What this is
- The study evaluates modified mRNA vaccines designed to protect against () in immunocompromised AG129 mice.
- Two vaccine candidates, ZA and ZB, were tested for their ability to induce immune responses and prevent viral infection.
- The ZA vaccine provided complete protection against lethal challenges, while ZB did not achieve full viral clearance.
Essence
- The modified mRNA ZA vaccine protects AG129 mice from lethal infection and multi-tissue viral dissemination, while also mitigating () of dengue virus (DENV) infection.
Key takeaways
- The ZA vaccine induced high levels of T cells secreting IFN-γ and provided complete protection against in AG129 mice.
- The ZA vaccine reduced the effect of DENV infection, demonstrating its potential to prevent complications associated with vaccination.
- The ZB vaccine showed good immunogenicity but failed to achieve complete viral clearance in AG129 mice.
Caveats
- The study's findings are based on AG129 mice, which may not fully represent immune responses in humans or other animal models.
- The long-term durability of the vaccine-induced immunity and its efficacy against vertical transmission of require further investigation.
Definitions
- Zika virus (ZIKV): A mosquito-borne virus linked to severe birth defects and neurological disorders in humans.
- Antibody-dependent enhancement (ADE): A phenomenon where pre-existing antibodies facilitate viral entry into cells, leading to increased infection severity.
Simplified
Introduction
The 2016 Zika virus (ZIKV) epidemic in Brazil garnered global attention due to its large-scale transmission [1]. Initially discovered in 1947 in Uganda's Sentinel Rhesus monkeys, ZIKV infection was considered a mild and sporadic disease for decades [2,3]. Phylogenetic analyses indicate that ZIKV has evolved into two lineages, Asian and African [4]. Notably, ZIKV has appeared the evolutionary enhancement of its infectivity in Aedes aegypti mosquitoes [5]. In addition, the effects of ZIKV on humans during its transmission suggest that the K101R substitution in the C protein or the S139N substitution in the prM protein may increase its infectivity and pathogenicity [6,7]. Clinical evidence in recent years demonstrated ZIKV infection was associated with Guillain–Barré syndrome in adults and congenital malformations in infants [8–10]. Although global ZIKV cases have declined since 2017, low-level transmission persists in several countries in the Americas and other endemic regions [11]. Therefore, we should attach importance to the research on ZIKV.
ZIKV is a mosquito-borne orthoflavivirus with a genome organization and protein structure similar to dengue virus (DENV) [12]. Its single-stranded positive RNA genome encodes three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) [13]. The E protein mediates viral attachment and entry by interacting with host cell receptors [14]. Upon infection, viral RNA is released into the cytoplasm, initiating the biosynthesis of viral polyproteins, which are subsequently cleaved into structural and nonstructural proteins. The NS5 protein acts as RNA-dependent RNA polymerase (RdRp) to generate offspring RNA [15]. In the cytoplasm, the C protein first assembles with offspring RNA into the viral RNA-protein complex. Then, the complex enters the endoplasmic reticulum lumen and obtains the lipid membrane with prM and E protein to form immature ZIKV (immZIKV) [16]. The surface of immZIKV is covered with 180 copies of prM-E heterodimers. The prM-E dimers undergo a low-pH mediated rearrangement at the trans-Golgi network, where the prM protein is cleaved, resulting in the formation of mature infectious ZIKV [16,17]. The surface of the mature virion is covered with 180 copies of M-E heterodimers. It is noteworthy that E protein is the main target antigen of neutralizing antibodies [18].
Currently, there are no specific treatments for ZIKV infection, making vaccination the most effective strategy for prevention and control. Previous studies have defined a single ZIKV serotype and indicated that infection or vaccination with a single ZIKV strain can provide immune protection against multiple strains [19]. According to previous studies of immunity to ZIKV, prM and E proteins are often selected as target antigens of ZIKV vaccines [20–23]. However, an important safety concern for ZIKV vaccine is the antibody-dependent enhancement (ADE) effect between ZIKV and its antigenically related dengue virus (DENV). The conserved fusion loop epitopes (FLEs) in their E protein could induce poorly neutralizing cross-reactive antibodies, which are considered the source of ADE. To address this, we designed two antigen sequences, ZA and ZB, introducing amino acid mutations to stabilize E protein dimers or disrupt the FLE. The latter has already been proven effective in reducing the ADE effect in the mRNA vaccine platform [24]. In the subunit vaccine platform, the former could abrogate antibody-dependent enhancement of dengue infection [25]. Given the timely and effective response of mRNA vaccines to epidemic outbreaks, we chose the mRNA-lipid nanoparticle (LNP) platform to evaluate the safety and efficacy of the ZIKV vaccine.
