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1.
Vaccine ; 34(37): 4378-85, 2016 08 17.
Article in English | MEDLINE | ID: mdl-27452864

ABSTRACT

In vivo vaccination studies are conventionally conducted in a single mouse strain with results, only reflecting responses to a single immunogenetic background. We decided to examine the immune response to an HIV transgene (gag, pol and nef fusion protein) in 3 strains of mice (CBA, C57BL/6 and BALB/c) to determine the spectrum of responses and in addition to determine whether the serotype of the adenoviral vector used (ChAd3 and ChAd63) impacted the outcome of response. Our results demonstrated that all three strains of mice responded to the transgene and that the magnitude of responses were different between the strains. The C57BL/6 strain showed the lowest range of responses compared to the other strains and, very few responses were seen to the same peptide pool in all three strains of mice. In CBA and BALB/c mice there were significant differences in IFNγ production dependent on the adenoviral vector used. Our results suggest that employing a single strain of mouse may underestimate the efficacy and efficiency of vaccine products.


Subject(s)
AIDS Vaccines/immunology , HIV Antigens/immunology , Immunogenicity, Vaccine , T-Lymphocytes/immunology , Adenoviridae , Animals , Female , Haplotypes , Interferon-gamma/immunology , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Pan troglodytes , Transgenes , gag Gene Products, Human Immunodeficiency Virus/immunology , nef Gene Products, Human Immunodeficiency Virus/immunology , pol Gene Products, Human Immunodeficiency Virus/immunology
2.
Vaccine ; 33(51): 7283-7289, 2015 Dec 16.
Article in English | MEDLINE | ID: mdl-26546736

ABSTRACT

Adenoviruses have been shown to be both immunogenic and efficient at presenting HIV proteins but recent trials have suggested that they may play a role in increasing the risk of HIV acquisition. This risk may be associated with the presence of pre-existing immunity to the viral vectors. Chimpanzee adenoviruses (chAd) have low seroprevalence in human populations and so reduce this risk. ChAd3 and chAd63 were used to deliver an HIV gag, pol and nef transgene. ELISpot analysis of T cell responses in mice showed that both chAd vectors were able to induce an immune response to Gag and Pol peptides but that only the chAd3 vector induced responses to Nef peptides. Although the route of injection did not influence the magnitude of immune responses to either chAd vector, the dose of vector did. Taken together these results demonstrate that chimpanzee adenoviruses are suitable vector candidates for the delivery of HIV proteins and could be used for an HIV vaccine and furthermore the chAd3 vector produces a broader response to the HIV transgene.


Subject(s)
AIDS Vaccines/immunology , Adenoviruses, Simian/immunology , Drug Carriers , T-Lymphocytes/immunology , gag Gene Products, Human Immunodeficiency Virus/immunology , nef Gene Products, Human Immunodeficiency Virus/immunology , pol Gene Products, Human Immunodeficiency Virus/immunology , AIDS Vaccines/administration & dosage , AIDS Vaccines/genetics , Adenoviruses, Simian/genetics , Animals , Enzyme-Linked Immunospot Assay , Female , Genetic Vectors , Interferon-gamma/metabolism , Mice, Inbred C57BL , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , gag Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/genetics , pol Gene Products, Human Immunodeficiency Virus/genetics
3.
Gene Ther ; 21(10): 855-65, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24989814

ABSTRACT

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, the limited cargo capacity of AAV prevents their use for therapy of those inherited retinopathies (IRs) due to mutations in large (>5 kb) genes. Viral vectors derived from adenovirus (Ad), lentivirus (LV) and herpes virus (HV) can package large DNA sequences, but do not target efficiently retinal photoreceptors (PRs) where the majority of genes responsible for IRs are expressed. Here, we have evaluated the mouse retinal transduction profiles of vectors derived from 16 different Ad serotypes, 7 LV pseudotypes and from a bovine HV. Most of the vectors tested transduced efficiently the retinal pigment epithelium. We found that LV-GP64 tends to transduce more PRs than the canonical LV-VSVG, albeit this was restricted to a narrow region. We observed more extensive PR transduction with HdAd1, 2 and 5/F35++ than with LV, although none of them outperformed the canonical HdAd5 or matched the extension of PR transduction achieved with AAV2/8.


