A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

doi:10.21203/rs.3.rs-89633/v1

Download PDF

Research Article

A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

https://doi.org/10.21203/rs.3.rs-89633/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 03 Feb, 2021

Read the published version in Nature Communications →

Version 1

posted

You are reading this latest preprint version

The rapid expansion of COVID-19 pandemic has made the development of a SARS-CoV-2 vaccine a global health and economic priority. Taking advantages of versatility and rapid development, three SARS-CoV-2 mRNA vaccine candidates has entered clinical trials with a two-dose immunization regimen. However, the waning antibodies response in convalescent patients after SARS-CoV-2 infection and the emergence of human re-infection have raised widespread concern about a short duration of SARS-CoV-2 vaccine protection. Here, we developed a nucleoside-modified mRNA vaccine in lipid-encapsulated form which encoded SARS-CoV-2 RBD, termed as mRNA-RBD. A single immunization of mRNA-RBD elicited both robust neutralizing antibody and cellular response, and conferred a near-complete protection against wild SARS-CoV-2 infection in lungs of hACE2 transgenic mice. Noticeably, high levels of neutralizing antibodies response induced by mRNA-RBD vaccination could maintain for at least 6.5 months and conferred a long-term remarkable protection for hACE2 transgenic mice against SARS-CoV-2 infection in sera transfer study. These data demonstrated that a single dose of mRNA-RBD provided long-term protection against SARS-CoV-2 challenge.

Vaccine Development

Virology

SARS-CoV-2

vaccine

mRNA

long-term protection

COVID-19

Coronavirus disease-19 (COVID-19) caused by SARS-CoV-2 has emerged as a severe global pandemic ^1-6. Since SARS-CoV-2 transmits efficiently from person to person, as of 25 August 2020 more than 23,000,000 cases and 800,000 deaths have been confirmed in 216 countries or territories worldwide. COVID-19 has symptoms ranging from mild disease to severe lung injury and multi-organ failure, eventually leading to death, especially in older patients with other co-morbidities ^7-9. The COVID-19 pandemic has leaded to not only the enormous burden of mortality and morbidity associated with SARS-CoV-2 infection, but also a global economy crisis because of the economic and societal lockdown in an attempt to thwart the progression of the disease. At present there are no available prophylactics or therapeutics against SARS-CoV-2 infections, highlighting a desperate need for safe and effective vaccine to finish the ongoing pandemic and prevent new potential outbreaks.

SARS-CoV-2 belongs to the genus Betacoronavirus of the family Coronavirdae ¹⁰. Like other human coronaviruses, SARS-CoV-2 surface spike glycoprotein (S) can be cleaved into S1 and S2 subdomains, where the receptor-binding domain (RBD), located at the C-terminal of S1 subdomain, engages human angiotensin-converting enzyme 2 (hACE2) as the receptor, and S2 mediates membrane fusion. Both the full-length S protein and RBD are capable of inducing highly potent neutralizing antibodies and cellular immunity ^11-14, therefore have been widely selected as antigens for SARS-CoV-2 vaccine development ^{12, 15-20}. As the vaccine antigen RBD can focus the immune response on interference of receptor binding and theoretically stands lower risk of inducing antibodies that readily mediates antibody-dependent enhancement of infection, compared with the full-length S ^{16, 21, 22}. A number of highly potent monoclonal antibodies have also been isolated that predominantly target the RBD ^23-28. The crystal structures of SARS-CoV-2 RBD in complex with hACE2 have been determined by us and other groups ^29-31, which further improved our understanding of this vaccine antigen. Therefore, RBD represents an ideal target for SARS-CoV-2 vaccine development.

mRNA vaccine takes advantage of safety, rapid development and potent immunogenicity, especially in lipid-encapsulated form, and multiple mRNA vaccine candidates against infectious disease or cancer are under clinical development ³². To date, three SARS-CoV-2 mRNA vaccine candidates have already advanced to clinical trials that include mRNA-1273 encoding the viral S protein from Moderna (the United States of America) ^{1, 33}, BNT162b1 expressing the RBD protein from BioNTech (Germany) ³⁴, and ARCoV encoding the RBD protein developed by Abogen (China) ²¹. Moreover, mRNA-1273 has advanced to Phase III testing on 27 July 2020 and remains one of the most leading vaccine candidates against SARS-CoV-2 in the world. All three clinical mRNA vaccines applied a two-dose immunization regimen ^{21, 33, 34}. Recently, a single nucleoside-modified mRNA vaccination has been shown to elicit strong cellular and humoral immune responses against SARS-CoV-2 ³⁵. However, protection by a single mRNA vaccination against wild SARS-CoV-2 in animal models is little investigated.

