A single dose of inactivated influenza virus vaccine expressing COBRA hemagglutinin elicits broadly-reactive and long-lasting protection

Abstract

Influenza virus infections present a pervasive global health concern resulting in millions of hospitalizations and thousands of fatalities annually. To address the influenza antigenic variation, the computationally optimized broadly reactive antigen (COBRA) methodology was used to design influenza hemagglutinin (HA) or neuraminidase (NA) for universal influenza vaccine candidates. In this study, whole inactivated virus (WIV) or split inactivated virus (SIV) vaccine formulations expressing either the H1 COBRA HA or H3 COBRA HA were formulated with or without an adjuvant and tested in ferrets with pre-existing anti-influenza immunity. A single dose of the COBRA-WIV vaccine elicited a robust and broadly reactive antibody response against H1N1 and H3N2 influenza viruses. In contrast, the COBRA-SIV elicited antibodies that recognized fewer viruses, but with R-DOATP, its specificity was expanded. Vaccinated ferrets were protected against morbidity and mortality following challenge with A/California/07/2009 at 14 weeks post-vaccination with reduced viral shedding post-infection compared to the naïve ferrets. However, the COBRA-IIVs did not block the viral transmission to naïve ferrets. The contact infection induced less severe disease and delayed viral shedding than direct infection. Overall, the COBRA HA WIV or the COBRA HA SIV plus R-DOTAP elicited broadly reactive antibodies with long-term protection against viral challenge and reduced viral transmission following a single dose of vaccine in ferrets pre-immune to historical H1N1 and H3N2 influenza viruses. IMPORTANCE The goal of the next-generation influenza vaccine is to provide broadly reactive protection against various drifted influenza strains. With the previous studies evaluating the COBRA HA-based vaccines, the breadth of antibody activities was confirmed following two or three vaccinations. However, for the commercial influenza vaccine, only one shot is required. In this study, only one shot was administrated to the pre-immune ferrets and the COBRA-WIV efficiently elicited broadly reactive antibodies and long-lasting protection against the pdm09 strain. Moreover, this study showed that different infection methods can lead to different disease severity, which emphasizes the significance of the model selection. The infection was conducted 14 weeks post-vaccination to evaluate the long-term protection elicited by only one vaccination. This is the first longevity study describing the immune responses elicited by COBRA-IIVs in ferrets and provides promising results for the potential clinical utilization.

Fig 1. Experimental timeline for vaccination and direct challenge.

42 naïve ferrets were pre-infected with Sing/86 and Pan99 (5*10⁵PFU/virus/ferret) 8 weeks prior to the vaccination. Sera samples were collected 2 weeks post-pre-infection. At week 0, ferrets were evenly divided into 6 vaccinated groups (n = 6/group) receiving vaccines named: COBRA-WIV, COBRA-WIV plus AddaVax, COBRA-WIV plus R-DOTAP, COBRA-SIV, COBRA-SIV plus AddaVax, and COBRA-SIV plus R-DOTAP; and mock-vaccinated group (n = 6) receiving PBS only. The detailed information of each vaccine’s formulation is shown in Table 1. Sera samples were collected on week 2, 10, and 14 to measure the antibody levels. Ferrets were challenged with CA/09 (1*10⁶PFU/ferret) at week 14, with 6 naïve mock-vaccinated ferrets serving as the mock control. Nasal washes were collected at 1DPI, 3DPI, and 5DPI to monitor the viral shedding. Body weight and clinical signs were closely recorded daily until 14DPI. DPI: day post-infection.

Fig 2. HAI titers against H1N1 and H3N2 panels for sera collected 2 weeks post vaccination.

The HAI titers elicited by COBRA-WIV (A), COBRA-WIV plus AddaVax (B), COBRA-WIV plus R-DOTAP (C), COBRA-SIV (D), COBRA-SIV plus AddaVax (E), and COBRA-SIV plus R-DOTAP (F) were measured. The X-axis represents different virus strains. The red bar indicates the H1N1 strains and the purple bar indicates the H3N2 strains. The Y-axis represents the Log2 HAI titers with absolute mean values ± SEM. The lower dotted line indicates the HAI titer of 1:40, and the upper dotted line indicates 1:80. 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).

