Naphthalene carboxamides as inhibitors of human cytomegalovirus DNA polymerase.

ortho-Hydroxynaphthalene carboxamides have been identified as inhibitors of HCMV DNA polymerase. SAR investigations have demonstrated that both the amide and hydroxy functionalities are required for activity. Substitution on the naphthalene ring has led to inhibitors with submicromolar IC50s against HCMV polymerase. These compounds have been found to be >100-fold selective for inhibition of HCMV polymerase versus human alpha polymerase and display antiviral activity in a cell-based plaque reduction assay.

New antiherpetic 1,3-phenylene derivatives, inhibitors of the interaction of the HSV-1 origin binding protein (OBP) with DNA.

The synthesis of new 1,3-phenylene derivatives and their preliminary evaluation as antivirals (Herpes simplex 1, HSV-1) whose antiherpetic activity can be related with the inhibition of the interaction of the origin binding protein (OBP) with the DNA are presented. The new compounds are adjusted to a previously defined common structural model, consisting of a central aromatic system, which presents two side chains of different lengths in relative position 1, 3; these chains are made up of atomic groups characterized by the alternation of positive and negative centers, situating differently substituted rings, preferably aromatic, at the ends of both chains. Some of these derivatives, such as N,N''-(4-methoxy-1,3-phenylene)bis[N'-(4-nitrophenyl)urea] (2c) or (1,3-phenylene)bis[N-(p-tolyl)aminosulfonyl] (11b), show antiherpetic activity related to the proposed mechanism.

Assembly of herpes simplex virus capsids using the human cytomegalovirus scaffold protein: critical role of the C terminus.

An essential step in assembly of herpes simplex virus (HSV) type 1 capsids involves interaction of the major capsid protein (VP5) with the C terminus of the scaffolding protein (encoded by the UL26.5 gene). The final 12 residues of the HSV scaffolding protein contains an A-X-X-F-V/A-X-Q-M-M-X-X-R motif which is conserved between scaffolding proteins found in other alphaherpesviruses but not in members of the beta- or gamma-herpesviruses. Previous studies have shown that the bovine herpesvirus 1 (alphaherpesvirus) UL26.5 homolog will functionally substitute for the HSV UL26.5 gene (E. J. Haanes et al., J. Virol. 69:7375-7379, 1995). The homolog of the UL26.5 gene in the human cytomegalovirus (HCMV) genome is the UL80.5 gene. In these studies, we tested whether the HCMV UL80.5 gene would substitute for the HSV UL26.5 gene in a baculovirus capsid assembly system that we have previously described (D. R. Thomsen et al., J. Virol. 68:2442-2457, 1994). The results demonstrate that (i) no intact capsids were assembled when the full-length or a truncated (missing the C-terminal 65 amino acids) UL80.5 protein was tested; (ii) when the C-terminal 65 amino acids of the UL80.5 protein were replaced with the C-terminal 25 amino acids of the UL26.5 protein, intact capsids were made and direct interaction of the UL80.5 protein with VP5 was detected; (iii) assembly of intact capsids was demonstrated when the sequence of the last 12 amino acids of the UL80.5 protein was changed from RRIFVA ALNKLE to RRIFVAAMMKLE; (iv) self-interaction of the scaffold proteins is mediated by sequences N terminal to the maturation cleavage site; and (v) the UL26.5 and UL80.5 proteins will not coassemble into scaffold structures. The results suggest that the UL26.5 and UL80.5 proteins form a scaffold by self-interaction via sequences in the N termini of the proteins and emphasize the importance of the C terminus for interaction of scaffold with the proteins that form the capsid shell.

Functional analysis of human cytomegalovirus polymerase accessory protein.

