Efficacy and safety studies of a recombinant chimeric respiratory syncytial virus FG glycoprotein vaccine in cotton rats.

Several formulations of a recombinant chimeric respiratory syncytial virus (RSV) vaccine consisting of the extramembrane domains of the F and G glycoproteins (FG) were tested in cotton rats to evaluate efficacy and safety. The FG vaccine was highly immunogenic, providing nearly complete resistance to pulmonary infection at doses as low as 25 ng in spite of inducing relatively low levels of serum neutralizing antibody at low vaccine doses. Upon RSV challenge animals primed with FG vaccine showed quite mild alveolitis and interstitial pneumonitis, which were eliminated by the addition of monophosphoryl lipid A to the formulation.

Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation.

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Ha-ras oncogene to transform primary rat embryo fibroblasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity. Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro.

Cytotoxic T cell immunity to human cytomegalovirus glycoprotein B.

Human cytomegalovirus (HCMV) is associated with significant morbidity and mortality following immunosuppression and in pregnancy. HCMV infection may be accompanied by acute disease but persists asymptomatically. Cytotoxic T lymphocytes (CTL) appear to be an important immune effector mechanism in maintaining the normal host-virus equilibrium. Glycoprotein B may be an important target for future subunit vaccines as it has been found to elicit both neutralising antibody and CTL responses. We therefore studied the ability of normal asymptomatic HCMV-seropositive individuals and women throughout pregnancy to determine the presence of HCMV and gB-specific CTL responses. CTL effector cells were induced by stimulation of peripheral blood mononuclear cells (PBMC) with AD169 HCMV-infected cells and gB-specific CTL were identified using chromium labeled, vac.gB-infected cells. In 7 HCMV-seropositive individuals, HCMV-specific CTL were identified. Three of the 7 individuals which lysed HCMV-infected cells lysed vac.gB-infected B cells. However, vac.gB-infected autologous fibroblasts, which only present MHC class I, were not killed. Using MHC class I single allele targets, no specific lytic response was observed, suggesting a MHC class II restricted CTL response. Flow cytometric analysis showed the gB-specific effector cell phenotype to be CD3+, CD4+, CD8-. In conclusion, a gB-specific CTL lytic response was identified in seropositive individuals which in most cases was MHC class II-restricted.

Vaccination with recombinant vaccinia viruses expressing ICP27 induces protective immunity against herpes simplex virus through CD4+ Th1+ T cells.

This study was designed to evaluate the efficacy and mechanisms of protection mediated by recombinant vaccinia viruses encoding immediate-early (IE) proteins of herpes simplex virus type 2 (HSV-2). Three mouse strains were immunized against the IE proteins ICP27, ICP0, and ICP4, and mice were challenged intracutaneously in the zosteriform model with HSV-2 strain MS. Protection was observed only following immunization with the ICP27 construct and then only in the BALB/c mouse strain. Protection in BALB/c mice was ablated by CD4+ T-cell suppression but remained intact in animals depleted of CD8+ T cells. Moreover, protection could be afforded to SCID nude recipients with CD4+ but not CD8+ T cells from ICP27-immunized mice. Only BALB/c mice developed a delayed-type hypersensitivity reaction to HSV-2, and in vitro measurements of humoral and cell-mediated immunity revealed response patterns to ICP27 and HSV that differed between protected BALB/c and unprotected mouse strains. Accordingly, BALB/c responses showed antigen-induced cytokine profiles dominated by type 1 cytokines, whereas C57BL/6 and C3H/HeN mice generated cytokine responses mainly of the type 2 variety. Our results may indicate that protection against zosterification is mainly mediated by CD4+ T cells that express a type 1 cytokine profile and that protective vaccines against HSV which effectively induce such T-cell responses should be chosen.

Subcellular localization of recombinant truncated Gag precursor proteins of HIV expressed in Saccharomyces cerevisiae.

