anti-VP7 Antibodies Research Articles

A comprehensive and single-use foot-and-mouth disease sero-surveillance prototype employing rationally designed multiple viral antigens

Foot-and-mouth disease (FMD) is a great havoc in agri-business-based countries like Bangladesh, for which existing detection system limits the identification and differentiation of serotypes. In this study, an engineered platform was introduced incorporating serotype-specific FMDV VP1 (structural), serotype-independent VP2 (structural) and 3AB (non-structural) proteins for holistic detection. VP1 sequences were engineered combining sequences of BAN/TA/Dh-301/2016 (serotype O), BAN/CH/Sa-304/2016 (serotype A) and BAN/DH/Sa-318/2016 (serotype Asia1). Consensus 3AB sequence was constructed from the selected prevalent viral genomes. Both VP1 and 3AB along with designed VP2 sequences were optimized for codon usage bias, stable mRNA, secondary and tertiary protein structure. Proteins were synthesized in pET-21a ( +) plasmid vector followed by transformation of Escherichia coli BL21(DE3) and IPTG-induced- expression. The western blot analysis of engineered proteins showed that purified VP1 prominently bound to anti-VP1 antibodies in vaccinated sera, whereas 3AB and VP2 bound anti-3AB and anti-VP2 antibodies, respectively from infected cattle sera, all previously collected during epidemiological investigation. Furthermore, dot blot hybridization confirmed efficient antibody capture ability of the membrane-immobilized proteins. This holistic diagnostic platform justifies a comprehensive prototype diagnostic kit that would be cost-effective and efficient for serotype specific and non-specific FMDV sero-surveillance.

Shedding Light on Viral Shedding: Novel Insights into Nuclear Assembly, Cytoplasmic Transformation and Extracellular Vesicle Release of the BK Virus.

Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.

Antibody-dependent enhancement and neutralization against CVB4 investigated in vitro and in silico through an agent-based model.

The infection with coxsackievirus B4 (CVB4) can be enhanced in vitro by antibodies directed against the viral capsid protein VP4. In peripheral blood mononuclear cells, antibody-dependent enhancement (ADE) of CVB4 infection leads to the production of interferon alpha (IFN-α). To investigate ADE of CVB4-induced production of IFN-α, an agent-based model was constructed with enhancing and neutralizing antibodies. The model recapitulates viral neutralization and ADE in silico. The enhancing and neutralizing activities of serum samples were evaluated in vitro to confront the model predictions with experimental results. Increasing the incubation time of CVB4 with serum samples improves virus neutralization in silico as well as in vitro. It also results in ADE at lower antibody numbers in silico, which is confirmed in vitro with IFN-α production at lower serum concentrations. Furthermore, incubation of CVB4 with serum at a low temperature does not induce IFN-α production in vitro. Thus, taken together our results suggest that enhancing antibodies bind cryptic epitopes, more accessible with longer incubation time and at higher temperature due to changes in capsid conformation, consistent with previous results indicating that enhancing antibodies are anti-VP4 antibodies.

Expression of VP1 protein of foot-and-mouth disease virus serotype O originated from Indonesia in mammalian cells as potential immunogen

In 2022, Indonesia experienced foot-and-mouth disease (FMD) outbreak resulting in the death of hundreds of cattle; after more than 30 years, the country maintained FMD-free status. The FMD viral polypeptides-1 (VP1) protein contains major antigenic sites, making the protein have an important role in diagnostic applications or vaccine development. In this study, a synthetic codon-optimized vp1 gene from FMD virus serotype O was integrated into pcDNA3.1 vector and transformed to Escherichia coli TOP10F’ for plasmid propagation. The pcDNA3.1-1D-VP1 FMDV plasmid was then verified using agarose gel, showing in 659 bp size of the DNA fragment. The VP1 recombinant plasmid was transfected into HEK293T cells in DMEM medium supplemented with 10% FBS. Protein profiles were determined with SDS-PAGE showing target protein at 33KDa. Protein expression was confirmed by in-cell western assay using anti-VP1 type O polyclonal antibody and IRDye® 800CW goat anti-rabbit IgG as the secondary antibody. The result revealed a high-intensity fluorescence signal, indicating a strong interaction between expressed protein and anti-VP1 antibody. Thus, the results of this study demonstrate the potential for VP1 protein to be used as an immunogen in vaccine or diagnostic development against FMD infection. Nonetheless, some additional studies should be conducted.

