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Article

Peptide Antigen Modifications Influence the On-Target and Off-Target Antibody Response for an Influenza Subunit Vaccine

by
Megan C. Schulte
1,
Adam C. Boll
1,
Agustin T. Barcellona
1,
Elida A. Lopez
2,
Adam G. Schrum
1,2,3,4 and
Bret D. Ulery
1,4,5,*
1
Department of Chemical and Biomedical Engineering, University of Missouri, Columbia, MO 65211, USA
2
Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211, USA
3
Department of Surgery, University of Missouri, Columbia, MO 65211, USA
4
NextGen Precision Health Institute, University of Missouri, Columbia, MO 65211, USA
5
Materials Science & Engineering Institute, University of Missouri, Columbia, MO 65211, USA
*
Author to whom correspondence should be addressed.
Vaccines 2025, 13(1), 51; https://doi.org/10.3390/vaccines13010051
Submission received: 8 October 2024 / Revised: 13 November 2024 / Accepted: 5 January 2025 / Published: 9 January 2025
(This article belongs to the Special Issue Peptide-Based Vaccines)
Browse Figures
Versions Notes

Abstract

:
Background/Objectives: Peptide amphiphile micelles (PAMs) are an exciting nanotechnology currently being studied for a variety of biomedical applications, especially for drug delivery. Specifically, PAMs can enhance in vivo trafficking, cell-targeting, and cell interactions/internalization. However, modifying peptides, as is commonly performed to induce micellization, can influence their bioactivity. In our previous work, murine antibody responses to PAMs containing the influenza antigen M22–16 were slightly incongruous with prior PAM vaccine studies using other antigens. In this current work, the effect of native protein linkages and non-native micellizing moieties on M2 immunogenicity was studied. Methods: PAMs were synthesized using an elongated M2 antigen (i.e., Palm2K-M21–24-(KE)4). The PAMs were characterized, then their immunogenicity was evaluated with bone marrow-derived dendritic cells and in mice. Results: Although the modification scheme yielded immunogenic PAMs, these PAMs induced a substantial amount of off-target antibody production compared to unmodified peptidyl micelles (PMs, M21–24 peptide). Conclusions: While the impact PAM-induced off-target antibodies had on vaccine efficacy remains to be elucidated, on-target antibodies from both PAM- and PM-vaccinated mice were excitingly able to recognize the M2 antigen within the context of the full M2 protein. This provides preliminary evidence that the PAM-induced on-target antibodies will at minimum be able to recognize the influenza virus upon exposure.

1. Introduction

Peptide amphiphile micelles (PAMs) are a lipid-based nanoparticle system that can deliver bioactive peptides for immunomodulation [1,2], cancer therapy [3] and atherosclerosis theranostics [4,5]. PAMs are self-assembled structures of lipopeptides (or peptide amphiphiles, PAs) held together by the hydrophobic intermolecular association of their lipid-portion. Unlike peptides that do not self-assemble, PAMs are less susceptible to protease-mediated degradation and are able to retain defined peptide secondary structures [6,7,8]. PAMs are also able to deliver high local concentrations of peptides to cells and improve peptide-cell association [9]. In particular, micelle size and morphology can be tuned to dictate their immunogenicity, which has important implications for their use in vaccinology [10]. Favorable micelle morphology for immunostimulation has been achieved by adding a dipalmitoyllysine on the N-terminus of the antigen and a zwitterion-like charge block of alternating lysines and glutamic acids on the C-terminus (i.e., Palm2K-peptide-(KE)4), which creates small spherical and/or cylindrical micelles that are ideal for trafficking through lymphatic vessels and being taken up by antigen presenting cells [10].
Despite the aforementioned benefits, the addition of lipids and non-native residues to a bioactive peptide (e.g., in order to induce micellization) can potentially affect the functionality and bioactivity of the peptide itself. Previously, we used the M22–16 antigen (a B cell antigen from the ectodomain of the influenza matrix 2 protein) to assess its potential as a universal subunit vaccine for influenza [11]. We showed that these PAMs elicited IgG titers that were comparable to the micelle-forming unmodified peptide group (i.e., peptidyl micelles; PMs), whereas, in studies using different antigens, PAMs elicited higher titers when compared to the unmodified peptide group [1,12].
In this work, we expanded the M2 antigen of focus from M22–16 to M21–24 (the entire external domain of the M2 protein), in order to investigate the flanking region effects on PAM immunogenicity.
Our hypothesis was that the addition of neighboring amino acids to the original M22–16 antigen, would act as native linkers between the non-native modifications and the antigen producing results that better aligned with prior PAM vaccine studies using other antigens [12,13]. Like the M22–16 antigen, the extended M21–24 antigen has been shown to be quite conserved, thus also making it a good candidate as a universal influenza vaccine [14,15,16]. Additionally, the entire M21–24 region has been used by several groups as an antigen in itself, further validating its viability as an alternative [17,18,19]. In addition to the antigen-containing peptide or PA, the adjuvant Pam2CSK4, which was previously shown to form heterogenous micelles with both M22–16 peptides and the Palm2K-M22–16-(KE)4 PAs and substantially increased antibody titers, was co-delivered to maximize antibody responses to the peptides and PAMs [11].

