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 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 vodka LAV and Shingrix vaccine. We found that aluminum-adjuvanted tgE was immunogenic as compared with vodka 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.

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varicella-zoster virus, glycoprotein E, Escherichia coli, vaccine

INTRODUCTION 

Varicella-zoster virus (VZV) is a pathogenic, human alpha herpesvirus that induces two main categories of disease: primary infection with varicella, a highly contagious disease characterized by a blister-like rash on the skin and mild systemic symptoms such as fever and malaise predominantly in children; and herpes zoster (HZ), the latent reactivation of the virus in response to a weakened cellular immune system caused by aging that causes a painful, localized vesicular rash (HZ) with or without several other complications (Cohen, 2013; Heininger and Seward, 2006; Zerboni et al., 2014). Varicella and HZ can both be prevented by live-attenuated virus vaccines (LAV). The first LAV for the prevention of varicella was developed by Takahashi et al. (1974) based on the vOka virus strain and was later licensed as Varivax for routine use in the United States. This was followed by the development and licensing of Zostavax for the prevention of HZ (Tseng et al., 2011). Unfortunately, in the 10 years since inoculations with Varivax began, almost 1/3 of HZ cases have been confirmed to be associated with the vOka strain (Galea et al., 2008), suggesting that vOka can infect and establish latency, like a wild-type virus, and then reactivate to cause HZ. In addition, the efficacy of Zostavax is inversely proportional to the age of the patient at the time of administration, with increased age associated with lower efficacy (Oxman et al., 2005). Previous studies have shown the VZV glycoprotein E (gE) to be the most abundant viral glycoprotein on the virion envelope and the infected cell surface. GE is also one of the most important VZV protective antigens capable of inducing cellular and humoral immunity during natural varicella infection as well as following vOka vaccination (Arvin, 1992; Oliver et al., 2016). Recently, a recombinant HZ subunit vaccine, Shingrix (HZ/su), was approved for the prevention of HZ and the associated postherpetic neuralgia (PHN) in adults aged≥50 years (Shah et al., 2019; Syed, 2018). In clinical trials, Shingrix induced an efficient, VZV-specific, cell-mediated immunity (CMI), with an overall vaccine efficacy of 97.2% among participants ≥50 years and a 91.3% protection rate in participants ≥70 years. These findings collectively indicate a significantly reduced risk of HZ in vaccinated individuals independent of age, and thus a more potent vaccine option as compared with ZOSTAVAX (Lal et al., 2015; Shah et al., 2019).

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Shingrix is designed from a recombinant gE antigen expressed in Chinese hamster ovary (CHO) cells and a liposome-based adjuvant system (AS01B) containing 3-Odesacyl-4′-monophosphoryl lipid A (MPL) and Quillaja saponaria Molina, fraction 21 (QS21) (Dendouga et al., 2012). However, recombinant CHO cells require a long production cycle and are associated with a complex purification process that results in a high manufacturing cost and a limited supply. As such, Shingrix is not an ideal vaccination solution for widespread use, particularly in developing countries (Kim et al., 2012). Microbial expression systems offer several advantages over mammalian expression systems, including rapid growth, ease of culture, and lower production costs (Overton, 2014), and are thus widely used in the expression and production of numerous biopharmaceuticals (Adkins and Wagstaff, 1998; Monie et al., 2008; Wu et al., 2012). However, several difficulties during production need to be overcome for the successful expression of recombinant eukaryotic proteins in prokaryotic systems, such as incorrect folding and a lack of post-translational modification (Singh and Panda, 2005). Despite these difficulties, microbial expression systems could offer an ideal system for widespread, cost-effective vaccine manufacturing, and it, therefore, warrants investigating antigens from an E. coli expression system for VZV vaccine design. In previous work, we showed the successful expression of a recombinant gE (rgE) protein in insect cells and obtained purified proteins for mouse immunization and mAb production (Liu et al., 2015). Building on this work, here we investigate whether the gE protein can be expressed in E. coli. We explored the non-fusion soluble expression of gE candidate antigens with a C-terminal truncation (hereafter referred to as truncated gE (tgE) or tgE) in E. coli and characterized the purified tgE proteins. We show that the purified tgE is antigenic similar to the native gE, and capable of inducing high antibody titers with vOka-neutralizing activity in mice. In comparing the immunogenicity of the tgE antigen generated in our study with the LAV and Shingrix, we find that our antigen not only exhibits an efficient humoral response but also induces an antigen-specific CMI. Overall, our E. coli strategy for the production of tgE protein may pave the way for an alternate industrially produced immunogen for both varicella and HZ vaccination. 

