Strain vOka Research Articles

Glycoprotein E-Displaying Nanoparticles Induce Robust Neutralizing Antibodies and T-Cell Response against Varicella Zoster Virus.

The Varicella zoster virus (VZV), responsible for both varicella (chickenpox) and herpes zoster (shingles), presents significant global health challenges. While primary VZV infection primarily affects children, leading to chickenpox, reactivation in later life can result in herpes zoster and associated post-herpetic neuralgia, among other complications. Vaccination remains the most effective strategy for VZV prevention, with current vaccines largely based on the attenuated vOka strains. Although these vaccines are generally effective, they can induce varicella-like rashes and have sparked concerns regarding cell virulence. As a safer alternative, subunit vaccines circumvent these issues. In this study, we developed a nanoparticle-based vaccine displaying the glycoprotein E (gE) on ferritin particles using the SpyCatcher/SpyTag system, termed FR-gE. This FR-gE nanoparticle antigen elicited substantial gE-specific binding and VZV-neutralizing antibody responses in BALB/c and C57BL/6 mice-responses that were up to 3.2-fold greater than those elicited by the subunit gE while formulated with FH002C, aluminum hydroxide, or a liposome-based XUA01 adjuvant. Antibody subclass analysis revealed that FR-gE produced comparable levels of IgG1 and significantly higher levels of IgG2a compared to subunit gE, indicating a Th1-biased immune response. Notably, XUA01-adjuvanted FR-gE induced a significant increase in neutralizing antibody response compared to the live attenuated varicella vaccine and recombinant vaccine, Shingrix. Furthermore, ELISPOT assays demonstrated that immunization with FR-gE/XUA01 generated IFN-γ and IL-2 levels comparable to those induced by Shingrix. These findings underscore the potential of FR-gE as a promising immunogen for the development of varicella and herpes zoster vaccines.

Development of a skin- and neuro-attenuated live vaccine for varicella

Varicella caused by the primary infection of varicella-zoster virus (VZV) exerts a considerable disease burden globally. Current varicella vaccines consisting of the live-attenuated vOka strain of VZV are generally safe and effective. However, vOka retains full neurovirulence and can establish latency and reactivate to cause herpes zoster in vaccine recipients, raising safety concerns. Here, we rationally design a live-attenuated varicella vaccine candidate, v7D. This virus replicates like wild-type virus in MRC-5 fibroblasts and human PBMCs, the carrier for VZV dissemination, but is severely impaired for infection of human skin and neuronal cells. Meanwhile, v7D shows immunogenicity comparable to vOka both in vitro and in multiple small animal species. Finally, v7D is proven well-tolerated and immunogenic in nonhuman primates. Our preclinical data suggest that v7D is a promising candidate as a safer live varicella vaccine with reduced risk of vaccine-related complications, and could inform the design of other herpes virus vaccines.

The comparison of seroconversion rates among different varicella vaccines administered Turkish children; MAV/06 and vOka

ABSTRACT Varicella is a vaccine-preventable disease, and the incidence of varicella has declined since the introduction of varicella vaccine campaigns. A wild type of varicella zoster virus (VZV) was isolated from a 33-month-old child with varicella in Korea in 1989, a different strain (MAV/06). A live-attenuated varicella vaccine containing strain (MAV/06), Suduvax®, was developed in South Korea in 1994. Turkey introduced the varicella vaccine containing the MAV/06 strain (Varicella Vaccine-GCC, Green Cross, South Korea) in January 2019. Therefore, we aimed to compare the seroconversion rates among MAV/06 vaccine- and vOka-administered children. We prospectively collected blood samples from 98 received vOKA and 98 received MAV/06 children 6 weeks after administration, and seroconversion rates were determined by an indirect fluorescence assay (Anti-VZV IIFT IgG, Euroimmun, Germany). Seroconversion rate was significantly higher in vOka group than MAV/06 group (82.7% vs. 64.3%; p = .004). Of the children vaccinated with vOka strain, 17 children did not develop antibodies, 12 were weakly positive, and the remaining 69 children were strongly positive. Of the children who were administered MAV/06 strain, 35 were negative, 20 were weakly positive, and 43 were strongly positive. In conclusion, this study demonstrated that MAV/06 varicella vaccine had lower seroconversion rates and the strong seropositive cases were less common than vOka-administered children. Larger and prospective studies are needed.

