Glycoproteins G1 Research Articles

Functionalized PLGA-Based Nanoparticles with Anti-HSV-2 Human Monoclonal Antibody: A Proof of Concept for Early Diagnosis and Targeted Therapy.

Background: Functionalized nanoparticles (NPs) represent a cutting edge in innovative clinical approaches, allowing for the delivery of selected compounds with higher specificity in a wider time frame. They also hold promise for novel theranostic applications that integrate both diagnostic and therapeutic functions. Pathogens are continuously evolving to try to escape the strategies designed to treat them. Objectives: In this work, we describe the development of a biotechnological device, Nano-Immuno-Probes (NIPs), for early detection and infections treatment. Human Herpes Simplex Virus 2 was chosen as model pathogen. Methods: NIPs consist of PLGA-PEG-Sulfone polymeric NPs conjugated to recombinant Fab antibody fragments targeting the viral glycoprotein G2. NIPs synthesis involved multiple steps and was validated through several techniques. Results: DLS analysis indicated an expected size increase with a good polydispersity index. Z-average and z-potential values were measured for PLGA-PEG-Bis-Sulfone NPs (86.6 ± 10.9 nm; -0.7 ± 0.3 mV) and NIPs (151 ± 10.4 nm; -5.1 ± 1.9 mV). SPR assays confirmed NIPs' specificity for the glycoprotein G2, with an apparent KD of 1.03 ± 0.61 µM. NIPs exhibited no cytotoxic effects on VERO cells at 24 and 48 h. Conclusions: This in vitro study showed that NIPs effectively target HSV-2, suggesting the potential use of these nanodevices to deliver both contrast agents as well as therapeutic compounds.

An Immunoinformatics Approach to Design a Potent Multi-Epitope Vaccine against Asia-1 Genotype of Crimean-Congo Haemorrhagic Fever Virus Using the Structural Glycoproteins as a Target.

Crimean-Congo haemorrhagic fever (CCHF), caused by Crimean-Congo haemorrhagic fever virus (CCHFV), is a disease of worldwide importance (endemic yet not limited to Asia, Middle East, and Africa) and has triggered several outbreaks amounting to a case fatality rate of 10-40% as per the World Health Organization. Genetic diversity and phylogenetic data revealed that the Asia-1 genotype of CCHFV remained dominant in Pakistan, where 688 confirmed cases were reported between the 2012-2022 period. Currently, no approved vaccine is available to tackle the viral infection. Epitope-based vaccine design has gained significant attention in recent years due to its safety, timeliness, and cost efficiency compared to conventional vaccines. In the present study, we employed a robust immunoinformatics-based approach targeting the structural glycoproteins G1 and G2 of CCHFV (Asia-1 genotype) to design a multi-epitope vaccine construct. Five B-cells and six cytotoxic T-lymphocytes (CTL) epitopes were mapped and finalized from G1 and G2 and were fused with suitable linkers (EAAAK, GGGS, AAY, and GPGPG), a PADRE sequence (13 aa), and an adjuvant (50S ribosomal protein L7/L12) to formulate a chimeric vaccine construct. The selected CTL epitopes showed high affinity and stable binding with the binding groove of common human HLA class I molecules (HLA-A*02:01 and HLA-B*44:02) and mouse major histocompatibility complex class I molecules. The chimeric vaccine was predicted to be an antigenic, non-allergenic, and soluble molecule with a suitable physicochemical profile. Molecular docking and molecular dynamics simulation indicated a stable and energetically favourable interaction between the constructed antigen and Toll-like receptors (TLR2, TLR3, and TLR4). Our results demonstrated that innate, adaptive, and humoral immune responses could be elicited upon administration of such a potent muti-epitope vaccine construct. These results could be helpful for an experimental vaccinologist to develop an effective vaccine against the Asia-1 genotype of CCHFV.