Epidemiology of bluetongue virus infection among small ruminants in Turkey: Seroprevalence and associated risk factors.

Bluetongue (BT) is an endemic disease of small ruminants in Turkey, and it has substantial socio-economic impact at national level. To reduce this impact, vaccination has been used for the control of BT but sporadic outbreaks have been reported. Although sheep and goat farming plays an important role in rural communities, little is known about the BT epidemiological situation in small ruminants in Turkey. Therefore, this study aimed to estimate the seroprevalence of the bluetongue virus (BTV) and to identify the potential risk factors associated with BTV seropositivity in small ruminants. This study was conducted in the Antalya Province in the Mediterranean region of Turkey, from June 2018 to June 2019. A total of 1026 blood samples, from clinically healthy goats (n=517) and sheep (n=509), obtained from randomly selected unvaccinated flocks (n=100) were tested for BTV anti-VP7 antibodies by using a competitive enzyme linked immunosorbent assay test. A questionnaire was administered to the flock owners to obtain data related to sampled flocks and animals. At the animal level, the true prevalence of BTV antibodies was 74.2% (n=651/1026, 95% CI = 70.7-77.7) with 85.3% (n=370/509, 95% CI = 80.6-89.9) seropositive sheep and 63.3% (n=281/517, 95% CI = 58.2-68.4) seropositive goats. The true flock-level seroprevalence of BTV was higher in goats (100.0%, 95% CI = 92.8-100.0) than in sheep (98.8%, 95% CI = 86.6-100.0). The intra-flock seroprevalence within seropositive flocks varied between 36.4% and 100%, with a mean value of 85.5% and 61.9% in sheep and goat flocks, respectively. The logistic regression model revealed that odds of seropositivity for sheep were significantly higher in female animals (OR: 1.8, 95% CI = 1.1-2.9), animals older than 24 months old (OR: 5.8, 95% CI = 3.1-10.8), Pirlak breed (OR: 3.3, 95% CI = 1.1-10.0) and Merino breed (OR: 4.9, 95% CI = 1.6-14.9), whereas for goats, it was higher in female animals (OR: 1.7, 95% CI = 1.0-2.6), animals older than 24 months old (OR: 4.2, 95% CI = 2.7-6.6) and Hair breed (OR: 5.6, 95% CI = 2.8-10.9). The use of insecticides was identified as a protective factor. The present study revealed that BTV infection is widespread in sheep and goats in the Antalya Province. It is recommended to implement biosecurity measures in flocks and use insecticides to mitigate the spread of infection and contact between hosts and vectors.

The role of the DE and EF loop of BKPyV VP1 in the serological cross-reactivity between subtypes

BK virus (BKPyV) is a causative agent of BKPyV-associated nephropathy and graft rejections in kidney transplant patients. It establishes persistent infection in the kidneys, which can lead to reactivation in an immunosuppressed state or transmission to kidney recipients. Complications in the case of donor-derived infections can be caused by differences between the four known BKPyV subtypes, as prior infection with one subtype does not guarantee protection against de novo infection with other subtypes. The recipient and donor pretransplant serotyping is not routinely performed since simple ELISA tests employing antigens derived from the major viral capsid protein 1 (VP1) are hindered by the high cross-reactivity of anti-VP1 antibodies against all subtypes. Identifying subtype-specific epitopes in VP1 could lead to the design of specific antigens and the improvement of serodiagnostics for kidney transplantation. We aimed to study the surface residues responsible for the interactions with the subtype-specific antibodies by focusing on the DE and EF loops of VP1, which have only a small number of distinct amino acid differences between the most common subtypes, BKPyV-I and BKPyV-IV.We designed two mutant virus-like particles (VLPs): we introduced BKPyV-I characteristic amino acid residues (either H139N in the DE loop or D175E and I178V changes in the EF loop) into the base sequence of a BKPyV-IV VP1. This way, we created BKPyV-IV mutant VLPs with the sequence of either the BKPyV-I DE loop or the BKPyV-I EF loop. These mutants were then used as competing antigens in an antigen competition assay with a panel of patient sera, and changes in antibody reactivity were assessed by ELISA.We found that the changes introduced into the BKPyV-IV VP1 EF loop restrict antibody recognition in most samples and that converting the BKPyV-IV DE loop into its BKPyV-I equivalent attracts anti-VP1 BKPyV-I antibodies. Although our results did not lead to the discovery of a subtype-specific epitope on the VP1, they suggested that the arrangement of the EF loop in VP1 might dictate the mode of interaction between virus and anti-VP1 antibodies in general and that the interactions between the antibodies and the viral capsid might be very complex. Consequently, an antigen competition assay as an assay to distinguish between BKPyV serotypes might prove difficult to interpret.