Varicella-zoster virus clades circulating in Spain over two decades

BackgroundDespite childhood universal VZV immunization was introduced in 2015, there are no data on VZV clade distribution in Spain. ObjectivesTo characterize the varicella-zoster virus strains circulating in Spain between 1997 and 2016. Study DesignIn this retrospective study, we determined the VZV clades in 294 patients with different pathologies (mainly encephalitis, zoster and varicella) by sequencing three fragments within ORF 22, ORF 21 and ORF 50 and, subsequently analyzing 7 relevant SNPs. ResultsAmong these 294 patients, 132(44.9%) patients were infected by clade 1, 42(14.3%) patients by clade 3, 19(6.5%) by clade 5, 29(9.9%) by clade VI and 3(1%) by clade 4. Four patients (1.4%) were infected by clade 2 vOKA strains, who received one dose of live-attenuated varicella vaccine. Putative recombinant clade 1/3 was identified in 6 cases (2.0%). Results obtained from partial sequences were assigned to clade 1 or 3 in 56(19%) patients and clade 5 or VI in 3(1.0%) patients. In the multivariate analysis, encephalitis was independently associated with clades 1 and 3 and age >14y.o. (P = 0.035 and P = 0.021, respectively). Additionally, Madrid had significant fewer cases of encephalitis compared with the rest of regions analyzed (P = 0.001). ConclusionsHigher prevalence of clades 1 and 3 and their relation with encephalitis and age >14y.o. suggest earlier introduction of this clades in Spain. Putative interclade 1 and 3 recombinants are circulating in patients with encephalitis, herpes zoster and varicella. Several cases were related to vOKA vaccination but vaccine strains do not seem to circulate in the general population.

Varicella-zoster virus inhibits autophagosome-lysosome fusion and the degradation stage of mTOR-mediated autophagic flux

Macroautophagy (herein referred to as autophagy) is a lysosomal degradation mechanism that is important for maintaining homeostasis and for coping with cellular stress such as nutrient deprivation. Previously, varicella-zoster virus (VZV) was reported to modulate the autophagy pathway in the host. However, how VZV affects the autophagy pathway is still unclear. In this study, we examined how wild-type rOka and attenuated vOka strains of cell-associated VZV affect autophagy in MRC-5 fibroblasts by using ratiometric flow cytometry and immunoblotting methods. While VZV does not prevent autophagosome formation, we demonstrate that, particularly when autophagy is upregulated, VZV inhibits late-stage autophagic flux, likely at the point where autophagosomes and lysosomes fuse or where vesicle contents are degraded. Importantly, inhibition of autophagy yields higher VZV titers. These results substantially contribute to the current view of the interaction between VZV and autophagy, and to a better understanding of VZV pathogenesis.

Titration of cell-associated varicella-zoster virus with the MV9G reporter cell line for antiviral studies

Titration of the cell-associated virus (CAV) of varicella-zoster virus (VZV) is essential for antiviral studies. A VZV reporter cell line, MV9G, generated in our previous study expresses firefly luciferase upon CAV infection in a dose-dependent manner, suggesting that use of the cell line for titration is feasible. In this study, MeWo cells infected with VZV vaccine Oka (vOka) strain or with clinical isolates obtained from patients with varicella or zoster were used as CAV. A co-culture of MV9G cells with the virus-infected MeWo cells were set up and optimized for titration of CAV. Luciferase activities of MV9G cells measured as relative light units (RLUs) of chemiluminescence correlated well (r > 0.9, p < 0.05) both with quantities of viral DNAs measured by TaqMan PCR and with numbers of viral foci detected by immunostaining with a monoclonal antibody against VZV IE62. In addition, the usefulness of MV9G for antiviral studies was exemplified by treatment of the VZV-infected cells with various concentrations of acyclovir. Thus, the reporter cell-based titration of CAV by measuring the induced RLUs may be a reliable way to estimate viral foci and viral DNAs.

