Chikungunya virus E2 B domain nanoparticle immunogen elicits homotypic neutralizing antibody in mice

Abstract

Alphaviruses are enveloped, positive-sense single-stranded RNA viruses that cause severe human and animal illness. Arthritogenic alphaviruses, such as Chikungunya virus (CHIKV) and Mayaro virus (MAYV), are globally distributed, transmitted by mosquitoes, and can cause rheumatic disease characterized by fever, rash, myalgia, and peripheral polyarthralgia that can persist for years post-infection. These infections can also result in more severe clinical manifestations such as hemorrhage, encephalopathy, and mortality. Several potent monoclonal antibodies (mAbs) with broad neutralizing activity have been shown to bind to the E2 B domain (E2-B) of the alphavirus glycoprotein, suggesting that E2-B epitopes are a site of susceptibility for multiple arthritogenic alphaviruses. However, it is unknown whether E2-B alone can elicit a broadly neutralizing humoral response. Here, we generate and characterize nanoparticle-based immunogens containing CHIKV and MAYV E2-B. Immunization with the CHIKV E2-B nanoparticle elicited sera that were cross-reactive toward CHIKV and MAYV E2-B, but had only homotypic neutralizing activity (serum titer of 1:256) against CHIKV vaccine strain 181/25. Furthermore, immunization with MAYV E2-B nanoparticles elicited non-neutralizing antibody, but sera were cross-reactive for both CHIKV and MAYV E2-B. Our findings suggest that the immunodominant epitopes within CHIKV and MAYV E2-B are bound by cross-reactive, but not cross-neutralizing antibody. Therefore, development of broad E2-B based vaccines that induce broadly neutralizing antibody responses will require engineering to alter the immunodominant landscape.

Introduction:

Alphaviruses are enveloped, positive-sense, single-stranded RNA viruses that cause significant human illness; they are classified into two clades: arthritogenic and encephalitic. Infection by encephalitic alphaviruses primarily affects the central nervous system, resulting in fever, severe headache, confusion, seizures, and severe neurological impairment (1), whereas infection by arthritogenic alphaviruses can lead to human musculoskeletal disease (2, 3). Symptoms of arthritogenic alphavirus infection include fever, rash, myalgia, and acute and chronic peripheral polyarthralgia (4, 5). The debilitating arthropathy can persist for months to years after infection, causing significant health complications over time (6).

Arthritogenic alphaviruses cause sporadic outbreaks in many different regions of the world. Of the 40 identified viral species, Chikungunya virus (CHIKV) has garnered the most global concern over the last two decades. CHIKV was originally identified in Tanzania in 1953 and was spread principally by the Aedes aegypti mosquito, causing limited outbreaks in Africa and Asia (7). However, between 2004 and 2007, a widespread CHIKV outbreak took place in islands throughout the Indian Ocean region. For example, in La Reunion, one-third of the island’s population contracted CHIKV, totaling to 255,000 reported cases and approximately 260 deaths (8). It was later determined that the circulating CHIKV strain harbored a point mutation in the E1 glycoprotein (A226V) (9). This single amino acid substitution was found to enable efficient CHIKV transmission in Aedes albopictus, a mosquito species with significant widespread distribution, thus leading to an epidemic of unprecedent scale (10). Spread of the virus was further exacerbated when infected travelers returned home, disseminating it through Central Africa, Asia, and Europe (11). Since then, CHIKV has continued to circulate worldwide and poses a growing threat to global public health. In the last decade since the first documented case of CHIKV was reported in the Americas in 2013, 3.7 million suspected and laboratory-confirmed cases have been reported across 50 countries and territories in North, Central, and South America (12).