The human cytomegalovirus (HCMV) UL44 gene product, polymerase accessory protein, was cloned and expressed in Escherichia coli as a 53,000 MW protein. The activity of HCMV DNA polymerase (Pol) alone and Pol/UL44 complex was evaluated in Pol assays designed specifically to elucidate Pol/UL44 interactions. Addition of UL44 to HCMV Pol with primed, single-stranded DNA resulted in increased incorporation of nucleotides into DNA, which was correlated with enhanced enzyme processivity. Several deletion mutants which span the UL44 sequence were constructed and examined for the ability to stimulate Pol activity and to bind double-stranded DNA. The functional domains of UL44 protein were determined to reside within the N-terminal 309 amino acids of the wild type sequence, since deletions within this region resulted in loss of DNA binding and the ability to stimulate Pol. Deletion of C-terminal amino acids 310-433 had no effect on the ability of UL44 protein to increase the processivity of HCMV DNA Pol.

Purification of a recombinant human respiratory syncytial virus chimeric glycoprotein using reversed-phase chromatography and protein refolding in guanidine hydrochloride.

FG glycoprotein is a recombinant chimeric protein consisting of the extracellular portions of human respiratory syncytial virus (RSV) F and G glycoproteins. In theory, highly purified FG glycoprotein may be effective as a RSV vaccine. Recombinant FG glycoprotein was expressed using the baculovirus/insect cell system. FG glycoprotein was isolated from cell culture supernatants using S Sepharose ion-exchange chromatography, Cu(2+)-immobilized metal affinity chromatography, preparative reversed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergent. The purified FG glycoprotein was concentrated on a S Sepharose column and exchanged into an appropriate buffer for vaccine formulation. Five batches of FG glycoprotein with protein purity of 92-99% were produced using this purification process. FG glycoprotein produced using reversed-phase chromatography and protein refolding was compared with nondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G glycoproteins. The results of these studies indicated that refolded FG glycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein.

Induction of local and systemic immunity against human respiratory syncytial virus using a chimeric FG glycoprotein and cholera toxin B subunit.

Local IgA and IgG antibodies against respiratory syncytial virus (RSV) were induced in the respiratory tract of mice following intranasal vaccination with an RSV chimeric FG glycoprotein and cholera toxin B (CTB) as a mucosal adjuvant. Local antibody production was not induced following parenteral immunization with FG administered in alum adjuvant. While both vaccination protocols induced serum antibodies against RSV and protected the lower respiratory tract from RSV infection, only intranasal FG/CTB afforded protection of the upper respiratory tract. These data suggest that vaccination via the mucosal route may be superior to vaccination by a parental route in providing complete protection against RSV.

Development of a novel subunit vaccine that protects cotton rats against both human respiratory syncytial virus and human parainfluenza virus type 3.

A cotton rat model of experimental human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (PIV-3) infection was used to examine the efficacy of FRHNP, a novel chimeric glycoprotein which contains the extracellular regions of the fusion glycoprotein of RSV and the attachment glycoprotein of PIV-3, as a single subunit vaccine against these two viruses. This work was prompted by previous cotton rat studies that demonstrated that the major protective antigens of the two viruses were these glycoproteins. FRHNP was expressed in insect cells using a recombinant baculovirus. Vaccination with FRHNP resulted in induction of both RSV and PIV-3 neutralizing antibody and doses of 200 ng completely protected rats from either RSV or PIV-3 challenge. These results demonstrate that in the cotton rat animal model a single chimeric glycoprotein can be an effective vaccine against both RSV and PIV-3.

Herpes simplex virus type 1 DNA cleavage and encapsidation require the product of the UL28 gene: isolation and characterization of two UL28 deletion mutants.