2. Materials and Methods

2.1. Peptide Synthesis and Purification

Pam2CSK4 was purchased from Invivogen (San Diego, CA, USA). Recombinant M2 protein was acquired from Abeomics (San Diego, CA, USA). All other peptides were synthesized by employing Fmoc solid-phase peptide synthesis on Sieber Amide resin with a Tetras Peptide Synthesizer (Louisville, KY, USA). Nα-Fmoc-L-amino acids were coupled to the resin (or peptide-on-resin) using a ratio of 3 equivalents of amino acid, 3 equivalents hydroxybenzotriazole (HOBt), 6 equivalents N, N-diisopropylethylamine (DIPEA), and 2.7 equivalents hexafluorophosphate benzotriazole tetramethyl uranium (HBTU) in N-methylpyrrolidone (NMP) to 1 molar equivalent resin. After coupling, 5% acetic anhydride and 7% DIPEA were added to the resin to cap any unreacted amines. Fmoc protecting groups were removed with 6% piperazine and 0.1 M HOBt in dimethylformamide (DMF). For lipidation, a non-native Fmoc-Lysine (Fmoc) was attached to the N-terminus of the peptide, followed by deprotection of both amines of the lysine and coupling of palmitic acid to both amines using the previously described coupling protocol. Peptides were cleaved from resin and deprotected using a cleavage cocktail of 2.5% each of water, phenol, triisopropylsilane, ethane-1,2-dithiol, and thioanisole in trifluoroacetic acid (TFA), followed by precipitation in diethyl ether. Peptides and PAs were purified to greater than 90% purity using LC-MS (Beckman Coulter, Brea, CA, USA) with a reverse-phase C18 column for peptides or a C4 column for PAs and a mobile phase of water, acetonitrile, and 0.1% TFA. Peptides and peptide amphiphiles eluted at the percent acetonitrile indicated in Table 1. LC-MS chromatograms of the purified compounds can be found in Figure S1. After purification, peptides and PAs were lyophilized and frozen until further use.

2.2. Micelle Characterization

Micelle formulations were characterized by critical micelle concentration (CMC), transmission electron microscopy (TEM), and circular dichroism (CD), as previously described [11]. To perform the CMC assay, a solution of 1 μM 1,6-diphenylhexatriene (DPH) was prepared by dissolving DPH in tetrahydrofuran, then diluting that 100-fold in deionized, distilled water (ddH2O), which was then diluted an additional 1000-fold in phosphate-buffered saline (PBS). A solution of 1 mM peptide or PA in water was serially diluted in the 1 μM DPH solution from 100 μM to 0.92 nM, incubated for 1 h in the dark, and then fluorescence was measured on a Cytation Biotek 5 Spectrophotometer (Santa Clara, CA, USA) at an excitation/emission of 350/428 nm. On a graph of fluorescence intensity versus peptide (or PA) concentration, CMCs were determined to be the intersection of two logarithmic regression lines roughly above and below, respectively, the concentration at which fluorescence intensity starts to increase significantly. To assess micelle morphology, peptides and PAs were imaged using TEM. Samples were diluted to 100 μM in PBS, then 5 μL were pipetted onto carbon-coated copper grids and incubated for 5 min. After wicking excess liquid from the grid with filter paper, grids were stained with Nano-Tungsten (2% methylamine tungstate) for 5 min, wicked, and imaged with a JEOL JEM-1400 Transmission Electron Microscope (Tokyo, Japan) at 15,000× and 25,000× magnification. Micelle size was determined using ImageJ Particle Size Analysis based on the TEM micrographs. Finally, CD was used to assess peptide secondary structure. Peptide and PA solutions were diluted to 250 μM in PBS, and then the CD of the solutions was measured from 190 to 250 nm with 1 nm intervals using a Jasco J-1500 Circular Dichroism Spectrophotometer (Oklahoma City, OK, USA). CD data (measured in triplicate) were fit to reference curves of poly(lysine) and poly(glutamine) to approximate α-helix, β-sheet, and random coil content.

2.3. Bone Marrow-Derived Dendritic Cell (BMDC) Studies

BMDCs were developed in tissue culture and tested for activation as previously described [11]. In brief, bone marrow was harvested from the femurs and tibias of 4-month-old BALB/c mice. Red blood cells were lysed using an ACK lysing buffer. Bone marrow cells were cultured in complete RPMI-1640 (i.e., 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μM β-mercaptoethanol) with 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF). Fresh media were added on days 3 and 8, and cells were split on day 6. After 10 days, cells were plated in untreated 24-well plates and cultured with peptides or PAs for 24 h (at dosages described in Table 2). The media supernatant was collected and frozen for later use. Cells were detached from the plates, blocked with Trustain FcX anti-mouse CD16/32 antibody (BioLegend, San Diego, CA, USA), and then stained with PE/Cyaninine7 anti-mouse CD11c (BioLegend), FITC anti-mouse CD40 (BioLegend), and APC anti-mouse MHC-II (BioLegend) to assess cell activation. Finally, cells were fixed with 4% paraformaldehyde. Cells were then analyzed using a BD LSR Fortessa Flow Cytometer (Franklin Lakes, NJ, USA) and gated using the strategy in Figure S2. To assess the cytokine content (i.e., TNF-α and IL-12/IL-23) in the media collected from the BMDCs, enzyme-linked immunosorbent assays (ELISAs) were completed using the protocols provided in the kits (BioLegend).

