
==== Front
Emerg Microbes Infect
Emerg Microbes Infect
Emerging Microbes & Infections
2222-1751
Taylor & Francis

39137287
2392661
10.1080/22221751.2024.2392661
Version of Record
Research Letter
Research Letter
Rational optimization of glycoprotein E (gE)-encoding mRNA for improved Varicella-zoster virus mRNA vaccine development
EMERGING MICROBES & INFECTIONS
L. HUANG ET AL.
Huang Lulu ab*
Zhang Shun c*
Zhao Tongyi ab*
Cai Ting c*
Bu Lingling ab
Di Zhenhua ab
Zhang Yujie ab
Yang Chen d
Yang Yong abe
Lin Ang abf
a Vaccine Center, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China
b Center for New Drug Safety Evaluation and Research, China Pharmaceutical University, Nanjing, People’s Republic of China
c Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo, People’s Republic of China
d School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, People’s Republic of China
e School of Pharmacy, Xuzhou Medical University, Xuzhou, People’s Republic of China
f School of Pharmaceutical Sciences, Institute of Immunopharmaceutical Sciences, Shandong University, Jinan, People’s Republic of China
CONTACT Ang Lin anglin@cpu.edu.cn Vaccine Center, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing 211198, People’s Republic of China; Center for New Drug Safety Evaluation and Research, China Pharmaceutical University, Nanjing 211198, People’s Republic of China; School of Pharmaceutical Sciences, Institute of Immunopharmaceutical Sciences, Shandong University, Jinan 250012, People’s Republic of China
* Authors contributed equally.

Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2024.2392661.

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https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

mRNA platform holds promise for next-generation Varicella-zoster Virus (VZV) vaccine development due to its high potency at inducing strong T-cell response. Built upon the design of our 1st-generation VZV mRNA vaccine that encodes for full-length gE antigen, in this study we reported on a novel combinatorial strategy to further optimize the gE-encoding mRNA sequence through signal peptide replacement, C-terminal modification, and insertion of mRNA-stabilizing motif, which collectively contributed to significantly improved vaccine immunogenicity. In adult mice, aged mice, and immunocompromised mice, this optimized VZV mRNA vaccine showed strong superiority in multiple aspects including the induction of gE-specific antibodies, specific memory B-cell response, as well as Th1-type T-cell response.

KEYWORDS

Varicella-zoster virus
mRNA vaccine
mRNA optimization
immunogenicity
T cell response
Natural Science Foundation of Jiangsu Province 10.13039/501100004608 BK20221031 National Science Foundation of China 10.13039/501100001809 32200764 Fundamental Research Funds for the Central Universities 10.13039/501100012226 This work was supported by the Natural Science Foundation of Jiangsu Province (BK20221031 to A.L.), the National Science Foundation of China (32200764, to A.L), the Fundamental Research Funds for the Central Universities (2632022YC01, to A.L.). We thank the Core Facility of China Pharmaceutical University for instrumental and technical support on this study.
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pmcThe past decades have seen increasingly rapid advances in the field of mRNA technology and its successful applications in prophylactic vaccine development [1,2]. Recently, we reported on the development of a novel Varicella-zoster virus (VZV) mRNA vaccine (named as ZOSAL) that contains mRNAs encoding for full-length gE immunogen (623 aa) encapsulated into a novel lipid nanoparticle (LNP) system [3]. In mice and rhesus macaques, ZOSAL induced superior virus-specific immunity over licensed subunit vaccine Shingrix, which potentiated the power of mRNA platform in next-generation VZV vaccine development [3].

However, the mRNA cargo is versatile and can be manipulated to increase its stability, translational efficiency, and more fundamentally, to alter the structure or cellular compartmentalization of the expressed antigens in a rationally designed format, which may contribute to improved vaccine efficacy [4]. To this end, we optimized the mRNA cargo of our 1st-generation vaccine (ZOSAL) using different strategies and generated four optimized mRNA sequences (Figure 1a), that is, ZOSAL-B that contains a signal peptide (SP) of kappa chain of human immunoglobulin (Igκ) in replacement of the original SP, ZOSAL-BQ that is inserted with a 21-mer motif (Exin 21) at the 3’-end of ZOSAL-B which was previously reported to increase mRNA stability [5], ZOSAL-BM that encodes for a C-terminal truncated form of gE depleted of 50 amino acids in combination with SP replacement and a site mutation (Y569A) to avoid trans-Golgi network (TGN) targeting thus facilitating antigen location on cell membrane [6], ZOSAL-BMQ that is modified on the basis of ZOSAL-BM with a Exin 21 motif insertion at the 3’-end. Figure. 1. Optimized VZV mRNA vaccine shows significantly improved vaccine immunogenicity. (a) Design of VZV gE-encoding mRNA sequences.(b) Representative images of immunofluorescence staining of VZV gE (green), Golgi membrane-related protein GM130 (red) in HEK-293 T cells upon transfection with different gE-mRNAs. Nuclei were stained using DAPI (blue). Scale bars: 40 μm (upper panel), 10 μm (lower panel). Cells not transfected with mRNA were used as negative control (NC). (c) 6-week old C57BL/6 mice (n = 6) were immunized i.m. with two doses of 1-µg or 5-µg mRNA vaccine candidates or PBS on day 0 and day 14. (d) Endpoint titer of anti-gE IgG was measured by ELISA on day 7 and day 28. (e) Frequencies of class-switched (IgD-IgM-) gE-specific MBCs in spleens from the PBS and 5-µg dosing groups were assessed by flow cytometry. (f) Frequencies of IFN-γ or IL-2-secreting T cells were measured by ELISpot. (g-h) 16-month old C57BL/6 mice (n = 6) or HBV-carrier mice (n = 6) were immunized i.m. with two doses of 5-μg ZOSAL-BMQ or 0.1 human dose of Shingrix at a 2-week interval. Splenocytes were stimulated with gE overlapping peptides pool (10 μg/ml) for 8 h in the presence of Brefeldin A. Frequencies of IFN-γ, IL-2 or TNF-secreting CD4+ T cells were analysed by flow cytometry. Data are shown as mean ± SEM. One-way ANOVA was used for statistical analysis in figure d-f. Mann-Whitney U test was used for statistical analysis in figure g-h. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

