Introduction
Influenza virus infection is a persistent public health challenge and causes serious respiratory illness each year resulting in hospitalizations with an estimated 290,000–650,000 deaths worldwide (Nair et al., 2011; Thompson et al., 2009) that strains healthcare systems and economies worldwide (Fragaszy et al., 2018; Willis et al., 2019). Seasonal influenza vaccination is currently the most effective approach for preventing influenza-associated illnesses (Guzmán, 2020; Xie et al., 2017). Influenza viruses contain a segmented, negative-sense RNA that allows for annual antigenic drift due to the error-prone RNA polymerase that lacks proofreading capability resulting in point mutations in all 8 influenza segmented RNA genes (Bouvier and Palese, 2008; McDonald et al., 2016; Palese and Shaw, 2007).
These evolutionary pressures are most pronounced in the major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The HA and NA proteins can accommodate amino acid substitutions without appreciable loss of function that allow influenza viruses to evade existing antibodies (Gouma et al., 2020; Hay et al., 2019; Knight et al., 2020).
Fluzone is a split-inactivated virus vaccine generated from embryonated chicken eggs manufactured by Sanofi Pasteur (Swiftwater, PA, USA) and is the most widely administered influenza vaccine each season. These vaccines are administered to people by intramuscular injection and are relatively inexpensive to produce compared to other influenza vaccine manufacturing platforms (Chen et al., 2020; Lee and McGlone, 2010). The goal of this study was to vaccinate mice with a single immunization of Fluzone mixed with adjuvant and compare the elicited immune responses to mice administered two doses of the same vaccine formulations.
Mice were vaccinated with Fluzone alone or mixed with one of two adjuvants. AddaVax is a squalene-based oil-in-water nanoemulsion with the same composition as MF59, which is a squalene emulsion adjuvant approved for human use in influenza vaccines that enhances antigen-specific antibody and T-cell responses (Gao et al., 2022; Kim et al., 2020; Ross et al., 2022). Following injection, a recombinant vaccine mixed with AddaVax creates a localized environment that recruits neutrophils, monocytes, and macrophages to the site of injection (O’Hagan et al., 2012; O’Hagan et al., 2021).
Overall, there is an enhancement of antigen presentation leading to production of protective antibodies and T cell responses against the vaccine antigens (Kim et al., 2020).
The second novel adjuvant, TRAC478, is a synthetic dual toll-like receptor (TLR) liposome delivery system combining a TLR4 agonist and TLR7/8 agonist (Lathrop et al., 2024; Short et al., 2022b; Siram et al., 2024). TLR4 is a pattern recognition receptor (PRR) that resides in the cell membrane and specifically recognizes lipopolysaccharide (LPS), the main cell wall component of Gram-negative bacteria (Abul et al., 2018; Kawai and Akira, 2011). TLR4 agonists activate antigen-presenting cells by engaging the MyD88-and TRIF-dependent pathways that stimulate the elicitation of pro-inflammatory cytokines, T cell responses (Goff et al., 2017), as well as enhanced humoral responses (Luo et al., 2024).
TLR7 is highly expressed on plasmacytoid dendritic cells and B cells where it drives the production of IFN-α for antiviral immunity (Kawai and Akira, 2011). TLR8 is highly expressed on myeloid dendritic cells and monocytes, where it stimulates pro-inflammatory cytokines such as IL-12 and TNF-α for the enhancement of CD8+ T cell responses (Kawai and Akira, 2011). Combining TLR7 and TLR8 agonists enhances dendritic cell activation and antigen presentation, resulting in a robust Th1 immune response (Kawai and Akira, 2011; Kayesh et al., 2024a; Miller et al., 2020).
Liposomes that contain TLR4 and TLR7/8 agonists synergistically stimulate a Th1/Th2 immune response (Napolitani et al., 2005; Short et al., 2019b, 2022b). Incorporating TRAC478 with Fluzone should engage innate signaling to drive more rapid and sustained humoral and cellular immune responses that results in enhanced immune potency compared to unadjuvanted Fluzone. Overall, this study demonstrated that a licensed human inactivated virus vaccine mixed with adjuvant can enhance systemic IgG responses and elicit protective antibody titers compared to unadjuvanted vaccine.
Vaccinated mice had anti-HA binding antibodies with HAI activity and survived influenza viral challenge following intramuscular delivery of vaccine with or without adjuvant.
Materials and methods
Viruses
The influenza viruses used in this study were passaged in embryonated chicken eggs following World Health Organization (WHO) protocols. Virus stocks were quantified with the hemagglutination assay and made into single use 1 mL aliquots that were stored at −80°C. The viruses used in this study were previous WHO recommended vaccine strains.
