Optimization of plasmid-only rescue of highly attenuated and temperature-sensitive respiratory syncytial virus (RSV) vaccine candidates for human trials.

Respiratory syncytial virus (RSV) is the most common cause of severe bronchiolitis in infants and young children in the U.S. No licensed RSV vaccines are currently available. Established techniques for recovering RSV from cDNA utilize mammalian cells, such as HEp-2 or BSR T7/5, that are not currently suitable for vaccine manufacture. When using HEp-2 cells, co-infection with an attenuated vaccinia virus that expresses T7 RNA polymerase is also required. For human clinical trials, processes that do not require the use of helper viruses and minimize the use of animal derived materials must be developed to reduce the potential theoretical risk of transmitting adventitious agents such as BSE. RSV was generated by electroporating Vero cells from a well characterized cell bank with 6 plasmids expressing T7 RNA polymerase, the full-length anti-genomic RSV and RSV N, P, M2-1 and L. The process was optimized such that highly attenuated and temperature-sensitive RSV vaccine candidates could be recovered in a system completely free of animal derived components. Efficiencies of virus recovery ranged from 30% to 100%. Human metapneumovirus was also readily recovered, suggesting that this protocol is applicable for the production of clinical trial material of other non-segmented negative sense RNA viruses.

A review of genetic differences between limited and extensively passaged human cytomegalovirus strains.

The complete genetic content of human cytomegalovirus (HCMV) has been difficult to determine, since most strains studied in the laboratory have been extensively passaged in human fibroblast cultures which can change the genetic content as well as the biological properties of the virus. Approximately 13 kb of novel DNA sequences located near the right edge of the unique long (UL) component of the genome has been discovered in Toledo, clinical isolates and certain stocks of Towne. This region of novel sequence, designated the UL/b' region, encodes several interesting proteins including vCXC-1, a potent IL-8 homologue, and UL144, a member of the TNF receptor family. This region is missing from the prototypic laboratory variants of Towne and AD169. In contrast to Toledo and other low passage isolates which have relatively small repeats bracketing the UL component, the Towne and AD169 laboratory variants contain large (>10 kb) b/b' repeats. The large size of these repeats in AD169 and Towne appear to have arisen as compensation for the loss of sequences from the UL/b' region that existed in less passaged variants of these strains. Consequently, many of the haploid genes at the left edge of the prototypic wild-type (wt) UL component are diploid in AD169 and Towne. We hypothesise that this plasticity of the genome at the right edge of the UL component results from extensive passage and adaptation to replication in fibroblasts in vitro. Further work will be required to understand the complete genetic content of wt HCMV.

Open reading frame S/L of varicella-zoster virus encodes a cytoplasmic protein expressed in infected cells.

We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3' untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation.

Expression of Epstein-Barr virus gp350 as a single chain glycoprotein for an EBV subunit vaccine.

There is currently no commercially available vaccine for Epstein Barr virus (EBV)-related disease in humans. Since the EBV glycoprotein gp350/220 is the primary target for EBV-neutralizing antibodies following natural infection in humans and some forms of gp350/220 have been shown to protect against EBV-related disease in animal models, it is a likely candidate for an EBV subunit vaccine. We have made gp350/220 gene constructs that facilitate gp350 secretion from CHO cells and created splice site mutations in the gene that effectively prevent production of the gp220 species. Recombinant CHO cell gp350 (MSTOP gp350) is recognized by several different anti-gp350/220 monoclonal antibodies, and is also competent to bind to the cellular EBV receptor, CD21, suggesting that the recombinant protein is conformationally similar to wild-type EBV gp350/220. The MSTOP gp350 antigen raises high antibody titers in rabbits and these antibodies neutralize wild-type EBV. These properties make MSTOP gp350 a realistic candidate for a subunit vaccine against EBV-related disease.

Evaluation of a live attenuated recombinant virus RAV 9395 as a herpes simplex virus type 2 vaccine in guinea pigs.

