Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in quadrivalent influenza vaccine vaccinated mice

Two LNP formulations were prepared by mixing an aqueous solution containing the in vitro transcribed SDI-RNA (or SDI) with an ethanolic solution containing (1) ionizable lipids, either K-Ac7-Dsa (comprising a ketal bond) or S-Ac7-Dog (comprising a disulfide bond; chemical structure outlined in Supplementary Figure 1), to interact with negatively charged SDI and mediate endosomal escape; (2) cholesterol, for structural stability; (3) dioleoylphosphatidylethanolamine (DOPE) phospholipid, as helper lipids to aid in nanoparticle formation; and (4) 1,2-distearoyl-rac-glycero-3-methylpolyethylene glycol (DSG-PEG; 2kDa PEG) to provide steric hindrance and thus avoiding aggregation and promoting mobility in vivo (23). The structure and composition of LNP incorporating SDI RNA is schematically represented in Figures 1A, B, respectively. The molar ratio of ionizable lipid (K-Ac7-Dsa or S-Ac7-Dog):cholesterol:DOPE : DSG-PEG was chosen to be 50:38.5:10:1.5, based on literature (24). Empty LNPs that did not contain SDI were prepared as control and hence referred to as empty LNP or LNP(−). Both LNPs encapsulating SDI [S-Ac7-Dog(SDI) and K-Ac7-Dsa (SDI)] and corresponding control empty LNPs [S-Ac7-Dog(−) and K-Ac7-Dsa (−)] were fabricated and characterized for their size and zeta potential (ZP) (Figures 1C, D). While K-Ac7-Dsa(−) and K-Ac7-Dsa(SDI) LNPs showed some differences in their size (78 and 189 nm, respectively), the S-Ac7-Dog(−) and S-Ac7-Dog(SDI) LNPs showed similar size distributions (91 and 101 nm, respectively) with low polydispersity indices (PDI < 0.25; as shown in Figure 1C) and a positive ZP of approximately 4–6.5 mV at physiological pH (Figure 1D).

We evaluated the potential of empty and SDI-incorporating S-Ac7-Dog and K-Ac7-Dsa LNPs to adjuvant a licensed QIV, with and without combination with IMDQ-PEG-Chol adjuvant, using our previously well-established preclinical vaccination-infection animal model (17, 30). The study is outlined in Figure 2A. We vaccinated 6- to 8-week female BALB/c mice with unadjuvanted QIV or in combination with SDI or IMDQ-PEG-Chol, or both, while mock animals received PBS. We further tested the adjuvant effect for QIV upon co-administration of SDI and/or IMDQ-PEG-Chol combined with one of the two LNPs containing different cationic lipids (S-Ac7-Dog or K-Ac7-Dsa) as described in the previous section. LNPs either were empty (−) or had SDI incorporated. The rationale of this setup is that LNP-formulated SDI can, besides endosomes, also be delivered to the cytosol, thereby promoting more efficient RIG-I-mediated innate immune sensing. On the other hand, IMDQ-PEG-Chol is expected to incorporate efficiently in LNPs via its cholesterol moiety. The animals were vaccinated only once via the intramuscular (IM) route. The serum collected 3 weeks post-vaccination was examined for the presence of H1 HA-specific IgG antibodies using enzyme-linked immunosorbent assay (ELISA) for total IgG, IgG1, and IgG2a (Figure 2B).

No virus-specific antibody titers were detected in the serum from the mock PBS group. The group that received unadjuvanted QIV was used as the reference to compare the IgG responses of other groups. QIV formulated with S-Ac7-Dog LNP (−) or K-Ac7-Dsa LNP(−), corresponding to empty S-Ac7-Dog and empty K-Ac7-Dsa LNPs, respectively, showed higher IgG1 titers but the lowest IgG2a titers compared with the corresponding S-Ac7-Dog LNP(SDI) or K-Ac7-Dsa LNP(SDI) groups (Figure 2B; Supplementary Figure 2), thereby illustrating the intrinsic adjuvant effect of LNPs. Additionally, QIV formulated with SDI-incorporated LNPs [S-Ac7-Dog LNP(SDI) and K-Ac7-Dsa LNP(SDI)] can induce a balanced IgG1 and IgG2a antibody response. Overall, the total IgG levels were found similar among all adjuvanted and LNP-formulated groups post-prime vaccination. However, the S-Ac7-Dog LNP(SDI) seemed to induce slightly higher total IgG compared to K-Ac7-Dsa LNP (SDI) in the corresponding SDI ± IMDQ-PEG-Chol combination adjuvanted groups. A similar observation was made for IgG1 antibody titers with S-Ac7-Dog LNPs inducing higher IgG1 titers than respective K-Ac7-Dsa LNP groups. Consistent with our previous findings (17), IMDQ-PEG-Chol administration, with either empty or SDI-incorporated S-Ac7-Dog or K-Ac7-Dsa LNPs, skews the antibody responses towards IgG2a with a significant reduction in IgG1 titers (Figure 2C). QIV+SDI and QIV+IMDQ-PEG-Chol groups, with no LNP formulations, were used as control vaccination groups for the study and to correlate with our previous study.

