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. Author manuscript; available in PMC: 2022 Sep 10.
Published in final edited form as: ACS Infect Dis. 2021 Aug 25;7(9):2723–2735. doi: 10.1021/acsinfecdis.1c00219

Tyrosine-based Crosslinking of Peptide Antigens to Generate Nanoclusters with Enhanced Immunogenicity: Demonstration Using the Conserved M2e Peptide of Influenza A

Logan R Wilks 1, Gaurav Joshi 1, Megan R Grisham 1, Harvinder Singh Gill 1,*
PMCID: PMC8521439  NIHMSID: NIHMS1740383  PMID: 34432416

Abstract

A method of creating nanoclusters (NCs) from soluble peptide molecules is described utilizing an approach based on a tyrosine-tyrosine crosslinking reaction. A reactive tag comprising histidine and tyrosine residues was introduced at the termini of the peptide molecules. The crosslinking reaction led to the creation of dityrosine bonds within the tag, which allowed for the generation of peptide NCs. We show that it is essential for the reactive tag to be present at both the ‘N’ and ‘C’ termini of the peptide for cluster formation to occur. Additionally, the crosslinking reaction was systematically characterized to show the importance of reaction conditions on final cluster diameter, allowing us to generate NCs of various sizes. To demonstrate the immunogenic potential of the peptide clusters, we chose to study the conserved influenza peptide, M2e, as the antigen. M2e NCs were formulated using the crosslinking reaction. We show the ability of the clusters to generate protective immunity in a dose, size, and frequency dependent manner against a lethal influenza A challenge in BALB/c mice. Taken together, the data presented suggests this new cluster formation technique can generate highly immunogenic peptide NCs in a simple and controllable manner.

Keywords: antigen, influenza, M2e, peptide, tyrosine crosslinking, vaccine

Graphical Abstract

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Peptide-based subunit vaccines have gained considerable popularity over recent years due to their ease of production, generation of focused epitope-specific immune responses, and overall safety profile, particularly compared to live vaccines.1, 2 Peptide immunogens have been investigated as tumor vaccines 3-5 and infectious disease vaccines for treating influenza,6-12 HIV,13, 14 malaria,15, 16 and even Alzheimer’s disease.17 One limitation of peptide-based immunogens is that they are often poorly immunogenic, and so must be formulated and presented in a way that enhances their immunogenicity. Presenting the peptides in the form of particles is a common strategy.1 For example, peptides have been attached to carrier proteins such as keyhole limpet hemocyanin, polymeric particles, virus-like particles, metal nanoparticles (NPs), and inorganic NPs such as silica, and liposomes.8, 9, 18-20 Additionally, emulsions, high molecular weight peptide nanoclusters, and dendrimers can be used to enhance the immune response to peptide immunogens.1, 2, 21, 22

Among the different approaches, peptide nanoclusters (NC) are particularly interesting because the peptide itself can be rendered into a particle, creating a particle made entirely of just the pure antigen. This simplifies the vaccine formulation and avoids any complications that the carrier material might present, such as carrier toxicity, carrier-specific off-target immune responses, and poor antigen loading.23 Moreover, NCs created from small peptides can allow for a particle with highly repetitive antigenic sequences reminiscent of viral envelopes that assist in generating robust immune responses.24 While transformation of small peptides into NCs without the use of a carrier system has not been widely studied, there are multiple reports of transforming larger proteins into NCs using desolvation,25-27 emulsion,28, 29 or electrospray techniques.30 In these approaches proteins are first converted into nano or microparticles, which are then stabilized by chemically crosslinking the proteins located in the particles through use of glutaraldehyde, or similar crosslinking agents.

In contrast to proteins, creation of pure peptide NCs is more challenging. This is because a peptide epitope may possess only a limited subset of the 20 essential amino acids while proteins are more likely to contain a broader selection of these residues. Therefore, thiols (cysteine) and primary amines (lysine), which are often the primary reaction centers used for crosslinking proteins, may be lacking in the peptides. Further, limited water solubility of some peptides can restrict the crosslinking reactions that can be performed.31, 32 In this study, we have sought to create a new cluster formation approach that can be used to generate NCs from small peptides in a controllable manner.

Previously, it has been shown that proteins containing tyrosine can be oligomerized by utilizing a simple reaction that employs nickel ions, hexahistidine tags, and an oxidizer. In this reaction, histidine (His) is thought to complex with nickel(II) (Ni(II)) ions to form a His-Ni(II) complex, which in turn reacts with an oxidizer such as magnesium monoperoxyphthalic acid (MMPP), which allows for the oxidation of the phenol group of closely situated tyrosine residues to form covalent dityrosine bonds.33-37 This reaction has been shown to be restricted to tyrosines located in the immediate vicinity of the histidine tags, which has important implications for avoiding off target crosslinks.36

We propose that instead of just forming oligomers, this reaction could be used to form peptide NCs. To evaluate this concept we selected the conserved ectodomain of the influenza membrane protein M2 (M2e) as a model peptide epitope.10 The reactive tag was designed based on work completed by Stayner et al.36 We placed the reactive tag on either just the ‘C’ terminus or on both ‘N’ and ‘C’ termini of the peptide, and the effect of reaction conditions on NC formation was subsequently analyzed. Once clusters were generated and characterized, we tested the efficacy of the M2e NCs as a vaccine candidate in a size, dose, and frequency dependent manner against a mouse adapted influenza A H1N1 strain in a murine model. By using this approach, we were able to successfully formulate peptide NCs of M2e, which suggests that the technique does not destructively alter the immunological activity of the epitope and this technique could be extended to other small peptide antigens.