Fig 3. The breadth of neutralizing antibodies elicited by COBRA-IIVs 2 weeks post vaccination.

The MNA titers elicited by CORBA-IIVs were measured against H1N1 strains CA/09 (A) and Vic/19 (B), and H3N2 strains Sing/16 (C), KS/17 (D), HK/19 (E), SA/19 (F), and TAS/20 (G). The X-axis represents different vaccines. The Y-axis represents the Log2 MNA endpoint with absolute mean values ± SEM. MNA: microneutralization assay.

Fig 4. IgG levels stimulated by COBRA-IIVs 2 weeks post vaccination.

A. Y2-specific total IgG level. B. J4-specific total IgG level. C. H1 HA stem-binding IgG level. D. H3 HA stem-binding IgG level. The X-axis represents different vaccines. The Y-axis represents the Log2 MNA endpoint with absolute mean values ± SEM. The X-axis represents the area under curve (AUC) obtained OD141 values from 3-fold serially diluted sera plus SEM. The Y-axis represents different vaccines. cH6/1: chimeric rHA with H6 head from H6 A/Mallard/Sweden/81/2002 and stalk from A/California/07/2009. cH7/3: chimeric rHA with H7 head from A/Anhui/1/2013 and H3 stalk from A/Texas/50/2012. 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).

Fig 5. Development of disease post-direct infection.

A: body weight loss curve. B: survival rate. C. Clinic scores. The legend shows the different vaccine groups with the naïve control with 3 ferrets in each group.

Fig 6. Viral shedding titer in nasal washes collected post-direct infection.

The X-axis represents the different time points for collecting the nasal washes. The Y-axis represents the viral titer PFU/ml nasal washes in the Log10 scale with absolute mean values ± SEM. The legend shows the different vaccine groups. 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).

Fig 7. Transmission study from vaccinated transmitters to naive receivers.

A. experimental design. Vaccinated transmitters were intranasally infected by CA/09 (1*10⁶PFU/ferret) on day 0. One day later, the naïve receivers were paired with the vaccinated transmitters and they were co-housed for 14 days. The transmission from naïve transmitters to naïve receivers functioned as the control in this study. The nasal washes from the receivers were collected on day 3 and 5, which were considered 2DPI and 4DPI for receivers. Body weight loss, clinical scores, and survival rate were closely monitored for 14 days since co-housing. B. Viral titers in nasal washes collected from receivers at 2DPI and 4DPI. The X-axis represents the different time points for collecting the nasal washes. The Y-axis represents the viral titer PFU/ml nasal washes in the Log10 scale with absolute mean values ± SEM. The legend shows the vaccine that the transmitters got. 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).

Fig 8. Development of disease post-direct infection.

A: body weight loss curve. B: survival rate. C. clinical sores. The legend shows the naïve ferrets co-housed with different vaccine groups with the naïve ferrets serving as a control with 3 ferrets in each group.

Fig 9. Transmission study from naïve transmitters to vaccinated receivers.

A. experimental design. Naïve ferrets were intranasally infected by CA/09 (1*10⁶PFU/ferret) on day 0. One day later, the naïve transmitters were paired with the vaccinated receivers and they were co-housed for 14 days. The transmission from naïve transmitters to naïve receivers functioned as the control in this study. The nasal washes from the receivers were collected on day 3 and 5, which were considered 2DPI and 4DPI for receivers. Body weight loss, clinical scores, and survival rate were closely monitored for 14 days since co-housing. B. Viral titers in nasal washes collected from receivers at 2DPI and 4DPI. The X-axis represents the different time points for collecting the nasal washes. The Y-axis represents the viral titer PFU/ml nasal washes in the Log10 scale with absolute mean values ± SEM. C. body weight loss for receivers post-contact infection. The legend shows the different vaccine groups. 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).