key: cord-0913511-m7y5optt
authors: Tai, Wanbo; Zhang, Xiujuan; Yang, Yang; Zhu, Jiang; Du, Lanying
title: Advances in mRNA and other vaccines against MERS-CoV
date: 2021-11-19
journal: Transl Res
DOI: 10.1016/j.trsl.2021.11.007
sha: af853efc9d0fda7d264011c73f70fede98c889be
doc_id: 913511
cord_uid: m7y5optt

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human coronavirus (CoV). Belonging to the same beta-CoV genus as severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1) and SARS-CoV-2, MERS-CoV has a significantly higher fatality rate with limited human-to-human transmissibility. MERS-CoV causes sporadic outbreaks, but no vaccines have yet been approved for use in humans, thus calling for continued efforts to develop effective vaccines against this important CoV. Similar to SARS-CoV-1 and SARS-CoV-2, MERS-CoV contains four structural proteins, among which the surface spike (S) protein has been used as a core component in the majority of currently developed MERS-CoV vaccines. Here, we illustrate the importance of the MERS-CoV S protein as a key vaccine target and provide an update on the currently developed MERS-CoV vaccines, including those based on DNAs, proteins, virus-like particles or nanoparticles, and viral vectors. Additionally, we describe approaches for designing MERS-CoV mRNA vaccines and explore the role and importance of naturally occurring pseudo-nucleosides in the design of effective MERS-CoV mRNA vaccines. This review also provides useful insights into designing and evaluating mRNA vaccines against other viral pathogens.

Coronaviruses (CoVs) belong to the Othocoronavirinae subfamily of Coronaviridae, which is a virus family in the order of Nidovirales. Othocoronavirinae consists of four genera: alpha-CoV, beta-CoV, gamma-CoV, and delta-CoV. 1, 2 Middle East respiratory syndrome (MERS)-CoV, severe acute respiratory syndrome (SARS)-CoV-1, and SARS-CoV-2 are three highly pathogenic human CoVs that are classified as beta-CoVs. [1] [2] [3] Phylogenetically, MERS-CoV is a member of lineage C of beta-CoV, whereas SARS-CoV-1 and SARS-CoV-2 are classified as members of lineage B of beta-CoV. 2,4 SARS-CoV-1 and SARS-CoV-2 were first reported in humans in 2002 and 2019, respectively, leading to the global outbreak or pandemic. [5] [6] [7] The first MERS-CoV infection in humans was reported in June 2012. 8 Since then, it has infected at least 2,578 people globally, including 888 associated deaths (34.4% case-fatality rate), with the majority of these cases identified from Saudi Arabia (37.2% case-fatality rate). 9 As a zoonotic virus, MERS-CoV is transmitted between animals and humans. 10 Similar to SARS-CoV-1, MERS-CoV originates from bats. [11] [12] [13] Different from SARS-CoV-1, however, human infections of MERS-CoV may occur through infected dromedary camels as an intermediate host. 14 Unlike SARS-CoV-2 that transmits efficiently among humans, MERS-CoV has limited human-to-human transmission, mostly limited to healthcare settings and household contacts. [15] [16] [17] [18] Both MERS-CoV and SARS-CoV-2 cause acute respiratory distress syndrome (ARDS), showing similar respiratory and ventilatory parameters post-intubation. 19 As MERS-CoV continues to cause localized outbreaks with a high fatality rate, it is imperative to develop safe and effective vaccines to prevent MERS.