Results and Discussion

Peptide Crosslinking as a Function of Reactive Tag Placement

The concept of the tyrosine-tyrosine crosslinking reaction is shown in Scheme 1. The reactive tag (t) of this system is H6(GY)2, and it comprises six histidines (H6) followed by a tyrosine-rich region (GY)2. Previously33-39, this chemistry has been used to form protein oligomers; however, whether the same chemistry can also be leveraged to form NCs has not been investigated. The advantage of this chemistry is that the tyrosine residues near the histidine tag participate in the dityrosine linkage, offering a degree of specificity for the location of crosslinks. The reaction is thought to proceed with Ni(II) ions forming a coordination bond with the imidazole ring of the histidine residues, which then facilitates dityrosine linkage of the nearby tyrosines in the presence of an oxidizer. It has previously been demonstrated that two tyrosine residues in the form of GYGY result in higher-order oligomers as compared to when a single tyrosine residue (GY) is used.36 Since making NCs would require greater degrees of crosslinking, we opted to use GYGY instead of GY as the reactive tag. We further postulated that placement of GYGY on both ‘N’ and ‘C’ termini would improve crosslinking between peptide molecules. To study this effect, we prepared two tagged M2e molecules, one with tags on both termini (t-M2e-t), and another with a tag on the ‘C’ terminus (M2e-t) only.

Scheme 1.

Scheme 1.

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(A) Possible crosslinking mechanism for the formation of dityrosine bonds. R’: represents the polypeptide chain in which tyrosine resides. Ni(II) is able to complex to the histidine residues in the reactive tag. The addition of MMPP is thought to oxidize the His-Ni(II) complex, which then extracts an electron from a nearby tyrosine, creating a tyrosyl radical. These tyrosyl radicals are then able to combine to form a covalent dityrosine bond. (B) Cartoon of the crosslinking reaction comparing M2e-t and t-M2e-t. The addition of the reactive tag at both the ‘N’ and ‘C’ termini allows for greater peptide crosslinking due to the presence of more tyrosine residues in the peptide.

We first studied the effect of reaction time on crosslinking. Reactions were carried out in 638 mM carbonate-bicarbonate buffer at pH 9.2 by varying reaction times in the range of 1 to 30 minutes. Crosslinking effect was studied using SDS gel electrophoresis. Figure 1 details the extent of crosslinking achieved. A comparison of Figure 1A with 1B shows that by placing the crosslinking tag on both the ‘N’ and ‘C’ termini (t-M2e-t) (Figure 1A), a superior crosslinking can be achieved as compared to when the tag is placed only on the ‘C’ terminus (M2e-t) (Figure 1B). Notably, after only one minute of reaction, oligomers can be seen in lane 3 of Figure 1A. These bands gradually fade as the crosslinking reaction proceeds, and within 10 minutes of reaction, the low molecular weight species are replaced with high molecular weight species, which are unable to migrate out of loading wells. This phenomenon is however absent in all lanes of Figure 1B in which only smears centering around 25 kDa are present. These results indicate that the reactive tag on just the ‘C’ terminus in M2e-t is insufficient to facilitate a high degree of crosslinking and, as a result, we chose to only consider t-M2e-t for further analysis.

Figure 1. Effect of reaction time on peptide crosslinking.

Figure 1.

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Reactions were carried out in a 638 mM carbonate buffer at pH 9.2, with (A) t-M2e-t (387 μM = 2 mg/ml), 4 mM Ni(OAc)2, 10 mM MMPP. C: 10 μg t-M2e-t as peptide alone control. (B) M2e-t (387 μM = 1.51 mg/ml), 4 mM Ni(OAc)2, 10 mM MMPP. C: 10 μg M2e-t as peptide alone control.

Peptide Crosslinking as a Function of Reaction pH

To fine-tune peptide crosslinking we next examined the effect of reaction pH. Reactions were completed in 638 mM carbonate-bicarbonate, phosphate-citrate, or glycine-HCl buffer at pH 9.2, pH 6.5, or pH 3.5, respectively, and the crosslinking efficiency was monitored using SDS gel electrophoresis. To determine if peptide concentration affects the degree of crosslinking, samples were run with varying peptide concentrations (2 mg/ml, 1.5 mg/ml, 1 mg/ml, 0.5 mg/ml) in each buffer system. Each reaction was run for 1.5 hours to provide adequate time for the reaction to occur.

Figure 2A shows that high molecular weight oligomers are obtained for each peptide concentration when run at pH 9.2. However, when the pH of the reaction was decreased to 6.5, the extent of crosslinking drastically decreased, which is evident from lanes 3 to 6 of Figure 2B where there is an absence of high molecular weight bands. When the pH was further reduced to 3.5, crosslinking of t-M2e-t was lowered even more as indicated by the lack of bands with molecular weight greater than 25 kDa (lanes 7-10 of Figure 2B). Interestingly, reactions occurring at a pH of 6.5 or 3.5 failed to produce highly crosslinked species, even when allowed to react for 1.5 hours. In contrast, a high degree of crosslinking was observed within 10 minutes of reaction time in the pH 9.2 buffer (Figure 1A). It can be concluded that reactions performed at a pH below 6.5 cannot adequately crosslink t-M2e-t, even at longer reaction times. Thus, the limiting condition of the reaction is the pH at which it is performed. To maximize crosslinking, all further reactions were performed at pH 9.2.