Subject(s)
Dependovirus/genetics , Herpesvirus 4, Bovine/genetics , Lentivirus/genetics , Retinal Pigment Epithelium/virology , Animals , Dependovirus/classification , Electroretinography , Epithelial Cells/virology , Genetic Vectors/administration & dosage , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Herpesvirus 4, Bovine/classification , Lentivirus/classification , Male , Mice , Mice, Inbred BALB C , Photoreceptor Cells, Vertebrate/metabolism , Retinal Pigment Epithelium/cytology , Transduction, Genetic
4.
Infect Immun ; 78(11): 4601-12, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20713623

ABSTRACT

Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.


Subject(s)
DNA Viruses/genetics , Erythrocytes/parasitology , Genetic Vectors , Malaria Vaccines , Malaria, Falciparum/prevention & control , Merozoite Surface Protein 1/metabolism , Adenoviruses, Human/genetics , Adenoviruses, Simian/genetics , Animals , Antibodies, Protozoan/blood , Chick Embryo , Drug Design , Female , Humans , Immunization , Immunization, Secondary , Malaria Vaccines/administration & dosage , Malaria Vaccines/genetics , Malaria Vaccines/immunology , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Merozoite Surface Protein 1/genetics , Merozoite Surface Protein 1/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Plasmodium falciparum/immunology , T-Lymphocytes/immunology , Vaccinia virus/genetics
5.
Gene Ther ; 13(14): 1088-96, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16554842

ABSTRACT

Efficient vaccination against viral agents requires a strong T-cell-mediated immune response to clear viral-infected cells. Optimal vaccination can be achieved by administration of recombinant viral vectors encoding phatogen antigens. Adenoviral vectors have attracted considerable attention as potential viral vectors for genetic vaccination owing to their favorable safety profile and potent transduction efficiency following intramuscular injection. However, the neutralizing antibody response against adenoviral capsid proteins following adenoviral vectors injection limits the success of vaccination protocols based on multiple administrations of the same adenoviral serotype. In this work, we describe efficient immunization of rhesus macaques, the preferred model for preclinical assessment, with an HCV candidate vaccine by heterologous priming-boosting with adenoviral vectors based on different serotypes. The induced responses are broad and show significant cross-strain reactivity. Boosting can be delayed for over 2 years after priming, indicating that there is long-term maintenance of resting memory cells.


Subject(s)
Genetic Therapy/methods , Genetic Vectors/administration & dosage , Hepacivirus/genetics , Hepatitis C/prevention & control , Viral Hepatitis Vaccines/administration & dosage , Adenoviridae/genetics , Animals , Antibodies, Viral/analysis , Genetic Engineering , Genetic Vectors/genetics , Genotype , Hepacivirus/immunology , Hepatitis C/immunology , Humans , Immunization Schedule , Immunization, Secondary , Interferon-gamma/immunology , Macaca mulatta , Mice , Mice, Inbred C57BL , T-Lymphocytes/immunology , Viral Hepatitis Vaccines/genetics
6.
Proc Natl Acad Sci U S A ; 96(6): 2615-20, 1999 Mar 16.
Article in English | MEDLINE | ID: mdl-10077559

ABSTRACT

Adenovirus (Ad) and adeno-associated virus (AAV) have attractive and complementary properties that can be exploited for gene transfer purposes. Ad vectors are probably the most efficient vehicles to deliver foreign genes both in vitro and in vivo. AAV exhibits the unique ability to establish latency by efficiently integrating at a specific locus of human chromosome 19 (AAVS1). Two viral elements are necessary for the integration at AAVS1: Rep68/78 and the inverted terminal repeats (AAV-ITRs). In this study, we report the development of two helper-dependent adenoviral (HD) vectors, one carrying the Rep78 gene, the other an AAV-ITR-flanked transgene. Although Rep proteins have been demonstrated to interfere with Ad replication, HD Rep78 vector was successfully amplified on serial passages in 293CRE4 cells with a yield of 50-100 transducing units per cell. DNA integration at the AAVS1 site also was demonstrated in hepatoma cells coinfected with the HD-expressing Rep78 and with the second HD vector carrying a transgene flanked by AAV-ITRs. The high transduction efficiency, large cloning capacity, and high titer of the HD, combined with the site-specific integration machinery provided by AAV-derived components, make the Ad/AAV hybrid viruses a promising vehicle for gene therapy.


