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PubMed2002

Parenteral and oral immunization with a plasmid DNA expressing the human papillomavirus 16-L1 gene induces systemic and mucosal antibodies and cytotoxic T lymphocyte responses.

The association of human papillomavirus (HPV) infection and cervical cancer has been demonstrated. The development of a prophylactic vaccine to protect against primary HPV infection may therefore be an efficient means to reduce the incidence of this cancer worldwide. To assess the capacity of a plasmid DNA that expresses the L1 gene of HPV type 16 to induce a protective immune response, mice were immunized by parenteral and oral routes. Animals that received the DNA vaccine intramuscularly, subcutaneously and orally, developed systemic anti-L1 IgG antibodies. Antibodies developed in mice vaccinated subcutaneously were detectable twelve months post-immunization. Specific IgA antibodies were also found in vaginal washes from immunized mice. Both systemic and local antibodies proved effective in a surrogate neutralization assay. Splenic T cells extracted from experimental mice showed cytotoxic T lymphocytes (CTL) activity mediated by CD8 + cells. Mice were challenged with a syngeneic melanoma cell line, engineered to express the HPV16-L1 protein, tumours in vaccinated animals showed slower growth rate, correlated directly with a longer survival of mice. The results suggest that the L1-based DNA vaccine may be useful for the prevention of primary infections by HPV16.

Animals↗
PubMed1993

The coat protein genes of squash mosaic virus: cloning, sequence analysis, and expression in tobacco protoplasts.

Complementary DNA of the middle-component RNA of the melon strain of squash mosaic comovirus (SqMV) was cloned. Clones containing the coat protein genes were identified by hybridization with a degenerate oligonucleotide synthesized according to the amino acid sequence of a purified peptide fragment of the SqMV large coat protein. A clone containing of 2.5 kbp cDNA insert of SqMV M-RNA was sequenced. The total insert sequence of 2510 bp included a 2373 bp open reading frame (ORF) (encoding 791 amino acids), a 123 bp 3'-untranslated region, and a poly(A) region. This ORF is capable of encoding both the 42 and 22 k SqMV coat proteins. Direct N-terminal sequence analysis of the 22 k coat protein revealed its presence at the 3' end of this ORF and the position of the proteolytic cleavage site (Q/S) used to separate the large and small coat proteins from each other. A putative location of the N-terminal proteolytic cleavage site of the 42 k coat protein (Q/N) was predicted by comparisons with the corresponding coat proteins of cowpea mosaic virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little similarity, their encoded coat proteins shared about 47% identity. The identity of the encoded 42 k and 22 k peptides was confirmed by engineering the respective gene regions for expression followed by transfer into tobacco protoplasts using the polyethylene glycol method. Both SqMV coat proteins were expressed in vivo as determined by their reactivity to SqMV coat protein specific antibodies.

Amino Acid Sequence↗
PubMed2004

Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas.

PURPOSE: To evaluate the feasibility and safety of intratumoral injection of autologous dendritic cells (DCs) transfected with an adenovirus encoding interleukin-12 genes (AFIL-12) for patients with metastatic gastrointestinal carcinomas. Secondarily, we have evaluated biologic effects and antitumoral activity. PATIENTS AND METHODS: Seventeen patients with metastatic pancreatic (n = 3), colorectal (n = 5), or primary liver (n = 9) malignancies entered the study. DCs were generated from CD14+ monocytes from leukapheresis, cultured and transfected with AFIL-12 before administration. Doses from 10 x 10(6) to 50 x 10(6) cells were escalated in three cohorts of patients. Patients received up to three doses at 21-day intervals. RESULTS: Fifteen (88%) and 11 of 17 (65%) patients were assessable for toxicity and response, respectively. Intratumoral DC injections were mainly guided by ultrasound. Treatment was well tolerated. The most common side effects were lymphopenia, fever, and malaise. Interferon gamma and interleukin-6 serum concentrations were increased in 15 patients after each treatment, as well as peripheral blood natural killer activity in five patients. DC transfected with AFIL-12 stimulated a potent antibody response against adenoviral capsides. DC treatment induced a marked increase of infiltrating CD8+ T lymphocytes in three of 11 tumor biopsies analyzed. A partial response was observed in one patient with pancreatic carcinoma. Stable disease was observed in two patients and progression in eight patients, with two of the cases fast-progressing during treatment. CONCLUSION: Intratumoral injection of DC transfected with an adenovirus encoding interleukin-12 to patients with metastatic gastrointestinal malignancies is feasible and well tolerated. Further studies are necessary to define and increase clinical efficacy.

