ABSTRACT In response to the increasingly evident need for herpes simplex virus (HSV) serotype-specific serologic assays that rely on proteins other than glycoprotein-G (gG), we developed a rapid serologic assay that is based on type-specific epitopes within the large subunit of HSV ribonucleotide reductase (R1). The assay (Au-2 enzyme-linked immunosorbent assay [ELISA]) uses an HSV type 2 (HSV-2) R1 peptide antigen. It provides a reliable method for detecting serotype-specific antibody to a protein other than gG-2. The Au-2 ELISA has high sensitivity and specificity as determined by direct comparison to Western blotting, a widely accepted “gold standard,” and to ELISA with an HSV-1 R1 peptide (Au-1). The use of the Au-2 ELISA in conjunction with the gG-2-based assays will improve the sensitivity and specificity of serologic diagnosis and patient management.

A growth compromised herpes simplex virus type 2 (HSV-2) mutant which is deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10ΔPK) protects from fatal HSV-2 challenge in the mouse model (Aurelian L, Kokuba H, Smith CC. Vaccine potential of a Herpes Simplex Virus type 2 mutant deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10). Vaccine 1999;17:1951–1963). Here we report the results of our studies with ICP10ΔPK in the guinea pig model of recurrent HSV-2 disease. ICP10ΔPK was also compromised for growth and disease causation in this model. It was not isolated from latently infected ganglia by explant co-cultivation. The proportions of latently infected ganglia were significantly lower for ICP10ΔPK than HSV-2 [3/25 (12%) and 7/10 (70%), respectively]. Similar results were obtained for the levels of viral DNA (8×103 and 2×105 molecules/ganglion for ICP10ΔPK and HSV-2, respectively]. ICP10ΔPK immunization caused a significant (P≤0.001) decrease in the proportion of animals with primary [1/14 (6%) and 16/16 (100%) for ICP10ΔPK and PBS, respectively) and recurrent [1/14 (6%) and 11/14 (79%) for ICP10ΔPK and PBS, respectively) HSV-2 skin lesions. It also protected from genital HSV-2 disease [1/10 and 10/10 for ICP10ΔPK and PBS, respectively] and decreased the severity of the lesions in both models. Quantitative PCR (Q-PCR) with primers that distinguish between HSV-2 and ICP10ΔPK indicated that immunization reduced the proportion of ganglia positive for HSV-2 DNA [8/25 (32%) and 7/10 (70%) for ICP10ΔPK and PBS, respectively) and its levels [3×103 and 2×105 molecules/ganglion for ICP10ΔPK and PBS, respectively]. The proportion of HSV-2 infected animals with recurrent disease was also significantly (P≤0.001) decreased by immunization with ICP10ΔPK [1/15 (7%) and 11/14 (79%) with recurrent disease for ICP10ΔPK and PBS, respectively], suggesting that ICP10ΔPK has prophylactic and therapeutic activity in the guinea pig.

Clinical Pharmacology & Therapeutics (1999) 65, 194–194; doi:

Evaluation of the elevation of host cell biosynthesis of deoxynucleoside triphosphates (dNTP's) induced by human cytomegalovirus (HCMV) infection as a target for antiviral therapeutics was carried out. The concentrations of all four intracellular dNTP's rose rapidly following HCMV infection, and were markedly above baseline by 8 h post infection (p.i.). All four deoxynucleoside triphosphates remained elevated above baseline for at least 72 h p.i. The effects of inhibitors of the de-novo pathway of pyrimidine biosynthesis on HCMV viral replication-were quantified by DNA dot blot. All pyrimidine biosynthesis inhibitors examined inhibited the HCMV DNA replication at concentrations that were non-toxic to the cell. These drugs were also more effective against HCMV, which is highly dependent on host denovo synthesis, than against HSV-1 which encodes enzymes capable of increasing the supply of dNTP's. The antiviral effect of brequinar, an inhibitor of one of the enzymes of the de-novo pathway (dihydroorotate dehydrogenase), was examined to determine if it coincided with a decrease in dNTP's. HCMV-infected fibroblasts and uninfected control cells were treated with a concentration of brequinar able to inhibit HCMV DNA levels 90%. It was found that brequinar markedly lowered the levels of dTTP found in treated cells compared to untreated cells in both HCMV-infected and uninfected cells.

