Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2023 Feb 7;41(11):1892–1901. doi: 10.1016/j.vaccine.2023.02.005

Recombinant measles virus encoding the spike protein of SARS-CoV-2 efficiently induces Th1 responses and neutralizing antibodies that block SARS-CoV-2 variants

Hye Won Kwak a,b,c,1, Hyo-Jung Park a,b,1, Seo-Yeon Jung d,1, Eun Young Oh e,1, Sang-In Park f, Yeonhwa Kim e, Hyeong-Jun Park a,b,c, Sohyun Park e, You-Jin Kim g, Hae Li Ko f, Jung-Ah Lee g, Hyeran Won g, Yun-Ho Hwang g, Seo Yeon Kim g, Se Eun Kim g, Seoung Eun Bae h, Minhyuk Yoon h, Jae-Ouk Kim h, Manki Song h, Su Jeen Lee d, Ki-Weon Seo d, Kunse Lee d, Dokeun Kim g, Hun Kim d, Sang-Myeong Lee e,⁎, So-Hee Hong i,⁎, Jae-Hwan Nam a,b,⁎
PMCID: PMC9902292  PMID: 36792434

Abstract

Owing to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, the development of effective and safe vaccines has become a priority. The measles virus (MeV) vaccine is an attractive vaccine platform as it has been administered to children for more than 40 years in over 100 countries. In this study, we developed a recombinant MeV expressing the full-length SARS-CoV-2 spike protein (rMeV-S) and tested its efficacy using mouse and hamster models. In hCD46Tg mice, two-dose rMeV-S vaccination induced higher Th1 secretion and humoral responses than one-dose vaccination. Interestingly, neutralizing antibodies induced by one-dose and two-dose rMeV-S immunization effectively blocked the entry of the α, β, γ, and δ variants of SARS-CoV-2. Furthermore, two-dose rMeV-S immunization provided complete protection against SARS-CoV-2 in the hamster model. These results suggest the potential of rMeV-S as a vaccine candidate for targeting SARS-CoV-2 and its variants.

Keywords: SARS-CoV-2, Measles virus vector, Neutralizing antibody

1. Introduction

In December 2019, a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in Wuhan, China; since then, it has spread rapidly worldwide. The World Health Organization (WHO) declared the coronavirus disease 2019 (COVID-19) outbreak a pandemic on March 11, 2020 [1]. Furthermore, the emergence of new variants of SARS-CoV-2 in the UK, Brazil, South Africa, and India has posed a major threat to global health and the economy [2], [3]. Approved COVID-19 vaccines were effective against the Wuhan strain, at the beginning of the pandemic. However, the emergence of the SARS-CoV-2 variants of concern (VOC) such as Delta (B.1.617.2) and Omicron (B.1.1.529) have caused large outbreaks even in vaccinated populations. Therefore, effective and safe vaccines that prevent the infection and transmission of SARS-CoV-2, as well as its variants, are urgently needed [4].

Most vaccines generally undergo several years of clinical trials, but the COVID-19 vaccine candidates have progressed to clinical phases at an unprecedented rate. Currently, approximately 64.2 % of the global population has received at least one dose of a COVID-19 vaccine, such as mRNA and viral vector vaccines [5].

The live attenuated measles virus (MeV) vaccine is considered one of the safest and most effective vaccines [6]. Over the past 40 years, it has been safely administered to more than 2 billion children without reversion. The MeV vaccine induces potent humoral and cellular immune responses and long-lasting memory responses [7], [8], [9]. The synthesis of mRNA and the translation and replication processes occur in the cytoplasm of host cells; moreover, the genome of MeV does not integrate into the DNA of host cells. In addition, the MeV vector may contain foreign genes of up to 6 kb or more because of helicoidal packaging [10].

The current MeV vaccine can be easily produced on a large scale in most countries and distributed at a low cost through an expanded immunization program. Thus, MeV vector-based vaccines can be rapidly scaled up at a low cost in response to the potential emergence of pandemics. In this milieu, recombinant MeV (rMeV) vectors are an attractive vaccine platform against emerging infectious viruses [10]. At present, several rMeV-based vaccines, including those against Zika, Lassa, and Chikungunya viruses, are in various stages of clinical trials [11], [12], [13], [14].

Most coronaviruses express the spike (S) protein on their surface, which is responsible for receptor binding and membrane fusion [15]. In SARS-CoV-2, the receptor-binding domain (RBD) in the S1 domain specifically recognizes angiotensin-converting enzyme 2 of host cells as its receptor, and the S2 domain mediates virus membrane fusion [16]. Therefore, the S protein of SARS-CoV-2 is a principal target in vaccine design, and several pharmaceutical organizations, including Moderna, Pfizer, and AstraZeneca, have selected the S protein as a target antigen for developing SARS-CoV-2 vaccines [17]. However, to date, only a few studies have demonstrated that an rMeV expressing the S protein of SARS-CoV-2 (rMeV-S) induces effective T helper type 1 (Th1) dominant responses and prevents SARS-CoV-2 infection [18], [19]. Additionally, none of the above studies have demonstrated that neutralizing antibodies induced by the rMeV-S vaccine can effectively block the entry of SARS-CoV-2 variants into host cells.

In this study, we generated an rMeV expressing the full-length S protein of SARS-CoV-2 (i.e., rMeV-S) and tested its potential as a COVID-19 vaccine using homologous or heterologous prime-boosting with the RBD of SARS-CoV-2 linked to the tetanus toxoid P2 (RBD-P2) protein, which has been reported to induce neutralizing antibody responses [20]. Furthermore, we confirmed whether one or two rMeV-S vaccination-induced antibodies could neutralize the α, β, γ, and δ variants of SARS-CoV-2.

