A Variant Allele in Varicella-Zoster Virus Glycoprotein B Selected during Production of the Varicella Vaccine Contributes to Its Attenuation

Abstract

Attenuation of the live varicella Oka vaccine (vOka) has been attributed to mutations in the genome acquired during cell culture passage of pOka (parent strain); however, the precise mechanisms of attenuation remain unknown. Comparative sequence analyses of several vaccine batches showed that over 100 single-nucleotide polymorphisms (SNPs) are conserved across all vaccine batches; 6 SNPs are nearly fixed, suggesting that these SNPs are responsible for attenuation. By contrast, prior analysis of chimeric vOka and pOka recombinants indicates that loci other than these six SNPs contribute to attenuation. Here, we report that pOka consists of a heterogenous population of virus sequences with two nearly equally represented bases, guanine (G) or adenine (A), at nucleotide 2096 of the ORF31 coding sequence, which encodes glycoprotein B (gB) resulting in arginine (R) or glutamine (Q), respectively, at amino acid 699 of gB. By contrast, 2096A/699Q is dominant in vOka (>99.98%). gB699Q/gH/gL showed significantly less fusion activity than gB699R/gH/gL in a cell-based fusion assay. Recombinant pOka with gB669Q (rpOka_gB699Q) had a similar growth phenotype as vOka during lytic infection in cell culture including human primary skin cells; however, rpOka_gB699R showed a growth phenotype similar to pOka. rpOka_gB699R entered neurons from axonal terminals more efficiently than rpOka_gB699Q in the presence of cell membrane-derived vesicles containing gB. Strikingly, when a mixture of pOka with both alleles equally represented was used to infect human neurons from axon terminals, pOka with gB699R was dominant for virus entry. These results identify a variant allele in gB that contributes to attenuation of vOka. IMPORTANCE The live-attenuated varicella vaccine has reduced the burden of chickenpox. Despite its development in 1974, the molecular basis for its attenuation is still not well understood. Since the live-attenuated varicella vaccine is the only licensed human herpesvirus vaccine that prevents primary disease, it is important to understand the mechanism for its attenuation. Here we identify that a variant allele in glycoprotein B (gB) selected during generation of the varicella vaccine contributes to its attenuation. This variant is impaired for fusion, virus entry into neurons from nerve terminals, and replication in human skin cells. Identification of a variant allele in gB, one of the essential herpesvirus core genes, that contributes to its attenuation may provide insights that assist in the development of other herpesvirus vaccines.

Keywords: attenuation mechanism; glycoprotein B; live attenuated varicella vaccine; varicella-zoster virus; virus fusogen.

FIG 1

Change in variant frequency of SNPs in pOka before and after axonal infection of human neurons. The percent of the frequency of variants of all the SNPs in pOka_P9 (A) and pOka_R5 (B) before axonal infection (input), at 24 h after infection (A) and mean of biological triplicates (B), and 14 days after infection (mean of biological triplicates [A] and quadruplicates [B]) are shown with nucleotide position based on pOka_AB097933.1 (see Table 1).

FIG 2

Characterization of gB699R and gB699Q expressed by plasmid transfection or by VZV infection. (A) Location of ORF31 in VZV genome (nucleotide numbering based on pOka_AB097933.1), location of the ORF31 SNP 2096G/2096A with corresponding nonsynonymous aa change in ORF31/gB (bottom). U_L, unique long; U_S, unique short; TR_L, terminal repeat long; IR_L, internal repeat long; IR_S, internal repeat short; TR_S, terminal repeat short; TM, transmembrane region. (B) Immunoblotting analysis of gB699R and gB699Q by transfection (left) and infection (right) in ARPE-19 cells using anti-gB polyclonal antibody and anti-α-tubulin MAb as internal control. DTT, dithiothreitol. Molecular mass standards (kDa) are shown at left. (C and D) Confocal microscopic analysis of gB699R and gB699Q localization by transfection (C) and infection (D) in ARPE-19 cells using anti-gB MAb and anti-gB polyclonal antibody along with anti-TGN46 polyclonal antibody. Nuclei were stained with DAPI. Images are representative of results from two independent experiments. Transfection efficiency of ARPE-19 cells was about 40% (B and C). Magnification; ×600 and ×2 digital zoom with 10 μm of white bars.