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RESULTS

Expression and purification of recombinant tgE in E. coli

DNA sequences encoding for the VZV gE extracellular domain were prepared as constructs with various N- or C-terminal truncations, and cloned into the pTO-T7 vector for protein expression in E. coli. The full-length extracellular domain of gE tended to form inclusion bodies in E. coli during expression, and this could be improved with a C-terminal truncation. Of the various truncated proteins, the best solubility was attained with the gE (aa 31–358) construct (Figure 1A), hereafter referred to as truncated gE or tgE. The candidate tgE was successfully expressed as a soluble recombinant protein and purified from the supernatant of bacteria lysates using three-step chromatography at the lab scale for further assessment and development as a potential vaccine. Strong anionic exchange chromatography was used for capture followed by ceramic hydroxyapatite and hydrophobic chromatography steps (Figure 1B). Successive stages of the purification process saw an increase in the purity of tgE on SDS-PAGE (Figure 1C). tgE migrated as an approximately 40 kD band and showed good reactivity with the specific antibody 1B11 (Figure 1D). The overall yield was about 500 μg of purified tgE per gram of bacteria. 

Characterization of the truncated gE 

High-performance size-exclusion chromatography (HPSEC) and analytical ultracentrifugation (AUC) were used to analyze the homogeneity and sedimentation coefficients of purified proteins. rgE protein purified from the insect cell system (Liu et al., 2015) was used as a control. In HPSEC, tgE appeared a sharp peak at the predominant retention time of 16.1 min (Figure 2A), reflecting high homogeneity for the purified tgE proteins. In AUC experiments, a predominant sedimentation peak was observed at ~2.3 S (Figure 2B), which equates to ~39.4 kD, indicating that tgE presents as a monomer in solution. Differential scanning calorimetry (DSC) was used to test the thermal stability of the tgE protein by monitoring the heat capacity during the thermal unfolding process. The DSC profiles showed only one thermal transition during protein unfolding at a transition temperature (Tm) value of 63.80°C for tgE, comparable to that of rgE (63.64°C) (Figure 2C). To investigate the conformational integrity of the purified tgE proteins, we analyzed the antigenicity of tgE using an ELISA assay, with purified rgE proteins expressed in insect cells serving as controls. Nine mAbs that could recognize conformational epitopes (mAb 1B11, 4G4, 14G1) or linear epitopes (4A2, 11B11, 11B12, 6H6, 10H6, 11E3) were used in the ELISA test (Figure 3). We found similar median effective concentration (EC50, ng mL−1) values for tgE and rgE proteins for most of the antibodies except for 1B11, which had an almost 10-fold lower reactivity, suggesting their similar antigenicity. These results suggest that key epitopes on the rgE are well preserved in the tgE protein.

Figure 1 (Color online) Expression and purification of the proteins. A Schematic representation of tgE construct design. E. coli cells were transformed with recombinant plasmids carrying the gE (aa 31–358) truncated gene. B, Schematic representation of the tgE purification process. tgE was purified from lysed supernatant using three-step chromatography. C, SDS-PAGE profile of tgE during the purification process. The molecular weight of tgE is approximately 40 kD. M: molecular weight marker. Lane 1: supernatant separated from cell lysate; lane 2: fraction containing tgE from the Q-FF chromatograph; lane 3: fraction containing tgE from CHT chromatograph; lane 4: fraction from the Butyl chromatograph, containing the tgE protein with high purity. D, Immunoblotting of the purified tgE.

