Recombinant Vaccinia Virus System Research Articles

A vaccinia virus expression system to study antigen processing of multiple recombinant proteins (130.43)

Abstract A major obstacle in designing effective vaccines is our limited knowledge of the mechanisms involved in eliciting a protective cell-mediated immune response. In recent years, research has focused on the development of recombinant vaccines, in which antigenic peptides derived from a specific pathogen are delivered to the cells via live vectors. For a vaccine to be effective in an out-bred population it must generate as many antigenic determinants as possible. One method that can be used to generate multiple peptide determinants is to express two or more recombinant proteins from a single live vaccine vector. To this end, we developed a recombinant vaccinia virus system that expresses two full-length influenza virus proteins; nucleoprotein (NP) and acidic polymerase (PA). In addition to the NP and PA proteins, our expression system is designed to produce yellow and red fluorescent proteins, which allow us to monitor, in a quantitative manner, recombinant protein expression both in vitro and in vivo. Using this novel expression system we have found that the route of immunization affects the immunodominance hierarchy of the host response to not only vector-derived peptides but also to recombinant peptides.

Functional Chimeras of Human Immunodeficiency Virus Type 1 gp120 and Influenza A Virus (H3) Hemagglutinin

In an attempt to produce a protein that will allow determination of the native human immunodeficiency virus type 1 (HIV-1) gp120 (Env) structure in its trimeric state, we fused the globular head of gp120 to the stalk region of influenza virus A (X31) hemagglutinin (HA). The chimeric protein (EnvHA) has been expressed by using a recombinant vaccinia virus system, and its functional characteristics were determined. EnvHA is expressed as a 120- to 150-kDa protein that can oligomerize to form dimers and trimers. It retains the low-pH (5.2 to 5.4) requirement of X31-HA to trigger membrane fusion but, unlike X31-HA, it is not absolutely dependent on exogenously added trypsin for protein processing to release the HA2 fusion peptide. In terms of receptor binding the chimeric protein retains specificity for human CD4 but, in relation to the membrane fusion event, it appears to lose the Env coreceptor specificity of the parental HIV-1 strains: NL43 for CXCR4 and JRFL for CCR5. These properties suggest that stable, functional EnvHAs are being produced and that they may be exploited in terms of structural studies. Further, the potential of introducing the envHA genes into influenza viruses, by use of reverse genetics, and their use as a therapeutic vaccine for HIV are discussed.

Early Selection of a New UL97 Mutant with a Severe Defect of Ganciclovir Phosphorylation after Valaciclovir Prophylaxis and Short-Term Ganciclovir Therapy in a Renal Transplant Recipient

We describe the emergence of a new ganciclovir resistance mutation in the UL97 gene of human cytomegalovirus, deletion of codon 601, after valaciclovir and short-term ganciclovir therapy following kidney transplantation. Its role in ganciclovir resistance was supported by decreased ganciclovir phosphorylation in a recombinant vaccinia virus system.

Regulation of HIV-1 env mRNA translation by Rev protein

We have examined the effect of Rev on the regulation of the expression of RRE containing mRNAs when they were synthesised in the nucleus or directly in the cytoplasm. In the nuclear expression system, Rev enhanced env mRNA transport by about 1.6-fold, while translation of this mRNA was increased more than a 100-fold. These findings indicate that the target of Rev activity is located mainly at the translational level. Synthesis of Env using a recombinant vaccinia virus system, which synthesised env mRNA directly in the cytoplasm, is also enhanced by Rev. Finally, RRE functioning was examined using a luciferase mRNA bearing this element. Rev stimulated the synthesis of Luciferase both when the luc mRNA was made in the nucleus or in cytoplasm. Our results indicate that the effect of Rev on env mRNA transport is low compared with the enhancement of translation of this mRNA.

Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins.

Nipah virus (NiV) and Hendra virus (HeV) are novel paramyxoviruses from pigs and horses, respectively, that are responsible for fatal zoonotic infections of humans. The unique genetic and biological characteristics of these emerging agents has led to their classification as the prototypic members of a new genus within the Paramyxovirinae subfamily called HENIPAVIRUS: These viruses are most closely related to members of the genus Morbillivirus and infect cells through a pH-independent membrane fusion event mediated by the actions of their attachment (G) and fusion (F) glycoproteins. Understanding their cell biological features and exploring the functional characteristics of the NiV and HeV glycoproteins will help define important properties of these emerging viruses and may provide new insights into paramyxovirus membrane fusion mechanisms. Using a recombinant vaccinia virus system and a quantitative assay for fusion, we demonstrate NiV glycoprotein function and the same pattern of cellular tropism recently reported for HeV-mediated fusion, suggesting that NiV likely uses the same cellular receptor for infection. Fusion specificity was verified by inhibition with a specific antiserum or peptides derived from the alpha-helical heptads of NiV or HeV F. Like that of HeV, NiV-mediated fusion also requires both F and G. Finally, interactions between the glycoproteins of the paramyxoviruses have not been well defined, but here we show that the NiV and HeV glycoproteins are capable of highly efficient heterotypic functional activity with each other. However, no heterotypic activity was observed with envelope glycoproteins of the morbilliviruses Measles virus and Canine distemper virus.

