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. 2021 Oct 26;118(43):e2111075118.
doi: 10.1073/pnas.2111075118.

Middle East respiratory syndrome coronavirus vaccine based on a propagation-defective RNA replicon elicited sterilizing immunity in mice

J Gutiérrez-Álvarez  1 , J M Honrubia  1 , A Sanz-Bravo  1 , E González-Miranda  1 , R Fernández-Delgado  1 , M T Rejas  2 , S Zúñiga  1 , I Sola  1 , L Enjuanes  3
Affiliations

Middle East respiratory syndrome coronavirus vaccine based on a propagation-defective RNA replicon elicited sterilizing immunity in mice

J Gutiérrez-Álvarez et al. Proc Natl Acad Sci U S A. .

Abstract

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.

Keywords: MERS-CoV; RNA replicon; coronavirus; vaccine.

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Conflict of interest statement

Competing interest statement: The authors declare patent applications (pending) filed by their institution.

Figures

Fig. 1.
Fig. 1.
Characterization of viral growth and morphogenesis of MERS-CoV lacking the E gene. (A) MERS-CoV-WT (WT) virus and rMERS-CoV-ΔE (ΔE) replicon growth were compared in Huh-7 cells transfected with plasmids constitutively (CONS; pcDNA-3.1-E-MERS-CoV) or inducibly (IND; TRE-Auto-rtTA-V10-2T-E-MERS) expressing E protein. Nontransfected cells (E−) were used as the control for viral growth in the absence of E protein expression. Results are expressed as the mean ± SD. (B) For morphogenesis studies, Huh-7 cells without E protein complementation were infected at two MOIs, and samples were processed 17 h after infection. Large vesicles with a high concentration of spherical virions can be seen in MERS-CoV-WT–infected cells, with greater morphological alterations observed in cells infected at an MOI of one. MERS-CoV-ΔE–infected cells showed the vesicles with virions are in both conditions and lower cytopathic effect.
Fig. 2.
Fig. 2.
In vivo evaluation of the EMC/2012 rMERS-CoV-ΔE replicon in a highly susceptible mouse model. K18-hDPP4 mice were infected with 5 × 103 FFU of MERS-CoV-WT virus or the rMERS-CoV-ΔE replicon and monitored for weight loss (A) and survival (B) for attenuation studies. After 21 dpim, nonimmunized and rMERS-CoV-ΔE–immunized mice were challenged with 5 × 104 FFU of MERS-CoV-WT per mouse, and (C) weight loss and (D) survival were monitored daily to evaluate the protection conferred by rMERS-CoV-ΔE against an MERS-CoV-WT lethal infection. Differences in weight loss are represented as mean ± SEM.
Fig. 3.
Fig. 3.
Engineering and in vitro characterization of MERS-MA30 mutants lacking different sets of viral genes. (A) Scheme of deletion mutants engineered using the MERS-CoV mouse-adapted infectious cDNA clone (MERS-MA30). Virus genes are indicated at the top of the figure. White boxes indicate deleted genes. A deletion and a stop codon (red asterisks) in ORF5 were present in MERS-MA30 and the infectious cDNA clone developed from this virus (66). (B and C) Growth kinetics of viruses and replicons derived from MERS-MA30 in the absence (B) or in the presence (C) of the E protein provided in trans. Huh-7 cells were infected at an MOI of 0.001, and the infection was followed for 72 h. Results are expressed as mean ± SD. (D) Titration of the rMERS-MA30-Δ[3,4a,4b,5,E] replicon passed in Huh-7 cells in the presence or absence of E protein. Virus passages were performed in Huh-7 cells every 24 h. The presence of the replicon at passage 1 in E− cells might be due to the initial inoculum. The detection limit was 50 FFU/mL (dashed black line). Results are expressed as mean ± SD. E+, replicon supplemented with E protein of MERS-CoV-WT virus; E−, replicon in the absence of E protein provided in trans; E− (F/T), cells not supplemented with E protein were subjected to three freeze–thaw cycles to mechanically release the replicon. (E) Scheme of the MERS-MA30-Δ[3,4a,4b,5,E] replicon indicating the amplified region by PCR for sequencing and electrophoretic analyses. (F) Agarose gel electrophoresis showing the PCR products from passages 1 to 5 in Huh-7 cells in the presence or absence of E protein provided in trans. S: spike gene; M: membrane gene; N: nucleocapsid gene; An: polyadenylation tail; MW: molecular weight; bp: base pairs.
