Intranasal Delivery of Quillaja brasiliensis Saponin-Based Nanoadjuvants Improve Humoral Immune Response of Influenza Vaccine in Aged Mice

Abstract

Increasing the effectiveness of vaccines against respiratory viruses is particularly relevant for the elderly, since they are prone to develop serious infections due to comorbidities and the senescence of the immune system. The addition of saponin-based adjuvants is an interesting strategy to increase the effectiveness of vaccines. We have previously shown that ISCOM matrices from Q. brasiliensis (IMXQB) are a safe and potent adjuvant. In this study, we evaluated the use of IMXQB as an adjuvant for the seasonal trivalent influenza vaccine (TIV) in an aged mice model. Herein, we show that subcutaneous injection of the adjuvanted vaccine promoted higher titers of IgM, IgG (and isotypes), and serum hemagglutination inhibition titers (HAI). Notably, aged mice immunized by intranasal route also produced higher IgG (and isotypes) and IgA titers up to 120 days after priming, as well as demonstrating an improvement in the HAI antibodies against the TIV. Further, experimental infected aged mice treated once with sera from adult naïve mice previously immunized with TIV-IMXQB subcutaneously successfully controlled the infection. Overall, TIV-IMXQB improved the immunogenicity compared to TIV by enhancing systemic and mucosal immunity in old mice conferring a faster recovery after the H1N1pdm09-like virus challenge. Thus, IMXQB nanoparticles may be a promising platform for next-generation viral vaccines.

Keywords: ISCOM matrices; IgA titers; Quillaja brasiliensis; adjuvant; aged mice; delivered intranasally; influenza virus; nanoparticle; viral challenge.

Figure 1

Schematic vaccination, blood sampling, immunogens, and delivery routes of each group. Aged female BALB/c mice were vaccinated by the subcutaneous or intranasal route on days 0 (priming) and 14 (booster) with either TIV-IMXQB or unadjuvanted commercial TIV vaccines. Blood samplings are represented by red drops, and the numbers indicate the days after the prime immunization. For each group, vaccine formulation, dose, and delivery routes are summarized in the table. Created with BioRender.com.

Figure 2

Subcutaneous injections of adjuvanted trivalent influenza vaccine (TIV-IMXQB) elicited higher antibody levels than commercial unadjuvanted vaccine (TIV). Aged female BALB/c mice were vaccinated by the s.c. route on days 0 (priming) and 14 (booster) with either TIV-IMXQB or unadjuvanted vaccine (commercial vaccine). Anti-TIV IgM (A) at day 14 and IgG (B) at days 14 (n = 19 animals per group, empty dots), 28 (n = 19 animals per group, strong-colored dots), and 120 (n = 5 animals per group, light-colored dots) are represented; the insert shows a zoom view of the IgG values at day 14. IgG1 (C) and IgG2a (D) antibody levels for 28 and 120 dpp, are shown. Avidity index of IgG antibodies at days 28 and 120 post-priming were assessed (E). The median value is indicated by a line, and the dots indicate individual values. The statistical analyses were performed using Kruskal–Wallis and uncorrected Dunn’s post hoc test, comparing the TIV-IMXQB against the TIV group. Statistically significant differences are indicated with the p specific value.

Figure 3

Subcutaneous injections of TIV-IMXQB-adjuvanted vaccine promoted a better protection than TIV. Aged female BALB/c mice. Hemagglutination inhibition antibody titers were measured on days 28 and 120 dpp (n = 19 and n = 5 animals per group, respectively). A line shows the median mean value, and the dots show individual values. Strong-colored dots: 28 dpp; light-colored dots: 120 dpp (A). One hundred twenty dpp, all animals in each group (n = 5 per group, light-colored dots) were intranasally challenged with 1 × 10⁶ TCID (median tissue culture infectious dose)/50 µL of A/Uruguay/897/2018 (H1N1)pdm09 influenza virus and followed during 14 days. Retained body-weight loss (mean and error) (B) and percent survival (C), are plotted vs. time. The statistical analyses were performed using Mann–Whitney test, comparing TIV-IMXQB against TIV group for each collected serum. Percentage of survival compared to TIV alone was determined by a log-rank (Mantel–Cox) test. Statistical analyses were performed using the non-parametric Kruskal–Wallis test with uncorrected Dunn’s post hoc test for multiple comparisons or a log-rank (Mantel–Cox) test, and each group was compared with the TIV mock group. Significant differences are indicated with the p specific value.

