Enhancement of protective humoral immune responses against Herpes simplex virus-2 in DNA-immunized guinea-pigs using protein boosting

Genital Herpes is a common sexually transmitted disease that is caused mostly by Herpes simplex virus type 2 (HSV-2). Its prevalence has increased in developing countries in spite of the availability of valuable antiviral drug therapy. Considering the importance of HSV-2 infections, effective vaccines remain the most likely hope for controlling the spread of HSV diseases. In the present study, the complete HSV-2 glycoprotein D gene was isolated and cloned into different plasmid vectors to construct a DNA vaccine and prepare recombinant subunit vaccines using a baculovirus expression system. The vaccines were tested alone or in combination to evaluate their ability to induce protective immunity in guinea-pigs against genital HSV infections. Immunization elicited humoral responses as measured by neutralization tests and enzyme-linked immunosorbent assay, and immunized animals had less severe genital skin disease as well as reduced replication of the challenging virus in the genital tract during experimental infection. Our results further demonstrate that DNA priming-protein boosting induced a neutralizing antibody titer higher than that obtained with DNA-DNA vaccination. The massive increase of antibody titer following DNA priming-protein boosting might be attributed to a recall of B cell memory.     

Inducing Ni sensitivity in the Ni hyperaccumulator plant <Emphasis Type="Italic">Alyssum inflatum</Emphasis> Nyárády (Brassicaceae) by transforming with <Emphasis Type="Italic">CAX1</Emphasis>, a vacuolar membrane calcium transporter

The importance of calcium in nickel tolerance was studied in the nickel hyperaccumulator plant Alyssum inflatum by gene transformation of CAX1, a vacuolar membrane transporter that reduces cytosolic calcium. CAX1 from Arabidopsis thaliana with a CaMV35S promoter accompanying a kanamycin resistance gene was transferred into A. inflatum using Agrobacterium tumefaciens. Transformed calli were sub-cultured three times on kanamycin-rich media and transformation was confirmed by PCR using a specific primer for CAX1. At least 10 callus lines were used as a pool of transformed material. Both transformed and untransformed calli were treated with varying concentrations of either calcium (1–15 mM) or nickel (0–500 µM) to compare their responses to those ions. Increased external calcium generally led to increased callus biomass, however, the increase was greater for untransformed callus. Further, increased external calcium led to increased callus calcium concentrations. Transformed callus was less nickel tolerant than untransformed callus: under increasing nickel concentrations callus relative growth rate was significantly less for transformed callus. Transformed callus also contained significantly less nickel than untransformed callus when exposed to the highest external nickel concentration (200 µM). We suggest that transformation with CAX1 decreased cytosolic calcium and resulted in decreased nickel tolerance. This in turn suggests that, at low cytosolic calcium concentrations, other nickel tolerance mechanisms (e.g., complexation and vacuolar sequestration) are insufficient for nickel tolerance. We propose that high cytosolic calcium is an important mechanism that results in nickel tolerance by nickel hyperaccumulator plants.     

The effects of BmNPV on biochemical changes in primary cultures of Bombyx mori embryonic tissue

The effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) on biochemical changes of TC-100 medium containing 10% fetal bovine serum (FBS) in embryonic primary cultures of silkworm was investigated. The primary cultures that reached 60% confluence were infected by 0.5, 1, and 2-ml viral inoculums (diluted with TC-100 medium representing multiplicity of infection (MOI) of 0.25, 0.5, and 1). Glucose, uric acid, urea, total protein, cholesterol, and alkaline phosphatase were measured in the medium of BmNPV-infected primary cultures. All biochemical compounds showed significant changes. Glucose decreased considerably by about 55 mg/ml, while different concentrations of the virus inoculums did not demonstrate significant differences among them. Total protein had only increased in 2 ml concentration and there were no changes in other concentrations. Uric acid as a by-product accumulated dramatically in all concentrations, while the amount of urea reduced in all treatments and this reduction was more evident in lower concentrations. Cholesterol consumption was high in cultures postinfection, while alkaline phosphatase (ALP) activity decreased in infected cells.     