Generation of CD8(+) T cell memory in response to low, high, and excessive levels of epitope.

The magnitude of a virus-specific memory CTL population can dramatically influence the outcome of secondary infections, yet little is known about the determinants of memory size. We investigated the impact of epitope levels on CTL memory generation by using a recombinant vaccinia virus system that allows for a broad range of epitope expression with the same infectious dose of virus. The size of the memory pool was examined using MHC class I/peptide tetramer staining and IFN-gamma ELISPOT analysis following priming with viruses expressing low, high, or excessive epitope levels. The size of the epitope-specific CD8(+) T cell memory population correlates with Ag dose at the low and high levels of epitope expression. However, at excessive epitope levels, the number of functional, IFN-gamma-producing, epitope-specific memory cells is significantly reduced compared with the number of tetramer(+) cells. These results demonstrate that the level of epitope expressed during an acute viral infection in vivo can dramatically influence CTL memory size. Furthermore, when epitope is overexpressed, the quality of the response can be adversely affected. Therefore, epitope expression level is an important consideration when developing approaches to optimize CTL memory induction.

Mutations in the UL97 ORF of ganciclovir-resistant clinical cytomegalovirus isolates differentially affect GCV phosphorylation as determined in a recombinant vaccinia virus system

Mutations in the human cytomegalovirus (HCMV) UL97 phosphotransferase have been associated with ganciclovir (GCV) resistance due to an impairment of GCV monophosphorylation. Vaccinia virus recombinants (rVV) were generated that encoded different HCMV UL97 proteins (pUL97) with mutations previously detected in resistant HCMV clinical isolates at codons 460, 520, 592, 594, 595, 598 and 607. These rVVs allowed quantification of GCV phosphorylation catalyzed by the different mutated pUL97s. When compared to rVV-UL97 wild type, mean levels of residual intracellular GCV phosphorylation differed by a factor of 10 for the mutated UL97 proteins ranging from 5.2 to 51.8%. Mutations M460V (located in a UL97 region homologous to domain VIb of protein kinases) and H520Q (located in a cytomegalovirus-specific, functionally critical domain) were responsible for the lowest levels of residual GCV phosphorylation (9.3 and 5.2%). Mutations in a region homologous to the domain IX had a lower impact on GCV phosphorylation (15.8–51.8%). The relevance of pUL97 mutation G598S in inducing GCV resistance was demonstrated for the first time.

PreproTRH178–199 and Two Novel Peptides (pFQ7 and pSE14) Derived from Its Processing, Which Are Produced in the Paraventricular Nucleus of the Rat Hypothalamus, Are Regulated during Suckling**This work was supported by the National Science Foundation (Grant No. IBN-9507952 to E.A.N.).

Suckling increases preproTRH messenger RNA in hypothalamic paraventricular neurons (PVN) and also markedly increases TRH release during the first period of lactation. Whether lactation alters preproTRH processing resulting in the generation of novel proTRH-derived peptides that may be involved in the regulation of PRL secretion lactation is not known. Therefore, in the present study we determine whether some other peptides derived from proTRH potentially contribute to lactation-induced PRL secretion. We have recently demonstrated that two members of the family of prohormone convertases PC1 and PC2 play a significant role in proTRH processing. PC1 is the major contributor in proTRH processing, whereas PC2 may have a specific role in cleaving TRH from its extended forms. In this study, we used a recombinant vaccinia virus system to coexpress rat preproTRH complementary DNA with PC1, PC2, and the neuropeptide 7B2 in GH4C1 cells (somatomammothophs, rat). We found that two novel peptides, preproTRH(178-184) (pFQ(7)), and preproTRH(186-199) (pSE(14)), were formed after the cleavage of their precursor preproTRH(178-199) (pFE(22)) by only PC2. Their formation was confirmed by microsequence analysis. Anatomical analyses revealed that these peptides are also found in the rat PVN. In addition, we found that pFE(22), pSE(14) and pFQ(7) produced a dose-dependent release of PRL from primary cultures of pituitary cells compared with one of the well studied secretagogues of PRL, TRH. To establish whether these peptides might play a role in vivo in the regulation of PRL release, we took rat litters on postnatal day 4, separated the pups from their mothers for 6 h, and then reunited the pups and mothers for 45 min. At the end of this period, the mothers were killed, acidic extracts of microdissected PVN were prepared and subjected to SDS-PAGE, followed by slicing and analysis by pFE(22) RIA. Forty-five minutes of suckling induced a marked 6-fold increase in serum levels of PRL. In addition, PVN levels of pFE(22) and pSE(14) increased approximately 5-fold during the same period in the acutely suckling females. Lactating animals that were separated from their litters and never reunited with their pups had low levels of PRL, and pFE(22) and pSE(14). These data provide the first evidence for alterations in proTRH processing in the PVN during lactation and suggest that the products of this altered processing may play a physiological role in the regulation of PRL secretion.