Figure 2. Effect of pH on crosslinking of t-M2e-t.

Figure 2.

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Reactions were performed with 4 mM Ni(OAc)2 and 10 mM MMPP at different pH conditions: (A) pH 9.2 in 638 mM carbonate buffer, (B) pH 6.5 (Lanes 3-6) in 638 mM phosphate-citrate buffer and pH 3.5 (Lanes 7-10) in 638 mM glycine-HCl buffer. The amount of t-M2e-t was varied and is listed below each lane. C: 10 μg t-M2e-t as peptide alone control.

The effect of pH on the oligomerization reaction can be explained by considering that, for the crosslinking reaction to proceed, Ni(II) ions must form a coordination bond with the imidazole ring of the histidine residues.33, 36 It is well known that the imidazole ring becomes protonated below pH 6.0, which would hinder the formation of the His-Ni(II) complex;40 accordingly, low crosslinking density would result when the pH is lowered from 9.2 to 6.5 and 3.5. Only when the pH is basic can the histidine residues become deprotonated and complex with Ni(II) to catalyze the reaction.

Effect of Ni(OAc)2 and MMPP Concentrations on Peptide Crosslinking

Ni(OAc)2 and MMPP are both essential to the crosslinking reaction because the peptide fails to crosslink in the absence of either of the two reactants (Figure S1). To better study the effect of Ni(OAc)2 and MMPP on the crosslinking reaction we systematically varied their respective concentrations.

Figure 3A shows a clear relationship between crosslinking and MMPP concentration. Monomeric t-M2e-t bands can be found when the MMPP concentration is below 5 mM but they disappear at higher MMPP concentrations with simultaneous appearance of high molecular weight species that are trapped in loading wells. MMPP does not act as a catalyst in the reaction but is irreversibly consumed. This inference is obtained based on previous work where it has been shown that after reduction of peracids only non-oxidizing species remain, indicating that the oxidizers are consumed in the reaction.41 Figure 3B shows crosslinking as a function of Ni(OAc)2 concentration (lanes 3-6). It can be seen that as the concentration of Ni(OAc)2 is increased the intensity of high molecular weight species in the loading well increases. Further, the peptide is not crosslinked in the presence of Ni(OAc)2 alone (Figure 3B lanes 7-10).

Figure 3. Effect of reactant concentrations on crosslinking of t-M2e-t.

Figure 3.

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All samples were run in 638 mM carbonate buffer at pH 9.2 with t-M2e-t concentration of 0.5 mg/ml. The reaction was run for 1.5 hours. (A) t-M2e-t crosslinking with varying MMPP concentrations but fixed Ni(OAc)2 concentration. C: 10 μg t-M2e-t as peptide alone control. (B) t-M2e-t crosslinking with varying Ni(OAc)2 concentrations but fixed MMPP concentration. Lanes 3-6: 10 mM MMPP. Lanes 7-10: No MMPP. C: 10 μg t-M2e-t as peptide alone control.

Effect of Other Bivalent and Trivalent Metal Ions on Peptide Crosslinking

We next evaluated the potential of other transition metals to facilitate the crosslinking reaction because histidine residues have the ability to complex with different transition metals.42 For example, Cu(II), Zn(II), and Fe(III) are known for their ability to complex with histidine residues in histag purification technologies,43 and Fe(II) is well-known for its interaction with histidine in hemoglobin. We also evaluated Mg(II) due to its presence in MMPP and we wanted to further clarify its role in crosslinking.

Use of Cu(OAc)2, Zn(SO4)2, MgCl2, FeCl2, and FeCl3 did not result in a high degree of crosslinking as compared to Ni(OAc)2, even when these metal salts were used at concentrations greater than 4 mM (Figure 4). Among the transition metals studied, FeCl3 at 8 mM concentration did produce high molecular weight species that remained in the loading well of the gel; however, a measurable amount of low molecular weight oligomers was also observed as a smear between 10 kDa and 25 kDa (lane 6 of Figure 4E). These low molecular weight species are absent when Ni(OAc)2 is used. Thus, besides Ni(OAc)2, none of the metal salts evaluated in this study provided good crosslinking. This phenomenon has also been noted elsewhere35 in the context of protein oligomer formation. While the reason for Ni(II) superiority over other metal ions remains unclear, possible explanations could be linked to the orientation of His-Ni(II) complexes, or the fact that the Ni(II)-Ni(III) transition is more energetically favorable compared to other transition metals.36, 44 Others hypothesize that the oxidation of tyrosine occurs rather indirectly; first MMPP oxidizes a Ni(II) ion to Ni(III), and then Ni(III) extracts an electron from the nearby phenol group of tyrosine, creating a tyrosyl radical, which then combines with another tyrosyl group, creating a dityrosine bond.34, 36 It is also possible that other metal ions beside Ni(II) create a His-Metal complex that energetically cannot outperform the His-Ni(II) complex in extracting the electron from the tyrosine ring. Additional studies are required to corroborate these hypothesis.

Figure 4. Effect of different metal salts on crosslinking of t-M2e-t.

Figure 4.

