A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors

Background

The gene delivery vector for DNA-based therapy should ensure its transfection efficiency and safety for clinical application. The Micro-Linear vector (MiLV) was developed to improve the limitations of traditional vectors such as viral vectors and plasmids.

Methods

The MiLV which contained only the gene expression cassette was amplified by polymerase chain reaction (PCR). Its cytotoxicity, transfection efficiency in vitro and in vivo, duration of expression, pro-inflammatory responses and potential application for Epstein-Barr virus (EBV) positive tumors were evaluated.

Results

Transfection efficiency for exogenous genes transferred by MiLV was at least comparable with or even greater than their corresponding plasmids in eukaryotic cell lines. MiLV elevated the expression and prolonged the duration of genes in vitro and in vivo when compared with that of the plasmid. The in vivo pro-inflammatory response of MiLV group was lower than that of the plasmid group. The MEKK1 gene transferred by MiLV significantly elevated the sensitivity of B95-8 cells and transplanted tumor to the treatment of Ganciclovir (GCV) and sodium butyrate (NaB).

Conclusions

The present study provides a safer, more efficient and stable MiLV gene delivery vector than plasmid. These advantages encourage further development and the preferential use of this novel vector type for clinical gene therapy studies.     

Safety and Tolerability of Conserved Region Vaccines Vectored by Plasmid DNA,Simian Adenovirus and Modified Vaccinia Virus Ankara Administered to Human Immunodeficiency Virus Type 1-Uninfected Adults in a Randomized,Single-Blind Phase I Trial

Trial Design

HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee) adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported.

Methods

Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination.

Results

Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1) and predominantly transient (<48 hours). Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range) of 633 (231-1533) post-vaccination, which is of no safety concern.

Conclusions

These data demonstrate safety and good tolerability of the pSG2.HIVconsv DNA, ChAdV63.HIVconsv and MVA.HIVconsv vaccines and together with their high immunogenicity support their further development towards efficacy studies.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01151319","term_id":"NCT01151319"}}NCT01151319     

Rapid high yield production of different glycoforms of Ebola virus monoclonal antibody

Background

Fc-glycosylation of monoclonal antibodies (mAbs) has profound implications on the Fc-mediated effector functions. Alteration of this glycosylation may affect the efficiency of an antibody. However, difficulties in the production of mAbs with homogeneous N-glycosylation profiles in sufficient amounts hamper investigations of the potential biological impact of different glycan residues.

Methodology/Principal Findings

Here we set out to evaluate a transient plant viral based production system for the rapid generation of different glycoforms of a monoclonal antibody. Ebola virus mAb h-13F6 was generated using magnICON expression system in Nicotiana benthamiana, a plant species developed for commercial scale production of therapeutic proteins. h-13F6 was co-expressed with a series of modified mammalian enzymes involved in the processing of complex N-glycans. Using wild type (WT) plants and the glycosylation mutant ΔXTFT that synthesizes human like biantennary N-glycans with terminal N-acetylglucosamine on each branch (GnGn structures) as expression hosts we demonstrate the generation of h-13F6 complex N-glycans with (i) bisected structures, (ii) core α1,6 fucosylation and (iii) β1,4 galactosylated oligosaccharides. In addition we emphasize the significance of precise sub Golgi localization of enzymes for engineering of IgG Fc-glycosylation.

Conclusion

The method described here allows the efficient generation of a series of different human-like glycoforms at large homogeneity of virtually any antibody within one week after cDNA delivery to plants. This accelerates follow up functional studies and thus may contribute to study the biological role of N-glycan residues on Fcs and maximizing the clinical efficacy of therapeutic antibodies.     

Immunogenicity of Live Attenuated B. pertussis BPZE1 Producing the Universal Influenza Vaccine Candidate M2e

Background

Intranasal delivery of vaccines directed against respiratory pathogens is an attractive alternative to parenteral administration. However, using this delivery route for inactivated vaccines usually requires the use of potent mucosal adjuvants, and no such adjuvant has yet been approved for human use.