Neutralizing antibodies are important defense mechanisms during viral infection. These antibodies bind to the viral antigens and change the conformation, thereby blocking the viral attachment to cells. As a surrogate for virus neutralizing antibody levels, we performed hemagglutination inhibition (HAI) assays with post-vaccination sera collected from all vaccinated animals at 3 weeks post-vaccination. As shown in Figure 3, the mice that received unadjuvanted QIV showed low HAI titers, which were not significantly different from the groups that received QIV formulated in either empty or SDI-containing S-Ac7-Dog or K-Ac7-Dsa LNPs. The group administered with QIV+IMDQ-PEG-Chol, without LNP formulations, showed significantly higher HAI titers compared with the unadjuvanted QIV group. HAI titers were significantly higher for the groups that received empty or SDI-containing S-Ac7-Dog in the LNP formulation when combined with the IMDQ-PEG-Chol adjuvant. However, the corresponding K-Ac7-Dsa LNP-formulated groups did not show any significant differences in HAI titers compared with the unadjuvanted or unformulated QIV group.

Helper CD4+ and cytotoxic CD8+ T cells play an important part in vaccination-mediated humoral and cellular responses by facilitating Ig class switch during B-cell maturation and direct killing of infected cells, respectively. T cells can recognize foreign antigens presented on the major histocompatibility complex (MHC) molecules on infected cells followed by the release of various cytokines, including IFN-γ and IL-4, two major cytokines corresponding to helper type 1 (Th1) and type 2 (Th2) T cells, respectively. IFN-γ and IL-4 can modulate class switching of B cells to IgG2a or IgG1 (19). To study the correlation between the two cytokines and antibody responses in our vaccination model, we examined the release of IFN-γ and IL-4 from splenocytes obtained from the mice 10 days post-vaccination (DPV) (outlined in Figure 4A), in the presence and absence of specific antigen (IVR-180 virus or H1 HA peptide) using enzyme-linked immunosorbent spot (ELISpot) assays.

As shown in Figure 4, antigen-specific IFN-γ (Figure 4B) or IL-4 (Figure 4C) release from the splenocytes was very low in the absence of a stimulant, except for QIV+S-Ac7-Dog(−) or QIV+K-Ac7-Dsa(−) groups, suggesting a basal level of non-specific stimulation after vaccination by the empty LNPs. Upon stimulation with specific H1-HA peptide or IVR-180 virus, the number of splenocytes producing antigen-specific IFN-γ as well as IL-4 were found to be higher in the mice that received QIV+IMDQ-PEG-Chol formulated in S-Ac7-Dog(SDI) or K-Ac7-Dsa(SDI) LNPs, suggesting an induction in both Th1 and Th2 immune response. The group administered with QIV+IMDQ-PEG-Chol, without LNP formulations, showed high IFN-γ release compared with the unadjuvanted QIV group, consistent with our previous study. Mice that received PBS were used as a reference for statistical comparison.

To further correlate B- and T-cell responses with the extent of protection against virus replication in vivo, all vaccinated and un-vaccinated mice were given a high-titer virus challenge with 18,000 plaque-forming units (PFU) of IVR-180 virus per animal. A single dose of vaccination was found effective in conferring protection from severe morbidity in challenged animals compared with mock-challenged mice in the initial 5 days of virus challenge. As shown in Figure 5A, the groups receiving unadjuvanted QIV and QIV with K-Ac7-Dsa(− or SDI) LNPs showed less than 10% body weight loss over 5 days post-infection. The unvaccinated PBS group lost approximately 20% body weight by 4 DPI. Interestingly, the mice vaccinated with S-Ac7-Dog (−), irrespective of the combination with IMDQ-PEG-Chol, showed drastic weight loss over 5 days, almost comparable to the unvaccinated PBS group. This is especially important to note because the same S-Ac7-Dog LNPs incorporating SDI did not show such extensive weight loss in vaccinated/challenged animals, suggesting higher morbidity in virus-infected lungs in case of empty S-Ac7-Dog LNP formulations.