Analysis of immune responses induced by tgE in mice 

To compare the immunogenicity of tgE with rgE, we formulated the proteins with two adjuvants commonly used for research work: aluminum-based adjuvant Al-001 and Freund's adjuvant. Mice were immunized thrice at weeks 0, 2, and 4 with a dosage of 1 μg for both adjuvanted tgE and rgE. gE-specific antibody titers and T-cell responses at week 5 post the first immunization were used to determine the immunogenicity of different adjuvant formulations. Mice immunized with the tgE vaccine formulated with Al-001 showed similar antibody profiles to mice in the rgE group, and Freund's adjuvant showed a better stimulation effect when formulated with tgE than with rgE (Figure 4A). Neutralization titers showed no differences in mice immunized with tgE or rgE vaccine, with neutralizing titers of ~103 for tgE formulated with the different adjuvants (Figure 4B). However, in the analysis of CD4+ T cells from splenocytes by intracellular cytokine staining (ICS), both tgE and rgE vaccines elicited similar levels of gE-specific IFN-γ as compared with the saline group (Figure 4C, ~0.1% for either vaccines groups or saline group), and only a moderate simulation of gE-specific IL-2 (Figure 4D, ~0.2% for vaccines groups vs. ~0.1% for saline group). Taken together, these results show that tgE/Al-001 and tgE/Freund's adjuvant elicit gE-specific immune responses comparable with those elicited by rgE, with a particularly strong humoral response. 

Evaluation of tgE varicella vaccine candidate as compared with LAV 

We next evaluated antibody production and the persistence of aluminum-adjuvanted tgE proteins in BALB/c mice. Two groups of mice (n=5 per group) were immunized thrice at weeks 0, 2, and 4 with different dosages of 0.5 μg and 5 μg of tgE/Al-001 (Figure 5A and B). The antibody response profiles showed that the primary inoculation of the tgE protein elicited high antibody titers, with a sustained increase after the two booster immunizations at weeks 2 and 4, and reaching a maximum level at week 6 before slowly dropping to more than 104 over monitoring period. Different dosage groups shared similar antibody titer profiles during the monitoring period, with similar high neutralizing titers at week 6. The immunogenicity of tgE was evaluated and compared with vodka. The half-effective dosage (ED50) in mice was used as a measure of seroconversion, with a lower ED50 indicative of better immunogenicity. Balb/c mice were separated into groups and intraperitoneally immunized with aluminum-adjuvanted tgE (1, 0.5, 0.25, 0.125, 0.0625, or 0.03125 μg; n=6) or vOka vaccine (1,000, 500, 250, 125, or 62.5 pfu; n=6) in serial dilutions. Sera were collected after 4 weeks and tested using ELISA. Seroconversion was analyzed according to the Reed and Muench method (Figure 5C). In the age groups, mice had seroconversion rates of 83%, 100%, 100%, 100%, 50%, and 17% for 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μg dosages, respectively. In the vodka groups, mice had lower seroconversion rates of 67%, 50%, 33%, 17%, and 0% for 1,000, 500, 250, 125, or 62.5 pfu dosages, respectively. The ED50 values were 0.063 μg for tgE and 449.425 pfu for vOka, indicating that the immunogenicity of tgE at a lower dosage of 0.3 μg was equivalent to that achieved with a single dose of varicella vaccine containing vOka (usually over 2,000 pfu per dosage).

Figure 2 Characterization of tgE and rgE proteins. A, HPSEC profiles of the purified tgE. The retention time of gE is indicated. B, Sedimentation velocity analysis of the purified tgE protein. The sedimentation coefficient and molecular weight of tgE were determined by c(s) and c(M) methods independently. In (A) and (B), tgE is shown in black and red in blue. C, Differential scanning calorimetry profiles of the purified tgE and rgE protein.

Figure 3 Reactivities of tgE and rgE with mAbs. mAbs were serial diluted and reacted with coated tgE or rgE proteins in an ELISA assay. Reactions were detected using HRP-labeled anti-mouse antibodies. Duplicate wells were measured on the same plate for each dilution. EC50 values were calculated by sigmoid trend fitting using GraphPad Prism software.

Figure 4 Immunogenicity of tgE and rgE tested in BALB/c mice 1 week after three-dose immunization. A, Antibody production in mice for tgE and rgE formulated with different adjuvants. B, Neutralizing antibody titers in mice for tgE and rgE formulated with different adjuvants. C and D, Evaluations of gEspecific IFN-γ and IL-2 responses induced by tgE or rgE vaccines.

Figure 5 Immunogenicity tests of aluminum-formulated tgE vaccine in BALB/c mice. A and B, Antibody persistence and neutralizing antibody response of tgE/Al-001. Titers were monitored for up to 16 weeks. C, ED50 in mice for aluminum-formulated tgE and vodka.