The herpes simplex virus type 1 UL28 gene contains a 785-amino-acid open reading frame that codes for an essential protein. Studies with temperature-sensitive mutants which map to the UL28 gene indicate that the UL28 gene product (ICP18.5) is required for packaging of viral DNA and for expression of viral glycoproteins on the surface of infected cells (C. Addison, F. J. Rixon, and V. G. Preston, J. Gen. Virol. 71:2377-2384, 1990; B. A. Pancake, D. P. Aschman, and P. A. Schaffer, J. Virol. 47:568-585, 1983). In this study, we describe the isolation of two UL28 deletion mutants that were constructed and propagated in Vero cells transformed with the UL28 gene. The mutants, gCB and gC delta 7B, contained deletions of 1,881 and 537 bp, respectively, in the UL28 gene. Although the mutants synthesize viral DNA, they fail to form plaques or produce infectious virus in cells that do not express the UL28 gene. Transmission electron microscopy and Southern blot analysis demonstrated that both mutants are defective in cleavage and encapsidation of viral DNA. Analysis by cell surface immunofluorescence showed that the UL28 gene is not required for expression of viral glycoproteins on the surface of infected cells. A rabbit polyclonal antiserum was made against an Escherichia coli-expressed Cro-UL28 fusion protein. This antibody reacted with an infected-cell protein having an apparent molecular mass of 87 kDa. The 87-kDa protein was first detected at 6 h postinfection and was expressed as late as 24 h postinfection. No detectable UL28 protein was synthesized in gCB- or gC delta 7B-infected Vero cells.

Comparison of soluble and secreted forms of human parainfluenza virus type 3 glycoproteins expressed from mammalian and insect cells as subunit vaccines.

Human parainfluenza virus type 3 (PIV-3) is one of the leading causes of paediatric viral respiratory disease. The PIV-3 genome encodes two envelope glycoproteins, F and HN, which are the major targets for the host antibody response. We have expressed secreted forms of the F and HN proteins and a novel chimeric FHN glycoprotein in insect cells using recombinant baculovirus vectors and secreted forms of the F and FHN glycoproteins in stably transformed Chinese hamster ovary (CHO) cells. Comparison of the mammalian cell- and insect cell-expressed F and FHN proteins by SDS-PAGE showed that the CHO cell-expressed proteins are several kilodaltons larger in size than the baculovirus-produced proteins. A partial characterization of the oligosaccharide structures of the F and FHN proteins revealed that the size difference is due to the different oligosaccharide structures added to these proteins by the two cell lines. The F, HN and FHN proteins were immunoaffinity-purified from the culture medium of baculovirus-infected Sf9 cells and the F and FHN proteins were immunoaffinity-purified from the culture medium of CHO cells. A comparison of the immunogenicity and efficacy of the mammalian cell- and insect cell-produced FHN proteins was tested in cotton rats. The CHO cell- and baculovirus-produced FHN proteins were found to induce similar levels of PIV-3-specific ELISA-positive and neutralizing antibodies and both proteins provided near complete protection when animals were vaccinated with low doses of the FHN protein.

Protection of cotton rats against human parainfluenza virus type 3 by vaccination with a chimeric FHN subunit glycoprotein.

A cotton rat model of experimental human parainfluenza virus type 3 (PIV-3) infection was used to examine the efficacy of FHN, a novel chimeric glycoprotein which contains the extracellular regions of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of PIV-3. The FHN protein was expressed in insect cells using a baculovirus vector system. FHN vaccination resulted in induction of neutralizing antibodies, was completely protective at doses of 100 ng, and was superior to vaccination with secreted forms F and HN proteins, or mixtures of the F and HN glycoproteins. In addition, FHN immunization induced lymphoproliferative responses in mice which were directed against both the F and HN glycoproteins. Fusion of the F and HN proteins into a single chimeric glycoprotein appeared to enhance the protective immune response compared to that elicited by the individual glycoproteins or mixtures of the two glycoproteins.

A human respiratory syncytial virus (RSV) primate model of enhanced pulmonary pathology induced with a formalin-inactivated RSV vaccine but not a recombinant FG subunit vaccine.