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All reactions were run for 1.5 hours in 638 mM carbonate buffer at pH 9.2 with 0.5 mg/ml t-M2e-t, and 10 mM MMPP (except *, where no MMPP was used). The metal salt used in the reaction are as follows and salt concentrations are listed below each image: (A) Cu(OAc)2, (B) Zn(SO4)2, (C) MgCl2, (D) FeCl2, (E) FeCl3. C: 10 μg t-M2e-t as peptide alone control.

* Indicates samples without MMPP present in the reaction

Peptide Crosslinking Results in Tunable Cluster Formation

To characterize the crosslinked material present in the loading wells, we began by collecting transmission electron microscopy (TEM) images. From Figure 5A, it can be seen that clusters are indeed created upon crosslinking of t-M2e-t, and at lower peptide concentrations, smaller clusters are formed. This was also corroborated by the fact that clusters made using 0.5 mg/ml remained suspended in solution, while those made using 1 mg/ml and 2 mg/ml t-M2e-t settled out of solution within a few minutes of stopping agitation (Figure S2).

Figure 5. Formation of tunable t-M2e-t NCs.

Figure 5.

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(A) TEM images of t-M2e-t NCs created using varying t-M2e-t concentrations during the crosslinking reaction (left to right: 0.5 mg/ml, 1.0 mg/ml, 2.0 mg/ml). (B) t-M2e-t NC size as a function of reaction peptide concentration: 0.1 mg/ml – 217 ± 183 nm, 0.2 mg/ml – 275 ± 238 nm, 0.5 mg/ml – 761 ± 659 nm, 1.0 mg/ml – peak 1: 240 ± 189 nm, peak 2: 3164 ± 3303 nm. (C) t-M2e-t NC size as a function of reaction time: 30 minutes – 322 ± 269 nm, 45 minutes – 210 ± 189 nm, 90 minutes – 275 ± 238 nm, 180 minutes – 274 ± 190 nm. (D) t-M2e-t NC size as a function of reaction stir speed: 300 RPM – 275 ± 238 nm, 650 RPM – 308 nm ± 237 nm, 1150 RPM – 170 ± 127 nm. (E) Relative fluorescence units to examine dityrosine bonds: Ex. λ = 323 nm. Fluorescence intensity of crosslinked t-M2e-t NCs and uncrosslinked t-M2e-t peptide each at a concentration of 1 mg/ml was measured.

We next used dynamic light scattering (DLS) to analyze the effect of peptide concentration, reaction time, and stir speed on cluster size. As seen in TEM images, upon increase in peptide concentration the average cluster size increased. Cluster sizes with average diameters of 217 ± 183 nm, 275 ± 238 nm, 761 ± 659 nm, and 240 ± 189 nm/3164 ± 3303 nm (first/second peaks) were obtained with peptide concentrations of 0.1 mg/ml, 0.2 mg/ml, 0.5 mg/ml, and 1.0 mg/ml, respectively (Figure 5B). Interestingly, clusters created with 1.0 mg/ml peptide not only exhibited a larger average cluster size but also a bimodal distribution, indicating an increase in polydispersity with increase in peptide concentration. Thus, lower peptide concentrations are more suitable to produce t-M2e-t clusters on the nanometer scale than higher peptide concentrations.

We also varied the crosslinking reaction time for clusters by keeping the peptide concentration constant at 0.2 mg/ml. Notably, no discernable trend was seen between cluster size and reaction time when crosslinked for 30 minutes to 180 minutes. This would indicate that the majority of crosslinking is complete within 30 minutes of the start of reaction (Figure 5C). Finally, solution stir speed was varied to determine its impact on cluster size (peptide concentration was kept constant at 0.2 mg/ml). Figure 5D shows that, as stir speed is increased, a marginal reduction in cluster size can be achieved. At 1150 rpm t-M2e-t NCs of 170 ± 127 nm were obtained versus sizes of 275 ± 238 nm when 300 rpm are used. The polydispersity also decreased with an increase in stir speeds.

Confirmation of Tyrosine-Tyrosine Bond Formation

To confirm involvement of tyrosine-tyrosine bond formation in crosslinking, we examined the crosslinking reaction using fluorescence spectroscopy for two representative cluster sizes: ‘small’ S t-M2e-t NCs (275 ± 238 nm) and ‘large’ L t-M2e-t NCs (761 ± 659 nm). Others have reported that dityrosine bonds have a fluorescence emission peak at 420 nm when excited at 323 nm. 34, 36, 37 From Figure 5E this signature emission peak at 420 nm wavelength can be observed for S t-M2e-t NCs and L t-M2e-t NCs but not uncrosslinked t-M2e-t peptide. This indicates that, as hypothesized in Scheme 1, dityrosine bonds are indeed present in the crosslinked NCs.

Crosslinked M2e Retains the Ability to Bind Anti-M2e Antibody

Next, to determine whether the crosslinking reaction can adversely affect the antigenic nature of the M2e peptide, an indirect (Figure 6A) and sandwich ELISA (Figure 6B) was performed. For the indirect ELISA, serum containing anti-M2e antibodies8-10 raised against non-crosslinked and non-tagged M2e was used for detection. Plates were coated with a constant amount (50 μl of 5 μg/ml) of either t-M2e-t, t-M2e-t NCs, or a negative control peptide (NCP) that contained reactive tags but not the M2e sequence. For both t-M2e-t and t-M2e-t NCs, similar absorbance readings were obtained indicating that the M2e epitope is accessible even after crosslinking. For the sandwich ELSIA, plates were coated with a purified anti-M2e monoclonal IgM antibody. The clusters generated a higher titer as compared to an equal mass of uncrosslinked peptide, further indicating that M2e is not destructively altered during the crosslinking reaction.