Evaluation of tgE Zoster vaccine candidate compared with Shingrix 

To compare the immunogenicity of our tgE vaccine with Shingrix, lyophilized tgE protein was formulated with the Shingrix adjuvant, AS01B. C57BL/6 mice were immunized with two doses of tgE/AS01B or Shingrix intramuscularly in the tibialis muscle at weeks 0 and 4. The antibody levels after the first immunization suggested that immunization with Shingrix was sufficient to induce high levels of anti-gE antibodies, significantly higher than those produced by tgE/ AS01B; after the second immunization, however, there was no significant difference in antibody production between the two groups (Figure 6A). As to the neutralizing antibody levels, a single-dose immunization of tgE/AS01B led to the production of negligible amounts of neutralizing antibody, significantly lower than that produced by Shingrix. However, again, after the booster immunization, we could see no significant difference in terms of neutralizing antibody production between the two groups (Figure 6B). To verify how immunization affects CMI, gE-specific IFN-γ, and IL-2 levels were detected after the first and the second immunizations using the enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining. The tgE/AS01B immunization induced a poor humoral immune response as compared with a single dose of Shingrix but showed comparable potency in terms of inducing IFN-γ- and IL-2-producing splenocytes (Figure 6C). Indeed, after the second immunization, a significant number of gE-specific cytokine-producing cells were detected in the tgE/AS01B group as compared with the saline control group, which were also comparable to or better than that for the Shingrix group (Figure 6D). Similar results were observed in the flow cytometry analysis, with observations of similar proportions of IFN-γ-expressing CD4+ T cells (Figure 6E), IL-2-expressing CD4+ T cells (Figure 6F), and IFN-γ-CD8+ T cells (Figure 6G) between the tgE/AS01B- and Shingriximmunized mice, regardless of a single or boost-dose immunization. These results demonstrate that the tgE protein derived from the bacterial expression system can induce both humoral and cellular immune responses when formulated with appropriate adjuvants, and thus may be considered as a candidate antigen for the generation of a vaccine against VZV.

Figure 6 Immunogenicity of tgE in C57BL/6 mice. Doses of 5 μg tgE formulated with 50 μL AS01B adjuvant were administered. This was 1/10 the dose of the Shingrix vaccine. Immunization was performed at weeks 0 and 4. A, Comparison of antibody production induced by tgE/AS01B vaccine compared with Shingrix. Serum titers were determined by ELISA assay using rgE-coated plates. B, Comparison of the neutralizing antibody response to vOka following vaccination with tgE/AS01B vaccine or Shingrix. The neutralization titers of antiserum at week 2 (left panel) and week 6 (right panel) are expressed as the reciprocal of the serum dilution resulting in 50% inhibition. C, Evaluation of gE-specific IFN-γ and IL-2 responses induced by tgE/AS01B vaccine or Shingrix at week 2. Numbers of IFN-γ- and IL-2-producing splenocytes were calculated from the ELISPOT assay after restimulation with a pool of gE peptides. D, Evaluation of gE-specific IFN-γ and IL-2 responses induced by tgE/AS01B vaccine or Shingrix at week 8. Numbers of IFN-γ- and IL-2-producing splenocytes were calculated from the ELISPOT assay after restimulation with a pool of gE peptides. E–G, Flow cytometry assays for gE-specific cytokine-expressing CD4+ and CD8+ cells. The proportion of IFN-γ-producing CD4+ T cells, IL-2-producing CD4+ T cells, and IFN-γ-producing CD8+ T cells among splenocytes was determined.

DISCUSSION 

The VZV gE is a major target of the humoral and cell-mediated immune response during natural varicella infection and following VZV Oka vaccination. Shingrix (GSK) is a recombinant subunit vaccine comprising a VZV gE expressed in CHO cells and the liposome-based adjuvant AS01B. Other work has sought to investigate subunit vaccines against HZ using gE proteins generated from CHO or insect cells formulated with novel adjuvants (Cao et al., 2021; Lee et al., 2020; Luan et al., 2022a, Luan et al., 2022b; Wang et al., 2021; Wui et al., 2019; Wui et al., 2021). Here, we investigated and showed that the gE protein could be solubly expressed and purified from E. coli, and then formulated as a subunit vaccine candidate with an appropriate adjuvant to elicit not only a robust neutralizing titer but also a strong CMI response.

