Historical H1N1 Influenza Virus Imprinting Increases Vaccine Protection by Influencing the Activity and Sustained Production of Antibodies Elicited at Vaccination in Ferrets

Abstract

Influenza virus imprinting is now understood to significantly influence the immune responses and clinical outcome of influenza virus infections that occur later in life. Due to the yearly cycling of influenza viruses, humans are imprinted with the circulating virus of their birth year and subsequently build a complex influenza virus immune history. Despite this knowledge, little is known about how the imprinting strain influences vaccine responses. To investigate the immune responses of the imprinted host to split-virion vaccination, we imprinted ferrets with a sublethal dose of the historical seasonal H1N1 strain A/USSR/90/1977. After a +60-day recovery period to build immune memory, ferrets were immunized and then challenged on Day 123. Antibody specificity and recall were investigated throughout the time course. At challenge, the imprinted vaccinated ferrets did not experience significant disease, while naïve-vaccinated ferrets had significant weight loss. Haemagglutination inhibition assays showed that imprinted ferrets had a more robust antibody response post vaccination and increased virus neutralization activity. Imprinted-vaccinated animals had increased virus-specific IgG antibodies compared to the other experimental groups, suggesting B-cell maturity and plasticity at vaccination. These results should be considered when designing the next generation of influenza vaccines.

Keywords: H1N1; Haemagglutinin; antibody titer; imprinting; influenza A; influenza virus; isotype; quadrivalent vaccine; split-virion; virus neutralization.

Figure 1

Study timeline and experimental group design for the investigation of influenza immune history and regulation of host responses to Sanofi FLUZONE^® QIV. (A) The study time shows the time course of infections and vaccinations in relation to the general dynamic trends of the innate and adaptive systems. (B) The Table depicts the experimental groups and corresponding preimmune virus, vaccine course, and challenge.

Figure 2

Previous imprinting with a historical H1N1 influenza virus increases influenza vaccine protection. Ferrets were infected with the historical H1N1 virus USSR/77 (10⁶ EID₅₀) to establish an influenza-specific immune background or were left naïve. After 67 and 105 days, the vaccine group were vaccinated with the Sanofi FLUZONE^® QIV vaccine. All groups were challenged at Day 123 with the contemporary Cal/07 H1N1 pandemic 2009 virus. (A) Temperature and (B) weight changes were monitored for 14 days pc. * indicates a p-value of less than 0.05.

Figure 3

Viral shedding following challenge was lower in previously infected and vaccinated ferrets. Nasal washes were collected from all groups Day 2 pc (Cal/09). Live viral load was calculated by TCID₅₀ titration assay using MDCK cells. Student’s t-tests were performed among groups to determine statistical significance. ** indicates a p-value of less than 0.001.

Figure 4

Histopathological analysis shows differential lung pathology on Day 14 post challenge dependent on influenza virus and vaccination history. Respiratory tissue was collected from ferrets Day 14 post challenge and the lungs were processed for histopathological assessment. Tissue morphology was assessed by hematoxylin & eosin staining. Lungs were analyzed by microscopy from at least three ferrets per group. Images were captured as described and representative images from the groups Naïve-Mock Vac; PreUSSR-Mock Vac; and PreUSSR-2x Vac are shown. High resolution scans were performed using an Aperio ScanScope XT (Leica Biosystems, Concord, Canada) at 40× magnification. Images of the scans were captured using the HALO program from UHN AOMF (Advanced Optical Microscopy Facility, Toronto, Canada) at 5× (low) or 20× (high) magnification of the scan.

Figure 5

Increased antibody titers directed toward vaccine antigens in previously infected ferrets. Haemagglutination inhibition assays were performed using serum collected from ferrets from each group throughout the time course. The reciprocal of the largest dilution capable of inhibiting red blood cell agglutination was determined to be the HAI titer. To calculate the HAI Units, a log2 was taken from the dilution factor. The mean HAI Units were calculated for each experimental group and the results were plotted in heat maps to visually represent changes in antibody titer and specificity over time. HAI assays were performed using specific whole virus (USSR/77) or BPL inactivated vaccine antigens with turkey red blood cells. Previously infected ferrets elicited greater antibody titer which were longer lived in circulation compared to naïve-vaccinated ferrets. Red asterisks indicate vaccination days.

Figure 6

Focused titer analysis of the H1-2009 specific antibodies elicited in the PreUSSR-Vac2x and Naïve-Vac2x groups. HAI assay utilizing serum collected throughout the time course for the preUSSR-Vac2x and Naïve-Vac2x groups against the 2009 H1N1 pandemic Cal/09 virus are graphed to better visualize quantitative and statistical differences based on the contribution of preimmunity. Black arrows denote time of vaccination. Red arrow denotes the challenge day. Day 0 is the time of infection with the seasonal H1N1 USSR/77 virus. Values are graphed in HAI Units. * indicates a p-value of less than 0.05 as determined by Student’s t-test.

Figure 7

Increased virus-specific IgG isotype serum antibody titers following challenge in the preimmune-vaccinated ferrets compared to naïve-vaccinated ferrets. Isotype ELISAs were performed using sera collected from each group throughout the time course to quantify the circulating virus-specific IgG and IgM antibodies. Only time points post vaccination and challenge are shown Post Vaccination, Day 2 pc, Day 7 pc, and Day 14 pc. A Student’s t-test was conducted to compare with results on Day 0, * p < 0.05.

Figure 8

Antibodies elicited post vaccination in the PreUSSR-Vac2x ferrets were detected earlier and had increased neutralization activity compared to other experimental groups. Serum was collected at early time points after primary vaccination (Days 0, 3, 7, and 14 post vaccination). (A) HAI assays were performed as previously described using sera isolated from blood collection post vaccination against the Cal/07 (pH1N1) virus to determine the dynamics of antibody generation after vaccination. (B) Microneutralization assays were performed by standard protocol on MDCK cells using serum from all experimental groups post vaccination and prior to challenge. For simplicity, only the PreUSSR-Vac2x, PreUSSR-MockVac, Naïve-Vac2x and Navie-Mock Vac groups are shown. Student’s t-test was conducted to compare results between each group, * p < 0.05.

Figure 9

Decrease in cross-reactive antibody titer over sequential influenza virus exposures of antigenically divergent strains. Serum was isolated from blood collected at end day (Day 137) from all ferrets. Serum was then used for HAI assays against an antigenically divergent influenza virus strain (A/Taiwan/1/1986, Taiwan/86) as a read-out for the production of cross-reactive antibodies. Standard HAI assays were conducted with turkey RBCs against Taiwan/86. Student’s t-test was conducted to compare results with the PreUSSR-Mock Vac group, * p < 0.05.