CoChat
Sign Up
Cited by 132RNA Interference and Gene DeliveryInfluenza Virus Research StudiesImmunotherapy and Immune Responses

The DOI matches its Crossref record; 28 of the 28 references it cites were found in a registry or an index.

mRNA Vaccines Encoding the HA Protein of Influenza A H1N1 Virus Delivered by Cationic Lipid Nanoparticles Induce Protective Immune Responses in Mice

Open access
Zhuang, Xinyu, Qi, Yanxin, Wang, Maopeng, Yu, Ning, Nan, Fulong, Zhang, He, Tian, Mingyao, Li, Chang, Lu, Huijun, Jin, Ningyi·2020·Vaccines
DOI 10.3390/vaccines8010123PMC PMC7157730
Open this paper in CoChat — free accountOpen PDF

Abstract

The design of the mRNA vaccine involves the selection of in vitro transcription (IVT) systems and nonviral delivery vectors. This study aimed to verify the effect of 5’ and 3’ untranslated region (UTR) sequences on the translation efficiency of mRNA. Three modes of IVT-mRNA systems (IVT-mRNA-n1/n2/n3) with diverse UTRs were constructed, and EGFP (enhanced green fluorescent protein) and HA (hemagglutinin) gene of H3N2 influenza virus were introduced into each of them. The results showed that the mode of 5’ and 3’ UTRs originating from human β-globulin was better than the mode of UTRs from human α-globulin, and the n3 mode was the best. mEGFP-n3, mH3HA-n3, and mLuciferease-n3 were prepared to compare the effect of cationic lipid nanoparticle (LNP) with that of mannose-conjugated LNP (LNP-Man) on the efficiency of gene delivery. The results showed that the effect of LNP-Man was better than that of LNP both in vitro and in vivo. Choosing appropriate ligands might help in vaccine design. After selecting the IVT-mRNA-n3 system and delivery vectors, mRNA vaccines were constructed against the H1N1 influenza virus, and C57BL/6 mice were immunized through intranasal administration. The results showed that mRNA vaccines could elicit both humoral and cellular immune responses and completely protect mice from the tenfold LD50 H1N1 influenza virus challenge.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.3390/vaccines8010123