The duration of neutralizing antibody (NAb) response in humans following coronavirus infection was vital for protection from re-infection. Whereas sustained IgG or NAb levels in individuals were usually maintained for more than 2 years against SARS-CoV or MERS-CoV infection ^36-38, according to recent studies NAb response in high proportion of humans who recovered from SARS-CoV-2 infection only lasted for 2-3 months after infection ^39-41. The transient NAb responses following SARS-CoV-2 infection reflect more the immune responses to endemic seasonal coronaviruses (those associated with common cold) which have also been reported to be transient ⁴². In addition, disease severity enhances the magnitude of the NAb response against SARS-CoV-2 infection but doses not impact on the kinetics of the NAb response ³⁹. For those patients with severe symptoms following SARS-CoV-2 infection, decline in NAb titers, ranged from 2- to 23- fold over an 18-65 day period, has been reported ³⁹. Noticeably, the first case of human re-infection just after a few months of recovery from the first infection is also recently documented ⁴³. The waning NAb response after SARS-CoV-2 infection has aroused widespread concern about a short duration of vaccine protection. Due to the recent emergence of SARS-CoV-2 in the human population, there is currently a paucity of information on the longevity of protection induced by vaccines. Here we evaluated protective efficacy of a single RBD-encoding mRNA immunization against wild SARS-CoV-2, investigated the kinetics of humoral response during 6.5 months following vaccination, and demonstrated the long-term protection of immune sera for hACE2 transgenic mice from SARS-CoV-2 challenge.

Construct and characterization of SARS-CoV-2 mRNA-RBD vaccine. We designed an mRNA vaccine which encodes RBD glycoprotein of SARS-CoV-2 strain HB-01 (Fig. 1a) and contains the modified nucleoside N₁-methylpseudouridine to prevent innate immune sensing and increase mRNA translation in vivo ⁴⁴. Then the expression profiles of the SARS-CoV-2 RBD-encoding mRNA (mRNA-RBD) were characterized by transfecting HEK293T cells. The results demonstrated that RBD was produced effectively and secreted into supernatant of HEK293T cells (Fig. 1b). To mediate efficient and prolonged antigen expression by mRNA in vivo, mRNA-RBD was further prepared for vaccination by encapsulation in lipid nanoparticles (LNPs) ⁴⁴. Encapsulation efficacy of mRNA-RBD was greater than 92%, based on a ribogreen fluorescence assay. The dynamic light scattering of LNPs in PBS demonstrated that the average particle size was 78 nm (Fig. 1c), with a narrow PDI of 0.117. Cryo-electron microscopy analysis showed that mRNA-RBD-encapsulated LNPs exhibited an electron dense core (Fig. 1d). Zeta potential measurement showed that intense ionization of the surface charge was observed as the zeta potential increased from -5.7 mV at pH 7.4 to +12.8 mV at pH 4.0 (Fig. 1e). These data suggested that LNPs could respond to pH changes, disrupt the endosomal membranes under an acidic pH, and release mRNA into the cytoplasm.

Immunogenicity of a single mRNA-RBD vaccination. The immune responses induced by SARS-CoV-2 mRNA-RBD vaccine candidate were analyzed in BALB/c mouse. Groups of mice (n=6) were immunized intramuscularly (i.m.) once with a high dose (15 μg) or low dose (2 μg). Polycytidylic acid (poly(C)) RNA encapsulated in LNP was used as a placebo control. Serum samples obtained at four weeks after vaccination were evaluated for SARS-CoV-2 RBD-specific IgG and IgG subtypes by enzyme-linked immunosorbent assay (ELISA). As a result, the mean endpoint titers of RBD-specific IgG in mice immunized with high dose rose to ＞10⁵ and were significantly higher than those observed in mice immunized with low dose (Fig. 2a). Additionally, both doses elicited IgG2a and IgG1 subclass RBD-specific antibodies, indicating a balanced Th1/Th2 response (Extended Data Fig. 1). Anti-SARS-CoV-2 neutralizing antibodies in serum of vaccinated mice were measured using two independent in vitro assays: pseudovirus neutralization assays and live SARS-CoV-2 neutralization assays. Pseudovirus neutralization assays showed that serum NT₉₀ (90% neutralization titers) in mice receiving low dose (mean value 193) was significantly lower than that in mice receiving high dose (mean value 727) (Fig. 2b). Live SARS-CoV-2 neutralization assays demonstrated that all vaccinated mice except one inoculated with low dose developed detectable neutralizing antibodies, and mean NT₅₀ (50% neutralization titers) value in high dose group approached 920 and was 8.5-fold higher than that observed in low dose group (Fig 2c). Therefore, we selected the high dose to immunize mice in the following studies.

Next, we investigated whether antibodies induced by SARS-CoV-2 mRNA-RBD could cross-react with or cross-neutralize the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle Eastern respiratory syndrome-related coronavirus (MERS-CoV). ELISA-based binding assays demonstrated that vaccinated serum obtained from BALB/c mice at four weeks following immunization exhibited a strong binding capacity to SARS-CoV-2 RBD, whereas they showed substantially lower affinity to SARS-CoV RBD and little (if any) cross-reactivity to MERS-CoV RBD, respectively (Extended Data Fig. 1a-c). Pseudovirus cross-neutralization showed that no detectable cross-neutralization activities against both pseudotyped SARS-CoV and MERS-CoV were observed, which were similar to placebo (Extended Data Fig. 1d). Therefore, these results indicated that although some cross-reactive antibodies elicited by SARS-CoV-2 mRNA-RBD exist, they may fail to block host cell entry mediated by SARS-CoV S protein.