Clinical Pharmacology & Therapeutics (1996) 59, 141–141; doi: 10.1038/sj.clpt.1996.65

We extend the observation that inhibitors of pyrimidine biosynthesis are active against human cytomegalovirus by demonstrating that methotrexate (MTX) has preferential activity against cytomegalovirus replication. The 50% and 90% inhibitory concentrations of MTX for inhibition of cytomegaloviral DNA replication at 3 days postinfection in MRC-5 cells were 0.05 and 0.2 microM, respectively. No cell toxicity was observed in uninfected confluent cells at the highest concentration tested (1 microM). Under similar conditions (3 days of treatment with 0.2 microM MTX), intracellular dTTP pools were diminished in cytomegalovirus-infected cells (87% decrease relative to untreated infected cells, P < 0.001) but were not reduced in uninfected cells. A potential explanation for the preferential antiviral effect of MTX was that human cytomegalovirus-infected cells preferentially accumulated MTX. Increased intracellular accumulation and increased polyglutamation of MTX were observed in cytomegalovirus-infected cells compared with uninfected cells. Increased uptake of [3H]MTX by cytomegalovirus-infected cells was first observed at 48 h postinfection, with threefold-higher accumulation within infected cells. By 96 h, accumulation had increased to approximately fourfold in comparison with uninfected cells. The uptake of [3H]MTX was saturable and was blocked by addition of unlabelled MTX. Intracellular MTX in infected cells was almost entirely in the polyglutamated form, as demonstrated by thin-layer chromatography, whereas intracellular MTX was almost exclusively in the parent form in uninfected cells.

We conducted a single-center, double-blind, placebo-controlled phase I study in HIV-positive subjects to ascertain the safety, tolerance, bioavailability, pharmacokinetics, and maximum tolerated dose of HPMPC (cidofovir). Five subjects were randomized to receive drug and two to receive placebo at each of three dosage tiers (1, 3, and 10 mg/kg) with a 2-week washout period between doses. Subjects at 1 and 3 mg/kg received single doses of HPMPC by subcutaneous (s.c.), intravenous (i.v.), and oral (p.o.) routes, while subjects at 10 mg/kg received only i.v. and p.o. doses. For subjects already taking zidovudine, zidovudine AUC values were determined before and then with HPMPC administration for each route. The AUC values of HPMPC were dose-proportional. Subcutaneous bioavailability was essentially equivalent to that of the intravenous route, but the development of transient local fibrosis and the volumes needed for subcutaneous dosing precluded higher subcutaneous dosing than 3 mg/kg. Oral bioavailability was poor, estimated to be less than 5%. Drug elimination was predominantly renal. Nephrotoxicity in one subject was the only serious adverse event observed. This subject had a significant lag period prior to oral absorption and also had the highest AUC values for both HPMPC and zidovudine. We found no consistent effect on zidovudine AUC by concomitant HPMPC.

The pharmacokinetics of cidofovir (HPMPC; (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine) were examined at five dose levels in three phase I/II studies in a total of 42 human immunodeficiency virus-infected patients (with or without asymptomatic cytomegalovirus infection). Levels of cidofovir in serum following intravenous infusion were dose proportional over the dose range of 1.0 to 10.0 mg/kg of body weight and declined biexponentially with an overall mean +/- standard deviation terminal half-life of 2.6 +/- 1.2 h (n = 25). Approximately 90% of the intravenous dose was recovered unchanged in the urine in 24 h. The overall mean +/- standard deviation total clearance of the drug from serum (148 +/- 25 ml/h/kg; n = 25) approximated renal clearance (129 +/- 42 ml/h/kg; n = 25), which was significantly higher (P < 0.001) than the baseline creatinine clearance in the same patients (83 +/- 21 ml/h/kg; n = 12). These data indicate that active tubular secretion played a significant role in the clearance of cidofovir. The steady-state volume of distribution of cidofovir was approximately 500 ml/kg, suggesting that the drug was distributed in total body water. Repeated dosing with cidofovir at 3.0 and 10.0 mg/kg/week did not alter the pharmacokinetics of the drug. Concomitant administration of intravenous cidofovir and oral probenecid to hydrated patients had no significant effect on the pharmacokinetics of cidofovir at a 3.0-mg/kg dose. At higher cidofovir doses, probenecid appeared to block tubular secretion of cidofovir and reduce its renal clearance to a level approaching glomerular filtration.

This phase I double-masked, randomized, placebo-controlled study evaluated the safety of single intravenous or subcutaneous doses of recombinant human nerve growth factor (rhNGF) in healthy human volunteers at doses ranging from 0.03 to 1 micrograms/kg. No life-threatening adverse events were seen at any dose. At doses above 0.1 microgram/kg, subjects reported mild to moderate muscle pain, primarily in the bulbar and truncal musculature. The duration and severity of these myalgias varied in a dose-dependent manner, and women appeared to be more susceptible than men. Intravenous rhNGF produced earlier and more pronounced systemic effects than did identical subcutaneous doses. Subjects receiving subcutaneous rhNGF noted hyperalgesia at the injection site, a local effect persisting up to 7 weeks, that also varied in a dose-dependent manner. Antibodies to NGF were not detected in any subject. These results indicate that systemically administered rhNGF exerts a characteristic and reproducible biological effect in healthy subjects at very low doses and in a dose-dependent manner.