Mayaro virus (MAYV) is a related alphavirus and is an emerging threat that has the potential to cause widespread disease like CHIKV. MAYV was first isolated in Trinidad in 1954 and has been identified as the cause of several outbreaks in mostly forested areas in Central and South America (13). However, in 2015, MAYV infection was reported in an 8-year-old boy in a semirural area of Haiti, indicating that MAYV is now circulating in a new type of environment and no longer isolated to the Amazonian jungle regions (14). Similarly to CHIKV, imported cases from travelers have also been reported in Europe, further increasing the risk for urban transmission (15-17). Although MAYV is mostly spread by the Haemagogus mosquitoes, studies have shown that the Anopheles and the Aedes mosquito are competent laboratory vectors for MAYV (18-20). Because MAYV and CHIKV have shared competent vectors with increasing worldwide distribution due to urbanization, travel, and global warming, there is substantial risk for widespread dissemination of MAYV. While there is a recently approved CHIKV vaccine, it is not known whether it can protect against other emerging alphaviruses, such as MAYV (21). Therefore, broadly protective vaccines and therapeutics would be highly beneficial for global preparedness.

The alphavirus surface glycoprotein is composed of two subunits, E1 and E2, each of which is anchored by a transmembrane domain. Together, these proteins mediate viral attachment and membrane fusion (22-24). The prefusion E1/E2 heterodimer forms a trimeric spike that is arranged in an icosahedral lattice on the virus particle surface. During viral replication, E2 is initially expressed as a precursor polypeptide, known as p62, that contains E2 and a small chaperone protein known as E3 joined by a linker region containing a furin site. During maturation, cellular furin cleaves p62, generating E2 and the peripheral E3 polypeptide. E3 remains bound to the E1/E2 heterodimer but is released during the final step of virus maturation and primes the glycoprotein for membrane fusion (25, 26).

CHIKV natural infection in humans elicits neutralizing antibodies, with a majority of cross-neutralizing antibodies targeting the B domain of E2 (E2-B) (27, 28). Furthermore, depleting E2-B reactive antibodies from convalescent sera decreased cross-neutralizing activity against MAYV (28). We and others have also isolated potent cross-neutralizing and cross-protective monoclonal antibodies (mAbs) with epitopes that map to the E2-B. For example, human mAbs DC2.M108, DC2.M16, and DC2.M357, were isolated from a CHIKV patient; the latter two have been shown to protect against both CHIKV- and MAYV-induced musculoskeletal disease in mice (29). Another human mAb, RRV-12, isolated from a patient with Ross River Virus (RRV), was shown to protect mice against RRV and MAYV infection (30). Similarly, CHIKV infection in mice also elicits a humoral response against the E2-B. In fact, murine mAb CHK265 has also been shown to bind to the E2-B and can protect against CHIKV, MAYV, and RRV challenge in mice (31). More recent studies involving recipients of the CHIKV virus-like particle (VLP) vaccine, PXVX0317, described several mAbs that were also E2-B reactive. In particular, human mAbs 506.A08 and 506.C01 were shown to bind to the apex of E2-B at different angles, suggesting that the angle of approach affects breadth, potency, and in vivo protection (32). The growing panel of protective E2-B reactive mAbs isolated from both natural infection and vaccination studies suggest E2-B is a critical target in protecting against alphavirus infection. However, it is currently not known whether E2-B-based immunogens elicit cross-neutralizing antibodies in vivo.

We sought to investigate and enhance the immunogenicity of the alphavirus E2-B by utilizing a self-assembling nanoparticle based on Aquifex aeolicus lumazine synthase (aaLS). Nanoparticle immunogens are an emerging strategy employed to induce strong immune responses against viral proteins (33). The multimeric and multivalent display of viral structural proteins on the surface of nanoparticles mimics the structure of viral particles and promotes cross-linking of B cell receptors, allowing for B cell activation and antibody maturation (34-36). This strategy has also been successfully implemented for a variety of pathogens (37-39). To investigate if the E2-B could elicit cross-neutralizing titers against CHIKV and MAYV, we engineered aaLS nanoparticles containing CHIKV E2-B or MAYV E2-B, evaluated their immunogenicity in vivo, and tested the resulting sera for neutralizing activity against CHIKV and MAYV. We found that the CHIKV E2-B nanoparticle (LS-CHIKV-B) elicited homotypic neutralizing antibody in mice, while the MAYV E2-B conjugated nanoparticle (LS-MAYV-B) elicited non-neutralizing antibody.

