The affinity adsorptive recovery of an infectious herpes simplex virus vaccine

The chromatographic purification of a recombinant Herpes Simplex Virus (type 2) from salt- and heparin-released harvests of infected complementing Vero (CR2) cells is addressed. Functionalized matrices and process operating conditions are identified that provide adequate virus titres in eluates that are significantly reduced in CR2 cell protein and DNA and possess a low level of HSV-2 protein. Virus from diluted salt-released harvests (0.14 M NaCl) was not appreciably adsorbed onto either heparin-Sepharose or Cellufine-heparin matrices but was virtually completely adsorbed onto Cellufine-sulfate and heparin-HP matrices. Virus was recovered by either a linear salt gradient elution (0.14-2 M NaCl) or by a single-step elution with 1.5 M NaCl in phosphate buffer. Recoveries of infectious virus with step elution were 21% and 89%, respectively, for these matrices. Virus from undiluted salt-released harvest (0.8 M NaCl) was substantially adsorbed onto Cellufine-sulfate gel (44% adsorption) and completely adsorbed onto heparin-HP matrices. This virus was recovered with high yield by either gradient or step elution with phosphate-buffered saline. Finally, heparin-harvested virus was fed directly to these matrices and quantitatively adsorbed. The virus could be completely recovered from the heparin-HP matrix with 1.5 M NaCl buffer to provide a purified preparation containing only 0.05 pg protein/pfu and 1.2 x 10(-4) pg DNA/pfu.     

Impact of valency of a glycoprotein B-specific monoclonal antibody on neutralization of herpes simplex virus

Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab')(2)] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibody-dependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug.     

Factors affecting recovery of latent herpes simplex virus from human trigeminal ganglia

The rate of recovery of herpes simplex virus (HSV) from human trigeminal ganglia explant monolayers is affected by two factors: (1) time elapsed from the death of an individual to the establishment of in vitro culture of ganglia and (2) surface area onto which ganglia are explanted. Spontaneous reactivation of HSV from human trigeminal ganglia can be maximized when ganglia are obtained within 12 h of death and explanted into surface area of 250 cm2. Viruses isolated by explantation of human trigeminal ganglia were found to be uniformly HSV type 1 by restriction enzyme analysis.     

Role of T-lymphocyte subsets in recovery from herpes simplex virus infection.

Our investigations probed the nature of different T-lymphocyte subsets effecting clearance of herpes simplex virus after infection of the pinna. Cell populations from animals recently infected subcutaneously or intraperitoneally (acute population) or from animals infected 6 weeks previously (primed population) or the latter cells reimmunized in vitro with virus (memory population) were studied. Viral clearance was a function of the Lyt 1+2- subset in the acute population, but with the memory population both Lyt 1+ and Lyt 2+ cells affected clearance. In primed populations, viral clearance was effected only by the Lyt 2+ subset. The ability of the various cell populations to adoptively transfer delayed-type hypersensitivity was also studied. Only acute population cells from animals infected subcutaneously and memory population cells transferred delayed-type hypersensitivity. In both cases, the cell subtype was Lyt 1+2-. Our results demonstrated that the delayed-type hypersensitivity response does not always correlate with immunity to herpes simplex virus. Multiple subsets of T cells participate in viral clearance, and their respective importances vary according to the stage of the virus-host interaction.     

Uncoating the herpes simplex virus genome

Initiation of infection by herpes simplex virus (HSV-1) involves a step in which the parental virus capsid docks at a nuclear pore and injects its DNA into the nucleus. Once "uncoated" in this way, the virus DNA can be transcribed and replicated. In an effort to clarify the mechanism of DNA injection, we examined DNA release as it occurs in purified capsids incubated in vitro. DNA ejection was observed following two different treatments, trypsin digestion of capsids in solution, and heating of capsids after attachment to a solid surface. In both cases, electron microscopic analysis revealed that DNA was ejected as a single double helix with ejection occurring at one vertex presumed to be the portal. In the case of trypsin-treated capsids, DNA release was found to correlate with cleavage of a small proportion of the portal protein, UL6, suggesting that UL6 cleavage may be involved in making the capsid permissive for DNA ejection. In capsids bound to a solid surface, DNA ejection was observed only when capsids were warmed above 4 degrees C. The proportion of capsids releasing their DNA increased as a function of incubation temperature with nearly all capsids ejecting their DNA when incubation was at 37 degrees C. The results demonstrate heterogeneity among HSV-1 capsids with respect to their sensitivity to heat-induced DNA ejection. Such heterogeneity may indicate a similar heterogeneity in the ease with which capsids are able to deliver DNA to the infected cell nucleus.     

