Structure Of Capsid Protein Research Articles

Characterization of capsid protein of porcine circovirus 4 by in-silico approach

Porcine Circovirus 4 (PCV4) has been detected in pigs from China only in 2019. PCV4 has been found to be associated with several forms of clinical syndromes in pigs. Like other Porcine Circoviruses, the only structural protein of PCV4 is the capsid protein. Therefore, it could be a key vaccine candidate or diagnostic marker. The present study aims to characterize the capsid protein of PCV4 by using several bioinformatics tools. The in-silico analysis revealed that the molecular weight of PCV4 capsid protein is about 27.3 kDa which is made up of 228 amino acids. The capsid protein of PCV4 contains a nuclear localization signal at N-terminal and several post-translational modifications could be predicted at different residues of the protein. The secondary structure of capsid protein contains coil, beta strand, and alpha helix. The 3D structure of the protein was predicted with a high level of confidence. The capsid protein of PCV4 also contains several potential B-cell epitopes. Thus, the in-silico characterization of the capsid protein will be beneficial in designing diagnostics and vaccine candidates in future to combat PCV4 infection in pigs.

Expression of virus-like particles (VLPs) of foot-and-mouth disease virus (FMDV) using Saccharomyces cerevisiae

We engineered Saccharomyces cerevisiae to express structural proteins of foot-and-mouth disease virus (FMDV) and produce virus-like particles (VLPs). The gene, which encodes four structural capsid proteins (VP0 (VP4 and VP2), VP3, and VP1), followed by a translational “ribosomal skipping” sequence consisting of 2A and protease 3C, was codon-optimized and chemically synthesized. The cloned gene was used to transform S. cerevisiae 2805 strain. Western blot analysis revealed that the polyprotein consisting of VP0, VP3, and VP1 was processed into the discrete capsid proteins. Western blot analysis of 3C confirmed the presence of discrete 3C protein, suggesting that the 2A sequence functioned as a “ribosomal skipping” signal in the yeast for an internal re-initiation of 3C translation from a monocistronic transcript, thereby indicating polyprotein processing by the discrete 3C protease. Moreover, a band corresponding to only VP2, which was known to be non-enzymatically processed from VP0 to both VP4 and VP2 during viral assembly, further validated the assembly of processed capsid proteins into VLPs. Electron microscopy showed the presence of the characteristic icosahedral VLPs. Our results clearly demonstrate that S. cerevisiae processes the viral structural polyprotein using a viral 3C protease and the resulting viral capsid subunits are assembled into virion particles.Key points• Ribosomal skipping by self-cleaving FMDV peptide in S. cerevisiae.• Proteolytic processing of a structural polyprotein from a monocistronic transcript.• Assembly of the processed viral capsid proteins into a virus-like particle.

Virus Neutralization Test for Detecting and Quantifying Serum-Neutralizing Antibodies to Epizootic Hemorrhagic Disease Virus (EHDV) (Serotypes 1, 2, and 4-8).

The virus neutralization test (VNT) is a functional immunoassay which detects the presence and quantity of neutralizing antibodies. It is a highly sensitive and specific test. As with most neutralization assays, the EHDV VNT does not react with all virus-targeting antibodies, but specifically with those antibodies that bind to VP2, the outermost capsid structural protein of the virus. The interaction between VP2 and neutralizing antibodies can block EHDV cell binding, neutralizing its infectivity. The detection and quantification of neutralizing antibodies are indicative of how protected an animal is against reinfection. The EHD VNT can therefore be a useful tool to monitor the efficacy of a vaccination campaign. VP2 is also the main determinant of EHDV serotype specificity, and so EHDV-neutralizing antibodies which target VP2 are also serotype-specific. Throughdetecting and quantifying neutralizing antibodies, the VNT can discriminate the EHDV serotype responsible for an infection and provides insights into the time of infection. It is considered the gold standard test for identifying and quantifying antibodies against EHDV serotypes present in test serum samples. The assay is performed in vitro and is based on inhibition of virus infectivity in the presence of neutralizing antibodies. A neutralizing antibody titer is determined through the presence or absence of cytopathic effect in a cell monolayer. The VNT is a relatively inexpensive assay using standard laboratory equipment; however, to perform the assay, cell cultures, significant time, intensive labor, and technical skill are required.

Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System.

The widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with optimized yield and transduction efficiency. The Baculovirus/Sf9 system is a promising platform for high yield production; however, a major drawback to using an invertebrate cell line compared to a mammalian system is a generally altered AAV capsid stoichiometry resulting in lower biological potency. Here, we introduce a term of the structural and biological "fitness" of an AAV capsid as a function of two interdependent parameters: (1) packaging efficiency (yield), and (2) transduction efficiency (infectivity). Both parameters are critically dependent on AAV capsid structural proteins VP1/2/3 stoichiometry. To identify an optimal AAV capsid composition, we developed a novel Directed Evolution (DE) protocol for assessing the structural and biological fitness of Sf9-manufactured rAAV for any given serotype. The approach involves the packaging of a combinatorial capsid library in insect Sf9 cells, followed by a library screening for high infectivity in human Cre-recombinase-expressing C12 cells. One single DE selection round, complemented by Next-Generation Sequencing (NGS) and guided by in silico analysis, identifies a small subset of VP1 translation initiation sites (known as Kozak sequence) encoding "fit" AAV capsids characterized by a high production yield and superior transduction efficiencies.

Genetic diversity and characterization of rhinoviruses from Chinese clinical samples with a global perspective.

Rhinovirus (RV) is a significant pathogen causing upper and lower respiratory diseases and is one of the most prevalent human respiratory viruses. However, research on the genetic diversity and evolutionary dynamics of RVs in China and worldwide remains limited. To address this knowledge gap, we utilized Chinese clinical RV samples as a starting point and detected and reported 22 types, including A9, B3, and C13, in China for the first time. Among these, A67, A76, and A106 were also detected and reported in Asia for the first time, characterizing their genetic diversity. We also identified A110 as a novel type of RVA and reported its distinctive characteristics in phylogeny, secondary structure, and capsid protein structure. Furthermore, by mining, refining, and annotating RV sequence data available worldwide, four previously unreported novel types, B107, C58, C59, and C60, were identified, and their genetic diversity was revealed. Furthermore, we observed variations in the guanine-cytosine (GC) content among different serotypes and clades. Also of note was that, based on a complete and refined VP1 data set, the evolutionary dynamics of RV were analyzed systematically and on a large scale for the first time. IMPORTANCE Based on clinical samples collected in China, we detected and reported 22 types for the first time in China, as well as three types for the first time in Asia, and reported their genetic characteristics and diversity. We identified a novel type of Rhinovirus (RV), A110, highlighting its unique genetic features. We annotated the genomic structure and serotype of all the existing RV sequences in the database, and four novel RV types were identified and their genetic diversity reported. Combined with the sequence annotation, we constructed a complete VP1 data set of RV and conducted the first large-scale evolutionary dynamics analysis of RV. Based on a high-quality data set, we conducted a comprehensive analysis of the guanine-cytosine (GC) content variations among serotypes of RVs. This study provides crucial theoretical support and valuable data for understanding RV's genetic diversity and developing antiviral strategies.

Cloning and Molecular Characterization of the Recombinant CVB4E2 Immunogenic Viral Protein (rVP1), as a Potential Subunit Protein for Vaccine and Immunodiagnostic Reagent Candidate.