Results

Generation and characterization of recombinant SpyTagged CHIKV and MAYV E2-B:

Our objective was to investigate whether CHIKV and MAYV E2-B immunogens elicit cross-neutralizing antibody in vivo. In order to elicit high antibody titers, B cell receptors (BCRs) must be cross-linked to signal proliferation and promote affinity maturation of reactive germline precursors (40). Generally, small proteins or domains are poorly immunogenic because they are unable to sufficiently cross-link and activate BCRs (41). One strategy to improve the immunogenicity of a monomeric protein is to present them on nanoparticles. Because nanoparticles can assemble into multimeric structures that are similar in size, shape, and symmetry configurations as viruses, they mimic the natural presentation of antigens on viral particles (35). The increased avidity afforded by nanoparticles also enables tighter and prolonged binding to BCRs, and promotes their cross-linking on the cell surface (35). Furthermore, nanoparticle immunogens are more readily trafficked to lymphoid tissue and are retained for longer in lymph node germinal centers (42). Nanoparticles have been shown to drive increased somatic hypermutation, leading to increased B cell clonal diversity and improved affinity maturation (33, 42, 43). In order to leverage the advantages of nanoparticles and enhance the immunogenicity of E2-B, we utilized the SpyCatcher/SpyTag (SpyC/SpyT) conjugation system to link CHIKV and MAYV E2-B to Aquifex aeolicus lumazine synthase (aaLS) nanoparticles. SpyC/SpyT is derived from a Streptococcus pyogenes protein domain, CnaB2 (44, 45). The “SpyTag” (SpyT) is a 13-residue peptide from the C-terminus of CnaB that spontaneously forms an isopeptide bond with a “SpyCatcher” tag (SpyC), which is the 116-residue N terminus of the remainder of CnaB. We designed E2-B nanoparticle immunogens to be expressed as two separate components —1) CHIKV and MAYV E2-B constructs linked to SpyT; 2) aaLS, self-assembling nanoparticle, fused to SpyC. We envision we could rapidly conjugate these components to create E2-B nanoparticle immunogens under mild conditions, preserving both protein integrity and functionality. We designed constructs of CHIKV E2-B (residues 234-301, numbering as per Voss et al. (46)) and MAYV E2-B (corresponding residues) with a SpyTag on the N terminus (CHIKV-B-SpyT, MAYV-B-SpyT). Our CHIKV-B-SpyT constructs also included 4 additional amino acids on the N-terminus and six amino acids on the C-terminus from the β-ribbon connector domain based on the available CHIKV p62-E1 crystal structure (3N42). Similarly, MAYV-B-SpyT constructs included five additional amino acids on the N-terminus and six amino acids on the C-terminus. The constructs were then codon-optimized for soluble expression in E. coli (Figure 1A). Both CHIKV-B-SpyT and MAYV-B-SpyT were expressed as E. coli inclusion bodies that were oxidatively refolded. Size exclusion chromatography was then used to obtain purified protein preparations at the expected molecular weight of approximately 10 kDa, with yields of 3-5 mg/L (Figure 1B-D).

Figure 1: Design, expression, and purification of recombinant CHIKV E2-B and MAYV E2-B.

(A) Schematic of alphavirus p62-E1 envelope glycoprotein (CHIKV shown as an example; PDB: 3N42) with E2-B domain highlighted in blue. Amino acid sequences of CHIKV E2-B and MAYV E2-B constructs with SpyTag in orange, E2-B in blue, and β-ribbon connector domain sequences in black. (B and C) Size exclusion chromatogram of CHIKV-B-SpyT and MAYV-B-SpyT domain. Sample was run on Sephacryl S-75 HR AKTA column. Red boxes indicate fractions that were collected. (D) Coomassie stained SDS-PAGE gel of CHIKV-B-SpyT and MAYV-B-SpyT. (E) ELISA reactivity of mAbs CHK265, DC2.M108, and DC2.M16 toward SpyTagged CHIKV B and MAYV B domain. EC₅₀ values listed in nM (mean ± SD). Created with Biorender.com

