Recombinant proteins and virus strains
rH1 (A/California/04/2009) with a C-terminal trimerization domain from fibritin foldon, used for immunizations, was expressed from baculovirus vectors in High Five cells (BTI-TN-5B1-4 cells; subclone from the Vienna Institute of Biotechnology[29, 30]). For serological detection of H1-specific and HA stalk-specific antibody responses, recombinant H1 (A/California/04/2009) and MiniHA #4900 (based on H1 A/Brisbane/59/2007) were expressed in Expi293F cells, as previously described[19, 21]. To avoid detection of antibodies binding the trimerization domain, both probes used for ELISA were engineered with an alternative C-terminal GCN4 isoleucine zipper trimerization domain from Saccharomyces cerevisiae. Recombinant HA1 domain (A/California/04/2009), was purchased from Sino Biologics does not feature a trimerization domain (#11055-V08H4). A wildtype pH1N1 influenza virus A/Netherlands/602/2009 (H1N1) (NL/09) was used for challenge experiments.
Mice
All animal studies were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee and were carried out at The University of Maryland School of Medicine Biohazard suite. Our study was carried out in compliance with the ARRIVE guidelines and all methods were carried out in accordance with all relevant guidelines and regulations.
Adjuvanted vaccine protection in aged mouse model
To show protection from homologous challenge, aged (~12-month-old), female, retired breeder, BALB/c mice, purchased from Taconic Biosciences (Rensselaer, NY), were immunized intramuscular (IM) in the hind, caudal thigh with100ml vaccine solution at day 0 and day 14 (prime-boost schedule) with 0.04 µg of rHA or day 0 only (prime only schedule) with 5 µg of rHA of influenza A/California/04/2009 (pandemic H1N1) (Cal/09) adjuvanted with either 100 µg of alum, 50 µg PHAD, 50 µg BECC438, or 50 µg BECC470 (HA antigen and BECC adjuvants previously reported in [16]). On day 28 all groups were inoculated i.n. with 32,000 PFU of homologous A/Netherlands/602/2009 (H1N1) (NL/09) virus and weighed daily for seven days. Mice that fell below 70% of starting weight were euthanized per IACUC protocol. On day 7 post-infection mice were harvested for blood and lung tissue following IACUC protocols.
Serum transfer protection from homologous challenge
As reported in Gregg et al[13], to assess the role of IgG in the protection induced by the vaccines with and without adjuvant, wild-type, 6-8 week-old, female, BALB/c mice (Charles River) were immunized IM in the hind, caudal thigh at day 0 and day 14 with 0.04 µg of rHA from influenza A/California/04/2009 (pandemic H1N1) (Cal/09) plus either 100 µg of alum, 50 µg PHAD, 50 µg BECC438, or 50 µg BECC470 or with unadjuvanted antigen or phosphate buffered saline (PBS). At day 28, mice were anesthetized using an isoflurane/oxygen mixture and bled via cardiac puncture. The serum within each group was pooled and frozen at -80°C. First, to assess antibody protection pre-infection, 100 µL of untreated sera was injected IP into each non-anesthetized, naïve mouse (by group). The mice were allowed to rest for two hours and then they were inoculated i.n. with 3200 PFU of homologous A/Netherlands/602/2009 (H1N1) (NL/09) virus and weighed daily for seven days. Mice that fell below 70% of starting weight were euthanized per IACUC protocol. On day 7 post-infection mice were harvested for blood and lung tissue following IACUC protocols. Next, to assess antibody protection from homologous challenge post-infection, we inoculated groups of mice i.n. with 3200 PFU of IAV NL/09 virus on day 0, then on day 1 and day 3 each post-infected, we IP-injected 100 µL serum from each above vaccination group (including non-immunized control groups) and weighed daily for seven days following IACUC protocols. On day 7 post-infection mice were harvested for blood and lung tissue following IACUC protocols.