Materials and methods
Animals, ethics and biosafety statement
All female C57BL/6 mice (aged 4∼6 weeks) were obtained from the Institute of Medical Biology, Chinese Academy of Medical Sciences (Kunming, China; Manufacturing licence: SYXK (DIAN) K2022-0006). Male and female AG129 mice (aged 4∼6 weeks) were obtained from the National Institutes for Food and Drug Control (NIFDC; Manufacturing licence: SCXK (JING) 2022-0002). All animal experiments were approved by the Institutional Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Science (Ethics number: DWSP202108008). In this study, AG129 mice are the engineered C57BL/6 mice with the double-knockout of α/β and γ interferon receptors.
Generation of modified mRNA and lipid nanoparticles (LNPs)
The mRNA was synthesized in vitro using the HiScribe® T7 mRNA Kit (NEB, E2080S), with UTP replaced by N1-Methyl-Pseudo-UTP (Novoprotein). The modified mRNAs incorporate a poly-A tail and 5′ and 3′ untranslated regions (UTRs) derived from the Pfizer-BioNTech coronavirus vaccine (BNT162b2). Additionally, the mRNAs encoded the signal peptide from human IgE (MDWTWILFLVAAATRVHS) and the prM and E proteins. We aligned over 300 amino acid sequences of ZIKV strains from Asian and African lineages for the conserved region. To reduce or eliminate the production of antibodies that enhance dengue infection, three mutations were introduced in the E protein of ZA (L107C, A264C, A319C) and four mutations in that of ZB (T76R, Q77E, W101R, L107C). We chose the conserved sequence without mutations as a control.
LNP was prepared following the Moderna coronavirus vaccine protocol. Lipids were dissolved in ethanol at a concentration of 12 mM at a molar ratio of 50:10:38.5:1.5 (SM102: DSPC: cholesterol: DMG-PEG-2000). Then, the lipid mixture was mixed with a 50 mM citrate buffer (pH 4.0) containing mRNA at an N/P ratio of 8:1 (lipids: RNA) using a microfluidic mixer (FluidicLab). The LNP formulations were then ultrafiltered and concentrated in 20 mM Tris-HCl buffer. All formulations were characterized for particle diameter (90–110 nm) and encapsulation efficiency (≥95%).
Transfection and protein expression analysis
To detect the protein expression, Vero cells were transfected with the mRNA using MessengerMAX reagent (Thermo, LMRNA003). At 12 and 24 h post-transfection, cells were lysed with RIPA buffer for Western Blot analysis. The PVDF membrane was blotted with ZIKV polyclonal prM antibody (GeneTex, GTX133305), ZIKV polyclonal E antibody (GeneTex, GTX133314) and orthoflavivirus monoclonal 4G2 antibody (GeneTex, GTX57154) at a dilution ratio of 1:1000. Meanwhile, we chose HRP-conjugated secondary antibodies (Affinity bioscience) for Western Blot.
Immunization and ZIKV challenge
We adopted a prime-boost vaccination strategy in C57BL/6 and AG129 mice. There were five female C57BL/6 mice (n = 5) and six male/female AG129 mice (n = 6) in each group. Mice were immunized intramuscularly with 2.5 and 5.0 μg of mRNA-LNPs. Booster immunizations were administered 21 days after the prime vaccination. Twenty-eight days post-boost, all AG129 mice were challenged subcutaneously with 103 TCID50 of ZIKV strain GZ01 (GenBank: KU820898). Mice were sacrificed eight days post-challenge.