Methodology/Principal Findings

We have developed a live attenuated Bordetella pertussis vaccine, called BPZE1, and show here that it can be used to present the universal influenza virus epitope M2e to the mouse respiratory tract to prime for protective immunity against viral challenge. Three copies of M2e were genetically fused to the N-terminal domain of filamentous hemagglutinin (FHA) and produced in recombinant BPZE1 derivatives in the presence or absence of endogenous full-length FHA. Only in the absence of FHA intranasal administration of the recombinant BPZE1 derivative induced antibody responses to M2e and effectively primed BALB/c mice for protection against influenza virus-induced mortality and reduced the viral load after challenge. Strong M2e-specific antibody responses and protection were observed after a single nasal administration with the recombinant BPZE1 derivative, followed by a single administration of M2e linked to a virus-like particle without adjuvant, whereas priming alone with the vaccine strain did not protect.

Conclusions/Significance

Using recombinant FHA-3M2e-producing BPZE1 derivatives for priming and the universal influenza M2e peptide linked to virus-like particles for boosting may constitute a promising approach for needle-free and adjuvant-free nasal vaccination against influenza.     

Plant-Generated Artificial Small RNAs Mediated Aphid Resistance

Background

RNA silencing is an important mechanism for regulation of endogenous gene expression and defense against genomic intruders in plants. This natural defense system was adopted to generate virus-resistant plants even before the mechanism of RNA silencing was unveiled. With the clarification of that mechanism, transgenic antiviral plants were developed that expressed artificial virus-specific hairpin RNAs (hpRNAs) or microRNAs (amiRNAs) in host plants. Previous works also showed that plant-mediated RNA silencing technology could be a practical method for constructing insect-resistant plants by expressing hpRNAs targeting essential genes of insects.

Methodology/Principal findings

In this study, we chose aphid Myzus persicae of order Hemiptera as a target insect. To screen for aphid genes vulnerable to attack by plant-mediated RNA silencing to establish plant aphid resistance, we selected nine genes of M. persicae as silencing targets, and constructed their hpRNA-expressing vectors. For the acetylcholinesterase 2 coding gene (MpAChE2), two amiRNA-expressing vectors were also constructed. The vectors were transformed into tobacco plants (Nicotiana tabacum cv. Xanti). Insect challenge assays showed that most of the transgenic plants gained aphid resistance, among which those expressing hpRNAs targeting V-type proton ATPase subunit E-like (V-ATPaseE) or tubulin folding cofactor D (TBCD) genes displayed stronger aphicidal activity. The transgenic plants expressing amiRNAs targeting two different sites in the MpAChE2 gene exhibited better aphid resistance than the plants expressing MpAChE2-specific hpRNA.

Conclusions/Significance

Our results indicated that plant-mediated insect-RNA silencing might be an effective way to develop plants resistant to insects with piercing-sucking mouthparts, and both the selection of vulnerable target genes and the biogenetic type of the small RNAs were crucial for the effectiveness of aphid control. The expression of insect-specific amiRNA is a promising and preferable approach to engineer plants resistant to aphids and, possibly, to other plant-infesting insects.     

Comparison on Virulence and Immunogenicity of Two Recombinant Vaccinia Vaccines,Tian Tan and Guang9 Strains,Expressing the HIV-1 Envelope Gene

Background

The vaccinia virus Guang9 strain (VG9), derived from the vaccinia virus Tian Tan strain (VTT) has been found to be less virulent than VTT.

Methodology/Principal Findings

To investigate whether VG9 could be a potential replicating virus vector, the TK genes in VG9 and VTT were replaced with the HIV-1 envelope gene via homologous recombination, resulting in the recombinant viruses, VG9-E and VTT-E. The biology, virulence, humoral and cellular immunological responses of VG9-E and VTT-E were evaluated. Our results indicated no obvious difference in range of host cells and diffusion between two recombinant viruses. Neurovirulence for VG9-E in weanling and suckling mice, and skin virulence in rabbits, were lower than that of VTT-E. The humoral immune responses, including binding antibody and neutralizing antibody responses, induced by VG9-E were not significantly different from those for VTT-E whilst IFN-γ response which represented cellular immune response induced by VG9-E was significantly higher than that did by VTT-E.

Conclusions/Significance

Our results indicated that VG9-E was less virulent, yet induced higher cellular immune response than VTT-E. Therefore, it could be an ideal replicating vaccinia vector for HIV vaccine research and development.     

Vertical Transmission of Respiratory Syncytial Virus Modulates Pre- and Postnatal Innervation and Reactivity of Rat Airways

Background

Environmental exposure to respiratory syncytial virus (RSV) is a leading cause of respiratory infections in infants, but it remains unknown whether this infection is transmitted transplacentally from the lungs of infected mothers to the offspring. We sought to test the hypothesis that RSV travels from the respiratory tract during pregnancy, crosses the placenta to the fetus, persists in the lung tissues of the offspring, and modulates pre- and postnatal expression of growth factors, thereby predisposing to airway hyperreactivity.