2. Methods

2.1. Experimental animals and the immunization schedule

In this study, 6–9-week-old B6.FVB-Tg(CD46)2Gsv/J male mice expressing human CD46 were purchased from Jackson Laboratory and inoculated intraperitoneally (i.p), twice or once (with homologous or heterologous prime-boost), with 1 × 106 plaque-forming units (PFUs) of rMeV-S in a volume of 200 µL (Groups 3 and 4), or subcutaneously (s.c.) with 10 µg of recombinant RBD-P2 adjuvant and 100 µg of alum hydroxide in a volume of 100 µL (Group 4, heterologous prime-boost), at an interval of 3 weeks. Control mice (Group 1) received saline in a volume of 100 µL. Group 2 mice were immunized i.p. only once with rMeV-S1. The mice were sacrificed using CO2 at 5 weeks (Groups 3 and 4) or 4 weeks (Group 2) after the first immunization.

The mice were housed in cages, in groups of five, under controlled conditions of humidity, temperature, and light (12-:12-h light/dark cycles). They were handled according to protocols approved by the Catholic University of Korea, which has an animal facility accredited by the Korean Association for Laboratory Animals (2018–027, August 24, 2018). All experimental procedures were conducted according to the guidelines of the Institutional Animal Care and Use Committee of the Catholic University of Korea (CUK-IACUC-2020–015). The study was conducted in compliance with the ARRIVE guidelines.

Six-week-old female golden Syrian hamsters were acclimatized at the Biosafety level 3 facility and maintained according to protocols approved by the Institutional Animal Care and Use Committee of Jeonbuk National University (No. CBNU 2020–56). The golden Syrian hamsters were immunized (i.p.) twice with a low (1 × 105/hamster) or high dose of rMeV (5 × 105/hamster) at an interval of 3 weeks. Two weeks after the last immunization, the hamsters were mildly anesthetized with isoflurane and intranasally inoculated with 1 × 105 PFUs of SARS-CoV-2 (NCCP43326, Wuhan strain) in a volume of 20 µL. In addition, five hamsters were intranasally inoculated with 20 μL of PBS and designated as uninfected controls. All hamsters were euthanized using CO2 at 4 days post-infection (dpi).

2.2. Cell lines

To generate BHK21 cell line stably expressing T7 RNA polymerase, T7 RNA polymerase plasmids were transfected into BHK21 cells, and T7 RNA polymerase-positive cells were screened using antibiotics-antimycotics. The BHK21 single-cell clones that expressed T7 RNA at high levels were selected using dilution methods. Vero 76 cell line (21587) purchased from the Korean Cell Line Bank was used for viral growth. In addition, Vero cell line (CCL-81) purchased from ATCC was used for the plaque assay. All cell lines were maintained in high-glucose Dulbecco Modified Eagle Medium (Lonza 12-604F) supplemented with 10 % Fetal bovine serum (Serana; S-FBS-US-015) and cultured at 37 °C under 5 % CO2.

2.3. Plasmid vector construction

An rMeV plasmid containing the cDNA sequence of the Moraten measles virus.

vaccine strain (GenBank Accession No.: AF266287.1) and a codon-optimized gene encoding the SARS-CoV-2 S protein (GenBank Accession No.: NC_045512.2) were obtained using gene synthesis (Invitrogen Life Technologies, Regensburg, Germany). The S gene inserted in the rMeV contains mutations at the furin cleavage site to maintain the pre-fusion form of the S protein. A full-length gene sequence of the SARS-CoV-2 S protein, flanked with MluI/AatII binding sites, was amplified using Polymerase Chain Reaction and fully sequenced.

2.4. Recovery of rMeV-S

T7-BHK21 cell line was transfected with 4 µg of MeV genome plasmids with the Open reading frame of the SARS-CoV-2 S protein and the helper plasmids pcDNA3.1-MeV-N (0.4 µg), pcDNA3.1-MeV-P (0.05 µg), and pcDNA3.1-MeV-L (0.1 µg), using TransIT LT1 reagent (Mirus; Catalog No. 2303). The transfected cells were incubated at 43 °C for 3 h to promote MeV cDNA rescue [21]. After 3 days, the transfected cells were trypsinized and overlaid with sub-confluent Vero 76 cells seeded in 10-cm dishes. A few days after the infection, syncytia formation was observed using phase-contrast microscopy, and a single syncytium was isolated to obtain monoclonal rMeV-S. In addition, individual plaques were isolated, and seed stocks were amplified in Vero 76 cells. The viral titer was determined using a plaque assay performed using Vero 76 cells.

2.5. MeV titration

Vero cells were seeded in six-well plates at 4.5 × 105 cells/well and cultured for 18 h. The medium was removed, and the cells were infected with serially diluted rMeV-S. After 1 h of adsorption in an incubator, the medium was removed and overlaid with Minimum Essential Media Eagle agarose medium (Gibco, Grand Island, NY, USA; 61100–061) containing 0.5 % agarose, 0.75 % sodium bicarbonate (Sigma S5761), 3 % FBS, 0.02 M HEPES, 2 mM l-glutamine, and NEAA solution (Sigma M7145). At 8 dpi, the plaques were fixed, and the cells were stained with crystal violet solution for visualization.

2.6. Immunocytochemistry

Vero 76 cells seeded on glass slides were infected with rMeV-S at the multiplicity of infection (MOI) of 1.0. Next, at 2 days post-transfection, the cells were fixed by incubation with 2 % paraformaldehyde in Phospahte Buffered Saline for 20 min at 20 °C–24 °C, and the nuclei were stained with 4,6-diamidino-2-phenylindole. The cells were subsequently incubated with a rabbit anti-SARS-CoV-2 S antibody (Sino Biological; #40150-T62-COV2) or rabbit anti-MeV nucleocapsid protein antibody (Abcam; #23974, 1:2000) for 1 h at 20 °C–24 °C, and then with goat anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher; A11034, 1:5000). Fluorescence images were recorded using an ECLIPSE Ts2-FL microscope (Nikon).

2.7. Western blotting

The detailed protocol of western blotting performed in this study is provided in the Supplementary Materials and Methods.

2.8. Flow cytometry

A detailed description of the flow cytometric analysis is provided in the Supplementary Materials and Methods.

2.9. Enzyme-Linked ImmunoSpot assays of mouse cells

Splenocytes of immunized mice were stimulated with 500 ng/well S1 protein (Sino Biological; Catalog No. 40591-V08B1), 1 µg/well S1 peptide scanning pool mixtures 1 and 2 (Mabtech; Catalog No. 3629–1), or SARS-CoV-2 peptide mixture (ST2, ST3, ST5, and ST7) for 48 h. IL-4- and IFN-γ-secreting cells were detected using the mouse IL-4 ELISpotbasic kit and the mouse IFN-γ ELISpotbasic kit (Mabtech, Nacka Strand, Sweden), respectively. The assays were performed per the manufacturer’s protocol.

