key: cord-0958392-i3nevqvz
authors: Elia, Uri; Rotem, Shahar; Bar-Haim, Erez; Ramishetti, Srinivas; Naidu, Gonna Somu; Gur, David; Aftalion, Moshe; Israeli, Ma’ayan; Bercovich-Kinori, Adi; Alcalay, Ron; Makdasi, Efi; Chitlaru, Theodor; Rosenfeld, Ronit; Israely, Tomer; Melamed, Sharon; Ionita, Inbal Abutbul; Danino, Dganit; Peer, Dan; Cohen, Ofer
title: A lipid nanoparticle RBD-hFc mRNA vaccine protects hACE2 transgenic mice against lethal SARS-CoV-2 infection
date: 2021-03-29
journal: bioRxiv
DOI: 10.1101/2021.03.29.436639
sha: 57db2035f84c6e5b302b3129a73e78c9650288ee
doc_id: 958392
cord_uid: i3nevqvz

The current global COVID-19 pandemic led to an unprecedented effort to develop effective vaccines against SARS-CoV-2. mRNA vaccines were developed very rapidly during the last year, and became the leading immunization platform against the virus, with highly promising phase-3 results and remarkable efficacy data. Since most animal models are not susceptible to SARS CoV-2 infection, pre-clinical studies are often limited to infection-prone animals such as hamsters and non-human primates. In these animal models, SARS-CoV-2 infection results in viral replication and a mild disease disease. Therefore, the protective efficacy of the vaccine in these animals is commonly evaluated by its ability to elicit immunologic responses, diminish viral replication and prevent weight loss. Our lab recently reported the design of a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). These experiments demonstrated the development of a robust and specific immunologic response in RBD-hFc mRNA-vaccinated BALB/c mice. In the current study, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18-hACE2 mouse model. We report that administration of RBD-hFc mRNA vaccine to K18-hACE2 mice led to a robust humoral response comprised of both binding and neutralizing antibodies. In accordance with the recorded immunologic immune response, 70% of vaccinated mice were protected against a lethal dose (3000 plaque forming units) of SARS-CoV-2, while all control animals succumbed to infection. To the best of our knowledge, this is the first non-replicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.

the virus, with highly promising phase-3 results and remarkable efficacy data. Since most animal models are not susceptible to SARS CoV-2 infection, pre-clinical studies are often limited to infection-prone animals such as hamsters and non-human primates. In these animal models, SARS-CoV-2 infection results in viral replication and a mild disease disease. Therefore, the protective efficacy of the vaccine in these animals is commonly evaluated by its ability to elicit immunologic responses, diminish viral replication and prevent weight loss.

Our lab recently reported the design of a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs) . These experiments demonstrated the development of a robust and specific immunologic response in RBD-hFc mRNA-vaccinated BALB/c mice.

In the current study, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18-hACE2 mouse model. We report that administration of RBD-hFc mRNA vaccine to K18-hACE2 mice led to a robust humoral response comprised of both binding and neutralizing antibodies. In accordance with the recorded immunologic immune response, 70% of vaccinated mice were protected against a lethal dose (3000 plaque forming units) of SARS-CoV-2, while all control animals succumbed to infection. To the best of our knowledge, this is the first nonreplicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), identified as the causative agent of coronavirus disease 2019 (COVID- 19) , developed in the last year into a global pandemic, causing (as of March 20 th , 2021) over 120 million cases and 2.7 million deaths worldwide. 1 Unprecedented international collaboration has led to record time development of several vaccine candidates, of which the mRNA vaccine platform has proven to be highly efficacious in phase 3 clinical studies. 2 An LNP RBD-hFc mRNA vaccine was designed, based on a previous lipid formulation screen that we recently reported. 6 The results of the screen led to the selection of lipid #14, which was highly potent in eliciting strong humoral and cellular responses in BALB/c mice immunization studies. A schematic representation of the hFc-fused RBD mRNA construct is shown in Figure 1a . Following LNP preparation, samples were analyzed for size and uniformity by dynamic light scattering (DLS). As can be seen in Figure 1b Control animals were administered with empty LNPs (prime-boost) (n=5). Serum samples were collected 23 ("pre boost") and 46 ("post boost") days after priming, and were assayed for SARS-CoV-2 spike-specific IgG antibodies (b) and neutralizing antibodies against SARS-CoV-2 (c) as described in the methods section. Animals were then intranasally infected with 3*10 3 PFU of SARS-CoV-2, and monitored for weight loss (d) and survival (e). Statistical analysis was performed using a two-way ANOVA with Bonferroni's multiple comparisons test (for ELISA data), one-way ANOVA followed by post hoc Newman-Keuls test (for neutralizing antibodies data) or log-rank (Mantel-Cox) test (for survival data) (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

As shown in Figure 3b , a single intramuscular immunization ("pre-boost") with either LNP RBD-hFc mRNA (n=10) or rRBD (n=11), did not elicit a significant humoral response, with most animals exhibiting anti-spike titers of <100. Conversely, a robust and statistically significant antibody response was observed in both LNP RBD-hFc mRNA (n=16) and rRBD (n=19) prime-boost immunization groups ("post-boost") compared with the empty LNPs control group (n=8). Within the immunization groups, the recorded anti-spike titers were significantly higher in mice administered with LNP RBD-hFc mRNA (mean log 10 titer=3.7) compared with rRBD-administered mice (mean log 10 titer =3.0). We next evaluated the post-boost in vitro neutralizing antibody response using a SARS-CoV-2 plaque reduction neutralization test (PRNT).

