Mastoparan-7 adjuvanted COBRA H1 and H3 hemagglutinin influenza vaccines

Abstract

Adjuvants enhance, prolong, and modulate immune responses by vaccine antigens to maximize protective immunity and enable more effective immunization in the young and elderly. Most adjuvants are formulated with injectable vaccines. However, an intranasal route of vaccination may induce mucosal and systemic immune responses for enhancing protective immunity in individuals and be easier to administer compared to injectable vaccines. In this study, a next generation of broadly-reactive influenza hemagglutinin (HA) vaccines were developed using the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology. These HA vaccines were formulated with Mastoparan 7 (M7-NH₂) mast cell degranulating peptide adjuvant and administered intranasally to determine vaccine-induced seroconversion of antibodies against a panel of influenza viruses and protection following infection with H1N1 and H3N2 viruses in mice. Mice vaccinated intranasally with M7-NH₂-adjuvanted COBRA HA vaccines had high HAIs against a panel of H1N1 and H3N2 influenza viruses and were protected against both morbidity and mortality, with reduced viral lung titers, following challenge with an H1N1 influenza virus. Additionally, M7-NH₂ adjuvanted COBRA HA vaccines induced Th2 skewed immune responses with robust IgG and isotype antibodies in the serum and mucosal lung lavages. Overall, this intranasally delivered M7-NH₂ -adjuvanted COBRA HA vaccine provides effective protection against drifted H1N1 and H3N2 viruses.

Keywords: Adjuvant; COBRA; Influenza; Mastoparan; Vaccine.

Figure 1

(a) DBA/2J (6–8 weeks old) female mice were randomly divided into six groups, with n = 11 mice in each group, and vaccinated intranasally (IN) with 3, 0.3, or 0.03 μg of COBRA HA proteins (Y2, J4, and TJ5) plus the addition of 28.436 μg of the adjuvant, M7-NH₂ (mast cell degranulating peptide), at a 1:1 ratio. As controls, some mice were vaccinated with COBRA HAs (3 μg) with 0.9% saline, with adjuvant only (0.9% saline plus M7-NH2) or without adjuvant (0.9% saline only). (b) Schematic of Study Timeline. DBA/2J mice were bled and prime vaccinated on day 0 follow by blood collection on days 14 and boost-vaccinated on day 28. On days 42 and 49, the mice were bled, followed by intranasal challenge on day 52 with A/Bris/02/2018 or with A/Swit/9715293/2013 viruses. Three days post-infections, lungs were harvested from 3 mice from each group and the remainder mice were monitored for 14 days post infection. Created with BioRender.com.

Figure 2

(a) BALB/c (6–8 weeks old) female mice were randomly divided into three groups, with n = 10 mice in each group, and vaccinated intranasally (IN) with 3 μg of COBRA HA proteins (Y2, J4, and TJ5) plus the addition of 28.436 μg of the adjuvant, M7-NH₂ (mast cell degranulating peptide), at a 1:1 ratio. As controls, some mice were vaccinated with COBRA HAs (3 μg) with 0.9% saline, or with adjuvant only (0.9% saline plus M7-NH₂). (b) Schematic of Study Timeline. BALB/c mice were bled and prime vaccinated (IN) on day 0 follow by blood collection on days 14 and 42. On day 28, all of the mice were boost-vaccinated as before. Seven days post-vaccinations (day 35), lungs were harvested from each mouse. Created with BioRender.com.

Figure 3

Quantification of total IgG in DBA/2J mice pooled sera collected on days 42 and 49, and in the lung lavages of BALB/c mice harvested on day 35, post two vaccinations. Mice were IN vaccinated with (Red dots) COBRA HAs (3 μg) with 0.9% saline or IN with (Blue dots) M7-NH₂ (28.436 μg) in the following formulations: with 0.9% saline only, or COBRA HAs (3, 0.3, or 0.03 μg). Antibody responses were measure against plates coated with (a) Y2, (b) J4, or (c) TJ5 COBRA HAs for DBA/2J mice day 42/49 sera, or WT IAV (d and e) Bris/18 H1N1, Tas/20 H3N2, or Sing/16 H3N2 HAs for (d) DBA/2J sera and (e) BALB/c mice lung lavages. The Y-axis represents the endpoint total IgG titers on a log 10 scale. The X-axis represents the different (a–c) COBRA HA vaccine formulations (3–0.03 μg) or no COBRA HAs, and (d,e) WT Bris/18, Tas/20, or Sing/16 HAs. Each column represents dots of 10, 11 or 5 mice per vaccine group and are expressed as the average ± standard error of the mean (SEM). IgG titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) by Prism 9 software (GraphPad Software, Inc., San Diego, CA, version 9.4.0, https://www.graphpad.com). 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).

Figure 4

Quantification of IgG1, IgG2a, and IgG2b isotypes in mice sera collected on days 42 and 49, and in lung lavages harvested on day 35, post two vaccinations. Mice were vaccinated IN with (Blue dots) M7-NH₂-Adjuvanted (28.436 μg) COBRA (3 μg) vaccines or (Red dots) COBRA (3 μg) plus 0.9% saline. Antibody responses were measure against plates coated with (a–f) WT Bris/18 H1N1, Tas/20 H3N2, or Sing/16 H3N2 HAs. The Y-axis represents the endpoint titers and the X-axis represents the different isotypes. Each column represents dots of 10, 11, or 5 mice per vaccine group and are expressed as the average + /- standard error of the mean (SEM).

Figure 5

Hemagglutinin-inhibition activity in mice vaccinated with M7-adjuvanted COBRA HA vaccines (Y2, J4, and TJ5). (a) Sera collected from individual mice vaccinated with COBRA (3 μg) + M7-NH₂ (28.436 μg) at days 42 and 49 (DBA/2J) post-boost, was pooled, or (b) sera collected at day 42 (BALB/c) post-boost, was tested against a panel of H1N1 (Left) and H3N2 (Right) IAVs. HAI titers (Y-axis) are displayed on a log 2 scale and the panel of H1N1 and H3N2 IAVs are displayed on the X-axis. The two dotted lines represent HAI titers of 1:40 (bottom) and 1:80 (top). Each column represents dots of 10, 11, or 5 mice per vaccine group and are expressed as the average ± standard error of the mean (SEM).

Figure 6

Weight loss, survival, and viral lung titers of DBA/2J mice vaccinated IN with M7-NH_2. Mice were challenged IN with (a–c) A/Bris/02/2018 (8 x10⁶ PFU/50 μL) or with (d–f) A/Swit/9,715,293/2013 (7 x10⁵ PFU/50 μL) and observed for 14 days post-infection. (a,d) Percent of original body weight, (b,e) percent survival, and (c,f) day 55 viral lung titers. The dotted line in panel a represents a 25% weight loss cutoff from the original body weights. The Y-axis in (a) represents the original body weights, (b) percent survival, and (c) the day 3 post-challenge lung viral titers (PFU/g of tissue). The X-axis represents (a,b,d,e) the days post-infection and (c,f) the vaccines with (3–0.03 μg) or without (0 μg) COBRA HAs, adjuvanted with (Blue) M7-NH₂ (28.436 μg) or (Red) 0.9% saline only. The dotted lines in panels c and f are representative of the limit of detection (LOD). In panel c, the circles represent distinct mice identified as number one in each group, squares represent distinct mice identified as number 2, and triangles represent distinct mice identified as number 3. Viral lung titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) by Prism 9 software (GraphPad Software, Inc., San Diego, CA, version 9.4.0, https://www.graphpad.com). 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).