To characterize the cellular immune responses induced by mRNA-RBD, enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) assays were performed. Spleens of C57BL/6 mice were harvested at four weeks post a single high-dose immunization. RBD-specific IgG and neutralizing antibodies were efficiently elicited in vaccinated mice (Fig. 2d,e). Splenocytes were isolated and evaluated for testing the capacity of secreting cytokines after re-stimulation with RBD peptide pools in vitro. ELISPOT assays demonstrated that T cells secreting gamma interferon (IFNγ) from mRNA-RBD-immunized mice were significantly more than those from placebo-immunized mice (Fig. 2f). Moreover, immune responses mediated by SARS-CoV-2 RBD-specific CD4⁺ and CD8⁺ T cells were detected on the basis of intracellular IFNγ production by splenocytes of mRNA-RBD-immunized mice as evidenced in ICS assays (Fig. 2g,h). Therefore, mRNA-RBD can induce a substantial T cell response against SARS-CoV-2 RBD antigens aside from humoral immune responses.

Protection efficacy of mRNA-RBD in hACE2 transgenic mice against SARS-CoV-2. To further explore the in vivo protection efficacy of mRNA-RBD against wild SARS-CoV-2 virus challenge, hACE2 transgenic mice (n=6) received one (prime group) or two (boost group) immunization of mRNA-RBD at 15 μg or placebo via i.m. route and pre-challenge sera were collected for detection of specific and neutralizing antibody titers (Fig. 3a). The results showed that a single immunization of mRNA-RBD induced robust production of RBD-specific IgG antibodies and neutralizing antibodies with a pseudovirus-neutralizing NT₉₀titer of ～170 (Fig. 3b,c). In addition, a second immunization at 2 weeks post initial inoculation resulted in a significant elevation in both IgG and neutralizing antibodies with NT₉₀titer reaching～2000, whereas no SARS-CoV-2 specific IgG and neutralizing antibody was detected in sera from mice vaccinated with placebo (Fig. 3b,c). Four weeks later, all mice were challenged with 1×10⁵ FFU of SARS-CoV-2 via intranasal (i.n.) route (Fig. 3a). We recorded the body weight for each mouse daily after infection for 5 days and found that the body weight of both prime and boost group mice showed a similar decrease at 1 day post infection (dpi) but a significantly faster increase during 3-5 dpi compared to the placebo group (Fig. 3d). Mice were euthanized at 5 dpi, and harvested lungs were analyzed for viral RNA loads (four mice per group) and histopathological and immunohistochemical assays (two mice per group). The results of lung viral RNA loads demonstrated that all placebo-vaccinated mice became infected, with high level of viral RNAs detected in lung (～10^8.5 RNA copies equivalents per gram) (Fig. 3e). By contrast, mRNA-RBD-vaccinated mice were highly protected against SARS-CoV-2 infection (Fig. 3e). Seven out of eight mice-including three in prime group and four in boost group-had no detectable viral RNA in lungs (Fig. 3e). A lower level of viral RNAs (～10⁷ RNA copies equivalents per gram) were detected in one mouse in prime group, representing a 97% reduction in lung viral load compared to the control mice (Fig. 3e). This mouse exhibited the lowest NT₉₀ titer of 34 in pseudovirus neutralizing assay (Fig. 3e). The significance of a low-level lung viral load in one mouse, including implications for a correlate of protection, warrants further study with a greater number of animals. Histopathological examination indicated that severe bronchopneumonia and interstitial pneumonia can be observed in placebo mice, with edema and bronchial epithelia cell desquamation and infiltration of lymphocytes within alveolar spaces (Fig. 3f). In contrast, only very mild bronchopneumonia was observed in prime group and no lesions were observed in boost group (Fig. 3f). Similarly, immunohistochemical assays detected SARS-CoV-2 viruses in the placebo mice, while no viral in lung from all mRNA-RBD vaccinated mice (Fig. 3g). These data demonstrated that a single vaccination of mRNA-RBD afforded remarkable prevention of SARS-CoV-2 replication in lung and efficiently protected mice from lung lesions.