Viral load
Viral RNA was quantified by RT-qPCR using TaqMan Fast Viral 1-Step Master Mix (Thermo Fisher, 4444432) on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA). Primers and probe targeting the ZIKV prM-E gene were used: Forward primer (5′-TTGGTCATGATACTGCTGATTGC-3′), reverse primer (5′-CCTTCCACAAAGTCCCTATTGC-3′) and probe (5′-FAM-CGGCATACAGCATCAGGTGCATAGGAG-BHQ1-3′). Primers and probe targeting the DENV-1 gene were used: Forward primer (5′-CGAAGCCAAAGAGGGACTAAA-3′), reverse primer (5′-TACAAGGTTCCTCTCCACAAAC-3′) and probe (5′-FAM-TTTAGCGGTTCCTCTCGACACTGC-BHQ1-3′). The reaction system consisted of 2.5 μL qPCR mix, 0.5 μL probe, 0.5 μL forward primer, 0.5 μL reverse primer, 2.5 μL RNA sample, and 3.5 μL water. The reaction conditions were: 50°C for 5 min, 95°C for 20 s, and 40 cycles of 95°C for 3 s and 60°C for 30 s. Viral copy numbers were calculated based on standard curves.
In vitro ADE assay
Serum from C57BL/6 mice was inactivated at 56°C for 30 min. Serum from the same vaccine group was pooled and diluted with DMEM at a ratio of 1:32. DENV or ZIKV, at a multiplicity of infection (MOI) of 0.1, was incubated with diluted serum or 4G2 antibody at 37°C for one hour. The serum-virus mixture was then added to 5×10⁴ K562 cells and incubated at 37℃ for three days. After cells were centrifuged and washed twice with PBS, we detected viral infectivity by One Step TB Green RT-PCR kit (Takara, RR096A). Primers targeting the DENV-1 gene were used: Forward primer (5′-CAAAAGGAAGTCGTGCAATA-3′), reverse primer (5′-CTGAGTGAATTCTCTCTACTGAACC-3′). Primers targeting the DENV-2 gene were used: Forward primer (5′-ACAAGTCGAACAACCTGGTCCAT-3′), reverse primer (5′-GCCGCACCATTGGTCTTCTC-3′). The reaction system consisted of 6.25 μL PCR buffer, 0.75 μL HS Mix, 0.25 μL RTase Mix, 0.5 μL forward primer, 0.5 μL reverse primer, 2.25 μL water, and 2 μL RNA sample. The reaction conditions were: 42°C for 5 min, 95°C for 10 s, and 40 cycles of 95°C for 5 s, 55°C for 30 s and 72°C for 30 s. Viral relative infectivity was calculated based on the following method: normalization RFU = RFUDENV/RFUActin; Relative infectivity = norRFUvaccine/norRFUPBS.
In vivo ADE assay
Serum from C57BL/6 mice was inactivated at 56°C for 30 min. Groups (n = 3) of AG129 mice were administered 10 μg mAb 4G2 or 20 μl immune serum (in 200 μL 1× PBS) intraperitoneally, and challenged subcutaneously 2 h later, with DENV-1 (105 TCID50/mouse).
Passive immunization
We collected immune serum from C57BL/6 mice 21 days after booster vaccination for passive transfer studies. Pooled mouse immune serum was transferred into groups of 3 AG129 mice at 200 μL per mouse using the intraperitoneal route of administration without dilution. The serum from C57BL/6 mice of the PBS group was used as a control. Within 1 h post-transfer of immune serum, mice were challenged with 103 TCID50 of ZIKV strain GZ01 (in 100 μL 1× PBS) subcutaneously. All mice were euthanized when the control group died of viral infection.
Data processing and statistics
Statistical analysis was performed using GraphPad Prism (8.0.2). The following statistical tests were used in this study: Dunnett's multiple comparisons test and two-way ANOVA. Quantitative data were presented as mean ± SD. P values < 0.05 were considered significant, with significance levels denoted as follows: NS, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.
