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.

Pharmacology of the biological response modifier bropirimine (PNU-54461) on experimental autoimmune encephalomyelitis (EAE) in mice.

In murine severe experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis (MS), we tested the efficacy of a 5-halo-6-phenyl pyrimidinone compound, bropirimine (PNU-54461). We observed that the compound is active in suppressing EAE when administered orally, a significant pharmacological advantage compared to some current therapies for the treatment of MS. Furthermore, bropirimine was most efficacious when dosing was begun 5-10 days after injection of myelin basic protein, the protein isolated from the central nervous system and used for inducing EAE in our model. This is a period of time following the initial immunological events leading to the disease, when large-scale leukocyte infiltration into the central nervous system begins. Following oral dosing, bropirimine peaked in the blood within 3 h and was cleared to undetectable concentrations within 16-18 h. Despite the pharmacokinetics in the blood, bropirimine was fully efficacious when dosed orally every two or three days. Surprisingly, bropirimine treatment did not result in a statistically significant decrease in leukocyte infiltration into the lower spinal cord, unless the compound was dosed daily at a high concentration. We also observed the concentration and time course of alpha-interferon in blood following oral dosing of bropirimine. The kinetics of interferon in the blood are similar to, but clearly distinguishable from, the pharmacokinetics of bropirimine in the blood. It is not clear whether or not the induction of interferon plays a key role in the efficacy of bropirimine. Nevertheless, the results using bropirimine in EAE suggest that the compound may be useful for the treatment of multiple sclerosis.

Pharmacokinetic properties, induction of interferon, and efficacy of selected 5-halo-6-phenyl pyrimidinones, bropirimine analogues, in a model of severe experimental autoimmune encephalomyelitis.

We showed previously that a 5-halo-6-phenyl-pyrimidinone, bropirimine (PNU-54461), inhibited progression of severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. In the work presented here, we examined the activity of a group of chemically-related bropirimine analogues. First, the pharmacokinetic properties of the bropirimine analogues were examined in normal mice following oral dosing. After equal oral doses, both PNU-56169 and PNU-63693 were found in the blood of normal mice at equal or higher concentrations than bropirimine, but PNU-54462 and PNU-56359 were present in blood only at very low concentrations. Next, we examined the bropirimine analogues for activity in our model of severe EAE. At a dose of 400 mg/kg administered orally every second day PNU-56169 nearly completely blocked EAE progression, but was ineffective at 100 mg/kg. PNU-63693 was effective in EAE at concentrations of 200 mg/kg, 100 mg/kg, 50 mg/kg, and as low as 25 mg/kg. Histopathology was examined by observing leukocyte infiltration into the lower spinal cords of the mice. Treatment with 400 mg/kg of PNU-56169 and doses of 25, 50, 100, and 200 mg/kg of PNU-63693 significantly inhibited leukocyte infiltration into the lower spinal cord of treated mice in a dose-dependent manner. Orally administered PNU-56169 and PNU-63693 also stimulated significant concentrations of IFNalpha in the serum of treated mice, which may be related to the efficacy of the compounds in EAE. However, the correlation between IFNalpha in the blood and efficacy in treating EAE was not exact. Thus, PNU-56169 and PNU-63693 were delivered to the blood following oral dosing, induced significant concentrations of IFNalpha in the blood, and were equally or more potent than PNU-54461 in inhibiting clinical signs of EAE. The results suggest that 5-halo-6-phenyl-pyrimidinones are an interesting class of compounds to investigate for development in the treatment of multiple sclerosis.

10th International Conference on Antiviral Research.

The 10th Annual International Conference on Antiviral Research held in Atlanta, GA was attended by nearly 500 participants representing 23 countries. The next meeting will be held in San Diego, CA in April 1998. The web site for additional information is www.ISAR-ICAR.com.

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.

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.

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.

Monoclonal antibodies that cross-react with the E1 glycoprotein of different alphavirus serogroups: characterization including passive protection in vivo.