MERS-CoV is a single-strand, positive-sense RNA virus. Similar to SARS-CoV-1 and SARS-CoV-2, MERS-CoV has a large genome that encodes four structural proteins ( open-reading frame (ORF) 1a and ORF 1b, and five accessory proteins (ORF 3, 4a, 4b, 5, and 8b) (Figs. 1A, B) . 1, 2, 20 These proteins have different functions in viral transcription, translation, assembly, infection, replication, and pathogenesis. 2 Among these, the S protein plays a critical role in viral infection and pathogenesis. 4, 21 During the course of viral evolution, mutations may be incorporated into the S protein that exhibit geographic differences in virulence. [22] [23] [24] 

The MERS-CoV S protein is presented as a native trimeric structure, and each S monomer consists of two subunits, S1 and S2 (Figs 1C and 2A) , as do other CoV S proteins. MERS-CoV infects host cells by first binding to a specific receptor on the cell surface via the receptorbinding domain (RBD) in the S1 subunit ( Fig. 2B ) and then mediating virus-membrane fusion through the S2 subunit, promoting virus entry into target cells. [25] [26] [27] Different from SARS-CoV-1 and SARS-CoV-2, which use angiotensin-converting enzyme 2 (ACE2) as their receptor, 28, 29 MERS-CoV recognizes dipeptidyl peptidase 4 (DPP4) as its receptor for virus entry. Formation of the RBD/DPP4 complex mediates MERS-CoV entry into DPP4-expressing host cells (Fig.   2C ). 25, 26, 30, 31 The MERS-CoV RBD consists of a core and a receptor-binding motif (RBM) region (Fig. 1C) . A set of amino acids in the RBM region has been identified as key residues required for DPP4 binding. 25, 27, 31 Therefore, mutations of these critical residues may result in a reduction of binding between the RBD and DPP4 receptor. In addition to the RBD, the S1 subunit of MERS-CoV S protein also contains an N-terminal domain (NTD) (Figs 1C and 2B), which may bind sialic acid and play a role in virion attachment. 32 Compared with other MERS-CoV proteins, the S protein, including its RBD, can induce highly potent neutralizing antibodies and/or cellular immune responses with protective efficacy against MERS-CoV in vaccinated animals. [33] [34] [35] [36] [37] [38] A number of neutralizing monoclonal antibodies (mAbs) targeting the RBD or S1-NTD of MERS-CoV S protein have been reported, and the structures for some of these antibodies in binding to the RBD or S1-NTD have be solved (Figs 2D, E) . [39] [40] [41] [42] [43] [44] Notably, Sspecific antibodies may be maintained in MERS-CoV-infected humans for a long period of time (up to 6 years post infection), with neutralizing activity against MERS-CoV infection. 45 Therefore, the S protein, including its fragments, presents an important target for the development of effective MERS-CoV vaccines. Notably, there is no appreciable cross-reactivity or cross-neutralizing activity among MERS-CoV and SARS-CoV-1/SARS-CoV-2 S proteins, particularly their RBD fragments and S1 subunits, in immunization studies. In contrast, SARS-CoV-1 and SARS-CoV-2 S proteins can elicit cross-neutralizing antibodies that react with both CoVs. [46] [47] [48] [49] 

Although MERS-CoV vaccines have been extensively developed, nearly all of them are still in preclinical stages. Several MERS vaccines (e.g., GLS-5300, ChAdOx1, and MVA-MERS-S) have undergone Phase 1 trials, 50-52 but no vaccines are yet approved for human use. Currently reported MERS-CoV vaccines include those based on DNAs, proteins, nanoparticles or viruslike particles (VLPs), viral vectors, and live-attenuated viruses. 2, 38, [53] [54] [55] [56] The majority of these vaccines are designed using the viral S protein, including its fragments, thereby protecting animals (e.g., mice, rabbits, camels, or nonhuman primates [NHPs]) against MERS-CoV infection (Table 1) . [57] [58] [59] [60] [61] MERS-CoV vaccines have been previously summarized in various review articles. 1, 2, 4, 20, [62] [63] [64] Here, we provide an updated summary with a focus on the newly developed vaccine candidates.