Characterization of interaction of gH and gL glycoproteins of varicella-zoster virus: their processing and trafficking.

Varicella-zoster virus (VZV) glycoproteins gH and gL were examined in a recombinant vaccinia virus system. Single expression of glycoprotein gL produced two molecular forms: an 18 kDa form and a 19 kDa form differing in size by one endoglycosidase H-sensitive N-linked oligosaccharide. Coexpression of gL and gH resulted in binding of the 18 kDa gL form with the mature form of gH, while the 19 kDa gL form remained uncomplexed. The glycosylation processing of gL was not dependent on gH; however, gL was required for the conversion of precursor gH (97 kDa) to mature gH (118 kDa). Subsequent analyses indicated that gL (18 kDa) was a more completely processed gL (19 kDa). Screening of the culture media revealed that gH and gL were secreted, but only if coexpressed and complexed together. The secreted form of gL was 18 kDa while that of gH was 114 kDa. The fact that secreted gH was smaller than intracytoplasmic gH suggested a proteolytic processing event prior to secretion. The 19 kDa form of gL was never secreted. These findings support a VZV gL recycling pathway between the endoplasmic reticulum and the cis-Golgi apparatus.

Improving proteolytic cleavage at the 3A/3B site of the hepatitis A virus polyprotein impairs processing and particle formation, and the impairment can be complemented in trans by 3AB and 3ABC.

The orchestrated liberation of viral proteins by 3C(pro)-mediated proteolysis is pivotal for gene expression by picornaviruses. Proteolytic processing is regulated either by the amino acid sequence at the cleavage site of the substrate or by cofactors covalently or noncovalently linked to the viral proteinase. To determine the role of the amino acid sequence at cleavage sites 3A/3B and 3B/3C that are essential for the liberation of 3C(pro) from its precursors and to assess the function of the stable processing intermediates 3AB and 3ABC, we studied the effect of cleavage site mutations on hepatitis A virus (HAV) polyprotein processing, particle formation, and replication. Using the recombinant vaccinia virus system, we showed that the normally retarded cleavage at the 3A/3B junction can be improved by altering the amino acid sequence at the scissile bond such that it matches the preferred HAV 3C cleavage sites. In contrast to the processing products of the wild-type polyprotein, 3ABC was no longer detectable in the mutant. VP0 and VP3 were generated less efficiently, implying that processing of the structural protein precursor P1-2A depends on the presence of stable 3ABC and/or 3AB. In addition, cleavage of 2BC was impaired in 3AB/3ABC-deficient mutants. Formation of HAV particles was not affected in mutants with blocked 3A/3B and/or 3B/3C cleavage sites. However, 3ABC-deficient mutants produced small numbers of HAV particles, which could be augmented by coexpressing 3AB or 3ABC. The hydrophobic domain of 3A that has been proposed to mediate membrane anchorage of the replication complex was crucial for restoration of defective particle formation. In vitro transcripts of the various cleavage site mutants were unable to initiate an infectious cycle, and no progeny viruses were obtained even after blind passages. Taken together, the data suggest that accumulation of uncleaved HAV 3AB and/or 3ABC is pivotal for both viral replication and efficient particle formation.

Expression and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes

Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preS1 and preS2 sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. However, the large (L) protein containing the entire preS region expressed in mammalian cells is not efficiently assembled into particles and secreted. Here we report an alternative approach to include the dominant epitopes of preS1 and preS2 to the small (S) protein as fusion proteins by the recombinant DNA technology. Three fusion proteins containing preS2(120–146) and preS1(21–47) at the N-terminus and/or truncated C-terminus of S protein were expressed using the recombinant vaccinia virus system. All these fusion proteins were efficiently secreted in the particulate form, and displayed S, preS1 and/or preS2 antigenicity. Further analysis showed that these chimeric HBsAg particles elicited strong antibody responses against S, preS1 and preS2 antigens in BALB/c mice, suggesting that they could be promising candidates for a new recombinant vaccine to induce broader antibody response required for protection against hepatitis B viral infection.