Cites

28 of 28 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 (a) Pattern diagram of the three modes of in vitro transcription systems. A549 cells transfected with enhanced green fluorescent protein (mEGFP)-n1/n2/n3 were observed 12 h after transfection under a fluorescence microscope (b), and the positive rates were detected by flow cytometry (c). Data are shown as means ± SDs (standard deviation) and analyzed using one-way ANOVA (analysis of variance) (n = 3, mEGFP-n3 vs. mEGFP-n1, *** p < 0.0001; mEGFP-n3 vs. mEGFP-n2, *** p < 0.0001). (d,e) Western blot analysis. A549 cells were harvested 12 h after transfection. The H3N2-HA protein was detected using rabbit anti-influenza A virus HA Mab (Sino Biological, Beijing, China). The gray value of the strips was analyzed using ImageJ software, and the bar chart was drawn using GraphPad Prism 8.0. (f) Fluorescence microscope images of various cell lines transfected with mEGFP-n3 after 12 h.
Figure 1 (a) Pattern diagram of the three modes of in vitro transcription systems. A549 cells transfected with enhanced green fluorescent protein (mEGFP)-n1/n2/n3 were observed 12 h after transfection under a fluorescence microscope (b), and the positive rates were detected by flow cytometry (c). Data are shown as means ± SDs (standard deviation) and analyzed using one-way ANOVA (analysis of variance) (n = 3, mEGFP-n3 vs. mEGFP-n1, *** p < 0.0001; mEGFP-n3 vs. mEGFP-n2, *** p < 0.0001). (d,e) Western blot analysis. A549 cells were harvested 12 h after transfection. The H3N2-HA protein was detected using rabbit anti-influenza A virus HA Mab (Sino Biological, Beijing, China). The gray value of the strips was analyzed using ImageJ software, and the bar chart was drawn using GraphPad Prism 8.0. (f) Fluorescence microscope images of various cell lines transfected with mEGFP-n3 after 12 h.CC BY · PubMed Central
Figure 2 Characterization of lipid nanoparticles (LNPs) and LNPs/mRNA. (a,b) TEM images of LNP and LNP-Man. (c) Gel retardation assay. LNPs/mRNA were run in the 1.2% nuclease-free agarose gel. LNPs/mRNA complexes were prepared at different N/P molar ratios. Naked mRNA was used as the negative control without any complexation. (d) Size and (e) zeta potential of LNPs and LNPs/mH3HA (N/P = 10:1). (f) Cytotoxicity of LNP and LNP-Man was tested on A549 cells. Untreated cells were defined as 100% viability cells. Data are shown as means ± SDs (n = 3).
Figure 2 Characterization of lipid nanoparticles (LNPs) and LNPs/mRNA. (a,b) TEM images of LNP and LNP-Man. (c) Gel retardation assay. LNPs/mRNA were run in the 1.2% nuclease-free agarose gel. LNPs/mRNA complexes were prepared at different N/P molar ratios. Naked mRNA was used as the negative control without any complexation. (d) Size and (e) zeta potential of LNPs and LNPs/mH3HA (N/P = 10:1). (f) Cytotoxicity of LNP and LNP-Man was tested on A549 cells. Untreated cells were defined as 100% viability cells. Data are shown as means ± SDs (n = 3).CC BY · PubMed Central
Figure 3 Functional verification of LNPs/mRNA. (a) Fluorescence microscope imaging. A549 cells transfected with LNP-Man/mEGFP at indicated N/P molar ratios were observed under a fluorescence microscope 12 h after transfection. (b) The EGFP positivity rates of cells transfected with LNPs/mEGFP at indicated N/P molar ratios were detected by flow cytometry. Data are shown as means ± SDs and were analyzed by two-way ANOVA (n = 3, ns p > 0.05; *** p < 0.0001). (c,d) Flow cytometric analysis of the dendritic cells’ (DC) maturation levels. Data are shown as means ± SDs and were analyzed by two-way ANOVA. (n = 3, ns p > 0.05; ***p < 0.0001; compared with Mock). (e,f) In vivo imaging. Images of lungs were acquired using an IVIS Lumina S5, and bioluminescence intensity from the region of interest was quantified using Living Image software. (g,h) In vivo imaging. Images of legs and the bioluminescence intensity.
Figure 3 Functional verification of LNPs/mRNA. (a) Fluorescence microscope imaging. A549 cells transfected with LNP-Man/mEGFP at indicated N/P molar ratios were observed under a fluorescence microscope 12 h after transfection. (b) The EGFP positivity rates of cells transfected with LNPs/mEGFP at indicated N/P molar ratios were detected by flow cytometry. Data are shown as means ± SDs and were analyzed by two-way ANOVA (n = 3, ns p > 0.05; *** p < 0.0001). (c,d) Flow cytometric analysis of the dendritic cells’ (DC) maturation levels. Data are shown as means ± SDs and were analyzed by two-way ANOVA. (n = 3, ns p > 0.05; ***p < 0.0001; compared with Mock). (e,f) In vivo imaging. Images of lungs were acquired using an IVIS Lumina S5, and bioluminescence intensity from the region of interest was quantified using Living Image software. (g,h) In vivo imaging. Images of legs and the bioluminescence intensity.CC BY · PubMed Central
Figure 4 Construction and verification of mRNA vaccine encoding the H1N1-HA protein. (a) Agarose gel electrophoresis of XhoI enzyme digestion products. 1 represents the intact plasmids of pGEM-H1N1-HA-n3 and 2 represents its linearized product of 5355 bp. M1: DL 5,000 DNA Marker (TaKaRa, Tokyo, Japan); M2: DL 2000 DNA Marker (TaKaRa, Tokyo, Japan). (b,c) Western blot and indirect immunofluorescence analyses. A549 cells were harvested 12 h and 48 h after transfection. The H1N1-HA protein was detected using rabbit anti-influenza A virus HA Mab. The H1N1-virus group was used as the positive control. Mock represents the negative control. DAPI was used to dye the nuclei.
Figure 4 Construction and verification of mRNA vaccine encoding the H1N1-HA protein. (a) Agarose gel electrophoresis of XhoI enzyme digestion products. 1 represents the intact plasmids of pGEM-H1N1-HA-n3 and 2 represents its linearized product of 5355 bp. M1: DL 5,000 DNA Marker (TaKaRa, Tokyo, Japan); M2: DL 2000 DNA Marker (TaKaRa, Tokyo, Japan). (b,c) Western blot and indirect immunofluorescence analyses. A549 cells were harvested 12 h and 48 h after transfection. The H1N1-HA protein was detected using rabbit anti-influenza A virus HA Mab. The H1N1-virus group was used as the positive control. Mock represents the negative control. DAPI was used to dye the nuclei.CC BY · PubMed Central
Figure 5 Immune responses in mice induced by LNPs/mH1HA vaccines. Serum samples were collected in the first, third, and fifth weeks after the initial immunization. (a) The flow chart of the animal experiment. (b) Hemagglutination inhibition (HI) assay. Data are shown as the geometric mean with 95% CI and were analyzed by two-way ANOVA (n = 6, ns p > 0.05; ** p < 0.01; *** p < 0.001). (c–e) Total IgG/IgG1/IgG2a levels in the serum were detected using ELISA kits. Data are shown as means ± SDs and were analyzed by two-way ANOVA (n = 3, ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). (f,g) The IL-4 and IFN-γ levels in sera were determined by ELISA. Data are shown as means ± SDs and were analyzed by two-way ANOVA (n = 3, ns p > 0.05; ** p < 0.01; **** p < 0.0001). (h) The percentages of CD3+CD4+ and CD3+CD8+ T lymphocyte subgroups were tested by flow cytometry. Data are shown as means ± SDs and were analyzed by two-way ANOVA (n = 3, ns p > 0.05; * p < 0.05; *** p < 0.0001, compared with the 0.9% NaCl group).
Figure 5 Immune responses in mice induced by LNPs/mH1HA vaccines. Serum samples were collected in the first, third, and fifth weeks after the initial immunization. (a) The flow chart of the animal experiment. (b) Hemagglutination inhibition (HI) assay. Data are shown as the geometric mean with 95% CI and were analyzed by two-way ANOVA (n = 6, ns p > 0.05; ** p < 0.01; *** p < 0.001). (c–e) Total IgG/IgG1/IgG2a levels in the serum were detected using ELISA kits. Data are shown as means ± SDs and were analyzed by two-way ANOVA (n = 3, ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). (f,g) The IL-4 and IFN-γ levels in sera were determined by ELISA. Data are shown as means ± SDs and were analyzed by two-way ANOVA (n = 3, ns p > 0.05; ** p < 0.01; **** p < 0.0001). (h) The percentages of CD3+CD4+ and CD3+CD8+ T lymphocyte subgroups were tested by flow cytometry. Data are shown as means ± SDs and were analyzed by two-way ANOVA (n = 3, ns p > 0.05; * p < 0.05; *** p < 0.0001, compared with the 0.9% NaCl group).CC BY · PubMed Central
Figure 6 Evaluation of the protective effects after the challenge. (a) Body weight changes and (b) Kaplan–Meier survival curves of H1N1-infected C57BL/6 mice (n = 6) treated with vaccines or vehicles by intranasal administration. Two weeks after the boost immunization, mice were infected with 10 × LD50 A/Jilin/JYT-01/2018(H1N1) influenza virus. Lungs were collected at 5th day post-infection. (c) Representative images of lung pathology in hematoxylin-and-eosin-stained sections from H1N1-infected C57BL/6 mice (n = 3). (d) Viral loads expressed as the fold change compared to noninfected lungs. Data are shown as means ± SDs and were analyzed by one-way ANOVA (n = 3, **** p < 0.0001, compared to 0.9% NaCl, LNP, and LNP-Man groups).
Figure 6 Evaluation of the protective effects after the challenge. (a) Body weight changes and (b) Kaplan–Meier survival curves of H1N1-infected C57BL/6 mice (n = 6) treated with vaccines or vehicles by intranasal administration. Two weeks after the boost immunization, mice were infected with 10 × LD50 A/Jilin/JYT-01/2018(H1N1) influenza virus. Lungs were collected at 5th day post-infection. (c) Representative images of lung pathology in hematoxylin-and-eosin-stained sections from H1N1-infected C57BL/6 mice (n = 3). (d) Viral loads expressed as the fold change compared to noninfected lungs. Data are shown as means ± SDs and were analyzed by one-way ANOVA (n = 3, **** p < 0.0001, compared to 0.9% NaCl, LNP, and LNP-Man groups).CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Cited by