The H1N1 viruses used in this study were: A/Brisbane/02/2018 (BR/18) (clade 6B.1a.1), A/Guangdong-Maonan/SWL/1536/2019 (GD/19) (clade 6B.1A.5a.2), A/Victoria/2570/2019 (VC/19) (clade 6B.1A.5a.2) and A/Victoria/4897/2022 (VC/22) (clade 6B.5a.2a.1). The H3N2 viruses used in this study were: A/Hong Kong/2671/2019 (HK/19) (clade 3c.2a1b.1b), A/Darwin/9/2021 (DR/21) (clade 3C.2a1b.2a.2), A/Massachusetts/18/2022 (MA/22) (clade 3C.2a1b.2a.3a.1_J), A/District of Columbia/27/2023 (DC/23) (clade 3C.2a1b.2a.3a.1_J.2). The influenza B viruses (IBVs) used in this study were B/Phuket/3073/2013 (PH/13) (Yamagata lineage) and B/Austria/1359417/2021 (AU/21) (Victoria lineage).
Vaccination and viral infection of mice
Influenza naïve female DBA/2J mice, Mus musculus, (6–8 weeks old) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were housed in microisolator caging, provided access to water and food ad libitum throughout the duration of the study, and cared for following the USDA guidelines for laboratory animals. The procedures used in this study were approved by the Cleveland Clinic Institutional Animal Care and Use Committee (no.
2935). On day 0 the mice were infected with a mixture of A/Singapore/6/1986 (SG/86) H1N1 influenza virus (2.5×105 plaque forming units (PFU)/50 μL) and A/Panama/2007/1999 (PN/99) H3N2 influenza virus (2.5×105 PFU/50 μL) diluted in sterile phosphate buffered saline (PBS) (Fig. 1).
The mice were weighed and monitored daily for 14 days following infection to monitor the progression of disease. On day 14 blood was collected from each mouse to confirm that the animals seroconverted against both influenza viruses. The mice were then allowed to rest to day 30 and then they were vaccinated intramuscularly with 750ng/HA of the 2023–2024 split-inactivated influenza virus vaccine, Fluzone (Sanofi Pasteur, Swiftwater, PA, USA) (Fig.
1). Mice (n = 22) were vaccinated with Fluzone plus the adjuvant TRAC478 (Inimmune, Missoula, MT, USA) in a 50 μL volume of 0.9 % sterile saline. Another group of mice (n = 22) were vaccinated with Fluzone plus the adjuvant Addavax (Thermo Fisher, Waltham, MA, USA) in a 50 μL volume of 0.9 % sterile saline.
A third group of mice (n = 22) were vaccinated with Fluzone without adjuvant in a 50 μL volume of 0.9 % sterile saline. Mice were vaccinated in a prime-boost regimen on day 30 and day 58, and 14 days after each vaccination, blood was collected from every mouse. On day 86, the mice were challenged with either 2.7×106 PFU/50 μL of the H1N1 influenza virus, BR/18, or 2.7×106 PFU/50 μL of the H3N2 influenza virus, SW/13 (Fig.
1). The mice were then monitored for 14 consecutive days for weight loss and clinical symptoms. On day 89 lungs were collected from 3 mice in each group to determine the amount of replicating virus present in the respiratory tissue 3 days post infection.
On day 100 the mice were humanely euthanized.
Enzyme linked immunosorbent assay (ELISA)
Immulon 4HBX 96-well microtiter plates (Thermo Fisher, Waltham, MA, USA) were coated with 1 μg/mL of Y2 or NG2 rHA, by mixing the protein with carbonate coating buffer pH 9.4 (2.65g Na2CO3, 2.1g NaHCO3, 450 mL diH2O) and adding 100 μL of the solution to each well. The plates were then incubated overnight at 4°C in a humidified chamber. Following incubation, the coating solution was removed and 200μL/well of blocking buffer (PBS, 4 % FBS (fetal bovine serum), 0.05 % Tween-20) was added to the plates at 37°C for 90 min.
Following incubation, the blocking buffer was decanted and 100 μL of serum (serially diluted 2-fold in blocking buffer starting at a dilution of 1:100) was transferred to the protein coated plate. The plates were then moved to a humidified chamber and incubated at 37°C for 90 min. Once this time had passed, the plates were decanted and washed five times with wash buffer (PBS + 0.05 % Tween-20).