The aim of the present study was, first, to clone the VP1 gene of the human coxsackievirus B4 strain E2 (CVB4E2) in the prokaryotic pUC19 plasmid expression vector then to compare it with the structural capsid proteins of the same strain using bioinformatic tools. PCR colony amplification followed through a restriction digestion analysis and sequencing process which affirmed the success of the cloning process. SDS-PAGE and Western Blotting were used to characterize the purified recombinant viral protein expressed in bacteria cells. The BLASTN tool revealed that the nucleotide sequence of the recombinant VP1 (rVP1) expressed by pUC19 highly matched the target nucleotide sequence of the diabetogenic CVB4E2 strain. Secondary structure and three-dimension structure prediction suggested that rVP1, such as wild-type VP1, is chiefly composed of random coils and a high percentage of exposed amino acids. Linear B-cell epitope prediction showed that several antigenic epitopes are likely present in rVP1 and CVB4E2 VP1 capsid protein. Additionally, phosphorylation site prediction revealed that both proteins may affect the signal transduction of host cells and can be involved in virus virulence. The present work highlights the usefulness of cloning and bioinformatics characterizations for gene investigation. Furthermore, the collected data are helpful for future experimental research related to the development of immunodiagnostic reagents and subunit vaccines based on the expression of immunogenic viral capsid proteins.

Identification of novel potential hepatitis E virus inhibitors as seen from molecular docking, free energy landscape and molecular dynamics simulation studies

ABSTRACT Hepatitis E virus (HEV), a non-enveloped and globular protein, is the primary cause of acute hepatitis. The structural capsid protein encoded by HEV is the open-reading frame 2 gene. However, there are presently no efficient and targeted therapies for HEV infections. This research aims to screen out the effective bioactive compounds from Paederia foetida, which may be novel potential inhibitors of Hepatitis E. Despite various research in recent decades, the molecular picture of the interaction between the Hepatitis E virus and iridoid glycosides from Paederia foetida remains vague. We performed in silico ADMET predictions of the phytochemicals of Paederia foetida, and the findings revealed a reasonably good ADMET profile for the twenty chosen phytochemicals. Herein, the twenty phytochemicals were submitted to an analysis of molecular docking studies with the HEV protein and the analysis suggested that asperuloside, ellagic acid, friedlin, lupeol and paederoside as the most potential HEV inhibitors among the selected phytochemicals. Subsequently, the stability of these phytochemical complexes was studied by using molecular dynamic (MD) simulation of 100 ns. Based on our computational study, lupeol and paederoside phytochemicals obtained from the paederia foetida plant may be used as potential drug candidates for the inhibition of hepatitis E.

Capsid structure of a fungal dsRNA megabirnavirus reveals its previously unidentified surface architecture.

Rosellinia necatrix megabirnavirus 1-W779 (RnMBV1) is a non-enveloped icosahedral double-stranded (ds)RNA virus that infects the ascomycete fungus Rosellinia necatrix, a causative agent that induces a lethal plant disease white root rot. Herein, we have first resolved the atomic structure of the RnMBV1 capsid at 3.2 Å resolution using cryo-electron microscopy (cryo-EM) single-particle analysis. Compared with other non-enveloped icosahedral dsRNA viruses, the RnMBV1 capsid protein structure exhibits an extra-long C-terminal arm and a surface protrusion domain. In addition, the previously unrecognized crown proteins are identified in a symmetry-expanded cryo-EM model and are present over the 3-fold axes. These exclusive structural features of the RnMBV1 capsid could have been acquired for playing essential roles in transmission and/or particle assembly of the megabirnaviruses. Our findings, therefore, will reinforce the understanding of how the structural and molecular machineries of the megabirnaviruses influence the virulence of the disease-related ascomycete fungus.