Recombinant vaccinia viruses. Design, generation, and isolation.

The technologies of recombinant gene expression have greatly enhanced the structural and functional analyses of genetic elements and proteins. Vaccinia virus, a large double-stranded DNA virus and the prototypic and best characterized member of the poxvirus family, has been an instrumental tool among these technologies and the recombinant vaccinia virus system has been widely employed to express genes from eukaryotic, prokaryotic, and viral origins. Vaccinia virus is also the prototype live viral vaccine and serves as the basis for well established viral vectors which have been successfully evaluated as human and animal vaccines for infectious diseases and as anticancer vaccines in a variety of animal model systems. Vaccinia virus technology has also been instrumental in a number of unique applications, from the discovery of new viral receptors to the synthesis and assembly of other viruses in culture. Here we provide a simple and detailed outline of the processes involved in the generation of a typical recombinant vaccinia virus, along with an up to date review of relevant literature.

Glycosylation and NH 2-terminal domain mutants of the tissue inhibitor of metalloproteinases-1 (TIMP-1)

Mutants in the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein have been created by site-directed mutagenesis and expressed in HeLa cells, using a recombinant vaccinia virus system. Removal of either or both glycosylation sites yielded proteins which retained wild-type inhibitory activity against both human fibroblast-type collagenase (FIB-CL) and M r 72 000 gelatinase (GL). However, the double glycosylation mutant protein was expressed at a level that was 2–4-fold lower than that of the wild-type or the single site glycosylation mutants. The ‘tiny-TIMP’ COOH-terminal deletion mutant that lacks the last 57 residues was also inhibitory, but the dose–response curve suggested that the interaction with the M r 72 000 gelatinase had been altered. A number of replacement mutants in the highly conserved NH 2-terminal domain, including replacement of P5A and P8A or a double mutation in the VIRAK sequence which is absolutely conserved in all TIMPs in all species (VIRAK to VIAAA), also yielded functional proteins capable of inhibiting FIB-CL and M r 72 000 GL and of forming SDS-resistant complexes with FIB-CL. None of the above manipulations abolished inhibitory function suggesting that binding of the inhibitor by the enzyme involves multiple interactions.

Recombinant hepatitis A virus antigen: improved production and utility in diagnostic immunoassays.

Hepatitis A virus (HAV) immunoassays use cell culture-derived HAV antigen to detect HAV-specific antibodies. The current method of production of HAV antigen in tissue culture is time-consuming and expensive. We previously expressed the HAV open reading frame in recombinant vaccinia viruses (rV-ORF). The recombinant HAV polyprotein was accurately processed and was assembled into subviral particles. These particles were bound by HAV-neutralizing antibodies and were able to elicit antibodies which were detected by commercial immunoassays. The present investigation compared the production of HAV antigen by standard tissue culture methods to the production of HAV antigen with the recombinant vaccinia virus system. In addition, HAV and rV-ORF antigens were assessed for their utility in diagnostic immunoassays. Serum or plasma samples from HAV antibody-positive and antibody-negative individuals were evaluated by immunoassay that used either HAV or rV-ORF antigen. All samples (86 of 86) in which HAV antibody was detected by a commercial enzyme-linked immunosorbent assay (ELISA) also tested positive by the recombinant antigen-based immunoassay (VacRIA). Similarly, all samples (50 of 50) that were HAV antibody negative also tested negative by the VacRIA. The lower limit of detection of HAV antibody was similar among immunoassays with either HAV or rV-ORF antigen. Thus, in the population studied, the sensitivity and specificity of the VacRIA were equivalent to those of the commercial ELISA. Since production of recombinant antigen is faster and less expensive than production of traditional HAV antigen, the development of diagnostic HAV antibody tests with recombinant HAV antigen appears warranted.

Expression of the potyvirus coat protein mediated by recombinant vaccinia virus and assembly of potyvirus-like particles in mammalian cells.

The coat protein of the potyvirus, Johnsongrass mosaic virus (JGMV), was expressed using a recombinant vaccinia virus (VV) system. Ultra-thin section electron microscopy demonstrated that the coat protein assembled into potyvirus-like particles (PVLPs) in recombinant VV infected cells. Infection of cells with two additional VV recombinants expressing coat protein plus N-terminal and N- and C-terminal extensions also resulted in the formation of PVLPs. These results suggest that the ability of VV to express the potyvirus coat protein at sufficient levels to allow PVLP formation in vitro, could make VV a suitable vector for the delivery of PVLPs displaying vaccine antigens in vivo without the need for particle purification and/or inclusion of adjuvant. Use of such a vaccine strategy would also benefit from the proven advantages of poxviruses as vaccines such as stability in a freeze dried form, resistance to environmental factors and the potential for oral administration.

