Expression and antigenic characterization of the major capsid proteins of human polyomaviruses BK and JC in Saccharomyces cerevisiae

doi:10.1016/S0166-0934(02)00036-8

Journal of Virological Methods

Volume 104, Issue 1, June 2002, Pages 93-98

https://doi.org/10.1016/S0166-0934(02)00036-8 Get rights and content

Abstract

BK and JC viruses are ubiquitous human polyomaviruses that are associated with post-transplant interstitial nephritis (BK virus) and progressive multifocal leucoencephalopathy (JC virus). The use of a yeast system to express the major capsid protein (VP1) of two antigenic variants of BKV (strains SB and AS) and JCV is described. VP1s of AS and JCV expressed in Saccharomyces cerevisiae produced proteins of expected molecular weight as determined by gel electrophoresis whereas that of SB appeared to be lower than anticipated. However, all VP1s self-assembled into virus-like particles (VLP) retaining sialic acid-binding and antigenic properties of native virions. This method is highly efficient for producing recombinant proteins and therefore provides an alternative to the baculovirus system.

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Acknowledgments

This work was partly supported by the Lithuanian Science Foundation, grant No. 273.

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In 2019, there were about 100,000 kidney transplants globally, with more than a quarter of them performed in the United States. Unfortunately, some engrafted organs are lost to polyomavirus-associated nephropathy (PyVAN) caused by BK and JC viruses (BKPyV and JCPyV). Both viruses cause brain disease and possibly bladder cancer in immunosuppressed individuals. Transplant patients are routinely monitored for BKPyV viremia, which is an accepted hallmark of nascent nephropathy. If viremia is detected, a reduction in immunosuppressive therapy is standard care, but the intervention comes with increased risk of immune rejection of the engrafted organ. Recent reports have suggested that transplant recipients with high levels of polyomavirus-neutralizing antibodies are protected against PyVAN. Virus-like particle (VLP) vaccines, similar to approved human papillomavirus vaccines, have an excellent safety record and are known to induce high levels of neutralizing antibodies and long-lasting protection from infection. In this study, we demonstrate that VLPs representing BKPyV genotypes I, II, and IV, as well as JCPyV genotype 2 produced in insect cells elicit robust antibody titers. In rhesus macaques, all monkeys developed neutralizing antibody titers above a previously proposed protective threshold of 10,000. A second inoculation, administered 19 weeks after priming, boosted titers to a plateau of $\geq$ 25,000 that was maintained for almost two years. No vaccine-related adverse events were observed in any macaques. A multivalent BK/JC VLP immunogen did not show inferiority compared to the single-genotype VLP immunogens. Considering these encouraging results, we believe a clinical trial administering the multivalent VLP vaccine in patients waiting to receive a kidney transplant is warranted to evaluate its ability to reduce or eliminate PyVAN.
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Although viruses are simple biological systems, they are capable of evolving highly efficient techniques for infecting cells, expressing their genomes, and generating new copies of themselves. It is possible to genetically manipulate most of the different classes of known viruses in order to produce recombinant viruses that express foreign proteins. Recombinant viruses have been used in gene therapy to deliver selected genes into higher organisms, in vaccinology and immunotherapy, and as important research tools to study the structure and function of these proteins.
Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome. They have been applied not only as prophylactic and therapeutic vaccines but also as vehicles in drug and gene delivery and, more recently, as tools in nanobiotechnology.
In this chapter, basic and advanced features of viruses and VLPs are presented and their major applications are discussed. The different production platforms based on animal cell technology are explained, and their main challenges and future perspectives are explored. The implications of large-scale production of viruses and VLPs are discussed in the context of process control, monitoring, and optimization. The main upstream and downstream technical challenges are identified and discussed accordingly.
JC virus/human immunodeficiency virus 1 co-infection in the Brazilian Amazonian region
2016, Brazilian Journal of Infectious Diseases
Citation Excerpt :
Two human polyomaviruses are important pathogens, JC virus (JCV) and BK virus (BKV), as they are able to cause a severe, usually fatal, neurological disease (progressive multifocal leukoencephalopathy, PML) and renal infection in immunosupressed patients, respectively.1–3 The viruses share approximately 72% of nucleotide homology and a high degree of identity (80%) of amino acid sequence of a capsid protein, VP1.4,5 JCV infects the human host and establishes a persistent replicative cycle (including latency) following the acute infection, which favors its dissemination within human populations and its carriage through generations.
JC virus (JCV) is a member of the Polyomaviridae family and is associated to a severe disease known as progressive multifocal leukoencephalopathy, PML, which is gradually increasing in incidence as an opportunistic infection among AIDS patients. The present study aimed to investigate the occurrence of JCV among HIV-1 carriers including their types and molecular subtypes and the possible association with disease. Urine samples from 66 HIV-1 infected subjects were investigated for the presence of the virus by amplifying VP1 (215 bp) and IG (610 bp) regions using the polymerase chain reaction. JCV was detected in 32% of the samples. The results confirmed the occurrence of type B (subtype Af2); in addition, another polyomavirus, BKV, was also detected in 1.5% of samples of the HIV-1 infected subjects. Apparently, there was no significant difference between mono- (HIV-1 only) and co-infected (HIV-1/JCV) subjects regarding their TCD4⁺/TCD8⁺ lymphocyte counts or HIV-1 plasma viral load. Self admitted seizures, hearing and visual loses were not significantly different between the two groups.