Detection of Antibodies in Serum and Lymphocytes of Patients with Hemorraghic Fever with Renal Syndrome

Hemorrhagic Fever with Renal Syndrome (HFRS) is zoonotic disease, endemic in the republic of Tatarstan. Increased endothelial barrier permeability, resulting in tissue edema, leukocyte migration and hemorrhages, is characteristic pathological finding in HFRS. Cytokine storm hypothesis was suggested to explain hantavirus pathogenesis, where over activation of pro-inflammatory cytokines was suggested to cause endothelial cell damage and increased leukocyte infiltration of target tissue. Hantavirus nucleocapsid (N) protein and glycoprotein proteins (G1 and G2) were suggested to be the most immunogenic viral proteins. However, information on immunogenic epitopes triggering humoral and cellular immune response is limited.Materials and methods. Blood and serum samples were collected from HFRS patients admitted to Republican Clinical Hospital for Infectious Disease named after Agafonov, Republic of Tatarstan along with controls matched for the region. The Institutional Review Board of the Kazan Federal University approved this study and informed consent was obtained from each study subject according to the guidelines approved under this protocol (article 20, Federal Law “Protection of Health Rights of Citizens of Russian Federation” N323- FZ, 11.21.2011).Peptide library consisting of overlapping peptides (15 aa; 5 aa overlap) of N, G1 and G2 of Puumala virus (PPUV) were generated. Total of 150 peptides were tested. PUUV was selected because it is endemic in Republic of Tatarstan causing HFRS.Immunogenic epitopes inducing antibody response in HFRS were analyzed using direct ELISA. Peptides were used to coat the plate (1 µg/well), followed by incubation with HFRS serum. Control serum samples were used to determine peptide non-specific reactivity. Statistical analysis was conducted using Minitab software; differences between medians of HFRS and control groups were analyzed using the Mann-Whitney test for non-parametric data. Data was considered significant at p ≤ 0.05.T-lymphocyte epitopes were analyzed using ELISpot assay. Blood from HFRS cases was collected in vacuum tubes containing sodium citrate (Vacuutest, Apexmed). Confirmation of PUUV infection was done by PCR (n=10). Peripheral blood mononuclear cells (PBMC) were isolated using ficoll density gradient separation (p=1,077 g/l). Isolated mononuclear leukocytes were cultured in 96-well plates coated with anti-IFNγ antibody. Leukocytes were incubated with each peptide for 48 hours. PBMC treated with phytohaemoagglutinine (PHA) served as a positive control. Spots were visualized using AEC substrate and counted by direct microscopy.Results. PUUV RNA was detected in all blood samples. Serum samples from 10 HFRS cases and 10 controls were analyzed on reactivity to the overlapping peptides coding nucleocapsid and G1 and G2 glycoproteins. Reactivity of eleven peptides (150 total) was statistically significant in HFRS serum as compared to controls (Figures 1a, 1b). Interestingly, the intensity of reactivity between HFRS serum and glycoprotein peptides was 1.5±0.2 times higher than that of N protein.Several G and N protein peptides were identified as inducing IFN-γ production by leukocytes. These peptides are M82 (YTRKACIQLGTEQTC), M69 (TDLELDFSLPSSASY), N3 (ARQKLKDAERAVEVY), N2 (ITRHEQQLVVARQKL), N1 (MSDLTDIQEEITRHE) and M104 (NLVRGQNTVHIVGKG) (Figure 1c).Collected data revealed that the most immunogenic peptides were M104 (NLVRGQNTVHIVGKG) and N3 (ARQKLKDAERAVEVY) from hantavirus G and N proteins respectively. These peptides revealed strong reactivity in ELISA and ELISpot assays suggesting that they play role in activation of humoral and immune responses.Conclusion. Data confirms that both N and G proteins of PUUV are immunogenic. New epitopes were identified on N and G proteins which can stimulate humoral and cellular immunity.This work was supported by Russian Science Foundation grant №15-14-00016. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A novel genetic lineage of Tula orthohantavirus in Altai voles (Microtus obscurus) from Turkey