In line with the binding antibody titers, a substantial neutralizing antibody response BALB/c, C57/BL6 mice). In order to examine the efficacy of the vaccine, SARS-CoV-2 infection-prone animal models are frequently used (e.g. hamsters and nonhuman primates). In many studies, these animals are vaccinated and then challenged with virulent SARS-CoV-2, leading to symptomatic disease, weight loss and extensive viral replication in different tissues, but not death. Therefore, the protective effect of the vaccine is commonly evaluated by its ability to diminish viral replication, elicit a robust immune response and reduce weight loss. [12] [13] [14] In the current study, we sought to evaluate the efficacy of the vaccine by using the K18-hACE2 mouse model, which has been shown to be highly susceptible to SARS- Sciences also employed a hACE2 mouse model for evaluation of vaccine protection efficacy. 16 The data in that study, however, is based on immune humoral response, weight loss, lung viral titer and lung immunohistochemistry analysis, and not on survival of immunized animals. In summary, to the best of our knowledge, the current study demonstrates for the first time SARS-CoV-2 mRNA vaccine-mediated protection of K18-hACE2 mice against an otherwise lethal viral infection. 

Samples were prepared in a closed chamber at a controlled temperature and at water 

LNPs-encapsulated RBD-hFc mRNA (1 g/ml) or empty LNPs were added to 3*10 5 HEK293T cells seeded in a 6-well plate with 2ml DMEM supplemented with 10% fetal bovine serum (FBS). For positive control, cells were transfected with RBD-hFc-expressing pcDNA3 vector (g/ml) using jetPEI (Polyplus-transfection) according to manufacturer's protocol.

In order to evaluate RBD-hFc expression in transfected cells, supernatants were collected, and cells were lysed on ice in RIPA buffer (Merck) at 24, 48 or 72 h after transfection. Lysates were nutated at 4°C for 10 min, then centrifuged at 20,000×g for 10 min at 4°C. RBD-hFc was purified from whole-cell lysates and supernatants using Protein A HP SpinTrap (GE) and eluted with 100ul IgG elution buffer (Pierce).

Samples were then separated by 4-12% polyacrylamide Bis-tris gel electrophoresis (Invitrogen) and blotted onto a nitrocellulose membrane. The membrane was blocked for 1 h in Intercept® Blocking Buffers (Li-COR). RBD-hFc was detected by incubation of the membrane with purified IgG fraction from serum of rabbit immunized with SARS-CoV-2 spike protein for over-night at 4°C, followed by a secondary antibody IRDye 680RD goat anti-rabbit (LIC-92668071) incubation of 1 h at RT. Reactive bands were detected by Odyssey CLx infrared imaging system (LI-COR).

Recombinant SARS-CoV-2 spike glycoprotein for ELISA and human Fc-RBD-fused protein for vaccination were designed and expressed as previously described. 19 Briefly, a stabilized soluble version of the spike protein (based on GenPept:QHD43416 ORF amino acids 1-1207) was produced using an ExpiCHO for evaluation of SARS-CoV-2 spike-specific humoral response.

All animal experiments involving SARS-CoV-2 were conducted in a BSL3 facility. 

Handling of SARS-CoV-2 was conducted in a BSL3 facility in accordance with the biosafety guidelines of the Israel Institute for Biological Research (IIBR). SARS-CoV-2 (GISAID accession EPI_ISL_406862) strain was kindly provided by Bundeswehr Institute of Microbiology, Munich, Germany. Virus stocks were propagated and tittered by infection of Vero E6 cells as recently described. 21 For PRNT, Vero E6 cells were plated overnight (as detailed above) at a density of (Serum dilution at which the plaque number was reduced by 50%, compared to plaque number of the control, in the absence of serum) was calculated using the Prism software version 8 (GraphPad Software Inc., USA).

All values are presented as mean plus standard error of the mean (SEM). Statistical analysis was performed using a two-way ANOVA with Bonferroni's multiple comparisons test (for ELISA data), one-way ANOVA followed by post hoc Newman-Keuls test (for neutralizing antibodies data) or log-rank (Mantel-Cox) test (for survival data) (*, p < 0.05; **, p < 0.01; ***, p < 0.001). All statistical analyses were performed using GraphPad Prism 8 statistical software.

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We thank Hila Cohen, Liat Bar-On, Shirley Lazar, Tal Noy-Porat, and Yinon Levi for their assistance and support and Shmuel Yitzhaki, Ohad Mazor, and Emanuelle Mamroud for their insightful discussions and encouragement. We also thank Amir Rosner, Tseela David and Beni Shareabi for animal husbandry.We thank Inbar Freilich for her professional contribution to the Cryo-EM analysis.

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