Chromatographic Purification and Polishing of AAV Particles.

The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids.

Peptide ligands for the affinity purification of adenovirus from HEK293 and vero cell lysates.

Adenovirus (AdVs) is the viral vector of choice in vaccines and oncolytic applications owing to its high transduction activity and inherent immunogenicity. For decades, AdV isolation has relied on ultracentrifugation and ion-exchange chromatography, which are not suitable to large-scale production and struggle to deliver sufficient purity. Immunoaffinity chromatography resins of recent introduction feature high binding capacity and selectivity, but mandate harsh elution conditions (pH 3.0), afford low yield (< 20%), and provide limited reusability. Seeking a more efficient and affordable alternative, this study introduces the first peptide affinity ligands for AdV purification. The peptides were identified via combinatorial selection and in silico design to target hexons, the most abundant proteins in the adenoviral capsid. Selected peptide ligands AEFFIWNA and TNDGPDYSSPLTGSG were conjugated on chromatographic resins and utilized to purify AdV serotype 5 from HEK293 and Vero cell lysates. The peptide-functionalized resins feature high binding capacity (> 1010 active virions per mL at the residence time of 2 min), provide high yield (> 50%) and up to 100-fold reduction of host cell proteins and DNA. Notably, the peptide ligands enable gentle elution conditions (pH 8) that prevent the "shedding" of penton and fiber proteins, thus affording intact adenovirus particles with high cell-transduction activity. The study of the peptide ligands by surface plasmon resonance and molecular docking and dynamics simulations confirmed the selective targeting of hexon proteins and elucidated the molecular-level mechanisms underlying binding and release. Collectively, these results demonstrate the strong promise of peptide ligands presented herein for the affinity purification of AdVs from cell lysates.

Membrane chromatography for AAV full capsid enrichment: Process development to scale up.

The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing "empty" capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model.

Virus-Based Separation of Rare Earth Elements.

The utilization of biomaterials for the separation of rare earth elements (REEs) has attracted considerable interest due to their inherent advantages, including diverse molecular structures for selective binding and the use of eco-friendly materials for sustainable systems. We present a pioneering methodology for developing a safe virus to selectively bind REEs and facilitate their release through pH modulation. We engineered the major coat protein of M13 bacteriophage (phage) to incorporate a lanthanide-binding peptide. The engineered lanthanide-binding phage (LBPh), presenting ∼3300 copies of the peptide, serves as an effective biological template for REE separation. Our findings demonstrate the LBPh's preferential binding for heavy REEs over light REEs. Moreover, the LBPh exhibits remarkable robustness with excellent recyclability and stability across multiple cycles of separations. This study underscores the potential of genetically integrating virus templates with selective binding motifs for REE separation, offering a promising avenue for environmentally friendly and energy-efficient separation processes.

Extraction of Modified Adeno-Associated Virus-Like Particles Displaying Peptides on Their Surface.

Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the surface of the AAV or AAV VLPs has been used to redirect the AAV to different target tissues and for vaccine development. Usually, the VLPs self-assemble intracellularly, and an extraction step must be performed before purification. Here, we describe the method we have used to extract AAV VLPs from insect cells successfully with peptide insertions on their surface.

Molecular dynamics simulation and purification of chimeric L1/L2 protein from human papillomavirus type 52 expressed in Escherichia coli BL21 (DE3).

The available prophylactic vaccines for human papillomavirus (HPV) in the market are only effective against specific types of HPV, rendering them ineffective for other types of HPV infections. The objective of this research is to investigate the stability of the recombinant protein constructed, namely chimeric L1/L2 protein from HPV type 52, with improved cross-neutralization ability. The 3D model, predicted using Alphafold, Robetta, I-Tasser, and refined with Galaxy Refinement, is validated using Ramachandran plot analysis. The stability is verified through molecular dynamics simulations, considering parameters such as RMSD, RMSF, Rg, and SASA, where stable conditions are observed. The chimeric L1/L2 protein from HPV type 52 is purified using affinity chromatography, and the His-tag is cleaved using SUMO protease to obtain pure chimeric protein with the size of ~ 55 kDa. Western blot analysis confirms binding to anti-L1 HPV type 52 polyclonal antibody. The obtained vaccine candidate can be utilized as an effective prophylactic vaccine against HPV.