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E. coli as an expression system offers several advantages: rapid cell growth rate, inexpensive culture conditions, and an efficient and versatile tool for producing recombinant proteins. Herpes virus-associated antigen research has had several successes using the E. coli-based expression system. A neutralizing linear epitope gE (aa 121–135) has been fused with 149 aa of the hepatitis B virus core (HBc) protein to produce chimeric VLPs that could induce VZV-specific neutralizing antibodies in mice (Zhu et al., 2016). In another study, a homologous gE protein from Simian varicella virus (SVV) was expressed in E. coli utilizing a GST fusion tag for generating gE antiserum (Gray et al., 2001). As yet, there have been no reports of a VZV gE protein without a fusion tag purified from E. coli as a vaccine candidate.

gE consists of three regions: a 544-aa hydrophilic ectodomain with a signal peptide (aa 1–30), a 17-aa hydrophobic transmembrane region, and a 62-aa cytoplasmic tail region. We investigated several candidate molecules with different truncations at the N- or C-terminal regions of the gE ectodomain and found that the exogenous expression of these proteins in E. coli was best using the truncated portion (data not shown). An intact gE ectodomain without a fusion tag tends to form inclusion bodies when expressed by E. coli and is difficult to purify. Among the truncation candidates tested, gE (aa 31–358) protein showed high soluble expression with minimal difficulties in its purification, as well as high purity and high homogeneity (Figure 1), Thus, this truncation construct was deemed as an ideal candidate vaccine antigen. This truncation strategy could have had a potential effect on the properties of the protein, particularly its antigenicity. We had previously expressed recombinant gE in insect cells and purified this construct to immunize BALB/c mice, obtaining 70 gE monoclonal antibodies for epitope analysis (Liu et al., 2015). Through our analysis, we identified aa 1– 194 as the immunodominant region (data not shown). Similar results were obtained elsewhere, with gE-specific mAbs pointing to aa 109–123 and 160–316 regions being the regions responsible for antibodies stable antibody binding among variant isolates (Vafai, 1994). In another study, using recombinant hybrid gE fragment-VLPs, the authors identified residues 1–134 as the most antigenic region of gE protein and showed that the C-terminal of gE (residues 303– 623) containing several cysteine residues failed to produce particles in yeast (Fowler et al., 1995), suggesting that heterologous expression of gE should consider not only the antigenicity but also the ease of expression. Here, we optimized the length of the gE protein for its efficient soluble expression with minimal reductions in antigenicity. The tgE showed similar reactivity to most of the antibodies in Figure 3 as did rgE produced in insect cells, suggesting that key epitopes are well preserved in the tgE protein purified from E. coli. However, one study showed that the immunodominant CD4+ T cell epitopes recognized by Shingrix vaccine donors were dispersed throughout the gE protein (Voic et al., 2020). The immunogenicity of the tgE in our study was identical to that found for gE derived from CHO cells in Shingrix when antigen combined with AS01B adjuvant, with similarly efficient humoral responses and CMI responses, as shown in Figure 5. These results indicated that the truncated gE derived from the prokaryotic system was as good as the intact gE ectodomain from the eukaryotic system in terms of antigenicity.

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There are considerable differences between gE proteins derived from different expression systems. The most prominent differences are noted with posttranslational modifications, particularly glycosylation. O-linked glycans from membrane glycoproteins have relevance in terms of T-cell and antibody recognition of viral glycopeptide epitopes, and this recognition has the potential of high biomedical significance in our attempts to mediate immune protection against viruses using subunit vaccines. Removal of the glycans in gE produced in CHO-K1 cells resulted in a 17% reduction in reactivity with VZV-positive sera. In contrast, O-glycosylation may interfere with the immunoreactivity of B cell epitopes: some important epitopes are blocked by additional O-linked glycans (Nordén et al., 2019). In our study, the tgE generated from E. coli seems to behave as well as the insect cell-derived rgE in terms of its immunoreactivity and immunogenicity, suggesting that the "naked" tgE protein has promise as a vaccine candidate. Vaccines with tgE as the antigen are likely to induce not only high IgG titers, indicative of its potential as a subunit varicella vaccine without a potential risk of virus latency, but also high CMI, suggestive of its potential as a zoster vaccine as well. Overall, the gE antigen derived from the bacterial expression system in this study offers a much more economical strategy for the development of VZV vaccines.

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