2.10. Enzyme-Linked immunosorbent assay

A detailed description of the procedure used to measure antigen-specific IgG1 and IgG2c levels in mouse or cytokines in the splenocyte culture supernatants is provided in the Supplementary Materials and Methods.

2.11. Plaque reduction neutralization assay

The plaque reduction neutralization assay was performed as previously described [20].

Sera from immunized mice or hamsters were serially diluted from 1:10 to 1:5120 in a serum-free medium. Virus–serum mixtures were prepared by mixing 100 PFUs (for hamster serum) or 50 PFUs (for mouse serum) of SARS-CoV-2 with the diluted serum samples, and the mixtures were incubated at 37 °C for 1 h. Vero cells were inoculated with the virus–serum mixtures, and the plates were incubated at 37 °C under 5 % CO2 for 1 h. After virus absorption, an agar overlay medium was added, and the plates were incubated at 37 °C under 5 % CO2 for 2–3 days. The cells were stained with 0.1 % crystal violet solution (Sigma, St. Louis, MO, USA) and the plaques were counted. The percentage of neutralization represented the reduction value, which was calculated as the number of plaques in 100 or 50 PFUs from virus-infected wells/number of plaques in the virus–serum mixture-infected wells (×100). All virus strains used in the experiments were officially purchased from the National Culture Collection for Pathogens (NCCP) of the National Institutes of Pathogens (NIP) affiliated with the National Institutes of Health (NIH) in Korea (Supplementary Table 1).

2.12. SARS-CoV-2 spike-pseudotyped vesicular stomatitis virus (VSV) neutralization assay

To construct the SARS-CoV-2 spike-pseudotyped VSV, the spike gene (SΔER, encoding amino acids 1–1254) of the Wuhan-Hu-1 strain (GenBank Accession No. YP_009724390.1) was synthesized using human codon optimization (GenScript, Piscataway, NJ, USA). Next, the S gene without 19C-terminal amino acids (SΔER) of the B.1.617.2 strain (T19R, G142D, del157/158, L452R, T478K, D614G, P681R, and D950N) was generated using site-directed mutagenesis (Agilent, Santa Clara, CA, USA). Each SΔER gene was cloned into the eukaryotic expression plasmid pCAGGS-Kan (Kerafast), and BHK-21/WI-2 cells (Kerafast, Boston, MA, USA) were transfected with 16 µg of these DNA plasmids in 100-mm dishes using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. After 24 h, the transfected cells were infected with G*ΔG-Luciferase (Kerafast) with an MOI of 4; at 1 h after infection, the cells were washed, and the medium was replaced with fresh DMEM (+5% FBS, without antibiotics). Next, at 24 h post-infection, culture supernatants containing SARS-CoV-2 spike-pseudotyped VSV were harvested, filtered (pore size: 0.45 μm; Millipore, Burlington, MA, USA), and stored at −80 °C until use [22]. First, the pseudovirus showing 2 × 106 RLU was incubated with serial dilutions of mouse serum for 1 h at room temperature. Then, the pseudovirus-serum mixture was added to HEK-293 T-hACE2 cells showing 90 % confluence in a 96-well plate. After 24 h of incubation at 37 °C under 5 % CO2, the cells were lysed, and luminescence was measured using a luciferase assay system (Promega, Madison, WI, USA) with a SpectraMax L Microplate Reader (Molecular Devices, San Jose, CA, USA). The percent neutralization was converted using GraphPad Prism 9.0 (GraphPad, San Diego, CA, USA).

2.13. Viral titer quantification

The viral titer in lung samples and nasopharyngeal swabs was quantified using reverse transcription-quantitative PCR (RT-qPCR), and the TCID50 value was determined. Lung samples were homogenized using a Precellys Homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) in RNAiso Plus (TAKARA, Japan). After centrifugation, the supernatants were directly used to inoculate Vero cells, which were incubated for 3 days at 37 °C to calculate the TCID50/mL. Viral RNA was extracted from the supernatants of swabs and tissue samples using a QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD, USA). RT-qPCR was conducted with a primer/probe set to detect ORF1b, as described previously [23]. Viral RNA copy number of the swab and tissue samples was determined using the SARS-CoV-2 RNA standard sample, which was run in parallel with RT-qPCR.

2.14. S peptide mixture

Detailed information on the peptides used in this study is provided in the Supplementary Materials and Methods.

2.15. Statistical analyses

Statistical analyses were performed using GraphPad Prism 8 (GraphPad) and SPSS for Windows (release 14.0 K; SPSS Inc., Chicago, IL, USA). The one-way analysis of variance (ANOVAs) with Tukey’s post hoc tests was used to compare multiple groups, and unpaired t-tests were used for comparison between groups. Differences were considered statistically significant at P less than 0.05. Data are expressed as mean ± standard deviation (SD).

3. Results

3.1. Generation and characterization of rMeV-S

The S protein of SARS-CoV-2 (Wuhan-1 strain) was selected as an antigen to be expressed by the rMeV because it has been shown to induce humoral and cellular immune responses potently. A codon-optimized full-length gene encoding the SARS-CoV-2 S protein, which has three mutations, was cloned downstream of the phosphoprotein sequence in the MeV genome (Fig. 1 a). The Furin cleavage site of the S gene was mutated from PRRAR (at 681–685 residues) to PGSAG to maintain a pre-fusion form of the spike protein. To increase the protein expression and thermal stability, the six amino acids at positions 817, 892, 900, 942, 986, and 987 were replaced with proline (F817P, A892P, A900P, A942P, A942P, K986P, and V987P). In addition, the residue of the spike protein was replaced from Asp to Gly (D614G mutation) at position 614 because this D614G mutation increases the affinity to the ACE receptor.

Fig. 1.