Recombinant virus RAV 9395 was constructed by deleting both copies of the gamma(1)34.5 gene, and the UL55 and UL56 open reading frames from herpes simplex virus type 2 (HSV-2) strain G. The potential use of RAV 9395 as an HSV-2 vaccine was investigated by evaluating the ability of RAV 9395 to protect guinea pigs from severe disease by HSV-2(G) challenge. RAV 9395 administered intramuscularly reduced both lesion development and severity in a dose-dependent manner in guinea pigs challenged with HSV-2(G). The frequency of reactivation of RAV 9395 from explanted dorsal root ganglia was low compared with that of HSV-2(G). Immunization with RAV 9395 at doses of 1 x 10(5) pfu and above generally precluded the establishment of latency by the challenge virus. The results presented in this report lend support for the development of genetically engineered live HSV vaccines.

Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains.

Nucleotide sequence comparisons were performed on a highly heterogeneous region of three human cytomegalovirus strains, Toledo, Towne, and AD169. The low-passage, virulent Toledo genome contained a DNA segment of approximately 13 kbp that was not found in the Towne genome and a segment of approximately 15 kbp that was not found in the AD169 genome. The Towne strain contained approximately 4.7 kbp of DNA that was absent from the AD169 genome, and only about half of this segment was present, arranged in an inverted orientation, in the Toledo genome. These additional sequences were located at the unique long (UL)/b' (IRL) boundary within the L component of the viral genome. A region representing nucleotides 175082 to 178221 of the AD169 genome was conserved in all three strains; however, substantial reduction in the size of the adjacent b' sequence was found. The additional DNA segment within the Toledo genome contained 19 open reading frames not present in the AD169 genome. The additional DNA segment within the Towne genome contained four new open reading frames, only one of which shared homology with the Toledo genome. This comparison was extended to five additional clinical isolates, and the additional Toledo sequence was conserved in all. These findings reveal a dramatic level of genome sequence complexity that may explain the differences that these strains exhibit in virulence and tissue tropism. Although the additional sequences have not altered the predicted size of the viral genome (230 to 235 kbp), a total of 22 new open reading frames (denoted UL133 to UL154), many of which have sequence characteristics of glycoproteins, are now defined as cytomegalovirus specific. Our work suggests that wild-type virus carries more than 220 genes, some of which are lost by large-scale deletion and rearrangement of the UL/b' region during laboratory passage.

Coexpression of truncated human cytomegalovirus gH with the UL115 gene product or the truncated human fibroblast growth factor receptor results in transport of gH to the cell surface.

The gH glycoprotein of herpesviruses is located on the cell surface in viral-infected cells but is retained in the endoplasmic reticulum (ER) when expressed separately from a recombinant expression vector. These observations suggested the requirement for either a viral function or a viral-induced cellular function which facilitates surface expression of gH. gL fulfills this role in the herpes simplex virus (HSV)-infected cell (J. Virol. 66, 2240-2250, 1992). We have identified the gene product of the UL 115 open reading frame (ORF) as the functional homologue of HSV gL in the human cytomegalovirus (CMV) genome. In addition, we have demonstrated that a cellular gene, the human basic fibroblast growth factor receptor (FGFr) will also facilitate some transport of CMV gH to the cell surface. Coexpression in Chinese hamster ovary cells of the gene product of the UL115 ORF or soluble FGFr with C-terminally truncated gH enhanced levels of secreted gH. These studies suggest that the coexpressed molecules act to mask an ER retention signal(s) exposed when recombinant gH is expressed outside of the context of the viral-infected cell.

5' end-dependent translation initiation of hepatitis C viral RNA and the presence of putative positive and negative translational control elements within the 5' untranslated region.