An Engineered Maturation Cleavage Provides a Recombinant Mimic of Foot-and-Mouth Disease Virus Capsid Assembly-Disassembly.

Picornavirus capsids are assembled from 60 copies of a capsid precursor via a pentameric assembly intermediate or ‘pentamer’. Upon completion of virion assembly, a maturation event induces a final cleavage of the capsid precursor to create the capsid protein VP4, which is essential for capsid stability and entry into new cells. For the picornavirus foot-and-mouth disease virus (FMDV), intact capsids are temperature and acid-labile and can disassemble into pentamers. During disassembly, capsid protein VP4 is lost, presumably altering the structure and properties of the resulting pentamers. The purpose of this study was to compare the characteristics of recombinant “assembly” and “disassembly” pentamers. We generated recombinant versions of these different pentamers containing an engineered cleavage site to mimic the maturation cleavage. We compared the sedimentation and antigenic characteristics of these pentamers using sucrose density gradients and reactivity with an antibody panel. Pentamers mimicking the assembly pathway sedimented faster than those on the disassembly pathway suggesting that for FMDV, in common with other picornaviruses, assembly pentamers sediment at 14S whereas only pentamers on the disassembly pathway sediment at 12S. The reactivity with anti-VP4 antibodies was reduced for the 12S pentamers, consistent with the predicted loss of VP4. Reactivity with other antibodies was similar for both pentamers suggesting that major antigenic features may be preserved between the VP4 containing assembly pentamers and the disassembly pentamers lacking VP4.

Humoral and cell-mediated immune responses to plant-produced African horse sickness virus VP7 quasi-crystals

African horse sickness (AHS) is a devastating viral disease affecting equines and has resulted in many disastrous epizootics. To date, no successful therapeutic treatment exists for AHS, and commercially used live-attenuated vaccines have various undesirable side effects. Previous studies have shown that mice inoculated with insoluble African horse sickness virus (AHSV) VP7 crystals are protected from live challenge with a lethal dose of AHSV. This study investigates the humoral and cell-mediated immune responses in guinea-pigs to a safer monovalent vaccine alternative based on AHSV-5 VP7 quasi-crystals produced in plants. Guinea-pigs received prime- and boost-inoculations of between 10 and 50 μg of purified plant-produced AHSV VP7. Western immunoblot analysis of the humoral response showed stimulation of high titres of anti-VP7 antibodies 28 days after the boost-inoculation in sera from three of the five experimental animals. In addition, RNA-seq transcriptome profiling of guinea-pig spleen-derived RNA highlighted thirty significantly (q ≤ 0.05) differentially expressed genes involved in innate and adaptive immunity. Differential expression of genes involved in Th1, Th2 and Th17 cell differentiation suggest a cell-mediated immune response to AHSV-5 VP7. Upregulation of several important cytokines and cytokine receptors were noted, including TNFSF14, CX3CR1, IFNLR1 and IL17RA. Upregulation of IL17RA suggests a Th17 response which has been reported as a key component in AHSV immunity. While further investigation is needed to validate these findings, these results suggest that AHSV-5 VP7 quasi-crystals produced in N. benthamiana are immunogenic and induce both humoral and cell-mediated responses.

Production and characterization of monoclonal antibodies specific for major capsid VP1 protein of trichodysplasia spinulosa-associated polyomavirus.

Trichodysplasia spinulosa-associated polyomavirus (TSPyV), a newly identified polyomavirus, has been implicated as a causative agent of trychodysplasia spinulosa (TS), a rare proliferative skin disease in severely immunocompromised hosts. Diagnosis using mAbs is a promising tool with high specificity towards the specific antigen. However, thus far, no suitable mAbs for diagnosing TS disease have been identified. In this study, mAbs specific for VP1 of TSPyV were developed and characterized. Wheat germ cell-free synthesized VP1 protein of TSPyV was used to immunize BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones resulted in selection of five strongly positive clones that produce mAbs that react with the TSPyV-VP1 antigen. Epitope mapping and bioinformatic analysis showed that these mAbs recognized epitopes located within highly conserved C-terminal region of all clinical isolates of TSPyV-VP1. Further, all these mAbs were highly effective for immunofluorescence and immunoprecipitation analysis. Three of the five mAbs exhibited no cross-reactivity with VP1 of other related polyomaviruses. In addition, one of our mAbs (#14) provided immunohistochemical staining of skin tissue of TS disease. It can be concluded that three of the mAbs in this panel of anti-VP1 antibodies may provide a useful set of tools for studying TSPyV infection and making the specific diagnosis.