Figure 6. Anti-M2e IgG can bind to t-M2e-t NCs, which shows that M2e sequence retains antigenicity during particle synthesis.

Figure 6.

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(A) An indirect ELISA was performed by coating maxisorp plates with a fixed amount of either t-M2e-t, t-M2e-t NCs, or a negative control peptide (NCP). Dilutions of a mouse serum collected from mice vaccinated with a Gold nanoparticle (AuNP) and M2e conjugate formulation were used to obtain the primary anti-M2e IgG antibodies. (B) A sandwich ELISA was performed by first coating maxisorp plates with a fixed amount of anti-M2e IgM monoclonal antibody. Dilutions of analyte (either t-M2e-t, t-M2e-t NCs, or a NCP) were then added to the wells. The anti-M2e IgG primary antibody (1:500 dilution) was obtained from mice vaccinated using a AuNP and M2e conjugate formulation.8-10 Cartoons of each ELISA procedure are shown below their respective graphs.

This result is perhaps not unexpected because M2e does not natively contain tyrosine residues. Therefore, crosslinks are not anticipated to form anywhere in the antigenic stretch of the peptide, which should leave the antigen unchanged post-crosslinking. It has previously been noted that in this reaction scheme, dityrosine bond formation occurs between tyrosine residues located in the proximity of histidine 33, 34, 36. Therefore, unless the peptide’s antigenic stretch has tyrosine and multiple histidine residues in proximity, there is a high probability that the crosslinking will occur only along the purposefully added reaction tags. Therefore, this particular reaction chemistry offers an advantage over the many other, less targeted, crosslinking reactions.

Crosslinked M2e NCs are Immunogenic and Protect Mice From Lethal Challenge With Influenza A

Having established that M2e NCs can be recognized by antibodies in vitro, we next proceeded to evaluate their in vivo immunogenicity. Smaller t-M2e-t (S t-M2e-t) NCs with an average size of 275 ± 238 nm were selected because it has been previously shown that nanoparticles around this size have the ability to migrate to the draining lymph node independent of dendritic cell uptake and can produce a better immune response compared to larger particles.45, 46 Mice received a total of three doses, where each dose contained 20 μg or 5 μg (S t-M2e-t NCs) with or without 20 μg CpG. Additionally, mice were also vaccinated in the same manner, but with uncrosslinked t-M2e-t in order to determine the immunogenicity of the monomeric peptide. As expected, mice receiving the monomeric uncrosslinked t-M2e-t peptide failed to generate significant levels of anti-M2e antibody titers (Figures 7A, 7B, and 7C).

Figure 7. Immune response and survival data of mice after immunization with t-M2e-t NCs.

Figure 7.

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Mice were immunized thrice, once each on day 0, 21, and 42. Blood was collected and analyzed for anti-M2e antibodies. . Within one week of the final serum collection, mice were challenged with 3x LD50 A/California/07/2009 (H1N1) and monitored for 14 days. Two antigens were used i) 20 μg or 5 μg t-M2e-t UCP (uncrosslinked peptide) with/without 20 μg CpG, and ii) 20 μg or 5 μg S t-M2e-t NCs (small NCs) with/without 20 μg CpG. (A, B, C) Anti-M2e IgG, IgG1 and IgG2a titers with t-M2e-t UCP as antigen. (D, E, F) Anti-M2e IgG, IgG1 and IgG2a titers with t-M2e-t NP as antigen. (G, H) Body weight and survival after virus challenge of t-M2e-t UCP groups. (I, J) Body weight and survival after virus challenge of t-M2e-t NP groups. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001

When, instead of uncrosslinked t-M2e-t peptide, crosslinked t-M2e-t NCs were used (no CpG), a higher IgG titer was observed for the 20 μg dose versus the 5 μg dose (Figure 7D). The IgG1 titer (Figure 7E) was slightly higher than the IgG2a titer (Figure 7F) although the difference was not statistically significant.

When CpG was added to NCs, the antibody titers increased significantly. Of all the treatment groups, mice receiving 20 μg S t-M2e-t NCs with CpG generated the highest total IgG (Figure 7D). Interestingly, with the addition of CpG to NCs, the relative trend between IgG1 and IgG2a titers reversed as compared to when no CpG was used. Specifically, IgG2a titers became significantly elevated across all groups as compared to IgG1 titers (Figure 7E and 7F).

It has been previously shown that CpG predominantly stimulates a type 1 T helper (Th1) immune response, which favors the production of the IgG2a subtype over IgG1 subtype.47 Notably, others have also documented the importance of IgG2a in clearing influenza infections, specifically in regard to non-neutralizing antibodies, such as those against M2e. The superior ability of IgG2a to interact with the family of Fcγ receptors and complement C3, leading to antibody dependent cellular cytotoxicity (ADCC) by NK cells and complement dependent cytotoxicity (CDC), respectively, has been reported to be the main pathway of viral clearance by anti-M2e antibodies.48-51 Therefore, the high IgG2a titers that can be generated by the S t-M2e-t NCs in conjunction with CpG speaks to the advantage of this system.