Orthohantaviruses (family Hantaviridae order Bunyavirales) are emerging pathogens with a significant impact on human health. They are transmitted via aerosolized excreta of rodents which also act as reservoir hosts, constituting a unique route for dispersion. Dobrava-Belgrade and Puumala orthohantaviruses have been previously reported from Anatolia, in rodents, case reports and occasional outbreaks. We have collected rodents at several locations during a surveillance study in eastern Anatolia. The specimens were morphologically-identified and various tissues were screened via a generic orthohantavirus reverse transcription polymerase chain reaction assay. DNA barcoding via mitochondrial cytochrome b sequencing was performed in rodents with detectable orthohantavirus sequences. High throughput sequencing was performed for viral genome characterization. Fifty rodents were collected and identified morphologically as Microtus spp. (96%) and Apodemus spp. (4%). Orthohantavirus sequences were detected in lung and spleen or liver tissues of 4 voles (8%), barcoded as Microtus obscurus. The virus sequences were identified as Tula orthohantavirus (TULV) and near-complete genomic segments of the prototype viral genome, tentatively named as the Tula orthohantavirus-Turkey (TULV-T), could be characterized. Putative open reading frames for viral nucleocapsid and a nonstructural protein on the S segment, glycoproteins G1 and G2 on the M segment and viral replicase on the L segment were identified on the TULV-T. Several minor sequence variants were further characterized. No evidence of recombination could be detected and pairwise comparisons displayed over 95% amino acid sequence identities to various Eurasian TULV strains. Phylogenetic analyses revealed distinct clustering of all genome segments from previously-characterized TULV strains via various approaches and models. Here, TULV-T constituted a novel lineage, forming an intermediate among Asian and European TULV lineages. This report describes the initial documentation of TULV circulation and its potential reservoir in Anatolia. The extent of virus dispersion, alternate hosts or outcomes of human exposure require elucidation.

Lujo virus: current concepts

Lujo virus: current concepts Nivesh H Sewlall,1,2 Janusz T Paweska2,3 1Department of Medicine, Morningside MediClinic, Johannesburg, South Africa; 2Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa; 3Department of Medicine, National Institute for Communicable Diseases of the National Health Laboratory Service, Johannesburg, South Africa Abstract: Lujo virus (LUJV), a novel Old World arenavirus, was found to cause a fulminant viral hemorrhagic fever syndrome in an outbreak in 2008. The primary patient was from Lusaka, Zambia, and subsequent nosocomial transmission occurred to four other patients in Johannesburg, South Africa, hence the name Lujo virus. Like all arenaviruses, LUJV is a segmented, single-stranded, negative-sense RNA virus. Genomic sequencing confirmed that LUJV G1 glycoprotein was novel, diverse and genetically equidistant from other arenaviruses. A clinical syndrome resembling severe, fulminant Lassa virus infection was responsible for a high case fatality rate of 80% (4/5 cases). This review describes briefly the clinical course of the disease, laboratory findings and diagnosis. Recent studies address the current aspects of epidemiology, pathophysiology and treatments, specifically comparing with Lassa virus. Keywords: Lujo virus, viral hemorrhagic fever, outbreak, epidemiology, pathophysiology, treatment

Linear Multiepitope (Glyco)peptides for Type-Specific Serology of Herpes Simplex Virus (HSV) Infections.

Detection of type-specific antibodies is an important and essential part of accurate diagnosis, even in silent carriers of herpes simplex virus (HSV)-1 (oral) and HSV-2 (genital) infections. Serologic assays that identify HSV-1 and HSV-2 type-specific antibodies have been commercially available for more than a decade but often face problems related to cross-reactivity and similar issues. Attempts to identify type-specific peptide epitopes for use in serology for both HSV-1 and HSV-2 have been limited. We recently demonstrated epitope mapping of envelope glycoprotein G2 and identified a type-specific glycopeptide epitope that broadly recognized HSV-2 infected individuals. In the present work we have performed a comprehensive glycopeptide synthesis and microarray epitope mapping of 14 envelope proteins from HSV-1 and HSV-2, namely, gB, gC, gD, gE, gG, gH, and gI, using sera from HSV-1- and HSV-2-infected individuals and control sera. Several unique type-specific peptide epitopes with high sensitivity were identified and synthesized as one large linear multiepitope sequence using microwave-assisted solid-phase (glyco)peptide synthesis. Microarray validation with clinically defined HSV and Varicella Zoster (VZV) sera confirmed excellent cumulative specificities and sensitivities.