After washing, the plates were decanted and 100 μL of secondary antibody diluted 1:4000 in blocking buffer was added to each well. Three different secondary antibodies were used in this study and were added to separate assay plates: Goat anti-mouse IgG HRP (1 mg/mL) (Cat. no. 1030-05, Southern Biotech, Birmingham, AL, USA), Goat anti-mouse IgG1 HRP (1 mg/mL) (Cat. no.
1070-05, Southern Biotech, Birmingham, AL, USA), Goat anti-mouse IgG2a HRP (1 mg/mL) (Cat. no. 1083-05, Southern Biotech, Birmingham, AL, USA). After the addition of secondary antibody, the plates were moved to a humidified chamber and incubated at 37°C for 90 min.
Following incubation, the plates were decanted and 100 μL of substrate (1 mg/mL ABTS diammonium salt (Sigma, Burlington, MA, USA), McIlvaine solution pH 5 (7.31g Na2HPO4 + 4.66g C6H8O7 + 500 mL diH2O), 3 % H2O2) was added to each well and allowed to develop for 12–15 min at 37°C in a humidified chamber. After colorimetric development, 50 μL of a 1 % SDS (sodium dodecyl sulfate) (Thermo Fisher, Waltham, MA, USA) solution was added to each well to stop the reaction. The plates were then read at 414 nm using a BioTek Epoch 2 plate reader (Agilent, Santa Clara, CA, USA) equipped with Gen6 software.
Optical density values were plotted in GraphPad Prism (San Diego, CA, USA) for each dilution point. Total area-under-the-curve (AUC) for each vaccination point was obtained, as were endpoint dilution titers determined by taking the reciprocal dilution of the most dilute well that contained more than twice the mean signal of the background (negative control) wells.
Hemagglutination inhibition assay (HAI)
The HAI assay was used to quantitate the presence of antibodies that bind to viral HA and block the binding of virus to the sialic acids present on the surface of red blood cells (RBCs). In this study, HAI assays performed with H1N1 viruses were run with turkey RBCs (Lampire Biological Laboratories, Pipersville, PA, USA) and assays performed with H3N2 viruses were performed with guinea pig RBCs (Lampire Biological Laboratories, Pipersville, PA, USA). The day before the assay, the serum samples were treated with receptor-destroying enzyme (RDE) (Denka, Seiken, Co., Japan) to inactivate non-specific inhibitors present in the samples according to the instructions provided by the manufacturer.
First, 300 μL of RDE reconstituted in sterile PBS was added to 100 μL of sera. This mixture was allowed to incubate at 37°C for 16 h. Following incubation, the samples were placed in a 56°C water bath for 30 min.
After heat treatment, 600 μL of sterile PBS was added to each serum sample. Following RDE treatment, 50 μL of sera was individually added to the first column of a 96-well V-bottom microtiter plate (Thermo Fisher, Waltham, MA, USA), and 25 μL of PBS was added to the remaining wells of the plate. The sera samples were then serially diluted two-fold across the plate by transferring 25 μL from the first column mixing it with the second column, and continuing this process across the plate, discarding the final 25 μL.
Then an equal volume, 25 μL, of influenza virus, which had been previously tittered to 8 hemagglutination units/50 μL in sterile PBS, was added to every well of the plate. Plates were incubated 20 min for H1N1 and IBVs, but 30 min for H3N2 viruses that were additionally supplemented with 20 nM Oseltamivir carboxylate (Aobious, Gloucester, MA, USA). Following incubation, 50μL/well of washed turkey RBCs diluted to 0.08 % in PBS, were added to the plates containing H1N1 or IBV viruses and incubated for 30 min.
In a similar fashion, 50μL/well of guinea pig RBCs were diluted to 0.08 % and added to plates containing H3N2 viruses and incubated for 1 h. Following incubation with RBCs the plates were titled to observe the presence or absence of hemagglutination, and the HAI titer of the serum was determined by taking the reciprocal of the dilution in the last well that contained non-agglutinated RBCs. In this study, a “protective” antibody titer is described as possessing an HAI titer of ≥1:40.
Microneutralization assay
Prior to the assay, pooled sera samples from each vaccine group were prepared and heat treated at 56°C for 45 min. On the day of the assay, each well of a 96-well flat bottom microtiter plate (Thermo Fisher, Waltham, MA, USA) was filled with 50 μL of virus diluent (Dulbecco’s Modified Eagle Medium (DMEM), 1 % Penicillin/Streptomycin, 13.5 % Bovine Serum Albumin (BSA), 2.5 % 1M HEPES buffer, 2 μg/mL TPCK trypsin (Thermo Fisher, Waltham, MA, USA). An additional 40 μL of virus diluent was added to the first column of microtiter plate, followed by the addition of 10 μL of pooled heat-treated serum for an initial 1:10 serum dilution.