Comparison of immune responses against FMD by a DNA vaccine encoding the FMDV/O/IRN/2007 VP1 gene and the conventional inactivated vaccine in an animal model

Foot-and-mouth disease virus (FMDV) is highly contagious and responsible for huge outbreaks among cloven hoofed animals. The aim of the present study is to evaluate a plasmid DNA immunization system that expresses the FMDV/O/IRN/2007 VP1 gene and compare it with the conventional inactivated vaccine in an animal model. The VP1 gene was sub-cloned into the unique Kpn I and BamH I cloning sites of the pcDNA3.1+ and pEGFP-N1 vectors to construct the VP₁ gene cassettes. The transfected BHKT7 cells with sub-cloned pEGFP-N1-VP1 vector expressed GFP-VP1 fusion protein and displayed more green fluorescence spots than the transfected BHKT7 cells with pEGFP-N1 vector, which solely expressed the GFP protein. Six mice groups were respectively immunized by the sub-cloned pcDNA3.1⁺-VP1 gene cassette as the DNA vaccine, DNA vaccine and PCMV-SPORT-GMCSF vector (as molecular adjuvant) together, conventional vaccine, PBS (as negative control), pcDNA3.1⁺ vector (as control group) and PCMV-SPORT vector that contained the GMCSF gene (as control group). Significant neutralizing antibody responses were induced in the mice which were immunized using plasmid vectors expressing the VP1 and GMCSF genes together, the DNA vaccine alone and the conventional inactivated vaccine (P<0.05). Co-administration of DNA vaccine and GMCSF gene improved neutralizing antibody response in comparison with administration of the DNA vaccine alone, but this response was the most for the conventional vaccine group. However, induction of humeral immunity response in the conventional vaccine group was more protective than for the DNA vaccine, but T-cell proliferation and IFN-γ concentration were the most in DNA vaccine with the GMCSF gene. Therefore the group that was vaccinated by DNA vaccine with the GMCSF gene, showed protective neutralizing antibody response and the most Th1 cellular immunity.     

The effect of DNA priming-protein boosting on enhancing humoral immunity and protecting mice against lethal HSV infections

Herpes simplex virus produces primary and latent infections with periodic recurrency. The prime-boost immunization strategies were studied using a DNA vaccine carrying the full-length glycoprotein D-1 gene and a baculovirus-derived recombinant glycoprotein D, both expressing herpes simplex virus glycoprotein D-1 protein. Immunization with recombinant DNAs encoding antigenic proteins could induce cellular and humoral responses by providing antigen expression in vivo. Higher immune response, however, occurred when the recombinant proteins followed DNA inoculation. While all groups of the immunized mice and positive control group could resist virus challenge, a higher virus neutralizing antibody level was detected in the animals receiving recombinant protein following DNA vaccination.     

A kinetic study of gamma interferon production in herpes simplex virus-1 DNA prime-protein boost regimen comparing to DNA or subunit vaccination

The vast majority of the world’s population is infected with Herpes simplex virus (HSV). Although antiviral therapy can reduce the incidence of reactivation and asymptomatic viral shedding and limits morbidity and mortality from active disease, it cannot cure infection. Therefore, the development of an effective vaccine is an important global health priority. In this study, the induction of IFN-γ production was compared in different herpes simplex virus 1 (HSV-1) vaccines. Glycoprotein D (gD1) as a major immunogenic HSV-1 glycoprotein was chosen to our study. Balb/c mice were administered with DNA vaccine encoding gD1, subunit glycoprotein vaccine including insect cells infected by a gD1 recombinant Baculovirus, prime DNA vaccine boosted by subunit glycoprotein vaccine, inactivated KOS strain as a positive control, pcDNA3 plasmid and Sf9 cells as negative controls. Evaluation tests showed that the amount of IFN-γ mRNA at 8, 16 and 32 hours after restimulation sharply decreased whereas, the IFN-γ protein level is significantly increased. Our results revealed that at 14 days after immunization IFN-γ secretion of stimulated cells in all of the vaccinated groups dramatically raised rather than secreted IFN-γ levels in mice that were analyzed at 7 days after vaccination. In comparison to other groups; Prime-Boost immunization dramatically caused vigorous and prompt IFN-γ production at 7 days after immunization and 8 hours after restimulation. The article is published in the original.     