In silico structural analysis of Hantaan virus glycoprotein G2 and conserved epitope prediction for vaccine development

<p>Hantaan virus (HNTV) is an etiological agent of potentially fatal hemorrhagic fever with renal syndrome (HFRS). The virus infects a large number of patients annually with a mortality rate more than 10%. However, no treatment option or vaccine is available against the virus. Between two envelope proteins, HNTV glycoprotein G2 has higher antigenicity making it a better target for vaccine development. However, 3-D structure of the protein is not available which is important for identifying epitopes that are essential for vaccine design. Therefore, this study was designed to predict a structural model of glycoprotein G2 and to predict peptide sequences for vaccine development containing conserved epitopes within the structure. Many of the physio-chemical and structural properties including secondary structure and di-sulfide linkage of the protein were predicted using a number of computational tools. <strong></strong>The 3D structure of the protein was modeled using I-TASSER online tool. The quality of the predicted models was evaluated with Ramachandran plot and Z-score. The structural and sequence information was used to predict B-cell and T-cell epitopes on glycoprotein G2. Using various bio-informatics and immuno-informatics tools, a total of 9 continuous B-cell and 22 T-cell epitopes were predicted having significant antigenicity. These antigenic epitopes were further analyzed for conservation and a total of 4 B-cells and 8 T-cell epitopes were found to be highly conserved in sequences from diverse origins. These epitopes revealed by the current study are recognized by immune system to protect host from HNTV infection can be potential targets for vaccine development.</p>

Immunoformatics and Molecular Simulation Study Unfold the Immunological Repertoire of Sabia Virus Glycoprotein G1

Sabia virus Glycoprotein G1 (GP1) is a peripheral membrane protein involved in viral adsorption and consequential pathogenic infection. Glycoprotein G1 (GP1) was subjected to immunoformatic and molecular simulation study to predict effective B-cell and T-cell epitopes with knowledge-based exploration of probable immunological responses the epitopes may elicit. The linear and conformational epitopes of GP1 for B-cell were predicted by immunoformatic tools housed in IEDB recourse. Both CD4+ and CD8+ T-cell epitopes were predicted exploiting IEDB recourse tools whereas CD8+ T cell epitopes were severed further by immune response evoking ability of epitope-MHC complex. Contribution of GP1 to innate antiviral response was evolved by interferon (IFN)-gamma inducing and imitation capacity by immunoformatic and docking study respectively. The results of the B-cell epitope analysis suggest the occurrence of potential linear and conformational epitope with cross-reactivity. A range of T-cell epitopes were assumed to be involved in MHC class I and MHC class II molecule dependent antigen presentation. Glycoprotein G1 (GP1) may induce the IFNgamma but its IFN-gamma mimicking ability (confirmed by docking study) that adduces a prompt immune defense. Therefore, inherent immunological repertoire of GP1 epitopes can be taken in to advantage in future immunization regiment development against Sabia virus.

Comparative performance of the Kalon and HerpeSelect enzyme-linked immunosorbant assays to determine the prevalence of herpes simplex virus type 2 in Papua New Guinea.

Background Infection with herpes simplex virus type 2 (HSV-2) is common worldwide and an important risk factor for HIV infection. Aetiological diagnosis of HSV-2 is typically determined with the use of commercially available type-specific enzyme-linked immunosorbent assays (ELISAs). This study aimed to determine the prevalence of HSV-2 among people attending sexual health clinics in the Highlands of Papua New Guinea. The study also aimed to compare the performance of two type-specific ELISA assays, the Kalon and HerpeSelect glycoprotein G2 assays, in this context. Participants were recruited as part of a longitudinal sexual health study. Participants attended four appointments over a 12-month period and had blood taken for HSV-2 serology at each time point. Both the Kalon and HerpeSelect assays were performed as per manufacturer's instructions. A total of 132 participants were tested for HSV-2 using the Kalon and HerpeSelect ELISAs. HSV-2 prevalence was 52% (95% CI, 43-60) and 61% (95% CI, 52-69) with Kalon and HerpeSelect assays respectively. There was high concordance (87%, ?=0.75, P<0.001, n=115) between the two assays at the manufacturer recommended index value cut-offs. For participants with discordant results at baseline, (n=16), three sero-conversions were observed over the 12-month period when sequential sera was tested. A high HSV-2 prevalence was observed in this clinic-based population. Our longitudinal data indicate the higher prevalence of HSV-2 detected with the HerpeSelect ELISA was likely due to false positives rather than a higher sensitivity in the early stages of infection.