Mass Spectrometry-Based System for Identifying and Typing Norovirus Major Capsid Protein VP1.

Norovirus-associated diseases are the most common foodborne illnesses worldwide. Polymerase chain reaction-based methods are the primary diagnostics for clinical samples; however, the high mutation rate of norovirus makes viral amplification and genotyping challenging. Technological advances in mass spectrometry (MS) make it a promising tool for identifying disease markers. Besides, the superior sensitivity of MS and proteomic approaches may enable the detection of all variants. Thus, this study aimed to establish an MS-based system for identifying and typing norovirus. We constructed three plasmids containing the major capsid protein VP1 of the norovirus GII.4 2006b, 2006a, and 2009a strains to produce virus-like particles for use as standards. Digested peptide signals were collected using a nano-flow ultra-performance liquid chromatography mass spectrometry (nano-UPLC/MSE) system, and analyzed by ProteinLynx Global SERVER and TREE-PUZZLE software. Results revealed that the LC/MSE system had an excellent coverage rate: the system detected more than 94% of amino acids of 3.61 femtomole norovirus VP1 structural protein. In the likelihood-mapping analysis, the proportions of unresolved quartets were 2.9% and 4.9% in the VP1 and S domains, respectively, which is superior to the 15.1% unresolved quartets in current PCR-based methodology. In summary, the use of LC/MSE may efficiently monitor genotypes, and sensitively detect structural and functional mutations of noroviruses.

Effect of N­terminal region of human parvovirus B19­VP1 unique region on cardiac injury in naïve mice.

A unique region of human parvovirus B19 virus­VP1 (B19V­VP1u) has been linked to a variety of cardiac disorders. However, the precise role of B19V­VP1u in inducing cardiac injury remains unknown. The present study investigated the effects of B19V­VP1u and different regions of B19V­VP1u, including B19V­VP1uA (residues 1­60), B19V­VP1uB (residues 61­129), B19V­VP1uC (residues 130­195) and B19V­VP1uD (residues 196­227), on inducing cardiac injury in naïve mice by zymography, immunoblotting, H&E staining and cytokine immunoassay. A significantly higher MMP­9/MMP­2 ratio and increased levels of inflammatory cytokines, including IL­6 and IL­1ß, were detected in the left ventricles of the mice injected with B19V­non­structural protein 1 (B19V­NS1) and B19V­VP1u, accompanied by increased expression levels of phosphorylated (p­)ERK and p­P38. Significantly upregulated expression levels of atrial natriuretic peptide (ANP), heart­type fatty acid­binding protein (H­FABP) and creatine kinase isoenzyme­MB (CK­MB), which are well­known cardiac injury markers, as well as increased infiltration of lymphocytes, were detected in the left ventricles of the mice injected with B19V­VP1, B19V­NS1 and B19V­VP1u. Moreover, a significantly higher MMP­9/MMP­2 ratio and increased levels of IL­6 and IL­1ß were observed in the left ventricles of the mice injected with B19V­VP1u, B19V­VP1u­A, B19V­VP1u­B and B19V­VP1u­C, accompanied by upregulated p­ERK and p­P38 expression. Notably, significantly lower levels of IL­6 and IL­1ß were observed in the left ventricles of the mice injected with B19V­VP1uD. Furthermore, significantly increased ANP, H­FABP and CK­MB expression levels were detected in the left ventricles of the mice injected with B19V­VP1u, B19V­VP1u­A and B19V­VP1u­B, along with enhanced infiltration of lymphocytes. Significantly higher serum IL­1ß, IL­6, TNF­α and IFN­Î³ levels were also detected in the mice injected with B19V­VP1u, B19V­VP1u­A and B19V­VP1u­B. To the best of our knowledge, the findings of the present study were the first to demonstrate that the N­terminal region (residues 1­129) of B19V­VP1u induces an increase in the levels of cardiac injury markers, thus providing evidence for understanding the possible functional regions within B19V­VP1u.

