Efficacy of intranasal vaccination with a multivalent vaccine containing temperature-sensitive modified-live bovine herpesvirus type 1 for protection of seronegative and seropositive calves against respiratory disease.

OBJECTIVE To evaluate efficacy and duration of immunity of the bovine herpesvirus type 1 (BHV-1) fraction of a trivalent vaccine also containing parainfluenza virus-3 and bovine respiratory syncytial virus fractions administered intranasally (IN) for protection of calves against infectious bovine rhinotracheitis (IBR). DESIGN Controlled challenge study. ANIMALS 120 dairy calves (3 to 8 days old) seronegative for antibody against BHV-1 (experiments 1 and 2) or seropositive for maternally derived antibody against BHV-1 (experiment 3). PROCEDURES In 3 separate experiments, calves were vaccinated IN via 2 nostrils (experiment 1) or 1 nostril (experiments 2 and 3) with a vaccine containing or not containing a BHV-1 fraction. For seronegative calves, the test vaccine contained a minimum immunizing dose of BHV-1; for seropositive calves, it contained a commercial dose of BHV-1. Calves were challenged IN with virulent BHV-1 on day 28 or 193 (seronegative calves) or day 105 (seropositive calves) after vaccination to evaluate vaccine efficacy. Frequency and duration of clinical signs, rectal temperatures, virus shedding, and serologic responses were compared between treatment groups within experiments. RESULTS In all experiments, BHV-1 vaccinated calves had lower frequencies or shorter durations of clinical signs of IBR than did control calves. Following viral challenge, peak rectal temperatures and degrees of virus shedding were lower and serologic responses were higher in vaccinated versus control calves. CONCLUSIONS AND CLINICAL RELEVANCE IN vaccination against BHV-1 protected all calves against clinical IBR disease, regardless of serologic status at the time of vaccination, and suppressed virus shedding. A single dose of this IN vaccine has the potential to protect seronegative calves for at least 193 days and override maternally derived antibody to protect seropositive calves for at least 105 days.

Modifications of C-2 on the pyrroloquinoline template aimed at the development of potent herpesvirus antivirals with improved aqueous solubility.

A series of C-2 pyrroloquinoline analogs designed to improve aqueous solubility were examined for herpesvirus polymerase and antiviral activity. Several analogs were identified that maintained the antiviral activity of the previous development candidate against HCMV, HSV-1 and VZV, but with significantly improved aqueous solubility.

The design and development of 2-aryl-2-hydroxy ethylamine substituted 1H,7H-pyrido[1,2,3-de]quinoxaline-6-carboxamides as inhibitors of human cytomegalovirus polymerase.

Discovery efforts were focused on identifying a non-nucleoside antiviral for treating infections caused by human cytomegalovirus (HCMV) with equal or better potency and diminished toxicity compared to current therapeutics. This Letter describes the HCMV DNA polymerase inhibition and in vitro antiviral activity of various 2-aryl-2-hydroxy ethylamine substituted 1H,7H-pyrido[1,2,3-de]quinoxaline-6-carboxamides.

Synthesis of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine-5-carboxamides with broad-spectrum human herpesvirus polymerase inhibition.

A versatile synthesis of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine-5-carboxylate esters has been developed which has lead to the identification of a new series of non-nucleoside inhibitors of human herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.

7-Oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamides: synthesis and biological activity of a new class of highly potent inhibitors of human cytomegalovirus DNA polymerase.

We report a new class of non-nucleoside antivirals, the 7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamides, some of which possess remarkable potency versus a broad spectrum of herpesvirus DNA polymerases and excellent selectivity compared to human DNA polymerases. A critical factor in the level of activity is hypothesized to be conformational restriction of the key 2-aryl-2-hydroxyethylamine sidechain by an adjacent methyl group.

2-Aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridines as broad-spectrum inhibitors of human herpesvirus polymerases.

A novel series of 2-aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have been identified as potent antivirals against human herpesviruses. These compounds demonstrate broad-spectrum inhibition of the herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.

Fetal protection following exposure to calves persistently infected with bovine viral diarrhea virus type 2 sixteen months after primary vaccination of the dams.

This study demonstrated that the bovine viral diarrhea virus (BVDV; types 1 and 2) fractions of a multivalent vaccine protected pregnant heifers and their fetuses at 149 to 217 days of gestation against exposure to calves persistently infected with BVDV type 2a. Eighty percent (eight of 10) of the control heifers were viremic at least 1 day following challenge, whereas all (20 of 20) BVDV-vaccinated heifers were virus isolation-negative on all postchallenge assessment days. Ninety percent (nine of 10) of the calves born to control heifers but only 5% (one of 20) of calves born to BVDV-vaccinated heifers seroconverted to BVDV type 2 before ingesting colostrum. One calf born to a control heifer was persistently infected. No calves from BVDV-vaccinated heifers were persistently infected.

4-Oxo-4,7-dihydrothieno[2,3-b]pyridines as non-nucleoside inhibitors of human cytomegalovirus and related herpesvirus polymerases.

