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. 2022 Jul 28:13:920256.
doi: 10.3389/fimmu.2022.920256. eCollection 2022.

Mucosal immunization with an adenoviral vector vaccine confers superior protection against RSV compared to natural immunity

Clara Maier  1 , Jana Fuchs  1 , Pascal Irrgang  1 , Michael Hermann Wißing  2 , Jasmin Beyerlein  1 , Matthias Tenbusch  1 , Dennis Lapuente  1
Affiliations

Mucosal immunization with an adenoviral vector vaccine confers superior protection against RSV compared to natural immunity

Clara Maier et al. Front Immunol. .

Abstract

Respiratory syncytial virus (RSV) infections are the leading cause of severe respiratory illness in early infancy. Although the majority of children and adults mount immune responses against RSV, recurrent infections are frequent throughout life. Humoral and cellular responses contribute to an effective immunity but also their localization at respiratory mucosae is increasingly recognized as an important factor. In the present study, we evaluate a mucosal vaccine based on an adenoviral vector encoding for the RSV fusion protein (Ad-F), and we investigate two genetic adjuvant candidates that encode for Interleukin (IL)-1β and IFN-β promoter stimulator I (IPS-1), respectively. While vaccination with Ad-F alone was immunogenic, the inclusion of Ad-IL-1β increased F-specific mucosal immunoglobulin A (IgA) and tissue-resident memory T cells (TRM). Consequently, immunization with Ad-F led to some control of virus replication upon RSV infection, but Ad-F+Ad-IL-1β was the most effective vaccine strategy in limiting viral load and weight loss. Subsequently, we compared the Ad-F+Ad-IL-1β-induced immunity with that provoked by a primary RSV infection. Systemic F-specific antibody responses were higher in immunized than in previously infected mice. However, the primary infection provoked glycoprotein G-specific antibodies as well eventually leading to similar neutralization titers in both groups. In contrast, mucosal antibody levels were low after infection, whereas mucosal immunization raised robust F-specific responses including IgA. Similarly, vaccination generated F-specific TRM more efficiently compared to a primary RSV infection. Although the primary infection resulted in matrix protein 2 (M2)-specific T cells as well, they did not reach levels of F-specific immunity in the vaccinated group. Moreover, the infection-induced T cell response was less biased towards TRM compared to vaccine-induced immunity. Finally, our vaccine candidate provided superior protection against RSV infection compared to a primary infection as indicated by reduced weight loss, virus replication, and tissue damage. In conclusion, our mucosal vaccine candidate Ad-F+Ad-IL-1β elicits stronger mucosal immune responses and a more effective protection against RSV infection than natural immunity generated by a previous infection. Harnessing mucosal immune responses by next-generation vaccines is therefore a promising option to establish effective RSV immunity and thereby tackle a major cause of infant hospitalization.