Methodology

Pregnant rats were inoculated intratracheally at midterm using recombinant RSV expressing red fluorescent protein (RFP). Viral RNA was amplified by RT-PCR and confirmed by sequencing. RFP expression was analyzed by flow cytometry and viral culture. Developmental and pathophysiologic implications of prenatal infection were determined by analyzing the expression of genes encoding critical growth factors, particularly neurotrophic factors and receptors. We also measured the expression of key neurotransmitters and postnatal bronchial reactivity in vertically infected lungs, and assessed their dependence on neurotrophic signaling using selective biological or chemical inhibition.

Principal Findings

RSV genome was found in 30% of fetuses, as well as in the lungs of 40% of newborns and 25% of adults. RFP expression was also shown by flow cytometry and replicating virus was cultured from exposed fetuses. Nerve growth factor and its TrkA receptor were upregulated in RSV- infected fetal lungs and co-localized with increased cholinergic innervation. Acetylcholine expression and smooth muscle response to cholinergic stimulation increased in lungs exposed to RSV in utero and reinfected after birth, and blocking TrkA signaling inhibited both effects.

Conclusions/Significance

Our data show transplacental transmission of RSV from mother to offspring and persistence of vertically transmitted virus in lungs after birth. Exposure to RSV in utero is followed by dysregulation of neurotrophic pathways predisposing to postnatal airway hyperreactivity upon reinfection with the virus.     

Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle

Background

Vaccination is a cost-effective counter-measure to the threat of seasonal or pandemic outbreaks of influenza. To address the need for improved influenza vaccines and alternatives to egg-based manufacturing, we have engineered an influenza virus-like particle (VLP) as a new generation of non-egg or non-mammalian cell culture-based candidate vaccine.

Methodology/Principal Findings

We generated from a baculovirus expression system using insect cells, a non-infectious recombinant VLP vaccine from both influenza A H5N1 clade 1 and clade 2 isolates with pandemic potential. VLPs were administered to mice in either a one-dose or two-dose regimen and the immune responses were compared to those induced by recombinant hemagglutinin (rHA). Both humoral and cellular responses were analyzed. Mice vaccinated with VLPs were protected against challenge with lethal reassortant viruses expressing the H5N1 HA and NA, regardless if the H5N1 clade was homologous or heterologous to the vaccine. However, rHA-vaccinated mice showed considerable weight loss and death following challenge with the heterovariant clade virus. Protection against death induced by VLPs was independent of the pre-challenge HAI titer or cell-mediated responses to HA or M1 since vaccinated mice, with low to undetectable cross-clade HAI antibodies or cellular responses to influenza antigens, were still protected from a lethal viral challenge. However, an apparent association rate of antibody binding to HA correlated with protection and was enhanced using VLPs, particularly when delivered intranasally, compared to rHA vaccines.

Conclusion/Significance

This is the first report describing the use of an H5N1 VLP vaccine created from a clade 2 isolate. The results show that a non-replicating virus-like particle is effective at eliciting a broadened, cross-clade protective immune response to proteins from emerging H5N1 influenza isolates giving rise to a potential pandemic influenza vaccine candidate for humans that can be stockpiled for use in the event of an outbreak of H5N1 influenza.     

Characterization of HCV interactions with Toll-like receptors and RIG-I in liver cells

Background and Aim

The aim of this study was to examine the mechanisms of IFN induction and viral escape. In order to accomplish the goal we compared our new hepatoma cell line LH86, which has intact TLR3 and RIG-I expression and responds to HCV by inducing IFN, with Huh7.5 cells which lack those features.

Methods

The initial interaction of LH86 cells, Huh7.5 cells or their transfected counter parts (LH86 siRIG-I, siTLR3 or siTLR7 and Huh7.5 RIG-I, TLR3 or TLR7) after infection with HCV (strain JFH-1) was studied by measuring the expression levels of IFNβ, TRAIL, DR4, DR5 and their correlation to viral replication.