The Major Determinant of Attenuation in Mice of the Candid1 Vaccine for Argentine Hemorrhagic Fever Is Located in the G2 Glycoprotein Transmembrane Domain

Candid1, a live-attenuated Junin virus vaccine strain, was developed during the early 1980s to control Argentine hemorrhagic fever, a severe and frequently fatal human disease. Six amino acid substitutions were found to be unique to this vaccine strain, and their role in virulence attenuation in mice was analyzed using a series of recombinant viruses. Our results indicate that Candid1 is attenuated in mice through a single amino acid substitution in the transmembrane domain of the G2 glycoprotein. This work provides insight into the molecular mechanisms of attenuation of the only arenavirus vaccine currently available.

Maporal Virus as a Surrogate for Pathogenic New World Hantaviruses and its Inhibition by Favipiravir

Pathogenic hantaviruses geographically distributed in the Old World cause haemorrhagic fever with renal syndrome (HFRS), whereas New World hantaviruses are the aetiological agents of hantavirus cardiopulmonary syndrome (HCPS). Ribavirin, a drug associated with toxicities, is presently indicated for treatment of HFRS, whereas treatment of the more frequently lethal HCPS is limited to supportive care. Because of the need for safe and effective antivirals to treat severe hantaviral infections, we evaluated favipiravir (T-705) against Dobrava and Maporal viruses as representative Old World and New World hantaviruses, respectively. Dobrava virus causes HFRS in Europe. Maporal virus (MPRLV), recently isolated from western Venezuela, is phylogenetically similar to Andes virus, the principal cause of HCPS in Argentina. Hantavirus replication in the presence of various inhibitors was measured by focus-forming unit assays and quantitative reverse transcriptase PCR. Phylogenetic relationships were assessed by the neighbour-joining and bootstrap consensus methods. Here, we show that infection of Vero E6 cells with MPRLV is dependent on β3 integrins, similar to that reported for pathogenic hantaviruses. Furthermore, by analysis of molecular determinants associated with the G1 glycoprotein cytoplasmic tail, we show the close genetic proximity of MPRLV to other HCPS-causing hantaviruses in these regions predictive of pathogenicity. We also demonstrate anti-hantavirus activity by favipiravir with inhibitory concentrations ranging from 65 to 93 μM and selectivity indices>50. Our data suggest that MPRLV may serve as a safer alternative to modelling infection caused by the highly lethal Andes virus and that hantaviruses are sensitive to the effects of favipiravir in cell culture.

Application and expression of HSV gG1 protein from a recombinant strain

According to the homologous sequence of glycoprotein G1 (gG1) genes from different strains of herpes simplex virus type 1 (HSV-1), a pair of primers was designed to amplify the gG1 gene fragment by PCR. Both the PCR product and the pGEX-4T-1 vector were digested with EcoR I and Sal I. The gG1 gene fragment was subcloned into the digested pGEX-4T-1 vector to construct a recombinant plasmid (pGEX-4T-1-gG1). The resultant plasmid was identified by dual-enzyme digestion and sequence analysis, and then transformed into Escherichia coli BL21 for expression under the induction of isopropyl β-D-1-thiogalactoside (IPTG). The expressed GST-gG1 fragment was detected by SDS-PAGE and purified by affinity chromatography. The properties of GST-gG1 fragment were evaluated by immunoblot analysis. Enzyme-linked immunosorbent assays (ELISAs) based on the GST-gG1 fragment were used for determining IgG or IgM to HSV-1. The GST-gG1 fragment-specific ELISA was also compared with ELISA with whole-HSV-1 antigen and commercial ELISA kits. The gG1-specific IgG and IFN-γ producing CD8+ T cells were induced in mice immunized with the GST-gG1 fragment. These results indicated that the GST-gG1 fragment could be used for replacing whole-virus antigen to detect IgM and IgG to HSV-1 in human sera, which provided a strategy for developing vaccines to protect HSV-1 infection using gG1 fragment.