Adenoviridae↗
PubMed2006

[Construction and identification of recombinant adenovirus containing the polyproteins coding regions of O type foot-and-mouth disease virus by homologous recombination in Escherichia coli].

The gene coding for the polyprotein (PP) of foot-and-mouth disease virus (FMDV)was obtained by PCR from recombinant plasmid rpMD18-T/PP. The PCR product was digested with Xba I and Not I and inserted into the cloning site of the adenovirus shuttle vector pAdTrack-CMV, previously digested with the same enzymes. This recombinant shuttle plasmid was designated rpAd-CMV/PP. The recombinant adenovirus vector rpAd/PP was obtained by homologous recombination of plasmid rpAd-CMV/PP and adenovirus skeletal vector pAdeasy-1 in E. coli. Plasmid rpAd/PP was linearized by Pme I and transformed into 293 competent cells to pack the adenovirus using liposome mediated gene transfer method and, as a result, the recombinant adenovirus rAd/PP that contained the polyprotein coding gene was obtained. Obvious CPE could be observed under an inverted microscope, the green fluorescence protein expression can be detected under fluorescence microscope and the empty capsid of FMDV was observed under electron microscope. These results indicated that the recombinant adenovirus rAd/PP expressed the PP protein and that this protein could be assembled into the empty capsid of FMDV. The recombinant adenovirus obtained in this study can be used for further research for making FMDV recombinant adenovirus vaccine.

Adenoviridae↗
PubMed1996

Immunobiology of pseudorabies (Aujeszky's disease).

Aujeszky's Disease (AD), a serious illness of pigs causing significant economic losses in the pig industry, is caused by Pseudorabies Virus (PrV). PrV belongs to the alphaherpesvirus subfamily of the herpesviruses with a double-stranded DNA genome in an enveloped capsid capable of encoding approximately 70 proteins. For disease control, vaccination with live and killed vaccines is performed. Recently, 'marked' vaccines have become available for use in eradication programs based on the differentiation between infected and vaccinated animals. PrV is also used as a viral vector for the development of multivalent vaccines. Despite the effectiveness of PrV vaccines, relatively little is known about the immune response against PrV infection. Several viral envelope glycoproteins have been shown to represent targets for antibody responses, and a number of isolated glycoproteins as well as genetically engineered proteins were able to elicit protective immunity. The nature of the cellular immune response is even less defined. Using viral mutants genetically engineered to lack specific antigens, it has been shown that glycoprotein C (gC) acts as a target for cytotoxic T-lymphocytes, and gB, gC, gD, and gH appear to be involved in stimulation of in vitro proliferation of PBMC from immune animals. In addition, gB and gC have been implicated in recognition of infected cells by lymphokine-activated killer (LAK) cells. In summary, the data indicate a prominent role for viral envelope glycoproteins in eliciting humoral and cellular immune responses in the animal host. A complicating factor is the ability of PrV to productively infect cells of the hematopoietic system, which may impair immune responses and might also play a role in persistent or latent infection.

Animals↗
PubMed2000

Role of matrix protein in the type D retrovirus replication cycle: importance of the arginine residue at position 55.

We previously reported that a mutant of Mason-Pfizer monkey virus (M-PMV), which has an amino acid substitution in the matrix (MA) protein at position 55, MA-R55W, showed altered viral morphogenesis, reduced glycoprotein incorporation, and loss of infectivity. In this report, we show that two additional amino acid substitutions at this site in MA, R55F and R55Y, also result in similar altered morphogenesis, Env incorporation, and infectivity, demonstrating that these changes are not specific for the substitution of tryptophan in place of arginine 55. Attempts to isolate second site infectious revertants from cells transfected with the R55W mutant genome resulted only in the recovery of infectious viruses in which the codon at position 55 had reverted to one encoding arginine. In contrast, no revertants were obtained from the phenylalanine and tyrosine mutants in which three nucleotide changes had been engineered into the arginine codon. These results confirm that the arginine residue at position 55 is critical for intracellular targeting of M-PMV Gag molecules and support the concept that as part of a cytoplasmic transport retention signal R55 interacts with cellular trafficking components rather than other regions of Gag.