A prospective study of renal transplant recipients reveals an absence of primary JC polyomavirus infections
2016, Journal of Clinical Virology
Citation Excerpt :
Both donor and recipient JCPyV and BKPyV IgG antibody status were determined using recombinant JC and BK VP1 virus-like particles (VLPs), as described previously [14,15]. The JC and BK VLPs used in this assay were produced in yeast Saccharomyces cerevisiae [16,17]. Nucleic acid was extracted from the prospective study samples using the Corbett X-tractor Gene and the routine audit study samples using the QIAGEN Bio Robot MDX (Qiagen, Crawley, UK).
Both JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) are acquired at an early age. JCPyV causes progressive multifocal leukoencephalopathy and has been described in association with nephropathy.
Urine and plasma samples from renal transplant recipients (RTRs) were examined for JCPyV to determine its involvement in causing infection and disease.
JCPyV testing was performed on 112 RTRs included in a randomised controlled study of steroid-sparing immunosuppressive regimens [1]. Urine and EDTA blood samples were collected pre- and post-transplantation and analysed for JCPyV using real-time PCR and sequencing to determine genotype and viral variation. Donor and recipient IgG antibody status to JCPyV was also determined.
Overall, 13.3% of RTRs were positive for JCPyV of which one patient developed viraemia without viruria. JCPyV DNA was detected early following transplantation (defined as five days post transplantation) from recipients with donors that were positive for JCPyV IgG antibodies. No dual cases of JCPyV and BKPyV were observed. One patient sample had sequence duplication in the non-coding control region.
Like BKPyV, JCPyV tends to occur early post transplantation but did not result in sustained viraemia. There was no deterioration of renal function in patients positive for JCPyV. As with other viruses, JCPyV donor serostatus was a risk factor for detection of JCPyV DNA. JCPyV appears to protect individuals from BKPyV infection, as recipients were twice as likely to develop BKPyV with a negative JCPyV donor.
Production and biomedical applications of virus-like particles derived from polyomaviruses
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The yield of recombinant VP1 was 5–10% of the total soluble protein (0.1–0.2 g per liter of medium) [24]. Later, the same group showed the expression of JCPyV, BKPyV [109], SV40, MPyV and avian polyomavirus (APyV) VLPs [110]. In all cases, DNA contamination was minor, and could be removed completely by dialysis and nuclease treatment without reassembly.
Virus-like particles (VLPs), aggregates of capsid proteins devoid of viral genetic material, show great promise in the fields of vaccine development and gene therapy. These particles spontaneously self-assemble after heterologous expression of viral structural proteins. This review will focus on the use of virus-like particles derived from polyomavirus capsid proteins. Since their first recombinant production 27 years ago these particles have been investigated for a myriad of biomedical applications. These virus-like particles are safe, easy to produce, can be loaded with a broad range of diverse cargos and can be tailored for specific delivery or epitope presentation. We will highlight the structural characteristics of polyomavirus-derived VLPs and give an overview of their applications in diagnostics, vaccine development and gene delivery.
Generation of recombinant metapneumovirus nucleocapsid protein as nucleocapsid-like particles and development of virus-specific monoclonal antibodies
2011, Virus Research
Citation Excerpt :
Since yeast are eukaryotes, their intracellular environment is more suitable for the correct folding of eukaryotic virus proteins as compared to bacterial expression systems (Böer et al., 2007). A considerable body of data on formation of homologous and chimeric virus-like and nucleocapsid-like particles (NLPs) of various viruses in yeast has been reported to date (Hofmann et al., 1996; Sasnauskas et al., 1999; Gedvilaite et al., 2000; Hale et al., 2002; Samuel et al., 2002; Juozapaitis et al., 2007; Freivalds et al., 2006; Kucinskaite et al., 2007). The expression of recombinant N proteins with spontaneous assembly into NLPs has been demonstrated for a number of paramyxoviruses using both prokaryotic and eukaryotic systems (Spehner et al., 1991; Hummel et al., 1992; Fooks et al., 1993; Meric et al., 1994; Warnes et al., 1995; Bhella et al., 2002).
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.

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Expression and antigenic characterization of the major capsid proteins of human polyomaviruses BK and JC in Saccharomyces cerevisiae

Abstract

Section snippets

Acknowledgments

Virology

Lancet

Clin. Diagn. Virol.

Lancet

Cell

Occurrence and significance of papovaviruses BK and JC in the urine

Prog. Med. Virol.

Class I major histocompatibility proteins as cell surface receptors for simian virus 40

J. Virol.

DNA sequences similar to those of simian virus 40 in ependymomas and choroid plexus tumors of childhood

N. Engl. J. Med.

Self-assembly of the JC virus major capsid protein, VP1, expressed in insect cells

J. Gen. Virol.

Cooperation of structural proteins during late events in the life cycle of polyomavirus

J. Virol.

Molecular cloning and expression of major structural protein VP1 of the human polyomavirus JC virus: formation of virus-like particles useful for immunological and therapeutic studies

J. Virol.