MERS-CoV DNA vaccines utilize different vectors to encode the full-length MERS-CoV S protein or the S1 subunit. 57, 61, 65, 66 The immunization of NHPs with a synthetic DNA vaccine encoding the full-length MERS-CoV S protein elicited S-specific cellular immune responses and antibodies specific to the full-length S protein, S1 or S2 subunit, and RBD fragment, with lower viral loads and less severity of pathological signs. 57 The vaccine-induced antibody response was able to neutralize MERS-CoV in vitro and protect against MERS-CoV infection in NHPs. The immunization of mice with a baculoviral-vectored DNA vaccine encoding the S1 subunit of MERS-CoV S protein (AcHERV-MERS-S1) induced S-specific immunoglobulin G (IgG) antibodies and neutralizing antibodies capable of protecting immunized hDPP4-transfenic (hDPP4-Tg) mice against MERS-CoV challenge. 65 Limited weight loss, 100% survival, and undetectable viral titers in the lungs were reported. Other DNA vaccines, such as those based on the pcDNA3.1 vector and encoding the S1 subunit of S protein (pcDNA3.1-S1), also elicited humoral and cellular immune responses and neutralizing antibodies in mice, protecting adenovirus 5 (Ad5)-hDPP4-transduced mice from MERS-CoV challenge. 66 

Subunit vaccines expressing MERS-CoV full-length S protein, particularly its RBD fragment, may induce highly potent antibody responses and neutralizing antibodies against multiple pseudotyped and live MERS-CoV infection with protective efficacy in immunized animals. 23,33,34,36,37,67-69 MERS-CoV-specific neutralizing antibodies were significantly enhanced in mice immunized with an alum-adjuvanted subunit vaccine encoding the RBD fragment of MERS-CoV S protein containing a C-terminal foldon trimeric motif and a protein adjuvant, rASP-1, in two separate injection sites. 70 Subunit vaccines can also be prepared by combining the genes encoding the MERS-CoV S protein and/or its fragments with those encoding an adjuvant, and then expressing the fusion proteins to increase the vaccine's immunogenicity and protection.

For example, MERS-CoV RBD protein fused with human β-defensin 2 (S RBD-HBD 2) adjuvant induced RBD-specific IgG antibodies and protective immunity against MERS-CoV infection in hDPP4-Tg mice. 59 Notably, the intranasal administration of this protein induced more mucosal IgA antibodies and conferred more effective protection against MERS-CoV challenge than immunization via the intramuscular route.

In addition to single DNA or protein vaccination, combined vaccinations with DNA and proteins may provide broader neutralizing activity. It has been shown that priming with DNA expressing a truncated MERS-CoV S protein (pSΔER) and boosting with a MERS-CoV S protein without the transmembrane region (pSΔTM) increased antibody and cellular immune responses and produced cross-neutralizing antibodies to the wildtype virus as well as pseudotyped variants. 71 This DNA-prime/protein-boost vaccine strategy protected hDPP4-Tg mice against MERS-CoV challenge.

MERS-CoV vaccines based on nanoparticles or virus-like particles have been developed and tested in animal models. A vaccine that was generated by coupling the MERS-CoV RBD to lumazine synthase (LS) nanoparticle (RBD-LS) induced antibodies specific to the RBD protein with neutralizing activity against diverse MERS-CoV clades and prevented MERS-CoV infection in rabbits. 72 