Hepatitis C virus (HCV) is a distant relative of pestiviruses and flaviviruses, but it has a 5' untranslated region (UTR) with some features structurally similar to that of picornaviruses. In order to test the role of the 5' UTR in controlling the expression of the HCV polyprotein, we fused full-length or deleted versions of the 5' UTR of HCV-1 RNA to chloramphenicol acetyl transferase (CAT) mRNA to monitor CAT activity in vivo. We found: (1) the full-length 5' UTR of HCV-1 RNA is translationally inactive while 5' deletions which mimic a 5' subgenomic RNA detected in vivo are active, (2) an efficient cis-acting element which represses translation is found at the 5' terminus, (3) a putative element which enhances translation is found near the 3' terminus of the 5' UTR, (4) additional cis-acting elements including small open reading frames (ORFs) upstream from the putative enhancer element downregulate translation. We did not find evidence supporting the existence of an internal ribosome entry site in the 5' UTR of HCV-1 RNA. These data suggest that HCV may employ a distinctive translation control strategy such as the generation of subgenomic viral mRNA in infected cells. Translational control of HCV might be responsible for some of the characteristic pathobiology seen in viral infection.

Primary chimpanzee skin fibroblast cells are fully permissive for human cytomegalovirus replication.

Cytomegaloviruses generally display a host range restricted to differentiated cell types from the species they infect. For human cytomegalovirus (HCMV) this has meant that with few exceptions tissue culture systems have relied on the use of primary foreskin fibroblast (HF) cells or primary human embryonic lung cells to study gene expression and virus replication functions. We have observed that primary skin fibroblast (CF) cells derived from the chimpanzee (Pan troglodytes) support the replication of a laboratory strain (Towne) of HCMV. The kinetics of gene expression of the Towne strain grown in CF or HF cells appeared to be equivalent. Titres of progeny virions grown in CF cells appeared to be reduced 10-fold relative to those of virus grown in HF cells. In contrast, replication of the Towne virus was not supported by growth in WES cells (ATCC no. CRL 1609), a chimpanzee skin fibroblast cell line transformed by an adenovirus 12-simian virus 40 hybrid. This study shows that HCMV is less parochial in its host range than previously thought.

Characterization of the hepatitis C virus E2/NS1 gene product expressed in mammalian cells.

Truncated and full-length versions of the hepatitis C virus protein domain encoding a presumptive envelope glycoprotein designated E2/NS1 were stably expressed in CHO cell lines. Characterization of the processing events involved in the maturation of E2/NS1 revealed that a high-mannose form resident in the endoplasmic reticulum was the most abundant form detected intracellularly. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that the E2/NS1 glycoprotein resided in the endoplasmic reticulum. The full-length form of E2/NS1 appeared to be cell-associated and could not be detected as a secreted product. C-terminal truncated molecules could be detected in the extracellular media as fully processed glycoproteins containing terminal sialic acid additions. These truncated glycoproteins are predicted to be biologically relevant targets of the host immune response and are therefore potential subunit vaccine candidates.

Sequence requirements for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne.

Truncated versions of the human cytomegalovirus (CMV) strain Towne glycoprotein B (gB) gene were stably expressed in CHO cell lines. The calcium-specific ionophore A23187 inhibited proteolytic cleavage of C-terminal-truncated gB expressed by cell line 67.77. These inhibition studies also showed that the 93-kilodalton cleavage product most likely represents the N-terminal cleavage fragment of gB. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that proteolytic cleavage of gB did not occur in the endoplasmic reticulum. Two-dimensional polyacrylamide gel electrophoresis demonstrated that the N- and C-terminal cleavage products of gB remained associated by disulfide linkages after cleavage. Expression studies using constructs in which 80% or all of the N terminus was deleted demonstrated that the N terminus was required for secretion of the gB molecule. The amino acid sequence at the site of cleavage was shown to be critical for cleavage by a cellular protease. Our results indicate that an arginine-to-threonine change at either amino acid 457 or 460, a lysine-to-glutamine change at amino acid 459, or all three substitutions together block gB cleavage. The effect on proteolysis of the arginine-to-threonine amino acid change at residue 457 (position -4 relative to the cleavage site) demonstrated that a basic pair of amino acids at the endoproteolytic processing site is not the only requirement in cis for gB cleavage.