The generation and characterisation of neutralising antibodies against the Theiler’s murine encephalomyelitis virus (TMEV) GDVII capsid reveals the potential binding site of the host cell co-receptor, heparan sulfate

The early stages of picornavirus capsid assembly and the host factors involved are poorly understood. Since the localisation of viral proteins in infected cells can provide information on their function, antibodies against purified Theiler's murine encephalomyelitis virus (TMEV) GDVII capsids were generated by immunisation of rabbits. The resultant anti-TMEV capsid antibodies recognised a C-terminal region of VP1 but not VP2 or VP3 by Western analysis. Examination of the sites of TMEV capsid assembly by indirect immunofluorescence and confocal microscopy showed that at 5h post infection, capsid signal was diffusely cytoplasmic with strong perinuclear staining and moved into large punctate structures from 6 to 8h post infection. A plaque reduction neutralisation assay showed that the anti-TMEV capsid antibodies but not anti-VP1 antibodies could neutralise viral infection in vitro. The VP1 C-terminal residues recognised by the anti-TMEV capsid antibodies were mapped to a loop on the capsid surface near to the putative receptor binding pocket. In silico docking experiments showed that the known TMEV co-receptor, heparan sulfate, interacts with residues of VP1 in the putative receptor binding pocket, residues of VP3 in the adjacent pit and residues of the adjoining VP1 C-terminal loop which is recognised by the anti-TMEV capsid antibodies. These findings suggest that the anti-TMEV capsid antibodies neutralise virus infection by preventing heparan sulfate from binding to the capsid. The antibodies produced in this study are an important tool for further investigating virus-host cell interactions essential to picornavirus assembly.

Immunogenicity of T7 bacteriophage nanoparticles displaying G-H loop of foot-and-mouth disease virus (FMDV)

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that causes severe economic losses worldwide. The G-H loop of the FMDV VP1 structural protein is the major neutralizing antigenic site. However, a fully protective G-H loop peptide vaccine requires the addition of promiscuous Th sites from a source outside VP1. Thus, we demonstrated the potential of T7 bacteriophage based nanoparticles displaying a genetically fused G-H loop peptide (T7-GH) as a FMDV vaccine candidate. Recombinant T7-GH phage was constructed by inserting the G-H loop coding region into the T7 Select 415-1b vector. Purified T7-GH phage nanoparticles were analyzed by SDS-PAGE, Western blot and Dot-ELISA. Pigs seronegative for FMDV exposure were immunized with T7-GH nanoparticles along with the adjuvant Montanide ISA206, and two commercially available FMDV vaccines (InactVac and PepVac). Humoral and cellular immune responses, as well as protection against virulent homologous virus challenge were assessed following single dose immunization. Pigs immunized T7-GH developed comparable anti-VP1 antibody titers to PepVac, although lower LPBE titers than was induced by InactVac. Antigen specific lymphocyte proliferation was detected in T7-GH group similar to that of PepVac group, however, weaker than InactVac group. Pigs immunized with T7-GH developed a neutralizing antibody response stronger than PepVac, but weaker than InactVac. Furthermore, 80% (4/5) of T7-GH immunized pigs were protected from challenge with virulent homologous virus. These findings demonstrate that the T7-GH phage nanoparticles were effective in eliciting antigen specific immune responses in pigs, highlighting the value of such an approach in the research and development of FMDV vaccines.

MTase Domain of Dendrolimus punctatus cypovirus VP3 Mediates Virion Attachment and Interacts with Host ALP Protein.