Results

HCV replicating RNA induces IFN in LH86 cells. The IFN induction system is functional in LH86, and the expression of the RIG-I and TLR3 in LH86 is comparable to the primary hepatocytes. Both proteins appear to play important roles in suppression of viral replication. We found that innate immunity against HCV is associated with the induction of apoptosis by RIG-I through the TRAIL pathway and the establishment of an antiviral state by TLR3. HCV envelope proteins interfere with the expression of TLR3 and RIG-I.

Conclusion

These findings correlate with the lower expression level of PRRs in HCV chronic patients and highlight the importance of the PRRs in the initial interaction of the virus and its host cells. This work represents a novel mechanism of viral pathogenesis for HCV and demonstrates the role of PRRs in viral infection.     

MECP2 Isoform-Specific Vectors with Regulated Expression for Rett Syndrome Gene Therapy

Background

Rett Syndrome (RTT) is an Autism Spectrum Disorder and the leading cause of mental retardation in females. RTT is caused by mutations in the Methyl CpG-Binding Protein-2 (MECP2) gene and has no treatment. Our objective is to develop viral vectors for MECP2 gene transfer into Neural Stem Cells (NSC) and neurons suitable for gene therapy of Rett Syndrome.

Methodology/Principal Findings

We generated self-inactivating (SIN) retroviral vectors with the ubiquitous EF1α promoter avoiding known silencer elements to escape stem-cell-specific viral silencing. High efficiency NSC infection resulted in long-term EGFP expression in transduced NSC and after differentiation into neurons. Infection with Myc-tagged MECP2-isoform-specific (E1 and E2) vectors directed MeCP2 to heterochromatin of transduced NSC and neurons. In contrast, vectors with an internal mouse Mecp2 promoter (MeP) directed restricted expression only in neurons and glia and not NSC, recapitulating the endogenous expression pattern required to avoid detrimental consequences of MECP2 ectopic expression. In differentiated NSC from adult heterozygous Mecp2^tm1.1Bird+/− female mice, 48% of neurons expressed endogenous MeCP2 due to random inactivation of the X-linked Mecp2 gene. Retroviral MECP2 transduction with EF1α and MeP vectors rescued expression in 95–100% of neurons resulting in increased dendrite branching function in vitro. Insulated MECP2 isoform-specific lentiviral vectors show long-term expression in NSC and their differentiated neuronal progeny, and directly infect dissociated murine cortical neurons with high efficiency.

Conclusions/Significance

MeP vectors recapitulate the endogenous expression pattern of MeCP2 in neurons and glia. They have utility to study MeCP2 isoform-specific functions in vitro, and are effective gene therapy vectors for rescuing dendritic maturation of neurons in an ex vivo model of RTT.     

Rapid Accumulation of Virulent Rift Valley Fever Virus in Mice from an Attenuated Virus Carrying a Single Nucleotide Substitution in the M RNA

Background

Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, while in livestock it causes fever and high abortion rates.

Methodology/Principal Findings

Sequence analysis showed that a wild-type RVFV ZH501 preparation consisted of two major viral subpopulations, with a single nucleotide heterogeneity at nucleotide 847 of M segment (M847); one had a G residue at M847 encoding glycine in a major viral envelope Gn protein, while the other carried A residue encoding glutamic acid at the corresponding site. Two ZH501-derived viruses, rZH501-M847-G and rZH501-M847-A, carried identical genomic sequences, except that the former and the latter had G and A, respectively, at M847 were recovered by using a reverse genetics system. Intraperitoneal inoculation of rZH501-M847-A into mice caused a rapid and efficient viral accumulation in the sera, livers, spleens, kidneys and brains, and killed most of the mice within 8 days, whereas rZH501-M847-G caused low viremia titers, did not replicate as efficiently as did rZH501-M847-A in these organs, and had attenuated virulence to mice. Remarkably, as early as 2 days postinfection with rZH501-M847-G, the viruses carrying A at M847 emerged and became the major virus population thereafter, while replicating viruses retained the input A residue at M847 in rZH501-M847-A-infected mice.

Conclusions/Significance

These data demonstrated that the single nucleotide substitution in the Gn protein substantially affected the RVFV mouse virulence and that a virus population carrying the virulent viral genotype quickly emerged and became the major viral population within a few days in mice that were inoculated with the attenuated virus.     

Respiratory Syncytial Virus (RSV) RNA loads in peripheral blood correlates with disease severity in mice

Background

Respiratory Syncytial Virus (RSV) infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity.

Methods

Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood.

Results

RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes.