After vaccination, mice were challenged with 3x LD50 A/California/07/2009 (H1N1). Body weight and survival data are given in Figure 7G and 7I and Figure 7H and 7J, respectively. Uncrosslinked M2e peptide failed to induce protective immunity even with CpG and led to only 40% survival and all groups behaved similarly to unvaccinated mice per average group body weights. On the other hand, complete protective immunity was achieved in the group receiving 20 μg S t-M2e-t NCs with CpG. This group also experienced the lowest average weight loss throughout the challenge compared to other groups, as expected. This demonstrates the importance of both NCs and CpG as an adjuvant.

Effect of NC Size on Immune Response

Next, to determine effect of NC size on immune response we selected S t-M2e-t NCs (275 ± 238 nm) and larger (L) t-M2e-t NCs (761 ± 659 nm) and examined their immunogenicity in vivo. NC amount was varied at 20 μg and 5 μg per dose with 20 μg CpG in each formulation. After two immunizations the antibody titers for the S t-M2e-t NC group were low. This is consistent with the previous experiment (Figure 7) where a third dose of S t-M2e-t NCs was required to increase the antibody titers. However, for the L t-M2e-t NC group, the 20 μg dose produced significantly higher anti-M2e IgG, IgG1 and IgG2a titers (Figure 8A, 8B, 8C). We further noticed that these antibody titers were similar to those stimulated in our previous work where M2e was attached to gold nanoparticles and which had provided full protection after just two immunizations.8-10 Therefore, to determine whether the L t-M2e-t group would also exhibit protection after two immunizations, the mice were challenged with 3x LD50 A/California/07/2009. Indeed, animals in the L t-M2e-L NC group exhibited lower weight loss (Figure 8D) and fully survived the challenge as compared to groups vaccinated with S t-M2e-t NCs (Figure 8E).

Figure 8. Effect of NC size on immune response and survival.

Figure 8.

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Mice were immunized twice, once each on day 0 and 21. Blood was collected and analyzed for anti-M2e antibodies. Within one week of the final serum collection, mice were challenged with 3x LD50 A/California/07/2009 (H1N1) and monitored for 14 days. Two antigens were used i) 20 μg or 5 μg S t-M2e-t NCs (small NCs) with 20 μg CpG, and ii) 20 μg or 5 μg L t-M2e-t NCs (large NCs) with 20 μg CpG. (A, B, C) Anti-M2e IgG, IgG1 and IgG2a titers for different groups. (D, E) Body weight and survival after virus challenge. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001

Others have previously noted that an increase in NP size can lead to better IgG immune responses through a variety of vaccination routes. 52 Larger particles have been reported to be cleared more slowly by the immune system, which can allow for longer presentation times of the antigenic clusters to antigen presenting cells.6, 52 This could possibly explain the higher immune response from L t-M2e-t NCs as compared to S t-M2e-t NCs, but further investigation is needed to fully evaluate this phenomenon.

To our knowledge, this is the first study that reports protective immunity generated from NCs synthesized from linear M2e peptides. Others have formulated M2e nanoclusters using tetrameric M2e, citing the need to present the peptide in its natural conformation.6, 11, 12, 53 Our t-M2e-t NCs, however, are randomly crosslinked peptides that have no specifically designed structure. Since there is no need for a carrier NP system, such as a metal or polymeric NCs, this greatly simplifies the vaccine formulation. Another advantage of this reaction system is that hexahistidine tags are very popular in recombinant protein technology. These tags are primarily used for protein and peptide purification from crude lysates.40, 43, 54 Others have also used hexahistidine tags to complex proteins to carrier nanoparticles in a safe manner and have noted low immunogenicity related to the polyhistidine tag.55-59 Additionally, tyrosine residues are often added to proteins and peptides to readily allow for concentration measurements using UV-Vis absorbance at 280 nm.60-62 Thus, there already exist important applications for both histidine and tyrosine and without additional modifications the same residues could also be used to make NCs, if required.

As a more general note, we hypothesize that this reaction system can be extended to work with any antigen by simply adding the H6(GY)2 tag on the ‘N’ and ‘C’ termini of the peptide. Since the reaction is specific to tyrosines located next to the hexahistidine sequence, presence of endogenous tyrosine in the peptide itself is not expected to participate in the crosslinking reaction, leaving the antigenic peptide unaltered. This offers the advantage that once the desired reaction conditions are optimized for a particular peptide or protein, it may be possible to use the same conditions to make NC vaccines out of a myriad of peptide antigens. This approach therefore adds another important tool in the NC creation toolkit.

Nickel Content in Vaccine Formulation

Although NC formulations were washed extensively, it is possible that some Ni(II) can remain within the NCs. To determine the Ni(II) content remaining in the washed NCs, energy-dispersive X-ray spectroscopy (EDS) analysis was performed on the clusters. It was determined that the S t-M2e-t NCs and L t-M2e-t NCs contained 2.2 ± 0.2 wt% and 1.6 ± 0.2 wt% Ni(II), respectively. These values correspond to approximately 400 ng Ni(II) per dose of 20 μg NCs and 100 ng Ni(II) per dose of 5 μg NCs. Others have reported on the safety of Ni(II) from intramuscular injections in mice, and as long as the Ni(II) dose remains below approximately 200 μg63 it is considered non-toxic. Moreover, nanogram levels of Ni(II) have been explored in creating nanoparticles for antigen decoration55, 56, and for use in anti-cancer therapy as a metal-chelated drug64, which have been tested in murine models and have been found to be safe as well. Thus, the Ni(II) content delivered per vaccination dose of the t-M2e-t NCs is well below what is generally considered toxic for mice.