Comparison between MDCK and MDCK-SIAT1 cell lines as preferred host for cell culture-based influenza vaccine production

Objectives

To evaluate MDCK and MDCK-SIAT1 cell lines for their ability to produce the yield of influenza virus in different Multiplicities of Infection.

Results

Yields obtained for influenza virus H1N1 grown in MDCK-SIAT1 cell was almost the same as MDCK; however, H3N2 virus grown in MDCK-SIAT1 had lower viral titers in comparison with MDCK cells. The optimized MOIs to infect the cells on plates and microcarrier were selected 0.01 and 0.1 for H1N1 and 0.001 and 0.01 for H3N2, respectively.

Conclusions

MDCK-SIAT1 cells may be considered as an alternative mean to manufacture cell-based flu vaccine, especially for the human strains (H1N1), due to its antigenic stability and high titer of influenza virus production.

     

Enhancement of osteogenic differentiation of adipose-derived stem cells by PRP modified nanofibrous scaffold

Recent developments in bone tissue engineering have paved the way for more efficient and cost-effective strategies. Additionally, utilization of autologous sources has been considered very desirable and is increasingly growing. Recently, activated platelet rich plasma (PRP) has been widely used in the field of bone tissue engineering, since it harbours a huge number of growth factors that can enhance osteogenesis and bone regeneration. In the present study, the osteogenic effects of PRP coated nanofibrous PES/PVA scaffolds on adipose-derived mesenchymal stem cells have been investigated. Common osteogenic markers were assayed by real time PCR. Alkaline phosphate activity, calcium deposition and Alizarin red staining assays were performed as well. The results revealed that the highest osteogenic differentiation occurred when cells were cultured on PRP coated PES/PVA scaffolds. Interestingly, direct application of PRP to culture media had no additive effects on osteogenesis of cells cultured on PRP coated PES/PVA scaffolds or those receiving typical osteogenic factors. The highest osteogenic effects were achieved by the simplest and most cost-effective method, i.e. merely by using PRP coated scaffolds. PRP coated PES/PVA scaffolds can maximally induce osteogenesis with no need for extrinsic factors. The major contribution of this paper to the current researches on bone regeneration is to suggest an easy, cost-effective approach to enhance osteogenesis via PRP coated scaffolds, with no additional external growth factors.     

Production of recombinant gG-1 protein of herpes simplex virus type 1 in a prokaryotic system in order to develop a type-specific enzyme-linked immunosorbent assay kit

The herpes simplex viruses are important causes of disease worldwide. Herpes simplex virus type 1 (HSV-1) is the primary cause of oral-facial and pharyngeal infections and may cause herpetic whitlow, eye infections as well as severe and sometimes dangerous infections of the eyes and brain. HSV-1 also accounts for 10-15% of all genital herpetic infections. Therefore, laboratory diagnosis of this virus and development of diagnostic serological techniques for HSV-1 is of particular importance. In the present study, pTrc His2A-gG1 plasmid, containing the full-length glycoprotein G (gG) protein, was produced in a prokaryotic system for the first time. Upon confirmation of a 37-kDa gG-1 protein production in a prokaryotic system based on western blotting and monoclonal antibodies, the protein was produced at a large scale and purified by ion-exchange chromatography using DEAE-sepharose. An HSV-1 type-specific diagnostic kit was designed and developed and the specificity and sensitivity of this kit were demonstrated to be 89.5% and 100%, respectively, as compared with a commercially available kit. A significant correlation was shown between the developed kit and the commercial kit.