FIG 3

Comparison of phenotype of VZV with different gB SNPs in different cell types. Infectious focus size (A, C, and E) and virus growth (B, D, and F) were compared in MRC-5 (A and B), ARPE-19 (C and D), and MeWo (E and F) cells. Representative data from two independent experiments is shown for each analysis. Infectious focus size is shown in Box and Whisker plots using the Tukey method (n = 30 to 50 foci) measured in each cell type. Red line, mean; gray circle, outliers. P value was calculated by one-way ANOVA with Fisher’s least significant difference (LSD) correction for multiple comparisons. Each virus titer is shown as a mean (symbol) with standard error of the mean (SEM; bar) of replicates.

FIG 4

Expression and fusion activity of gB/gH/gL in a cell-based quantitative fusion assay. Total protein expression (A), cellular localization (B), and cell surface expression (C) of gB and gB/gH/gL were compared in HEK-293T cells used as effector cells for the cell-based fusion assay. (A) Immunoblotting by anti-gB polyclonal antibody, anti-gH Mab, and anti-α-tubulin MAb in the presence of DTT (dithiothreitol). Molecular mass standards (kDa) are shown at left. (B) Confocal microscopic analysis using anti-gB polyclonal antibody and anti-gB MAb or anti-gH MAb along with anti-TGN46 polyclonal antibody. Nuclei were stained with DAPI. Magnification is ×600 and ×2 digital zoom; white bars represent = 10 μm. (C) Flow cytometry using anti-gB MAb or anti-gH MAb. Anti-ORF62 MAb was used as a negative control. Cell surface expression level is shown by mean fluorescent intensity (MFI) obtained from 50,000 events. (A to C) Representative data from two independent experiments is shown for each analysis. (D) Quantitative luciferase-based cell-to-cell fusion assay. Effector HEK-293T cells expressing VZV glycoprotein(s) with firefly luciferase under the control of the T7 promoter, and target ARPE-19 cells (left) or target MeWo cells (right) expressing T7 RNA polymerase were coincubated for the indicated time and luciferase activity (LUC units) was recorded as a measure of cell-to-cell fusion activity. Representative data from three independent experiments are shown with mean and SEM (standard error of the mean) of four biological replicates. P value was calculated by one-way ANOVA with Fisher’s LSD correction. Transfection efficiency of HEK-293T cells by PEImax was more than 80% as shown in panel B. Transfection efficiency in ARPE-19 cells by nucleofection was 70 to 80% by FACS using pCAG_EGFP plasmid.

FIG 5

Reduction of infection by membrane protein enriched extracellular vesicles (MPEEVs) expressing gB. (A) Comparison of gB699R and gB699Q from MPEEVs expressing gB (left) and rpOka cell-free viruses (middle) by immunoblotting using anti-gB polyclonal antibody. Other virion components were compared between rpOka_gB699R and rpOka_gB699Q by immunoblotting using anti-gH MAb, anti-pORF63 polyclonal antibody and anti-pORF49 polyclonal antibody (right). DTT, dithiothreitol. Molecular mass standards (kDa) are shown at left. Image is representative of results from three independent experiments. (B) Number of infectious foci generated by each virus after infection in the presence of MPEEVs expressing gB or MPEEV-empty in APRE-19 cells is shown. Biological triplicate data is shown with the mean (red line). P value was calculated by one-way ANOVA with Fisher’s LSD correction for multiple comparisons. (C) Relative numbers of viral genomes transported to neuronal soma after axonal virus infection in the presence of MPEEV expressing gB or MPEEV-empty are compared between rpOka_gB699R and rpOka_gB699Q. Four biological replicates data are shown with the mean (red line). P values were calculated by one-way ANOVA with Fisher’s LSD correction for multiple comparisons.

FIG 6

Comparison of the phenotype of VZV with different gB SNPs in human primary skin cells. Infectious focus size after infection of HEKn_P3 cells (A), Hs68 cells (B), and HEKn_P6 cells (C) with different VZV isolates. Infectious focus size after infection of HEKn_P3 and HEK_P6 cells with the same VZV isolate (D). Data were derived from panels A and C. Representative data from two independent experiments are shown for each analysis. Infectious focus size is shown in box and whisker plots using the Tukey method (n = 20 to 50 foci) measured in each cell type. Red line, mean; gray circle, outliers. The P value was calculated by one-way ANOVA with Fisher’s LSD correction for multiple comparisons.