Human anti-varicella-zoster virus (VZV) recombinant monoclonal antibody produced after Zostavax immunization recognizes the gH/gL complex and neutralizes VZV infection.

Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.

Human trigeminal ganglionic explants as a model to study alphaherpesvirus reactivation.

Varicella zoster virus (VZV) latency is characterized by limited virus gene expression and the absence of virus DNA replication. Investigations of VZV latency and reactivation have been hindered by the lack of an in vitro model of virus latency. Since VZV is an exclusively human pathogen, we used naturally infected human trigeminal ganglia (TG) obtained at autopsy to study virus latency. Herein, we report optimization of medium to maintain TG integrity as determined by histology and immunohistochemistry. Using the optimized culture medium, we also found that both herpes simplex virus-1 (HSV-1) and VZV DNA replicated in TG explants after 5 days in culture. The increase in HSV-1 DNA was fourfold greater than the increase in VZV DNA. Overall, we present a model for alphaherpesvirus latency in human neurons in which the key molecular events leading to virus reactivation can be studied.

Synthesis and decay of varicella zoster virus transcripts.

Varicella zoster virus (VZV) is highly cell-associated. At least 68 VZV open reading frames (ORFs) are transcribed in varying amounts that increase as infection progresses. Using reverse transcriptase PCR, quantification of total and newly synthesized mRNA showed that ongoing VZV DNA replication is required for continued accumulation of VZV ORF 63, 9, and 40 transcripts. Analysis of stability of 4-thiouridine-labeled transcripts of nine VZV ORFs revealed a similar half-life for all VZV ORFs tested. Thus, difference in mRNA synthesis, and not mRNA decay, is the major factor contributing to the difference in the relative abundance of VZV transcripts in infected cells.

Molecular characterization of varicella zoster virus in latently infected human ganglia: physical state and abundance of VZV DNA, Quantitation of viral transcripts and detection of VZV-specific proteins.

Varicella zoster virus (VZV) establishes latency in neurons of human peripheral ganglia where the virus genome is most likely maintained as a circular episome bound to histones. There is considerable variability among individuals in the number of latent VZV DNA copies. The VZV DNA burden does not appear to exceed that of herpes simplex type 1 (HSV-1). Expression of VZV genes during latency is highly restricted and is regulated epigenetically. Of the VZV open reading frames (ORFs) that have been analyzed for transcription during latency using cDNA sequencing, only ORFs 21, 29, 62, 63, and 66 have been detected. VZV ORF 63 is the most frequently and abundantly transcribed VZV gene detected in human ganglia during latency, suggesting a critical role for this gene in maintaining the latent state and perhaps the early stages of virus reactivation. The inconsistent detection and low abundance of other VZV transcripts suggest that these genes play secondary roles in latency or possibly reflect a subpopulation of neurons undergoing VZV reactivation. New technologies, such as GeXPS multiplex PCR, have the sensitivity to detect multiple low abundance transcripts and thus provide a means to elucidate the entire VZV transcriptome during latency.