Subject(s)
Adenoviridae , DNA-Binding Proteins , Dependovirus , Gene Transfer Techniques , Genetic Vectors , Base Sequence , DNA Helicases/genetics , HeLa Cells , Humans , In Situ Hybridization , Molecular Sequence Data , Reassortant Viruses , Trans-Activators/genetics
7.
Anal Biochem ; 266(2): 192-7, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9888975

ABSTRACT

A novel radiometric in vitro assay for discovery of inhibitors of hepatitis C viral protease activity, suitable for high-throughput screening, was developed. The NS3 protein of hepatitis C virus (HCV) contains a serine protease, whose function is to process the majority of the nonstructural proteins of the viral polyprotein. The viral NS4A protein is a cofactor of NS3 protease activity in the cleavage of NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions. To establish an in vitro assay system we used NS3 proteases from different HCV strains, purified from Escherichia coli and a synthetic radiolabeled peptide substrate that mimics the NS4A-NS4B junction. Upon incubation with the enzyme the substrate was separated from the radiolabeled cleavage product by addition of an ion exchange resin. The assay was performed in a microtiter plate format and offered the potential for assaying numerous samples using a laboratory robot. Taking advantage of these features, we used the assay to optimize reaction conditions by simultaneously varying different buffer components. We showed that physicochemical conditions affect NS3 protease activity in a strain-specific way. Furthermore, the sensitivity of the assay makes it suitable for detection and detailed mechanistic characterization of inhibitors with low-nanomolar affinities for the HCV serine protease.


Subject(s)
Radiometry/methods , Viral Nonstructural Proteins/analysis , Buffers , Endopeptidases/metabolism , Kinetics , Peptides/pharmacology , Protease Inhibitors/pharmacology , Sensitivity and Specificity , Time Factors , Viral Nonstructural Proteins/antagonists & inhibitors
8.
J Virol ; 66(11): 6819-23, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1357192

ABSTRACT

The hepatitis B virus genome contains a unique polyadenylation (TATAAA) signal which is differentially utilized in the formation of the various hepatitis B virus transcripts. A head-to-tail multiple-copy insertion of a viral fragment comprising the viral enhancer, the X promoter, the X open reading frame, and the viral poly(A) signal in transgenic mice allowed us to monitor tissue-specific differences in the expression of transcripts initiating from the X promoter. These transcripts are efficiently processed at the first polyadenylation site in the liver, while in the kidney, the brain, and the testis, a portion of the transcripts covers two copies of the transgene, since only the second polyadenylation site is properly recognized. As discussed in this article, this observation suggests a tissue-specific distribution of cellular factors involved in polyadenylation.


Subject(s)
Hepatitis B virus/genetics , Poly A/biosynthesis , RNA Processing, Post-Transcriptional , RNA, Messenger/biosynthesis , Regulatory Sequences, Nucleic Acid/genetics , Animals , Chromosome Mapping , Gene Expression Regulation, Viral , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Open Reading Frames , Structure-Activity Relationship , Tissue Distribution
9.
J Clin Pathol ; 42(11): 1206-10, 1989 Nov.
Article in English | MEDLINE | ID: mdl-2584433

ABSTRACT

A rapid and simplified technique for detecting hepatitis B virus (HBV) DNA by spot hybridisation in the sera of patients with different clinical forms of HBV infection was investigated using enzyme conjugated synthetic oligodeoxyribonucleotides as probes. These are able to hybridize to the S and C regions of the HBV L(-) DNA strand. When compared with a complete 32P-labelled HBV DNA probe, the synthetic oligonucleotides provided a sensitive and quick method for the routine survey of HBV infection. Moreover, the DNA extraction procedure used allowed the spot hybridisation technique to be applied and read easily and the results obtained within a few hours. It is concluded that synthetic cold probes can be used in hybridisation assays HBV DNA detection as part of current clinical laboratory procedures.


Subject(s)
DNA Probes , DNA, Viral/blood , Hepatitis B virus/genetics , Oligodeoxyribonucleotides , Carrier State/blood , Carrier State/therapy , Hepatitis B/blood , Hepatitis B/diagnosis , Hepatitis B/therapy , Humans , Interferon Type I/therapeutic use , Nucleic Acid Hybridization , Recombinant Proteins
10.
J Med Virol ; 28(3): 169-75, 1989 Jul.
Article in English | MEDLINE | ID: mdl-2754427