Duration and long-term protection of humoral response induced by mRNA-RBD. To evaluate the durability and long-term protection of antibodies response elicited by a single mRNA-RBD immunization, we performed an immunization and passive transfer study (Fig. 4a). BALB/c mice (n=10) were immunized with mRNA-RBD-H or placebo via i.m. route. Half mice per group (n=5) were euthanized at 8 weeks post vaccination and sera were collected for further passive transfer as short-term group. The other half mice per group (n=5) were bled at desired time to explore the kinetics of induced humoral response and finally euthanized at 26 weeks post vaccination, and massive sera were collected for further passive transfer as long-term group. The time-course study of RBD-specific IgG and pseudovirus neutralizing antibodies demonstrated that both IgG and neutralizing NT₉₀ titers in serum from vaccinated mice increased to peak at～10⁵ and～10³ at week 8, respectively (Fig. 4b,c). Noticeably, neutralizing NT₉₀ titers was relatively stable during weeks 10-26 and remained approximately 600 at 26 weeks post vaccination (Fig. 4c). Pooled immune or placebo sera (350 μl per mouse) were transferred to hACE2 mice (n=5) by intraperitoneal injection (Fig. 4a). One day later, mice were infected with 1×10⁵ FFU of SARS-CoV-2 via i.n. route (Fig. 4a). Mice body weight was monitored daily after challenge. The results showed that compared to placebo sera-transferred mice, both short-term and long-term sera-transferred mice displayed a similar weight loss during 1-2 dpi but then a significantly rapid increase to a normal level from 3 to 5 dpi (Fig. 4d). Lung tissue were harvested at 5 dpi and detected for viral RNA loads. As expected, lungs from animals treated with mRNA-RBD immune sera from short-term and long-term group animals showed substantially reduced infectious virus burden (Fig. 4e). Consistent with the high neutralizing titers, a lower (but not significantly) viral RNA level was also observed in the lung of animals receiving short-term sera compared to animals receiving long-term sera (Fig. 4e). The detection of viral RNA loads in hearts showed that only a very low level of virus RNAs was detected in two mice from placebo group, while no measurable viral RNAs were detected in all other mice (Extended Data Fig. 3). Together, these data suggest that a single immunization of mRNA-RBD conferred a long-term remarkable protection against SARS-CoV-2 infection by eliciting a durable humoral response.

The ongoing pandemic of COVID-19 necessitates an urgent need for a safe and effective SARS-CoV-2 vaccine. Here, we developed a nucleoside-modified RBD-encoding mRNA vaccine against SARS-CoV-2, and demonstrated that a single immunization with mRNA-RBD induced robust NAb responses and provided near-complete protection against wild SARS-CoV-2 challenge in hACE2 transgenic mice model. Noticeably, we also showed that a single dose of mRNA-RBD could induce a durable NAb response and thus conferred a long-term protection in sera passive transfer study. These data extend recent preclinical studies of inactivated virus, DNA, RNA, subunit and adenovirus-vectored vaccines for SARS-CoV-2.

Whereas SARS-CoV-2 mRNA vaccine candidates in clinical trials applied a two-dose regimen, a single nucleoside-modified mRNA vaccination has been shown to be sufficient to protect non-human primates against zika virus challenge ⁴⁴. Complete protection conferred by a single dose of Ad5-nCoV or Ad26-vectored vaccine against SARS-CoV-2 challenge in animal models is also currently published ^{45, 46}. It has been shown that adenovirus-vectored ChAdOx1 nCoV-19 vaccine elicits specific IgG titer of 400-6400 and neutralizing titers of 5-40 ⁴⁷, whereas the DNA vaccine induced a neutralizing antibody titer of 74-170 against live virus infection in immunized monkeys ¹². Moreover, SARS-CoV-2 neutralizing antibody geometric mean titers for human convalescent sera were reported to be approximately 100 ^{1, 34}. Here we found a single dose of mRNA-RBD elicited RBD-specific IgG titer of 10⁵ and live virus-neutralizing antibody titer of 920, respectively. Consistent with the high IgG and neutralizing antibody titers compared to those reported, a single dose of mRNA-RBD conferred a near-complete protection against SARS-CoV-2 infection in vivo. Moreover, a single-shot SARS-CoV-2 vaccine would have important logistic and practical advantages compared with a two-dose vaccine for mass vaccination campaigns and pandemic control.

To our knowledge, our study demonstrated for the first time that high level of NAb responses elicited by SARS-CoV-2 vaccine could maintain for at least 6.5 months, and probably much longer, since NAb titers were stable, and therefore conferred a long-term protection against SARS-CoV-2 infection as evidenced in sera passive transfer study. However, several previous studies reported that antibody-mediated immunity in convalescent humans from SARS-CoV-2 infection only persisted for 2-3 moths ^{41, 48}. One explanation for the difference of duration of humoral responses elicited by vaccination and infection may be that compared with immune response to vaccination, the immune response to SARS-CoV-2 infection will target not only RBD, the major target for potent neutralizing antibodies, but also non-RBD regions of S and various other viral proteins which may readily induce transient immune response. However, to elucidate the difference, further studies such as investigation of memory B cells and antibody repertoire induced by vaccination and infection are obviously warranted. Another limitation of our study is that we exclude contributions of memory B cell and cellular immunity induced by vaccination in evaluation of long-term protection against SARS-CoV-2 by challenging sera-transferred rather than directly-vaccinated mice, further studies are also needed to investigate that question. Overall, our study proved near-complete protection of a single mRNA vaccination against SARS-CoV-2 challenge, and provided valuable information about durability of protective immune response induced by SARS-CoV-2 vaccines.