Dendrolimus punctatus cypovirus (DpCPV) is an important pathogen of D. punctatus, but little is known about the mechanisms of DpCPV infection. Here, we investigated the effects of VP3, VP4 and VP5 structural proteins on the viral invasion. Both the C-terminal of VP3 (methyltransferase (MTase) domain) and VP4 (A-spike) bound to Spodoptera exigua midgut brush border membrane vesicles (BBMVs) in a dose-dependent manner, and the binding was inhibited by purified DpCPV virions. Importantly, anti-MTase and anti-VP4 antibodies inhibited viral binding to S. exigua BBMVs. Using far-Western blots, a 65 kDa protein in Bombyx mori BBMVs, identified as alkaline phosphatase protein (BmALP) by mass spectrometry, specifically interacted with DpCPV MTase. The interaction between MTase and BmALP was verified by co-immunoprecipitation in vitro. Pretreatment of B. mori BBMVs with an anti-ALP antibody or incubation of DpCPV virions with prokaryotically expressed BmALP reduced viral attachment. Additionally, BmALP inhibited DpCPV infection in S. exigua larvae. Our data provide evidence that the MTase domain and A-spike function as viral attachment proteins during the DpCPV infection process, and ALP is the ligand that interacts with DpCPV via the MTase domain. These results augment our understanding of the mechanisms used by cypoviruses to enter their hosts.

Engineering and expression of a human rotavirus candidate vaccine in Nicotiana benthamiana.

BackgroundHuman rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties.ResultsWe report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2.ConclusionOur results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher overall efficacy for the existing vaccines. The production of rotavirus proteins in plants would probably result in lower manufacturing costs, making it more affordable for developing countries. Further investigation is required to evaluate the immunogenic potential of the VLPs and fusion proteins created in this study.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0436-8) contains supplementary material, which is available to authorized users.

Development of a Specific Latex Agglutination Test to Detect Antibodies of Enterovirus 71

A latex agglutination test (LAT) was developed for the rapid detection of antibodies against the VP1 or VP1 proteins of Enterovirus 71 (EV71). The proteins of interest including prokaryotically expressed VP1 and two strains of anti-VP1 monoclonal antibody (McAb) against EV71 were covalently linked to carboxylated latex using ethyl-dimethyl-amino-propyl carbodiimide (EDC) to prepare sensitized latex beads. LAT was evaluated by an enzyme-linked immunosorbent assay (ELISA) as a reference test. The VP1-LAT showed a sensitivity of 87.0%, specificity of 88.9%, and an agreement ratio of 90.0% in detecting VP1 in 100 serum samples from experimentally infected mice, whereas these values were 86.8, 96.7, and 93.3%, respectively, for 608 clinical human serum samples. The VP1-LAT has advantages over other assays in terms of low cost, rapidity, chemical stability, high sensitivity, repeatability, and specificity. The LAT established in the present study is a rapid and simple test suitable for field monitoring of antibodies against VP1-EV71.

Monocytes of Patients with Type 1 Diabetes Harbour Enterovirus RNA.

Intracellular enterovirus (EV) RNA was detected in blood of patients with type 1 diabetes (T1D). The presence of EV RNA in subsets of peripheral blood mononuclear cells (PBMCs) of patients, and the in vitro infection of these cells with an EV, was investigated. Blood was collected from 42 patients with T1D, PBMCs were isolated and monocytes were purified. Interferon alpha (IFNα) mRNA and EV RNA were investigated using RT-PCR. Levels of IFNα in plasma were measured using an immunoassay. Cells were inoculated with Coxsackievirus B4 (CBV4) in vitro, and infection was assessed by indirect immunofluorescence (IFI). Interferon alpha mRNA was detected in blood and in monocytes of 12 of 42 patients with T1D, but not in monocyte-depleted PBMCs of the same individuals. Significant plasma levels of IFNα (≥ 5 IU/mL) were found in six patients. EV RNA was detected in whole blood and in monocytes of seven patients and negative-strand EV RNA was found in monocytes of 6 of them. When monocytes of patients with IFNα and/or EV RNA in their blood were inoculated with CVB4, the proportion of cells stained by an anti-VP1 antibody was 8.8 ± 1%, whereas no VP1 was detected in the monocytes of IFNα, EV RNA negative patients. Nevertheless, when CBV4 was mixed with plasma, VP1 was detected in monocytes of all patients with T1D (staining ranging from 12 to 36%). Our data indicate that monocytes of patients with T1D can harbor EV RNA and IFNα mRNA and can be infected with an EV in vitro.

Identification of the neutralizing epitopes of Merkel cell polyomavirus major capsid protein within the BC and EF surface loops.

Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops.

