Varicella-zoster Virus gE Research Articles

Structures of the Varicella Zoster Virus Glycoprotein E and Epitope Mapping of Vaccine-Elicited Antibodies.

Background: Varicella zoster virus (VZV) is the causative agent for chickenpox and herpes zoster (HZ, shingles). HZ is a debilitating disease affecting elderly and immunocompromised populations. Glycoprotein E (gE) is indispensable for viral replication and cell-to-cell spread and is the primary target for anti-VZV antibodies. Importantly, gE is the sole antigen in Shingrix, a highly efficacious, AS01B-adjuvanted vaccine approved in multiple countries for the prevention of HZ, yet the three-dimensional (3D) structure of gE remains elusive. Objectives: We sought to determine the structure of VZV gE and to understand in detail its interactions with neutralizing antibodies. Methods: We used X-ray crystallography and cryo-electron microscopy to elucidate structures of gE bound by recombinant Fabs of antibodies previously elicited through vaccination with Zostavax, a live, attenuated vaccine. Results: The 3D structures resolve distinct central and C-terminal antigenic domains, presenting an array of diverse conformational epitopes. The central domain has two beta-sheets and two alpha helices, including an IgG-like fold. The C-terminal domain exhibits 3 beta-sheets and an Ig-like fold and high structural similarity to HSV1 gE. Conclusions: gE from VZV-infected cells elicits a human antibody response with a preference for the gI binding domain of gE. These results yield insights to VZV gE structure and immunogenicity, provide a framework for future studies, and may guide the design of additional herpesvirus vaccine antigens. Teaser: Structures of varicella zoster virus glycoprotein E reveal distinct antigenic domains and define epitopes for vaccine-elicited human antibodies.

Heterologous Prime-Boost Immunization Strategies Using Varicella-Zoster Virus gE mRNA Vaccine and Adjuvanted Protein Subunit Vaccine Triggered Superior Cell Immune Response in Middle-Aged Mice.

Heterologous immunization using different vaccine platforms has been demonstrated as an efficient strategy to enhance antigen-specific immune responses. In this study, we performed a head-to-head comparison of both humoral and cellular immune response induced by different prime-boost immunization regimens of mRNA vaccine and adjuvanted protein subunit vaccine against varicella-zoster virus (VZV) in middle-aged mice, aiming to get a better understanding of the influence of vaccination schedule on immune response. VZV glycoprotein (gE) mRNA was synthesized and encapsulated into SM-102-based lipid nanoparticles (LNPs). VZV-primed middle-aged C57BL/6 mice were then subjected to homologous and heterologous prime-boost immunization strategies using VZV gE mRNA vaccine (RNA-gE) and protein subunit vaccine (PS-gE). The antigen-specific antibodies were evaluated using enzyme-linked immunosorbent assay (ELISA) analysis. Additionally, cell-mediated immunity (CMI) was detected using ELISPOT assay and flow cytometry. Besides, in vivo safety profiles were also evaluated and compared. The mRNA-loaded lipid nanoparticles had a hydrodynamic diameter of approximately 130 nm and a polydispersity index of 0.156. Total IgG antibody levels exhibited no significant differences among different immunization strategies. However, mice received 2×RNA-gE or RNA-gE>PS-gE showed a lower IgG1/IgG2c ratio than those received 2×PS-gE and PS-gE> RNA-gE. The CMI response induced by 2×RNA-gE or RNA-gE>PS-gE was significantly stronger than that induced by 2×PS-gE and PS-gE> RNA-gE. The safety evaluation indicated that both mRNA vaccine and protein vaccine induced a transient body weight loss in mice. Furthermore, the protein vaccine produced a notable inflammatory response at the injection sites, while the mRNA vaccine showed no observable inflammation. The heterologous prime-boost strategy has demonstrated that an mRNA-primed immunization regimen can induce a better cell-mediated immune response than a protein subunit-primed regimen in middle-aged mice. These findings provide valuable insights into the design and optimization of VZV vaccines with the potentials to broaden varicella vaccination strategies in the future.

A nanoparticle vaccine displaying varicella-zoster virus gE antigen induces a superior cellular immune response than a licensed vaccine in mice and non-human primates.