We have previously shown that an oligo(nucleoside methylphosphonate) (deoxynucleoside methylphosphonate residues in italics) complementary to the acceptor splice junction of herpes simplex virus type 1 (HSV-1) immediate-early (IE) pre-mRNAs 4,5 [d(TpTCCTCCTGCGG)], causes sequence-specific inhibition of virus growth in infected cell cultures (Smith et al., 1986; Kulka et al., 1989). Here we report a similar inhibition of HSV-1 growth by oligo(nucleoside methylphosphonates) complementary to the splice donor site of HSV-1 IE pre-mRNAs 4,5 [d(GpCTTACCCGTGC)] and to the translation initiation site of IE4 mRNA [d(ApATGTCGGCCAT)]. An oligomer complementary to the translation initiation site of IE5 mRNA [d(GpGCCCACGACAT)] or an unrelated oligomer [d(GpCGGGAAGGCAC)] did not inhibit virus growth. IC50 values were 20, 25 and 20 μM for d(TpTCCTCCTGCGG), d(GpCTTACCCGTGC) and d(ApATGTCGGCCAT) respectively. In infected BALB/c mice d(TpTCCTCCTGCGG) caused a significant decrease in HSV-1 growth (82% inhibition at 500 μM). A psoralen-derivative of d(TpTCCTCCTGCGG) that binds covalently to complementary sequences after exposure to 365 nm irradiation, inhibited HSV-1 growth (86–91%) at a 10-fold lower concentration than the non-derivatized oligomer. The inhibition was sequence-specific and significantly lower (27%) for HSV-2 that differs from HSV-1 in 7 of the 12 bases targeted by d(pTCCTCCTGCGG). Virus growth was not inhibited by d(GpGCCCACGACAT). The data suggest that oligo(nucleoside methylphosphonates) may be effective antiviral agents.

Immunization of mice with a vaccinia recombinant (VP176) that expresses a fully glycosylated herpes simplex virus (HSV) glycoprotein D (gD) induces long-term (greater than or equal to 50 days) HSV-specific lymphoproliferation and delayed type hypersensitivity (DTH) responses, the ability to eliminate a high challenge dose of HSV-2 from the epidermis and protection from fatal disease due to HSV replication in the nervous system. Adoptive transfer studies indicate that protection is mediated by the DTH functions of L3T4+ cells and requires the contribution of a non-specific irradiation-sensitive cell. Long-term protection (defined as that seen at greater than or equal to 50 days after immunization) from fatal HSV-2 challenge, virus clearance from the epidermis, and HSV-specific T-cell responses are not induced by a partially glycosylated gD expressed by a vaccinia recombinant (VP254) in which gD is controlled by a late vaccinia virus promoter. However, mice immunized with VP254 are protected from HSV-2 challenge early (day 10) after immunization. The VP254-induced protection is HSV-specific, but it is not mediated by L3T4+ and Lyt2+ cells. The findings are discussed within the context of future developments of anti-HSV vaccines.

Vaccinia virus recombinants containing the herpes simplex virus (HSV) gene for glycoprotein D type 1 (gD-1) under control of an early (VP176) or late (VP254) vaccinia virus promoter or for HSV glycoprotein type 2 (gD-2) under control of the early promoter (VP221) were studied for their ability to induce protective immunity to HSV-2 in the guinea pig model of cutaneous recurrent disease and the mouse model of fatal disease. Titers of HSV-specific neutralizing antibody were similar in the two groups of immunized animals, but HSV-specific T cell responses were significantly higher in VP176-immunized than in VP254-immunized animals, as determined by lymphoproliferation (P less than .005) and delayed-type hypersensitivity (P less than .01) responses. The reduced responses correlated with poor expression of the gD protein and its impaired processing in infected antigen-presenting cells (splenic adherent and epidermal cells). VP176 immunization protected against primary (P much less than .001) and recurrent (P much less than .001) cutaneous HSV-2 lesions and ganglionic latency (62% protection) in the guinea pig and against zosteriform skin lesions and fatal disease in the mouse. Immunization with VP254 was not protective. In guinea pigs VP221 did not protect against primary HSV-2 cutaneous disease but did reduce the proportion of animals with recurrent disease (P less than .05). This partial protection appears to be associated with the role of type-specific antigenic determinants in gD-2 immunoregulation.

Large intradermal injections of crude sea nettle (Chrysaora quinquecirrha) venom in normal saline produced immunosuppression in a healthy human adult male. This response persisted several days and was homologous against that coelenterate antigen but also heterologous against antigens contained within vaccinia and herpes simplex viruses and tetanus bacillus. This down-regulation of immunity was probably mediated by cells with functional properties of suppressor cells and could be reversed by indomethacin suggesting a role for prostaglandins. This result suggests the possibility that naturally occurring environmental agents, other than sunlight, may influence the human immune response.