Irrespective of the body weight loss differences attributed by LNPs, all groups that received adjuvanted QIV showed a lower amount of replicating virus in their lungs at 5 DPI (Figure 5B), compared with the unadjuvanted QIV group. QIV formulated in either of the two empty LNPs [S-Ac7-Dog(−) or K-Ac7-Dsa(−)] did not provide significant protection in the initial days of infection compared with the unadjuvanted QIV group, contrary to the higher IgG1 induction. The groups that received QIV with either S-Ac7-Dog(− or SDI) or K-Ac7-Dsa(− or SDI) LNPs, irrespective of IMDQ-PEG-Chol combination, resulted in significantly better control of lung virus replication, with very low detectable titers in their lungs, and, therefore, correlated with enhanced vaccine responses observed in mice. Interestingly, S-Ac7-Dog LNPs resulted in lower body weight loss in the first 5 days of infection, compared to K-Ac7-Dsa LNPs in animals when compared between corresponding adjuvant groups.

The body weight data over 5 DPI suggested that the administration of empty S-Ac7-Dog LNP as vaccine ± adjuvant formulation, although protective, resulted in higher body weight loss, comparable to PBS-vaccinated control mice. The lungs of all infected animals were examined for their cytokine profiles at 5 DPI. As shown in Figure 6 ; Supplementary Figure 3, the cytokine profiles in the infected lungs of animals from vaccinated or unvaccinated/PBS groups were different. The PBS group showed high levels of inflammatory cytokines including IL-6, IL-18, IL-12 p70, IFN-γ, and TNF-α as well as chemokine GMCSF, which may suggest enhanced vascular permeability and increased infiltration of innate immune cells in response to infection in unvaccinated animals. As shown in Figure 6A, these cytokine levels were significantly lower in all vaccinated groups, implying a better control over inflammation and morbidity in response to the virus. Some of the cytokines were very low in the PBS group, especially the signatures for T-cell responses such as IL-4, IL-5, and IL-13, which is in line with the typical type 1 skewed host immune response to influenza infection. Interestingly, pro-inflammatory cytokines and chemokines, including IL-1β, GMCSF, and the type 2 cytokines IL-4, IL-5, IL-6, and IL-13, were significantly elevated in unadjuvanted QIV and QIV± IMDQ-PEG-Chol formulated with empty LNPs. This was not the case for the corresponding groups with LNP(SDI), especially for those LNP groups with S-Ac7-Dog lipids. The PCA plot in Figure 6B clusters different vaccinated and unvaccinated groups based on their differences in the production of inflammatory cytokines and chemokines in lungs post-infection. The unvaccinated PBS group clusters far away from all vaccinated animals, corresponding to high inflammation and chemokine production in lungs post-infection with lower interleukins. Moreover, the QIV+ S-Ac7-Dog(−) vaccinated group clustered separately from the other vaccinated groups. Additionally, the group that received QIV+ S-Ac7-Dog(−) combined with IMDQ-PEG-Chol is clustered together with unadjuvanted QIV, suggesting that the use of empty S-Ac7-Dog is disadvantageous as it reduces the protective effect of IMDQ-PEG-Chol when used without any lipid formulation. The other vaccinated groups showing low inflammation in lungs are clustered together.

The Madin-Darby canine kidney (MDCK) cell line was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1× antibiotics (penicillin/streptomycin).

Quadrivalent inactivated influenza vaccine (FLUCALVEX 2018/2019 season Lot 252681) was obtained from Seqirus. The vaccine consists of MDCK-grown two IAV and two IBV- A/Singapore/GP1908/2015 IVR-180 (H1N1) (A/Michigan/45/2015-like virus), A/North Carolina/04/2016 (H3N2) (A/Singapore/INFIMH-16-0019/2016-like virus), B/Iowa/06/2017 (B/Colorado/06/2017-like virus), and B/Singapore/INFTT-16-0610/2016 (B/Phuket/3073/2013-like virus).

The SDI-RNA (or SDI) was in vitro transcribed as described in our recent study (17).