Development of a Specific Latex Agglutination Test to Detect Antibodies of Enterovirus 71

A latex agglutination test (LAT) was developed for the rapid detection of antibodies against the VP1 or VP1 proteins of Enterovirus 71 (EV71). The proteins of interest, including prokaryotically expressed VP1 and two strains of anti-VP1 Monoclonal antibody (McAb) against EV71 were covalently linked to carboxylated latex using Ethyl-dimethyl-aminopropyl carbodiimide (EDC) to prepare sensitized latex beads. The LAT was evaluated by an enzyme-linked immunosorbent assay (ELISA) as a reference test. The VP1-LAT showed a sensitivity of 87.0%, a specificity of 88.9%, and an agreement ratio of 90.0% in detecting VP1 in 100 serum samples from experimentally infected mice, while these values were 86.8%, 96.7%, and 93.3%, respectively, for 608 clinical human serum samples. The VP1-LAT has advantages over other assays in terms of low cost, rapidity, chemical stability, high sensitivity, repeatability, and specificity. The LAT established in the present study is a rapid and simple test suitable for field monitoring of antibodies against VP1-EV71.

T Cell Epitope Mapping of JC Polyoma Virus-Encoded Proteome Reveals Reduced T Cell Responses in HLA-DRB1*04:01+Donors

JC polyomavirus (JCV) infection is highly prevalent and usually kept in a persistent state without clinical signs and symptoms. It is only during immunocompromise and especially impaired CD4(+) T cell function in the brain, as seen in AIDS patients or natalizumab-treated multiple sclerosis patients, that JCV may cause progressive multifocal leukoencephalopathy (PML), an often life-threatening brain disease. Since CD4(+) T cells likely play an important role in controlling JCV infection, we here describe the T cell response to JCV in a group of predominantly HLA-DR-heterozygotic healthy donors (HD) by using a series of overlapping 15-mer peptides spanning all JCV-encoded open reading frames. We identified immunodominant epitopes and compared T cell responses with anti-JCV VP1 antibody production and with the presence of urinary viral shedding. We observed positive JCV-specific T cell responses in 28.6% to 77.6%, humoral immune response in 42.6% to 89.4%, and urinary viral shedding in 36.4% to 45.5% of HD depending on the threshold. Four immunodominant peptides were mapped, and at least one immunogenic peptide per HLA-DRB1 allele was detected in DRB1*01(+), DRB1*07(+), DRB1*11(+), DRB1*13(+), DRB1*15(+), and DRB1*03(+) individuals. We show for the first time that JCV-specific T cell responses may be directed not only against JCV VP1 and large T antigen but also against all other JCV-encoded proteins. Heterozygotic DRB1*04:01(+) individuals showed very low T cell responses to JCV together with normal anti-VP1 antibody levels and no urinary viral shedding, indicating a dominant-negative effect of this allele on global JCV-directed T cell responses. Our data are potentially relevant for the development of vaccines against JCV.

Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice

Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni–NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.

Reduced rhinovirus-specific antibodies are associated with acute exacerbations of chronic obstructive pulmonary disease requiring hospitalisation

BackgroundAcute exacerbations of chronic obstructive pulmonary disease (AECOPD) are often linked to respiratory infections. However, it is unknown if COPD patients who experience frequent exacerbations have impaired humoral immunity. The aim of this study was to determine if antibodies specific for common respiratory pathogens are associated with AECOPD.MethodsPlasma was obtained from COPD patients when clinically stable. AECOPD requiring hospitalisation were recorded. IgG1 antibodies to H. Influenzae outer membrane protein 6 (P6), pneumococcal surface protein C (PspC) and the VP1 viral capsid protein of rhinovirus were measured.ResultsCOPD patients who had an AECOPD (n = 32) had significantly lower anti-VP1 IgG1 antibody levels when stable compared to COPD patients who did not have an AECOPD (n = 28, p = 0.024). Furthermore, the number of hospitalisations was inversely proportional to anti-VP1 antibody levels (r = −0.331, p = 0.011). In contrast, antibodies specific for P6 and PspC were present at similar concentrations between groups. Plasma IL-21, a cytokine important for B-cell development and antibody synthesis, was also lower in COPD patients who had an AECOPD, than in stable COPD patients (p = 0.046).ConclusionDeficient humoral immunity specific for rhinoviruses is associated with AECOPD requiring hospitalisation, and may partly explain why some COPD patients have an increased exacerbation risk following respiratory viral infections.