Journal Article Clinical and subclinical HSV infection resulting from exposure to asymptomatic patients Get access L. Aurelian, L. Aurelian Virology/Immunology Laboratories, Department of Pharmacology and Experimental Therapeutics, School of Medicine Baltimore, MD 21201Divisions of Comparative Medicine, The John Hopkins Medical Institutions, Baltimore, MD 21205, U.S.A.Divisions of Biophysics, The John Hopkins Medical Institutions, Baltimore, MD 21205, U.S.A. Search for other works by this author on: Oxford Academic Google Scholar M. Wachsman, M. Wachsman Virology/Immunology Laboratories, Department of Pharmacology and Experimental Therapeutics, School of Medicine Baltimore, MD 21201 Search for other works by this author on: Oxford Academic Google Scholar J.W. Burnett J.W. Burnett Division of Dermatology, Department of Medicine, School of Medicine Baltimore, MD 21201 Search for other works by this author on: Oxford Academic Google Scholar British Journal of Dermatology, Volume 122, Issue 1, 1 January 1990, Pages 117–119, https://doi.org/10.1111/j.1365-2133.1990.tb08252.x Published: 01 January 1990

In an open study, continuous oral therapy with cyclooxygenase inhibitors (ibuprofen or indomethacin) reduced the incidence and frequency of recurrent eruptions in nine of 16 patients with herpes simplex virus infection. Retrospective analysis of seven cases indicated that natural killer cell enhancement assays might allow us to predict the clinical outcome of such treatment.

The effect of regulation of herpes simplex virus (HSV) type 1 glycoprotein D (gD-1) gene expression on HSV-specific immune response and protection from cutaneous HSV-2 disease was studied using vaccinia virus recombinants containing gD-1 under the control of early (VP176) or late (VP254) vaccinia virus promoters. Expression of gD-1 in VP176-infected cells was first observed at 2 h after infection. It did not depend on viral DNA replication. In VP254-infected cells, gD-1 was first observed at 24 h after infection and its expression depended on DNA replication. Immunized guinea pigs had similar titers of HSV-specific neutralizing antibody. However, HSV-specific T cell responses were significantly higher in VP176- than in VP254-immunized animals as determined by lymphoproliferation (P less than .005) and delayed type hypersensitivity (P less than .01). The reduced T cell responses of VP254-immunized guinea pigs correlated with poor gD-1 expression in VP254-infected antigen presenting cells (splenic adherent and epidermal cells). Immunization with VP176, but not with VP254, protected guinea pigs from primary (P less than .0005) and recurrent (P less than .0005) cutaneous HSV-2 lesions.

We studied the effect of the temporal regulation of herpes simplex virus (HSV) type 1 glycoprotein D (gD-1) expression in Ia+ epidermal cells (EC) and macrophages on virus specific immunity and protection from HSV-2 challenge. gD-1 was expressed on the surface of cells infected with a vaccinia recombinant containing gD-1 under the control of an early vaccinia virus promoter (VP176). It was not expressed in cells infected with a recombinant (VP254) in which gD-1 is controlled by a late vaccinia virus promoter. BALB/c mice immunized with both recombinants seroconverted to HSV-2 as determined by neutralization. However, HSV specific delayed type hypersensitivity (DTH) responses were significantly (p<0.025) higher in VP176 than VP254 immunized animals. Both VP176 and VP254 immunized mice were protected from severe neurological disease due to HSV-2 challenge at 14 days post immunization, but long term protection was observed only in VP176 immunized mice.

We studied the effect of an analogue of polyinosinic acid:polycytidylic acid, the mismatched poly(rI).poly(rC12U), on herpes simplex virus type 2 (HSV-2)-induced cutaneous disease in the guinea-pig. Recurrence patterns and HSV-2-induced immune responses were also defined. Intranasal administration (1.5 micrograms/g body weight, five doses at 48 h intervals) of poly(rI).poly(rC12U) during initial HSV-2 infection caused a significant (P less than 0.05) reduction in virus titres in the skin and decreased (P less than 0.01) the duration and severity of the primary cutaneous lesions. The incidence and frequency of subsequent recurrent episodes were also significantly (P less than 0.01) reduced. Titres of serum neutralizing antibody were identical in treated and untreated animals. Interferon (IFN) activity was detectable in the sera from poly(rI).poly(rC12U)-treated animals. Peripheral blood mononuclear (PBL) and spleen cells from treated animals had enhanced cytotoxic activity for HSV-2-infected and uninfected target cells. The cytotoxic activity of the PBL was enhanced by treatment in vitro with poly(rI).poly(rC12U) or IFN.