Fig. 1

Open in a new tab

Characterization of recombinant measles virus (rMeV) expressing the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (a) Strategy for the insertion of the SARS-CoV-2 S gene into the measles virus (MeV) genome. The codon-optimized full-length S sequence was amplified using polymerase chain reaction (PCR) and inserted into the same position at the gene junction between sequences encoding the P and M proteins in the genome of the Moraten MeV vaccine strain. (b) Plaque morphology of rMeV expressing SARS-CoV-2 S antigens. Plaques developed after 5 days of incubation in Vero CCL-81 cells. (c) Analysis of SARS-CoV-2 S protein expression in cell lysate and supernatants using western blotting. (d) Analysis of SARS-CoV-2 S protein expression using immunohistochemistry.

Recombinant viruses were successfully generated and amplified until Passage 10 in Vero cells, with titers of up to 3 × 106 PFUs/mL. The peak titer of rMeV-S was observed at 4 dpi in Vero 76 cells (Supplementary Fig. 1). The stability of rMeV-S genome was demonstrated by sequencing the PCR product of rMeV-S in Passage 2 or Passage 10. However, no mutation was observed until P10 (data not shown). Several plaques were detected in the rMeV-S- and Moraten measles strain-infected groups (Fig. 1b). To verify the SARS-CoV-2 S protein expression level, Vero 76 cells infected with rMeV-S were subjected to western blot analysis. As indicated in Fig. 1c, the S protein (160 kDa) was produced by the respective rMeV vector-infected cells, consistent with the predicted molecular weight (Fig. 1c). The expression of the S protein of SARS-CoV-2 and the nucleocapsid protein of MeV in Vero 76 cells was also confirmed using immunofluorescence staining (Fig. 1d).

3.2. rMeV-S immunization induces effective neutralizing antibodies against SARS-CoV-2 α, β, γ, and δ variants

First, we analyze the humoral responses induced by rMeV-S immunization. To compare the immunization efficacy of rMeV-S depending on the dose of immunization, human CD46 transgenic B6 male mice, which are known to exhibit semi-permissiveness to MeV [24], were injected i.p. once (mG2) or twice (mG3) with 1 × 106 PFU of rMeV-S. Although immune-compromised interferon-α-receptor-deficient mice expressing hCD46 (IFNARCD46tg) are usually used for MeV research, we used the immune-competent hCD46tg mice model in this study to evaluate meaningful immune responses induced by vaccination. As MeV immunization is primarily passive and the virus does not replicate in CD46Tg mice, we used rMeV-S at high PFUs. In addition, one group (mG4) of mice was boosted subcutaneously with RBD-P2 at 3 weeks after rMeV-S priming to evaluate the efficacy of the heterologous prime-boost strategy (Fig. 2 a). Interestingly, high IgG2c levels were observed in three groups of mice (mG2, mG3, and mG4) at 3 or 4 weeks after the first immunization with rMeV-S. However, the IgG1 levels were relatively lower than the IgG2c levels (Fig. 2b), even after the second immunization (Fig. 2c). We quantified the S specific-IgG1 and IgG2c antibody levels by serial dilution of serum. As shown in supplement Fig. 2, mG3, which received the two-dose of rMeV-S immunization, showed slightly increased S1-specific IgG1 and IgG2c antibody levels than mG2.

Fig. 2.

Fig. 2

Open in a new tab

Analysis of humoral responses induced by recombinant measles virus (rMeV) expressing the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (a) Overview of the experimental groups and immunization schedule of mice; 6–9-week-old B6.FVB-Tg(CD46)2Gsv/J male mice expressing human CD46 were inoculated intraperitoneally (i.p.), twice or once (homologous or heterologous prime-boost), with 1 × 106 plaque-forming units (PFUs) of the rMeV vector expressing the S protein of SARS-CoV-2 (rMeV-S) in a volume of 200 µL (Group 3 and 4), or subcutaneously (s.c.) with 10 µg of recombinant receptor-binding domain (RBD)-P2 protein adjuvanted with 100 µg of alum hydroxide in a volume of 100 µL (Group 4, heterologous prime-boost), at an interval of 3 weeks. Group 2 mice were immunized i.p. only once with rMeV-S. The mice were sacrificed at 5 weeks (Groups 3 and 4) or 4 weeks (Group 2) after the first immunization. (b) The spike protein-specific IgG1 and IgG2 levels in 1:50 diluted serum were measured at 2 or 3 weeks after priming. (c) The spike protein-specific IgG1 and IgG2 levels in 1:50 diluted serum were measured at 2 weeks after the boost. (d) GL7+CD19 population in the spleen was assessed using flow cytometry. (e) SARS-CoV-2-neutralizing activity of serum from immunized mice against the Wuhan, α, β, γ, and δ strains (reduction rate) was determined using the standard plaque reduction neutralization test (PRNT50). The five samples were pooled and analyzed in triplicates. Data represent mean ± standard deviation (SD). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.

We also quantified GL7+ germinal center B cells using flowcyometry, as they play critical roles in the generation of high-affinity antibody-producing cells. The frequency of GL7+CD19+ was also the highest in mice that received two doses of rMeV-S (mG3; Fig. 2d).

Next, we confirmed whether rMeV-S could induce neutralizing antibodies against the SARS-CoV-2 Wuhan-1 strain and other variants (α, β, γ, and δ). High PRNT50 titers were observed in all groups of mice immunized with rMeV-S against the Wuhan-1 strain. However, there was a difference in the magnitude (Fig. 2e and Supplementary Fig. 2). Notably, the PRNT50 titers of mice that received one dose (mG2) or two-doses of rMeV-S(mG3) against the Wuhan-1 strain were not significantly different (Fig. 2e, and Supplementary Fig. 3). We also tested the neutralizing antibody titers against the Wuhan-1 and Omicron strains using a pseudovirus neutralizing assay. Both one- and two-dose rMeV-S immunization induced similar high level neutralization titers against Wuhan-1 strain, but boosting with RBD-P2 marginally reduced the neutralization antibody titers (Supplementary Fig. 4). Similar to other approved COVID-19 vaccines targeting the Wuhan-1 strain, such as BNT 162b2 and mRNA1273, neutralization titers against Omicron were significantly reduced in both one- and two-dose rMeV-S-immunized groups. The VNT50 titers of one-dose immunized group was 200 and slightly higher level of VNT50 titers were detected in two-dose rMEV-S immunized group although it is not statistically significant (GMT = 46.4 against mG1, 200 against mG2, 251.9 against mG3, 125.9 against mG4)(Supplementary Fig. 5).Although the PRNT50 titers against the β, γ, and δ variants were reduced in mice that received two doses of rMeV-S (mG3), they were still significant (average PRNT50 = 640 against β, 533 against γ, and 1,066 against δ; Fig. 2e). Compared with the mG3 group, the mG2 and mG4 groups exhibited a relatively small decrease in PRNT50 titers against the β, γ, and δ strains (Fig. 2e and Supplementary Fig. 3).