Screening for HSV-2 Infection in STD Clinics and Beyond: A Few Answers But More Questions

Screening for HSV-2 Infection in STD Clinics and Beyond: A Few Answers But More Questions

A duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of California serogroup and Cache Valley viruses

A duplex TaqMan real-time reverse transcriptase polymerase chain reaction (PCR) assay was developed for the detection of California (CAL) serogroup viruses and Cache Valley virus (CVV), for use in human surveillance. The targets selected for the assay were the sequences encoding the nucleocapsid protein of CAL and the G1 glycoprotein of CVV. Conserved regions were selected by aligning genetic sequences from various strains available in the GenBank database. Primers and probes were selected in conserved regions. The assay sensitivity was 75 gene copies (gc)/reaction for CAL serogroup viruses and 30 gc/reaction for CVV. The performance of the assay was linear over at least 6 log 10 gc. The assay was specific, given that it did not cross-react with a variety of pathogens. It did, however, detect 11 viruses within the CAL serogroup and 12 CVV isolates. The use of an internal control ensured that possible inefficiency in nucleic acid extraction or PCR inhibition would be detected.

Identification of oligopeptides mimicking the receptor-binding domain of Hantaan virus envelope glycoprotein from a phage-displayed peptide library

Attachment of enveloped viruses to cells is triggered by the receptor-binding domain (RBD) on envelope glycoproteins (GP) binding to receptors located on the cell surface. To date, recognized receptors and RBD of hantaan virus (HTNV) have not been exactly defined. In this study, one monoclonal antibody (MAb) 3G1 possessing high neutralizing activity, which is directed against HTNV envelope glycoprotein G2, was used to determine the crucial motif of RBD. Peptide ligands binding to MAb 3G1 were selected from a 12 amino acid peptide library displayed on filamentous phages. After 3 rounds of selection, the binding capacity between phages and MAb 3G1 was examined byELISA. Afterwards the positive phage clones with high binding activity to MAb 3G1 were chosen and sequenced. The peptide sequences of positive phage clones were compared with that of HTNV 76-118 strain G2. A motif Y/F/WPW(X)HX1-2HY, aligned to the primary sequences of G2 96YPWHTAKCHY105, was identified from the peptide inserts in the 9 positive clones. Positive phages and synthesized peptide containing the motif were bound significantly to virus-susceptible cell (Vero-E6) membranes by ELISA and immunofluorescence assay, respectively. Therefore, the sequence on G2 between amino acid 96 and 105 may be a key motif of HTNV RBD recognized by viral receptors on target cell membranes. Further characterization of the motif would provide useful information in understanding of the cellular entry of HTNV.

Genetic Detection and Characterization of Lujo Virus, a New Hemorrhagic Fever–Associated Arenavirus from Southern Africa

Lujo virus (LUJV), a new member of the family Arenaviridae and the first hemorrhagic fever–associated arenavirus from the Old World discovered in three decades, was isolated in South Africa during an outbreak of human disease characterized by nosocomial transmission and an unprecedented high case fatality rate of 80% (4/5 cases). Unbiased pyrosequencing of RNA extracts from serum and tissues of outbreak victims enabled identification and detailed phylogenetic characterization within 72 hours of sample receipt. Full genome analyses of LUJV showed it to be unique and branching off the ancestral node of the Old World arenaviruses. The virus G1 glycoprotein sequence was highly diverse and almost equidistant from that of other Old World and New World arenaviruses, consistent with a potential distinctive receptor tropism. LUJV is a novel, genetically distinct, highly pathogenic arenavirus.

Serological microarray for detection of HSV-1, HSV-2, VZV, and CMV antibodies

The seroprevalence of human herpesviruses is high and reactivations occur frequently. A microarray was designed and tested for the detection of IgG and IgM antibodies for Puumala hantavirus (PUUV) and IgG antibodies against four herpesviruses. Initially, a microarray platform was set up using an unrelated in-house antigen, PUUV recombinant nucleocapsid protein, to optimize the protocol for the detection of antibodies. Detection of the four herpesviruses was set up in a microarray using the recombinant proteins of herpes simplex virus (HSV) glycoprotein G1 and G2, varicella-zoster virus (VZV) glycoprotein E, and cytomegalovirus (CMV) pp150 phosphoprotein. The results of the PUUV panel were in good agreement with the PUUV IgG immunofluorescent assay and IgM enzyme immunoassay (EIA). Seropositive and negative clinical reference panels were tested for herpesviruses by the serological microarray, and the results were compared to those of individual EIAs used for standard diagnostic purposes. The serologic microarray for HSV, VZV and CMV antibody detection gave good specificities for IgG. However, sensitivities of the assay varied depending on the herpesvirus detected. The serological microarray showed potential for screening purposes. The microarray based analyses were easy to perform, and HSV-1, HSV-2, VZV, and CMV antibodies could be detected on the same microarray.