Herpes zoster (HZ), also known as shingles, remains a significant global health issue and most commonly seen in elderly individuals with an early exposure history to varicella-zoster virus (VZV). Currently, the licensed vaccine Shingrix, which comprises a recombinant VZV glycoprotein E (gE) formulated with a potent adjuvant AS01B, is the most effective shingles vaccine on the market. However, undesired reactogenicity and increasing global demand causing vaccine shortage, prompting the development of novel shingles vaccines. Here, we developed novel vaccine candidates utilising multiple nanoparticle (NP) platforms to display the recombinant gE antigen, formulated in an MF59-biosimilar adjuvant. In naïve mice, all tested NP vaccines induced higher humoral and cellular immune responses than Shingrix, among which, the gEM candidate induced the highest cellular response. In live attenuated VZV (VZV LAV)-primed mouse and rhesus macaque models, the gEM candidate elicited superior cell-mediated immunity (CMI) over Shingrix. Collectively, we demonstrated that NP technology remains a suitable tool for developing shingles vaccine, and the reported gEM construct is a highly promising candidate in the next-generation shingles vaccine development.

Varicella zoster virus glycoprotein E facilitates PINK1/Parkin-mediated mitophagy to evade STING and MAVS-mediated antiviral innate immunity

Viruses have evolved to control mitochondrial quality and content to facilitate viral replication. Mitophagy is a selective autophagy, in which the damaged or unnecessary mitochondria are removed, and thus considered an essential mechanism for mitochondrial quality control. Although mitophagy manipulation by several RNA viruses has recently been reported, the effect of mitophagy regulation by varicella zoster virus (VZV) remains to be fully determined. In this study, we showed that dynamin-related protein-1 (DRP1)-mediated mitochondrial fission and subsequent PINK1/Parkin-dependent mitophagy were triggered during VZV infection, facilitating VZV replication. In addition, VZV glycoprotein E (gE) promoted PINK1/Parkin-mediated mitophagy by interacting with LC3 and upregulating mitochondrial reactive oxygen species. Importantly, VZV gE inhibited MAVS oligomerization and STING translocation to disrupt MAVS- and STING-mediated interferon (IFN) responses, and PINK1/Parkin-mediated mitophagy was required for VZV gE-mediated inhibition of IFN production. Similarly, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-mediated mitophagy induction led to increased VZV replication but attenuated IFN production in a three-dimensional human skin organ culture model. Our results provide new insights into the immune evasion mechanism of VZV gE via PINK1/Parkin-dependent mitophagy.

Enhancing cell-scale performance via sustained release of the varicella-zoster virus antigen from a microneedle patch under simulated microgravity.

The immune system of astronauts might become weakened in the microgravity environment in space, and the dormant varicella-zoster virus (VZV) in the body might be reactivated, seriously affecting their work and safety. For working in orbit for the long term, there is currently no efficient and durable delivery system of general vaccines in a microgravity environment. Accordingly, based on the previous foundation, we designed, modified, and synthesized a biodegradable and biocompatible copolymer, polyethylene glycol-polysulfamethazine carbonate urethane (PEG-PSCU) that could be mainly adopted to fabricate a novel sustained-release microneedle (S-R MN) patch. Compared with conventional biodegradable microneedles, this S-R MN patch could not only efficiently encapsulate protein vaccines (varicella-zoster virus glycoprotein E, VZV gE) but also further prolong the release time of VZV gE in a simulated microgravity (SMG) environment. Eventually, we verified the activation of dendritic cells by VZV gE released from the S-R MN patch in an SMG environment and the positive bioeffect of activated dendritic cells on lymphocytes using an in vitro lymph node model. This study is of great significance for the exploration of long-term specific immune responses to the VZV in an SMG environment.

Immunogenicity in Mice Immunized with Recombinant Adenoviruses Expressing Varicella-Zoster Virus Envelope Glycoprotein E.