SDI or SDI equivalent (1 μg) was encapsulated in LNPs by rapid mixing under vigorous stirring of an acetate buffer (5 mM, pH 4.5) containing SDI with an ethanolic solution containing the ionizable lipid S-Ac7-Dog or K-Ac7-Dsa (to obtain S-Ac7-Dog and K-Ac7-Dsa LNPs respectively), cholesterol, 1,2-dioleoyl-n-glycero-3-phosphoethanolamine (DOPE), and a poly(ethylene glycol)-lipid (DSG-PEG; PEG had an MW of 2 kDa) at a 50:38.5:10:1.5 ratio. After mixing, LNPs were dialyzed against 1× PBS to get rid of ethanol and the pH was adjusted to 7.4. An N/P (ionizable nitrogen atoms of the ionizable lipid to anionic phosphor atoms of SDI) molar ratio of 5:1 was targeted.

The diameter and polydispersity index (PDI) of LNPs were measured with dynamic light scattering (DLS) at physiological pH. Each sample was measured in triplicate and a cumulative average of z average and PDI was calculated. For ELS, each sample was appropriately diluted in HEPES buffer and measurements were taken in triplicate. The Zeta potential was calculated for all samples based on the Smoluchowski equation.

For each animal, 1.5 mg of HA equivalent of QIV was mixed with empty or SDI-encapsulating S-Ac7-Dog or K-Ac7-Dsa LNPs, with or without 100 μg of IMDQ-PEG-Chol (equivalent to 10 μg of core IMDQ), and vortexed for 30 s. Adjuvant doses were chosen based on our previously published work with these adjuvants (17, 30). Unadjuvanted or adjuvanted QIV, with or without LNP formulations, was administered intramuscularly in a total of 50 μL per mouse, in the right hind leg. The control group was administered with equal volume of PBS instead of vaccine or vaccine ± adjuvant ± LNP mixture. All animals received only one dose of vaccine without any further boosters.

A/Singapore/GP1908/2015 IVR-180 (H1N1) was grown in 8-day-old embryonated chicken eggs and was titrated by plaque assay on pre-seeded MDCK cells.

The study was performed on 6- to 8-week-old female BALB/c mice strains obtained from Jackson Laboratories, CT. The mice were housed with food and water ad libitum in a pathogen-free facility at Icahn School of Medicine at Mount Sinai, New York. All mice were vaccinated intramuscularly (50 μL; hamstring muscles of the left hind leg; per mouse) and infected intranasally (in 50 μL total volume per mouse) under ketamine/xylazine anesthesia. All procedures were performed according to the protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC-PROTO202100007).

Mice blood was collected by submandibular bleed 3 weeks post-vaccination from all animals. The blood was allowed to clot at 4°C for overnight. The serum was collected after a brief centrifugation and was heat inactivated at 56°C for 30 min. The samples were stored at −20°C until further use.

ELISA was performed to quantify the vaccine-specific IgG titers in mice sera. Briefly, ELISA plates were coated with recombinant trimeric HA derived from the A/Michigan/45/2015 H1N1 virus, which is closely related to IVR-180, as previously described (17), equivalent to 2 µg H1N1-HA/mL, in bicarbonate buffer and left overnight at 4°C. Plates were washed three times with 1× PBS and incubated in 100 μl of blocking buffer per well [5% fat-free milk in PBST (1× PBS + 0.1% Tween20)] for 1 h at room temperature (RT). In the meantime, the serum samples were serially diluted 3-fold, starting with 1:100 dilution, in blocking buffer and 50 μL of each sample dilution was incubated on HA-coated ELISA plates overnight at 4°C. The following day, the plates were washed three times with PBST and incubated with 100 μL of diluted horseradish peroxidase (HRP)-conjugated anti-mouse secondary total IgG (1:5,000) or IgG1 (1:2,000) or IgG2a (1:2,000) antibodies, for 1 h at RT. Finally, the plates were washed three times in PBST and incubated with 100 µl of tetramethylbenzidine (TMB) substrate at RT until the blue color appeared. The reaction was terminated with 50 µl of 1 M sulfuric acid (H₂SO₄), and the absorbance was measured at 450 nm and 650 nm wavelengths using BIOTEK ELISA plate reader.

Mice sera collected 3 weeks post-vaccination were treated with four volumes of receptor destroying enzyme (RDE) at 37°C overnight, followed by treatment with five volumes of 1.5% sodium citrate at 56°C, 30 min. The thus obtained 1:20-diluted serum samples were further serially diluted in a transparent V-bottom 96-well plate and incubated with 4 HA units per well of IVR-180 virus for 30 min at RT, followed by the addition of 0.5% chicken red blood cells for 40 min at 4°C. The results were recorded as HAI titers.