Sorted by citations

See all 132

Related

See all

Metadata

Authors
Zhuang, X., Qi, Y., Wang, M., Yu, N., Nan, F., Zhang, H., Tian, M., Li, C., Lu, H., & Jin, N.
Affiliations
  • Academy of Military Medical Sciences (CN)
  • Chinese Academy of Sciences (CN)
  • Wenzhou University (CN)
  • Yanbian University (CN)
  • Jilin University (CN)
Corresponding author
Jin, Ningyi
ORCID
Venue
Vaccines
Publisher
MDPI AG
Published
2020-03-10
Volume
8
Issue
1
Pages
123
ISSN
2076-393X (ISSN-L)
DOI
10.3390/vaccines8010123
PMC
PMC7157730
OpenAlex
W3011203304
Cited by
132
References
28
Citations by year
2026: 4 · 2025: 27 · 2024: 26 · 2023: 23 · 2022: 29 · 2021: 18 · 2020: 5
FWCI
6.08
Type
journal article
Language
en
Open access
Licence
Locations
  • Vaccines (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Science and Technology Major Project of the Ministry of Science and Technology of China (2018ZX10102-001)
  • Changchun Science and technology plan project (19SS016)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Health Sciences › Medicine › Epidemiology › Influenza Virus Research Studies
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
mRNA vaccines, influenza A (H1N1) virus, hemagglutinin, cationic lipid nanoparticles, 5' and 3' untranslated regions, in vitro transcription, gene delivery, intranasal immunization, humoral immune response, cell-mediated immunity, protective immunity

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review