2.4. Vaccination Schedule

In vivo studies were conducted in accordance with protocol 32,204, as approved by the Animal Care and Use Committee of the University of Missouri. BALB/c mice (4 females and 3–4 males per group) approximately 10 weeks in age were subcutaneously injected in the nape of the neck with the vaccine formulations. Vaccines consisted of 100 μL PBS containing 20 nmol peptide or PA with or without 2.22 nmol Pam2CSK4 (Table 3). Vaccinations were administered on days 0 and 21 with blood collection on day 35. Blood was collected by cardiac puncture (after CO2 asphyxiation), according to ACUC guidelines.

2.5. Serum Enzyme-Linked Immunosorbent Assay (ELISA)

The serum was collected by centrifuging blood at 9400× g for 10 min and harvesting the supernatant, after which it was frozen at −80 °C until further use. ELISAs were conducted to quantify antibody content in the serum. To coat the plates, 1.69 μM of the coating peptide in a carbonate buffer was incubated in the wells at 4°C overnight. Plates were washed with 0.05% Tween-20 in PBS and then blocked with 10% FBS in PBS for 1 h. The serum was then added to wells at an initial 100× dilution, then serially diluted in assay diluent across the plate 21 times and serially diluted across the plate before being incubated at 4°C overnight. The next day, plates were washed, incubated with a 1:3000 dilution of goat anti-mouse IgG-HRP (Invitrogen, Waltham, MA, USA) for 1 h, washed again, then incubated with TMB (3,3′,5,5′ tetramethylbenzidine, BioLegend). After 30 min, the absorbance of the wells was measured at 650 nm using a Biotek Cytation 5 Spectrophotometer. The samples were normalized across plates by subtracting the absorbance of assay-diluent only wells from the absorbance of each serum-containing well. Antibody titers were calculated as the lowest serum dilution with an absorbance at least twice the absorbance of the serum samples from PBS-vaccinated mice at a given serum dilution. For the M2 protein ELISA study, the M2 protein and M21–24 peptide were coated on the plates at a concentration of 0.09 μM in a carbonate buffer after which the rest of the protocol remained unchanged.

2.6. Statistics

One-way analysis of variance (ANOVA) and Tukey’s Honestly Significant Difference (HSD) tests were performed using GraphPad Prism software (Version 7.02). Within a graph, groups that are labeled with the same letter have similar means (p > 0.05), whereas pairwise comparisons without the same letter possess statically significantly different means (p ≤ 0.05).

3. Results and Discussion

3.1. M21–24 and Palm2K-M21–24-(KE)4 Formed Micelles with Vaccine Favorable Characteristics

Peptides and PAs were synthesized on-resin and purified by LC-MS. The M21–24 peptide and Palm2K-M21–24-(KE)4 PA were characterized for their ability to micellize at sufficiently low concentrations and form small micelles that are favorable for trafficking and cell uptake. The CMC of the M21–24 peptide was 1.2 ± 0.9 μM (Figure 1a), and TEM micrographs showed that the M21–24 peptides formed small spherical micelles (Figure 1b), which will be referred to as peptidyl micelles (PMs). M21–24 PMs had an estimated average maximum caliper diameter of 28 ± 15 nm, based on particle analysis using ImageJ (Figure S3a). Micellization was also confirmed for the Palm2K-M21–24-(KE)4 PA. The CMC of Palm2K-M21–24-(KE)4 was 0.038 ± 0.018 μM (Figure 1c), which was sufficiently low to ensure micellization in vivo, even when considering their dissemination into lymphatic vessels or more diffusely in the blood for which an estimated 1.5–2 mL total volume per mouse was used [20]. In the Palm2K-M21–24-(KE)4 PAMs, the strong hydrophobic contribution of the dipalmitoyllysine probably caused the lower CMC (relative to the unmodified M21–24 PMs) [21,22,23]. Micrographs from TEM (Figure 1d) showed that Palm2K-M21–24-(KE)4 formed a combination of spherical and short cylindrical micelles, which have been shown to be ideal for trafficking to draining lymph nodes and uptake by antigen-presenting cells [10]. Employing ImageJ to perform particle analysis, Palm2K-M21–24-(KE)4 PAMs were found to have an estimated average maximum caliper diameter of 37 ± 24 nm (Figure S3b).
The differences in micelle morphology between the unmodified M21–24 PMs and the Palm2K-M21–24-(KE)4 PAMs can potentially be explained by a difference in the hydrophobicity/amphipathicity between the micelle types [24]. The PAMs had strong amphipathicity caused by the dipalmitoyllysine on the N-terminus and the set of charged residues on the C-terminus (Figure 1e), which would allow for the PAs in the micelles to have a lipid core and a corona consisting of the (KE)4 charge block, with the M21–24 sequence most likely between the two. This relatively linear conformation of the PA in the PAMs could facilitate tight packing at a low concentration, leading to the low CMC and the development of cylindrical micelles (in which individual PAs have tighter packing angles) than in spheres (as was found for the M21–24 PMs) [24].
To further characterize the micelles, CD was conducted to evaluate the secondary structure of M21–24 and the peptide content within Palm2K-M21–24-(KE)4 (Figure 2). M21–24 PMs had substantial random coil content (32%) but were still predominantly β-sheet (67%). Palm2K-M21–24-(KE)4 PAMs were nearly entirely β-sheet (87%) with the rest split between α-helix and random coil. The β-sheet-dominant structure of the PAMs aligns well with what has been observed for other PAMs using the Palm2K-peptide-(KE)4 configuration, including in Palm2K-M22–16-(KE)4 (97% β-sheet), Palm2K-OVABT-(KE)4 (91% β-sheet), and Palm2K-VIP-(KE)4 (67% β-sheet) [2].
Vaccines 13 00051 g001aVaccines 13 00051 g001b
Figure 1. Both M21–24 and Palm2K-M21–24-(KE)4 self-assemble into small micelles with low critical micelle concentrations. (a) A representative graph of M21–24 shows a CMC of 0.92 μM, denoted by ∗. (b) A TEM micrograph of M21–24 indicates the presence of small spherical micelles. (c) A representative graph of Palm2K-M21–24-(KE)4 shows a CMC of 0.04 μM, denoted by ∗. (d) A TEM micrograph of Palm2K-M21–24-(KE)4 indicates the presence of spherical and short cylindrical micelles. (e) The hydropathy chart of M21–24-(KE)4 (dipalmitoyllysine not shown) shows the hydrophobic and hydrophilic amino acids are interspersed throughout M21–24, whereas the (KE)4 region imparts the strong hydrophilicity to the C-terminus of Palm2K-M21–24-(KE)4 [25].
Figure 1. Both M21–24 and Palm2K-M21–24-(KE)4 self-assemble into small micelles with low critical micelle concentrations. (a) A representative graph of M21–24 shows a CMC of 0.92 μM, denoted by ∗. (b) A TEM micrograph of M21–24 indicates the presence of small spherical micelles. (c) A representative graph of Palm2K-M21–24-(KE)4 shows a CMC of 0.04 μM, denoted by ∗. (d) A TEM micrograph of Palm2K-M21–24-(KE)4 indicates the presence of spherical and short cylindrical micelles. (e) The hydropathy chart of M21–24-(KE)4 (dipalmitoyllysine not shown) shows the hydrophobic and hydrophilic amino acids are interspersed throughout M21–24, whereas the (KE)4 region imparts the strong hydrophilicity to the C-terminus of Palm2K-M21–24-(KE)4 [25].
Vaccines 13 00051 g001aVaccines 13 00051 g001b

