The Central Conserved Peptides of Respiratory Syncytial Virus G Protein Enhance the Immune Response to the RSV F Protein in an Adenovirus Vector Vaccine Candidate
<p>Designing and characterizing the Ad-based vaccines. (<b>a</b>) Schematic of Ad5-PreF, Ad5-Gcc, Ad5-PreF-P2A-Gcc and Ad5-PreF-Qa-P2A-Gcc. (<b>b</b>,<b>c</b>) Western blot analysis of F expression in cell lysis (<b>b</b>) and supernatant (<b>c</b>). (<b>d</b>,<b>e</b>) Western blot analysis of Gcc expression in cell lysis (<b>d</b>) and supernatant (<b>e</b>). (<b>f</b>) Identification of the conformation of F protein. Ad5-empty was used as control. Site Ø represents the unique antigenic sites of PreF.</p> "> Figure 2
<p>Immune responses in immunized Balc/c mice. (<b>a</b>) Schedule of Ad5 vaccines immunization and bleeding strategies in female Balb/C mice. (<b>b</b>) Kinetics of PreF-specific total IgG reciprocal endpoint titers (log10) were measured within 8 weeks of initial vaccination. (<b>c</b>) Kinetics of G-specific total IgG reciprocal endpoint titers (log10) were measured within 8 weeks after vaccination. (<b>d</b>) Endpoint titer of PreF-specific total IgG was measured in serum at week 6. (<b>e</b>) Endpoint titer of PreF-specific total IgA was measured in serum at week 6. (<b>f</b>) Endpoint titer of G-specific total IgG was measured in serum at week 6. (<b>g</b>) Endpoint titer of G-specific total IgA was measured in serum at week 6. (<b>h</b>) Endpoint titer of PreF-specific total IgG was measured in BALF at week 8. (<b>i</b>) Endpoint titer of PreF-specific total IgA was measured in BALF at week 8. (<b>j</b>) Endpoint titer of G-specific total IgG was measured in BALF at week 8. (<b>k</b>) Endpoint titer of G-specific total IgA was measured in BALF at week 8. ns = not significant; * <span class="html-italic">p</span> < 0.05, **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 3
<p>Intranasal immunization induced a balanced Th1/Th2 immune response in mice vaccinated with Ad-PreF-Qa-P2A-Gcc. (<b>a</b>–<b>c</b>) Anti-PreF antibody subtype IgG1 (<b>a</b>) and IgG2a (<b>b</b>) were detected in serum at week 6, and the ratio of IgG2a/IgG1 (<b>c</b>) was calculated. (<b>d</b>–<b>f</b>) Anti-G antibody subtypes IgG1 (<b>d</b>) and IgG2a (<b>e</b>) were detected in serum at week 6, and the ratio of IgG2a/IgG1 (<b>f</b>) was calculated. ns = not significant; *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 4
<p>Mice vaccinated with Ad5-PreF-Qa-Gcc induced superior neutralizing antibody. (<b>a</b>,<b>b</b>) Serum-neutralizing antibody response against RSV Long (<b>a</b>) and RSV B1 (<b>b</b>) at week 6. All data are shown as means ± SEM. <span class="html-italic">p</span>-values were analyzed with one-way ANOVA (* <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; **** <span class="html-italic">p</span> < 0.0001).</p> "> Figure 5
<p>Immunogenicity and protective efficacy in BALB/c mice. (<b>a</b>) Schematics of vaccine immunization and challenge in BALB/c mice (6–8 weeks old). Mice were challenged with 2 × 10<sup>6</sup> pfu RSV Long via the i.n. route at 4 weeks after boost vaccination. (<b>b</b>) Body weight change over 5 days post-infection. (<b>c</b>,<b>d</b>) Copies of RSV viral L gene in lung tissue (<b>c</b>) and nasal tissue (<b>d</b>). The limit of detection for the RT-qPCRs is 10 copies/mL. (<b>e</b>–<b>j</b>) Tissue sections of the lung tissues were stained with H&E and PAS for pathological examination. The magnification of all images is 20x. All data are shown as means ± SEM. <span class="html-italic">p</span>-values were analyzed with one-way ANOVA (* <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001; **** <span class="html-italic">p</span> < 0.0001).</p> "> Figure 6