ABSTRACT

To clarify the relationship between the pre-S antigens and other serological markers of hepatitis B virus (HBV) replication, we followed up 27 patients: 21 presented with symptoms of acute hepatitis (two progressed to chronicity) and six suffered from chronic hepatitis. Pre-S1, pre-S2, HBV DNA, IgM antihepatitis core antigen (HBc), hepatitis B e antigen (HBeAg), and anti-HBe were detected in about 200 sera serially collected at different times for at least 6-12 months from the onset of clinical observation. In the early symptomatic phase of acute hepatitis, the pre-S1 and pre-S2 antigens were present in 95% of the cases and correlated well with high levels of alanine-transferase (ALT) and IgM anti-HBc, while HBV DNA was present in the sera of only six (28.6%) patients (P less than 0.0001). This was the first marker to disappear (1 month after the initial stage). All of the HBV DNA-positive patients were also HBeAg positive, whereas no HBeAg-negative subjects were found with serum HBV DNA. In the six chronic patients, pre-S antigens were always present independently of the HBeAg/anti-HBe status; HBV DNA was detected in three of them, even if transiently, and in two of these it reappeared together with pre-S2 epitope. The follow-up data suggest that, in acute hepatitis, the clearance of pre-S antigens can be considered as a prognostic index of clinical resolution and that, in chronic hepatitis, the persistence of pre-S antigens seems to indicate progression of the disease. In particular, pre-S2, in patients in whom it is intermittent, can be considered as an index of reactivation.


Subject(s)
DNA, Viral/analysis , Hepatitis B Surface Antigens/analysis , Hepatitis B/immunology , Protein Precursors/analysis , Acute Disease , Biomarkers , Enzyme-Linked Immunosorbent Assay , Follow-Up Studies , Hepatitis B virus/genetics , Hepatitis B virus/immunology , Hepatitis B virus/physiology , Hepatitis, Chronic/microbiology , Humans , Immunoglobulin M/immunology , Reagent Kits, Diagnostic , Virus Replication
11.
J Med Virol ; 21(1): 15-23, 1987 Jan.
Article in English | MEDLINE | ID: mdl-3794671

ABSTRACT

DNA of hepatitis B virus (HBV DNA) in sera from HBeAg-positive carriers is now the most important and reliable marker of infectivity, but its significance in the progression of chronic hepatitis in anti-HBe carrier status is still under discussion. In this study, viral DNA was tested by a simplified spot hybridization method in sera of 206 HBeAg-negative Italian subjects. In a group of 153 HBsAg carriers, we found that 15.6% of anti-HBe-positive and 10.5% of anti-HBe-negative samples contained viral DNA. No HBV DNA was revealed in 38 HBsAg-negative nor in 15 anti-HBs-positive subjects with different serological markers of HBV. Viral DNA in sera of HBeAg-negative patients with severe chronic liver disease was correlated with increased alaninetransferase activity and IgM anti-HBc. Thus the presence of HBV DNA in these sera not only predicts which subjects are potentially infectious but also indicates chronic progression of hepatitis. Finally, viral DNA extracted from Dane particles of nine anti-HBe-positive sera was characterized by the Southern blot technique. The hybridization pattern shows bands indicating the presence of replicative intermediates.


Subject(s)
Carrier State/diagnosis , DNA, Viral/analysis , Hepatitis B/diagnosis , Alanine Transaminase/blood , Hepatitis B Surface Antigens/analysis , Hepatitis B e Antigens/analysis , Humans , Nucleic Acid Hybridization
12.
Arch Virol ; 87(1-2): 97-105, 1986.
Article in English | MEDLINE | ID: mdl-3942507

ABSTRACT

The presence of Hepatitis B virus (HBV) DNA in sera of patients with HBV related diseases is considered a reliable marker of virus active replication. In this paper HBV DNA was assayed by the molecular hybridization method with a 32P labeled nick translated HBV probe. The assay was positive in sera of 21 out of 22 HBeAg-positive and 4 out of 8 HBeAg-negative asymptomatic HBsAg carriers. 15 HBsAg-negative sera obtained from healthy donors showed no HBV DNA. Almost 80 per cent of HBeAg and HBV DNA positive sera revealed a DNA-polymerase activity. In order to determine the infectivity of HBsAg carriers, it appears that, whenever possible, the HBV DNA spot hybridization should be performed in conjunction with the DNA-polymerase, HBsAg and HBeAg tests.


Subject(s)
DNA, Viral/analysis , Hepatitis B virus/analysis , Hepatitis B/diagnosis , Carrier State , DNA-Directed DNA Polymerase/blood , Hepatitis B/blood , Hepatitis B Antibodies/analysis , Hepatitis B Surface Antigens/analysis , Hepatitis B e Antigens/analysis , Hepatitis B virus/enzymology , Humans , Italy
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