Sequence Analysis and Comparison of the Key Genes of Domestic Live Attenuated Varicella Vaccine.

Objective To analyze the safety and consistency of domestic live attenuated varicella vaccines (LAVVs) at gene level.Methods The key genes (ORF38,ORF54,and ORF62) of LAVVs produced by four Chinese manufacturers were amplified by polymerase chain reaction (PCR) and sequenced.The sequencing results were compared with the sequences of Dumas,P-Oka,and V-Oka strains in GenBank and with the sequences of Varilrix (GSK) and Varivax (Merck).Results The ORF38 and ORF54 gene sequences of four domestic LAVVs were the same as each other and completely consistent with the sequences of V-Oka and Varilrix;however,it was different from Varivax (Merck) at one site.The ORF62 gene sequences of four domestic LAVVs were similar,and had individual nucleotide differences with V-Oka,Varilrix(GSK),and Varivax (Merck).Conclusions The sequences of ORF38,ORF54,and ORF62 of four domestic LAVVs are almost the same,showing good stability.They have small differences with V-Oka,Varilrix(GSK),and Varivax (Merck),without introducing new mutations.

Recombination of Globally Circulating Varicella-Zoster Virus.

Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpes virus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

Varicella Zoster Virus and the Neuro-Attenuated Vaccine Necessity

Varicella Zoster Virus and the Neuro-Attenuated Vaccine Necessity

Characterization of immediate early gene ORF62 of live attenuated varicella vaccine Oka strains pro-duced in China

Objective To investigate mutations in immediate early （ IE） gene ORF62 of three varicella vaccine Oka strains （ vOka ） including two strains produced in China and their parental Oka strain （pOka）, and then to further elucidate its possible roles in attenuation mechanism by comparing their ORF 62 promoter sequences and its activities , ORF62 coding regions and its transactivities .Methods ORF62 promoter-reporter plasmids and ORF62-expressing plasmids of pOka and three vOka strains （ vOka-BK from Changchun BCHT Biotechnology Co ., vOka-SH from Shanghai Institute of Biological Product Co .Ltd., and vOka-GSK from GlaxoSmithKline plc, as control） were constructed, respectively.ORF62 promoter regions and coding regions of the four strains were sequenced and then compared with each other .Differences of activities of the ORF62 promoter, and transactivities of the ORF62-encoded IE62 upon immediate early （ORF4）, early （ORF28） and late （ORF67） gene promoters between pOka and vOka strains were assayed with transient transfection technique .Results Compared with pOka strain , three vOka strains had a consistent T deletion mutation at site 110 050 in ORF62 promoters, which did not result in any change of tran-scription factor binding motif .However , activities of ORF62 promoters from three vOka strains were significantly lower than those of pOka strain .Three consistent substitution mutations were observed in ORF 62 coding regions of three vOka strains and three new enzyme restriction sites including SmaⅠ, NaeⅠand BssHⅡwere generated, respectively.Transactivities of IE62 from three vOka strains upon ORF4, ORF28 and ORF67 promoters were significantly higher than those of pOka both in CV-1 and MeWo cells , except that vOka-SH IE62 showed significantly lower transactivities upon ORF 4 promoter than those of pOka strain in CV-1 cells.Conclusion Consistent T deletion mutation at site 110 050 in ORF62 promoters of three vOka strains might be responsible for the reduced promoter activities and the changes of IE 62 transactivities .However , it seemed that cell types have no significant effect on ORF 62 promoter activity or IE 62 transactivity between pOka and vOka strains . Key words: Varicella-zoster virus; Promoter activity; Transactivity

Cycling probe technology to quantify and discriminate between wild-type varicella-zoster virus and Oka vaccine strains

Rapid differentiation between wild-type varicella zoster virus (VZV) and Oka-vaccine (vOka) strains is important for monitoring side reactions of varicella vaccination. To develop a high-throughput molecular diagnostic method for the differentiation of wild-type VZV and vOka strains based on cycling probe technology. The primers were designed to amplify common sequences spanning a single nucleotide polymorphism (SNP) in gene 62 of VZV. DNA–RNA chimeric probes (cycling probes) were designed to detect the SNP at nucleotide 105705. The cycling probe real-time PCR assays for VZV wild-type and vOka strains specifically amplified plasmids containing target sequences that ranged between 10 and 1×106 copies per reaction. The inter- and intra-assay coefficients of variation were less than 5%. After initial validation studies, the clinical reliability of this method was evaluated using 38 swab samples that were collected from patients suspected of being zoster. Compared to the loop mediated isothermal amplification method, which is defined as the gold standard, cycling probe real-time PCR was highly sensitive and specific. The cycling probe real-time PCR technology is a reliable tool for differentiating between wild-type VZV and vOka strains in clinical samples.