Cryo-EM structure of ssDNA bacteriophage ΦCjT23 provides insight into early virus evolution

The origin of viruses remains an open question. While lack of detectable sequence similarity hampers the analysis of distantly related viruses, structural biology investigations of conserved capsid protein structures facilitate the study of distant evolutionary relationships. Here we characterize the lipid-containing ssDNA temperate bacteriophage ΦCjT23, which infects Flavobacterium sp. (Bacteroidetes). We report ΦCjT23-like sequences in the genome of strains belonging to several Flavobacterium species. The virion structure determined by cryogenic electron microscopy reveals similarities to members of the viral kingdom Bamfordvirae that currently consists solely of dsDNA viruses with a major capsid protein composed of two upright β-sandwiches. The minimalistic structure of ΦCjT23 suggests that this phage serves as a model for the last common ancestor between ssDNA and dsDNA viruses in the Bamfordvirae. Both ΦCjT23 and the related phage FLiP infect Flavobacterium species found in several environments, suggesting that these types of viruses have a global distribution and a shared evolutionary origin. Detailed comparisons to related, more complex viruses not only expand our knowledge about this group of viruses but also provide a rare glimpse into early virus evolution.

Confirmation of the Rabbit Hemorrhagic Disease Virus Type 2 (GI.2) Circulation in North Africa

Abstract Rabbit hemorrhagic disease (RHD) is a highly contagious viral disease that causes fatal acute hepatitis in domestic and wild lagomorphs. It has taken on major economic importance in countries like Morocco. In addition to the classical virus (RHDV), a novel emerged genotype (RHDV2) is circulating, especially in the north shore of the Mediterranean basin since 2010. Many small animal farmers reported clinical cases from several rabbitries in Agadir (Morocco) despite systematic vaccination against the RHDV. The main objective was to characterize the current RHDV strains circulating in the studied area to help to choose an adequate vaccine. For that, we extracted viral RNA from rabbit livers, carried out the PCR analyses, and we sequenced the viral structural capsid protein (VP60) of the RHDV. The phylogenetic analysis results allowed us to state that the novel genotype (RHDV2) is circulating in the studied geographical area, and to characterize the isolated sequences. As a conclusion, we recommend updating RHD epidemiological relating data and reviewing the vaccine protocols by both targeting RHDV (GI.1) and RHDV2 (GI.2) in any future preventive program.

Prediction of promiscuous epitopes in ORF2 of Hepatitis E virus: an In-Silico approach.

Vaccine development against emerging infections is essentially important for saving people from increasing viral infections. In developing countries, Hepatitis E (HEV) is a common infection affecting millions of people worldwide. Based on In-silico analysis, different approaches have been targeted. Rationale of this study is to design an epitope-based vaccine candidates with the help of immunoinformatics that can predict promiscuous B-cell and T-cell epitopes of the most antigenic HEV-ORF2 capsid protein. This study suggests potential T-cell and B-cell epitopes of the highly antigenic HEV ORF2 capsid protein while using various In-silico tools such as NCBI-BLAST, Expassy, CLC workbench, Ellipro and Discotope. Potential antigenic and immunogenic CD8+ T-cell epitopes were predicted from the global consensus sequence of ORF2-HEV. Furthermore, twenty-two linear B-cell epitopes were predicted. Among these, "SLGAGPV" at position 587-593 and "LEFRNLTPGNTNTRVSRYSS" at position 306-325 were most antigenic with antigenicity score 1.4206 and 1.3600 respectively. Discontinuous B-cell epitopes were found by three-dimensional capsid protein structure. Epitopes predicted in this study reveal high antigenicity and promiscuity for HLA classes. Collectively, our data suggests promiscuous epitopes that can potentially acts as new candidates for the design of HEV peptide vaccine.

Dominance of phage particles carrying antibiotic resistance genes in the viromes of retail food sources