3.3. rMeV-S immunization elicits potent Th1 cell-mediated immune responses

As T-cell-mediated immune responses are crucial for the effective clearance of SARS-CoV-2 infection, we evaluated whether rMeV-S provokes T-cell immune responses. hCD46 Tg mice were immunized as described in Fig. 3 a. As shown in Fig. 3b, the number of IFN-γ-producing cells was the highest in mice that received two doses of rMeV-S (mG3) upon S1 protein, four-peptide mixture, and S1-1 and S1-2 peptide pool stimulation, elucidated in a previous report and the Methods section [25]. A one-dose or heterologous rMeV-S prime and RBD-P2 boost also increased the number of IFN-γ-producing cells, but to a less extent than the two-dose rMeV-S prime (Fig. 3b). Compared with that induced by one-dose rMeV-S administration, boosting with RBD-P2 did not significantly increase the number of IFN-γ-producing cells (Fig. 3b). Similarly, the frequency of IFN-γ-producing cells among CD8+ T cells increased only in mice that received two doses of rMeV-S upon four-peptide mixture stimulation (Supplementary Fig. 6a). No significant increase was observed in the number of IFN-γ-producing cells among CD4+ T cells (data not shown). However, the number of IL-4-producing cells increased only in mice administered heterologous rMeV-S and RBD-P2 upon S1-2 peptide pool stimulation (Fig. 3c). Next, we assessed the frequency of granzyme B- and perforin-producing CD8+ T cells to determine whether rMeV-S induces cytotoxic T-cell responses. Notably, the granzyme B- and perforin-producing CD8+ T cell populations were significantly increased only in mice that received two doses of rMeV-S upon four-peptide mixture stimulation (Fig. 3d and 3e). The expression of CD25, an activation marker of T cells, increased in the CD8+ T cells of mice that received two doses of rMeV-S upon four-peptide mixture stimulation (Fig. 3f). Interestingly, CD69 expression in CD4+ T cells increased in mice that received heterologous rMeV-S priming and RBD-P2 boosting upon S1 protein stimulation, although the difference was not significant. CD69 expression in CD8+ T cells increased marginally in mice that received two doses of rMeV-S (mG3) and mice subjected to heterologous rMeV-S priming/RBD-P2 boosting (mG4) upon protein stimulation. Upon peptide stimulation, a significant increase of CD69 expression in CD8+ T cells was only detected in mice that received two doses of rMeV-S. (Supplementary Fig. 6b and 6c). The levels of IL-6 in the culture supernatants of splenocytes increased substantially in mice that received two doses of rMeV-S upon stimulation with the S1 protein or four-peptide mixture (Supplementary Fig. 7a). The levels of TNF-α in the supernatants also significantly increased in mice that received two doses of rMeV-S upon four-peptide mixture stimulation (Supplementary Fig. 7b). The IL-2 level in the supernatants increased in mice that received two doses of rMeV-S, but this increase was not four-peptide mixture or S1 protein specific (Fig. 3c).

Fig. 3.

Fig. 3

Open in a new tab

Analysis of T-cell responses induced by recombinant measles virus (rMeV) expressing the spike protein of SARS-CoV-2. (a) Overview of the experimental groups and immunization schedule of mice. (b) The number of SARS-CoV-2-S1 proteins (ST2, ST3, ST5, and ST7 pool peptides, and S1-1 and S1-2 pool peptides specific to IFN-γ-producing cells in splenocytes) was measured using the ELISPOT assay (n = 5 mice). (c) The number of SARS-CoV proteins and S1 and S2 peptides specific to IL-4-producing cells in splenocytes was determined using the ELISPOT assay (n = 5 mice). (d, e) Granzyme B- and perforin-producing CD8+ T cells in the spleen analyzed using flow cytometry. (f) CD25 expression in CD8+ T cells in the spleen examined using flow cytometry. Data represent mean ± standard deviation (SD). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.

3.4. rMeV-S immunization provides complete protection against SARS-CoV-2 challenge in hamsters

Finally, we tested the protective capacity of rMeV-S-induced immune responses against SARS-CoV-2. Syrian golden hamsters were immunized i.p. with 1 × 105 PFU (low dose) or 5 × 105 PFU (high dose) rMeV-S twice at 3-week intervals and challenged with 1 × 105 PFUs of SARS-CoV-2 at 2 weeks after the booster dose (Fig. 4 a). As the viral load in the lungs was observed to peak at approximately 2–4 dpi in previous studies [26], [27], we sacrificed the Syrian golden hamsters 4 days after the challenge. As shown in Fig. 4b, the lung weight of low- or high-dose rMeV-S-immunized mice did not increase compared with that of non-vaccinated mice. Importantly, rMeV-S vaccination provided complete protection against SARS-CoV-2, even at a low dose (1 × 105 PFUs of rMeV-S). As weight loss became apparent only 6–7 days after infection, we could not observe significant weight loss in this experiment.

Fig. 4.