Next, we confirmed that the SpyTagged E2-B proteins were refolded properly by testing their reactivity to mAbs, CHK265, DC2.M108, and DC2.M16, by enzyme linked immunosorbent assay (ELISA). All three of these mAbs were previously described to bind to the E2-B. The footprint of CHK265 was identified via cryo-electron microscopy (cryo-EM) to encompass 19 residues in the E2 B domain (amino acids 180-220) and 4 residues in the E1 A domain (24). On the E2-B, CHK265 binds a 5-stranded β-sheet structure on the apical tip of the E2-B. The contact residues for DC2.M108 and DC2.M16 were identified via escape mutants and were mapped to G209 and K215 of the E2-B, epitopes that are distal in sequence but proximal to each other in the E2-B structure (29). Therefore, CHK265, DC2.M108, and DC2.M16 bind conformational epitopes and binding of CHIKV and MAYV E2-B to these mAbs would provide evidence that E2-B is properly refolded. All three mAbs, CHK265, DC2.M108, and DC2.M16, reacted strongly with CHIKV-B-SpyT (EC₅₀ = 0.05 nM, 0.11 nM, 0.04 nM, respectively) and MAYV-B-SpyT domain (EC₅₀ = 0.06 nM, 0.01 nM, 0.02 nM, respectively), confirming that the recombinant proteins retained an antigenically relevant conformation (Figure 1E).

Production and characterization of CHIKV and MAYV E2-B nanoparticle immunogens:

AaLS is an enzyme derived from Aquifex aeolicus that naturally assembles into a highly thermostable 60-mer nanoparticle in an icosahedral configuration (47). AaLS has also been previously used for vaccine design against pathogens, such as HIV and SARS-COV-2 (48, 49). Furthermore, we have previously presented viral antigens on aaLS to elicit broadly reactive and cross-neutralizing responses in vivo against other arthropodborne viruses, such as Powassan virus and Zika virus (50, 51).

We engineered aaLS constructs containing a C-terminal SpyCatcher sequence (aaLS-SpyC) for soluble expression in E. coli. We also incorporated an N-terminus His tag for purification via Ni-NTA. After purification, we coupled either CHIKV-B-SpyT or MAYV-B-SpyT to aaLS-SpyC (Figure 2A). After overnight conjugation, there was no unconjugated aaLS-SpyC remaining, indicating that all the aaLS-SpyC was converted to E2-B decorated nanoparticles (Figure 2B, C). The conjugated nanoparticles (“LS-CHIKV-B” and “LS-MAYV-B”) were purified by size-exclusion chromatography and purity assessed by SDS-PAGE (Figure 2D-F). A polypeptide of approximately ~45 kDa was observed under denaturing conditions, consistent with a conjugated protein containing CHIKV-B-SpyT or MAYV-B-SpyT (~10 kDa) and aaLS-SpyC monomer (32 kDa).

Figure 2: Expression and characterization of LS-CHIKV-B and LS-MAYV-B nanoparticle immunogen.

(A) Schematic representation of LS-CHIKV-B and LS-MAYV-B nanoparticle. (B, C) Coomassie stained SDS-PAGE gel of LS-CHIKV-B and LS-MAYV-B nanoparticle conjugation via SpyCatcher/SpyTag system. (D, E) Size exclusion chromatograms of LS-CHIKV-B (D) and LS-MAYV-B (E). Sample was run on Sephacryl S-400 HR AKTA column. Red boxes indicate fractions collected. (F) Coomassie stained SDS-PAGE gel of purified LS-CHIKV-B (left) and LS-MAYV-B (right). (G) ELISA reactivity of mAbs CHK265, DC2.M108, and DC2.M16 toward LS-CHIKV-B and LS-MAYV-B with EC₅₀ values listed in nM. ELISAs were performed three times independently in duplicate wells (mean ± SD). Representative data shown. (H, I) Negative-stain transmission EM images of LS-CHIKV-B (H) and LS-MAYV-B (I). Scale is 50 nm. Created with Biorender.com

After purification, we tested LS-CHIKV-B and LS-MAYV-B for binding to CHK265, DC2.M108, and DC2.M16 to confirm whether the SpyTagged E2 B domains retained conformational integrity when presented on a nanoparticle (Figure 2G). We observed binding of all three mAbs against both nanoparticle immunogens, indicating that the SpyTagged E2 B domains were successfully coupled to aaLS and that E2-B epitopes were still accessible when displayed multivalently (EC₅₀ values ranging from 0.21 nM to 0.87 nM). However, in general the EC₅₀ values for binding to the nanoparticle-mounted E2-B were higher than was observed for free (unconjugated) E2-B, by ~2.4-26-fold. This lower binding may indicate that, while the E2-B epitopes for CHK265, DC2.M108, and DC2.M16 may be accessible, there me some steric blocking from the nanoparticle that weakens binding.