The growth of antibiotic resistance has stimulated interest in understanding the mechanisms by which antibiotic resistance genes (ARG) are mobilized. Among them, studies analyzing the presence of ARGs in the viral fraction of environmental, food and human samples, and reporting bacteriophages as vehicles of ARG transmission, have been the focus of increasing research. However, it has been argued that in these studies the abundance of phages carrying ARGs has been overestimated due to experimental contamination with non-packaged bacterial DNA or other elements such as outer membrane vesicles (OMVs). This study aims to shed light on the extent to which phages, OMVs or contaminating non-packaged DNA contribute as carriers of ARGs in the viromes. The viral fractions of three types of food (chicken, fish, and mussels) were selected as sources of ARG-carrying phage particles, whose ability to infect and propagate in an Escherichia coli host was confirmed after isolation. The ARG-containing fraction was further purified by CsCl density gradient centrifugation and, after removal of DNA outside the capsids, ARGs inside the particles were confirmed. The purified fraction was stained with SYBR Gold, which allowed the visualization of phage capsids attached to and infecting E. coli cells. Phages with Myoviridae and Siphoviridae morphology were observed by electron microscopy. The proteins in the purified fraction belonged predominantly to phages (71.8% in fish, 52.9% in mussels, 78.7% in chicken sample 1, and 64.1% in chicken sample 2), mainly corresponding to tail, capsid, and other structural proteins, whereas membrane proteins, expected to be abundant if OMVs were present, accounted for only 3.8–21.4% of the protein content. The predominance of phage particles in the viromes supports the reliability of the protocols used in this study and in recent findings on the abundance of ARG-carrying phage particles.

The oncogenic roles of JC polyomavirus in cancer.

JC polyomavirus (JCPyV) belongs to the human polyomavirus family. Based on alternative splicing, the early region encodes the large and small T antigens, while the late region encodes the capsid structural proteins (VP1, VP2, and VP3) and the agnoprotein. The regulatory transcription factors for JCPyV include Sp1, TCF-4, DDX1, YB-1, LCP-1, Purα, GF-1, and NF-1. JCPyV enters tonsillar tissue through the intake of raw sewage, inhalation of air droplets, or parent-to-child transmission. It persists quiescently in lymphoid and renal tissues during latency. Both TGF-β1 and TNF-α stimulates JCPyV multiplication, while interferon-γ suppresses the process. The distinct distribution of caspid receptors (α-2, 6-linked sialic acid, non-sialylated glycosaminoglycans, and serotonin) determines the infection capabilities of JCPyV virions, and JCPyV entry is mediated by clathrin-mediated endocytosis. In permissive cells, JCPyV undergoes lytic proliferation and causes progressive multifocal leukoencephalopathy, while its DNA is inserted into genomic DNA and leads to carcinogenesis in non-permissive cells. T antigen targets p53, β-catenin, IRS, Rb, TGF-β1, PI3K/Akt and AMPK signal pathways in cancer cells. Intracranial injection of T antigen into animals results in neural tumors, and transgenic mice develop neural tumors, lens tumor, breast cancer, gastric, Vater’s, colorectal and pancreatic cancers, insulinoma, and hepatocellular carcinoma. Additionally, JCPyV DNA and its encoded products can be detected in the brain tissues of PML patients and brain, oral, esophageal, gastric, colorectal, breast, cervical, pancreatic, and hepatocellular cancer tissues. Therefore, JCPyV might represent an etiological risk factor for carcinogenesis and should be evaluated for early prevention, diagnosis, and treatment of cancers.

Echovirus 9 genetic diversity detected in whole-capsid genome sequences obtained directly from clinical specimens using next generation sequencing.

Echovirus 9 (E9) has been detected in an increased number of symptomatic patient samples received bythe National Enterovirus Reference Laboratory in Hungary during 2018 compared to previously reported years.Formerly identified E9 viruses from different specimen types detected from patients of various ages and showing differing clinical signs were chosen for the detailed analysis of genetic relationships and potential variations within the viral populations. We used next generation sequencing (NGS) analysis of 3,900 nucleotide long amplicons covering the entire capsid coding region of the viral genome without isolation, directly from clinical samples.Compared to the E9 reference strain, the viruses showed about 79% nucleotide and around 93% amino acid sequence similarity. The four new viral genome sequences had 1-20 nucleotide differences between them also resulting in 6 amino acid variances in the coding region, including 3 in the structural VP1 capsid protein. One virus from a patient with hand, foot, and mouth disease had two amino acid changes in the VP1 capsid protein. An amino acid difference was also detected in the non-structural 2C gene of one virus sequenced from a throat swab sample from a patient with meningitis, compared to the faecal specimen taken two days later. Two amino acid changes, one in the capsid protein, were found between faecal samples of meningitis patients of different ages.Sequencing the whole capsid genome revealed several nucleotide and amino acid differences between E9 virus strains detected in Hungary in 2018.