3.2. Bone Marrow-Derived Dendritic Cell Activation Was Driven by the Adjuvant Pam2CSK4

Because both M21–24 peptide and Palm2K-M21–24-(KE)4 PA produced micelles with favorable properties, the immunogenicity of the PMs and PAMs was evaluated with BMDCs. Dendritic cells (DCs) play a significant role in antigen presentation and activation of CD4+ T cells, which triggers downstream effects on B cells and antibody production, highlighting the importance of DCs in vaccine-induced immune responses [26,27,28]. BMDCs were processed for flow cytometry and identified by CD11c expression, whereas the markers CD40 and MHC-II were used as markers of BMDC stimulation [29,30]. BMDC activation was primarily dictated by the presence or absence of the TLR2 agonist Pam2CSK4, with some minor influence by the presence of micelles, as seen in Figure 3. Treatment with M21–24 PMs or Palm2K-M21–24-(KE)4 PAMs resulted in a slight increase in the percentage of CD11c+ cells expressing CD40 (Figure 3a). There was a much greater increase observed when the adjuvant Pam2CSK4 was added to treatment, dwarfing the influence of micelle presence. There was no treatment effect on MHC-II expression of CD11c+ cells compared to untreated cells. The median fluorescence intensity (MFI) in the FITC (CD40) and APC (MHC-II) channels of CD11c+CD40+ and even CD11c+MHC-II+ populations, respectively, was heightened in response to treatment with an adjuvant, but not due to exposure to PMs or PAM alone (Figure 3b). Clearly, all treatment groups containing Pam2CSK4 activated the BMDCs, based on CD40 expression. The less pronounced differences in MHC-II expression between treatment groups were likely due to MHC-II being constitutively expressed on most BMDCs, even in the basal state of the assay observed in Figure 3a. Also, previous research like that from Dearman and colleagues has found a less pronounced increase in MHC-II expression above baseline compared to other markers, including CD40, CD80, and CD86 in BMDCs treated with lipopolysaccharide (LPS), in accordance with our observations in Figure 3b [30].
To evaluate the secretion of pro-inflammatory cytokines, TNF-α, and IL-12/IL-23, sandwich ELISAs were conducted using media collected from the treated BMDCs. Specifically, IL-12/IL-23 was quantified using an ELISA specific for the p40 monomer, which is present in both IL-12 and IL-23 heterodimers and has been frequently used to assess BMDC activation [30,31,32,33]. As with the upregulation of activation markers, the elevated secretion of TNF-α and IL-12/IL-23 p40 was largely driven by the presence of Pam2CSK4 (Figure 3c,d). It was not surprising that the primary effect on pro-inflammatory cytokine secretion was the adjuvant, as TNF-α, IL-12, and IL-23 expression have all been shown to be elevated in activated BMDCs upon initiation of the MyD88-NF-κB pathway, which is triggered by Pam2CSK4 via TLR2 [31,34,35,36,37]. Beyond the adjuvant effect on cytokine secretion, there was a further statistically significant increase in TNF-α and IL-12/IL-23 p40 secretion in both adjuvant-loaded micelle treatment groups (i.e., M21–24/Pam2CSK4 and Palm2K-M21–24-(KE)4/Pam2CSK4), suggesting an additional effect of co-delivery that was not observed with CD40 nor MHC-II expression. Like Pam2CSK4, Palm2K-M21–24-(KE)4 has two 16-carbon lipids at its N-terminus, which previous studies have shown can effectively stimulate TLR2 [38]. However, these lipids alone are not sufficient for TLR2 activation, which could explain the less consistent immunogenic effect of PMs and PAMs delivered alone compared to when Pam2CSK4 is present [12,39,40].