Novel Genetic Variation Identified at Fixed Loci in ORF62 of the Oka Varicella Vaccine and in a Case of Vaccine-Associated Herpes Zoster

The live attenuated Oka varicella vaccine (vOka), derived from clade 2 wild-type (wt) virus pOka, is used for routine childhood immunization in several countries, including the United States, which has caused dramatic declines in the incidence of varicella. vOka can cause varicella, establish latency, and reactivate to cause herpes zoster (HZ). Three loci in varicella-zoster virus (VZV) open reading frame 62 (ORF62) (106262, 107252, and 108111) are used to distinguish vOka from wt VZV. A fourth position (105705) is also fixed for the vOka allele in nearly all vaccine batches. These 4 positions and two vOka mutations (106710 and 107599) reportedly absent from Varivax were analyzed on Varivax-derived ORF62 TOPO TA clones. The wt allele was detected at positions 105705 and 107252 on 3% and 2% of clones, respectively, but was absent at positions 106262 and 108111. Position 106710 was fixed for the wt allele, whereas the vOka allele was present on 18.4% of clones at position 107599. We also evaluated the 4 vOka markers in an isolate obtained from a case of vaccine-caused HZ. The isolate carried the vOka allele at positions 105705, 106262, and 108111. However, at position 107252, the wt allele was present. Thus, all of the ORF62 vOka markers previously regarded as fixed occur as the wt allele in a small percentage of vOka strains. Characterization of all four vOka markers in ORF62 and of the clade 2 subtype marker in ORF38 is now necessary to confirm vOka adverse events.

Transmission of Varicella Vaccine Virus, Japan

Transmission of Varicella Vaccine Virus, Japan

Discriminating between Varicella-Zoster Virus Vaccine and Wild-Type Strains by Loop-Mediated Isothermal Amplification

The loop-mediated isothermal amplification (LAMP) method was developed to distinguish between the varicella-zoster virus (VZV) vaccine (vOka) strain and wild-type strains. Two single nucleotide polymorphisms (SNPs) (nucleotide [nt] 105705 for VR-1 VZV LAMP and nt 106262 for VR-2 VZV LAMP) located in the open reading frame 62 gene were selected as LAMP targets. Amplified vOka DNA demonstrated a typical ladder pattern; however, no LAMP product was detected in reactions performed with DNAs from other human herpesviruses by either VR-1 VZV LAMP or VR-2 VZV LAMP. This result was confirmed by a turbidity assay. The sensitivities of both VR-1 and VR-2 VZV LAMP determined by either the turbidity assay or agarose gel electrophoresis were 100 copies per reaction. To discriminate the vOka strain from wild-type strains, VR-1 and VR-2 VZV LAMP products were digested with the appropriate restriction enzymes (SacII for VR-1 LAMP and SmaI for VR-2 LAMP). The digested products were clearly different in the vOka strain and wild-type strains. To evaluate the utility of the LAMP methods for rapid differentiation, viral DNA (without DNA extraction) in swab samples was directly tested. Wild-type VZV DNA was detected in 20 swab samples by either VR-1 VZV LAMP or VR-2 VZV LAMP. Sequence analysis confirmed the expected SNPs in the LAMP products amplified from the vOka strain and the five wild-type strains.