Transiently Transfected Mammalian Cell Cultures: An Adaptable and Effective Platform for Virus-like Particle-Based Vaccines against Foot-and-Mouth Disease Virus.

RNA viruses, such as foot-and-mouth disease virus (FMDV), have error-prone replication resulting in the continuous emergence of new viral strains capable of evading current vaccine coverage. Vaccine formulations must be regularly updated, which is both costly and technically challenging for many vaccine platforms. In this report, we describe a plasmid-based virus-like particle (VLP) production platform utilizing transiently transfected mammalian cell cultures that combines both the rapid response adaptability of nucleic-acid-based vaccines with the ability to produce intact capsid epitopes required for immunity. Formulated vaccines which employed this platform conferred complete protection from clinical foot-and-mouth disease in both swine and cattle. This novel platform can be quickly adapted to new viral strains and serotypes through targeted exchanges of only the FMDV capsid polypeptide nucleic acid sequences, from which processed structural capsid proteins are derived. This platform obviates the need for high biocontainment manufacturing facilities to produce inactivated whole-virus vaccines from infected mammalian cell cultures, which requires upstream expansion and downstream concentration of large quantities of live virulent viruses.

In vitro bacteriophage-mediated synthesis of silver nanoparticles for antibacterial applications and heavy metal detection

The risk of multidrug-resistant bacteria and heavy metals contaminating the environment raises a drastic public issue. In this report, we succeeded in introducing a novel synthesis technique of silver nanoparticles (AgNPs) with antibacterial, anti-biofilm activities and detection of cadmium in water. Furthermore, we managed to reduce silver nitrate (AgNO3) using the capsid structural proteins of bacteriophage, a naturally occurring bacterial virus. The formulated AgNPs were regular in shape and ranged from 10 to 30 nm in size, as indicated from the transmission electron micrographs (TEM). Moreover, the formulated AgNPs highlighted biofilm deformation capabilities against the model bacteria, Staphylococcussciuri. Furthermore, the newly synthesized AgNPs can rapidly sense the heavy metal in a concentration of 100 µM. Thus, this study reveals the outstanding ability of the bacteriophage-mediated synthesis of AgNPs and suggests its promising activity over the traditional synthesis methods.

Identification of acridinedione scaffolds as potential inhibitor of DENV-2 C protein: An in silico strategy to combat dengue.

Dengue is a prominent viral disease transmitted by mosquitoes to humans that affects mainly tropical and subtropical countries worldwide. The global spread of dengue virus (DENV) is mainly occurred by Aedes aegypti and Aedes albopictus mosquitoes. The dengue virus serotypes-2 (DENV-2) is a widely prevalent serotype of DENV, that causes the hemorrhagic fever and bleeding in the mucosa, which can be fatal. In the life cycle of DENV-2, a structural capsid (DENV-2 C) protein forms the nucleocapsid assembly and bind to the viral progeny RNA. For DENV-2 maturation, the nucleocapsid is a vital component. We used virtual ligand screening to filter out the best in-house synthesized acridinedione analogs (DSPD molecules) that could efficiently bind to DENV-2 C protein. The molecular docking and dynamics simulations studies were performed to analyze the effect of DSPD molecules on DENV-2 C protein after binding. Our findings showed that DSPD molecules strongly interacted with DENV-2 C protein, as evident from molecular interactions and several time-dependent molecular dynamics-driven analyses. Moreover, this study was also supported by the thermodynamic binding free energy and steered molecular dynamics simulations. Therefore, we intend to suggest that the DSPD3 molecule could be used as a potential therapeutic molecule against dengue complications as compared to the cocrystallized inhibitor ST-148. However, further studies are required to demonstrate the ability of DSPD3 to induce DENV-2 C tetramer formation.

