Intranasal Immunization with DNA Vaccine HA-CCL19/Polyethylenimine/Chitosan Composite Provides Immune Protection Against H7N9 Infection

Abstract

Background/objectives: The H7N9 avian influenza virus (AIV) constitutes a novel subtype of influenza virus that has emerged within the past decade. Empirical studies have demonstrated that H7N9 AIV holds the potential to trigger a human pandemic. Vaccines constitute the sole armament available to humanity in combating influenza epidemics. DNA vaccines present numerous merits; however, substantial conundrums persist regarding how to augment their immunogenicity and implement their delivery through mucosal immunization.

Methods: In this study; BALB/c mice were utilized as a model to investigate the effect of CCL19 as a molecular adjuvant and to determine the immune response elicited by polyethylene imine (PEI) and chitosan (CS) as adjuvants during the delivery of a DNA vaccine through the nasal mucosal route.

Results: Our results revealed that the CCL19 molecular adjuvant exerts a substantial immunomodulatory enhancement effect on the H7N9-HA DNA vaccine, inducing more pronounced cellular and humoral immunity. Additionally, our results indicated that the composite formed by the HA-CCL19 DNA in combination with PEI and CS effectively activates local mucosal immunity as well as systemic humoral and cellular immunity, offering 100% protection against lethal doses of homologous virus challenges.

Conclusions: CCL19 conspicuously augments the immunogenicity of the influenza virus HA DNA and conserves the integrity of the vaccine antigen. Simultaneously, CS and PEI proficiently facilitate the mucosal delivery of DNA, thereby eliciting mucosal immunity related to DNA vaccines. This study investigated the feasibility of utilizing nasal mucosa for DNA vaccine immunization, which holds significant implications for the advancement and application of DNA vaccines in public health.

Keywords: CCL19; HA; HA-CCL19/poly-ethylenimine/chitosan; chitosan; influenza; polyethyleneimine.

Figure 1

Schematic diagram of schedule of DNA vaccine immunization and following bioassays and challenges with virus. (A) mouse vaccinated by intramuscular electroporation; (B) mouse vaccinated by intranasal delivery.

Figure 2

T cell immune response elicited by either the HA DNA vaccine or the HA-CCL19 DNA vaccine through the intramuscular administration approach. Five mice in each group were intramuscularly immunized with disparate doses of either the HA DNA vaccine or the HA-CCL19 DNA vaccine, as delineated. The interval between primary immunization and booster immunization was two weeks. Two weeks subsequent to booster immunization, spleen lymphocytes of immunized and control mice were isolated and cultivated. The expression levels of IL-2 (A) or IFN-γ (B) in the supernatant of the culture medium were determined by ELISA. A one-way analysis of variance (ANOVA) was utilized for the statistical significance assessment between the vaccinated cohorts and the control group. **** p < 0.0001 represents a significant difference between the vaccinated group and the control group. ** p < 0.01 represents a significant difference between the HA group and the HA-CCL19 group.

Figure 3

The protective efficacy of diverse DNA vaccines against the challenge of lethal influenza virus subsequent to the intramuscular administration approach. As depicted in the figure, the HA DNA or HA-CCL19 DNA vaccine was administered to mice at dosages of 5 µg or 30 µg, respectively, while the control mice were immunized with an empty plasmid in the same manner, with 15 mice in each group. The interval between primary immunization and booster immunization was two weeks. Two weeks following the booster immunization, the mice were challenged with lethal H7N9 virus at a dose of 5 LD₅₀. On the third day after the challenge, five mice from each group were randomly selected for the collection of lung tissue and the determination of the viral titer in the lung tissue; the rest of the mice were observed daily for the morbidity record. When the mice underwent a body weight reduction surpassing 25%, they were subjected to euthanasia. (A): body weight loss; (B): mortality rates; (C): viral titers in the lung tissue. A one-way analysis of variance (ANOVA) was utilized for the statistical significance assessment between the vaccinated cohorts and the control group. **** p < 0.0001 represents a significant difference between the vaccinated group and the control group. ** p < 0.01 represents a significant difference between the HA group and the HA-CCL19 group.

Figure 4

T cell immune response elicited by the HA-CCL19 DNA vaccine or the HA-CCL19/PEI or HA-CCL19/PEI/CS composite through the intranasal administration approach. Five mice in each group were intranasally immunized with the HA-CCL19 DNA vaccine, HA-CCL19/PEI or HA-CCL19/PEI/CS composite, as delineated. The application dose of HA-CCL19 in different formulas was 30 µg per mouse. The interval between primary immunization and booster immunization was two weeks. Two weeks subsequent to booster immunization, spleen lymphocytes of immunized and control mice were isolated and cultivated. The concentration of IL-2 (A) or IFN-γ (B) in the supernatant of the culture medium were determined by ELISA. A one-way analysis of variance (ANOVA) was utilized for the statistical significance assessment between the vaccinated cohorts and the control group. **** p < 0.0001 represents a significant difference between the HA-CCL19/PEI or HA-CCL19/PEI/CS composite-vaccinated group and the naked HA-CCL19 DNA vaccine group. *** p < 0.001 represents a significant difference between the HA-CCL19/PEI group and the HA-CCL19/PEI/CS group.

Figure 5

The protective efficacy of the HA-CCL19 DNA vaccine and the HA-CCL19/PEI or HA-CCL19/PEI/CS composite against the challenge of lethal influenza virus subsequent to the intranasal administration approach. Fifteen mice in each group were vaccinated with the HA-CCL19 DNA vaccine or the HA-CCL19/PEI or HA-CCL19/PEI/CS composite, respectively. The application dose of HA-CCL19 in different formula was 30 µg per mouse. The interval between primary immunization and booster immunization was two weeks. Two weeks following the booster immunization, the mice were challenged with a lethal H7N9 virus at a dose of 5 LD₅₀. On the third day after challenge, five mice from each group were randomly selected for the collection of lung tissue and the determination of the viral titer in the lung tissue; the rest of the mice were observed daily for the morbidity record. When the mice underwent a body weight reduction surpassing 25%, they were subjected to euthanasia. (A): body weight loss; (B): mortality rates; (C): viral titers in the lung tissue. A one-way analysis of variance (ANOVA) was utilized for the statistical significance assessment between the vaccinated cohorts and the control group. *** p < 0.001 represents a significant difference between the HA-CCL19/PEI or HA-CCL19/PEI/CS composite-vaccinated group and the naked HA-CCL19 DNA vaccine group. ** p < 0.01 represents a significant difference between the HA-CCL19/PEI group and the HA-CCL19/PEI/CS group.