A novel one-step amplification refractory mutation system PCR (ARMS-PCR) for differentiation of canine parvovirus-2 variants.

Enteritis caused by CPV-2 antigenic variants (CPV-2a, 2b, and 2c) is frequently reported in dogs worldwide leading to significant morbidity and mortality. Here, we describe about a simple, single-step, ARMS-PCR strategy targeting the mutant 426 amino acid of VP2 to differentiate CPV-2 antigenic types. A total of 150 fecal samples were subjected to ARMS-PCR of which 18 were typed as CPV-2a, 79 were typed as CPV-2b, and 6 were typed as CPV-2c. The ARMS-PCR results were validated by randomly sequencing partial VP2 gene of 14 samples. Phylogenetic analysis of partial VP2 gene sequencing of each of the CPV-2 variants revealed that CPV-2a and CPV-2b isolates formed a separate clade of Indian lineage, while CPV-2c shared common evolutionary origin with Asian lineage. The developed technique is first of its kind, one-step, rapid, sequencing independent method for typing of CPV-2 antigenic variants.

Detection of diarrhoea associated rotavirus and co-infection with diarrhoeagenic pathogens in the Littoral region of Cameroon using ELISA, RT-PCR and Luminex xTAG GPP assays.

BACKGROUND: Despite the global roll-out of rotavirus vaccines (RotaTeq/Rotarix / ROTAVAC/Rotasiil), mortality and morbidity due to group A rotavirus (RVA) remains high in sub-Saharan Africa, causing 104,000 deaths and 600,000 hospitalizations yearly. In Cameroon, Rotarix™ was introduced in March 2014, but, routine laboratory diagnosis of rotavirus infection is not yet a common practice, and vaccine effectiveness studies to determine the impact of vaccine introduction have not been done. Thus, studies examining RVA prevalence post vaccine introduction are needed. The study aim was to determine RVA prevalence in severe diarrhoea cases in Littoral region, Cameroon and investigate the role of other diarrheagenic pathogens in RVA-positive cases. METHODS: We carried out a study among hospitalized children < 5 years of age, presenting with acute gastroenteritis in selected hospitals of the Littoral region of Cameroon, from May 2015 to April 2016. Diarrheic stool samples and socio-demographic data including immunization and breastfeeding status were collected from these participating children. Samples were screened by ELISA (ProSpecT™ Rotavirus) for detection of RVA antigen and by gel-based RT-PCR for detection of the VP6 gene. Co-infection was assessed by multiplexed molecular detection of diarrheal pathogens using the Luminex xTAG GPP assay. RESULTS: The ELISA assay detected RVA antigen in 54.6% (71/130) of specimens, with 45, positive by VP6 RT-PCR and 54, positive using Luminex xTAG GPP. Luminex GPP was able to detect all 45 VP6 RT-PCR positive samples. Co-infections were found in 63.0% (34/54) of Luminex positive RVA infections, with Shigella (35.3%; 12/34) and ETEC (29.4%; 10/34) detected frequently. Of the 71 ELISA positive RVA cases, 57.8% (41/71) were fully vaccinated, receiving two doses of Rotarix. CONCLUSION: This study provides insight on RVA prevalence in Cameroon, which could be useful for post-vaccine epidemiological studies, highlights higher than expected RVA prevalence in vaccinated children hospitalized for diarrhoea and provides the trend of RVA co-infection with other enteric pathogens. RVA genotyping is needed to determine circulating rotavirus genotypes in Cameroon, including those causing disease in vaccinated children.

Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer.

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

Comparative evaluation of integrated purification pathways for bacterial modular polyomavirus major capsid protein VP1 to produce virus-like particles using high throughput process technologies.

Modular virus-like particles and capsomeres are potential vaccine candidates that can induce strong immune responses. There are many described protocols for the purification of microbially-produced viral protein in the literature, however, they suffer from inherent limitations in efficiency, scalability and overall process costs. In this study, we investigated alternative purification pathways to identify and optimise a suitable purification pathway to overcome some of the current challenges. Among the methods, the optimised purification strategy consists of an anion exchange step in flow through mode followed by a multi modal cation exchange step in bind and elute mode. This approach allows an integrated process without any buffer adjustment between the purification steps. The major contaminants like host cell proteins, DNA and aggregates can be efficiently removed by the optimised strategy, without the need for a size exclusion polishing chromatography step, which otherwise could complicate the process scalability and increase overall cost. High throughput process technology studies were conducted to optimise binding and elution conditions for multi modal cation exchanger, Capto™ MMC and strong anion exchanger Capto™ Q. A dynamic binding capacity of 14 mg ml-1 was achieved for Capto™ MMC resin. Samples derived from each purification process were thoroughly characterized by RP-HPLC, SEC-HPLC, SDS-PAGE and LC-ESI-MS/MS Mass Spectrometry analytical methods. Modular polyomavirus major capsid protein could be purified within hours using the optimised process achieving purities above 87% and above 96% with inclusion of an initial precipitation step. Purified capsid protein could be easily assembled in-vitro into well-defined virus-like particles by lowering pH with addition of calcium chloride to the eluate. High throughout studies allowed the screening of a vast design space within weeks, rather than months, and unveiled complicated binding behaviour for CaptoTM MMC.

Virus-Based Nanoreactors with GALT Activity for Classic Galactosemia Therapy.

Enzymatic nanoreactors were obtained by galactose-1-phosphate uridylyl-transferase (GALT) encapsulation into plant virus capsids by a molecular self-assembly strategy. The aim of this work was to produce virus-like nanoparticles containing GALT for an enzyme-replacement therapy for classic galactosemia. The encapsulation efficiency and the catalytic constants of bio-nanoreactors were determined by using different GALT and virus coat protein ratios. The substrate affinity of nanoreactors was slightly lower than that of the free enzyme; the activity rate was 16 % of the GALT free enzyme. The enzymatic nanoreactors without functionalization were internalized into different cell lines including fibroblast and kidney cells, but especially into hepatocytes. The enzymatic nanoreactors are an innovative enzyme preparation with potential use for the treatment of classic galactosemia.

[Phylodynamic characteristics of the Russian population of rotavirus А (Reoviridae: Sedoreovirinae: Rotavirus) based on the VP6 gene].

INTRODUCTION: Rotavirus A is one of the leading causes of acute gastroenteritis in children in the first years of life. Rotavirus infection is currently classified as a preventable infection. The most abundant rotavirion protein is VP6. MATERIAL AND METHODS: Phylogenetic analysis and calculation of phylodynamic characteristics were carried out for 262 nucleotide sequences of the VP6 gene of rotavirus species A, isolated in Russia, using the BEAST v.1.10.4 software package. The derivation and analysis of amino acid sequences was performed using the MEGAX program. RESULTS: This study provides phylodynamic characteristics of the rotaviruses in Russia based on the sequences coding VP6 protein. Bayesian analysis showed the circulation of rotaviruses of three sublineages of genotype I1 and three sublineages of genotype I2 in Russia. The level of accumulation of mutations was established, which turned out to be similar for genotypes I1 and I2 and amounted to 7.732E-4 and 1.008E-3 nucleotides/site/year, respectively. The effective population sizes based on nucleotide sequences of the VP6 I1 and I2 genotypes are relatively stable while after the 2000s there is a tendency of its decreasing. Comparative analysis of the amino acid sequences in the region of the intracellular neutralization sites A (231-260 aa) and B (265-292 aa) made it possible to reveal a mutation in position V252I in a proportion of Russian strains of genotype I1 some strains of genotypes I1 and I2 had mutation I281V. These substitutions were not associated with any sublineages to which the strains belong. The analysis of three T-cell epitopes revealed four amino acid differences (in aa positions 305, 315, 342, 348) that were associated with the first or second genogroup. CONCLUSION: Based on the phylodynamic characteristics and amino acid composition of antigenic determinants, it was concluded that the VP6 protein is highly stable and could potentially be a good model for development of a rotavirus vaccine.

