Multi-COBRA hemagglutinin formulated with cGAMP microparticles elicits protective immune responses against influenza viruses

Abstract

In humans, seasonal influenza viruses cause epidemics. Avian influenza viruses are of particular concern because they can infect multiple species and lead to unpredictable and severe disease. Therefore, there is an urgent need for a universal influenza vaccine that provides protection against all influenza strains. The cyclic GMP-AMP (cGAMP) is a promising adjuvant for subunit vaccines, which promotes type I interferons' production through the stimulator of interferon genes (STING) pathway. The encapsulation of cGAMP in acetalated dextran (Ace-DEX) microparticles (MPs) enhances its intracellular delivery. In this study, the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology was used to generate H1, H3, and H5 vaccine candidates. Monovalent and multivalent COBRA HA vaccines formulated with cGAMP Ace-DEX MPs were evaluated in mice for protective antibody responses. cGAMP MPs adjuvanted COBRA HA vaccines elicited robust antigen-specific antibodies following vaccination. Compared with COBRA HA vaccine groups with no adjuvant or blank MPs, the cGAMP MPs enhanced HAI activity elicited by COBRA HA vaccines. The HAI activity was not significantly different between cGAMP MPs adjuvanted monovalent or multivalent COBRA HA vaccines. The cGAMP MPs adjuvanted COBRA vaccine groups had higher antigen-specific IgG2a-binding titers than the COBRA vaccine groups with no adjuvant or blank MPs. The COBRA vaccines formulated with cGAMP MPs mitigated diseases caused by influenza viral challenge and decreased pulmonary viral titers in mice. Therefore, the formulation of COBRA vaccines plus cGAMP MPs is a promising universal influenza vaccine that elicits protective immune responses against human seasonal and pre-pandemic strains.

Importance: Influenza viruses cause severe respiratory disease, particularly in the very young and the elderly. Next-generation influenza vaccines are needed to protect against new influenza variants. This report used a promising adjuvant, cyclic GMP-AMP (cGAMP), to enhance the elicited antibodies by an improved influenza hemagglutinin candidate and protect against influenza virus infection. Overall, adding adjuvants to influenza vaccines is an effective method to improve vaccines.

Keywords: COBRA; acetalated-dextran (Ace-DEX); cGAMP; influenza; mice; microparticles (MPs); vaccine.

Fig 1

Experimental design. (A) Vaccine formulation. Antigens J4, Y1, and IAN5 COBRAs were co-administrated with cGAMP encapsulated in Ace-DEX microparticles (MPs). (B) Experimental groups. (C) Schedule for vaccination and challenge. Female DBA/2 J mice were vaccinated three times, 3 weeks apart, with the same vaccine formulation. Sera samples were collected after each vaccination for analysis. The mice were challenged with either H1N1, H5N6, or H3N2 influenza viruses at 3 weeks after the final boost. Lung samples were collected on day 3 post-infection for pulmonary viral loads. Over the course of infection, weights and clinical signs were monitored for 14 days.

Fig 2

Total IgG antibody response after vaccination. Sera samples collected 2 weeks after the final boost were used in ELISA to determine antibody responses against each strain-specific HA after vaccination. The following antigens were used: (A) Bris/18 rHA, (B) Tas/20 rHA, and (C) Sich/14 rHA. ELISA data were statistically analyzed using nonparametric one-way ANOVA by Prism nine software (GraphPad Software, Inc., San Diego, CA). A P value of less than 0.05 was defined as statistically significant (*, P < 0.05; **, P < 0.01). Data are presented as average ± standard deviation. The dashed line on the graph indicates the limit of detection as 1:500.

Fig 3

IgG isotype antibody response after vaccination. Sera samples collected 2 weeks after the final boost were used in ELISA to determine antibody responses after vaccination. The following antigens were used: (A and B) Bris/18 rHA, (D and E) Tas/20 rHA, and (G and H) Sich/14 rHA. (C, F, and I) IgG1: IgG2a ratio. The dashed line on graphs A-B, D-E, and G-H indicates limit of detection as 1:500. The solid line on graphs C, F, and I indicates IgG1: IgG2a at 1:1 ratio. ELISA titers were statistically analyzed using nonparametric one-way ANOVA. A P value of less than 0.05 was defined as statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Data are presented as average ± standard deviation.

Fig 4

Hemagglutinin inhibition assays. Individual mice serum collected after the final boost were used in HAI assay against a panel of historical H1N1, H3N2, and H5Nx influenza viruses. The title of each figure indicates the virus name. The x-axis indicates the experimental group. The y-axis indicates HAI titer in Log₂. The lower dashed line indicates 1:40, and the higher dashed line indicates 1:80. HAI titers were statistically analyzed using nonparametric one-way ANOVA. A P value of less than 0.05 was defined as statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Data are presented as average ± standard deviation.

Fig 5

Focal reduction assay (FRA). Pooled mice serum for each group collected after the final boost were used in FRA against H3N2, H1N1, and H5N6 influenza viruses. The title of each figure indicates virus name. (A–D) Raw data obtained in FRA. The x-axis indicates the serum dilution in Log₂, and the y-axis indicates the percentage of infected cells by the virus. The lower dotted line represents 80% neutralization (Neut₈₀), the middle-dotted line represents 50% neutralization (Neut₅₀), and the upper dotted line represents no neutralization of viral infection. Data are presented as average ± standard deviation. (E–H) Neut₅₀ titers against each of the indicated viruses. The x-axis indicates the experimental group, and the y-axis indicates titer at 50% inhibition in Log₂. The dashed line on the graph indicates the limit of detection as 1:20.

Fig 6

Mice challenged with Bris/18 H1N1 influenza virus. (A) The weight loss curves, (B) clinical scores, (C) survival, and (D) pulmonary viral loads on day 3 post-infection. Colors indicate experimental groups given in D. Data are given as average ± standard deviation. Statistical analysis was conducted using nonparametric one-way ANOVA. A P value of less than 0.05 was defined as statistically significant (**, P < 0.01).

Fig 7

Mice challenged with Sich/14 H5N6 influenza virus. (A) The weight loss curves, (B) clinical scores, (C) survival, and (D) pulmonary viral loads on day 3 post-infection. Colors indicate experimental groups given in D. Data are given as average ± standard deviation. Statistical analysis was conducted using nonparametric one-way ANOVA. A P value of less than 0.05 was defined as statistically significant (*, P < 0.05).