Results
mRNA design and in vitro protein expression
Design of ZIKV mRNA vaccines and in vitro expression. (A) Schematic illustration of ZIKV antigen sequences design and mRNA-LNPs (created with BioRender.com). (B) Results of structure prediction of prM and E proteins from ZA, ZB, and WT sequences (created with AlphaFold 3). (C) Agarose gel electrophoresis maps of linearized plasmids and transcribed mRNAs (5000 bp DNA marker). (D) Western-blot results of cellular protein expression of ZA, ZB, and WT mRNAs in Vero cells. (E) Particle size distribution of freshly prepared mRNA-LNPs
mRNA vaccines induced humoral and cellular immune responses in female C57BL/6 mice
The immunogenicity of ZIKV mRNA vaccines in female C57BL/6 mice. (A) The timeline of vaccine immunization and sampling in female C57BL/6 mice (created with BioRender.com). (B) E protein-specific binding antibody levels at each time point after immunization in each group. Data are presented as mean ± SD (error bars). Two-way ANOVA: NS, not significant, > 0.05; * < 0.05; ** < 0.01; *** < 0.001; and **** < 0.0001. (C) The result of T cells secreting IL-2 and IFN-γ in each group at 21 days after booster immunization (n = 3). Data are presented as mean ± SD (error bars). Two-way ANOVA: NS, not significant. (D) The GMT of neutralization antibodies in each group at 21 days after booster immunization against GZ01, MR766, FSS13025, and PRVABC59 (n = 5). P P P P P
mRNA vaccines protected male and female AG129 mice from lethal ZIKV challenge at low levels of neutralizing antibodies
ZIKV mRNA vaccines protect male/female AG129 mice from lethal ZIKV challenge at low levels of neutralizing antibodies. (A) The timeline of vaccine immunization and ZIKV challenge in male/female AG129 mice (created with BioRender.com). (B) The GMT of neutralization antibodies of male/female AG129 mice in each group at 21 days after booster immunization against GZ01 (n = 6). (C) Weight change of male/female AG129 mice in each group after ZIKV challenge (n = 6). (D) Viral load in the blood of male/female AG129 mice in each group after the ZIKV challenge (n = 6). (E) Changes in survival rate of male/female AG129 mice in each group within 8 days of ZIKV infection (n = 6).
ZA vaccine protected male and female AG129 mice from multi-tissue infection by ZIKV
ZIKV mRNA vaccines protect male/female AG129 mice from multi-tissue infection by ZIKV. (A) Viral load in the heart, liver, spleen, lung, kidney, cerebrum, cerebellum, spinal cord, and uterus of female AG129 mice in each group (n = 3) at 8pi. (B) Viral load in the heart, liver, spleen, lung, kidney, cerebrum, cerebellum, spinal cord, and testicle of male AG129 mice in each group (n = 3) at 8 dpi. (C) Detection of infectious Zika virus in heart, liver, spleen, lung, kidney, cerebrum, cerebellum, spinal cord, and uterus of female AG129 mice in each group (n = 3) at 8 dpi. (D) Detection of infectious Zika virus in heart, liver, spleen, lung, kidney, cerebrum, cerebellum, spinal cord, and testicle of male AG129 mice in each group (n = 3) at 8 dpi.
mRNA vaccine reduces pathological lesions caused by ZIKV challenge in male and female AG129 mice
ZIKV mRNA vaccines could alleviate pathological lesions of the heart, kidney, lung, cerebrum, uterus or testicle of AG129 mice at 8 days post-ZIKV challenge (n = 3). The scale bar represents 200 μm.
Immune serum showed reduced ADE activities
ZA and ZB vaccines reduced the enhancement of DENV infection. (A) Enhancing effects of ZIKV immune serum on DENV-1, DENV-2, and ZIKV infection on K562 cells. (B) Enhancing effects of ZIKV immune serum on DENV-1 infection in AG129 mice. Data are presented as mean±SD (error bars). Dunnett's multiple comparisons test with PBS group as the control: NS, not significant, > 0.05; * < 0.05. P P
Passive transfer of immune serum afforded partial protection against lethal ZIKV challenge
Passive transfer of immune serum afforded partial protection against lethal ZIKV challenge. (A) The timeline of passive transfer of immune serum in female AG129 mice (created with BioRender.com). (B) Weight change of female AG129 mice in each group after ZIKV challenge (n = 3). (C) Viral load in the blood of female AG129 mice in each group after ZIKV challenge (n = 3). (D) Viral load in the heart, liver, spleen, lung, kidney, cerebrum, cerebellum, spinal cord, and uterus of female AG129 mice in each group at 8 dpi (n = 3). Data are presented as mean±SD (error bars). Dunnett's multiple comparisons test with PBS group as the control: NS, not significant, > 0.05; * < 0.05; ** < 0.01; *** < 0.001; and **** < 0.0001. P P P P P
Discussion
In this study, we evaluated the protective efficacy of two mRNA vaccines encoding prM and E proteins in male and female AG129 mice. A prime-boost vaccination strategy protected AG129 mice from lethal ZIKV challenge and multi-tissue infection, indicating that our vaccine has the potential to respond rapidly to future ZIKV epidemics. Additionally, our ZIKV vaccines reduced the severity of dengue ADE induced by immunization.