Intrauterine herpes simplex virus infection

Neonatal herpes simplex virus (HSV) infection usually is the consequence of intrapartum infection, with disease onset between 5 and 15 days of life. More recently, intrauterine HSV infection has been identified. Intrauterine infection is apparent within the first 24-48 hr of life and is associated with skin vesicles/scarring, chorioretinitis, and/or hydraencephaly. The recognition that babies with these findings can have disease caused by HSV should prompt enhanced physician awareness in the evaluation of newborns with suspected intrauterine infection. The frequency of intrauterine infection appears to be about 5% of all babies with neonatal HSV infection.     

Cellular proteins expressed in herpes simplex virus transformed cells also accumulate on herpes simplex virus infection.

The cell proteins expressed in rat embryo cells transformed by herpes simplex virus (HSV) have been analysed by immunoprecipitation assays to determine those polypeptides which can be identified by immunoprecipitation with the sera of tumour-bearing animals and also with antisera to herpes simplex infected cells. Cell polypeptides commonly recognised by both these sera have been further characterised using a monoclonal antibody directed against a cellular polypeptide which accumulates on HSV-2 lytic infection. This monoclonal antibody recognises in HSV-transformed cells polypeptides of mol. wts. 90 000, 40 000 and 32 000. Further studies show that the accumulation of these polypeptides in HSV-transformed cells is not HSV specific but is a common feature of transformation or of cells which have been immortalised. We suggest that cellular polypeptides accumulating as a result of HSV infection may be of importance in the initiation of transformation by HSV, i.e., at the level of immortalisation of cells.     

Mapping of the herpes simplex virus DNA sequences present in herpes simplex virus type-1 thymidine kinase-transformed cells

Analyses of the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase-transformed (HSVtk+) mouse cell lines have revealed that these cells contain relatively large and variable portions of the viral genome. Two of these cell lines do not contain the viral DNA sequences known to encode the early viral genes normally responsible for regulating tk gene expression during lytic HSV infections. This finding suggests that cell-associated viral tk gene expression may be regulated by cellular rather than viral control mechanisms. In addition, we have compared the viral DNA sequences present in one unstable HSVtk+ cell line to those present in tk- revertant and tk+ rerevertant cell lines sequentially derived from it. Our results have shown that within the limits of sensitivity of our mapping approach, these three related cell lines contain the same set of viral DNA sequences. Thus, gross changes in viral DNA content do not appear to be responsible for the different tk phenotypes of these cells.     

Inhibitory effect of heparin on herpes simplex virus

Nahmias, André J. (Boston University School of Medicine, Boston, Mass.), and Sidney Kibrick. Inhibitory effect of heparin on herpes simplex virus. J. Bacteriol. 87:1060-1066. 1964.-A substance inhibitory to herpes simplex virus was observed during experiments with leukocyte cultures. The component in the cultures responsible for this inhibition was identified as heparin. The minimal inhibitory concentration required to inhibit 30 to 300 tcd(50) of the virus in human amnion tissue culture was found to be 1 to 2 units per ml (10 to 20 mug/ml). This effect was confirmed with other strains of herpes simplex virus, other tissue-culture systems, and other media. The inhibitory activity of the heparin was found to be related to the sulfate groupings on the molecule. The effect of heparin appears to be on the virus, rather than on the cell. The virus is not inactivated, however, and the heparin-virus "complex" is readily dissociable on dilution. Heparin was shown to affect viral infection in its earliest phase, probably at the primary electrostatic attachment of virus to cell. The import of these and related observations on common virological laboratory procedures and the possible biological significance of our findings are discussed.