Conclusions

RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.     

Construction of an expression plasmid (vector) encoding Brucella melitensis outer membrane protein,a candidate for DNA vaccine

Background:

DNA vaccination with plasmid encoding bacterial, viral, and parasitic immunogens has been shown to be an attractive method to induce efficient immune responses. Bacteria of the genus Brucella are facultative intracellular pathogens for which new and efficient vaccines are needed.

Methods:

To evaluate the use of a DNA immunization strategy for protection against brucellosis, a plasmid containing the DNA encoding the Brucella melitensis (B. melitensis) 31 kDa outer membrane protein, as a potent immunogenic target, was constructed.

Results:

The constructed plasmid, pcDNA3.1+omp31, was injected intramuscularly into mice and the expression of omp31 RNA was assessed by RT-PCR. The integrity of the pcDNA3.1+omp31 construct was confirmed with restriction analysis and sequencing. Omp31 mRNA expression was verified by RT-PCR.

Conclusion:

Our results indicate that the pcDNA3.1+omp31 eukaryotic expression vector expresses omp31 mRNA and could be useful as a vaccine candidate.Key Words: Brucella melitensis, omp31, DNA Vaccine, pcDNA3.1     

Involvement of C4 Protein of Beet Severe Curly Top Virus (Family Geminiviridae) in Virus Movement

Background

Beet severe curly top virus (BSCTV) is a leafhopper transmitted geminivirus with a monopartite genome. C4 proteins encoded by geminivirus play an important role in virus/plant interaction.

Methods and Findings

To understand the function of C4 encoded by BSCTV, two BSCTV mutants were constructed by introducing termination codons in ORF C4 without affecting the amino acids encoded by overlapping ORF Rep. BSCTV mutants containing disrupted ORF C4 retained the ability to replicate in Arabidopsis protoplasts and in the agro-inoculated leaf discs of N. benthamiana, suggesting C4 is not required for virus DNA replication. However, both mutants did not accumulate viral DNA in newly emerged leaves of inoculated N. benthamiana and Arabidopsis, and the inoculated plants were asymptomatic. We also showed that C4 expression in plant could help C4 deficient BSCTV mutants to move systemically. C4 was localized in the cytosol and the nucleus in both Arabidopsis protoplasts and N. benthamiana leaves and the protein appeared to bind viral DNA and ds/ssDNA nonspecifically, displaying novel DNA binding properties.

Conclusions

Our results suggest that C4 protein in BSCTV is involved in symptom production and may facilitate virus movement instead of virus replication.     

An antiviral defense role of AGO2 in plants

Background

Argonaute (AGO) proteins bind to small-interfering (si)RNAs and micro (mi)RNAs to target RNA silencing against viruses, transgenes and in regulation of mRNAs. Plants encode multiple AGO proteins but, in Arabidopsis, only AGO1 is known to have an antiviral role.

Methodology/Principal Findings

To uncover the roles of specific AGOs in limiting virus accumulation we inoculated turnip crinkle virus (TCV) to Arabidopsis plants that were mutant for each of the ten AGO genes. The viral symptoms on most of the plants were the same as on wild type plants although the ago2 mutants were markedly hyper-susceptible to this virus. ago2 plants were also hyper-susceptible to cucumber mosaic virus (CMV), confirming that the antiviral role of AGO2 is not specific to a single virus. For both viruses, this phenotype was associated with transient increase in virus accumulation. In wild type plants the AGO2 protein was induced by TCV and CMV infection.

Conclusions/Significance

Based on these results we propose that there are multiple layers to RNA-mediated defense and counter-defense in the interactions between plants and their viruses. AGO1 represents a first layer. With some viruses, including TCV and CMV, this layer is overcome by viral suppressors of silencing that can target AGO1 and a second layer involving AGO2 limits virus accumulation. The second layer is activated when the first layer is suppressed because AGO2 is repressed by AGO1 via miR403. The activation of the second layer is therefore a direct consequence of the loss of the first layer of defense.     

GFAP-Driven GFP Expression in Activated Mouse Müller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 Vectors

Background

Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.

Methodology/Principal Findings

We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1^−/− retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.

Conclusions/Significance

Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.     

Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin

Background

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis.

Methodology/Principal Findings

The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus.

Conclusions/Significance

Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.     

Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide

Background and Aims

Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied.

Methods

Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin.

Results

The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease.

Conclusions

The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.