High- and low-molecular-weight chitosan act as adjuvants during single-dose influenza A virus protein vaccination through distinct mechanisms

Abstract

The investigation of new adjuvants is essential for the development of efficacious vaccines. Chitosan (CS), a derivative of chitin, has been shown to act as an adjuvant, improving vaccine-induced immune responses. However, the effect of CS molecular weight (MW) on this adjuvanticity has not been investigated, despite MW having been shown to impact CS biological properties. Here, two MW variants of CS were investigated for their ability to enhance vaccine-elicited immune responses in vitro and in vivo, using a single-dose influenza A virus (IAV) protein vaccine model. Both low-molecular-weight (LMW) and high-molecular-weight (HMW) CS-induced interferon regulatory factor pathway signaling, antigen-presenting cell activation, and cytokine messenger RNA (mRNA) production, with LMW inducing higher mRNA levels at 24 h and HMW elevating mRNA responses at 48 h. LMW and HMW CS also induced adaptive immune responses after vaccination, indicated by enhanced immunoglobulin G production in mice receiving LMW CS and increased CD4 interleukin 4 (IL-4) and IL-2 production in mice receiving HMW CS. Importantly, both LMW and HMW CS adjuvantation reduced morbidity following homologous IAV challenge. Taken together, these results support that LMW and HMW CS can act as adjuvants, although this protection may be mediated through distinct mechanisms based on CS MW.

Keywords: IRF signaling; adaptive immunity; adjuvants; chitosan; innate immunity.

Figure 1

LMW and HMW CS significantly enhances cytokine production by BMDCs 24 h after treatment. BMDCs (n = 3) were treated with varying doses of two varieties of CS or 0.01 µg/ml MPLA as a positive control. BMDCs were treated for 24 (a–c) or 48 h (d–f) before cells were harvested, placed in TRIzol reagent, and RNA was isolated. qRT‐PCR was performed on complementary DNA generated from the isolated RNA. qRT‐PCR was performed for Il6 (a, d), Ifnb1 (b, e), and Cxcl10 (c, f). *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001 by one‐way ANOVA with Sidak's multiple comparisons test compared to untreated BMDCs or indicated comparison. The dotted line indicates untreated BMDC (media alone) reference control set to 1. Error bars represent SEM. ANOVA, analysis of variance; BMDC, bone marrow‐derived dendritic cell; CS, chitosan; HMW, high molecular weight; LMW, low molecular weight; MPLA, monophosphoryl lipid A; mRNA, messenger RNA; qRT‐PCR, quantitative reverse transcription‐polymerase chain reaction [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

HMW CS treatment significantly increases the activation status of BMDCs. BMDCs (n = 3) were treated with varying doses of two varieties of CS for 24 h or with MPLA at 1 µg as a positive control for activation. Untreated cells (media alone) were used as negative control. After treatment, BMDCs were harvested and CD11b⁺/CD11c⁺ cells were analyzed for CD80 (a), CD86 (b), CD40 (c), and I‐A/I‐E major histocompatibility complex (MHC) class II (d) using flow cytometry. Cells were examined for the level of expression using mean fluorescent intensity (MFI). *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001 between indicated comparisons by one‐way ANOVA with Sidak's multiple comparisons test. Error bars represent SEM. ANOVA, analysis of variance; BMDC, bone marrow‐derived dendritic cell; CS, chitosan; HMW, high molecular weight; LMW, low molecular weight; MPLA, monophosphoryl lipid A [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