Generation of a recombinant Oka varicella vaccine expressing mumps virus hemagglutinin-neuraminidase protein as a polyvalent live vaccine

We constructed a recombinant varicella-zoster virus (VZV) Oka vaccine strain (vOka) that contained the mumps virus (MuV) hemagglutinin-neuraminidase (HN) gene, inserted into the site of the ORF 13 gene by using the bacterial artificial chromosome (BAC) system in Escherichia coli. Insertion of the HN gene into the VZV genome was confirmed by PCR and Southern blot. The infectious virus reconstituted from the vOka-HN genome (rvOka-HN) had a growth curve similar to the original recombinant vOka without the HN gene. The mumps virus HN protein expressed in rvOka-HN infected cells was expressed diffusely in the cytoplasm, and modification of the protein was similar to that seen in MuV-infected cells. Electron microscopic examination of infected cells revealed that HN was expressed on the plasma membrane of the cells but not in the viral envelope, suggesting that the tropism of rvOka-HN would be unchanged from that of the original vOka strain. Immunization of guinea pigs with rvOka-HN-induced VZV- and HN-specific antibodies. Interestingly, the induced antibodies had a strong neutralizing activity against virus-cell infections of both MuV and VZV. Therefore, the novel varicella vaccine expressing MuV HN protein is suitable as a polyvalent live attenuated vaccine against VZV and MuV infections.

Comparison of Virus Transcription during Lytic Infection of the Oka Parental and Vaccine Strains of Varicella-Zoster Virus

The attenuated Oka vaccine (V-Oka) strain of varicella-zoster virus (VZV) effectively reduces disease produced by primary infection and virus reactivation. V-Oka was developed by propagation of the Oka parental (P-Oka) strain of VZV in guinea pig and human embryo fibroblasts. Complete DNA sequencing of both viruses has revealed 63 sites that differ between P-Oka and V-Oka, 37 of which are located within 21 unique open reading frames (ORFs). Of the ORFs that differ, ORF 62 contains the greatest number (10) of mutated sites. ORF 62 encodes IE 62, the major immediate-early transactivator of virus genes, and is essential for lytic virus replication. To determine whether a disproportionate number of mutations in ORF 62 might account for virus attenuation, we compared the global pattern of V-Oka gene expression to that of P-Oka. Transcription of ORFs 62, 65, 66, and 67 was suppressed, whereas ORF 41 was elevated in V-Oka-infected cells compared to P-Oka-infected cells (P < 0.01; z test). Suppression of ORF 62, 65, and 66 transcription was confirmed by quantitative dot blot and Western blot analyses. Transient-transfection assays to determine whether mutations within V-Oka-derived IE 62 affected its ability to transactivate VZV gene promoters revealed similar IE 62 transactivation of VZV gene 20, 21, 28, 29, 65, and 66 promoters in both P-Oka and V-Oka. Together, our results indicate that mutations in V-Oka IE 62 alone are unlikely to account for vaccine virus attenuation.

An evaluation of single nucleotide polymorphisms used to differentiate vaccine and wild type strains of varicella‐zoster virus

Rashes following immunization with the vaccine strain (vOka) of varicella-zoster virus (VZV) may occur in up to 5% of children and 10% of adults. In 40% of cases, the causative virus is the vaccine strain and in 60% wild type virus is found. Several reports have identified three restriction site polymorphisms in ORF 62 and the loss of one in ORF 6, which differentiate vOka from wild type VZV, including the parental wild type strain from which vOka, is derived. Using polymerase chain reaction (PCR), restriction enzyme analysis, and sequencing, we analyzed the presence of these markers in the GlaxoSmithKline (GSK, UK) and Merck vaccine preparations as well as in 15 vaccine virus rashes and 15 wild type UK viruses. Our data suggest that a Sma1 positive and an Nae1 positive site in ORF 62 are present in the GSK and Merck vaccine preparations and all vaccine virus rashes. By contrast, a BssHII positive vaccine virus restriction site in ORF 62 and an Alu1 negative site in ORF 6 were mixed in the GSK and Merck vaccines and absent in some of the vaccine rashes. The BssHII site was also present in the European wild type C viruses in UK. The data suggest that unlike the Biken vaccine preparation, the Merck and GSK vaccine preparations are polymorphic for the BssHII and Alu1 restriction sites. These sites are also present variably in the vaccine viruses causing rashes following vaccination, and are therefore unreliable markers for differentiating vOka and wild type VZV strains.