognize hMPV N protein variants containing either N- or C-terminal truncations. The reactivity of recombinant hMPV N protein with hMPV-positive serum specimens and the ability of MAbs to recognize virus-infected cells confirms the antigenic similarity between yeast-expressed hMPV N protein and native viral nucleocapsids. In conclusion, recombinant hMPV N protein and hMPV-specific MAbs provide new diagnostic reagents for hMPV infection.

Production and characterization of monoclonal antibodies against vaginolysin: mapping of a region critical for its cytolytic activity.

Vaginolysin (VLY) is a protein toxin released by Gardnerella vaginalis. VLY belongs to the group of cholesterol-dependent cytolysins (CDCs). We have generated a panel of novel monoclonal antibodies (MAbs) against VLY. For the generation of MAbs, we have used recombinant VLY expressed in Escherichia coli. The functional activity of recombinant VLY was confirmed by an in vitro hemolytic assay using human erythrocytes. The MAbs raised against recombinant VLY were reactive with VLY from G. vaginalis both by Western blot and ELISA. The cross-reactivity of MAbs with other CDCs was investigated. For this purpose, recombinant cytolysins perfringolysin, listeriolysin, intermedilysin, pneumolysin and streptolysin were expressed in E. coli. The MAbs were specific exclusively to VLY and did not react with other CDCs. All MAbs were studied for the ability to neutralize hemolytic activity of VLY in vitro and several neutralizing MAbs were identified. The MAb produced by clone 9B4 showed the most potent neutralizing activity. The epitope for this MAb was localized near the N-terminus of VLY, between amino acid (aa) residues 112 and 268. The region recognized by the neutralizing MAb 9B4 includes the conserved motif (VAARMQYD, aa 189-196) supposed to be involved in VLY oligomerization. Selected MAbs were employed to develop a sandwich ELISA for VLY quantification. The MAb-based immunoassay was suitable for the detection of VLY in the cultures of G. vaginalis. In conclusion, the MAbs described in the current study may be useful for structural and functional studies of VLY as well as immunodetection of VLY in biological specimens.

Novel monoclonal antibodies against Menangle virus nucleocapsid protein.

Menangle virus (MenV) is a member of the family Paramyxoviridae isolated in Australia that causes a reproductive disease of pigs. There is a need for specific immunoassays for virus detection to facilitate the diagnosis of MenV infection. Three novel monoclonal antibodies (MAbs) of the IgG1 subtype were generated by immunizing mice with recombinant yeast-expressed MenV nucleocapsid (N) protein self-assembled to nucleocapsid-like structures. One MAb was cross-reactive with recombinant N protein of Tioman virus. The epitopes of MAbs were mapped using a series of truncated MenV N proteins lacking the 29-119 carboxy-terminal amino acid (aa) residues. The epitopes of two MAbs were mapped to aa 430-460 of the MenV N protein, whilst the epitope of one MAb was mapped to residues 460-490. All three MAbs specifically recognized MenV, as indicated by immunohistochemical staining of brain tissue isolated from a field case (a stillborn piglet) of MenV infection. The MAbs against MenV N protein may be a useful tool for immunohistological diagnosis of MenV infection.