<p>Treg cells and Th17 cells in the lung of vaccinated mice induced by RSV challenge infection. Vaccinated mice were challenged i.n. with RSV Long at 6 weeks after the immunization. (<b>a</b>) The percentage of CD4<sup>+</sup>CD25<sup>+</sup>Foxp3<sup>+</sup> Treg cells. (<b>b</b>) The percentage of IL-17A<sup>+</sup> CD4<sup>+</sup> T cells. All data are shown as means ± SEM. <span class="html-italic">p</span>-values were analyzed with one-way ANOVA (** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001; **** <span class="html-italic">p</span> < 0.0001).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Virus
2.2. Vaccine Preparation
2.3. Western Blot
2.4. Characterization of RSV PreF
2.5. Immunization and Challenge of Mice
2.6. Antibody ELISAs
2.7. Neutralization Assay
2.8. Histological Analysis
2.9. Treg Cells and Th17 Cells
2.10. Quantitative RT-PCR Assay
2.11. Statistical Analysis
3. Results
3.1. A Heptapeptide, Qa, Can Boost Protein Expression and Improve Self-Cleaving Efficiency
3.2. Intranasal Immunization with Ad5-PreF-Qa-Gcc Induces a High-Level Antibody Response in Mice
3.3. The Addition of the Gcc Antigen Improved the Neutralizing Antibody Titers against RSV Long and RSV B1
3.4. Intranasal Immunization Provided Protection against RSV Long In Vivo
3.5. PreF-Gcc Immunization Enhanced Treg Cells in the Post-Challenge Mice
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primer | Nucleotides Sequence (5′-3′) |
---|---|
RSV Long q-PCR-F | GAACTCAGTGTAGGTAGAATGTTTGCA |
RSV Long q-PCR-R | TTCAGCTATCATTTTCTCTGCCAAT |
RSV Long probe | 5′FAMATTTGAACCTGTCTGAACATTCCCGGTTGCAT 3′BHQ1 |
Inoculum | Alveolitis a | Interstitial Pneumonia a | Perivascular Bronchitis a |
---|---|---|---|
PBS | 0 | 0 | 0 |
Ad5-empty | 1.6 ± 0.08 | 1.6 ± 0.15 | 2.25 ± 0.09 |
FI-RSV | 2.12 ± 0.13 | 2.21 ± 0.12 | 2.9 ± 0.1 |
Ad5-PreF | 0 | 0.6 ± 0.08 | 1.02 ± 0.05 |
Ad5-Gcc | 0.82 ± 0.08 | 1.51 ± 0.05 | 1.51 ± 0.08 |
Ad5-PreF-Qa-Gcc | 0 | 0.8 ± 0.12 | 1.15 ± 0.06 |
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Chai, P.; Shi, Y.; Yu, J.; Liu, X.; Li, D.; Li, J.; Li, L.; Li, D.; Duan, Z. The Central Conserved Peptides of Respiratory Syncytial Virus G Protein Enhance the Immune Response to the RSV F Protein in an Adenovirus Vector Vaccine Candidate. Vaccines 2024, 12, 807. https://doi.org/10.3390/vaccines12070807
Chai P, Shi Y, Yu J, Liu X, Li D, Li J, Li L, Li D, Duan Z. The Central Conserved Peptides of Respiratory Syncytial Virus G Protein Enhance the Immune Response to the RSV F Protein in an Adenovirus Vector Vaccine Candidate. Vaccines. 2024; 12(7):807. https://doi.org/10.3390/vaccines12070807
Chicago/Turabian StyleChai, Pengdi, Yi Shi, Junjie Yu, Xiafei Liu, Dongwei Li, Jinsong Li, Lili Li, Dandi Li, and Zhaojun Duan. 2024. "The Central Conserved Peptides of Respiratory Syncytial Virus G Protein Enhance the Immune Response to the RSV F Protein in an Adenovirus Vector Vaccine Candidate" Vaccines 12, no. 7: 807. https://doi.org/10.3390/vaccines12070807