Amino Acid Substitution↗
PubMed2002

The capsid of infectious bursal disease virus contains several small peptides arising from the maturation process of pVP2.

The capsid proteins VP2 and VP3 of infectious bursal disease virus, a birnavirus, are derived from the processing of a large polyprotein: NH2-pVP2-VP4-VP3-COOH. Although the primary cleavage sites at the pVP2-VP4 and VP4-VP3 junctions have been identified, the proteolytic cascade involved in the processing of this polyprotein is not yet fully understood, particularly the maturation of pVP2. By using different approaches, we showed that the processing of pVP2 (residues 1 to 512) generated VP2 and four small peptides (residues 442 to 487, 488 to 494, 495 to 501, and 502 to 512). We also showed that in addition to VP2, at least three of these peptides (residues 442 to 487, 488 to 494, and 502 to 512) were associated with the viral particles. The importance of the small peptides in the virus cycle was assessed by reverse genetics. Our results showed that the mutants lacking the two smaller peptides were viable, although the virus growth was affected. In contrast, deletions of the domain 442 to 487 or 502 to 512 did not allow virus recovery. Several amino acids of the peptide 502 to 512 appeared essential for virus viability. Substitutions of the P1 and/or P1" position were engineered at each of the cleavage sites (P1-P1": 441-442, 487-488, 494-495, 501-502, and 512-513). Most substitutions at the pVP2-VP4 junction (512-513) and at the final VP2 maturation cleavage site (441-442) were lethal. Mutations of intermediate cleavage sites (487-488, 494-495, and 501-502) led to viable viruses showing different but efficient pVP2 processing. Our data suggested that while peptides 488 to 494 and 495 to 501 play an accessory role, peptides 442 to 487 and 502 to 512 have an unknown but important function within the virus cycle.

Amino Acid Sequence↗
PubMed1994

Insertion of a 27 amino acid viral peptide in different zones of Escherichia coli beta-galactosidase: effects on the enzyme activity.

Seven internal, putatively exposed regions of Escherichia coli beta-galactosidase have been explored regarding their tolerance to insertions of large foreign peptides. Small sequence modifications, including amino acid substitutions and small deletions, were introduced into the lacZ gene to generate unique BamHI restriction sites. By using these mutant genes, a 27 amino acid stretch reproducing the hypervariable loop of foot-and-mouth disease virus VP1 protein (site A) was further inserted in predefined regions of the enzyme. Among the 13 resulting engineered proteins only three, carrying sequence modifications within a short region, are active, with only moderate reduction of their specific activities. The identified permissive region, which involves amino acids 275 to 279, seems to be a flexible area that could be appropriate to incorporate and study biological properties of heterologous peptides in correctly folded beta-galactosidase chimeric proteins.

Base Sequence↗
PubMed2004

Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo.

The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research.

Adenoviridae↗
PubMed1993

Characterization of the foot-and-mouth disease virus 3C protease expressed in Escherichia coli.

We have constructed a clone encoding the foot-and-mouth disease virus (FMDV) 3C protease gene (p3C) using the polymerase chain reaction. The construct was engineered to contain initiation and termination codons and cloned into a plasmid under the control of the bacteriophage T7 promoter. The p3C gene was expressed both in an in vitro transcription-translation system and in vivo in an Escherichia coli system containing an inducible T7 RNA polymerase gene. In both systems the expressed products were of the appropriate molecular weight and immunologically reactive with bovine convalescent serum. E. coli-expressed 3C protein was mainly found in the insoluble fraction of cell lysates. The E. coli-expressed protease was assayed in an in vitro system with radiolabeled P1 capsid precursor protein and P2 precursor protein as substrates. E. coli-expressed 3C completely processed the P1 and P2 precursors into mature capsid and nonstructural proteins, respectively. The kinetics of processing of P1 by E. coli-expressed 3C revealed the following order of cleavage: VP3-VP1, VP0-VP3, VP1-2A.

3C Viral Proteases↗
PubMed2004

Display of biologically functional insecticidal toxin on the surface of lambda phage.