The ZA vaccine (5.0 μg) demonstrated the best protection against ZIKV. Previously published anti-ZIKV vaccines have shown efficacy in mice or non-human primates (NHPs), protecting against viremia, tissue viral load, or lethal challenge [20,22,24,28,29]. Two ZIKV mRNA vaccines have entered Phase I trials, mRNA-1325 and mRNA-1893 from ModernaTX [23]. In mice experiment, mRNA-1325 showed poor immunogenicity. Although mRNA-1893 showed superior immunogenicity and efficacy compared with mRNA-1325 in rhesus macaques, both of them had no published experimental data about ADE evaluation. Additionally, ZIKV infection in mice or NHPs often leads to viral persistence in multiple tissues [30,31]. Unlike previous studies, which primarily detected viral infection in the blood or a few tissues, our study provided a more comprehensive analysis across nine tissues. The results demonstrated that the ZA vaccine (5.0 μg) achieved complete clearance of viral infection in AG129 mice.
It is noteworthy that ZA or ZB vaccines in AG129 mice produced higher levels of nAbs than those in C57 mice. This discrepancy may be attributed to the immune deficiency of AG129 mice. Compared to WT vaccine, ZA and ZB vaccine induced significantly lower levels of nAbs. The possible explanation is the substitution of amino acids in vaccine design, which caused insufficient exposure of neutralizing epitopes. When challenged with a lethal dose of ZIKV, AG129 mouse model exhibited sex-based differences in protection efficacy, particularly at the ZA vaccine (2.5 μg) and ZB vaccine (2.5 μg). Females exhibited the complete viral clearance, possibly due to estrogen-enhanced immunity or stronger Th2-type immune responses [32,33]. Additionally, the ZA vaccine (5.0 μg) only partially alleviated pathological lesions in certain tissues. According to our previous findings [31], ZIKV infection triggers a massive release of cytokines in AG129 mice, suggesting that pathological damage may be linked to excessive immune cell activation following ZIKV challenge.
To better understand the humoral responses and correlates of protection induced by the mRNA vaccines, we performed passive protection studies in AG129 mice. The results demonstrated that passive transfer of immune serum could not protect AG129 mice from lethal ZIKV infection. The main reason for this phenomenon might be that T-cell immunity of the vaccine played a major antiviral protective role [34,35]. In C57BL/6 mice, ZA vaccines induced higher levels of T cells secreting IFN-γ by ELISpot assay than ZB vaccines. In this study, passive immunization could only provide low levels of nAbs in immune serum, without the cooperation of T cell immunity. During active immunity, ZIKV-specific T cell responses could compensate for low levels of humoral immunity. Additionally, passive antibody injection, without IFN signal amplification, finds it difficult to suppress viral infection in AG129 mice.
The ZA and ZB vaccines showed reduced ADE in vitro. Previous studies have described that certain mutations, such as W101R, in ZIKV vaccines significantly reduced the ADE of dengue infection [24,25]. However, these mutations often compromise neutralizing antibody titres and provide incomplete protection against viremia and tissue infection [24,25]. The W101 mutation disturbed E-dimer formation and the E-dimer epitope (EDE), which is targeted by potent ZIKV nAbs [25,36]. Maintaining the E-dimer conformation is likely critical for vaccine efficacy. Therefore, we introduced three disulphide bonds in the ZA antigen. Although the ZA vaccine (5.0 ug) also induced low nAb levels in mice, it provided complete protection and prevented multi-tissue infection in AG129 mice without enhancing dengue infection. Although the NS1-based vaccine could avoid ADE, the viral clearance efficiency in the mouse model is significantly lower than that of prM-E vaccine [37,38]. However, the ZA mRNA vaccine has some limitations. First, the protection efficacy has only been tested in AG129 mice. Although AG129 mice have been used extensively for the evaluation of short-term protection efficacy of ZIKV vaccine, they could not fully simulate the pathological processes of immunocompetent hosts. Since non-human primates (NHPs) are the natural hosts of ZIKV, further evaluation in NHPs is necessary. Second, maternal ZIKV infection during pregnancy is associated with congenital malformations in the fetus [39], so it is essential to determine whether the vaccine could block vertical transmission of ZIKV. Finally, we only reported the vaccine's short-term protection. The durability of vaccine-induced immunity requires further investigation. Future studies will address these issues. In conclusion, the modified mRNA vaccine elicited sufficient immunity, protecting against ZIKV infection and disease in AG129 mice.