We also visualized LS-CHIKV-B and LS-MAYV-B particles by negative-stain electron microscopy (nsEM) and identified spherical molecules approximately ~20 nm in diameter (Figure H, I). The nanoparticles appeared uniform and non-aggregated. Thus, CHIKV and MAYV E2-B can be conjugated and presented with conformational integrity on aaLS nanoparticles.

Immunization of mice with LS-CHIKV-B and LS-MAYV-B nanoparticles:

We next immunized 6-8 week old female BALB/c mice with three doses of 15 ug of 1) LS alone, 2) LS-CHIKV-B, or 3) LS-MAYV-B nanoparticle, spaced 3 weeks apart, using Addavax as an adjuvant. (Figure 3A). Serum samples were collected two weeks after the final booster on day 56 and we characterized the IgG responses against CHIKV and MAYV E2-B domains. Immunization with LS-CHIKV-B elicited robust binding antibody titers against the CHIKV E2-B (mean EC₅₀ ~ 1:140,000) with strong cross-reactivity to MAYV E2-B (mean EC₅₀ ~1:66,000; Figure 3B). Similarly, LS-MAYV-B elicited sera that was cross-reactive for both MAYV and CHIKV E2-B proteins (mean EC₅₀ 1:28,000 and 1:105,000, respectively; Figure 3B).

Figure 3: Binding and neutralization of antibodies induced by CHIKV E2-B and MAYV E2-B immunogens.

(A) Immunization schedule of mice with CHIKV E2-B and MAYV E2-B immunogens. Mice were immunized on Day 0 (prime), Day 21 (1^st boost), and Day 42 (2^nd boost) with either LS (15 ug), LS-CHIKV-B (15 ug), or LS-MAYV-B (15 ug). Sera was collected two weeks after the second boost. (B) ELISA reactivity of sera from immunized mice toward CHIKV-B-SpyT and MAYV-B-SpyT. Groups shown are LS alone (n=5), LS-CHIKV-B (n=5), LS-MAYV-B (n=5) at day 56. Full curve ELISAs were performed twice independently in duplicate wells, shown as −log₁₀ (EC₅₀ mean) ± SD for each group. EC₅₀ values were determined using non-linear regression analysis. (C, D) Serum neutralization of CHIKV 181/25 (C) and MAYV TRVL-4675 (D) by FRNT. Groups shown are LS alone (n=5), LS-CHIKV-B (n=10), and LS-MAYV-B (n=9). Neutralization curves were performed twice independently in triplicate wells, shown as −log₁₀ (EC₅₀ mean) ± SD. IC₅₀ values were determined using non-linear regression analysis. Representative data is shown. One-way ANOVA with Tukey’s multiple comparison test was done. (*, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, p < 0.0001). Created with Biorender.com

Next, we tested serum neutralization against CHIKV vaccine strain 181/clone 25 and MAYV strain TRVL-4675 by focus reduction neutralization test (FRNT). LS-CHIKV-B antisera neutralized CHIKV 181/clone 25 (mean IC₅₀=1:512), but notably did not neutralize MAYV TRVL-4675 (Figure 3C, D). Interestingly, LS-MAYV-B antisera did not neutralize either CHIKV 181/clone 25 or MAYV TRVL-4675, despite demonstrating high serum reactivity to both B domains. Thus, we demonstrate that nanoparticles displaying CHIKV E2-B elicit homotypic neutralizing antibody, while nanoparticles displaying MAYV E2-B elicit non-neutralizing antibody.