3.3. Peptide Amphiphile Micelles Induced On- and Off-Target Antibody Production

To investigate the immunogenicity of the micelles in vivo, mice were vaccinated with the M21–24 PM or Palm2K-M21–24-(KE)4 PAM formulations. The peptides and vaccine dosages used in this study are outlined in Table 3. The serum collected from the mice was used to assess antibody titers by ELISA. IgG titers were first tested using the M21–24 peptide as the plate coating, as that most closely represented the antigen within the context of the influenza virus. Again, the presence of adjuvant was found to be the primary driving force in inducing a productive immune response, with both PM and PAMs eliciting high IgG titers when co-delivered with Pam2CSK4 (Figure 4a). While there were more responders in mice vaccinated with only PMs or PAMs when compared to mice vaccinated with PBS, there were no statistical differences found between these groups. These results aligned with previous results using the M22–16 antigen, in which Palm2K-M22–16-(KE)4 PAMs did not produce titers above the unmodified M22–16 peptides, which is an effect that had not been observed with other antigens [1,11,12]. To investigate the cause of these results, another ELISA was conducted using K-M21–24-(KE)4 as the plate coating, which would capture antibody specificity against the entire peptidyl component of the PA. ELISAs completed using this new coating antigen produced an interesting result in that the IgG titers of both PAM-containing vaccine groups (i.e., Palm2K-M21–24-(KE)4 and Palm2K-M21–24-(KE)4/Pam2CSK4) increased considerably whereas the titers of the PM-containing groups (i.e., M21–24 and M21–24/Pam2CSK4) stayed relatively similar to what was seen with the M21–24 coating (Figure 4b). This result showed that overall IgG titers elicited by the PAMs actually were higher than by the PMs (especially when comparing between groups without adjuvant), which was more in line with what had been previously observed using other antigens like J8 and OVABT [1,12]. A likely explanation for the difference in results between the M21–24 and K-M21–24-(KE)4 ELISAs was that the PAMs were eliciting the production of off-target antibodies that were not entirely specific to the M21–24 region, but to the modifications on the N- and/or C-terminus of the PA.
To investigate this off-target antibody production effect further, a series of “peptide walk” ELISAs were conducted in order to elucidate the sequence specificity of the antibodies in the vaccinated mice. A series of 15-mer peptides were synthesized from K-M21–14 to M218–24-(KE)4, shifting 2–5 amino acids towards the C-terminus with each peptide. These peptides were used individually as the coating antigen in an ELISA to test antibody specificity from day 35 serum (Figure 5). Mice vaccinated with Palm2K-M21–24-(KE)4 PAMs (with and without Pam2CSK4) mice had higher titers than M21–24 PM groups (with and without Pam2CSK4) against the coating antigens of K-M21–14 (Figure 5a), M212–24-KE (Figure 5d), M216–24-(KE)3 (Figure 5e), and M218–24-(KE)4 (Figure 5f) (i.e., all coating antigens containing non-native residues on the N- or C-terminus). The fact that PAMs elicited higher titers against both the N-terminal and C-terminal portions of the PA suggests a polyclonal antibody response. The especially high titers in Figure 5e,f provides strong evidence for off-target antibody production towards the C-terminus. Titers were most similar when comparing the coating antigens M27–21 (Figure 5c) and M21–24 (Figure 4a), while, interestingly, the titers of the PAM groups against M24–18 (Figure 5b) were substantially lower than the PM groups. The stark increase in relative PAM titers against the K-M21–14, and even M21–24, coating antigen compared to those against the M24–18 coating antigen suggests that at least one of the first three amino acids of the M21–24 antigen was critical for binding of PAM vaccine-induced antibodies. The fact that Palm2K-M21–24-(KE)4/Pam2CSK4 titers against M24–18 were low, despite previous work showing good immunogenicity of Palm2K-M22–16-(KE)4/Pam2CSK4 PAMs against M21–24, further suggests the importance of the second and/or third residues in M2 for PAM-elicited antibody binding [11].
The off-target antibody responses were unexpected given that the (KE)4 motif is found in multiple places throughout the murine proteome as determined by a peptide sequence search of the UniProt database, which would suggest that (KE)4 should be non-immunogenic [41]. Yet, there was a clear antibody response against (KE)4 shown here. There is some evidence in the literature of peptides that can exert an adjuvanting effect when paired with an antigenic peptide, even without being inherently immunogenic themselves (i.e., KFE8 and Q11) [42,43], which can likely be at least partially attributed to the self-assembly driven by these peptides. Given that the production of off-target antibodies seen here has not been previously observed in PAMs using other antigens and that (KE)4 should be non-immunogenic, it is possible that the M21–24 sequence itself was acting as an adjuvant for the (KE)4 region of the PA. Further testing will be required to investigate this hypothesis; however, there are some exciting implications that could result from this. Namely, that the (KE)4 moiety could be replaced with a biologically relevant peptide, such as a CD8 or CD4 antigen to further expand the immunogenicity of the vaccine.