Fig. 4

Open in a new tab

Immunization with recombinant measles virus expressing the spike protein of SARS-CoV-2 (rMeV-S) provides complete protection to hamsters against SARS-CoV-2 infection. (a) Overview of the experimental groups and the immunization and virus challenge schedule. (b) Weights of the lungs from each group of hamsters. (c, d) Real-time polymerase chain reaction (PCR) of viral genomic RNA in the nasal wash and lung tissues. (e) The SARS-CoV-2-neutralizing activity of serum from immunized hamsters at the sacrifice time point was analyzed using the 7-standard plaque reduction neutralization test (PRNT50). (f) Representative images of hematoxylin and eosin-stained lung tissue sections. B, bronchus or bronchi; V, blood vein; P, pulmonary emphysema. Arrows indicate inflammatory cells (neutrophils and lymphocytes), arrowheads indicate hemorrhage, and asterisks indicate exudate.(g) Pathological scores of the lungs at 4 days after the challenge. (h) Representative histological images of hematoxylin and eosin-stained tracheal tissues. L, lumen of the trachea; C, hyaline cartilage; E, respiratory epithelium. Arrows indicate inflammatory cells (neutrophils and lymphocytes), arrowheads indicate hyperplasia, asterisks indicate tracheal mucosa, and crosses indicate focal erosion. (i) Pathological scores of the trachea at 4 days after the challenge. Data represent mean ± standard deviation (SD). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.

The viral titers in the nasal and lung samples of the vaccinated hamsters (hG3 and hG4), which received low or high doses of rMeV-S, were not detected (Fig. 4c and 4d). Both low- and high-dose rMeV-S-immunized groups showed significant levels of neutralizing antibody titers (average PRNT50: 320; Fig. 4e).

Next, we assessed the histological changes in all lung and trachea samples from the hamster challenge study. At 4 days post-challenge, all lung tissues from the SARS-CoV-2-inoculated control group (hG2) showed extremely severe histopathological changes, characterized by parenchymal inflammation, bronchiolitis, lymphocyte infiltration, and epithelial hyperplasia (Fig. 4f and 4g). In contrast, no significant changes were observed in the lung and trachea tissues between low-dose or high-dose rMeV-S-immunized hamsters (hG3 and hG4) (Fig. 4h and 4i).

4. Discussion

Here, we demonstrated the potential of an rMeV expressing the full-length SARS-CoV-2 S protein as a SARS-CoV-2 vaccine candidate. As reported previously, rMeV-S induced strong Th1-biased immune responses. While high levels of IgG2c were observed, relatively lower levels of IgG1 were observed in mice that received one or two dose(s) of rMeV-S. We also tested the efficacy of heterologous rMeV-S prime and RBD-P2 protein boost strategies, as we observed in a previous study that RBD-P2 immunization induces protective immunity in nonhuman primates [20]. Boosting with RBD-P2 appears insufficient for reversing Th1-biased humoral immune responses because the IgG1 level did not increase significantly in heterologous prime-boosting groups, although boosting with RBD-P2 increased the number of IL-4-producing cells in the spleen. The S protein has been shown to induce high immunogenicity in mice. The comparatively poor immunogenicity of RBD is considered to be associated with limited germinal center and T follicular helper cell activities. In contrast, both S and RBD vaccines showed comparable immunogenicity in macaques, eliciting serological neutralizing activity [28]. Therefore, boosting with a higher dose of RBD-P2 or the full-length S protein may promote T helper type 2 (Th2) immune responses. Reportedly, patients with moderate COVID-19 symptoms have higher proportions of IFN-γ-producing Th1 cells than patients with a severe disease [29]. Although it has been shown that SARS-CoV-1 vaccine-induced Th2 cell responses are associated with the enhancement of lung disease owing to the induction of antibody-dependent enhancement (ADE) [30], the role of Th2 responses in patients with severe COVID-19 remains unclear, and patients with a mild disease may exhibit normal Th2 responses [31]. Thus, further studies must confirm whether biased Th1 responses would be advantageous for vaccine-induced protection.

We also demonstrated that rMeV-S-induced antibodies could neutralize α, β, δ, γ, and Omicron SARS-CoV-2 variants to varying degrees. Several variants of SARS-CoV-2 have emerged, which is a concern because of the potential for increased transmissibility, virulence, and resistance to neutralization [32]. Evasion of neutralizing antibodies induced by mRNA1273 or BNT162b2 is less propound in early variants, but greater in later variants [4]. Especially Omicron escapes the majority of neutralizing antibodies, and it has been reported that at least three doses of BNT162b2 are required to induce neutralizing capacity against Omicron [33]. Similarly, antibodies induced by our rMeV-S showed a significantly reduced neutralizing ability against Omicron. Thus, we expect that three-dose rMeV-S immunization is required to induce more potent neutralizing antibodies against Omicron.

We expect that the rMeV-S vaccine, which contains the S sequence of Omicron, would be more effective in the current pandemic situation as it induces neutralizing antibodies specific to the Omicron strain. However, if another variant with a substantially different S1 region emerges, it could escape the neutralizing antibodies. Thus, it would be worthy to develop rMeV-S that contains a conserved neutralizing epitope of S1 to induce broad-spectrum protection. Furthermore, the rMeV-vaccine that contains rarely mutated proteins, such as the nucleocapsid protein, is another alternative [34].

Herein, we showed that a two-dose rMeV-S immunization regimen confers complete protection against SARS-CoV-2 (Wuhan) in the hamster model.

We also tested the protective efficacy of intramuscularly administrated one dose of rMeV-S (data not shown). Interestingly, one-dose rMeV-S immunization significantly reduced the viral titers and expression of the nucleocapsid protein of SARS-CoV-2 in the lungs after the SARS-CoV-2 challenge. In addition, the viral titers and N protein expression in one-dose rMeV-immunized hamsters were not significantly different from those in two-dose rMeV-immunized hamsters.

Another strength of rMeV-S as an SARS-CoV-2 vaccine is its strong T-cell immunogenicity. A recent study revealed that some variants could partially escape humoral immunity induced by SARS-CoV-2 infection or BNT162b2 vaccination, but S-specific CD4+ T-cell activation is unaffected by mutations in B.1.1.7 and B.1.351 variants [35]. Furthermore, while SARS-CoV-1-specific antibodies are undetectable at 6 years post-infection in individuals who recovered from SARS-CoV-1 infection, SARS-CoV-specific memory T cells persist for up to 11 years following recovery [36]. Therefore, rMeV-S-induced strong T-cell immune responses may countervail SARS-CoV-2 variants with a long-term memory.