Ethics statement. This study was carried out in strict accordance with the recommendations described in the Guide for the Care and Use of Laboratory Animals of the Institute of Microbiology, Chinese Academy of Sciences (IMCAS) Ethic Committee. All animal experiments were reviewed and approved by the Committee on the Ethics of Animal Experiments of IMCAS.

Cells, viruses and animals. HEK293T cells (ATCC CRL-3216), Huh7 cells (3111C0001CCC000679) and Vero E6 cells (ATCC CRL-1586) were cultured at 37℃ in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). BALB/c and C57BL/6 mice were purchased from Beijing Vital River Animal Technology Co., Ltd. (licensed by Charles River), and housed in specific-pathogen-free (SPF) mouse facilities in IMCAS. hACE2 transgenic mice were kindly provided by Professor Zhengli Shi from Wuhan Institute of Virology, CAS. SARS-CoV-2 virus strain HB-01 used in this study was provided by Professor Wenjie Tan from Chinese center for disease control and prevention. Vero E6 cells were applied to the amplification and titer titration of the virus stocks.

mRNA production. mRNA was produced using T7 RNA polymerase on linearized plasmids (synthesized by Genescript) encoding codon-optimized SARS-CoV-2 RBD glycoproteins (residues 319-541, GISAID: EPI_ISL_402119). mRNA was transcribed to contain 104 nucleotide-long poly(A) tail. 1-methylpseudourine-5’-triphosphate was used instead of UTP to generate modified nucleoside-containing mRNA. After transcription and purification, mRNA was analyzed by agrose gel electrophoresis and stored frozen at -20℃.

mRNA transfection. Transfection of HEK293T cells was performed with TransIT-mRNA (Mirus Bio) according to the manufacturer’s instructions. In briefly, mRNA (0.5 μg) was combined with TransIT-mRNA regent (1 μl) and boost regent (1 μl) in 50 μl of serum-free medium, and the complex was added to 2.5 × 10⁵ cells in 500 μl complete medium. Supernatant was collected and concentrated, and cells were lysed on ice in RIPA buffer (Beyotime) at 48 h after transfection.

Western blot. Whole-cell lysates and supernatants from cells transfected with mRNA-RBD were assayed for SARS-CoV-2 RBD expression by western blot. Samples were combined with loading buffer with dithiothreitol (DTT) and separated by 12% SDS-PAGE. Transfer to PVDF membrane was performed using a semi-dry apparatus (Ellard Instrumentation). The membrane was blocked with non-fat milk in TBS buffer containing 0.5% Tween-20. RBD protein was detected using mouse serum immunized with SARS-CoV-2 S1 proteins (Sino Biological) for 1 h, followed by secondary goat anti-rabbit IgG HRP (Santa Cruz) for 1 h. The membrane was developed by SuperSignal West Pic chemiluminescent substrate (Thermo Fisher Scientific).

Lipid-nanoparticle encapsulation of mRNA. SARS-CoV-2 RBD-encoded mRNA (mRNA-RBD) was encapsulated in LNPs using a self-assembly process in which an aqueous solution of mRNA at pH=4.0 was rapidly mixed with a solution of lipids dissolved in ethanol. LNPs used in this study contained an ionizable cationic lipid, phosphatidylcholine, cholesterol and PEG-lipid with a ratio of 50:10:38.5:1.5 mol/mol and were encapsulated at an mRNA to lipid ratio of around 0.05 (wt/wt). mRNA-RBD LNPs were stored at 4℃ at a concentration of RNA of about 1 mg/ml.

Particle size, zeta potential and encapsulation efficiency. Zetasizer was used to determine the particle size and zeta potential. Zeta potential was measured on particles after suspending LNPs in deionized water at pH 4.0 and 7.4. The free and total mRNA concentrations in LNPs were determined using the Quant-iT Ribogreen RNA assay kit according to the manufacturer’s instructions. The encapsulation efficiency (EE, %) was calculated as follows: EE (%) = (1- free mRNA concentration /total mRNA concentration) ×100.

Cryo-electron microscopy of LNPs. LNPs were transferred onto a glow-discharged ultrathin carbon-coated copper grid (Zhongjingkeyi Company) followed by 60 s of waiting and blotted for 2 s with filter paper before plugging into liquid ethane using the Vitrobot Mark IV. The frozen grids were transferred at liquid nitrogen temperature and loaded into a Talos transmission electron microscope (Thermo Fisher Scientific) equipped with a field emission gun operated at 200 kV. The images were recorded on a direct electron detector (ED20) at a total electron dose of ~50 e−/Å2.