Two-fold serial dilutions of 50 μL were transferred across the plate to column 10, discarding the final 50 μL. Next, 50 μL of virus which has been previously tittered to 100x the 50 % tissue culture infectious dose (100x TCID50) was added to each well excluding column 12. An extra 50 μL of virus diluent was added to column 12 to establish negative control wells.
The plates were then incubated at 37 °C for 1 h. Following incubation, 100 μL of Madin-Darby canine kidney (MDCK)-SIAT-1 cells at a concentration of 1.5×105-2.0×105 cells/mL in virus diluent were added to every well of the plate. The plates were then incubated at 37 °C + 5 % CO2 for 18–20 h.
The next day, the media was removed and each well was washed with 200 μL of PBS. The PBS was decanted and the wells were fixed with ice-cold fixative solution (80 % Acetone (Thermo Fisher, Waltham, MA, USA) diluted in PBS for 10 min at room temperature. The fixative solution was decanted and the plates were dried for 1 h.
Following incubation, 100 μL of Madin-Darby canine kidney MDCK-SIAT-1 cells at a concentration of 1.5×105-2.0×105 cells/mL in virus diluent were added to every well of the plate. The plates were then incubated at 37°C + 5 % CO2 for 18–20 h. The next day, the media was removed from the plates and each well was washed with 200 μL of PBS.
The PBS was then removed, and the wells were fixed with ice-cold fixative solution (80 % Acetone diluted in PBS and incubated at room temperature for 10 min. The fixative solution was then decanted, and the plates were allowed to air dry for 1 h. The plates were then washed three times with 200 μL wash buffer (PBS + 0.03 % Tween-20).
The plates were then decanted and 100 μL of rabbit anti-influenza-A-NP polyclonal antibody (1 mg/mL, Cat. no. PA5–81661, Thermo Fisher, Waltham, MA, USA) diluted 1:3000 in blocking buffer (PBS + 0.03 % Tween-20 + 14.5 % BSA) was added to each well. The plates were then moved to a humidified chamber to incubate at RT for 1 h.
The plates were then washed three times with 200 μL wash buffer. After washing, the plates were decanted and 100 μL of goat-anti-rabbit IgG (H + L) HRP (1 mg/mL, Thermo Fisher, Waltham, MA, USA) diluted 1:5000 in blocking buffer was added to each well. After the addition of the secondary antibody, the plates were moved to a humidified chamber and incubated at room temperature for 1h.
Following incubation, the plates were washed five times with 200 μL wash buffer. The wash buffer was then decanted and 100 μL of substrate (one phosphate citrate tablet (Sigma, Saint Louis, MO, USA) dissolved in 100 mL diH2O, followed by the addition of five o-phenylenediamine dihydrochloride (OPD) tablets (Sigma, Saint Louis, MO, USA), and 50 μL of 30 % H2O2) was added to each well and allowed to develop for 3–5 min at room temperature. Once a color change started to occur in the negative control wells, column 12, 100 μL of stop solution (2N H2SO4 (Thermo Fisher, Waltham, MA, USA)) was added to each well.
The plates were then read at 492 nm using a BioTek Epoch 2 plate reader (Agilent, Santa Clara, CA, USA) equipped with Gen6 software. The 50 % neutralization titer was determined by averaging the optical density (OD) values of the virus control wells, column 11, minus the background signal, average of column 12, and using that value as the 100 % infection titer. The values in the experimental wells were then compared to the 100 % infection OD value, and a 50 % inhibition titer was calculated for each experimental group using a linear interpolation of the Log2 serum titers above and below the 50 % neutralization OD value.
All plates were run in duplicate, and the neutralization titers were averaged between the plates.
Influenza virus plaque assay
Influenza virus plaque assays were used to determine the quantity of replicating virus present in mouse respiratory tracts at 3 days post infection. In brief, 1×106 MDCK cells (Sigma, Saint Louis, MO, USA) were added to each well of a 6-well culture plate (Thermo Fisher, Waltham, MA, USA) and allowed to incubate overnight at 37°C. Lung samples were collected from 4 mice in each group at day 3 post infection and stored at −80°C for future use.
On the day of the assay, lung samples were thawed, weighed, and homogenized in 1 mL of DMEM containing 1 % Penicillin/Streptomycin (DMEM + P/S) (Thermo Fisher, Waltham, MA, USA). The supernatants were then serially diluted 10-fold in DMEM + P/S. On the day of the assay the samples were thawed and serially diluted 10-fold in DMEM + P/S.