Fig. 4.
Fig. 4.
Evaluation of rMERS-MA30–derived mutants and replicons attenuation in hDDP4-KI mice. Mice were infected with 1 × 104 FFU of each virus or replicon, and (A) weight loss and (B) survival were monitored. Differences in weight loss are represented as mean ± SEM. (C) Titers of MERS-MA30 virus or the rMERS-MA30-Δ[3,4a,4b,5,E] replicon in the lungs of infected mice. (D) Viral genomic RNA and (E) subgenomic RNA N in the lungs of infected mice. The results are expressed as mean ± SD. **Student’s t test: significance level is lower than 0.01. (F) Histopathology induced by MERA-MA30 or the rMERS-MA30-Δ[3,4a,4b,5,E] replicon in the lungs of infected mice. In the lungs of the mice infected with rMERS-MA30, cell infiltrates and thickening of the alveoli walls (yellow arrows) were observed at 3 dpi, with the appearance of edema (red asterisks) and general lung infiltration at 6 dpi. The lungs of mice infected with the rMERS-MA30-Δ[3,4a,4b,5,E] replicon remained healthy and looked similar to the lungs of the uninfected mice.
Fig. 5.
Fig. 5.
Protection conferred by rMERS-MA30–derived mutants and replicons in hDPP4-KI mice. Mice were infected with the indicated viruses or replicons and challenged 21 d later with 105 FFU of MERS-MA30 per mouse. (A) Weight loss and (B) survival were monitored. Differences in weight loss are represented as mean ± SEM. (C) Titers of MERS-MA30 challenge virus in the lungs of nonimmunized mice and mice immunized with the rMERS-MA30-Δ[3,4a,4b,5,E] replicon. (D) Viral replication and (E) transcription in challenged mice. The results are expressed as mean ± SD. *Student's t test: significance level lower than 0.05; **Student's t test: significance level lower than 0.01. (F) Histopathology of immunized and nonimmunized mice challenged with MERS-MA30 virus. The lungs of the mice immunized with the rMERS-MA30-Δ[3,4a,4b,5,E] replicon looked healthy throughout the experiment. In the lungs of nonimmunized mice, peribronchial cuffing and alveolar thickening (yellow arrows) could be seen at 2 dpc. At 4 dpc, edema (red asterisks) could be observed in some air spaces, while at 6 dpc, highly evident edema, general cell infiltration, and focal hemorrhage could be observed. (G) Levels of neutralizing antibodies in the serum of immunized mice. Blood samples were taken from nonimmunized and rMERS-MA30-Δ[3,4a,4b,5,E]–immunized mice at 0 and 21 dpim to quantify neutralizing antibodies. Titers were expressed as the highest serum dilution showing complete neutralization of the cytopathic effect in 50% of the wells (TCID50). *Student's t test: P value = 0.0102919.
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References

    1. Gorbalenya A. E., et al. ., Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, The species Severe acute respiratory syndrome-related coronavirus: Classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 5, 536–544 (2020). - PMC - PubMed
    1. de Groot R. J., et al. ., Middle East respiratory syndrome coronavirus (MERS-CoV): Announcement of the Coronavirus Study Group. J. Virol. 87, 7790–7792 (2013). - PMC - PubMed
    1. International Committee on Taxonomy of Viruses, ICTV Master Species List 2019.v1 (2019). https://talk.ictvonline.org/files/master-species-lists/m/msl/9601. Accessed 9 June 2021.
    1. Colina S. E., Serena M. S., Echeverría M. G., Metz G. E., Clinical and molecular aspects of veterinary coronaviruses. Virus Res. 297, 198382 (2021). - PMC - PubMed
    1. Corman V. M., Muth D., Niemeyer D., Drosten C., Hosts and sources of endemic human coronaviruses. Adv. Virus Res. 100, 163–188 (2018). - PMC - PubMed

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