Targeting the Virus Capsid as a Tool to Fight RNA Viruses.

Several strategies have been developed to fight viral infections, not only in humans but also in animals and plants. Some of them are based on the development of efficient vaccines, to target the virus by developed antibodies, others focus on finding antiviral compounds with activities that inhibit selected virus replication steps. Currently, there is an increasing number of antiviral drugs on the market; however, some have unpleasant side effects, are toxic to cells, or the viruses quickly develop resistance to them. As the current situation shows, the combination of multiple antiviral strategies or the combination of the use of various compounds within one strategy is very important. The most desirable are combinations of drugs that inhibit different steps in the virus life cycle. This is an important issue especially for RNA viruses, which replicate their genomes using error-prone RNA polymerases and rapidly develop mutants resistant to applied antiviral compounds. Here, we focus on compounds targeting viral structural capsid proteins, thereby inhibiting virus assembly or disassembly, virus binding to cellular receptors, or acting by inhibiting other virus replication mechanisms. This review is an update of existing papers on a similar topic, by focusing on the most recent advances in the rapidly evolving research of compounds targeting capsid proteins of RNA viruses.

New Progress in Pharmacochemistry of Anti Enterovirus 71

Although great progress has been made in the research and development of new drugs against enterovirus 71, the prevention and treatment of enterovirus 71 still relies on vaccines in most cases.Therefore, it is an urgent task to speed up the research and development of anti enterovirus 71 drugs. With the development and integration of multiple disciplines, the research and development directions of anti enterovirus 71 drugs are more diverse.At present, there are further research results on the crystal structure of capsid protein and some key proteases in clinical research. In the future development of anti enterovirus drugs, we can continue to design reasonably on this basis.In this paper, through consulting the relevant literature in recent years, the chemical research of antienterovirus 71 was summarized and analyzed, in order to provide reference for related people.

Cytotoxic CD4+ T-cells specific for EBV capsid antigen BORF1 are maintained in long-term latently infected healthy donors.

Epstein Barr Virus (EBV) infects more than 95% of the population whereupon it establishes a latent infection of B-cells that persists for life under immune control. Primary EBV infection can cause infectious mononucleosis (IM) and long-term viral carriage is associated with several malignancies and certain autoimmune diseases. Current efforts developing EBV prophylactic vaccination have focussed on neutralising antibodies. An alternative strategy, that could enhance the efficacy of such vaccines or be used alone, is to generate T-cell responses capable of recognising and eliminating newly EBV-infected cells before the virus initiates its growth transformation program. T-cell responses against the EBV structural proteins, brought into the newly infected cell by the incoming virion, are prime candidates for such responses. Here we show the structural EBV capsid proteins BcLF1, BDLF1 and BORF1 are frequent targets of T-cell responses in EBV infected people, identify new CD8+ and CD4+ T-cell epitopes and map their HLA restricting alleles. Using T-cell clones we demonstrate that CD4+ but not CD8+ T-cell clones specific for the capsid proteins can recognise newly EBV-infected B-cells and control B-cell outgrowth via cytotoxicity. Using MHC-II tetramers we show a CD4+ T-cell response to an epitope within the BORF1 capsid protein epitope is present during acute EBV infection and in long-term viral carriage. In common with other EBV-specific CD4+ T-cell responses the BORF1-specific CD4+ T-cells in IM patients expressed perforin and granzyme-B. Unexpectedly, perforin and granzyme-B expression was sustained over time even when the donor had entered the long-term infected state. These data further our understanding of EBV structural proteins as targets of T-cell responses and how CD4+ T-cell responses to EBV change from acute disease into convalescence. They also identify new targets for prophylactic EBV vaccine development.