3.4. Antibodies Elicited by PMs and PAMs Recognized M21–24 Within the Full M2 Protein

To assess whether the off-target antibody production in the PAM-vaccinated mice affected the ability of the antibodies to recognize the M21–24 epitope as a part of the entire M2 protein, an ELISA was conducted using the entire M2 protein as the plate coating. This was compared to an ELISA performed again using the M21–24 peptide, but at a lower coating concentration for a more accurate comparison of the results between the different plate coatings (Figure 6). Another notable result was that in the context of the M2 protein ELISA (Figure 6) especially, the range of antibody titers for the adjuvanted Palm2K-M21–24-(KE)4 PAM-vaccinated group was quite a bit narrower than that of the adjuvanted PM vaccinated-group (M21–24/Pam2CSK4). A potential explanation for this result is that the PAMs were likely more ordered (and have a more consistent antigen display) than the PMs because of the stronger hydrophobic association effect conveyed by the lipids in the PAMs. It is also worth noting that although there was evidently off-target antibody production caused by the PAMs, there was still a comparable M2 protein-specific titer between the PAMs and PMs. If in a future study off-target antibody production can be prevented in the PAMs, the on-target titers in PAM-vaccinated mice could possibly be higher than in the PM-vaccinated mice, as was observed in studies employing PAMs with other antigens (i.e., J8 and OVABT) [1,12].

4. Conclusions

PMs and PAMs from the M21–24 antigen were generated that were of sufficiently small size and low enough CMCs to be good candidates as vaccine delivery vehicles. Both M21–24 PMs and Palm2K-M21–24-(KE)4 PAMs, (with the aid of an adjuvant), elicited strong antibody titers specific to the M21–24 antigen in vivo. However, upon further investigation, it became apparent that there was considerable off-target antibody production in the PAM-vaccinated mice, especially against the non-native lysine on the N-terminus and the (KE)4 region on the C-terminus of the antigen. Unfortunately, the addition of native residues to both sides of the M22–16 antigen (one amino acid on the N-terminus and eight amino acids on the C-terminus) was insufficient to prevent the production of off-target antibodies. This information, and the fact that this phenomenon was not seen in studies of other epitopes, suggested an antigen-specific effect, specifically, that the M21–24 antigen was able to exert an adjuvanting effect on the (KE)4 peptide moiety. It remains to be seen whether this off-target antibody production has any immune distraction or synergistic effects in terms of vaccine efficacy, but this proposed adjuvanting effect could have exciting implications for linking multiple antigens in a single PA. Regardless, it was promising that antibodies from PM- and PAM-vaccinated mice were still able to recognize their target antigen in the context of the entire M2 protein. Future work to advance micelle technology for influenza vaccine applications will compare the immune responses elicited by PMs and PAMs against publicly available influenza vaccines.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/vaccines13010051/s1, Figure S1: Peptides were purified to greater than 90% purity using high performance liquid chromatography-mass spectrometry (LC-MS); Figure S2: Bone-marrow derived dendritic cells were gated using the above strategy; Figure S3: ImageJ was used to analyze micelle size.

Author Contributions

M.C.S.—writing, editing, experimental design, experimental work (characterization, in vitro work, and in vivo work). A.C.B.—experimental work (ELISAs). A.T.B.—experimental work (in vivo work). E.A.L.—experimental work (ELISAs). A.G.S.—editing and conceptualization. B.D.U.—writing, editing, conceptualization, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by funding provided by the University of Missouri System to the Center for Vector-Borne and Emerging Infectious Diseases (CVBEID).

Institutional Review Board Statement

The animal study protocol used in this research was approved by the Animal Care and Use Committee of the University of Missouri (protocol code 32204 approved 7 December 2021).

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are contained within the article and Supplementary Materials.

Acknowledgments

The authors would like to thank the Molecular Interactions Core at the University of Missouri for use of the Tetras Peptide Synthesizer, LC-MS, and J-1500 circular dichroism spectrophotometer, and Fabio Gallazzi for his assistance and guidance with peptide synthesis. Additionally, the authors would like to thank the Electron Microscopy Core at the University of Missouri for use of the JEOL JEM-1400 Transmission Electron Microscope.

Conflicts of Interest

The authors declare that there are no known conflicts of interest.