We first assessed translation of the different mRNAs and the cellular location of the translated proteins in vitro. Upon transfection into HEK-293 T cells, all the mRNAs were efficiently translated into gE antigens (Figure 1b). Notably, antigens expressed by the mutated ZOSAL-BM and ZOSAL-BMQ mRNAs were largely localized on plasma membrane. ZOSAL, ZOSAL-B and ZOSAL-BQ mRNAs-translated antigens were distributed in both plasma membrane and cytoplasm co-localizing with Golgi apparatus (Figure 1b), which was also seen in HepG2 cells following mRNA transfection (Figure S1a). The Y569A site mutation clearly altered cellular distribution of gE antigen resulting in cell membrane enrichment. This was further confirmed by detection of surface gE antigen using flow cytometry (Figure S1b).

Next, we prepared five different mRNA vaccine candidates using a novel ionizable lipid-based LNP delivery system [3,7] and evaluated their immunogenicity in C57BL/6 mice administered with two doses of 1-µg or 5-µg vaccines at a two-week interval (Figure 1c). 7 days after the prime dose, ZOSAL-BM and ZOSAL-BMQ induced a significantly higher level of anti-gE IgG than other vaccine candidates (Figure 1d). The Ab responses were further boosted following the 2nd dose in all vaccine groups, which was more prominent in the ZOSAL-BMQ group. Moreover, ZOSAL-BMQ elicited the highest frequencies of class-switched IgD-IgM- gE-specific memory B cells (MBC) in spleens two weeks after booster (Figure 1e). Considering that T cell responses are dominantly critical to prevent VZV from reactivating [8], we assessed gE-specific T cell responses and found that ZOSAL-BMQ elicited significantly higher levels of IFN-γ or IL-2-secreting T cells than other four vaccine candidates (Figure 1f). We also detected a robust induction of gE-specific AIM+ (activation-induced marker, CD137 and OX40) CD4+ T cells in all vaccinated mice, particularly in the ZOSAL-BMQ group (Figure S2).

Given the fact that herpes zoster occurs mainly in elderly and immunocompromised individuals [9], we next performed comprehensive evaluation of ZOSAL-BMQ and benchmarked it to Shingrix in aged mouse model and chronic HBV infection mouse model. In 16-month old C57BL/6 mice, two doses of 5-µg ZOSAL-BMQ elicited significantly higher frequencies of Th1-type CD4+ memory T cells in spleens when compared with Shingrix (Figure 1g). Moreover, a stronger induction of cytokine-producing CD8+ memory T cells in spleens of BMQ-vaccinated mice was detected (Figure S3). The superior ability of ZOSAL-BMQ over Shingrix in eliciting higher T cell responses in aged mice was further validated by ELISpot assay (Figure S4). We also detected higher levels of anti-gE IgG, Th1-prone IgG2c, and gE-specific MBCs induced by ZOSAL-BMQ than Shingrix (Figure S5a-c). Of note, the higher level of IgG2c elicited by ZOSAL-BMQ was associated with enhanced Fc-mediated Ab functions including Ab-dependent complement deposition (ADCD) and Ab-dependent neutrophil phagocytosis (ADNP) assessed by systems serology assays (Figure S5d).

A pAAV-HBV1.2-transduced HBV carrier mouse model with a systemic immune tolerance status [7] was next used for further assessment of ZOSAL-BMQ (Figure 1h). Consistent with what we have found earlier in aged mice, ZOSAL-BMQ demonstrated remarkably stronger capability in inducing higher levels of Th1-type cytokine-producing T cells, anti-gE IgG and IgG2c, ADCD and ADNP functions, as well as gE-specific MBCs when compared with Shingrix (Figure 1h, S6-7). These data altogether demonstrated that ZOSAL-BMQ outperformed in multiple aspects associated with vaccine immunogenicity over other candidates including the 1st generation vaccine ZOSAL that has previously been shown to induce superior immunity over Shingrix in both mice and rhesus macaques [3].