Conclusion

We have successfully synthesized t-M2e-t NCs solely from a small peptide using a tyrosine-based crosslinking scheme. These NCs are able to elicit protective immunity against a lethal influenza A H1N1 virus challenge in BALB/c mice. This reaction system can be used to form tunable clusters in a facile manner by the crosslinking of tyrosine residues in an engineered reactive tag. We have sought to characterize and optimize the reaction in order to obtain clusters most apt for vaccinations, though clusters with varying characteristics can be formulated to meet the needs of other applications as well. Importantly, given the mechanism governing the crosslinking reaction, we hypothesize that this system is suitable for peptide antigens other than M2e. As such, the data presented here show the development of a method that can be added to the already existing repertoire used for creating peptide NCs.

Methods

Chemicals

Magnesium monoperoxyphthalate hexadyrate (MMPP) (69868-50G), L-tyrosine (T3754-50G), hydrochloric acid (HCl) (258148-4L), tween 20 (P1379-1L), phosphate-citrate buffer (P4809-100TAB), o-phenylenediamine (OPD) (P9029-50G), sodium bicarbonate (S5761-500G), sodium carbonate monohydrate (230952-100G), sodium phosphate dibasic (71640-250G), citric acid (251275-100G), and glycine (G7126-100G) were purchased from Millipore Sigma (MO, USA). 10x Tris/Glycine/SDS Buffer (1610732) and QC colloidal coomassie stain (1610803) were acquired from BioRad (CA, USA). Hydrogen peroxide (H325-4) and fraction V bovine albumin at pH 7 (AAJ1085722) were obtained from Fisher Scientific (PA, USA). Nickel (II) acetate tetrahydrate (Ni) (A13026-22) was purchased from Alfa Aesar. Orthophosphoric acid (BDH3104-2.5LPC) was purchased from VWR (PA, USA). Phosphate buffered saline (PBS) 10x was purchased from Corning (NY, USA).

Synthetic Peptides, Secondary Antibodies, and Oligonucleotides

t-M2e-t (acetylated-HHHHH HGYGY SLLTE VETPI RNEWG SRSND SSDGY GYHHH HHH-amidated; Mw: 5160.42 Da), M2e-t (acetylated-SLLTE VETPI RNEWG SRSND SSDGY GYHHH HHH-amidated; Mw: 3897.10), and the negative control peptide (acetylated-HHHHH HGYGY ILRTQ ESECY GYGHH HHHH-amidated; Mw: 3645.82) were synthesized by AAPPTec (KY, USA) at >95% purity. All peptides were end-terminal acetylated and amidated in order to increase peptide stability against degradation.65-67 Goat anti-mouse IgG (1030-05), goat anti-mouse IgG1 (1070-05), and goat anti-mouse IgG2a (1080-05) secondary antibodies with HRP were obtained from Southern Biotech (AL, USA). CpG ODN 1826 (5’-T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T-3’) was obtained from IDT (IA, USA) with endotoxin levels less than 0.100 EU/mg.

Peptide Nanocluster Synthesis and Characterization

To a 0.5 mg/ml t-M2e-t solution in carbonate-bicarbonate buffer (638 mM pH 9.2, unless otherwise noted) Ni(OAc)2 was added to attain a final concentration of 4 mM and allowed to sit at room temperature for 5 minutes. MMPP was then added for a final concentration of 10 mM and the reaction was allowed to proceed for 1.5 h with stirring (300 rpm) unless otherwise stated. In samples using M2e-t, molar concentrations of M2e-t were kept equal to that of t-M2e-t. The reaction was stopped with the brief addition of tyrosine (dissolved in 1M HCl) to the reaction solution to give a final tyrosine concentration of 18 mM. All solutions were sterile filtered through a 0.22 μm syringe filter (03-337-155) purchased from Fisher Scientific.

Degree of peptide crosslinking was assessed using SDS Gel Electrophoresis. Samples were mixed with reducing sample buffer (catalogue no. 39000, ThermoFisher), heated at 95°C for 5 min, and loaded (10 μg per well) onto 4-20% Mini-PROTEAN TGX gels (catalogue no. 4561094, BioRad, CA, USA).

For clusters used in vaccinations, the crosslinking reaction was run for 1.5 h using 0.2 mg/ml or 0.5 mg/ml t-M2e-t, 4 mM Ni(OAc)2, and 10 mM MMPP in carbonate-bicarbonate buffer pH 9.2. Upon completion of the reaction, the solution was dialyzed (Slide-A-Lyzer™ dialysis cassette, 10K MWCO, 66810, Thermo Fisher Scientific, MA, USA) for 24 h against deionized water, with regular water changes. The clusters were then collected, lyophilized, and the mass determined gravimetrically using a Mettler Toledo XP105 Delta Range balance. The recovery efficiency was 71.2% and 68.9% for the S t-M2e-t NCs and L t-M2e-t NCs, respectively. The clusters were then suspended in a 0.1% tween 20 solution and thoroughly mixed.