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Figure 2. Circular dichroism of M21–24 and Palm2K-M21–24-(KE)4 shows substantial differences between peptides and peptide amphiphiles. Circular dichroism spectra show that M21–24 was predominantly β-sheet, although with significant random coil character. Palm2K-M21–24-(KE)4 was almost entirely β-sheet with minimal amounts of α-helix and random coil.
Figure 2. Circular dichroism of M21–24 and Palm2K-M21–24-(KE)4 shows substantial differences between peptides and peptide amphiphiles. Circular dichroism spectra show that M21–24 was predominantly β-sheet, although with significant random coil character. Palm2K-M21–24-(KE)4 was almost entirely β-sheet with minimal amounts of α-helix and random coil.
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Figure 3. BMDC activation was largely driven by treatment with Pam2CSK4, with pro-inflammatory cytokine secretion further enhanced by PM- or PAM-mediated delivery of Pam2CSK4. (a) The percentage of cells expressing substantial amounts of CD40 was significantly increased in cells treated with Pam2CSK4-containing formulations. There was not an appreciable increase in MHC-II expression. (b) The MFIs in the FITC and APC channels were significantly elevated among CD11c+CD40+ and CD11c+MHC-II+ cells, respectively, for those treated with Pam2CSK4. Treatment with PMs or PAMs alone did not have a stimulatory effect. (c) TNF-α and (d) IL-12/IL-23 secretion was above baseline for all Pam2CSK4-containing groups. Interestingly, micelles alone did not induce pro-inflammatory cytokine secretion, but micelle-mediated adjuvant delivery did enhance this effect above Pam2CSK4 alone. Within a graph, groups that possess different letters have a statistically significant difference in mean (p ≤ 0.05) whereas those that possess the same letter have similar means (p > 0.05). Only treatment groups within the same channel and cell population were compared to each other.
Figure 3. BMDC activation was largely driven by treatment with Pam2CSK4, with pro-inflammatory cytokine secretion further enhanced by PM- or PAM-mediated delivery of Pam2CSK4. (a) The percentage of cells expressing substantial amounts of CD40 was significantly increased in cells treated with Pam2CSK4-containing formulations. There was not an appreciable increase in MHC-II expression. (b) The MFIs in the FITC and APC channels were significantly elevated among CD11c+CD40+ and CD11c+MHC-II+ cells, respectively, for those treated with Pam2CSK4. Treatment with PMs or PAMs alone did not have a stimulatory effect. (c) TNF-α and (d) IL-12/IL-23 secretion was above baseline for all Pam2CSK4-containing groups. Interestingly, micelles alone did not induce pro-inflammatory cytokine secretion, but micelle-mediated adjuvant delivery did enhance this effect above Pam2CSK4 alone. Within a graph, groups that possess different letters have a statistically significant difference in mean (p ≤ 0.05) whereas those that possess the same letter have similar means (p > 0.05). Only treatment groups within the same channel and cell population were compared to each other.
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Figure 4. Day 35 IgG Titers against M21–24 and K-M21–24-(KE)4 peptide coatings showed PAMs induced on-target and off-target antibody production. (a) Vaccine groups with Pam2CSK4 produced similar, high IgG titers against the M21–24 coating antigen whereas those without Pam2CSK4 did not produce appreciable titers above baseline. (b) K-M21–24-(KE)4-specific titers in both PAM groups were elevated relative to their respective PM groups, though only statistically significant differences were observed for the adjuvant-free groups. Within a graph, groups that possess different letters have a statistically significant difference in mean (p ≤ 0.05), whereas those that possess the same letter have similar means (p > 0.05).
Figure 4. Day 35 IgG Titers against M21–24 and K-M21–24-(KE)4 peptide coatings showed PAMs induced on-target and off-target antibody production. (a) Vaccine groups with Pam2CSK4 produced similar, high IgG titers against the M21–24 coating antigen whereas those without Pam2CSK4 did not produce appreciable titers above baseline. (b) K-M21–24-(KE)4-specific titers in both PAM groups were elevated relative to their respective PM groups, though only statistically significant differences were observed for the adjuvant-free groups. Within a graph, groups that possess different letters have a statistically significant difference in mean (p ≤ 0.05), whereas those that possess the same letter have similar means (p > 0.05).
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Figure 5. Peptide walk ELISA showed that PAM-vaccinated mice produced higher titers when the non-native amino acids of the PA were present. (a) The PAM groups elicited higher titers against the coating antigen K-M21–14 than the PM groups. (b) M21–24/Pam2CSK4 produced the highest titer against M24–18, although not statistically significantly higher than the PAM groups. (c) The titers against M27–21 were highest in groups with Pam2CSK4. (d) The PAM groups, (especially with Pam2CSK4), elicited the highest titers against M212–24-KE. (e) The PM groups produced almost no titers against M216–24-(KE)3, while PAM titers were several orders of magnitude higher. (f) PAM groups elicited high titers against M218–24-(KE)4, while PM group titers were at baseline. The darker orange letters underneath the graphs indicate the area of the coating antigen within the context of the entire peptide amphiphile Palm2K-M21–24-(KE)4 sequence. Within a graph, groups that possess different letters have a statistically significant difference in mean (p ≤ 0.05) whereas those that possess the same letter have similar means (p > 0.05).