LMW and HMW CS induce IRF pathway signaling. J774‐Dual™ cells (n = 12–17) were treated with two varieties of CS at the indicated dose. J774 dual cells use the Lucia luciferase gene to report IRF activity. Treatment durations included 8, 12, 18, 24, 36, and 48 h. Reporter output normalized to total protein and fold change in relative light units (RLU) calculated over untreated cells (media alone). *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001 compared to MPLA‐treated (red and blue asterisk) and untreated (black asterisk) cells by two‐way ANOVA with Tukey's multiple comparisons test. Error bars represent SEM. ANOVA, analysis of variance; CS, chitosan; HMW, high molecular weight; IRF, interferon regulatory factor; LMW, low molecular weight; MPLA, monophosphoryl lipid A [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Mice vaccinated with LMW CS as an adjuvant produce significantly more anti‐OVA IgG compared to mice receiving unadjuvanted vaccines. Mice (n = 4–5) were vaccinated i.m. with 5 µg OVA protein (a, b) or 1 µg pdm09 HA (c, d) ± CS as an adjuvant at the indicated dose. Vaccination with antigen alone served as the negative control. As positive control mice were vaccinated with antigen with MPLA (a, b) or mice were infected with a low‐dose (500 EID₅₀) PR8 virus (c, d). At 3 (a) and 4 weeks (b–d), serum was collected from the mice and ELISA performed to assess levels of anti‐OVA (a, b) or anti‐PR8 (c, d) antibodies induced by vaccination. Antigen‐specific IgG (a, b), IgG2a (c), and IgG1 (d) subtypes were investigated. *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001 between indicated comparisons by one‐way ANOVA with Sidak's multiple comparisons test (a, b). *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001 between indicated comparisons by one‐way ANOVA with Tukey's multiple comparisons test (c, d). ANOVA, analysis of variance; CS, chitosan; EID, egg infective dose; ELISA, enzyme‐linked immunosorbent assay; HA, hemagglutinin; HMW, high molecular weight; IgG, immunoglobulin G; i.m., intramuscularly; LMW, low molecular weight; MPLA, monophosphoryl lipid A; OVA, ovalbumin [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Mice vaccinated with formulations that contain CS as an adjuvant experience significantly less weight loss after homologous infection compared to mice receiving unadjuvanted vaccines. Mice (n = 5) were vaccinated i.m. with 1 µg H1N1 pdm09 HA ± 40 µg CS as an adjuvant. Four weeks after vaccination mice were challenged with 6.3e5 CEID₅₀ H1N1 pdm09 virus. Following the viral challenge, weight loss (a) and survival (b) were recorded daily. Five mice were included in each treatment group. *p ≤ .05 by mixed‐effects analysis using Tukey's multiple comparisons test compared to HA alone. Error bars represent SEM. CEID, chicken embryo infectious dose; CS, chitosan; EID, egg infective dose; HA, hemagglutinin; HMW, high molecular weight; i.m., intramuscularly; LMW, low molecular weight [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

HMW CS as an adjuvant increases the frequency of CD4 and CD8 lung T_RM 5 days after challenge compared to LMW CS. Mice (n = 5) were vaccinated i.m. with 1 µg H1N1 PR8 HA ± 40 µg CS as an adjuvant. Four weeks after vaccination, mice were challenged with 5000 EID₅₀ PR8 viruses. At 5 days after the challenge, mice were killed, and lung T‐cell populations examined using flow cytometry. CD4 (a) and CD8 (b) T cells were CD69/CD103 expressing populations. *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001 by one‐way ANOVA with Tukey's multiple comparisons test. ANOVA, analysis of variance; CS, chitosan; EID, egg infective dose; HA, hemagglutinin; HMW, high molecular weight; i.m., intramuscularly; LMW, low molecular weight; T_RM, T resident memory cell [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7

HMW CS as an adjuvant increases IL‐4 production by CD4 T cells in the lung 5 days after challenge. Mice (n = 5) were vaccinated i.m. with 1 µg H1N1 PR8 HA ± 40 µg CS as an adjuvant. Four weeks after vaccination mice were challenged with 5000 EID₅₀ H1N1 PR8 virus. Five days after the challenge, mice were killed, and lung T‐cell cytokine production was examined using flow cytometry. CD4 (a) and CD8 (b) T cells were examined for IL‐4 production. *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001 by one‐way ANOVA with Tukey's multiple comparisons test. ANOVA, analysis of variance; CS, chitosan; EID, egg infective dose; HA, hemagglutinin; HMW, high molecular weight; IL‐4, interleukin 4; i.m., intramuscularly; LMW, low molecular weight [Color figure can be viewed at wileyonlinelibrary.com]

Figure 8

HMW CS as an adjuvant increases IL‐2 production by DLN CD4 T cells 2 days after challenge. Mice (n = 5) were vaccinated i.m. with 1 µg H1N1 PR8 HA ± 40 µg CS as an adjuvant. As positive control mice were infected with low‐dose PR8 (500 EID₅₀). Four weeks after vaccination mice were challenged with 5000 EID₅₀ H1N1 PR8 virus. Two days after the challenge, mice were killed, and DLN T‐cell cytokine production was examined using flow cytometry. IFN‐γ (a), IL‐4 (b), and IL‐2 (c) production by CD44+/CD4+ T cells was examined. *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001 by one‐way ANOVA with Tukey's multiple comparisons test. ANOVA, analysis of variance; CS, chitosan; DLN, draining lymph node; EID, egg infective dose; HA, hemagglutinin; HMW, high molecular weight; IFN‐γ, interferon‐γ; IL‐4, interleukin 4; i.m., intramuscularly; LMW, low molecular weight [Color figure can be viewed at wileyonlinelibrary.com]