Cluster sizes and standard deviations were determined by Dynamic Light Scattering (DLS). Data were collected on a Wyatt Mobius and data was analyzed using Dynamics version 7.3.1.15. Values determined via DLS are reported as mean ± standard deviation. Curves were plotted using GraphPad Prism 8.4.3. All clusters analyzed by DLS were created using a peptide concentration of 0.2 mg/ml, 4 mM Ni(OAc)2, 10 mM MMPP, 1.5 hour reaction time, 300 rpm stir speed, and 638 mM pH 9.2 carbonate buffer, unless otherwise stated.

Fluorescence spectra were collected using a BioTek Synergy H1 microplate reader. Samples were excited at 323 nm and emissions spectra were collected. Data was then plotted using GraphPad Prism 8.4.3.

Energy dispersity X-ray microanalysis (EDS) measurements were collected using a Hitachi S-4300 E/N (FESEM) in conjunction with the EDAX Pegasus 4040 analysis system. Both negative and positive controls were used to verify reported results. No measureable nickel was reported in the negative control (pure OVA) while the positive control (OVA containing 5.9 wt% Ni(II)) reported 6.0 ± 0.3 wt% nickel.

Immunizations

All animal experiments and protocols were approved by the Texas Tech University (TTU) Institutional Animal Care and Use Committee (IACUC). Female BALB/c mice (Charles River Laboratory, 6-8 weeks old, n = 5 mice/group) were immunized either twice (day 0 and day 21) or thrice (day 0, 21, 42) via intramuscular injection of 50 μl formulation. Blood was collected one day prior to vaccination, and again 21 days after the last dose (day 42 or day 63 for two and three vaccinations, respectively). The NC stock was sonicated twice for 30 min and thoroughly mixed before creating the vaccine formulations. In CpG containing groups, CpG was mixed with the clusters just prior to vaccination.

Serum Antibody Measurement and Evaluation of Antigenic Nature of t-M2e-t in NCs

Serum antibodies against M2e were measured using an ELISA protocol.10 Briefly, 96 well MaxiSorp plates (catalogue no. 442404, ThermoFisher) were coated with 50 μl of a 5 μg/ml solution of M2e in PBS and stored at 4 °C overnight. The plates were then washed using a Biotek ELx405 Microplate washer (VT, USA) with a 0.05% tween 20 solution in PBS (PBST) and then incubated for 2 hours with 100 μl of 3% bovine serum albumin in PBST. Plates were washed and incubated for 1.5 hours with serum diluted in PBST. After another wash, 50 μl of a 1:4000 dilution of secondary antibody in PBST was added to each well and incubated for an additional 1.5 hours. Plates were washed before the addition of an OPD/H2O2 solution in phosphate-citrate buffer to generate colorimetric changes in each well. The reaction was stopped with a 3 M phosphoric acid solution. Plate OD values were read at 492 nm (SpectraMax Plus 384 Microplate reader, Molecular Devices, CA, USA). Endpoint titers were determined as the highest dilution of immune sera with an OD value above the average OD value plus three standard deviations of the naïve group (at 50x dilution). For the indirect ELISA in Figure 6A, plates were coated with 50 μl 5 mg/mL t-M2e-t, 5 mg/mL t-M2e-t NCs, or 7 mg/mL NC peptide. All other steps follow those listed above.

To examine antigenic nature of t-M2e-t in NCs a sandwich ELISA was performed by coating 96 well MaxiSorp plates with 50 μl 50 μg/ml anti-M2e IgM monoclonal antibody (prepared in house). Plates were blocked for 2 hours with a 3% BSA solution in PBST and then washed. Serial dilutions of either soluble t-M2e-t, t-M2e-t NCs, or a negative control peptide were added to wells for 1.5 hours and plates were then washed again. A 1:500 dilution of mouse serum in PBST from mice vaccinated with gold nanoparticles conjugated to M2e was then added and incubated for an additional 1.5 hours.8-10 The steps of secondary antibody addition, OPD/H2O2 addition, H3PO4 addition to stop reaction, and collection of OD value were the same as above.

Virus Challenge

Mice were intranasally inoculated with 30 μl of mouse adapted 3x LD50 A/California/07/2009 (H1N1). Body weights were monitored for 14 days after inoculation. Mice were euthanized if their body weight dropped below 25% of their initial weight for more than 24 hours.

Statistical Analysis

All data and figures, as well as statistical analysis, were produced and analyzed using GraphPad Prism 8.4.3. Statistics for the ELISA endpoint titers were obtained using two-way ANOVA with post-hoc Tukey tests.

Supplementary Material

SupplementaryMaterial

Acknowledgments

All research completed in this publication was funded by the National Institutes of Health (NIH) award number R01AI137846 and by funds from the Texas Tech University Whitacre Endowed Chair in Science and Engineering to H. Gill.

We would also like to thank Dr. Bo Zhao (Texas Tech University) for her help in running and collecting EDS measurements and Dr. Maninder Kumar for providing the anti-M2e IgM monoclonal antibody used for the sandwich ELISA.

Footnotes

Supporting Information

All supporting information is available free of charge at http://pubs.acs.org.

Crosslinking of t-M2e-t using only MMPP or Ni(OAc)2 (Figure S1); Colloidal stability of t-M2e-t NCs in solution as a function of NC size (Figure S2); DLS curves for S t-M2e-t NCs and L t-M2e-t NCs used for vaccinations (Figure S3).

Financial disclosures: The authors have no conflict of interest related to this study.

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