Some non-S-protein-based or combined antigen-based MERS-CoV vaccines have been explored and/or tested in animal models. For example, a multiepitope (MEP) vaccine was designed by combining the conserved epitopes predicted from several MERS-CoV structural (M, E, or N) and non-structural (ORF 1a, 3, 4a, or 8) proteins with an N-terminal adjuvant (β-defensin) in order to increase its immunogenicity potential. 79 Further studies need to be conducted to demonstrate the immunogenicity and/or protection of this predicted peptide vaccine in vivo. In addition, a genetically engineered live-attenuated MERS-CoV vaccine (MERS-CoV-E*Δ2in), which was generated by mutation with partial deletion of the C-terminal domain of E protein using a reverse genetics system, reduced virulence and protected hDPP4-Tg mice from MERS-CoV challenge, without causing apparent histopathological damages in the lungs. 56 Unlike viral vectors or VLPs, mRNA eliminates the possibility to elicit vector/carrier-specific immunogenicity. 92, 93 Generally, mRNA can be readily prepared with rapid production and manufacturing capability. Both non-replicating and self-amplifying approaches have been proposed to generate mRNA vaccines. The former only encodes the target protein antigen, whereas the latter also encodes the RNA genome of an RNA virus (e.g., alphavirus, flavivirus, or picornavirus), which enables RNA replication. 94, 95 Despite numerous advantages, mRNA vaccines face some potential challenges. 96 Unmodified mRNA is intrinsically unstable and can be readily degraded by nucleases. Appropriate strategies have been employed to address mRNA degradation. For example, the addition of a 5'-cap, adjustment of the length and structure of 3'-poly(A) tails, the optimization of nucleoside sequences, and the modification of nucleosides can significantly reduce mRNA degradation. [97] [98] [99] mRNA can also be encapsulated with lipid nanoparticles (LNPs) and other delivery vehicles based on polymers, peptides, and cationic nanoemulsions (CNEs) to increase its stability and prevent degradation. 100, 101 In addition to its instability, unmodified mRNA may activate innate immune systems through the endosomal recognition of Toll-like receptors (TLRs) 7, 8 and 9 and the RIG-1-like receptor family, such as RIG-1, MDA5, and LGP2, resulting in the production of proinflammatory cytokines and type I interferons. 92, 102 To address this concern and further increase translational activity, naturally occurring pseudo-nucleosides, such as pseudouridine, N6-methyladenosine (m6A), and 5-methylcytosine (m5C), may be inserted into the mRNA during synthesis. 103, 104 In addition to these technical challenges in mRNA vaccine production, some adverse effects caused by LNPs were reported for mRNA-based coronavirus disease 2019 (COVID-19) vaccines, 105, 106 highlighting the need for further optimization of the LNP delivery platform.

Since the COVID-19 pandemic began, tremendous efforts have focused on the development of vaccines against SARS-CoV-2, resulting in the emergency use authorization (EUA) of at least three COVID-19 vaccines, two of which are mRNA vaccines, for the prevention of SARS-CoV-2 infection among various ages. [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] However, the mRNA vaccine technology is still in its infancy. So far, only a limited number of studies have reported the design and evaluation of mRNA vaccines against MERS-CoV. Because the RBD fragment in the S1 subunit is a main target for inducing potent neutralizing antibodies that protect against MERS-CoV infection, it can be used as the "core antigen" for mRNA vaccine development. In the following paragraphs, we illustrate the construction, expression, and delivery of RBD-based mRNA vaccines for MERS-CoV.

The genes encoding the MERS-CoV RBD (Fig. 3A) and an N-terminal signal peptide, tissue plasminogen activator (tPA), were amplified using polymerase chain reaction (PCR) and a codon-optimized MERS-CoV S plasmid as a template and inserted into a pCAGGS-mCherry vector, which contains essential elements for the efficient transcription of mRNA in vitro. (Fig. 3E) . The synthesis and encapsulation of MERS-CoV RBD-mRNAs are summarized in Figure 3 .

To illustrate the presentation of mRNA in target cells, mCherry-RBD-mRNA was synthesized as (Fig. 4Aa) . Additionally, a fluorescent (violet) signal was detected in Cy5-labeled mCherry-RBD-mRNA-incubated cells but not in cells incubated with the empty LNPs (Fig. 4Ab) . A timeline of the presence of the Cy5 signal in cells is shown in Figure 4Ac . These findings suggest that mCherry-RBD-mRNA can enter target cells with high efficiency and may be present in target cells for a long period of time, potentially increasing the time for the mRNA to be transcribed into the target protein antigen.

To determine whether mRNA can durably express the target protein in cells, mCherry-RBD-mRNA was incubated with cells for different periods of time, and the fluorescent mCherry signal was measured. Indeed, mCherry-RBD-mRNA had long-term (> 68 h) expression of the target protein in cells, reaching the highest levels at 27-60 h post-incubation, whereas no fluorescence was detected in the empty LNP control (Fig. 4Ba) . Notably, the mCherry signal was detectable in cells transcribed with mCherry-RBD-mRNA but not in cells incubated with the empty LNP control. Furthermore, the mCherry signal co-existed with lysosomes but did not colocalize with nuclei (Fig. 4Bb) , suggesting that RBD-mRNA does not cross the nucleus and may be resistant to lysosomal degradation. A timeline of the expression of mRNA-encoding mCherry protein in cells is shown in Figure 4Bc .