Virus-like particle preparation is improved by control over capsomere-DNA interactions during chromatographic purification.

Expression of viral capsomeres in bacterial systems and subsequent in vitro assembly into virus-like particles is a possible pathway for affordable future vaccines. However, purification is challenging as viral capsomeres show poor binding to chromatography media. In this study, the behavior of capsomeres in unfractionated bacterial lysate was compared with that for purified capsomeres, with or without added microbial DNA, to better understand reasons for poor bioprocess behavior. We show that aggregates or complexes form through the interaction between viral capsomeres and DNA, especially in bacterial lysates rich in contaminating DNA. The formation of these complexes prevents the target protein capsomeres from accessing the pores of chromatography media. We find that protein-DNA interactions can be modulated by controlling the ionic strength of the buffer and that at elevated ionic strengths the protein-DNA complexes dissociate. Capsomeres thus released show enhanced bind-elute behavior on salt-tolerant chromatography media. DNA could therefore be efficiently removed. We believe this is the first report of the use of an optimized salt concentration that dissociates capsomere-DNA complexes yet enables binding to salt-tolerant media. Post purification, assembly experiments indicate that DNA-protein interactions can play a negative role during in vitro assembly, as DNA-protein complexes could not be assembled into virus-like particles, but formed worm-like structures. This study reveals that the control over DNA-protein interaction is a critical consideration during downstream process development for viral vaccines.

Asymmetrizing an icosahedral virus capsid by hierarchical assembly of subunits with designed asymmetry.

Symmetrical protein complexes are ubiquitous in biology. Many have been re-engineered for chemical and medical applications. Viral capsids and their assembly are frequent platforms for these investigations. A means to create asymmetric capsids may expand applications. Here, starting with homodimeric Hepatitis B Virus capsid protein, we develop a heterodimer, design a hierarchical assembly pathway, and produce asymmetric capsids. In the heterodimer, the two halves have different growth potentials and assemble into hexamers. These preformed hexamers can nucleate co-assembly with other dimers, leading to Janus-like capsids with a small discrete hexamer patch. We can remove the patch specifically and observe asymmetric holey capsids by cryo-EM reconstruction. The resulting hole in the surface can be refilled with fluorescently labeled dimers to regenerate an intact capsid. In this study, we show how an asymmetric subunit can be used to generate an asymmetric particle, creating the potential for a capsid with different surface chemistries.

Broad-range and effective detection of human noroviruses by colloidal gold immunochromatographic assay based on the shell domain of the major capsid protein.