Herpes zoster (HZ) is a disease caused by the reactivation of latent varicella-zoster virus (VZV). The subunit vaccine, Shingrix®, and live attenuated vaccine, Zostavax®, could be used as an HZ vaccine that prevents HZ from being developed due to the reactivation of latent VZV in the sensory ganglia due to aging, stress or immunosuppression. In this study, the recombinant adenoviruses rChAd63/gE expressing glycoprotein E (gE) of VZV based on chimpanzee adenovirus serotype 63 (ChAd63) were constructed and investigated for the immunogenicity of different immune pathways in C57BL/6 mice. The results showed similar CD4+ T and CD8+ T cell responses to Shingrix® were induced in mice vaccinated using rChAd63/gE via different immune pathways. This study elucidates that recombinant adenoviruses expressing VZV gE could be appropriate for further development as a new HZ vaccine candidate via different immune pathways.

EPID-17. ANTIBODY REACTIVITY TO VARICELLA ZOSTER VIRUS IS ASSOCIATED WITH IMPROVED GLIOMA SURVIVAL AND IS MODIFIED BY GERMLINE HLA POLYMORPHISMS

Abstract Antigenic reactivity towards varicella zoster virus (VZV) has shown a consistent reduction in glioma risk across multiple studies on multiple continents. To our knowledge this is the virus to be robustly associated with reduced risk of any cancer. Our recent work has shown that high VZV reactivity is also assumed with improved glioma prognosis. Using the UKBioBank we have previously shown that germline HLA polymorphism, rs9273325, significantly effects reactivity to VZV. In this study we investigate the VZV glioma survival association in the context of germline HLA polymorphisms. METHODS: We measured quantitative IgG reactivity towards VZV gE, and conducted genome wide array based genotyping in 891 adults with glioma. Associations of patient IgG levels (1st quartile vs. quartiles 2,3,4) with overall survival were estimated using Cox models adjusted for age, sex, self-reported race, surgery type, dexamethasone usage at blood draw. RESULTS: VZV seroreactivity was significantly associated with reduced mortality (HR=0.83, 95% CI: 0.70-0.99; p=0.034), as expected from our previous study. Among patients with rs9273325-GG, higher levels of VZV antibodies were associated better prognosis (HR=0.75, 0.62-0.92; p=0.005), but in the rs9273325-GA/AA strata this relationship with VZV antibodies was not observed (HR=0.94, 0.66-1.35; p=0.75). Similar results were observed in grade IV gliomas, IDHwt GBMs. CONCLUSIONS: Strong antibody response to VZV is associated with improved glioma prognosis and the effect of VZV reactivity on glioma survival is modified by a specific HLA polymorphism, a Gene x Virus interaction, that is unlikely due to chance. This study highlights the importance of accounting for HLA variation in immune related studies, including oncolytic viral therapies. Our findings also point towards a potentially MHC mediated mechanism by which VZV could be priming an immune response targeting glioma cells. Further investigations into this mechanism and VZV/shingles vaccination as a potential glioma therapeutic are warranted.

Rapid detection of varicella-zoster virus based on an immunochromatographic strip

Varicella-zoster virus (VZV) is a highly infectious DNA virus that can cause varicella (chickenpox) and herpes zoster (HZ). A simple, sensitive and specific detection method is desirable for the VZV infection. In this study, VZV gE protein, expressed in CHO cells, was used to immunize BALB/c mice for the generation of monoclonal antibodies (mAbs). For the first time, we developed a colloidal gold-based immunochromatographic strip for rapid detection of VZV using a pair of mAbs against gE protein. The limit of detection (LOD) of the strip was 30 ng mL−1 of purified VZV gE antigen, and it could specifically test VZV without cross-reactivity with Enterovirus 71 (EV-71), Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2). The coincidence rate between the strip and commercial real-time PCR diagnostic kit was 100% using vesicle as the clinical sample. Our strip provided a technical support for rapid and specific detection of VZV.

Truncated glycoprotein E of varicella-zoster virus is an ideal immunogen for Escherichia coli-based vaccine design.