Figure 4

Intranasal delivery of adjuvanted trivalent influenza vaccine (TIV-IMXQB) elicited higher antibody levels than commercial unadjuvanted vaccine (TIV). Aged female BALB/c mice were vaccinated via i.n. on days 0 (priming) and 14 (booster) with either TIV-IMXQB or TIV alone. Anti-TIV IgM (A) at day 14 and IgG (B) at days 14 (n = 19 animals per group, empty dots), 28 (n = 19 animals per group, strong-colored dots), and 120 (n = 5 animals per group, light-colored dots) are represented; the insert shows a zoom view of the IgG values at day 14. IgG1 (C) and IgG2a (D) antibody levels for 28 and 120 dpp are shown. Avidity index of IgG antibodies at days 28 and 120 post-priming were assessed (E). The median value is indicated by a line, and the dots indicate individual values. The statistical analyses were performed using Kruskal–Wallis and uncorrected Dunn’s post hoc test, comparing the TIV-IMXQB against the TIV group. Statistically significant differences are indicated with the p-specific value.

Figure 5

Intranasal delivery of adjuvanted trivalent influenza vaccine (TIV-IMXQB) elicited a significant increase of IgA antibody levels compare to commercial unadjuvanted vaccine (TIV). Anti-TIV IgA were measured on days 28 and 120 dpp (n = 19 and n = 5 animals per group, respectively). A line shows the median mean value, and the dots show individual values. Strong colored dots: 28 dpp; light colored dots: 120 dpp (A). The avidity index of IgA antibodies was evaluated. A line shows the IgA avidity index median value, for de TIV group undetected value, and the dots show individual values (B). Statistical analyses were performed using the Mann-Whitney test. Significant differences are indicated with the p specific value.

Figure 6

Intranasal delivery of TIV-IMXQB-adjuvanted vaccine induced superior protection than TIV. Hemagglutination inhibition antibody titers were measured on days 28 and 120 dpp (n = 19 and n = 5 animals per group, respectively). A line shows the median mean value, and the dots show individual values. Strong-colored dots: 28 dpp; light-colored dots: 120 dpp (A). One hundred twenty dpp, all animals in each group (n = 5 per group, light-colored dots) were intranasally challenged with 1 × 10⁶ TCID (median tissue culture infectious dose)/50 µL of A/Uruguay/897/2018 (H1N1)pdm09 influenza virus and followed during 14 days. Retained body-weight loss (mean and error) (B) and percent survival (C), are plotted vs. time. The statistical analyses were performed using Mann–Whitney test, comparing TIV-IMXQB against TIV group for each collected serum. Percentage of survival compared to TIV alone was determined by a log-rank (Mantel–Cox) test. Statistical analyses were performed using the non-parametric Kruskal–Wallis test with uncorrected Dunn’s post hoc test for multiple comparisons or a log-rank (Mantel–Cox) test, and each group was compared with the TIV mock group. Significant differences are indicated with the p specific value.

Figure 7

Passive immunization with pooled sera from immunized adult mice with TIV-IMXQB-adjuvanted vaccine contributed to faster recovery of aged mice against sublethal challenge with A/Uruguay/897/2018 (H1N1)pdm09-like virus. Mice aged 15 months old were intranasally infected with a sublethal dose (1 × 10⁴ TCID₅₀/50 µL) of A/Uruguay/897/2018 (H1N1)pdm09-like virus. Twenty-four hours after that, the mice received, intraperitoneally, pooled sera from adult animals immunized with TIV (n = 7), TIV-IMXQB (n = 7), IMXQB (only nanoparticle adjuvant) (n = 5), or saline (n = 3) and monitored daily for clinical signs of disease, weight loss, and mortality for 21 days. The plot shows body weight retained in each group vs. time (represented by the mean and error). Statistical analyses were performed using the non-parametric Kruskal–Wallis test with uncorrected Dunn’s post hoc test for multiple comparisons, and each group was compared with the TIV-IMXQB group. Significant differences are indicated with the p-specific value.