OBJECTIVE: To determine signals contained in the HIV-1 Gag precursor implicated in protein transport. DESIGN: To study the localization of truncated Gag proteins expressed in Saccharomyces cerevisiae. METHODS: Thin-section immunoelectron microscopy studies were performed on S. cerevisiae cells producing myristoylated or non-myristoylated Pr55gag, the core protein (p24) and several truncated Gag proteins. RESULTS: Pr55gag and the carboxy-terminal truncated Gag proteins were myristoylated and localized at the plasma membrane. p24 was localized in the nucleus or perinuclear membrane. However, addition of a myristoyl group to p24 targeted this molecule to the plasma membrane. CONCLUSIONS: The myristoylated amino-terminal 214 amino acids are sufficient to target Pr55gag to the plasma membrane. Subcellular signals implicated in protein transport are present in the core p24 polypeptide which may become dominant or accessible in the absence of the amino-myristoyl group.

Plasmodium falciparum: recombinant baculoviruses direct the expression of circumsporozoite proteins in Spodoptera frugiperda cell cultures.

The DNA coding for the circumsporozoite protein (CPS) of Plasmodium falciparum has been cloned into the baculovirus expression vector pAcYM1 and expressed in Spodoptera frugiperda (Sf9) insect cells. Three DNA constructs have been made: the first one directs the synthesis of the complete CSP (aa 1-412), the second leads to the production of a species devoid of the anchor domain (aa 1-391) and the third one to a molecule lacking both signal and membrane anchor sequences (aa 18-391). All three recombinant CPS were produced at about 3 micrograms per 10(6) infected cells and were characterized in terms of immunoreactivity and apparent molecular weight. Analytical purification of the recombinant proteins was achieved by a combination of heat treatment, acidification, isoelectric focusing and ion exchange chromatography. The purified material, when injected into mice, generated only modest antibody responses, although antisera from immunized mice reacted with control CSP antigens carrying or not the major immunodominant repeat region.

Synthesis of functional bovine leukemia virus (BLV) p34tax protein by recombinant baculoviruses.

The bovine leukemia virus (BLV) p34tax (also called tat, p34, XLOR gene product) is a 34-kDa polypeptide encoded in the 3'-terminal region of the virus. This protein is responsible for positive transcriptional trans-activation of promoter elements located within the BLV long-terminal repeat. We introduced the protein-coding region of BLV p34tax into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus. After infection of the insect Spodoptera frugiperda (SF9) cell line, this recombinant strain of baculovirus produced approximately 100 to 150 mg of p34tax per 2 X 10(9) cells. This protein, when introduced into mammalian fibroblasts by using a cell-to-cell fusion technique, functionally trans-activated the BLV long-terminal repeat. Analysis of 32P-labeled proteins of SF9 cells expressing BLV tax by two-dimensional gel electrophoresis indicated that the BLV p34tax was phosphorylated.

Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells.

The unprocessed Gag precursor from HIV-1, when expressed in recombinant baculovirus-infected insect cells, is targeted to the plasma membrane and assembles in 100-120 nm particles budding from the cell surface. This process mimics HIV immature particle formation and is dependent on myristoylation of the N-terminal glycine, as deletion of the latter results in particle accumulation in the cytoplasm and, interestingly, in the nucleus, pointing to a potential role of this non-fatty-acid-acylated species in the viral life cycle. Inclusion of the pol gene in the construct results in efficient processing of Pr55gag and a pronounced decrease in particle formation. Deletion of the C terminus (p16) of the Gag precursor, including the finger domains, abolishes particle assembly, but membrane targeting and evagination still occur. Heterologous expression in insect cells may prove very useful for the study of the molecular events leading to retroviral particle morphogenesis.

The GAG precursor of simian immunodeficiency virus assembles into virus-like particles.

To examine the potential role of the GAG precursor polyprotein in morphogenesis and assembly of the simian immunodeficiency virus (SIV), we have expressed the gag gene of SIVMac using a baculovirus expression vector. Infection of insect cells with recombinant virus containing the entire gag gene results in high expression of the GAG precursor protein, Pr57gag. The recombinant protein is myristylated and is released in the culture supernatant in an insoluble particulate form. A point mutation in the N-terminal glycine codon (Gly----Ala) inhibits myristylation. This mutated product is highly expressed but is not found in the culture supernatant. Electron microscopy and immunogold labelling of infected cells show that the native Pr57gag protein assembles into 100-120 nm virus-like particles that bud from the cell plasma membrane and are released in the culture supernatant. The unmyristylated protein also assembles into particulate structures which only accumulate inside the cells. These results demonstrate that the unprocessed GAG precursor of SIV can spontaneously assemble into particles in the absence of other viral proteins. Myristylation of the Pr57gag precursor is necessary for its association with the cell plasma membrane, for budding and for extracellular release.