When the MDCK cells reached 90–95 % confluency in the 6-well plates they were rinsed two times with DMEM + P/S. Following the wash steps, the media was removed, and 100 μL of each dilution of mouse lung homogenate was added to individual wells of the plates. Each sample was run in duplicate.
The plates were then allowed to incubate at room temperature for 1 h. During this hour the plates were shaken every 15 min to ensure that the plates did not dry. Once the incubation was complete the media was removed, and the wells were washed two times with DMEM + P/S.
The media was then removed and 2 mL of a mixture (50:50 v/v) of 2x MEM and 1.6 % agarose supplemented with 1 μg/mL of TPCK trypsin (Thermo Fisher, Waltham, MA, USA) was added to each well. The solution was allowed to solidify and cool to RT and the plates were incubated at 37°C + 5 % CO2 for approximately 72 h. Following the 72-h incubation, the agarose gels were physically removed, and the cells were fixed by adding 1 mL of 10 % buffered formalin (Thermo Fisher, Waltham, MA, USA) to each well.
After 10 min the formalin was removed and each well was stained with 1 % crystal violet (Thermo Fisher, Waltham, MA, USA) for 10 min. The crystal violet was then removed from each well and the plates were rinsed five times with water. The plates were then allowed to air dry.
Once the plates fully dried, the viral plaques were enumerated and reported as the number of plaques present in the reciprocal of each dilution. The mouse lung viral titers were reported as plaque forming units per gram (PFU/g) of lung tissue by dividing the number of plaques by the starting weight of each lung.
Hematoxylin and eosin staining of lung tissue
For pathological analysis, lung sections were subjected to hematosylin and eosin (H&E) staining as previously described (Li et al., 2021). The fixed lungs were embedded in paraffin and 5 μm thick transverse sections were mounted in Apex superior adhesive slides (Leica biosystem Inc., Buffalo Grove, IL, USA). Sections were deparaffinized in xylene and hydrated using ethanol.
Deparaffinized and hydrated lung sections were stained with hematoxylin (Millipore Sigma, Burlington, MA, USA) for 8 min at RT, differentiated in 1 % acid alcohol for 10 s, and then counterstained with eosin (Millipore Sigma) for 30 s. Slides were then dehydrated with 95 % and 100 % ethanol, cleared by Xylene, and mounted using Permount mounting media (Thermo Fisher Scientific, Waltham, MA, USA). The tissues were examined for inflammatory cell infiltration and inflammation scoring (Sont et al., 1996), which was performed as previously described (Wang et al., 2018).
The severity of inflammation was assessed using a 0–5 scale (Song et al., 2024). A score of 0 represented normal tissue with no inflammation. A score of 1 indicated mild inflammation with small clusters of inflammatory cells.
A score of 2 corresponded to mild ring-shaped infiltration of inflammatory cells, forming a layer one cell in thickness. A score of 3 was given for moderate ring-shaped infiltration with a layer two to four cells in thickness. A score of 4 represented severe ring-shaped infiltration with a layer four cells in thickness.
Finally, a score of 5 indicated very severe inflammation with extensive infiltration and a loss of the normal tissue structure.
Statistical analysis
A nonparametric one-way analysis of variance (ANOVA) was used to determine the statistical differences between groups using GraphPad Prism software (GraphPad, San Diego, CA, USA). For this study, a p value < 0.05 was defined as statistically significant (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).
Results
Vaccinations
Mice (female, DBA2/J; n = 22, 6–8 weeks of age) were infected with historical H1N1 and H3N2 influenza viruses to induce immune responses and establish a pre-immune anti-influenza state prior to vaccination (Fig. 1). All mice had HAI activity against each virus 14 days post-infection (Supplemental Fig.
1). Mice were then vaccinated with 2023–2024 Fluzone (750 ng dose) manufactured by Sanofi Pasteur (Swiftwater, PA, USA), a split-inactivated commercial influenza virus vaccine, at day 0 and half the mice were boosted at day 28 with or without an adjuvant (Fig. 1).
Each vaccine was formulated with one of two adjuvants, TRAC478 (Inimmune, Missoula, MT, USA) or Addavax (ThermoFisher, Waltham, MA, USA) in a 1:1 ratio in a 50 μl solution. Mock vaccinated animals received adjuvant only in 0.9 % buffered saline (Fig. 1).
Blood was collected from each mouse at 0, 14, 44, 51, 72, and 79 days post-vaccination.
Serological assessment
All vaccinated mice had anti-HA antibodies that bound to rHA proteins representing the H1, H3, and B HA proteins in the vaccine (Fig. 2). Mice vaccinated with Fluzone without adjuvant had low to undetectable anti-HA antibodies two weeks following the first vaccination (Fig.