The human cytomegalovirus strain Towne glycoprotein H gene encodes glycoprotein p86.

The gene encoding the glycoprotein H (gH) homologue of CMV strain Towne was cloned, sequenced, and expressed. The predicted 742 amino acid gH protein had characteristics typical of a membrane glycoprotein including hydrophobic signal and transmembrane domains and six possible N-linked glycosylation sites. The CMV (Towne) gH gene had a 95% nucleotide identity and a 96.6% amino acid identity with the CMV (AD169) gH gene, as described by M. P. Cranage, G. L. Smith, S. E. Bell, H. Hart, C. Brown, A. T. Bankier, P. Tomlinson, B. G. Barrell, and T. C. Minson (1988, J. Virol. 62, 1416-1422). Transcriptional analysis of the gH gene revealed that the 2.9-kilobase (kb) gH transcript was not detected until late after CMV infection, indicating that the kinetics of gH expression were typical of the late class of CMV genes. The gH gene was expressed in COS cells using a vector in which transcription was driven by the SV40 early promoter. The expression of gH was detected by immunofluorescence using the virus neutralizing murine monoclonal antibody 1G6, which is specific for an 86-kilodalton (kDa) CMV virion membrane protein (p86). Amino acid sequence analysis of p86 tryptic peptides revealed sequence identity with peptides from the deduced gH amino acid sequence, confirming that the gH gene encodes p86. These results indicate that CMV gH can induce virus neutralizing antibodies and establishes gH as a candidate antigen for a subunit vaccine against CMV.

Human cytomegalovirus strain Towne glycoprotein B is processed by proteolytic cleavage.

The gene encoding glycoprotein B of human cytomegalovirus (CMV) strain Towne was cloned, sequenced, and expressed in order to study potential targets for viral neutralization. Secondary structure analysis of the 907 amino acid protein predicted a 24 amino acid N-terminal signal sequence and a potential transmembrane region composed of two domains, 34 and 21 amino acids. The CMV (Towne) gB gene had a 94% nucleotide similarity and a 95% amino acid similarity to the CMV (AD169) gB gene [as described by M.P. Cranage et al. (1986, EMBO J. 5, 3057-3063)]. Transcriptional analysis of the CMV (Towne) gB coding strand revealed that the gB message (3.9 kb), was transcribed from this region as early as 4 hr postinfection, and well in advance of gB protein synthesis. Full-length and truncated versions of the gB gene were expressed in COS cells using expression vectors where transcription was driven by the SV40 early promoter or the CMV major immediate early promoter. Expression was detected by immunofluorescence and ELISA using the virus neutralizing murine monoclonal antibody 15D8 (L. Rasmussen, J. Mullenax, R. Nelson, and T.C. Merigan, 1985, J. Virol. 55, 274-280). This antibody had been shown previously to recognize a 55-kDa CMV virion protein and a related 130-kDa intracellular precursor. Amino acid sequence analysis of the N-terminus of the 55-kDa viral glycoprotein (gp55) showed that gp55 is derived from gB (gp130) by proteolytic cleavage and represents the C-terminal region of gp130. The truncated version of gB expressed in COS and CHO cells was also processed by proteolytic cleavage as demonstrated by Western blotting. Our study localizes the epitope recognized by 15D8 to within a 186 amino acid fragment of the gp55 protein. These results indicate that CMV gB is a target for neutralization and establishes gp55 as a candidate component for use in a subunit vaccine.

Insertion and deletion mutagenesis of the human cytomegalovirus genome.

Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. We have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major beta gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this beta gene within the L-component repeats of CMV DNA. We observed high-level expression of beta-galactosidase by the recombinant in a temporally authentic manner, with levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl beta-D-galactoside, we generated random endpoint deletion mutants. Analysis of these mutants revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, we have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.