Varicella-zoster virus (VZV) is a highly infectious agent responsible for both varicella and herpes zoster disease. Despite high efficacy, there remain safety and accessibility concerns with the licensed vaccines. Here, we sought to produce a VZV gE immunogen using an E. coli expression system. We found that the soluble expression and yield of gE protein could be enhanced via C-terminal truncations to the protein, thereby facilitating a robust and scalable purification process for the purpose of vaccine manufacturing. The lead truncated gE (aa 31-358), hereafter referred to as tgE, was a homogenous monomer in solution and showed excellent antigenicity. Finally, we assessed and compared the immunogenicity of tgE with commercial vOka LAV and Shingrix vaccine. We found that aluminum-adjuvanted tgE was immunogenic as compared with vOka LAV. When adjuvanted with AS01B, a two-dose immunization of tgE showed comparable or better potency in antibody responses and cell-mediated immunity with those of the Shingrix vaccine at the same dosage, especially in terms of the proportion of IFN-γ-expressing CD4+ T cells. In conclusion, this method of E. coli-mediate tgE expression offers a cost-effective and scalable strategy to generate an ideal VZV gE immunogen for the development of both varicella and zoster vaccines.

Rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of varicella-zoster virus immunoglobulin G antibodies in fingerstick blood

Varicella zoster virus (VZV) causes childhood chickenpox, becomes latent in sensory ganglia and reactivates years later to cause shingles (Zoster) and postherpetic neuralgia in the elderly and immunosuppressed individuals. Serologic IgG tests can be used to determine if a person has antibodies to VZV from past varicella infection or had received varicella or zoster (shingles) vaccination. Commercial enzyme-linked immunosorbent assays (ELISAs) are currently used for the detection of VZV IgG antibodies in patient serum samples. However, ELISA tests require collection and processing of blood samples in a CLIA laboratory to separate serum or plasma for further testing. In this paper, we describe the development and testing of an antibody based Lateral Flow Immunochromatographic assay (LFA) device for the detection of VZV IgG in fingerstick whole blood. Analytical and clinical analyses were performed to compare the performance characteristics of the Viro VZV IgG LFA (VZV LFA) and the Diamedix VZV IgG ELISA. Analytical studies demonstrated the higher sensitivity of the VZV LFA compared to the ELISA by testing dilutions of the WHO VZV IgG serum International Standard. Clinical performance characteristics of the VZV LFA fingerstick whole blood assay were assessed at three point of care (POC) facilities by untrained users testing samples from 300 prospectively enrolled study subjects. VZV LFA results were compared with results obtained by testing serum samples obtained from the same study participants by the Diamedix VZV IgG ELISA. Two specimens with invalid results by the LFA assay were not included in the LFA performance calculations and nine equivocal ELISA results were included as positive for IgG results. The results from all three POC clinical sites demonstrated the higher sensitivity/positive percent agreement (PPA) (99.26%, 95% CI: 97.34–99.80) of the VZV LFA compared to the Diamedix VZV IgG ELISA (94.08%, 95% CI: 90.72–96.27). The specificity/negative percent agreement (NPA) of the VZV LFA compared to the ELISA test was calculated initially to be 39.29% (95% CI: 23.57–57.59) with 19 discordant test results out of 298 test results between the two assays (17 LFA positive/ELISA negative and two LFA negative/ELISA positive). The PPA and true NPA of the VZV LFA were determined by testing all 298 samples, including the discordant (19) and all concordant negative and positive (279) study subject serum samples, before and after blocking VZV gE antibody sites in the samples by spiking with VZV LFA gE capture antigen. The NPA improved to 100% (95% CI: 74.12–100) after the procedure when compared to the ELISA test results. The comparator ELISA PPA based on the spiking/blocking study remained as 94.08%, (95% CI: 90.72–96.27), comparable to test results from untreated samples.The VZV LFA has been demonstrated to be simple and sufficiently robust for use in CLIA-waived POC facilities by untrained healthcare professionals and to detect VZV IgG in 20 min from fingerstick whole blood. The VZV LFA therefore provides a fast, reliable, and highly sensitive method of determining prior VZV viral infection or varicella and zoster vaccination status.

Novel BC02 Compound Adjuvant Enhances Adaptive and Innate Immunity Induced by Recombinant Glycoprotein E of Varicella-Zoster Virus.