HA specific antibody titers by ELISA
Briefly (as described in detail in [16]), total IgG, IgG2a and IgG1 antibody titers were determined by ELISA in sera collected on day 28 of the vaccination schedule (pre-infection). Depending on the experiment, 50 μL of either full trimer Cal/09 rHA or group 1 H1 stalk Mini-HA or HA1 Subunit (Sino Biologicals #11055-V08H4) protein-coated 96-well plates (Nunc Maxisorp #44240421) were incubated at 4°C overnight, washed, and then blocked in goat serum/milk/PBS-T for one hour[19, 21]. Blocking buffer was then removed and duplicates of 100 μL of serum, diluted in blocking buffer, was added in a ten point, two-fold dilution series starting with a 1:100 dilution. After two hours of incubation at RT, plates were washed and secondary antibody for either total IgG (goat anti-mouse IgG, KPL-474-1802, Kirkegaard and Perry Laboratories, Gaithersburg, MD), IgG1 (1070-05, Southern Biotech, Birmingham, AL) or IgG2a (1080-05, Southern Biotech, Birmingham, AL) was added for one hour at RT. Plates were washed again and 50 μL of room temperature BD OptEIA 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose, CA) was added to the plates, incubated for 10 minutes at RT and the reaction stopped by adding 50 μL of 3M HCl. An OD450 value was determined using a BioTek Instruments Synergy HTX plate reader using BioTek Instruments Gen5 software (BioTek Instruments, Winooski, VT). All analyses and graphs were made using Prism 9 for Mac OS X (GraphPad Software, Inc., La Jolla, CA). Antibody titer (Mean + SEM) and area under the curve (AUC) (Mean +/- Standard Error, SEM) are individual mice readings with the blank subtracted from the readout. Prism computes the area under the curve using the Gagnon trapezoid rule with mean and SEM reported as a standard error and confidence interval for the AUC.
Lung tissue histology
Lung tissue fixed in 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO) was processed for hematoxylin and eosin (H&E) slides (University of Maryland - Baltimore EM and Histology Laboratory) to assess the histopathology and potential resolution of viral infection. Representative stained tissue sections were selected to most accurately portray the condition of the lungs within each group of vaccinated mice (Supplemental Figures 1 - 4). A single pathologist assessed the selected sections for the presence of alveolar consolidations, peribronchial and perivascular inflammatory infiltrate, and bronchial necrosis then scored them for overall inflammation using the following scoring parameters: 0 = no inflammation, 1 = scant inflammation (<5% multifocal or one small focus); 2 = prominent inflammation, <25% parenchyma; 3 = pronounced inflammation, 25-50% parenchyma; 4 = diffuse inflammation, 50-75% parenchyma; 5 = widely diffuse inflammation, >75% parenchyma. The scores of each group of vaccinated mice were averaged for comparison. All analyses and graphs (mean + SEM) were made using Prism 9 for Mac OS X (GraphPad Software, Inc., La Jolla, CA).
Plaque assays for lung virus titer
Plaque assays were used to determine the lung IAV titers from mice euthanized on day 7 post infection. Madin-Darby canine kidney (MDCK) cells were grown to confluence overnight at 37°C on 6-well plates and a dilution series from 1x10-1 – 1x10-6 of lung homogenate were added to the wells and allowed to infect the cells for 1 hour with period rocking. After one hour, cells were washed with Dulbecco's Modified Eagle Medium (DMEM) (Quality Biological, Gaithersburg, MD) supplemented with 1x penicillin/streptomycin (Gemini Bio- Products, Sacramento, CA) and 1% L-glutamine (Gibco/Life Technologies, Grand Island, NY). A 3-mL overlay of SeaKem LE Agarose (Lonza, Rockland, ME) mixed with 1:500 dilution of Tosyl phenylalanyl chloromethyl ketone(TPCK)-treated trypsin (Sigma-Aldrich, St Louis, MO) in 2x MEM (Quality Biological, Gaithersburg, MD) was used to cover the cells in each well and the plates were incubated at 37°C for three days. After this incubation period the agarose was removed, cells were fixed with 70% ethanol then stained with 0.05% crystal violet. Plaques were visualized and counted manually. All analyses and graphs (mean + SEM) were made using Prism 9 for Mac OS X (GraphPad Software, Inc., La Jolla, CA).
Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.