A novel series of 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have been identified as potential antivirals against human herpesvirus infections resulting from human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV). Compounds 10c and 14 demonstrated broad-spectrum inhibition of the herpesvirus polymerases HCMV, HSV-1, and VZV. High specificity for the viral polymerases was observed compared to human alpha polymerase. The antiviral activity of 10c and 14, as determined by plaque reduction assay, was comparable or superior to that of existing antiherpes drugs, ganciclovir (for HCMV) and acyclovir (for HSV-1 and VZV). Drug resistance to compound 14 correlated to point mutations in conserved domain III of the herpesvirus DNA polymerase, but these mutations do not confer resistance to existing nucleoside therapy. In addition, compound 14 maintained potent antiviral activity against acyclovir-resistant HSV-1 strains. Substitution to the pyridone nitrogen (N7) was found to be critical for enhanced in vitro antiviral activity.

Amino acid changes within conserved region III of the herpes simplex virus and human cytomegalovirus DNA polymerases confer resistance to 4-oxo-dihydroquinolines, a novel class of herpesvirus antiviral agents.

The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.

Identification of phenylisoxazolines as novel and viable antibacterial agents active against Gram-positive pathogens.

A new and promising group of antibacterial agents, collectively known as the oxazolidinones and exemplified by linezolid (PNU-100766, marketed as Zyvox), have recently emerged as important new therapeutic agents for the treatment of infections caused by Gram-positive bacteria. Because of their significance, extensive synthetic investigations into the structure-activity relationships of the oxazolidinones have been conducted at Pharmacia. One facet of this research effort has focused on the identification of bioisosteric replacements for the usual oxazolidinone A-ring. In this paper we describe studies leading to the identification of antibacterial agents incorporating a novel isoxazoline A-ring surrogate. In a gratifying result, the initial isoxazoline analogue prepared was found to exhibit in vitro antibacterial activity approaching that of the corresponding oxazolidinone progenitor. The synthesis and antibacterial activity profile of a preliminary series of isoxazoline analogues incorporating either a C-C or N-C linkage between their B- and C-rings will be presented. Many of the analogues exhibited interesting levels of antibacterial activity. The piperazine derivative 54 displayed especially promising in vitro activity and in vivo efficacy comparable to the activity and efficacy of linezolid.

Broad-spectrum antiviral activity of PNU-183792, a 4-oxo-dihydroquinoline, against human and animal herpesviruses.

We identified a novel class of 4-oxo-dihydroquinolines represented by PNU-183792 which specifically inhibit herpesvirus polymerases. PNU-183792 was highly active against human cytomegalovirus (HCMV, IC(50) value 0.69 microM), varicella zoster virus (VZV, IC(50) value 0.37 microM) and herpes simplex virus (HSV, IC(50) value 0.58 microM) polymerases but was inactive (IC(50) value >40 microM) against human alpha (alpha), gamma (gamma), or delta (delta) polymerases. In vitro antiviral activity against HCMV was determined using cytopathic effect, plaque reduction and virus yield reduction assays (IC(50) ranging from 0.3 to 2.4 microM). PNU-183792 antiviral activity against both VZV (IC(50) value 0.1 microM) and HSV (IC(50) ranging from 3 to 5 microM) was analyzed using plaque reduction assays. PNU-183792 was also active (IC(50) ranging 0.1-0.7 microM) in cell culture assays against simian varicella virus (SVV), murine cytomegalovirus (MCMV) and rat cytomegalovirus (RCMV). Cell culture activity was compared with the appropriate licensed drugs ganciclovir (GCV), cidofovir (CDV) and acyclovir (ACV). PNU-183792 was also active against both GCV-resistant and CDV-resistant HCMV and against ACV-resistant HSV. Toxicity assays using four different species of proliferating mammalian cells indicated PNU-183792 was not cytotoxic at relevant drug concentrations (CC(50) value >100 microM). PNU-183792 was inactive against unrelated DNA and RNA viruses indicating specificity for herpesviruses. In animals, PNU-183792 was orally bioavailable and was efficacious in a model of lethal MCMV infection.

Broad-spectrum antiherpes activities of 4-hydroxyquinoline carboxamides, a novel class of herpesvirus polymerase inhibitors.

Through broad screening of the compound library at Pharmacia, a naphthalene carboxamide was identified as a nonnucleoside inhibitor of human cytomegalovirus (HCMV) polymerase. Structure-activity relationship studies demonstrated that a quinoline ring could be substituted for naphthalene, resulting in the discovery of a 4-hydroxyquinoline-3-carboxamide (4-HQC) class of antiviral agents with unique biological properties. In vitro assays with the 4-HQCs have demonstrated potent inhibition of HCMV, herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV) polymerases but no inhibition of human alpha, delta, and gamma polymerases. Antiviral cell culture assays have further confirmed that these compounds are active against HCMV, HSV-1, HSV-2, VZV, and many animal herpesviruses. However, these compounds were not active against several nonherpesviruses representing different DNA and RNA virus families. A strong correlation between the viral DNA polymerase and antiviral activity for this class of compounds supports inhibition of the viral polymerase as the mechanism of antiviral activity. Northern blot analysis of immediate-early and late viral transcripts also pointed to a block in the viral life cycle consistent with inhibition of viral DNA replication. In vitro HCMV polymerase assays indicate that the 4-HQCs are competitive inhibitors of nucleoside binding. However, no cross-resistance could be detected with ganciclovir-resistant HCMV or acyclovir-resistant HSV-1 mutants. The unique, broad-spectrum activities of the 4-HQCs may offer new opportunities for treating many of the diseases caused by herpesviruses.