Additionally, rMeV-S can be applied to children less than 18 years of age. The safety of the Measle, mumps and rubblla vaccine has been confirmed by administering it to infants. Although the FDA authorized the Pfizer COVID-19 vaccine for use in children aged 12–15 years, rare cases of myocarditis and pericarditis have been recorded mainly in male adolescents and young adults [37]. As the safety of the MeV platform has already been confirmed in children, we expect that rMeV-S will be suitable for use in children and young adults. In summary, these preliminary results warrant further evaluation of rMeV-S as a COVID-19 vaccine candidate.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Acknowledgments

This work was supported by the Research Investment for Global Health Technology Fund [RIGHT Fund]; SK Bioscience [grant numbers M-2020-D0731-0001, M-2020-D0732-0003]; the Korea Health Industry Development Institute (KHIDI) funded by the Ministry for Health and Welfare, Republic of Korea [grant number KHIDIHV21C0020020021]; the National Research Foundation (NRF) funded by the Ministry of Science and ICT, Republic of Korea [grant number 2021M3E5E3080558]; and the Brain Korea 21 Plus Program.

Author contributions

J.-H. N., S.-M. L., and S.-H.H. conceived and supervised the research, designed the experiments, and edited the manuscript. S.-Y.J., H.-J.P., H.W.K., E.Y.O., Y.K., S.Y.P., Y.-J.K., S.-I.P., H.L.K., H.-J.P., J.-A.L., H.W., Y.-H.H., S.Y.K., S.E. K., S.E.B., M.Y., J.-O.K., M.S., S.J. L., K.-W.S., K.L., D.K., H.K., and S.-M. L performed data acquisition. S.-H.H. wrote the manuscript, and H.-J. P. assisted in drafting the manuscript. All authors have read and approved the final version of the manuscript.

Data availability

All major data are available in the text and the Supplementary Figures. Certificates of analysis and origins, material data sheets, and detailed procedures are available from the corresponding author on request.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2023.02.005.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (124.7KB, docx)
Supplementary data 2
mmc2.docx (3.5MB, docx)

Data availability

Data will be made available on request.

