RSV Vaccine with Nanoparticle-Based Poly-Sorbitol Transporter (PST) Adjuvant Improves Respiratory Protection Against RSV Through Inducing Both Systemic and Mucosal Humoral Immunity

Abstract

Background/Objectives: Respiratory syncytial virus (RSV) causes symptoms similar to a mild cold for adults, but in case of infants, it causes bronchitis and/or pneumonia, and in some cases, mortality. Mucosal immunity within the respiratory tract includes tissue-resident memory T (T_RM) cells and tissue-resident memory B (B_RM) cells, which provides rapid and efficient protection against RSV re-infection. Therefore, vaccine strategies should aim to generate mucosal immune responses. However, the interactions between RSV vaccines and mucosal immune responses within the respiratory tract are poorly understood. We evaluated a mucosal immune system following immunization by RSV vaccine with poly-sorbitol transporter (RSV-PST), a nanoparticle adjuvant. Methods: We intranasally immunized the RSV-PST and identified the systemic and mucosal immune responses. Furthermore, we challenged with RSV A2 strain after immunization and investigated the protective effects. Results: Consequently, antigen-specific CD8⁺ T_RM cells were markedly elevated in the lung parenchyma, yet exhibited impaired cytokine expression. In contrast, humoral immunity, with systemic antibody production from serum, but not in the respiratory tract, was significantly increased by RSV-PST immunization. Interestingly, the production of respiratory mucosal antigen-specific IgG after RSV A2 challenge dramatically increased in the bronchoalveolar lavage fluid (BALF) of the RSV-PST immunized group in the presence of FTY720, and the lung-infected RSV titer was significantly lower in this group. Furthermore, after RSV A2 challenge, CD69⁺ IgG⁺ B_RM cells were significantly increased in lung tissues in the RSV-PST group. Conclusions: The RSV-PST vaccine has protective effects against RSV infection by promoting both systemic and local humoral immunity rather than cellular immunity.

Keywords: adjuvant; mucosal immunity; nanovaccine; poly-sorbitol transporter (PST); respiratory syncytial virus (RSV); systemic humoral immune response; vaccination.

Figure 1

Intranasal RSV-PST immunization generates bona fide RSV-F specific CD8⁺ T_RM cells in the respiratory tract. BALB/c mice were intranasally immunized with PBS, RSV-F protein only, RSV-PST, or RSV-CT at 2-week intervals. Mice were i.v. injected with α-CD45 antibody 5 min before sacrifice on day 35 post first immunization. (A) Schematic of the experimental design. (B) Representative dot plots, (C) frequencies, and cell numbers of lung RSV-F-specific circulating T_MEM cells (CD45_i.v.⁺ CD8⁺ CD69⁻ RSV-F Tetramer⁺). (D) Representative dot plots of lung RSV-F-specific effector T_MEM (CD45_i.v.⁻ CD8⁺ CD69⁻ RSV-F Tetramer⁺) or resident T_RM cells (CD45_i.v.⁻ CD8⁺ CD69⁺ RSV-F Tetramer⁺). Frequencies and cell numbers of lung RSV-F-specific (E) effector T_MEM and (F) resident T_RM cells. (G) Representative dot plots of BALF RSV-F-specific effector T_MEM or resident T_RM cells. Frequencies and cell numbers of BALF RSV-F-specific (H) effector T_MEM cells and (I) resident T_RM cells. (J) Representative histogram (left) and expression of tissue resident markers (right) in lung RSV-F-specific circulating T_MEM or resident T_RM cells induced by intranasal RSV-PST immunization. (K) Representative histogram (left) and expression of tissue resident markers (right) in lung RSV-F-specific resident T_RM cells induced by intranasal RSV-PST or RSV-CT immunization. Data in (C,E,F,H,I) were pooled from two independent experiments (n = 4–5 per group). p values in (C,E,F,H,I) were analyzed by ordinary one-way ANOVA. Dot plots or histograms in (B,D,G,J,K) were representative data from two independent experiments (n = 4–5 per group). p values in the bar graphs of (J,K) were analyzed by unpaired two-tailed Student’s t-test. Data are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 2

Deficient cytokine production in the lung RSV-F-specific CD8⁺ and CD4⁺ T cells induced by intranasal RSV-PST immunization. After intranasal immunizations with PBS, RSV-F protein only, RSV-PST or RSV-CT, single cells from lung tissue were re-stimulated with RSV-F protein for 18 h and the cytokine production of T cells was analyzed by flow cytometry. Frequencies and cell numbers of lung (A) IFN-γ⁺ or (B) IL-17A⁺ activated CD8⁺ T cells and lung (C) IFN-γ⁺, (D) IL-4⁺ or (E) IL-17A⁺ activated CD4⁺ T cells. (F) The levels of cytokine gene expression in whole lung tissues were measured by qRT-PCR. All data were pooled from two independent experiments (n = 4–5 per group). p values were analyzed by ordinary one-way ANOVA. Data are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 3

Intranasal RSV-PST immunization induces the production of systemic RSV-F-specific IgG. BALF and serum were harvested on day 35 from the intranasally immunized mice with PBS, RSV-F protein only, RSV-PST, or RSV-CT at 2-week intervals. The production levels of RSV-F-specific IgG and IgA in serial diluted (A) BALF and (B) serum were measured by ELISA. All data were pooled from two independent experiments (n = 4–5 per group). p values were analyzed by ordinary two-way ANOVA with Tukey’s multiple comparisons test (single pooled variance). Symbol * indicates a comparison to control group. Data are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 4

Intranasal RSV-PST immunization provides mucosal protection against RSV A2 challenge by inducing systemic and local antigen-specific IgG production. BALB/c mice were immunized with intranasal administration of PBS, RSV-PST, or RSV-CT at 2-week intervals and challenged with RSV A2 strain (starting at day 35 post first immunization, intranasal route) in the presence or absence of FTY720 treatment (starting at day 34 post first immunization, i.p. route). The lung tissues were harvested and analyzed at 4 d.p.i. (A) Schematic of the experimental design. (B) Virus titers of RSV A2 strain within the lung tissues were measured by plaque assay. Frequencies and cell numbers of lung (C) IFN-γ⁺, (D) TNF-α⁺, (E) granzyme B⁺ (GrB), or (F) IL-17A⁺-activated CD8⁺ T cells that were re-stimulated with RSV-F protein for 18 h. The production levels of RSV-F-specific IgG and IgA in serial diluted (G) BALF and (H) serum were measured by ELISA. All data were pooled from two independent experiments (n = 4–5 per group). p values in (B–F) were calculated by ordinary one-way ANOVA. p values in (G,H) were analyzed by ordinary two-way ANOVA with Tukey’s multiple comparisons test (single pooled variance). A red or purple symbol * indicates a comparison to the control group. A black symbol * indicate a comparison between the RSV-PST and RSV-CT groups. Data are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.