Keywords: IgA; RSV (respiratory syncytial virus); TRM; adenoviral (Ad) vector; mucosal; natural immunity; tissue-resident memory T cells; vaccine.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Vaccine-induced antibody responses. BALB/cJRj mice were immunized with 2x108 particles Ad-F and 1x109 particles Ad-empty, Ad-IPS-1, or Ad-IL-1β. Antibody levels were characterized in sera 35 days and in BAL samples 42 days post-immunization. (A, B) FACS-based analysis for F-specific antibodies was performed for Ig in sera (A, 1:100 dilution, detection with anti-Ig-FITC) and for IgA in BAL samples (B, 1:20 dilution, detection with anti-IgA-FITC) with HEK 293A cells expressing the F protein. Shown are the MFIs for each sample and the mean of the values for each group. (C) 75% plaque reduction neutralization titers (PRNT75) were analyzed in sera by RSV microneutralization assay (detection limit at a titer of <1:12). Bars represent group means (A, B) or medians (C) overlaid with individual data points. n=10-11 for sera and n=4-5 for BALs. Data were analyzed by one-way ANOVA followed by Tukey´s post test (values in C were log-transformed before analysis). *p<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001.
Figure 2
Figure 2
Vaccine-induced T cell responses in the lung. BALB/cJRj mice were immunized as described above and lung lymphocytes were analyzed 49 days later. Counts of F85-93 pentamer+ CD8+ T cells per lung (A) and counts of specific memory T cell subsets (B) are shown; effector T cells, TEFF, KLRG1+CD127-; effector memory T cells, TEM: KLRG1+CD127+; central memory T cells, TCM: KLRG1-CD69-CD103-CD127+; TRM: KLRG1-CD127+/-CD69+CD103+; gating scheme in Supplementary Figure 2 . (C, D) Lung lymphocytes were restimulated with MHC-class I and class II peptides derived from the F protein and the functionality of CD8+ T cells (C) and CD4+ T cells (D) was detected via extracellular staining for CD107a and intracellular cytokine staining (poly; for CD8+: CD107a+IFNγ+IL-2+TNFα+; for CD4+: IFNγ+IL-2+TNFα+; gating strategy in Supplementary Figure 3 ). Bars represent group means overlaid with individual data points. n=4-5. Data were analyzed by one-way ANOVA followed by Tukey´s post Test. *p<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001.
Figure 3
Figure 3
Vaccine efficacy against RSV infection. Mice were experimentally infected with 1x106 PFU RSV-A2 48 days after the immunization. (A) The initial weight was set as 100% (dotted line) and weight changes were monitored for five days. (B) Viral RNA copies were quantified by qRT-PCR in lung homogenates five days post-infection. The dotted line represents the lower limit of quantification. Data represent group means with SEM (A) or group medians overlaid with individual data points (B). n=5-6. Data were analyzed by one-way ANOVA followed by Tukey´s post test (values in B were log-transformed before analysis). For (A): *p<0.05 vs. naive, # p<0.05 vs. F+IL-1β. For (B): *p<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001.
Figure 4
Figure 4
Humoral responses induced by mucosal vaccination or primary RSV infection. BALB/cJRj mice were infected with 5x105 PFU RSV-A2 or immunized with 2x108 particles Ad-F and 1x109 particles Ad-IL-1β. Antibody levels were characterized in sera 35 days and in BAL samples 42 days post-immunization. (A, B) Virus-specific IgG1 and IgG2a in sera (A, 1:1000 dilution) and Ig and IgA in BAL (B, 1:10 dilution) were detected via virus-coated ELISA. (C) 75% plaque reduction neutralization titers (PRNT75) in sera (detection limit at a titer of <1:12, dotted line) and BAL (detection limit at a titer of <1:2, dotted line) were tested by microneutralization assay. (D, E) FACS-based analysis for F- (D) and G-specific (E) antibodies was performed in sera (1:100 dilution) and BAL samples (1:20 dilution). Bars represent group medians (A-C) or means (D, E) overlaid with individual data points. n=8-12 for sera and n=4-6 for BALs. Data were analyzed by one-way ANOVA followed by Tukey´s post test (values in A-C were log-transformed before analysis). *p<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001. Relative light units per second, RLU/s.
Figure 5
Figure 5
T cell phenotypes induced by mucosal vaccination and a primary RSV infection. BALB/cJRj mice were immunized or infected as described above and isolated lung lymphocytes were analyzed seven weeks later. Counts of F85-93 pentamer+ (A) and M282-90 pentamer+ CD8+ T cells (C) per lung and of specific memory T cell subsets (B, D) are shown; effector T cells, TEFF, KLRG1+CD127-; effector memory T cells, TEM, KLRG1+CD127+; central memory T cells, TCM, KLRG1-CD69-CD103-CD127+; TRM, KLRG1-CD127+/-CD69+CD103+; gating scheme in Supplementary Figure 2 . (E) Pentamer+ CD8+ T cells (M282-90-spec. in RSV group, F85-93-spec. in Ad-F+Ad-IL-1β group) that were not stained by the intravenous staining were further characterized for their expression of CD11a, CD69, and CD103. Bars represent group means overlaid with individual data points. n=4-6. Data were analyzed by one-way ANOVA followed by Tukey´s post test (A-D) or two-tailed Student’s t-test (E). *p<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001.
Figure 6
Figure 6
Mucosal T cell functionality induced by mucosal vaccination and a primary RSV infection. Lung lymphocytes were restimulated with MHC-class I peptides derived from the F (A) and the M2 protein (B) and the functionality of CD8+ T cells was detected via extracellular staining for CD107a and intracellular cytokine staining (poly; CD107a+IFNγ+IL-2+TNFα+; gating strategy in Supplementary Figure 3 ). Bars represent group means overlaid with individual data points. n=4-6. Data were analyzed by one-way ANOVA followed by Tukey´s post Test. *p<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001.
Figure 7
Figure 7
Protection against RSV infection mediated by vaccination and a primary RSV infection. Mice were experimentally infected with 1x106 PFU RSV-A2 48 days after the immunization or primary infection. (A) The initial weight was set as 100% (dotted line) and weight changes were monitored for five days. (B) Viral RNA copies were quantified by qRT-PCR in lung homogenates five days post-infection. The dotted line represents the lower limit of quantification. (C) Tissue damage was assessed by measuring the total concentration of protein in BAL samples via bicinchoninic acid assay. Data represent group means with SEM (A), group medians overlaid with individual data points (B), or group means overlaid with individual data points (C). n=4-6. Data were analyzed by one-way ANOVA followed by Tukey´s post test (values in B were log-transformed before analysis). For (A): # p<0.05 vs. F+IL-1β. For (B, C): *p<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001.
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