References

  • 1.Hu B., Guo H., Zhou P., Shi Z.L. Characteristics of SARS-CoV-2 and COVID-19. Nat Rev Microbiol. 2021;19:141–154. doi: 10.1038/s41579-020-00459-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Davies N.G., Abbott S., Barnard R.C., Jarvis C.I., Kucharski A.J., Munday J.D., et al. Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England. Science. 2021;372:eabg3055. doi: 10.1126/science.abg3055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Gonzalez-Candelas F., Shaw M.A., Phan T., Kulkarni-Kale U., Paraskevis D., Luciani F., et al. One year into the pandemic: short-term evolution of SARS-CoV-2 and emergence of new lineages. Infect Genet Evol. 2021;92 doi: 10.1016/j.meegid.2021.104869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Sean Wei Xiang Ong TC, Young BE. SARS-CoV-2 variants of concern and vaccine escape, from Alpha to Omicron and beyond. Expert Rev Respir Med. 2022;16(5):499-502. 10.1080/17476348.2022.2057299. [DOI] [PMC free article] [PubMed]
  • 5.https://covid19.trackvaccines.org/; 2022 [accessed 2020].
  • 6.Frantz P.N., Teeravechyan S., Tangy F. Measles-derived vaccines to prevent emerging viral diseases. Microbes Infect. 2018;20:493–500. doi: 10.1016/j.micinf.2018.01.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.De Quadros C.A., Izurieta H., Carrasco P., Brana M., Tambini G. Progress toward measles eradication in the region of the Americas. J Infect Dis. 2003;187(Suppl 1):S102–S110. doi: 10.1086/368032. [DOI] [PubMed] [Google Scholar]
  • 8.Ovsyannikova I.G., Dhiman N., Jacobson R.M., Vierkant R.A., Poland G.A. Frequency of measles virus-specific CD4+ and CD8+ T cells in subjects seronegative or highly seropositive for measles vaccine. Clin Diagn Lab Immunol. 2003;10:411–416. doi: 10.1128/cdli.10.3.411-416.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Griffin D.E., Lin W.H., Pan C.H. Measles virus, immune control, and persistence. FEMS Microbiol Rev. 2012;36:649–662. doi: 10.1111/j.1574-6976.2012.00330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Tangy F., Naim H.Y. Live attenuated measles vaccine as a potential multivalent pediatric vaccination vector. Viral Immunol. 2005;18:317–326. doi: 10.1089/vim.2005.18.317. [DOI] [PubMed] [Google Scholar]
  • 11.Ramsauer K., Schwameis M., Firbas C., Mullner M., Putnak R.J., Thomas S.J., et al. Immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial. Lancet Infect Dis. 2015;15:519–527. doi: 10.1016/S1473-3099(15)70043-5. [DOI] [PubMed] [Google Scholar]
  • 12.Reisinger E.C., Tschismarov R., Beubler E., Wiedermann U., Firbas C., Loebermann M., et al. Immunogenicity, safety, and tolerability of the measles-vectored chikungunya virus vaccine MV-CHIK: a double-blind, randomised, placebo-controlled and active-controlled phase 2 trial. Lancet. 2019;392:2718–2727. doi: 10.1016/S0140-6736(18)32488-7. [DOI] [PubMed] [Google Scholar]
  • 13.Mateo M., Reynard S., Carnec X., Journeaux A., Baillet N., Schaeffer J., et al. Vaccines inducing immunity to Lassa virus glycoprotein and nucleoprotein protect macaques after a single shot. Sci Transl Med. 2019;11:eaaw3163. doi: 10.1126/scitranslmed.aaw3163. [DOI] [PubMed] [Google Scholar]
  • 14.Nurnberger C., Bodmer B.S., Fiedler A.H., Gabriel G., Muhlebach M.D. A measles virus-based vaccine candidate mediates protection against Zika virus in an allogeneic mouse pregnancy model. J Virol. 2019;93:e01485–e01518. doi: 10.1128/JVI.01485-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Huang Y., Yang C., Xu X.F., Xu W., Liu S.W. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin. 2020;41:1141–1149. doi: 10.1038/s41401-020-0485-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Chambers J.P., Yu J., Valdes J.J., Arulanandam B.P. SARS-CoV-2, early entry events. J Pathog. 2020;2020:9238696. doi: 10.1155/2020/9238696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kyriakidis N.C., Lopez-Cortes A., Gonzalez E.V., Grimaldos A.B., Prado E.O. SARS-CoV-2 vaccines strategies: a comprehensive review of phase 3 candidates. npj Vaccines. 2021;6:28. doi: 10.1038/s41541-021-00292-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Horner C., Schurmann C., Auste A., Ebenig A., Muraleedharan S., Dinnon K.H., 3rd, et al. A highly immunogenic and effective measles virus-based Th1-biased COVID-19 vaccine. Proc Natl Acad Sci U S A. 2020;117:32657–32666. doi: 10.1073/pnas.2014468117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lu M, Dravid P, Zhang Y, Trivedi S, Li A, Harder O, et al. A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike. Proc Natl Acad Sci U S A. 2021;118:e2026153118. 10.1073/pnas.2026153118. [DOI] [PMC free article] [PubMed]
  • 20.Hong S.H., Oh H., Park Y.W., Kwak H.W., Oh E.Y., Park H.J., et al. Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates. Sci Adv. 2021;7:eabg7156. doi: 10.1126/sciadv.abg7156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Parks C.L., Lerch R.A., Walpita P., Sidhu M.S., Udem S.A. Enhanced measles virus cDNA rescue and gene expression after heat shock. J Virol. 1999;73:3560–3566. doi: 10.1128/JVI.73.5.3560-3566.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Whitt M.A. Generation of VSV pseudotypes using recombinant DeltaG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines. J Virol Methods. 2010;169:365–374. doi: 10.1016/j.jviromet.2010.08.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Chu D.K.W., Pan Y., Cheng S.M.S., Hui K.P.Y., Krishnan P., Liu Y., et al. Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia. Clin Chem. 2020;66:549–555. doi: 10.1093/clinchem/hvaa029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Blixenkrone-Moller M., Bernard A., Bencsik A., Sixt N., Diamond L.E., Logan J.S., et al. Role of CD46 in measles virus infection in CD46 transgenic mice. Virology. 1998;249:238–248. doi: 10.1006/viro.1998.9301. [DOI] [PubMed] [Google Scholar]
  • 25.Lee Y.S., Hong S.H., Park H.J., Lee H.Y., Hwang J.Y., Kim S.Y., et al. Peptides derived from S and N proteins of severe acute respiratory syndrome coronavirus 2 induce T cell responses: a proof of concept for T cell vaccines. Front Microbiol. 2021;12 doi: 10.3389/fmicb.2021.732450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Sia S.F., Yan L.M., Chin A.W.H., Fung K., Choy K.T., Wong A.Y.L., et al. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters. Nature. 2020;583:834–838. doi: 10.1038/s41586-020-2342-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Yang M.S., Oh B.K., Yang D., Oh E.Y., Kim Y., Kang K.W., et al. Ultra- and micro-structural changes of respiratory tracts in SARS-CoV-2 infected Syrian hamsters. Vet Res. 2021;52:121. doi: 10.1186/s13567-021-00988-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Tan H.X., Juno J.A., Lee W.S., Barber-Axthelm I., Kelly H.G., Wragg K.M., et al. Immunogenicity of prime-boost protein subunit vaccine strategies against SARS-CoV-2 in mice and macaques. Nat Commun. 2021;12:1403. doi: 10.1038/s41467-021-21665-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Chen G., Wu D., Guo W., Cao Y., Huang D., Wang H., et al. Clinical and immunological features of severe and moderate coronavirus disease 2019. J Clin Invest. 2020;130:2620–2629. doi: 10.1172/JCI137244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Bolles M., Deming D., Long K., Agnihothram S., Whitmore A., Ferris M., et al. A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge. J Virol. 2011;85:12201–12215. doi: 10.1128/JVI.06048-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Laing AG, Lorenc A, Del Molino Del Barrio I, Das A, Fish M, Monin L, et al. A dynamic COVID-19 immune signature includes associations with poor prognosis. Nat Med. 2020;26:1623-35. 10.1038/s41591-020-1038-6. [DOI] [PubMed]
  • 32.Khateeb J., Li Y., Zhang H. Emerging SARS-CoV-2 variants of concern and potential intervention approaches. Crit Care. 2021;25:244. doi: 10.1186/s13054-021-03662-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Wratil P.R., Stern M., Priller A., Willmann A., Almanzar G., Vogel E., et al. Three exposures to the spike protein of SARS-CoV-2 by either infection or vaccination elicit superior neutralizing immunity to all variants of concern. Nat Med. 2022;28:496–503. doi: 10.1038/s41591-022-01715-4. [DOI] [PubMed] [Google Scholar]
  • 34.Zhao F., Zai X., Zhang Z., Xu J., Chen W. Challenges and developments in universal vaccine design against SARS-CoV-2 variants. npj Vaccines. 2022;7:167. doi: 10.1038/s41541-022-00597-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Geers D., Shamier M.C., Bogers S., den Hartog G., Gommers L., Nieuwkoop N.N., et al. SARS-CoV-2 variants of concern partially escape humoral but not T-cell responses in COVID-19 convalescent donors and vaccinees. Sci Immunol. 2021;6:eabj1750. doi: 10.1126/sciimmunol.abj1750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Ng O.W., Chia A., Tan A.T., Jadi R.S., Leong H.N., Bertoletti A., et al. Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection. Vaccine. 2016;34:2008–2014. doi: 10.1016/j.vaccine.2016.02.063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Das B.B., Moskowitz W.B., Taylor M.B., Palmer A. Myocarditis and pericarditis following mRNA COVID-19 vaccination: what do we know so far? Children (Basel) 2021;8 doi: 10.3390/children8070607. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary data 1
mmc1.docx (124.7KB, docx)
Supplementary data 2
mmc2.docx (3.5MB, docx)

Data Availability Statement

Data will be made available on request.

Articles from Vaccine are provided here courtesy of Elsevier

RESOURCES