6] Functional identification of vesicular monoamine and acetylcholine transporters

Active transport of monoamines by vesicular monoamine transporter (VMAT1) and VMAT2 is performed in permeabilized fibroblasts transiently expressing these proteins by the recombinant T7 vaccinia virus system. Active transport of acetylcholine (ACh) by human vesicular ACh transporter (VAChT) is performed in postnuclear homogenates containing secretory vesicles from stably transfected neuroendocrine PC-12 cells. Neurotransmission depends on the regulated release of transmitter molecules, such as the biogenic amines and ACh. This requires the packaging of these molecules into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific vesicular transporters. Active transport of neurotransmitters into secretory organelles requires the presence of a transmembrane H + electrochemical gradient, established and maintained by a vacuolar-type H + -ATPase and a vesicular transporter molecule, which catalyzes the exchange of H + ions for neurotransmitter. A characteristic feature of the VMATs is their broad range of substrate specificity. The chapter describes the development of active transport and binding assay in permeabilized cells.

Deletion mapping of functional domains in the rotavirus capsid protein VP6.

VP6, the major capsid protein of rotavirus, oligomerizes into trimers that constitute the intermediate shell of the virions. In order to map functional domains in this protein, we introduced seven internal in-frame deletions within the coding region of gene 6 of human rotavirus strain Wa. Regions of homology among the VP6 proteins of group A and group C rotaviruses were targeted for deletion mutagenesis. The mutant VP6 proteins were expressed in mammalian cells using the recombinant vaccinia virus system and were examined for their ability to oligomerize into trimers as well as to assemble into double-layered virus-like particles upon coexpression with the rotavirus core protein VP2. Deletions that abolished trimerization defined a domain (residues 246 to 314) that maps within a larger region previously found to be critical for oligomerization (amino acids 105 to 328). When the capacity of each mutant to assemble into double-layered virus-like particles was analysed, three different assembly phenotypes were observed. Phenotype I was represented by two deletion mutants lacking residues 246 to 250 and 308 to 314 that produced particles with efficiencies similar to that of wild-type VP6. Phenotype II, characterized by a moderate decrease in the efficiency of particle assembly with respect to that of wild-type VP6, included two mutants with deletions at the C terminus of the protein. Phenotype III was exhibited by three mutants whose abilities to assemble into double-layered virus-like particles were drastically impaired. Two of these mutants define a previously unidentified assembly domain (amino acids 122 to 147) at the N terminus of rotavirus VP6.

Poliovirus protein 2BC increases cytosolic free calcium concentrations.

Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis.

Design and construction of recombinant vaccinia viruses.

AbstractThe structural and functional analyses of proteins have benefited enormously from the use of technologies of recombinant gene expression. The recombinant vaccinia virus system has been widely employed to express genes from eukaryotic, prokaryotic, and viral origins (for reviews, see refs. 2–7) and several detailed protocols for the generation, identification, isolation, and characterization of recombinant vaccinia viruses have been published (8-10).KeywordsVaccinia VirusRecombinant VirusVirus StockRecombinant Vaccinia VirusDialyze Fetal Bovine SerumThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Presentation of the hydrophilic domains of hepatitis C viral E2 envelope glycoprotein on hepatitis B surface antigen particles.

Subviral particles of hepatitis B virus have been used to present foreign epitopes. We attempted to present the hydrophilic domains of E2 envelope protein of hepatitis C virus (HCV) as a fusion protein with hepatitis B virus surface antigen (HBsAg). The five hydrophilic domains of HCV E2 antigen were inserted into HBsAg such that the inserted hydrophilic domains were presented on the outer surface of HBV subviral particles. In addition, a fusion encoding the hypervariable region (HVR) of E2 antigen was also made. Cell lysate and culture medium were analyzed for the synthesis and secretion of the fusion proteins by immunoprecipitation with polyclonal anti-HBsAg antibody using recombinant vaccinia virus system. The results showed that the fusion proteins containing these six E2 domains were made in the cell, but only two out of six fusion proteins were secreted into culture medium. Further, cesium chloride density gradient analysis and electron microscopy revealed that these fusions were secreted into culture media as particles. It will be of interest to test immunogenicity of the HBsAg fusion particles containing the HCV E2 domains in animal model.