2A–D), but these titers rose ~5–7 fold following the second vaccination (Fig. 2E–H). Mice vaccinated with Fluzone plus Addavax had 4–5 fold higher anti-HA antibody titers against the H1 and H3 rHA proteins following the first vaccination compared to mice vaccinated with Fluzone without adjuvant.
Similar results were observed with mice vaccinated with Fluzone mixed with TRAC478 (5–7 fold higher titers) compared to mice vaccinated with Fluzone without adjuvant. Following the first vaccination, anti-HA antibodies were low to undetectable against the B/Yamagata (PH/13) HA, but titers against the B/Victoria (AU/21) HA were similar to the titers detected against the two influenza A components. Following vaccination with or without adjuvant, the anti-HA titers against the two influenza B HA components rose ~2-fold against AU/21 and 4–10 fold against PH/13 (Fig.
2G and H). Following a booster vaccination, mice vaccinated with Fluzone plus Addavax or unadjuvanted Fluzone vaccine had a CD4+ T helper type 2 (Th2) bias as indicated by the significantly higher titer of IgG1 anti-HA antibodies compared to IgG2a (Fig. 3).
Mice vaccinated with Fluzone mixed with either adjuvant had higher IgG2b anti-HA antibodies against the H1 and H3 components (Fig. 3). Mice vaccinated with unadjuvanted Fluzone or Fluzone plus Addavax had higher IgG1-IgG2a ratios indicating a Th2 bias, whereas mice vaccinated with Fluzone plus TRAC478 had a ratio 1 or less indicating a T helper 1 (Th1) or mixed response (Supplemental Fig.
2).
To assess the ability of the vaccine elicited antibodies that block HA receptor binding, serum samples were assessed in an hemagglutination-inhibition (HAI) assay against H1N1, H3N2, and influenza B viruses. Mice vaccinated with unadjuvanted Fluzone had sera with HAI activity against the H1N1 component, VC/19, that ranged from 1:10 to 1:640 following the initial vaccination with an average HAI titer of 1:20 (Fig. 4A).
These same serum samples had low to undetectable HAI activity against the H1N1 VC/22 (Fig. 4B). Mice vaccinated with Fluzone plus Addavax had, on average, an HAI titer of 1:80 against VC/19 and mice vaccinated with Fluzone plus TRAC478 had an average HAI titer of 1:40 against VC/19 (Fig.
4A). HAI titers were ~2-fold lower against VC/22 (Fig. 4B).
Following a second vaccination, mice vaccinated with Fluzone with or without adjuvant had average HAI titers of 1:320 or greater against VC/19 (Fig. 4G) with a 6–8 fold increase in HAI activity against the drifted VC/22H1N1 virus following the second vaccination (Fig. 4H).
Mice vaccinated with Fluzone plus Addavax had statistically similar anti-HA titers to mice vaccinated with unadjuvanted Fluzone following one vaccination (Fig. 4C). These ant-HA against DR/21 rose 4–6 fold following the second vaccination (Fig.
4I). Mice vaccinated with Fluzone plus Addavax had significantly higher HAI titers against drifted H3N2 virus, MA/22, compared to unadjuvanted Fluzone, albeit the average HAI titers were low (Fig. 4D).
Following a second vaccination, there was a significant rise in HAI activity with average HAI titers of 1:80 or higher against the MA/22 strain (Fig. 4J). Similar results were observed using these same serum samples against an extended panel of H1N1 and H3N2 viruses (Supplemental Fig.
3). These same mice had HAI activity against both influenza B viruses following the prime vaccination with average HAI titers of 1:80 or higher (Fig. 4E and F) with titers that rose 2–4-fold following the second vaccination (Fig.
4K and L).
Following the first vaccination, mice vaccinated with unadjuvanted Fluzone or Fluzone mixed with Addavax had low neutralization titers against VC/19 and DR/21 influenza viruses with higher titers against the two influenza B viruses (Fig. 5A–D). Mice vaccinated with Fluzone plus TRAC478 had 2–6 fold higher neutralization titers after one vaccination against the 4 influenza vaccine strains than mice vaccinated with the other vaccine formulations (Supplemental Fig.
4). Following a second booster vaccination, neutralizing titers rose against all viruses, regardless of the vaccine formulation used (Fig. 5E–H), although the lowest neutralization titers were against the H3N2 virus (Fig.
5F).
Influenza virus challenge
To determine if each vaccine and adjuvant combination could protect against viral infection, mice were challenged with H1N1 or H3N2 influenza viruses (Fig. 6). Mock vaccinated mice lost on average 15 % of their body weight by day 5 following infection with signs of morbidity and began to recover following challenge with the H1N1 influenza virus BR/18 (Fig.