Unmodified MERS-CoV mRNA vaccines can induce a strong, inflammatory-type innate immune response, as previously observed for mRNA vaccines against other pathogens 92, 102 . Thus, naturally occurring pseudo-nucleosides, such as pseudouridine, can be incorporated into mRNA synthesis to reduce the unwanted innate response. For the MERS-CoV RBD-mRNA vaccine, a modified RBD-mRNA-pseudoU was synthesized using pseudouridine as described above and compared with the unmodified RBD-mRNA synthesized using UTP for the ability to reduce inflammatory cytokines in mice (Fig. 5A) . Mice were injected with each mRNA or an empty LNP control, and sera were collected at different time points post-injection to detect cytokine levels. Significantly lower levels of interleukin-6 (IL-6), interferon- (IFN-α), and tumor necrosis factor- (TNF-α) cytokines were detected in mice that received modified RBD-mRNA-pseudoU than the mice that received unmodified RBD-mRNA at 2 or 6 h post-injection, which were similar to the baseline signals observed for the control group in which mice received empty LNPs without mRNA (Fig. 5B ).

To identify an optimal ratio of pseudouridine and UTP in reducing mRNA-associated innate immune activation, pseudouridine was added at 100%, 50%, 25%, 12.5%, and 0%, respectively (with corresponding ratios of 1:0, 1:1, 1:3, 1:7, and 0:1 for pseudouridine and UTP), during the synthesis of RBD-mRNA, and related cytokine levels were detected as described above.

Pseudouridine at 100% (pseudouridine:UTP, 1:0) resulted in the lowest levels of IL-6 and IFN-α, whereas unmodified RBD-mRNA (0% pseudouridine) led to the highest secretion of these cytokines (Fig. 5C) . Similarly, pseudouridine at 100%, 50%, and 25% (pseudouridine:UTP, 1:0-1:3) only had a background level of TNF-α secretion, whereas unmodified RBD-mRNA (0% pseudouridine) led to the highest expression of this cytokine (Fig. 5C) .

The above data suggest that the addition of pseudouridine to mRNA during synthesis completely blocked or significantly reduced the ability of unmodified MERS-CoV RBD-mRNA to activate innate immune pathways. Moreover, the pseudouridine:UTP ratio affected the secretion of inflammatory cytokines. These data confirm the important role of pseudo-nucleosides in reducing or blocking MERS-CoV RBD-mRNA-associated innate immune activation. (Figs 6A, B) . Similarly, RBD-mRNA-pseudoU with 100% pseudouridine elicited MERS-CoV-specific IgG antibodies, which potently neutralized pseudotyped MERS-CoV with titers that were significantly higher than those induced by the unmodified and modified MERS-CoV RBD-mRNAs with varying percentages of pseudouridine (Figs 6C, D) . These data suggest that pseudouridine in MERS-CoV RBD-mRNA vaccines plays an important role in the induction of effective MERS-CoV-specific adaptive immune responses.

A potential working model of the MERS-CoV RBD-mRNA vaccine is summarized in Figure 7 .