BACKGROUND: Human noroviruses (HuNoVs) are a major cause of nonbacterial gastroenteritis in all age groups worldwide. HuNoVs can be detected in vitro using molecular assays such as RT-PCR and RT-qPCR. However, these molecular-based techniques require special equipment, unique reagents, experienced personnel, and extended time to obtain results. Besides, the diversity of viral genotypes is high. Therefore, methods that are rapid, broad-range and effective in the detection of HuNoVs are desiderated for screening the feces or vomit of infected people during outbreaks. RESULTS: In this study, a colloidal-gold-based immunochromatographic assay (ICA) was developed for effective detection of HuNoVs in clinical samples. Monoclonal antibodies (MAbs) against the shell (S) domain in the major capsid protein of HuNoVs were used in the ICA. The limitations of detection for HuNoVs in clinical samples were 1.2 × 106 genomic copies per gram of stool sample (gc/g) and 4.4 × 105 gc/g for genogroup I and II (GI and GII) HuNoVs, respectively. A total of 122 clinical samples were tested for HuNoVs by ICA and compared against RT-qPCR. The relative sensitivity, specificity and agreement of ICA was 84.2% (95% CI: 83.6-84.8%), 100.0% (95% CI: 98.5-100.0%) and 87.7% (95% CI: 85.6-89.8%), respectively. No cross-reaction with other common enteric viruses or bacteria was observed. The ICA detected a broad range of genotypes, including GI.1, GI.3, GI.4, GI.6, GI.14, GII.2, GII.3, GII.4, GII.6, GII.13, and GII.17 HuNoVs. CONCLUSIONS: This study demonstrates that ICA targeting the S domain of VP1 is a promising candidate for effectively identifying the different genotypes of HuNoVs in clinical samples with high sensitivity and specificity.

Production and characterization of a fusion form of hepatitis E virus tORF2 capsid protein in Escherichia coli.

Hepatitis E virus (HEV) is a nonenveloped virus causing an emerging zoonotic disease posing a severe threat to the public health in the world, especially to pregnant women. In this study, a truncated form (aa 368-606) of the open reading frame 2 of the capsid protein (tORF2-HEV), a major structural protein of HEV, was expressed in Escherichia coli. This work characterizes for the first time, the fused Glutathione-S-Transferase-tagged tORF2 (GST-tORF2) and tORF2-HEV forms in E. coli. The fusion protein was purified by affinity chromatography with a purity higher than 90% and to yield about 27% after thrombin digestion. The purified GST-tORF2 protein was then characterized by western blot, using anti-GST antibodies, and CD spectroscopy. The GST-tORF2 and tORF2-HEV proteins were shown to be efficient to develop an ELISA test to detect anti-HEV IgG in mice sera immunized with a recombinant full length ORF2 protein. Sera showed a significant increase of the absorbance signal at 450 nm, in plate wells coated with a quantity of 0.5, 1 and 2 µg of proteins. ELISA plates coated with the purified GST-tORF2 and tORF2-HEV showed similar response when compared to the HEV ELISA where total insect cell lysate, infected with the recombinant baculovirus expressing full ORF2, was used as positive control.

Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.

Association of host proteins with the broad host range filamentous phage NgoΦ6 of Neisseria gonorrhoeae.

All Neisseria gonorrhoeae strains contain multiple copies of integrated filamentous phage genomes with undefined structures. In this study, we sought to characterize the capsid proteins of filamentous N. gonorrhoeae bacteriophage NgoΦ6 and phagemids propagated in different bacteria. The data demonstrate that purified phage contain phage-encoded structural proteins and bacterial host proteins; host proteins consistently copurified with the phage particles. The bacterial host proteins associated with the phage filament (as identified by mass spectrometry) tended to be one of the predominant outer membrane components of the host strain, plus minor additional host proteins. We were able to copurify a functional ß-lactamase, a phagemid-encoded protein, with phage filaments. We used protein modeling and immunological analysis to identify the major phage encoded structural proteins. The antigenic properties of these proteins depended on the bacterium where the phages were propagated. Polyclonal antibodies against N. gonorrhoeae phage NgoΦ6 recognized phage-encoded proteins if the phage was propagated in N. gonorrhoeae or H. influenzae cells but not if it was propagated in Salmonella or E. coli. We show that the phage filaments isolated from gonococci and Haemophilus are glycosylated, and this may explain the antigenic diversity seen. Taken en toto, the data demonstrate that while the neisserial filamentous phage are similar to other Inovirus with respect to overall genomic organization, their ability to closely associate with host proteins suggests that they have unique surface properties and are secreted by a here-to-fore unknown secretory pathway.