Animal experiments. LNPs-encapsulated mRNA-RBD was diluted with PBS. Female BALB/c or C57BL/6 mice aged 6-8 weeks were inoculated intramuscularly (i.m.) with a high (15 μg) or low (2μg) dose of mRNA-RBD LNP formulations or poly(C) LNP formulations as placebo control. Serum samples were collected at indicated time after vaccination, inactivated at 56°C for 30 min and stored at -20℃.

For SARS-CoV-2 challenge experiments, hACE2 transgenic mice (n=6) were immunized with one (prime group) or two (boost group) doses of mRNA-RBD via i.m. route. Four weeks post initial immunization, mice were infected with 1 × 10⁵ FFU of SARS-CoV-2 (strain HB- 01) in a total volume of 50 μl of DMEM medium via intranasal (i.n.) route. Animals were monitored daily for survival and weight loss. The mice were enthanized 5 days following challenge. Lung tissues were harvested for virus load detection (four mice per group), and pathological and immunohistochemical examination (two mice per group).

For passive immunization and SARS-CoV-2 challenge experiments, groups of BALB/c mice (n=10) received 15 μg of mRNA-RBD or placebo. Half mice per group (n=5) were euthanized at 8 weeks (short term) post vaccination. The other mice per group (n=5) were euthanized at 26 weeks (long term) post vaccination. hACE2 transgenic mice (n=5) were administered 350 μl per mouse of pooled immune sera collected from placebo or mRNA-RBD vaccinated mice via i.p. route. One day following serum transfer, mice were challenged with 1× 10⁵ FFU of SARS-CoV-2 via i.n. route. Animals were monitored daily for survival and weight loss. Lung tissues were harvested for virus load detection at 5 days following challenge. All animal experiments with SARS-CoV-2 challenge were conducted under animal biosafety level 3 (ABSL3) facilities in IMCAS.

Enzyme-linked immunosorbent assay (ELISA). ELISA plates (Corning) were coated overnight with 2 μg/ml of SARS-CoV-2 RBD, SARS-CoV RBD or MERS-CoV RBD recombinant protein in 0.05 M carbonate-bicarbonate buffer, pH 9.6, and blocked in 5% skim milk in PBS. Serum samples were 2-fold serially diluted and added to each well. Plates were incubated with goat anti-mouse IgG-HRP, IgG1-HRP or IgG2a-HRP antibodies and developed with 3, 3’, 5, 5’-tetramethylbenzidine (TMB) substrate. Reactions were stopped with 2 M hydrochloric acid, and the absorbance was measured at 450 nm using a microplate reader (PerkinElmer, USA). The endpoint titers were defined as the highest reciprocal dilution of serum to give an absorbance greater than 2.1-fold of the background values. Antibody titer below the limit of detection was determined as half the limit of detection.

Pseudovirus neutralization assay. SARS-CoV-2, SARS-CoV and MERS-CoV pseudovirus preparation and titration determination were performed as described previously ⁴⁹. Briefly, the plasmids of 14 μg pCAGGS-SARS-CoV-2-S, pCAGGS-SARS-CoV-S or pCAGGS-MERS-CoV-S and 7 μg pNL4-3.luc.RE were cotransfected into HEK293T cells. After 48 h, the supernatant containing pseudovirus was harvested, centrifuged and filtered through a 0.45μm sterilized membrane. Single use aliquots were stored at -80℃. The TCID₅₀ was determined by infection of Huh7 cells. For the neutralization assay, 100 TCID₅₀/well was incubated with 2-fold serially diluted mouse sera for 30 min at 37℃. The mixtures were then used to infect Huh7 cells seeded in 96-well plates. After 5 h incubation, the medium was replaced with DMEM containing 10% FBS, and the samples were incubated for an additional 24 h at 37℃. Luciferase activity was measured using a GloMax 96 Microplate luminometer (Promega). The neutralization endpoint was defined as the fold-dilution of serum necessary for 90% inhibition of luciferase activity in comparison with virus control samples.

Live SARS-CoV-2 neutralization assay. The neutralizing activity of mice sera was assessed using a SARS-CoV-2 microneutralization assay. Briefly, heat-inactivated serum was 2-fold serially diluted and incubated with SARS-CoV-2 strain 01 (100TCID₅₀) for 1 h at 37℃. The mixture was added to pre-seeded Vero E6 cell monolayers in 96-well plates. After incubation for 48 h at 37℃, the supernatant was collected for virus detection. The neutralization titers were defined as serum dilution required for 50% neutralization of viral infection.