LNP-encapsulated, pseudouridine-modified mRNA is delivered into the target cell, where the mRNA is released into the cytoplasm (Fig. 7A) . The mRNA is subsequently translated into the target protein, which is presented into antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages (Fig. 7A ). This process activates MHC class II and MHC class I responses, resulting in the production of antigen-specific CD4 + and CD8 + T cell responses to help B-cell maturation and kill virus-infected cells, respectively. GC B and Tfh cell responses are induced in the GC, and the activated B cells may differentiate into plasma cells to generate antigen-specific neutralizing antibodies (Fig. 7B ). In the absence of MERS-CoV RBD-specific neutralizing antibodies, the MERS-CoV RBD binds to its cellular receptor DPP4, mediating virus entry and subsequent membrane fusion processes (virus infection) (Fig. 7C) . Nevertheless, in the presence of MERS-CoV RBD-specific neutralizing antibodies, these antibodies block binding of the MERS-CoV RBD to the DPP4 receptor, thereby inhibiting subsequent virus entry into the target cell (i.e., virus neutralization) (Fig. 7C) . subunit, and RBD fragment, were found less effective in neutralizing variants with S mutations (e.g., in the RBD and NTD of the S1 subunit). 90, [121] [122] [123] [124] [125] [126] Similarly, MERS-CoV vaccines targeting the prototypic S sequence may have a lower ability to neutralize variant strains. In addition, some MERS-like CoVs that share the same DPP4 receptor as MERS-CoV have been identified in bats, 127 which may have potential to cause infection in humans. Therefore, effective vaccines with broad-spectrum activity against multiple MERS-CoV strains, including their variants in humans, camels, and bats, are needed to prevent MERS-CoV infection.

Different from S1 subunit fragments (e.g., NTD and RBD), the S2 subunit region of MERS-CoV is relatively conserved among different isolates, underscoring its potential as a target for the development of universal MERS-CoV vaccines with broad-spectrum activity. Unlike the MERS-CoV full-length S protein or its domain fragments (S1 and RBD), the S2 region alone generally has low immunogenicity to induce strong immune responses with neutralizing activity against MERS-CoV infection. Thus, novel approaches, such as structure-based design, may be explored for the development of effective S2-based MERS-CoV vaccines to improve immunogenicity and neutralizing activity. Moreover, T cell-based vaccines that target the conserved S2 or other regions in the S protein or other proteins of MERS-CoV, such as the N protein, may have potential to improve the coverage and efficacy of MERS-CoV vaccines against diverse strains.

Such strategies have proven effective in protecting against variant strains of other viral pathogens, including Zika virus and SARS-CoV-2. [128] [129] [130] Similar to the mRNA-based COVID-19 vaccines, which have been developed to target the viral S protein, S1 subunit, and/or RBD fragment, and shown efficacy against SARS-CoV-2 infection, 48, 90, 118, 131 the mRNA-based MERS-CoV vaccines can be designed based on the fulllength S protein or its fragments (e.g., S1) of this protein, in addition to the RBD, and tested for their broad-spectrum ability against multiple virus strains. Additionally, mRNA vaccines synthesized using other pseudo-nucleosides (except pseudouridine) or combinations of different pseudo-nucleosides may be tested to identify the optimal strategy to maximally reduce mRNAassociated innate immune activation, enhance mRNA stability, and increase immunogenicity and neutralizing activity of mRNA vaccines against MERS-CoV infection. Furthermore, elements of mRNA components and coding sequences of MERS-CoV proteins, as well as delivery systems, may be further optimized to improve mRNA stability and overall immunogenicity. MERS-CoV vaccines based on other S fragments (such as S1 subunit) DNA AcHERV-MERS-S1 Induced S1/RBD-specific T-cell Protected hDPP4-Tg mice from 61, 65, 66 

Subunit vaccines against emerging pathogenic human coronaviruses

An overview of Middle East respiratory syndrome coronavirus vaccines in preclinical studies

Therapeutic antibodies and fusion inhibitors targeting the spike protein of SARS-CoV-2

Current advancements and potential strategies in the development of MERS-CoV vaccines

The spike protein of SARS-CoV--a target for vaccine and therapeutic development

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China

Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia

Middle East respiratory syndromes (MERS) situation update

An emerging zoonotic virus

Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus

Bat origins of MERS-CoV supported by bat coronavirus HKU4 usage of human receptor CD26

Replication and shedding of MERS-CoV in Jamaican fruit bats (Artibeus jamaicensis)

Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation

First known person-to-person transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the USA

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Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor

Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4

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Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26

Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein

Identification of an ideal adjuvant for receptorbinding domain-based subunit vaccines against Middle East respiratory syndrome coronavirus