Both adaptive and innate immunity responses are necessary for the efficient elimination of different pathogens. However, the magnitude, quality and desired type of immune response specific to the co-administered antigen is largely determined by adjuvants. BC02 (BCG CpG DNA compound adjuvants system 02) is a novel compound adjuvant with independent intellectual properties, which is composed of BCG CpG DNA biological adjuvant with Al(OH)3 inorganic salt adjuvant acting as a delivery system. Its safety and strong adjuvant efficacy have been effectively verified in preclinical and clinical trials (Phase Ib, ClinicalTrials.gov Identifier: NCT04239313 and Phase II, ClinicalTrials.gov Identifier: NCT05284812). In this study, we report the level of cell-mediated immunity (CMI) and humoral immune response induced by the BC02 novel adjuvant combined with different doses of varicella-zoster virus (VZV) glycoprotein E (gE) in a mouse model. In addition, we conducted preliminary in vitro experiments to explore the enhancement of RAW264.7 cell immune activity by BC02 adjuvanted-gE experimental vaccine to activate innate immune response. The results showed that the BC02-adjuvanted low, medium or high dose of gE were highly effective in eliciting both CMI and humoral immune responses to the immunized mice, respectively. The production of gE-specific IFN-γ and IL-2-specific T cells was established within 28 days after booster immunization. In particular, the effect of BC02-adjuvanted medium dose of gE has been shown to be more prominent. Meanwhile, fluorescent antibody to membrane antigen (FAMA) and serum antibody plaque reduction tests have also shown that the BC02 adjuvanted-medium dose of gE antigen could induce the secretion of neutralizing antibodies against clinically isolated VZV strains in mice. In addition, our findings have shown that 1/25 dose of gE+BC02 medium dose experimental vaccine can significantly induce the secretion of innate immune cytokines TNF-A, MCP-1, IL-6 and GM-CSF and up-regulate the costimulatory molecules CD40, CD80 and I-A/I-E on RAW264.7 cells; and it has also been activated to form M2 macrophages. At the same time, RAW264.7 cells were stimulated for 12 h, and their phagocytosis was significantly enhanced. Taken together, these results suggest that the BC02 compound adjuvant offers a strategy to induce an appropriate innate and adaptive immunity against the different doses of the VZV gE protein to improve subunit vaccine efficacy, and BC02 may be a promising adjuvant candidate for subunit HZ vaccines.

Baculovirus Display of Varicella-Zoster Virus Glycoprotein E Induces Robust Humoral and Cellular Immune Responses in Mice.

Varicella–zoster virus (VZV) is the causative agent of varicella and herpes zoster (HZ) and can pose a significant challenge to human health globally. The initial VZV infection—more common in children—causes a self-limiting chicken pox. However, in later life, the latent VZV can become reactivated in these patients, causing HZ and postherpetic neuralgia (PHN), a serious and painful complication. VZV glycoprotein E (gE) has been developed into a licensed subunit vaccine against HZ (Shingrix). However, its efficacy relies on the concomitant delivery of a robust adjuvant (AS01B). Here, we sought to create a new immunogen for vaccine design by displaying the VZV–gE on the baculovirus surface (Bac–gE). Correct localization and display of gE on the engineered baculovirus was verified by flow cytometry and immune electron microscopy. We show that Bac–gE provides excellent antigenicity against VZV and induces not only stronger gE-specific CD4+ and CD8+ T cell responses but also higher levels of VZV–specific neutralizing antibodies as compared with other vaccine strategies in mice. Collectively, we show that the baculovirus display of VZV–gE confers ideal humoral and cellular immune responses required for HZ vaccine development, paving the way for a baculovirus-based vaccine design.

Development of an Indirect ELISA Kit for Rapid Detection of Varicella-Zoster Virus Antibody by Glycoprotein E.

Varicella-zoster virus (VZV), a highly infectious agent that causes varicella (chickenpox), can also cause zoster (shingles), a disorder that is frequently associated with severe neuralgia. A reliable serological VZV diagnostic assay would be useful for identifying unprotected individuals and for surveilling post-vaccination immunoprotection status. Toward this goal, VZV membrane glycoprotein E (gE), the immunodominant VZV protein, served as target antigen in an indirect ELISA kit developed here to detect anti-VZV antibodies in clinical samples. For target antigen preparation, Chinese hamster ovary (CHO) cells were modified to express and secrete the VZV gE ectodomain, which was subsequently purified and used as coating antigen in an indirect ELISA. Ultimately, the optimal purified gE coating antigen concentration was determined to be 2 μg.ml−1 and the OD450nm detection cutoff value was 0.286. The coefficient of variation (CV) of intra-assay and inter-assay were <10 and 15%, respectively. A comparative test of 66 clinical samples showed that the coincidence rate was 93.9% between the indirect ELISA and a commercial varicella-zoster virus IgG ELISA kit. Thus, the indirect ELISA kit developed here may be useful for achieving rapid, sensitive, and specific detection of anti-VZV antibodies.