Enzyme-linked immunosorbent spot (ELISPOT) assay. To detect antigen-specifific T lymphocyte responses, an IFN-γ-based ELISPOT assay was performed. Briefly, spleens were removed from vaccinated C57BL/6 mice at 4 weeks post immunization and splenocytes were isolated. Flat-bottom, 96-well plates were pre-coated with 10 g/ml anti-mouse IFNγ Ab (BD Biosciences, USA) overnight at 4°C and then blocked for 2 h at RT. Mouse splenocytes were added to the plate (1 × 10⁵/well). Then, a peptide pool (2 μg/ml of individual peptide) consisting of 18-mers (overlapping by 10 amino acids) spanning the entire SARS-CoV-2 RBD proteins was added to the wells. Phytohemagglutinin (PHA) was added as a positive control. Cells incubated without stimulation were employed as a negative control. After 18 h of incubation, the cells were removed, and the plates were processed in turn with biotinylated IFNγ-detection antibody, streptavidin-HRP conjugate, and substrate. When the colored spots were intense enough to be visually observed, the development was stopped by thoroughly rinsing samples with deionized water. The numbers of the spots were determined using an automatic ELISPOT reader and image analysis software (Cellular Technology Ltd.).

Intracellular cytokine staining (ICS) assay. An ICS assay was performed to characterize antigen-specific CD4⁺ and CD8⁺ immune response. Briefly, mouse splenocytes were added to the plate (1 × 10⁶ cells/well) and then stimulated with the peptide pool (2 μg/ml of individual peptide) for 4h. The cells were incubated with Golgiplug (BD Biosciences) for additional 14 h at 37℃. Then the cells were harvested and stained with anti-CD3, anti-CD4 and anti-CD8α surface makers (Biolegend). The cells were subsequently fixed and permeabilized in permeabilizing buffer (BD Biosciences) and stained with anti-IFN-γ (Biolegend). All fluorescent lymphocytes were gated on a FACSCanton flow cytometer (BD Biosciences).

Viral RNA extraction and RT-PCR. Viral RNA was extracted from 100 μl of samples using the automated nucleic acid extraction system (TIANLONG, China) and under the manufacturer’s instructions. Detection of the SARS-CoV-2 virus was performed using the One Step Prime Script RT-PCR kit (TaKaRa, Japan) on the Light Cycler 480 Real-Time PCR system (Roche, Rotkreuz, Switzerland) with primers. The following sequences were used:

forward primer: 5ʹ-AGAAGATTGGTTAGATGATGATAGT-3ʹ;

reverse primer:5ʹ-TTCCATCTCTAATTGAGGTTGAACC-3ʹ;

and probe:5ʹ-FAM-TCCTCACTGCCGTCTTGTTG ACCA-BHQ1-3ʹ.

Real-time RT-PCR was performed using the following conditions: 50°C for 15 min and 95°C for 3 min, 50 cycles of amplification at 95°C for 10 s and 60°C for 45 s. All experiments were conducted in triplicates.

Histopathology and immunohistochemistry. The lungs were fixed in 4% (v/v) paraformaldehyde solution for 72 hours, and the paraffin sections (3-4 μm) were prepared routinely. Hematoxylin and Eosin (H＆E) stain was used to identify histopathological changes in the lungs. The histopathology of the lung tissue was observed by light microscopy. For immunohistochemistry, SARS-CoV-2 N protein was detected using monoclonal antibody clone 019 (Sino Biological, China). Images were captured using LEICA Versa 200 and were processed using software HALO v3.1.1076.379.

Statistical analysis. All data are expressed as the means ± standard errors of the means (SEM). For all analyses, P values were analyzed with one-way analysis of variance or Student’s t test. All graphs were generated with GraphPad Prism, version 7.0, software.

Data availability

Source data for Fig. 1-4 and Extended Data Figs. 1-3 are available within the paper. All other data are available from the corresponding author upon reasonable request.

Acknowledgments

We are grateful to X. Qu and Q. Peng (Institute of Microbiology, CAS) for their help in performing cryo-electron microscopy of LNPs. This work is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29040201), the National Natural Science Foundation of China (NSFC) (81901680), and the Technological Innovation Project of Shanxi Transformation and comprehensive Reform demonstration Zone (2017KJCX01).

Author contributions

J.Y. and Q.H. designed the study; Q.H., K.J., S.T., F.W., B.H. conducted assays. Q.H., K.J., S.T., Z.T., W.T., J.H. and J.Y. analyzed and interpreted the data. Q.H. wrote the manuscript. Q.H., S.T., Q.W., G.F.G. and J.Y. discussed and edited manuscript.

Competing interests:

The authors declare no competing interests.