Mice were vaccinated with different combinations of QIV ± adjuvant ± LNPs and spleens were harvested at 10 DPV from all vaccinated as well as unvaccinated animals. Spleens were collected in 5 mL of RPMI-1640 media supplemented with 2% FBS and 1× penicillin/streptomycin and kept on ice. A single-cell suspension of splenocytes was obtained by homogenizing the spleens against a 70-μm strainer. Interferon gamma (IFN-γ) or interleukin-4 (IL-4) ELIspot assays were performed using 10⁵ splenocytes per well in a 96-well polyvinylidene difluoride (PVDF) ELIspot plates provided in the ELIspot kits (Supplementary Table), precoated with IFN-γ or IL-4 capture antibodies, respectively, according to the manufacturer's protocol. Splenocytes were left unstimulated or restimulated either with hemagglutination (HA-H1N1) overlapping 15-mer peptides or whole live IVR-180 (H1N1) virus and incubated overnight in 37°C incubator. The wells were washed thrice with water to get rid of cells and incubated with 100 μL of biotinylated polyclonal detection antibody against IFN-γ or IL-4 for 1.5 h at RT, followed by an incubation with streptavidin-HRP-conjugated antibody for 1 h at RT. The plates were finally incubated with 100 μL of the substrate for 1 h in dark, followed by thorough washing under tap water multiple times. The plates were air-dried in the dark and the number of spots in each well was manually counted using a dissection microscope. The results were represented as number of IFN-γ- or IL-4-producing splenocytes per million splenocytes.

A high-titer dose of IVR-180 H1N1 virus of 18,000 PFU, which corresponds to 100× lethal dose that kills 50% of female BALB/c mice, per animal was used for intranasal infection in a final volume of 50 µL per mouse. The virus challenge was performed under mild anesthesia with ketamine/xylazine (intraperitoneal) as recommended by IACUC. The unvaccinated but challenged group was used as a control in the experiment. Body weights were recorded every day post-infection until lung harvest. The lungs were collected at 5 DPI in 500 μL of 1× PBS and homogenized using a tissue homogenizer. The lysate thus obtained was stored at −80°C for viral titrations.

Virus titrations were performed by plaque assays to quantify the replicating virus titers in the lungs of vaccinated versus unvaccinated mice. The lung homogenate (or lysate) was 10-fold serially diluted in 1× PBS and incubated on pre-seeded and pre-washed monolayers of MDCK cells for 1 h in an incubator, at 37°C, 5% CO₂ with gentle shaking every 5 min. The diluted samples were then removed, and the monolayers were again briefly washed with 1 mL of 1× PBS. Lastly, 1 mL of the overlay mixture [2% oxoid agar and 2× minimal essential medium (MEM) supplemented with 1% diethyl-aminoethyl (DEAE)-dextran and 1 mg/mL tosylamide-2- phenylethyl chloromethyl ketone (TPCK)-treated trypsin] was added on top of the monolayers and incubated for 48 h in the incubator, at 37°C, 5% CO₂. The plates were finally fixed in 4% formaldehyde. The overlay was removed, and the plaques were immune-stained with IVR-180-postchallenge polyclonal serum, diluted 1:1,000 in blocking buffer. The plates were washed and incubated with 1:5,000 dilution of HRP-conjugated anti-mouse secondary antibody for 1 h at RT with gentle shaking. Followed by a brief washing in 1× PBST, the plaques were finally visualized with True Blue substrate and the number of plaques was counted and presented as PFU/mL.

Luminex-based cytokine ELISA was performed for simultaneous measurements of different cytokines in the lung homogenates from IVR-180-infected mice using the Th1/Th2 Cytokine 11-Plex Mouse ProcartaPlex™ kit (Invitrogen; EPX110-20820-901). Lungs were harvested at 5 DPI, homogenized in 500 μL of PBS, and centrifuged at 5,000 g for 5 min. Twenty-five microliters of each lysate was used for the assay. The following cytokines were measured: Granulocyte macrophage colony-stimulating factor (GMCSF), interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-12p70, IL-13, IL-18, and interferon gamma (IFN-γ). The assay was performed according to the manufacturer's guidelines and the readings were recorded using the Luminex 100/200 system.

The schematic figures were created with BioRender.com. GraphPad Prism version 10 was used for data visualization, analysis, graph plotting, and statistical analysis. Principal component analysis was performed using the statistical software package R: R Core Team (2023). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/↗.