6A). Mock vaccinated mice had high viral lung titers (~1×10^5 pfu/g) at day 3 post-challenge (Fig. 6B).
All mice were vaccinated with a single vaccination were protected from an BR/18H1N1 viral challenge with little weight loss (Fig. 6A). Mice vaccinated with unadjuvanted Fluzone had detectable viral lung titers in 2 of 3 mice at day 3 post-challenge, but no virus was detected in mice vaccinated with Fluzone plus Addavax or TRAC478 (Fig.
6B). In contrast, there were no detectable viral titers in mice vaccinated two times with vaccine and challenged with BR/18.
A second set of mice were challenged with the H3N2 influenza virus, SW/13 (Fig. 6C and D). Mock vaccinated mice lost ~22 % of their original body weight by day 4 post-challenge with 30 % of mice surviving challenge (Fig.
6C) and had an average viral lung titer of 1×10^4.5 pfu/g (Fig. 6D). Vaccinated mice lost 10–12 % of their body weight by day 3 post-challenge and then began to regain weight and recover over the 14 days of observation.
Mice vaccinated with unadjuvanted Fluzone had statistically similar viral lung titers as mock vaccinated mice, but all mice survived challenge (Fig. 6D). In contrast, mice vaccinated with Fluzone and either adjuvant had 2–2.5 logs lower viral lung titers at day 3 than mice administered unadjuvanted Fluzone vaccine (Fig.
6D).
Lung pathology
The lungs of BR/18 challenged mice vaccinated a single time were assessed for lung damage and cellular infiltration by H&E staining (Fig. 7A–D). Mock vaccinated mice challenged with BR/18 had lungs with severe diffuse pathology, resulting in a pathological score of 5 (Fig.
7D). The alveoli were filled with inflammatory exudate and the lungs had severe inflammation with extensive infiltration and loss of normal tissue structure with influenza virus induced pneumonia. The tissue destruction and immune cell exudate exceeded damage observed in the lungs of vaccinated mice.
Mice vaccinated with unadjuvanted Fluzone had a score of ~3 with a moderate level of interstitial inflammation in the lungs with many alveoli partially filled with mononuclear cells and septal thickening, however the alveolar architecture remained intact (Fig. 7A). Mice vaccinated with Fluzone plus Addavax had lungs with mild cellular infiltration with a few clusters of inflammatory cells in the airways, but the alveolar space was preserved.
The lungs from these mice were scored as a 2 (Fig. 7C). Mice vaccinated with Fluzone plus TRAC478 and challenged with BR/18 had close to normal lungs with few infiltrating inflammatory cells and a score of 1 (Fig.
7B). Similar results were observed following SW/13 challenge, with the lungs of mice vaccinated with unadjuvanted Fluzone with a pathological score of 3 (Fig. 7E), the lungs of mice vaccinated with Fluzone plus Addavax scoring a 2 (Fig.
7F), the lungs of mice vaccinated with Fluzone plus TRAC478 scoring a 1, and the lungs of mock-vaccinated mice scoring a 5 (Fig. 7H).
Statistical analysis
The nonparametric Kruskal-Wallis test, unpaired t-test or one-way analysis of variance (ANOVA) were used to analyze the statistical differences between groups using GraphPad Prism 10 software (GraphPad, San Diego, CA, USA). For this study, a p value < 0.05 was defined as statistically significant (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).
Discussion
Current influenza vaccines primarily target the surface glycoprotein hemagglutinin (HA), since the elicitation of protective antibodies against HA is crucial to neutralize virus infections (Skehel and Wiley, 2000). Several commercial seasonal inactivated influenza vaccines (IIV) are on the market (Cowling et al., 2020) that contain H1N1, H3N2, and influenza B strains. Fluzone Standard Dose (SD), produced by Sanofi Pasteur in embryonated chicken eggs, is a split inactivated virus vaccine contains 15 μg of HA per strain without the inclusion of an adjuvant (Abreu et al., 2021; Grohskopf et al., 2019).
In this report, adjuvants were formulated with Fluzone influenza vaccine and assessed for the elicitation of immune responses following one or two vaccinations. Vaccination with recombinant HA and NA proteins mixed with Addavax elicits a systemic Th2 antibody response (AboulFotouh et al., 2022; Huang et al., 2024; Nagashima et al., 2023; Ross et al., 2022), which was also observed in this study using split-inactivated Fluzone influenza virus vaccine. The adjuvant TRAC478 is a combination adjuvant consisting of unique synthetic TLR4 (INI-2002) and TLR7/8 (INI-4001) agonists (Miller et al., 2023) that stimulates robust immune responses (Uno et al., 2024; Uno and Ross, 2024).