Jackson, L.A. et al. An mRNA Vaccine against SARS-CoV-2 - Preliminary Report. The New England journal of medicine (2020).
Bao, L. et al. The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice. Nature (2020).
Jiang, R.D. et al. Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing Human Angiotensin-Converting Enzyme 2. Cell 182, 50-58.e58 (2020).
Sun, S.H. et al. A Mouse Model of SARS-CoV-2 Infection and Pathogenesis. Cell host & microbe 28, 124-133.e124 (2020).
Sheahan, T. et al. An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Science translational medicine 12 (2020).
Boni, M.F. et al. Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic. Nature microbiology (2020).
Zhu, N. et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. The New England journal of medicine 382, 727-733 (2020).
Bulut, C. & Kato, Y. Epidemiology of COVID-19. Turkish journal of medical sciences 50, 563-570 (2020).
Liu, Y. et al. Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals. Nature (2020).
Gao, Q. et al. Rapid development of an inactivated vaccine candidate for SARS-CoV-2. Science, eabc1932 (2020).
Lu, J. et al. A COVID-19 mRNA vaccine encoding SARS-CoV-2 virus-like particles induces a strong antiviral-like immune response in mice. Cell research, 1-4 (2020).
Yu, J. et al. DNA vaccine protection against SARS-CoV-2 in rhesus macaques. Science (2020).
Ravichandran, S. et al. Antibody signature induced by SARS-CoV-2 spike protein immunogens in rabbits. Science translational medicine 12 (2020).
Hsieh, C.L. et al. Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes. bioRxiv : the preprint server for biology (2020).
Tai, W. et al. A novel receptor-binding domain (RBD)-based mRNA vaccine against SARS-CoV-2. Cell research, 1-4 (2020).
Dai, L. et al. A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS. Cell 182, 722-733.e711 (2020).
Yang, J. et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature (2020).
Zhu, F.C. et al. Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial. Lancet (London, England) 395, 1845-1854 (2020).
Zhu, F.C. et al. Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet (London, England) 396, 479-488 (2020).
Folegatti, P.M. et al. Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. Lancet (London, England) 396, 467-478 (2020).
Zhang, N.N. et al. A Thermostable mRNA Vaccine against COVID-19. Cell (2020).
Lee, W.S., Wheatley, A.K., Kent, S.J. & DeKosky, B.J. Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies. Nature microbiology (2020).
Shi, R. et al. A human neutralizing antibody targets the receptor binding site of SARS-CoV-2. Nature (2020).
Wu, Y. et al. A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2. Science (2020).
Cao, Y. et al. Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients' B Cells. Cell 182, 73-84.e16 (2020).
Zost, S.J. et al. Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature 584, 443-449 (2020).
Erasmus, J.H. et al. An Alphavirus-derived replicon RNA vaccine induces SARS-CoV-2 neutralizing antibody and T cell responses in mice and nonhuman primates. Science translational medicine 12 (2020).
Lv, Z. et al. Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody. Science (2020).
Wang, Q. et al. Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell (2020).
Walls, A.C. et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281-292.e286 (2020).
Yuan, M. et al. A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV. Science 368, 630-633 (2020).
Pardi, N., Hogan, M.J., Porter, F.W. & Weissman, D. mRNA vaccines - a new era in vaccinology. Nature reviews. Drug discovery 17, 261-279 (2018).
Corbett, K.S. et al. Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates. The New England journal of medicine (2020).
Mulligan, M.J. et al. Phase 1/2 study of COVID-19 RNA vaccine BNT162b1 in adults. Nature (2020).
Laczkó, D. et al. A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice. Immunity (2020).
Wu, L.P. et al. Duration of antibody responses after severe acute respiratory syndrome. Emerging infectious diseases 13, 1562-1564 (2007).
Choe, P.G. et al. MERS-CoV Antibody Responses 1 Year after Symptom Onset, South Korea, 2015. Emerging infectious diseases 23, 1079-1084 (2017).
Payne, D.C. et al. Persistence of Antibodies against Middle East Respiratory Syndrome Coronavirus. Emerging infectious diseases 22, 1824-1826 (2016).
Seow, J. et al. Longitudinal evaluation and decline of antibody responses in SARS-CoV-2 infection. medRxiv, 2020.2007.2009.20148429 (2020).
Wang, K. et al. Longitudinal dynamics of the neutralizing antibody response to SARS-CoV-2 infection. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America (2020).
Long, Q.X. et al. Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections. Nature medicine 26, 1200-1204 (2020).
Gorse, G.J., Donovan, M.M. & Patel, G.B. Antibodies to coronaviruses are higher in older compared with younger adults and binding antibodies are more sensitive than neutralizing antibodies in identifying coronavirus-associated illnesses. Journal of medical virology 92, 512-517 (2020).
To, K.K. et al. COVID-19 re-infection by a phylogenetically distinct SARS-coronavirus-2 strain confirmed by whole genome sequencing. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America (2020).
Pardi, N. et al. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature 543, 248-251 (2017).
Wu, S. et al. A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge. Nature communications 11, 4081 (2020).
Mercado, N.B. et al. Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques. Nature (2020).
van Doremalen, N. et al. ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques. bioRxiv : the preprint server for biology (2020).
Juno, J.A. et al. Humoral and circulating follicular helper T cell responses in recovered patients with COVID-19. Nature medicine (2020).
Li, Y. et al. A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein. Cell research 25, 1237-1249 (2015).

ExtendedDataFig.docx

Download PDF

Journal Publication

published 03 Feb, 2021

Read the published version in Nature Communications →

Version 1

posted

You are reading this latest preprint version

A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Discussion

Methods

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1