An adjuvanted zoster vaccine elicits potent cellular immune responses in mice without QS21

Herpes zoster (HZ) is caused by reactivation of latent varicella-zoster virus (VZV) when VZV-specific cellular immunity is insufficient to control reactivation. Currently, Shingrix, which contains the VZV gE protein and GSK’s AS01B adjuvant composed of liposomes formulated with cholesterol, monophosphoryl lipid A (MPL) and QS21, is used for prevention of HZ. However, reactogenicity to Shingrix is common leading to poor patient compliance in receiving one or both shots. Here, we evaluated the immunogenicity of a newly formulated gE protein-based HZ vaccine containing Second-generation Lipid Adjuvant (SLA), a synthetic TLR4 ligand, formulated in an oil-in-water emulsion (SLA-SE) without QS21 (gE/SLA-SE). In VZV-primed mouse models, gE/SLA-SE-induced gE-specific humoral and cellular immune responses at comparable levels to those elicited by Shingrix in young mice, as both gE/SLA-SE and Shingrix induce polyfunctional CD4+ T-cell responses. In aged mice, gE/SLA-SE elicited more robust gE-specific T-cell responses than Shingrix. Furthermore, gE/SLA-SE-induced T-cell responses were sustained until 5 months after immunization. Thus, QS21-free, gE/SLA-SE is a promising candidate for development of gE-based HZ vaccines with high immunogenicity—particularly when targeting an older population.

Enhanced Potency and Persistence of Immunity to Varicella-Zoster Virus Glycoprotein E in Mice by Addition of a Novel BC02 Compound Adjuvant

Herpes zoster (HZ) is one of two distinct syndromes caused by Varicella-zoster virus (VZV). A primary infection with VZV causes varicella in susceptible young children. After resolution of the primary infection, VZV establishes a lifelong latency within the cranial or dorsal root ganglia. With increasing age, family history of shingles, immunosuppression or other risk factors, there is a decline in the virus-specific T-cell-mediated immune (CMI) response which allows reactivation of latent VZV in the root ganglia resulting in HZ. There are currently two vaccines that have been approved to prevent HZ and postherpetic neuralgia (PHN) but one is a live attenuated vaccine, the protective effect of which is considered to decrease significantly with the age of the recipient. However, a recombinant subunit vaccine may provide targeted VZV-specific cellular and humoral immunity, giving it a more potent and longer-lasting protective effect against HZ. The current study reports the development of a novel adjuvant, BC02 (BCG CpG DNA compound adjuvants system 02), composed of Al(OH)3 inorganic salt adjuvant and BC01 (BCG CpG DNA compound adjuvants system 01), a Toll-like receptor 9 (TLR9) agonist. Immunogenicity and compatibility with recombinant VZV glycoprotein E (gE) in mice were studied. The BC02-adjuvanted gE experimental vaccine was highly effective in eliciting both humoral and cellular immune responses to the recombinant gE glycoprotein and VZV-Oka in a mouse model. The efficient production and long-term persistence of gE and VZV-Oka-specific IFN-γ, IL-2-specific T cells and memory B cells in the early (1W), middle (7W), middle-late (15W), and final (27W) immune stages were established. Results of fluorescent antibody to membrane antigen (FAMA) and serum antibody plaque reduction tests also showed that the BC02 adjuvanted-gE experimental vaccine induced mice to secrete neutralizing antibodies against clinically isolated VZV strains. In combination, the current data suggest that the BC02 compound adjuvant offers a strategy to induce an appropriately strong cellular and humoral immunity against the VZV gE protein subunit to improve vaccine efficacy.

Culture media optimization for Chinese hamster ovary cell growth and expression of recombinant varicella-zoster virus glycoprotein E.