Figure 5. Peptide walk ELISA showed that PAM-vaccinated mice produced higher titers when the non-native amino acids of the PA were present. (a) The PAM groups elicited higher titers against the coating antigen K-M21–14 than the PM groups. (b) M21–24/Pam2CSK4 produced the highest titer against M24–18, although not statistically significantly higher than the PAM groups. (c) The titers against M27–21 were highest in groups with Pam2CSK4. (d) The PAM groups, (especially with Pam2CSK4), elicited the highest titers against M212–24-KE. (e) The PM groups produced almost no titers against M216–24-(KE)3, while PAM titers were several orders of magnitude higher. (f) PAM groups elicited high titers against M218–24-(KE)4, while PM group titers were at baseline. The darker orange letters underneath the graphs indicate the area of the coating antigen within the context of the entire peptide amphiphile Palm2K-M21–24-(KE)4 sequence. Within a graph, groups that possess different letters have a statistically significant difference in mean (p ≤ 0.05) whereas those that possess the same letter have similar means (p > 0.05).
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Figure 6. Both peptidyl micelles and peptide amphiphile micelles were able to induce the production of antibodies capable of recognizing the M2 protein. To better compare results between titers against M21–24 and M2 protein, a reduced coating concentration of 0.09 μM was used for both coating antigens. (a) Titers were most influenced by the presence of Pam2CSK4 in the vaccine regardless of micelle type. (b) M2 protein-specific IgG titers were again more influenced by the presence of Pam2CSK4 in the micelle vaccine formulation. Titers, spread, and number of responders aligned well between the peptide and protein coatings. Within a graph, groups that possess different letters have a statistically significant difference in mean (p ≤ 0.05), whereas those that possess the same letter have similar means (p > 0.05).
Figure 6. Both peptidyl micelles and peptide amphiphile micelles were able to induce the production of antibodies capable of recognizing the M2 protein. To better compare results between titers against M21–24 and M2 protein, a reduced coating concentration of 0.09 μM was used for both coating antigens. (a) Titers were most influenced by the presence of Pam2CSK4 in the vaccine regardless of micelle type. (b) M2 protein-specific IgG titers were again more influenced by the presence of Pam2CSK4 in the micelle vaccine formulation. Titers, spread, and number of responders aligned well between the peptide and protein coatings. Within a graph, groups that possess different letters have a statistically significant difference in mean (p ≤ 0.05), whereas those that possess the same letter have similar means (p > 0.05).
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Table 1. Percent Acetonitrile at Peptide and Peptide Amphiphile Elution Time.
Table 1. Percent Acetonitrile at Peptide and Peptide Amphiphile Elution Time.
PeptideApproximate % Acetonitrile at Elution
M21–2430%
K-M21–24-(KE)425%
Palm2K-M21–24-(KE)460%
K-M21–1425%
M24–1825%
M27–2125%
M212–24-KE15%
M216–24-(KE)310%
M218–24-(KE)410%
Where M21–24 is MSLLTEVETPIRNEWGCRCNDSSD, subscripts after M2 denote the amino acid positions within the M2 ectodomain, Palm2K is dipalmitoyllysine, and (KE)x is a repeat of lysine-glutamic acid for a total of x number of times.
Table 2. Vaccine Formulations Used in BMDC Activation Assessment.
Table 2. Vaccine Formulations Used in BMDC Activation Assessment.
Vaccine GroupVaccine Dosage
No TreatmentNo Peptide nor PA
Pam2CSK40.2 μM Pam2CSK4
M21–241.8 μM M21–24
M21–24/Pam2CSK41.8 μM M21–24 and 0.2 μM Pam2CSK4
Palm2K-M21–24-(KE)4 1.8 μM Palm2K-M21–24-(KE)4
Palm2K-M21–24-(KE)4/Pam2CSK41.8 μM Palm2K-M21–24-(KE)4 and 0.2 μM Pam2CSK4
Table 3. Vaccine formulations used for the in vivo immunization study.
Table 3. Vaccine formulations used for the in vivo immunization study.
Vaccine GroupVaccine Dosage
PBSNo Peptide nor PA
M21–2420 nmol M21–24
M21–24/Pam2CSK420 nmol M21–24 and 2.22 nmol Pam2CSK4
Palm2K-M21–24-(KE)420 nmol Palm2K-M21–24-(KE)4
Palm2K-M21–24-(KE)4/Pam2CSK420 nmol Palm2K-M21–24-(KE)4 and 2.22 nmol Pam2CSK4
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Schulte, M.C.; Boll, A.C.; Barcellona, A.T.; Lopez, E.A.; Schrum, A.G.; Ulery, B.D. Peptide Antigen Modifications Influence the On-Target and Off-Target Antibody Response for an Influenza Subunit Vaccine. Vaccines 2025, 13, 51. https://doi.org/10.3390/vaccines13010051

AMA Style

Schulte MC, Boll AC, Barcellona AT, Lopez EA, Schrum AG, Ulery BD. Peptide Antigen Modifications Influence the On-Target and Off-Target Antibody Response for an Influenza Subunit Vaccine. Vaccines. 2025; 13(1):51. https://doi.org/10.3390/vaccines13010051

Chicago/Turabian Style

Schulte, Megan C., Adam C. Boll, Agustin T. Barcellona, Elida A. Lopez, Adam G. Schrum, and Bret D. Ulery. 2025. "Peptide Antigen Modifications Influence the On-Target and Off-Target Antibody Response for an Influenza Subunit Vaccine" Vaccines 13, no. 1: 51. https://doi.org/10.3390/vaccines13010051

APA Style

Schulte, M. C., Boll, A. C., Barcellona, A. T., Lopez, E. A., Schrum, A. G., & Ulery, B. D. (2025). Peptide Antigen Modifications Influence the On-Target and Off-Target Antibody Response for an Influenza Subunit Vaccine. Vaccines, 13(1), 51. https://doi.org/10.3390/vaccines13010051

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