Advax-SM™-Adjuvanted COBRA (H1/H3) Hemagglutinin Influenza Vaccines

Abstract

Adjuvants enhance immune responses stimulated by vaccines. To date, many seasonal influenza vaccines are not formulated with an adjuvant. In the present study, the adjuvant Advax-SM™ was combined with next generation, broadly reactive influenza hemagglutinin (HA) vaccines that were designed using a computationally optimized broadly reactive antigen (COBRA) methodology. Advax-SM™ is a novel adjuvant comprising inulin polysaccharide and CpG55.2, a TLR9 agonist. COBRA HA vaccines were combined with Advax-SM™ or a comparator squalene emulsion (SE) adjuvant and administered to mice intramuscularly. Mice vaccinated with Advax-SM™ adjuvanted COBRA HA vaccines had increased serum levels of anti-influenza IgG and IgA, high hemagglutination inhibition activity against a panel of H1N1 and H3N2 influenza viruses, and increased anti-influenza antibody secreting cells isolated from spleens. COBRA HA plus Advax-SM™ immunized mice were protected against both morbidity and mortality following viral challenge and, at postmortem, had no detectable lung viral titers or lung inflammation. Overall, the Advax-SM™-adjuvanted COBRA HA formulation provided effective protection against drifted H1N1 and H3N2 influenza viruses.

Keywords: Advax-SM™; COBRA; adjuvant; influenza; vaccine.

Figure 1

(a) Mice were randomly divided into four groups, with n = 18 mice in each group, and immunized intramuscularly (IM) with 3 μg of each COBRA HA proteins, Y2, and NG2 (total 6 μg), alone or formulated with AddaVax SE adjuvant at a 1:1 ratio or 1 mg Advax-SM™. As controls, a group of mice were immunized with 1 mg Advax-SM™ adjuvant only without antigen. (b) Schematic of study timeline. Mice were bled and prime-vaccinated on day 0 and boosted on days 28 and 56. On day 61, three mice from each group were sacrificed and spleens harvested for B cell FluoroSpot assays. Bleeds on remaining mice were performed on days 70 and 76, and an intranasal virus challenge was performed on day 82. Three days post-infection (day 85), lungs were harvested from three mice per group for lung viral titers and pathology, and the remaining mice were monitored for clinical illness and mortality for 14 days post infection.

Figure 2

Mice were all challenged intranasally (IN) with the H1N1 strain, A/Brisbane/02/2018 (8 × 10⁶ PFU/50 μL), and observed for 14 days post-infection. (a) Percent of original body weight loss, (b) clinical scores, and (c) percent survival. The dotted line in (a) represents the 20% weight loss endpoint cutoff. The dotted line in (b) represents a mean clinical score of 3. Each line (a,b) is conveyed as the average +/− standard error of the mean (SEM). p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.

Figure 3

Lung viral titers of mice three days following challenge with A/Brisbane/02/2018. The Y-axis represents the day 3 post-challenge lung viral titers (PFU/g of tissue) and the X-axis represents the vaccine groups. Advax-SM™ alone control (Grey symbols), COBRA HA alone (Green symbols), COBRA HA plus Advax-SM™ (Blue symbols), COBRA HA plus SE adjuvant (Red symbols). The dotted line represents the limit of detection (LOD).

Figure 4

Histopathology of mice lung tissue harvested at three days post-infection with H1N1 Brisbane/02/18 influenza virus. The mice were humanely euthanized and their left lungs were infused with 10% formalin for fixing the tissue. Hematoxylin and Eosin (H&E) staining on lung slices measuring 5 µm was used to visualize pathology in the lungs. The lungs are represented at 20× magnification—the lower bar represents 200 um scale. COBRA HA plus (a) saline, (b) SE adjuvant, or (c) Advax-SM^TM. Saline plus (d) Advax-SM^TM, Mock plus (e) saline, or (f) uninfected.

Figure 5

Serum anti-influenza total IgG, IgA and IgG1, IgG2a, and IgG2b in vaccinated mice. (a) Serum anti-influenza total IgG against Bris/02/18 and Sing/16 rHA, (b) serum IgA against WT Bris/18 and Sing/16 rHA, (c) serum IgG isotype titers against WT Bris/18, and (d) serum IgG isotype titers against Sing/16 rHA. Represented on the Y-axis are the endpoint titers. Represented on the X-axis are the WT rHAs (a,b) or the Ig isotypes (c,d). Each bar corresponds to 15 individual mice according to vaccine regimens and are conveyed as the average +/− standard error of the mean (SEM). p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.

Figure 6

Hemagglutination inhibition activity in mice vaccinated with COBRA HA vaccines (Y2 and NG2). Sera collected from immunized mice at days 70 and 76 were pooled (3 weeks post the third vaccine dose) and tested for HAI activity against two H1N1 viruses, Cal/09 and Bris/18. COBRA HA alone (Green symbols), COBRA HA + SE adjuvant (1:1) (Red symbols), COBRA HA + Advax-SM™ (Blue symbols), or Mock + Advax-SM™ (Grey symbols). Represented on the Y-axis are the HAI titers on a log 2 scale. Represented on the X-axis is the panel of H1N1 influenza viruses. The top dotted line indicates a 1:80 HAI titer and the bottom dotted line indicates a 1:40 HAI titer. Each column represents 15 individual mice pertaining to each vaccine regimen and are conveyed as the average +/− standard error of the mean (SEM). p < 0.05 *, p < 0.0001 ****, p < 0.9999 not significant (ns).

Figure 7

Hemagglutinin inhibition responses in mice immunized with COBRA HA antigens (Y2 and NG2). (a–c) Sera collected from immunized mice at days 70 and 76 were pooled (3 weeks post the third vaccination) and tested against two H3N2 influenza viruses. (a) Advax-SM™ alone controls, (b) COBRA HA + SE adjuvant (1:1), (c) COBRA HA + Advax-SM™, or (d) Mock + Advax-SM™. Represented on the Y-axis are the HAI titers on a log 2 scale. Represented on the X-axis is the panel of H3N2 influenza viruses. The top dotted line indicates a 1:80 HAI titer and the bottom dotted line indicates a 1:40 HAI titer. Statistical differences shown are of group (a) compared to group (b) or (c). Each column represents 15 individual mice pertaining to each vaccine regimen and are conveyed as the average +/− standard error of the mean (SEM). p < 0.0001 ****.

Figure 8

Quantification of total IgG and antigen-specific IgG1, IgG2a, and IgG2b isotypes from antibody secreting cells (ASCs). Spleens from DBA/2J female mice (6–8 weeks old) were harvested five days following the third vaccination with COBRA HA+ SE adjuvant (Red), COBRA HA+ Advax-SM™ (Blue), Mock + Advax-SM™ (Grey), or COBRA HA alone (Green). Plates coated with anti-Igκ/λ (a), Y2 (b), or NG2 (c) were used to detect IgG1, IgG2a, or IgG2b ASCs. The Y-axis represents the number of ASCs (spots) per million input cells. The Ig isotypes from immunized mice are represented on the X-axis. Each column represented 3 individual mice per the indicated vaccine regimens and expressed as the average +/− standard error of the mean (SEM). p < 0.05 *.