Herpes zoster (HZ) is caused by reactivation of varicella-zoster virus (VZV) latent in the sensory ganglia and causes severe pain, often leading to postherpetic neuralgia (PHN). Two prophylactic vaccines against HZ are currently licensed for human use, a live attenuated vaccine and a subunit vaccine containing recombinant VZV glycoprotein E (gE) as antigen. The latter has superior protective efficacy against HZ and PHN. During HZ subunit vaccine development, we obtained Chinese hamster ovary (CHO) cell clones expressing VZV gE. This study was performed to optimize culture media conditions for CHO cell growth and gE production. Using a high-throughput culture system, three CHO cell clones were cultured in microtiter plates containing 24 different basal media, and three basal media were selected. The clone with the highest gE expression was fed-batch cultured in each of the three basal media in combination with 13 different feed media. A pair of media, BalanCD CHO Growth A and EX-CELL Advanced CHO Feed 1, with the highest productivity was selected for gE production. Scale-up fed-batch cultures of the selected clone cultured in a wave bag bioreactor containing the optimized media yielded 2440 mg gE protein/L culture, a 11.5-fold increase compared to original culture conditions (batch culture in CD OptiCHO medium). The optimized media condition is used to produce VZV gE antigen for an HZ subunit vaccine, which is under phase I clinical trial. This study would provide valuable insights on culture media optimization for CHO cells expressing a recombinant vaccine antigen.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10616-021-00468-1.

The Effect of a TLR4 Agonist/Cationic Liposome Adjuvant on Varicella-Zoster Virus Glycoprotein E Vaccine Efficacy: Antigen Presentation, Uptake, and Delivery to Lymph Nodes.

Adjuvant CIA09, composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based cationic liposomes and the toll-like receptor 4 agonist de-O-acylated lipooligosaccharide (dLOS), has been shown to enhance antibody and cellular immune responses to varicella-zoster virus (VZV) glycoprotein E (gE), recombinant tuberculosis vaccine antigen, and inactivated Japanese encephalitis vaccine. In this study, we investigated its modes of action using VZV gE as a model antigen. Liposomes adsorbed gE and cooperatively with dLOS promoted endocytosis-mediated cellular uptake of gE by mouse dendritic cells in vitro. CIA09 increased the stability and cellular uptake of the antigen at the muscle site of injection, and induced immune cell recruitment and cytokine and chemokine production, which led to efficient antigen delivery to draining lymph nodes. Mouse bone marrow-derived dendritic cells, pulsed with CIA09-adjuvanted gE, efficiently presented gE to antigen-specific T cells, inducing Th1-type biased immunity, as shown by high IFN-γ production. The data indicate that liposomes and dLOS cooperate in the adjuvant activity of CIA09 by promoting antigen uptake and delivery to lymph nodes as well as antigen presentation to T cells.

Immunogenicity generated by mRNA vaccine encoding VZV gE antigen is comparable to adjuvanted subunit vaccine and better than live attenuated vaccine in nonhuman primates

Shingles is a painful, blistering rash caused by reactivation of latent varicella-zoster virus (VZV) and most frequently occurs in elderly and immunocompromised individuals. Currently, two approved vaccines for the prevention of shingles are on the market, a live attenuated virus vaccine ZOSTAVAX® (Merck & Co., Inc., Kenilworth, NJ, USA) and an AS01B adjuvanted subunit protein vaccine Shingrix™ (Glaxo Smith Kline, Rockville, MD, USA). Human clinical immunogenicity and vaccine efficacy data is available for these two benchmark vaccines, offering a unique opportunity for comparative analyses with novel vaccine platforms and animal model translatability studies.The studies presented here utilized non-human primates (NHP) to evaluate humoral and cellular immune response by three vaccine modalities: the new platform of lipid nanoparticle (LNP) formulated mRNA encoding VZV gE antigen (VZV gE mRNA/LNP) as compared with well-established platforms of live attenuated VZV (VZV LAV) and adjuvanted VZV gE subunit protein (VZV gE protein/adjuvant). The magnitude of response to vaccination with a single 100–200 μg mRNA dose or two 50 μg mRNA doses of VZV gE mRNA/LNP were comparable to two 50 μg protein doses of VZV gE protein/adjuvant, suggesting the VZV gE mRNA/LNP platform has the potential to elicit a robust immune response, and both modalities generated markedly higher responses than VZV LAV. Additionally, the slopes of decay for VZV-specific antibody titers were roughly similar across all three vaccines, indicating the magnitude of peak immunogenicity was the driving force in determining immune response longevity. Finally, vaccine-induced immunogenicity with VZV LAV and VZV gE protein/adjuvant in NHP closely resembled human clinical trials immune response data for ZOSTAVAX® and Shingrix™, helping to validate NHP as an appropriate preclinical model for evaluating these vaccines.