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Structural definition of a neutralization epitope on the N-terminal domain of MERS-CoV spike glycoprotein

Structural definition of a unique neutralization epitope on the receptor-binding domain of MERS-CoV spike glycoprotein

Longevity of Middle East respiratory syndrome coronavirus antibody responses in humans, Saudi Arabia

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Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen

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Cross-protection against MERS-CoV by prime-boost vaccination using viral spike DNA and protein

Particulate multivalent presentation of the receptor binding domain induces protective immune responses against MERS-CoV

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Characterization of the immune response of MERS-CoV vaccine candidates derived from two different vectors in mice

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An orthopoxvirus-based vaccine reduces virus excretion after MERS-CoV infection in dromedary camels

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Structural overview of MERS-CoV spike (S) protein. (A) Cryo-EM structure of MERS

The three S monomers are colored in yellow, blue and light coral, respectively. (B) Close-up views of MERS-CoV receptor-binding domain (RBD) and Nterminal domain (NTD) in the S1 subunit of S protein. (C) Crystal structure of MERS-CoV RBD in complex with the receptor human dipeptidyl peptidase 4 (DPP4) (PDB 4KR0). The human DPP4 is colored in orchid. (D) Crystal structure of MERS-CoV RBD in complex with neutralizing monoclonal antibody (mAb) m336 Fab (PDB 4XAK). The m336 Fab is colored in dark green and blue. (E) Crystal structure of MERS-CoV S1-NTD in complex with neutralizing mAb G2 Fab (PDB 6PXH)

MERS-CoV virion, showing RBD in the spike (S) protein. (B-D) Design of unmodified RBD-mRNA (B), nucleoside (pseudoUTP: pseudoU)-modified RBD-mRNA (RBD-mRNA-pseudoU) (C), and nucleoside (pseudoU)-modified RBD-mRNA containing an Nterminal mCherry sequence (mCherry-RBD-mRNA) (D)

Presentation of mRNA in 293T cells. (a) Cy5-labeled mCherry-RBD-mRNA (1 μg/ml) was incubated with 293T cells at 37°C. The cells were collected at different times post-incubation and analyzed for Cy5-positive cells by flow cytometry. (b) Representative images by confocal microscopy at 27 h post-incubation, with nuclei in blue, cell membrane (pan-Cadherin) in green, and Cy5-mRNA in violet. (c) Schematic images showing the time period of Cy5-labeled mRNA in 293T cells. (B) Detection of expression of mRNA-encoding protein in target cells. (a) Cy5-labeled mCherry-RBD-mRNA (1 μg/ml) was incubated with 293T cells at 37°C. The cells were collected at different times post-incubation and detected for mCherry fluorescence (mRNA expression) by flow cytometry. (b) Representative confocal microscopy images of the expression of mRNA-encoding protein

Lipid nanoparticle (LNP)-encapsulated, nucleoside-modified MERS-CoV RBD-mRNA (pseudoU) was delivered into target cells, where it was released and then translated into the target receptor-binding domain (RBD) protein pcDNA3.1-S1 pS1 responses and/or S-specific IgG and neutralizing Abs in mice against infection of multiple pseudotyped MERS-CoV strains with human and camel origins or live MERS-CoV strain MERS-CoV challenge

Protected Ad5-hDPP4-transduced mice from MERS-CoV infection

Note: Abs, antibodies; BLP, bacterium-like particle; hDPP4, dipeptidyl peptidase 4; hDPP4-KI, human DPP4-knock-in mice; hDPP4-Tg, human DPP4-transgenic mice; LS, lumazine synthase

not available; NHPs, nonhuman primates; PIV5, parainfluenza virus 5; rAd, recombinant adenovirus; RBD, receptor-binding domain

RV, rabies virus

This work was supported by NIH grants (R01AI139092 and R01AI137472). The authors thanks Michael Arends for proofreading the manuscript.The authors have read and agreed to the journal's authorship statement and policy on disclosure of potential conflicts of interest. The authors declare no conflicts of interest. The manuscript has been reviewed by and approved by all authors.