The successful use of Bacillus thuringiensis insecticidal toxins to control agricultural pests could be undermined by the evolution of insect resistance. Under selection pressure in the laboratory, a number of insects have gained resistance to the toxins, and several cases of resistance in the diamondback moth have been reported from the field. The use of protein engineering to develop novel toxins active against resistant insects could offer a solution to this problem. The display of proteins on the surface of phages has been shown to be a powerful technology to search for proteins with new characteristics from combinatorial libraries. However, this potential of phage display to develop Cry toxins with new binding properties and new target specificities has hitherto not been realized because of the failure of displayed Cry toxins to bind their natural receptors. In this work we describe the construction of a display system in which the Cry1Ac toxin is fused to the amino terminus of the capsid protein D of bacteriophage lambda. The resultant phage was viable and infectious, and the displayed toxin interacted successfully with its natural receptor.

Aminopeptidases↗
PubMed2001

Preexisting immunity to poliovirus does not impair the efficacy of recombinant poliovirus vaccine vectors.

Recombinant viruses are attractive candidates for the development of novel vaccines. A number of viruses have been engineered as vaccine vectors to express antigens from other pathogens or tumors. Inoculation of susceptible animals with this type of recombinant virus results in the induction of both humoral and cellular immune responses directed against the foreign antigens. A general problem to this approach is that existing immunity to the vector can diminish or completely abolish the efficacy of the viral vector. In this study, we investigated whether poliovirus recombinants are capable of inducing effective immunity to the foreign antigen in previously vaccinated animals. Antipoliovirus immunity was induced in susceptible mice by intraperitoneal immunization with live poliovirus. Immunized mice developed antibodies directed against capsid proteins that effectively neutralized poliovirus in vitro and protected animals from a lethal challenge with a high dose of pathogenic poliovirus. To test whether preexisting immunity reduces the efficacy of vaccination with recombinant poliovirus, immunized mice were inoculated with a recombinant poliovirus expressing the C-terminal half of chicken ovalbumin (Polio-Ova). Animals developed ovalbumin-specific antibodies and cytotoxic T lymphocytes (CTL). While the antibody titers observed in preimmune and naive mice were similar, the overall CTL response appeared to be reduced in preimmune mice. Importantly, vaccination with Polio-Ova was able to effectively protect preimmune mice against lethal challenge with a tumor expressing the antigen. Thus, preexisting immunity to poliovirus does not compromise seriously the efficacy of replication-competent poliovirus vaccine vectors. These results contrast with those observed for other viral vaccine vectors and suggest that preexisting immunity does not equally affect the vaccine potential of individual viral vectors.

Animals↗
PubMed2000

A novel method for the preparation of large cDNA fragments from dengue-3 RNA genome by long RT-PCR amplification.

For many years, dengue viruses were among the most difficult flaviviruses to isolate and to identify, but technical advances in the past 20 years have facilitated this process. Dengue viruses are usually recovered from specimens by the infection of mosquito-cell cultures. The virus may be passaged several times in cell cultures until a sufficient infectivity titer is attained. The viral nucleocapsid consists of capsid protein and an RNA genome. The dengue genome is a single stranded messenger (positive) sense RNA of approximately 11 kb in length. The isolation of dengue genomic RNA from various sources requires precautions to avoid RNases. RNases are released during cell disruption, and their activity must be inhibited as quickly as possible by using guanidinium thiocyanate in the presence of 2-mercaptoethanol. There has recently been a revolution in molecular biology with the development of the powerful reverse transcriptase (RT) and polymerase chain reaction (PCR) technology. Advanced studies on RT technique lead to much further improvement of the reverse transcriptase enzyme by genetic engineering. The Superscript II RNase H- RT (GIBCO BRL, USA) is genetically engineered DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA. DNA or an RNA-DNA hybrid. This enzyme is produced from a cloned M-MLV RT gene constructed by the introduction of point mutation in the RNase H active center. The selective mutations within the RNase H domain maintain full polymerase activity. This structural modification eliminates degradation of RNA molecules during the first strand cDNA synthesis. The combination of thermostable DNA polymerase with and without proofreading activity (3'-exonuclease activity), improved buffer conditions and thermal cycling profiles overcome the length limitation of PCR. On the basis of these findings, we have developed a long RT-PCR system for preparing large cDNA fragments of dengue 3 virus (H-87) by using the Superscript II RNase H- RT for reverse transcription and a mixture of Taq and Pwo DNA polymerases for PCR. Three large cDNA fragments covered the full genomic RNA from the 5'-end to the 3'-end of dengue-3 virus (H-87; 10,696 bps) could be successfully prepared as the lengths of 2.437 bps, 3,980 bps and 4,337 bps respectively. The ability of our developed long RT-PCR will bring speed and simplicity to genomic mapping and sequencing and facilitate studies in molecular genetics of dengue viruses.