The HIV-1 Gag precursor Pr55gag synthesized in yeast is myristoylated and targeted to the plasma membrane.

Myristoylation of the Pr65gag protein from Moloney murine leukemia virus has been shown to be essential for virus particle formation [Rein et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7246-7250], and by analogy, myristoylation of the human immunodeficiency virus (HIV) Gag precursor could possibly play a similar role. We have investigated the expression and myristoylation of the complete HIV Gag precursor Pr55gag in yeast, the subcellular localization of that protein, and the contribution of the myristoyl-glycine residue to this localization. Immunogold labelling of myristoylated Pr55gage with antibodies directed against HIV Gag products was apparent in the vicinity of the plasma membrane. On the contrary, non-myristoylated derivatives of Pr55gag were only detected in relatively well-defined regions of the cytoplasm. These results show that targeting and accumulation of the HIV Gag precursor, Pr55gag, at the plasma membrane occurs in yeast in the absence of other viral components and requires the N-myristoyl-glycine residue.

Characterization of a chimaeric plasminogen activator obtained by insertion of the second kringle structure of tissue-type plasminogen activator (amino acids 173 through 262) between residues Asp130 and Ser139 of urokinase-type plasminogen activator.

A chimaeric recombinant plasminogen activator (rscu-PA- K2) obtained by insertion of the second kringle (K2) of tissue-type plasminogen activator (t-PA) (amino acids 173-262) between residues Asp130 and Ser139 of single chain urokinase-type plasminogen activator (scu-PA) was purified from the conditioned medium of mouse myeloma cells transfected with the previously described plasmid pULB9137 (Piérard et al., J. Biol. Chem. 262, 11771-11778, 1987). Approximately 22 micrograms of purified protein was obtained per liter of conditioned medium with a yield of approximately 25 percent. On sodium dodecylsulfate gel electrophoresis under reducing conditions, rscu-PA- K2 migrated with an apparent Mr of 65,000. Plasmin caused a time- and concentration-dependent conversion to an amidolytically active two chain derivative (rtcu-PA- K2) with a specific activity of 45,000 IU/mg. Both rscu-PA- K2 and rtcu-PA- K2 activated plasminogen directly with Km = 2.0 microM and k2 = 0.00063 s-1 and Km = 100 microM and k2 = 4.1 s-1 respectively. rscu-PA- K2 did not bind extensively to fibrin. It caused concentration-dependent lysis of 125I-fibrin-labeled plasma clots immersed in human plasma with a comparable specific activity and fibrin-specificity as rscu-PA. It is concluded that insertion in scu-PA of the second kringle of t-PA, which is believed to be involved in its fibrin affinity, does not significantly alter the enzymatic properties of scu-PA, but does not confer marked fibrin-affinity to the molecule.

Mutant and chimeric recombinant plasminogen activators. Production in eukaryotic cells and preliminary characterization.

Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-PA have been designed to direct the synthesis of new plasminogen activators and to investigate the structure-function relationship in these molecules. The following classes of constructs were made starting from cDNA encoding human t-PA or u-PA: 1) u-PA mutants in which the Arg156 and Lys158 were substituted with threonine, thus preventing cleavage by thrombin and plasmin; 2) hybrid molecules in which the NH2-terminal regions of t-PA (amino acid residues 1-67, 1-262, or 1-313) were fused with the COOH-terminal region of u-PA (amino acids 136-411, 139-411, or 195-411, respectively); and 3) a hybrid molecule in which the second kringle of t-PA (amino acids 173-262) was inserted between amino acids 130 and 139 of u-PA. In all cases but one, the recombinant proteins, produced by transfected eukaryotic cells, were efficiently secreted in the culture medium. The translation products have been tested for their ability to activate plasminogen after in situ binding to an insolubilized monoclonal antibody directed against urokinase. All recombinant enzymes were shown to be active, except those in which Lys158 of u-PA was substituted with threonine. Recombination of structural regions derived from t-PA, such as the finger, the kringle 2, or most of the A-chain sequences, with the protease part or the complete u-PA molecule did not impair the catalytic activity of the hybrid polypeptides. This observation supports the hypothesis that structural domains in t-PA and u-PA fold independently from one to another.

Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator.

Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system. The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration. Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900. On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid. Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively. Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma. It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.

Simian virus 40 mutants carrying extensive deletions in the 72-base-pair repeat region.

Simian virus 40 mutants were constructed with deletions at the late side of the origin of DNA replication by partial Bal 31 digestion at the SphI site or at the PvuII site. Some of these mutants lost virtually all of both 72-base-pair repeat segments ("enhancer" sequences) and exhibited a decrease in viability from 20-to 300-fold; one particular mutant, dl1852, even showed a reduction of almost 10(4)-fold. The very poorly growing deletion mutants were unstable and gave rise to DNA rearrangements upon further growth. An essential region for viability, at least in the absence of a 72-base-pair repeat, was revealed at the distal side of the 72-base-pair elements (L250 through L272). The effect of the deletions on T-antigen expression was measured, and the decreased viability of the mutants correlated with the impairment of T-antigen expression in all cases. The study of these mutants also revealed that the 72-base-pair repeats are not required for late transcription.

Deletion mapping of the inducible promoter of human IFN-beta gene.

Human fibroblast cells produce beta-type interferon only in response to viral infection or treatment with an inducer such as poly(rI) X poly(rC); this event is most probably controlled at the transcriptional level (for review see ref. 1). To study the induction process, we inserted the human fibroblast interferon (IFN-beta) gene, with or without its promoter region, into recombinant simian virus 40 (SV40) plasmid vectors which subsequently were transfected into monkey AP-8 cells. We report here that upon induction with poly(rI) X poly(rC) there was a 10-30-fold increase in IFN-beta synthesis. This inducer had no effect on interferon production when the coding region only was inserted into the vector plasmid, which indicates that the promoter region is required for inducibility of this gene. Deletion mapping implicates the region between nucleotides -144 and -186 from the mRNA initiation site in the specific regulation of the IFN-beta gene. This region contains a sequence that is remarkably homologous with a consensus sequence found in the 5' flanking region of steroid hormone responsive genes, which might be involved in binding the progesterone-receptor complex.

The human fibroblast and human immune interferon genes and their expression in homologous and heterologous cells.

The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.

The human fibroblast interferon gene(s) and their expression in heterologous cells.

A clone has been identified which contains the genetic information specifying human fibroblast interferon (beta 1) and which was derived from a messenger RNA population. On the basis of the nucleotide sequence the complete amino acid sequence (166 residues) of human fibroblast interferon was deduced. It is preceded by a 21 amino acids long signal sequence. Also, a genomic clone was isolated and characterized in detail; on this basis we can conclude that the interferon beta 1 gene does not contain introns. The nucleotide sequence of the putative promoter region was deduced and compared with other eukaryotic promoters. So far, no evidence has been obtained for other human interferons of the fibroblast type. The interferon beta 1 was expressed in the mature form of Escherichia coli without glycosylation. Fully processed interferon was obtained by transfection of monkey CV1-cells with an SV40-derivative, in which the interferon gene replaced the major structural protein VP1.

Expression and excretion of human fibroblast beta 1 interferon in monkey cells after transfection with a recombinant SV40 plasmid vector.

We have constructed a eukaryotic expression vector designed to express a gene under late SV40 transcriptional control. From this chimeric plasmic-SV40 vector, virtually all the sequences which code for the major capsid protein VP1 have been deleted and instead, the human fibroblast interferon beta 1 cDNA gene has been inserted. After transfection of monkey cells with this recombinant, substantial quantities of human beta 1 interferon (up to 2 x 10(-4) IU/ml) were excreted in the culture medium. Transfection of nonpermissive mouse L cells or rat cells yielded virtually undetectable quantities of human beta 1 interferon (5 x 10(3) to 10(4) times less than that in monkey cells). The recombinant SV40 vector may serve as a model vehicle for the efficient expression of other eukaryotic genes and might also be used as a direct screening vector for cloning of eukaryotic or prokaryotic cDNA genes.

High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter.

Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter (pL) of bacteriophage lambda. Upon temperature induction the best of our constructions expressed a small-t-related 19 000-dalton polypeptide in an amount corresponding to approx. 2.5% of total de novo protein synthesis. This 19 000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis. In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent Mr of 14 500. This 14 500-dalton polypeptide was shown to be related to authentic small-t. Presumably the secondary structure of the mRNA starting at pL is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon.