Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene.

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-(4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl)-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.

Enhanced immunogenicity of hepatitis B surface antigen by insertion of a helper T cell epitope from tetanus toxoid.

The currently marketed hepatitis B vaccines in the U.S. are based on the recombinant major hepatitis B surface antigen (HBsAg) of hepatitis B virus. Although a large majority of individuals develop protective immunity to HBV-induced disease after three immunizations, routinely a small but a significant percentage of the human population does not respond well to these vaccines. In this report, we describe the generation of a novel HBsAg molecule containing a Th epitope derived from tetanus toxoid (TT). Using recombinant DNA technology. the TT Th epitope (TTe) was inserted into the HBsAg coding sequence. Using a recombinant adenovirus expression system, HBsAg TTe chimeric protein was produced in A549 cells and found to be secreted into culture medium as 22 nm particles. The chimeric HBsAg particles were readily purified by immunoaffinity chromatography and their immunogenicity was evaluated relative to native HBsAg produced in an adenovirus expression system. When evaluated in inbred and outbred strains of mice, HBsAg TTe was shown to enhance several-fold the anti-HBs response relative to native HBsAg. Further enhanced responses were observed in mice primed with TT. This highly immunogenic form of HBsAg has promise as an improved HBsAg subunit vaccine.

Establishment and hormonal regulation of a conditionally transformed preosteocytic cell line from adult human bone.

Osteocytes are differentiated forms of osteoblasts that arise upon entrapment within the bone matrix. In this report, we describe the establishment and hormonal regulation of the first conditionally transformed human preosteocytic cell line. Primary adult bone cells were obtained from protease cell line. Primary adult bone cells were obtained from protease digestion of cancellous chips. The cells were infected with adenovirus-ori- SV40 tsA 209, which encodes for a temperature-sensitive large T-antigen. After immortalization, we isolated a clone designated HOB-01-C1. This cell line expressed the mutant T-antigen and proliferated at the permissive temperature (34 C) but stopped dividing at the nonpermissive temperature (39-40 C). Electron microscopy of cells incubated at 39 C demonstrated the presence of preosteocytic cellular processes, some of which appeared to form gap junctions or were rich in microfilaments. The clone expressed alpha 1 type (I) procollagen messenger RNA (mRNA) and secreted type I procollagen C peptide at both temperatures, and this expression was elevated 1.6-fold to 1.8-fold at 40 degrees C. The cells expressed very low basal levels of alkaline phosphatase activity (approximately 0.02 nmol/min.mg), which was increased 2- to 5-fold in a dose-dependent manner by 0.1-100 nM 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures. Vitamin D3 also increased osteocalcin secretion in a dose-dependent manner when the clone was maintained at 34 C (approximately 6-fold), and this stimulation was enhanced > 5 fold at 40 C. In contrast to the low expression of alkaline phosphatase, the cells secreted high amounts of osteocalcin in response to vitamin D3 (approximately 15 ng/mg cell protein); this biochemical profile also resembled that of preosteocytes. Alizarin red-S histochemical staining demonstrated that these cells rapidly produced mineralized nodules at both temperatures. PTH (10 and 100 nM) had no effect on the intracellular accumulation of cAMP at 34 C but stimulated a 14- to 18-fold increase in the production of this second messenger at 40 C. In contrast, 100 nM prostaglandin E2 and 1 microM forskolin stimulated cAMP synthesis better at 34 C. Western blot analysis indicated that the cells expressed CD44, a putative osteocytic marker, at both temperatures. Finally, interleukin-1 beta and tumor necrosis factor-alpha (1-1000 pM) stimulated dose-dependent increases in the secretion of interleukin-6 and monocyte chemoattractant protein-1 at 34 C and 40C. We conclude that the HOB-01-C1 cell line has a preosteocytic phenotype. Moreover, these cells respond to calcitropic hormones and bone resorbing cytokines.

Immunogenicity of recombinant human adenovirus-human immunodeficiency virus vaccines in chimpanzees.

Recombinant human adenovirus (Ad) type 4-, 5-, and 7-vectored vaccines expressing either the HIV env or gag-protease genes were tested for immunogenicity in three chimpanzees. The first phase of the vaccination protocol consisted of a primary and two booster immunizations with Ad-HIVs by the oral route of administration, followed by a single booster immunization with Gag and/or Env subunit vaccines. The second phase of the vaccination protocol consisted of intranasal administration of Ad-HIVs previously administered by the oral route. Following the first phase adenovirus was shed into stools for only 1-7 days and modest type-specific anti-adenovirus neutralizing antibody titers were induced. Strong anti-Env binding antibody responses were detected in all three animals following the second oral booster immunization. One chimpanzee responded with a low-titered type-specific neutralizing antibody response to HIV. Cell-mediated immune responses to Env were not detected after the primary vaccination, but were detected following all booster immunizations. Administration of the Gag subunit vaccine boosted both humoral and cell-mediated immune responses to Gag antigens. In contrast, the Env subunit vaccine boosted cellular but not humoral immune responses. In the second phase of the vaccination protocol, both virus shedding and anti-adenovirus responses were enhanced. All three chimpanzees responded to the intranasal administration of Ad7-HIVs with boosted anti-HIV serum responses, including low-titered type-specific neutralizing antibodies, elicited anti-HIV antibodies at secretory sites, and stimulated cell-mediated immune responses to both Gag and Env antigens.

Ultrastructural characterization of human immunodeficiency virus type 1 Gag-containing particles assembled in a recombinant adenovirus vector system.

The human immunodeficiency virus type 1 (HIV-1) Gag protein was expressed in A549 cells infected with recombinant adenovirus types 4 and 7, each carrying the HIV-1 gag and pro genes. The Gag protein was assembled into enveloped virus-like particles that budded from plasma and vacuolar membranes. The particles, isolated by precipitation and isopycnic density centrifugation, contained both processed and unprocessed Gag-associated proteins.

Distinctive properties of the hepatitis B virus envelope proteins.

Using recombinant adenoviral vectors, we expressed and characterized the large, middle, and major envelope proteins of hepatitis B virus (HBV). Cells infected with the recombinant adenovirus which contained the large envelope gene (HS1.HP) expressed predominantly large envelope and small but detectable quantities of middle (4%) and major (6%) envelope proteins in the cell lysate. No HBV envelope proteins were detected in the culture medium from HS1.HP-infected cells. Cells infected with recombinant adenovirus which contained the middle envelope gene (HS2.HP) expressed and secreted the middle and major envelope proteins in a molar ratio of 3:1. Cells infected with the recombinant adenovirus which contained the major envelope gene (HS.HP) expressed and secreted major envelope proteins. The HBV envelope proteins secreted by cells infected with either HS2.HP or HS.HP were assembled in 22-nm particles, as shown by velocity sedimentation rate determination, buoyant densities, and electron microscopy. Cells coinfected with a recombinant adenovirus which contained the large envelope gene and with either HS2.HP or HS.HP expressed similar quantities of the large, middle, and major envelope proteins in the cell lysates. Secretion of the major and middle envelope proteins was inhibited more than 95% by the presence of the large envelope proteins. These results suggest that differential biosynthesis, transport, and processing of the envelope proteins occur during HBV infection, allowing efficient assembly and secretion of virions and hepatitis B surface antigen particles.

A new hamster model for adenoviral vaccination.

Both adult and baby hamsters infected intranasally with human adenovirus type 5 exhibited virologic, serologic, and histologic evidence of infection. When 8-day old hamsters were infected with 4 x 10(6) pfu, concentrations of virus up to 2 x 10(6) pfu/animal were detected in the lung, peaking on day 2. The minimum infectious dose was 1 x 10(3) pfu/animal. This model may be useful in studies of conventional and recombinant adenoviral vaccines for humans.

Clinical and serologic responses to acellular pertussis vaccine in infants and young children.

We administered diphtheria, tetanus, and pertussis (DTP) vaccine containing acellular (lymphocytosis promoting factor and filamentous hemagglutinin) pertussis vaccine to three groups of 20 children each (4 to 6 years, 17 to 21 months, and 5 to 9 months of age). All the children tolerated the vaccine well; no reactions occurred that contraindicated further immunization. Older children had significantly more local (redness or swelling) and systemic (fever or fretfulness) reactions than younger children. Eighty percent to 90% of the children in the two older age groups had fourfold or greater increases in antibody titers to DTP antigens one month after vaccination. The postvaccine concentrations of antibody to tetanus and diphtheria were greater than 0.01 IU/mL in all children. Serologic responses to lymphocytosis promoting factor and filamentous hemagglutinin varied with age; significantly more older children than younger children had four-fold or greater increases. Acellular pertussis DTP vaccine was antigenic in young children and was less reactogenic than standard whole cell DTP vaccine according to rates reported in previous studies.

Morphological components of herpesvirus. III. Localization of herpes simplex virus type 1 nucleocapsid polypeptides by immune electron microscopy.

Herpes simplex virus type 1 (HSV-1) nucleocapsids were observed in the electron microscope after their reaction with IgG's purified from the sera of rabbits immunized with the individual nucleocapsid polypeptides. The combining sites of NC1, the major capsid protein (mol. wt. 154K), were distributed over the entire capsid surface. This result provides further evidence that NC1 represents the major hexamer constituent. NC2 (mol. wt. 50K) was less widely distributed and appeared to be located at capsid vertices; that antigen may be a constituent of the pentamers or of peripentameric hexamers. One or both of NC3 and NC4 (mol. wt. 40K and 38K) were also located all over the capsid, possibly at positions interior to those of NC1. One or both may represent the intercapsomeric fibrils, hexamer-associated protein or material associated with the pericore. The locations of the other nucleocapsid polypeptides could not be determined.

Structural analysis of the capsid polypeptides of herpes simplex virus types 1 and 2.

Capsids of herpes simplex virus (HSV) types 1 and 2 contain seven polypeptides ranging in molecular weight from 154,000 to 12,000 (termed NC-1 through NC-7 in order of descending molecular weight). Antibodies prepared to HSV-1 capsid polypeptides isolated from sodium dodecyl sulfate-polyacrylamide gels reacted in an immunofluorescence assay against HSV-1-infected KB cells. Three of the antibodies (anti-NC-1, anti-NC-2, and anti-NC-3,4) also reacted with HSV-2-infected cells. Tryptic peptide analysis showed that each of the HSV-1 capsid polypeptides had a unique methionine peptide profile, and none appeared to be derived from the major capsid polypeptide. Comparative peptide analysis of HSV-1 and HSV-2 showed that one polypeptide (NC-7, 12,000 molecular weight) had an identical methionine peptide profile and a very similar arginine peptide profile in both virus types. The arginine peptide profile of NC-7 of HSV-1 was very different from the arginine profile of KB histone H4. Although there were certain intertypic similarities in the methionine peptide profiles of the other capsid components especially in NC-1 (the major capsid protein), there was no case where the tryptic peptides were identical in the two virus types.

Morphological components of herpesvirus. II. Preservation of virus during negative staining procedures.

The disruption of envelopes and the fragmentation of capsids of equine herpes-virus type I observed in negatively stained samples were attributed to viral dehydration on carbon films during preparation for electron microscopy. Prior fixation of virus with OSO4 or glutaraldehyde and subsequent application of negative stain before drying minimized envelope disruption and virtually eliminated the occurrence of capsomere sheets and broken capsids. This sample procedure significantly improves electron microscopic evaluation of herpesvirus samples.

Cultivation and survival studies of Neisseria gonorrhoeae in a human diploid cell strain.

Neisseria gonorrhoeae was cultivated in a human diploid cell strain (WI-38). Eighty percent of the cultures contained viable gonococci for at least 4 m at 36°C, as evidenced by subculture to brain heart infusion broth. Monthly subcultures of bacteria could be made to fresh WI-38 cultures for at least 11 monthly passages with a 69% survival rate. The identity of gonococci was confirmed by morphology, gram staining, oxidase testing and fermentation reactions. Viability in brain heart infusion broth, minimum essential medium (Eagle), and WI-38 spent fluid was of much shorter duration. The organisms grown in WI-38 cultures appeared to orient largely in the vicinity of the WI-38 cells as well as within the cytoplasm of the cells.

Morphological components of herpesvirus. I. Intercapsomeric fibrils and the geometry of the capsid.

Highly purified equine herpesvirus type 1 (EHV-1) and herpes simplex virus type 1 (HSV-1) were observed by electron microscopy in various stages of disintegration. Under the conditions used, envelopes of virions usually were fragmented, and virus capsids collapsed in situ into flattened sheets of capsomeres. A matrix of intercapsomeric fibrils was observed. Evidence suggesting that the capsid hexamer has threefold structural symmetry is presented.