Structure and variability of the a sequence in the genome of human cytomegalovirus (Towne strain).

We have defined the boundaries of the a sequence from human cytomegalovirus (CMV) strain Towne, characterized internal variability and determined the position of the cleavage site used to generate genomic termini. The cleavage site is positioned a fixed distance from two stretches of sequence homology that have been observed near the ends of many herpesvirus genomes. Unlike a comparable region in CMV (AD169), the CMV (Towne) a sequence has a relatively low level of variability within the a sequence and its structure is stable through repeated virus passage.

A cis-acting element within the 5' leader of a cytomegalovirus beta transcript determines kinetic class.

During cytomegalovirus (CMV) infection, gene expression is regulated by transcriptional and posttranscriptional events. Although recent studies have established that posttranscriptional controls are important determinants of gene expression in several eukaryotic systems, the precise signals and mechanisms have not been clearly identified. We present evidence for a cis-acting signal, contained within the 5' leader region of a CMV beta (or early) gene, that acts posttranscriptionally in the regulation of gene expression. Addition of this signal to an alpha (or immediate early) gene construct converted expression of the indicator protein to the beta class, even though the gene remained under alpha transcriptional control. Deletion of a portion of the beta gene leader sequence (nucleotides +62 to +142) reverted expression to the alpha class. This cis-dominant signal appears to act by blocking expression posttranscriptionally until a viral function activates full gene expression at the appropriate time in infection.

Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts.

We have fused immediate (alpha) and delayed (beta) early promoter-regulatory sequences taken from the cytomegalovirus (CMV) genome to Escherichia coli lacZ (beta-galactosidase) as an indicator gene to study regulated expression of these promoters. After transfection of human fibroblast cells with plasmid constructs carrying beta-galactosidase fusions, and subsequent infection with CMV, we have demonstrated that viral trans-acting functions up-regulate the expression of these genes in a temporally authentic manner. The alpha promoter is activated even when de novo protein synthesis is blocked and when UV-inactivated virus is used, suggesting that, as for herpes simplex virus type 1 (HSV-1), a virion structural protein is responsible for its up-regulation. We have found that HSV-1, as well as CMV, is capable of trans activating the CMV alpha promoter. The beta promoter is activated by CMV but is completely unresponsive to HSV-1 infection. The temporal synthesis of the alpha and beta promoters in the transient expression system conforms with their natural regulation during viral replication. The beta-galactosidase fusions we describe provide a most exquisitely sensitive indicator system for the study of cis- and trans-acting viral regulatory functions.

The alpha sequence of the cytomegalovirus genome functions as a cleavage/packaging signal for herpes simplex virus defective genomes.

Although herpes simplex virus (HSV) 1 and human cytomegalovirus (CMV) differ remarkably in their biological characteristics and do not share nucleotide sequence homology, they have in common a genome structure that undergoes sequence isomerization of the long (L) and short (S) components. We have demonstrated that the similarity in their genome structures extends to the existence of an alpha sequence in the CMV genome as previously defined for the HSV genome. As such, the alpha sequence is predicted to participate as a cis-replication signal in four viral functions: (i) inversion, (ii) circularization, (iii) amplification, and (iv) cleavage and packaging of progeny viral DNA. We have constructed a chimeric HSV-CMV amplicon (herpesvirus cis replication functions carried on an Escherichia coli plasmid vector) substituting CMV DNA sequences for the HSV cleavage/packaging signal in a test of the ability of this CMV L-S junction sequence to provide the cis signal for cleavage/packaging in HSV 1-infected cells. We demonstrate that the alpha sequence of CMV DNA functions as a cleavage/packaging signal for HSV defective genomes. We show the structure of this sequence and provide a functional demonstration of cross complementation in replication signals which have been preserved over evolutionary time in these two widely divergent human herpesviruses.