Aedes↗
PubMed2006

Small RNA viruses of insects: expression in plants and RNA silencing.

Interest in insect small RNA viruses (SRVs) has grown slowly but steadily. A number of new viruses have been analyzed at the sequence level, adding to our knowledge of their diversity at the level of both individual virus species and families. In particular, a number of possible new virus families have emerged. This research has largely been driven by interest in their potential for pest control, as well as in their importance as the causal agents of disease in beneficial arthropods. At the same time, research into known viruses has made valuable contributions to our understanding of an emerging new field of central importance to molecular biology-the existence of RNA-based gene silencing, developmental control, and adaptive immune systems in eukaryotes. Subject to RNA-based adaptive immune responses in their hosts, viruses have evolved a variety of genes encoding proteins capable of suppressing the immune response. Such genes were first identified in plant viruses, but the first examples known from animal viruses were identified in insect RNA viruses. This chapter will address the diversity of insect SRVs, and attempts to harness their simplicity in the engineering of transgenic plants expressing viruses for resistance to insect pests. We also describe RNA interference and antiviral pathways identified in plants and animals, how they have led viruses to evolve genes capable of suppressing such adaptive immunity, and the problems presented by these pathways for the strategy of expressing viruses in transgenic plants. Approaches for countering these problems are also discussed.

Animals↗
PubMed2003

Role of the UL28 homologue of bovine herpesvirus 1 in viral DNA cleavage and packaging.

In the aim to study the function of the bovine herpesvirus 1 (BoHV-1) UL28 protein during the replicative cycle, we characterized a UL28 deletion mutant of BoHV-1, BoHV-1 Delta UL28. Productive growth of BoHV-1 Delta UL28 was only observed in a specifically engineered complementing cell line expressing the native UL28 protein, demonstrating that UL28 is essential for virus replication. UL28 deficiency did not compromised viral protein synthesis of the late class as shown by the detection of the viral alpha gene trans-inducing factor protein encoded by UL48, a gene of the gamma2 class. Southern blotting analyses of total DNA extracted from BoHV-1 Delta UL28-infected normal cells revealed that viral DNA replication was not compromised but the process of cleavage of the newly synthesized DNA was defective. Transmission electron microscopy of non-complementing BoHV-1 Delta UL28-infected cells revealed an accumulation of capsids devoid of DNA, suggesting that the DNA packaging was impaired. We conclude that the BoHV-1 UL28 protein is essential for viral replication and is necessary for the formation of mature capsid.

Animals↗
PubMed2006

The role of the S-S bridge in retroviral protease function and virion maturation.

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.

Amino Acid Sequence↗
PubMed2006

Assembly of trans-encapsidated recombinant viral vectors engineered from Tobacco mosaic virus and Semliki Forest virus and their evaluation as immunogens.

RNA virus vectors are attractive vaccine delivery agents capable of directing high-level gene expression without integration into host cell DNA. However, delivery of non-encapsidated RNA viral vectors into animal cells is relatively inefficient. By introducing the tobacco mosaic virus (TMV) origin of assembly into the RNA genome of Semliki Forest virus (SFV), we generated an SFV expression vector that could be efficiently packaged (trans-encapsidated) in vitro by purified TMV coat protein (CP). Using cellular assays, pseudovirus disassembly, RNA replication and reporter gene expression were demonstrated. We also evaluated the immune response to trans-encapsidated recombinant SFV carrying a model antigen gene (beta-galactosidase) in C57/B6 mice. Relative to RNA alone, vector encapsidation significantly improved the humoral and cellular immune responses. Furthermore, reassembly with recombinant TMV CPs permitted the display of peptide epitopes on the capsid surface as either genetic fusions or through chemical conjugation, to complement the immunoreactivity of the encapsidated RNA genetic payload. The SFV vector/TMV CP system described provides an alternative nucleic acid delivery mechanism that is safe, easy to manufacture in vitro and that also facilitates the generation of unique nucleic acid/protein antigen compositions.

Animals↗