Evaluation of glycoprotein E subunit and live attenuated varicella-zoster virus vaccines formulated with a single-strand RNA-based adjuvant.

IntroductionVaricella‐zoster virus (VZV), a human alphaherpesvirus 3, elicits both chickenpox and shingles and/or postherpetic neuralgia. A live attenuated vaccine (LAV) and glycoprotein E (gE) subunit vaccine were developed to prevent VZV‐induced diseases. We recently reported that single‐strand RNA (ssRNA) based on the intergenic region of the internal ribosome entry site of cricket paralysis virus (CrPV) is an effective adjuvant for protein‐based and virus‐like particle‐based vaccines. Here, Chinese hamster ovary expression system and an LAV from Oka/SK strains.MethodsWe appraised the adjuvant effect of the same CrPV ssRNA encoding the gE gene formulated in the two vaccines using VZV‐primed C57BL/6 mice and guinea pigs. Humoral immunity and cell‐mediated immunity were assessed by enzyme‐linked immunosorbent assay (ELISA) and ELISPOT in gE subunit vaccine and by ELISA and fluorescent antibody to membrane antigen in LAV.ResultsThe gE subunit vaccine‐induced gE‐specific antibodies and CD4+ T‐cell responses (indicated by interferon‐γ [IFN‐γ] and interleukin‐2 secretion) in the ssRNA‐based adjuvant containing the VZV gE gene. Therefore, an ssRNA adjuvant combined with gE antigen can trigger the innate immune response and induce an adaptive immune response to ultimately activate humoral and cell‐mediated responses. VZV LAV could also induce VZV‐specific antibodies and IFN‐γ stimulated by LAV, whereas the effect of ssRNA as a vaccine adjuvant could not be confirmed. However, the ssRNA adjuvant increased VZV‐specific neutralizing antibody response.ConclusionsTaken together, these results highlight that the gE subunit vaccine and LAV developed in this study can be functional VZV vaccines, and ssRNAs appear to function better as adjuvants in a subunit vaccine than in an LAV.

Varicella-Zoster Virus infected human neurons are resistant to apoptosis.

Varicella-zoster virus (VZV) is a pathogenic human herpesvirus that causes varicella (chickenpox) as a primary infection following which it becomes latent in ganglionic neurons. Following viral reactivation many years later VZV causes herpes zoster (shingles) as well as a variety of other neurological syndromes. The molecular mechanisms of the conversion of the virus from a lytic to a latent state in ganglia are not well understood. In order to gain insights into the neuron-virus interaction, we studied virus-induced apoptosis in cultures of both highly pure terminally differentiated human neurons and human fetal lung fibroblasts (HFL). It was found that (a) VZV DNA did not accumulate in infected human neurons; (b) VZV transcripts were present at lower levels at all days studied post-infection in neurons; (c) Western blot analysis showed less VZV IE 63 and very little detectable VZV gE proteins in infected neurons compared with HFL; (d) lower levels of the apoptotic marker cleaved Caspase-3 protein were detected in VZV-infected neurons compared with HFL, and higher levels of the known anti-apoptotic proteins Bcl2, Bcl-XL and also the mitochondrial MT-CO2 protein were found in VZV-infected neurons compared with uninfected cells; and (e) both the MT-CO2 protein and VZV IE 63-encoded protein were detected in infected neurons by dual immunofluorescence. These findings showed that neurons are resistant to VZV-induced apoptosis, which may have relevance to the switching of VZV from a lytic to latent ganglionic neuronal infection.