Human respiratory syncytial virus (RSV) is the leading cause of severe bronchiolitis and pneumonia in infants. RSV vaccine development has been stifled for the past 23 years because infants vaccinated with formalin-inactivated (FI) RSV have experienced exacerbated disease upon RSV infection. This exacerbated disease phenomenon is poorly understood, in part because of the lack of a primate model that exhibits a similar exacerbated disease state. Vaccination of African green monkeys with either FI RSV or a genetically engineered subunit vaccine termed FG glycoprotein reduced replication of challenge virus. However, only vaccination with FI RSV induced an enhanced pulmonary pathologic response to RSV infection. Pulmonary inflammatory scores in the FG glycoprotein-vaccinated monkeys were no greater than in monkeys vaccinated with adjuvant alone. This is the first demonstration of RSV vaccine-induced enhanced pathology in a primate and illustrates that a subunit vaccine has the potential of circumventing this exacerbated disease phenomenon.

Vaccination with a heterologous respiratory syncytial virus chimeric FG glycoprotein demonstrates significant subgroup cross-reactivity.

A subunit vaccine candidate, termed FG, is a chimeric glycoprotein composed of the extracellular domains of the fusion (F) glycoprotein and the attachment (G) glycoproteins of a subgroup A respiratory syncytial virus (RSV). Two subgroups, A and B, of RSV differ primarily within the G glycoprotein. Therefore, it has been suggested that a subunit vaccine composed of the G glycoprotein would need to contain the G glycoproteins from both RSV subgroups. We have engineered a second chimeric glycoprotein, FGB, which is composed of the F glycoprotein from RSV subgroup A and the G glycoprotein from RSV subgroup B and is expressed in baculovirus. A comparison of protection between the two subunit vaccines (FG and FGB) was performed in cotton rats after homologous and heterologous virus challenge. FG and FGB appeared to afford the same degree of protection against either homologous or heterologous challenge. Serum neutralization titres against homologous or heterologous virus were nearly equivalent following FG or FGB vaccination. Radioimmunoprecipitation using sera from rats immunized with FG or FGB revealed cross-reactivity between the two G glycoproteins. Adsorption of anti-F antibody from serum of rats immunized with FG significantly reduced the RSV neutralizing activity of the serum suggesting that enhanced neutralization previously observed with FG antisera compared with F antisera alone may not be entirely attributed to antibodies against the G glycoprotein but may be attributed to a function associated with the G glycoprotein portion of FG which enhances the immunogenicity of the F portion of FG.

A chimeric glycoprotein of human respiratory syncytial virus termed FG induces T-cell mediated immunity in mice.

Popliteal lymph node cells taken from mice vaccinated with the FG glycoprotein were exposed in vitro to respiratory syncytial virus (RSV) antigens. Proliferation to FG or RSV antigens was blocked with anti-CD4 monoclonal antibody treatment. FG-vaccinated mice developed classical late delayed type hypersensitivity (DTH) reactions when exposed to FG antigen in vivo.

Vaccination of cotton rats with a chimeric FG glycoprotein of human respiratory syncytial virus induces minimal pulmonary pathology on challenge.

The cotton rat model of human respiratory syncytial virus (RSV) infection was used to study the safety and efficacy of a chimeric FG glycoprotein that was expressed in insect cells using a baculovirus vector. Histologic and virologic examination of vaccinated rat lungs was done after challenge with RSV. When rats were challenged 1 month after vaccination, severe pulmonary inflammation characterized by both a mononuclear and polymorphonuclear cell infiltrate and 30%-40% involvement of lung tissue was observed with a formalin-inactivated RSV vaccine. The FG glycoprotein induced minimal lung inflammation (involving 2%-5% of the lung), while negative controls had 1%-3% lung involvement. Two doses with as little as 20 ng of FG glycoprotein formulated in an aluminum hydroxide adjuvant completely protected the cotton rats from RSV challenge. Thus the chimeric FG glycoprotein is highly immunogenic and induces minimal pulmonary inflammation in the cotton rat model.

Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9.

The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with [3H]glucosamine and infected with a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Gal beta 1-3GalNAc constituting 17 and 66% of the total number of structures, respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked alpha 1-6 to the asparagine-linked N-acetylglucosamine.

Validation of an immunoradiometric assay to measure plasma levels of active renin.

An immunoradiometric assay (IRMA) for active renin in human plasma was analytically and clinically validated. Analytical validation established 1) precision, 2) recovery, 3) linearity, 4) cross-reactivity, 5) sample stability, and 6) the validity and specificity of the 125I-labeled anti-renin monoclonal in the Diagnostics Pasteur immunoradiometric renin kit. Clinical validation included 1) establishing normal reference range for renin, 2) comparing plasma renin activity (PRA) results to immunoreactive renin levels in subjects on Upjohn research protocols, and 3) comparing the renin responsiveness of sodium replete subjects to that of sodium deplete subjects prior to, during, and after infusion with Upjohn renin inhibitory peptide, ditekiren. This study was undertaken to demonstrate the research validity of an assay tool for the differentiation of enzymatically active renin from inactive renin or a form of prorenin.

Production, characterization, and utility of monoclonal antibodies which react with a novel chimeric glycoprotein of human respiratory syncytial virus termed FG.

Primary immunization of mice with recombinant vaccinia virus expressing the F or G glycoprotein of human respiratory syncytial virus followed by an intravenous boost with crude FG chimeric glycoprotein resulted in the generation of hybridomas each specific for either the F or G portion of FG. Characterization of each MAb was determined following binding to various viral and glycoprotein antigens, by immunoprecipitation, by competition binding and by subclass determination. Relative affinity was determined for each MAb following inhibition of binding by ammonium thiocyanate.

Characterization of a novel human respiratory syncytial virus chimeric FG glycoprotein expressed using a baculovirus vector.

Human respiratory syncytial virus (RSV) codes for two glycoproteins (F and G) which have been shown to the major targets for the host antibody response. We have expressed a novel chimeric glycoprotein (FG) in insect cells using a baculovirus vector. The chimeric glycoprotein contains the signal and extracellular regions of the RSV F glycoprotein linked to the extracellular region of the RSV G glycoprotein. Beginning at the amino terminus, the chimeric glycoprotein consists of amino acids 1 to 489 from RSV F followed by amino acids 97 to 279 from RSV G. The chimeric FG glycoprotein did not contain an anchor region and was efficiently secreted into the medium of recombinant baculovirus-infected insect cells. The FG glycoprotein ranged in size from 69K to 91K and was heterogeneous with respect to isoelectric point. The cleavage site present on the F glycoprotein was recognized on the chimeric FG, and the glycoprotein appeared to be antigenically similar to the native RSV F and G glycoproteins.

Protection of cotton rats against human respiratory syncytial virus by vaccination with a novel chimeric FG glycoprotein.

The cotton rat model of experimental human respiratory syncytial virus (RSV) infection was used to study the efficacy of FG, a novel chimeric glycoprotein which was expressed in insect cells using a baculovirus vector. FG contained the extracellular regions of the F (fusion) and G (attachment) glycoproteins of RSV. Vaccination with FG resulted in induction of neutralizing antibody and was correlated with protection of lung tissue from RSV challenge against both serogroup A and B virus strains. Both crude FG taken from supernatants of insect cells and affinity-purified FG were immunogenic and active against RSV. FG vaccination was effective by three routes of administration, following a single dose, and when administered with different adjuvants.

Immunization of cotton rats with the human respiratory syncytial virus F glycoprotein produced using a baculovirus vector.

The F glycoprotein of respiratory syncytial virus in insect cells was produced using a baculovirus expression vector to examine its potential as a subunit vaccine. Two different forms of the F glycoprotein were expressed: the intact F (F) glycoprotein and the truncated (Ft) glycoprotein, in which the COOH-terminal anchor region was deleted. The F glycoprotein remained cell associated, whereas the Ft glycoprotein was secreted into the media of infected cells. In contrast to the processing of the F0 precursor into its F1 and F2 subunits that was observed in mammalian cells, a second cleavage site within the F1 subunit was recognized by the insect cell proteases and resulted in the formation of two F1 subunits. The baculovirus-expressed Ft glycoprotein induced neutralizing antibodies in cotton rats and protected vaccinated animals from challenge with respiratory syncytial virus.