A panel of four monoclonal antibodies (mAb) were produced that cross-react with representatives of two different togavirus serogroups, namely sindbis (SIN) and Semliki Forest (SF) viruses, by ELISA and ADCMC assays. Three of these mAb, IgG2a and IgG2b isotypes, passively protected C3H/Hej mice against 10 and 100 LD50 of SF challenge and one, IgM, did not protect against either challenge dose, or even at 1 LD50. All these mAb were cross-reactive with the E1 glycoprotein of the viruses by immunoblotting in which three different patterns of reactivity were evident, suggesting that three epitopes were involved. The patterns depended upon whether the mAb recognized E1 extracted from purified virions or infected cells and whether SDS-PAGE and immunoblotting were done in the presence or absence of beta-mercaptoethanol. One mAb (IgM) reacted with nonreduced or reduced E1 from either virions or cells suggesting recognition of a linear epitope. The other three mAb reacted with nonreduced but not reduced E1 from virions suggesting that recognition depends upon conformational epitopes. These three mAb reacted also with nonreduced E1 extracted from SF-infected cells whereas only one reacted with nonreduced E1 extracted from SIN-infected cells.

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.

Pyrimidinone (bropirimine) mediated alteration of T lymphocyte subsets during murine cytomegalovirus infection.

Murine cytomegalovirus (MCMV) infection of mice resulted in suppression of mitogen induced proliferation and interleukin 2 (IL-2) responses of splenic cells. This suppression was also evident as a reduction in the number of cells expressing Thy-1 or L3T4 and a reduction in the ratio of T helper/T suppressor cells. The pyrimidinone compound, bropirimine, when administered to MCMV infected mice was able to restore mitogen-induced proliferative and IL-2 responses of splenic cells, to increase the number of cells expressing Thy-1 or L3T4, to restore the ratio of T helper/T suppressor cells and to increase the number of cells inducible for expression of IL-2 receptors.

During recovery from cytomegalovirus infection T-lymphocyte subsets become selectively responsive to activation and have depressed interleukin 2 (IL2) secretion and IL2 receptor expression.

The ability to induce lymphocyte activation with Concanavalin A (Con A) was suppressed in spleen cell cultures derived from BALB/c mice acutely infected with murine cytomegalovirus (MCMV) when compared to cultures derived from control, uninfected mice. This immunosuppression was observed as a reduced incorporation of 3H-thymidine in the lymphocyte proliferative response to Con A, was highly correlated with reduced secretion of interleukin 2 (IL2), and could not be augmented by addition of exogenous IL2. The degree of suppression of both the proliferative response and IL2 secretion was directly dependent on the infecting virus dose and on the time post-infection. At weekly intervals during infection, fluorescein-labeled monoclonal antibodies to various T-lymphocyte surface markers were used to stain spleen cells from control or MCMV-infected mice before or after Con A activation. Analysis of stained spleen cells by flow cytometry revealed several unusual responses to activation signals in lymphocytes derived from mice with acute MCMV-induced immunosuppression. At 4 days post-infection T-lymphocytes of each subset [Thy 1.2 (pan T), L3T4 (T-helper/inducer), Lyt 2 (T-cytotoxic/suppressor) and Lyt 1 (subset of Thy 1.2)] were each present in the spleen but each was in reduced percentage of the total spleen T cell population compared to control (52% to 75% of controls). At this time post-infection these cells were non-responsive to Con A activation as shown by inability to induce 3H-thymidine uptake, IL2 secretion or IL2 responsiveness and an inability to demonstrate an IL2 receptor-positive (IL2R+) population. By 11 days post-infection all tested subsets of T-lymphocytes (Thy 1.2+, L3T4+, Lyt 2+ and Lyt 1+) were in normal control range in fresh spleen. However, only Thy 1.2+L3T4+ (T-helper/inducer) cells were responsive to Con A activation. 3H-thymidine uptake and IL2 secretion were at levels of about 60% of the control but, surprisingly, cells were unresponsive to addition of exogenous IL2 and few, if any, of these cells were found to express IL2 receptors. By 18 days post-infection, when 3H-thymidine uptake could be induced at control level, Thy 1.2+, L3T4+, and Lyt 2+ (T-cytotoxic/suppressor) cells were each responsive to Con A activation at levels comparable to control but Lyt 1+ and IL2 R+ cells were still substantially suppressed (ca. 35% to 40% of control value). Detection of Lyt 1+ subset and IL2 R+ cells after Con A activation did not near control levels until very late in the recovery process (day 25).(ABSTRACT TRUNCATED AT 400 WORDS)