It remains largely unclear regarding the rationales behind enhanced immunogenicity of ZOSAL-BMQ. We next preliminarily investigated into some aspects of the potential mechanisms, and particularly focused on the in-cell mRNA stability and degree of innate immune activation early after vaccine administration (Figure S8). 36 h after transfection into HEK-293 T and DC2.4 cells, ZOSAL-BMQ mRNA demonstrated much higher in-cell stability than ZOSAL mRNA (Figure S8a), which may potentially play a role in the enhanced vaccine immunogenicity. Moreover, considering that vaccine-specific T cell responses are determined by the degree of antigen presentation by antigen-presenting cells like dendritic cell (DC), we evaluated cDC1 and cDC2 activation in draining lymph nodes and spleens 12 h following vaccine administration. In general, ZOSAL-BMQ vaccine induced a comparable but slightly higher level of DC activation than ZOSAL (Figure S8b), which may partly contribute to the enhanced immunogenicity. However, more in-depth underlying mechanisms await further investigation.

The safety profile of a novel vaccine is undoubtedly a key parameter that should be assessed. We preliminarily assessed the cytotoxicity and safety profiles of ZOSAL-BMQ vaccine. HEK-293 T cells incubated with escalating concentrations of mRNA vaccines for 24 h maintained high viability, which suggested a very limited cytotoxicity (Figure S9a). In mice that were administered with two doses (5μg and 20μg) of ZOSAL or ZOSAL-BMQ vaccines, body weight of mice in all vaccine groups showed normal fluctuation with steady increase and no obvious loss during the period of observation (Figure S9b). Some key serological parameters including serum ALT (alanine transaminase), AST (aspartate aminotransferase), BUN (blood urea nitrogen), CREA (creatinine), as well as the frequencies of major white blood cell subsets were monitored longitudinally. A transient and rapid increase of circulating neutrophils and monocytes, and a rapid decrease of total lymphocytes were noticed 24 h after prime and boost vaccination (Figure S9c), which was in line with previous studies indicating innate immune activation [10,11]. We also noticed a transient increase of serum ALT and AST levels early after vaccination, but both two parameters quickly returned to baseline 7 days after vaccination, which suggested a limited liver toxicity (Figure S9d). In addition, levels of BUN and CREA showed normal fluctuation after vaccination indicating a very limited kidney toxicity. Overall, ZOSAL-BMQ vaccine is well-tolerated and demonstrates comparable safety profiles to our 1st-generation VZV vaccine ZOSAL that was previously reported to have superior safety profiles over licensed vaccine Shingrix [3].

Discussion

mRNA vaccine is a complex and versatile platform that can be rationally manipulated in multifaceted aspects. Built upon the design of our 1st generation VZV mRNA vaccine ZOSAL [3], in this study we reported a novel combinatorial strategy to optimize the gE-encoding mRNA sequence through signal peptide replacement, C-terminal modification, and insertion of mRNA-stabilizing motifs, which collectively contributed to a significantly improved vaccine immunogenicity. However, it remains largely unanswered that how the modifications affect vaccine efficacy from a mechanistic perspective. Previous study indicated that replacement of authentic SP of viral antigen with the one from human Igκ could improve the immunogenicity of mRNA vaccine by affecting protein translation [12]. Insertion of Exin21 motif into the 3’end of mRNA sequence was shown to increase mRNA stability [5]. In addition to the modifications made to increase mRNA stability and translational efficiency, ZOSAL-BMQ contains a Y569A site mutation that resulted in increased antigen enrichment on cell membrane [6]. This likely contributed to the enhanced immunogenicity of ZOSAL-BMQ vaccine since previous studies have reported that membrane-bound mRNA immunogen construct could elicit more potent vaccine responses than soluble immunogen construct [13,14]. Overall, we propose that ZOSAL-BMQ mRNA has an improved stability, translational efficiency and surface-prone expression of translated antigens, which may together contribute to better vaccine efficacy.

This research letter provided solid evidence gained from multiple mouse models showing a remarkably improved vaccine immunogenicity upon three modifications on the VZV gE-encoding mRNA and desirable safety profiles, which we believe will provide critical guidance for improved VZV mRNA vaccine development. However, there are some unanswered questions that remain to be further addressed, such as more in-depth mechanistic investigations into the rationales behind the optimization, durability of vaccine responses, as well as the potential impact of immunization schedule on vaccine efficacy.

Supplementary Material

Revised Supplementary Materials.docx

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

A.L. designed the project. L.H., T.Z., S.Z., T.C., Z.D., L.B. performed experiments and analysis; C.Y., S.Z. and T.C. provided methodological and technical support; A.L., C.Y., Y.Y. and Y.Z. discussed the data; A.L. and L.H. wrote the manuscript. All authors have read and approved the manuscript.

Data availability

All data are available upon reasonable request to the corresponding authors.
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