Both these synthetic TLR agonists signal through TRIF and MyD88 pathways that ultimately drive the production of pro-inflammatory cytokines for improved T cell priming, as well as enhanced humoral responses (Luo et al., 2024). TLR7 and TLR8 each primarily recognize ssRNA within the endosome (Kayesh et al., 2024b). Co-encapsulating TLR4 and TLR7/8 within liposomes results in synergistic innate and adaptive immune responses characterized by a Th1 or a balanced Th1/Th2 response depending on route of administration or antigen used in the formulations (Bretscher, 2014; Maroof et al., 2014; PreventionC.f.D.C.a., 2019; Reis and Ross, 2025; Short et al., 2022a; Short et al., 2019a).
Mice vaccinated with Fluzone plus TRAC478 elicited higher neutralizing antibodies than mice vaccinated with unadjuvanted Fluzone or Fluzone mixed with Addavax after the first vaccination but had statistically similar titers against most influenza virus strains with lower HAI titers against some drifted H1 and H3 strains not included in the vaccine compared to mice vaccinated with Fluzone plus Addavax. The activation of T-helper 1 CD4+ cells stimulates the release of IFN-gamma that results in enhanced antigen presentation to B cells, stimulating B cells differentiation and antibody production, as well as the activation of CD8+ T cells that target infected cells for clearance (Janeway et al., 2001). Adjuvants that effectively stimulate T-helper CD4+ cells can result in IL-4/IL-5 secretion and thereby enhancing B cell antibody secretion (Kopf et al., 1993). in this study TRAC478 stimulated a mixed T helper response when formulated with rHA, this adjuvant was more effective at eliciting neutralizing antibodies after a single vaccination against influenza viruses compared to mice vaccinated with Addavax that elicited primarily at T helper 2 response.
Following challenge with an H1N1 or H3N2 influenza viruses, there were little statistical differences in weight loss or survival, regardless if the mice were vaccinated with Fluzone with or without adjuvant. Therefore, despite any differences in elicited immune responses between vaccine formulations. mice vaccinated one time with unadjuvanted Fluzone were completely protected against morbidity or weight loss. Regardless of the vaccine administered, mice had similar viral lung titers at 3 days following H1N1 influenza virus challenge.
Mock vaccinated mice had high viral lung titers 3 days post-challenge, whereas mice vaccinated with unadjuvanted. Mice immunized with unadjuvanted Fluzone had ~2–3 logs higher viral lung titers than mice vaccinated with Fluzone plus adjuvant but had ~2 logs lower viral lung titers than mock vaccinated mice. In contrast there were undetectable viral titers in mice vaccinated with Fluzone and mixed with either adjuvant.
However, following a second vaccination, all mice vaccinated with Fluzone with or without adjuvant had no detectable viral lung titers.
Fluzone is approved in the U.S. for people age 6 months or older with higher dose vaccine available for older adults The vaccine is administered as a single dose vaccination each influenza season, with some exceptions for children under the age of 9 and the elderly (Prevention, C. f.D.C.a., 2024). The vaccine effectiveness of this single shot vaccine ranged between 29 and 56 % over the past 9 seasons (2016–2017 to 2024–2025) and can be influenced by several factors, including antigenic match of the vaccine strains to circulating strains, a person's age and health status, and status of previous influenza vaccinations (Carlock et al., 2024; Hammerton et al., 2025; Nunez et al., 2017; Pavia-Ruz et al., 2013). Despite these limitations, influenza vaccination can enhance and maintain anti-influenza immunity and reduce the risk of severe illness, hospitalization, and death (Munoz et al., 2018, 2020; Net et al., 2021).
In this study, the addition of adjuvant with Fluzone enhanced the vaccine effectiveness and protected mice following a single vaccination. This highlights how the addition of an effective adjuvant with a currently available commercial influenza vaccine can elicit higher protective antibody responses and reduce morbidity and mortality following influenza virus infection. FluAd is a commercial standard-dose, inactivated influenza vaccine manufactured from eggs and adjuvanted with MF59 (homologous to Addavax), that is approved for use in elderly populations.
A single dose of FluAd is consistently more effective than standard dose unadjuvanted vaccines in the elderly (Squarcione et al., 2003). Overall, the addition of adjuvants to influenza vaccines may be an effective strategy to enhance immunity and reduce the dose of the vaccine and maintain a single dose regimen each season.
Supplementary Material