Seroprevalence of Herpes Simplex Virus 1 and 2 in a Population-Based Cohort in Japan

BackgroundThere have been few population-based studies of the seroprevalence and correlates of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in Japan.MethodsWe enrolled a total of 1244 adults, aged 18–59 years, from a population-based cohort in southern Japan, and tested their serum samples using an ELISA kit containing HSV type-specific antigens to glycoproteins G1 and G2.ResultsThe seroprevalence of HSV-1 and HSV-2 was 55.4% and 7.4% in men and 63.3% and 9.3% in women, respectively. Overall, 4 percent of the participants (2.3% of men and 5.0% of women) were co-infected with HSV-1 and HSV-2. The seroprevalences of both HSV-1 and HSV-2 increased with age in both sexes, and were always higher among women than among men in each age bracket. The prevalence of HSV-2 infection among HSV-1 infected individuals was lower than that among uninfected individuals, both in men and women. Male current drinkers, and male and female current smokers, were more likely to be infected with HSV-1 and HSV-2, as compared to never drinkers and never smokers, respectively.ConclusionIt is hoped that the estimates produced in this study will help in understanding the burden of these infections in Japan.

Genetic immunization with Hantavirus vaccine combining expression of G2 glycoprotein and fused interleukin-2

In this research, we developed a novel chimeric HTNV-IL-2-G2 DNA vaccine plasmid by genetically linking IL-2 gene to the G2 segment DNA and tested whether it could be a candidate vaccine. Chimeric gene was first expressed in eukaryotic expression system pcDNA3.1 (+). The HTNV-IL-2-G2 expressed a 72 kDa fusion protein in COS-7 cells. Meanwhile, the fusion protein kept the activity of its parental proteins. Furthermore, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response.- The results showed that the chimeric gene could simultaneously evoke specific antibody against G2 glycoprotein and IL-2. And the immunized mice of every group elicited neutralizing antibodies with different titers. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to G2 and IL-2 were significantly higher than that of other groups. Our results suggest that IL-2-based HTNV G2 DNA can induce both humoral and cellular immune response specific for HTNV G2 and can be a candidate DNA vaccine for HTNV infection.

Investigating recombinant protein exudation from roots of transgenic tobacco

It is widely acknowledged that plant-made pharmaceuticals (PMPs) offer numerous benefits, including inexpensive production, biological safety and the facility for production at agricultural scale. At the same time, it is important to minimize any potential risk associated with this new technology, including the potential release of bioactive proteins into the environment. To address this issue, we studied transgenic Nicotiana benthamiana and Nicotiana tabacum plants expressing two recombinant single-chain variable fragment (scFv) antibodies, respectively scFvB9 and scFvH10. ScFvB9 was raised against glycoprotein G1 of Tomato spotted wilt virus (TSWV), and scFvH10 was raised against human tumor-associated antigen tenascin-C. Both antibodies were targeted to the secretory pathway using the N-terminal signal peptide from Phaseolus vulgaris polygalacturonase-inhibiting protein (PGIP), and scFvH10 carried in addition a C-terminal KDEL tetrapeptide for retention in the endoplasmic reticulum (ER). Sterile hydroponic cultures were established, allowing us to investigate whether scFvB9 and scFvH10 were present in root exudates. Intercellular fluids extracted from different plant tissues were analyzed by western blotting revealing the presence of scFvB9. Successful secretion of scFvB9 in hydroponic medium was also demonstrated, whereas no scFvH10 could be detected in the leaf, stem or root apoplast, nor secreted into the hydroponic medium. Our results show that scFvH10 release or diffusion from the roots of transgenic plants was not occurring, suggesting that the KDEL signal might contribute to the environmental biosafety of crops producing PMPs.