Discussion

Recent work has described E2-B mAbs that neutralize and protect against multiple arthritogenic alphaviruses in mice, but the capacity for E2-B to act as an immunogen that induces cross-neutralizing titers has not been studied. We therefore designed CHIKV and MAYV E2-B nanoparticle immunogens and characterized the antibody response to them in mice. We utilized aaLS, a commonly used self-assembling protein nanoparticle, for multivalent display of CHIKV and MAYV E2-B. Nanoparticles have been shown to exhibit notable advantages for vaccine development because they augment the immunogenicity of recombinant monomeric proteins by extending half-life, increase trafficking to lymph nodes and enhance crosslinking of B cells to stimulate robust B cell activation (34). In particular, we have previously demonstrated that use of aaLS as an antigen carrier for orthoflavivirus domain III proteins from Zika virus and Powassan virus results in induction of up to ~2 logs more potent neutralizing antibody titers relative to weight-adjusted molar equivalent of free antigen (51, 52).

Despite a 68% sequence similarity between CHIKV and MAYV E2-B, our data showed that LS-CHIKV-B nanoparticles elicited high levels of binding antibody with homotypic neutralizing activity against CHIKV 181/25, but not against MAYV TRVL-4675. Notably, the neutralizing antibody titer observed here (1:512) for CHIKV 181/25 is in a similar range of the known protective threshold (PRNT₅₀ = 1:150) elicited by Ixchiq in humans (53).

We also show that LS-MAYV-B nanoparticles elicit non-neutralizing antibody against CHIKV 181/25 or MAYV TRVL-4675. In both cases, sera had high cross-reactive binding activity to both CHIKV E2-B and MAYV E2-B. Interestingly, the sequence of the MAYV E2-B utilized here is based on the sequence of MAYV TRVL-4675, and yet the highly reactive LS-MAYV-B antisera was unable to neutralize this MAYV strain. Previous findings with E2-B mAbs suggest that the structural location of the epitopes they target within the domain may affect the breadth and potency. For example, mAb 506.A08, which was isolated from recipients of the CHIKV VLP vaccine PXVX0317, binds an epitope at the tip of E2-B that is apically located on the E1/E2 prefusion spike and affords more potent cross-protection against CHIKV, MAYV, and RRV viral dissemination in mice than mAb 506.C01, which was isolated from the same donor but binds more laterally on the E1/E2 spike (32). This is corroborated by comparing the cross-neutralizing activity of DC2.M108 versus DC2.M357, another broadly neutralizing mAb isolated from the same CHIKV convalescent patient as DC2.M108 using single B cell sorting technology (29). Based on escape mutant data, DC2.M357 engages epitopes toward the apex of the crown formed by the three E2 subunits on the prefusion trimer, whereas DC2.M108 binds more laterally. Moreover, DC2.M108 is only able to neutralize CHIKV, MAYV, and O’nyong’nyong virus (ONNV), whereas DC2.M357 neutralizes CHIKV, MAYV, ONNV, RRV, and Semliki Forest Virus (SFV).

One possible explanation for the lack of cross-neutralizing activity in LS-CHIKV-B sera is that these apical epitopes are not immunodominant. For example, it is plausible that the configuration in which the E2-B apical epitopes are presented on aaLS renders them less accessible to BCRs than the lateral epitopes that are bound by mAbs with a more restricted breadth of activity. Therefore, only B cells that express antibodies with a narrow range of neutralizing activity would be activated. This hypothesis is supported by the higher EC₅₀ values for binding of CHK265, DC2.M108, and DC2.M16 to the nanoparticle-mounted E2-B relative to the unconjugated (free E2-B), which may be due to some level of steric occlusion of these epitopesby the nanoparticle. Such steric blocking of BCR engagement may render these epitopes subdominant. Additionally, LS-MAYV-B elicited non-neutralizing titers, suggesting that the non-neutralizing epitopes against MAYV are immunodominant, while neutralizing epitopes against MAYV are subdominant.

The shape and size of the nanoparticle could also be a factor to consider. The alphavirus particle has a T=4 icosahedral symmetry, whereas aaLS has a T=1 icosahedral symmetry. Alphaviruses are also larger structures (60-70 nm) compared to aaLS (~20 nm). Therefore, alphaviruses would present 240 copies of the B domain, whereas aaLS, as a 60mer particle, would only present 60 copies. It is possible that the E2-B epitopes must be presented in a particular orientation or that more copies of B domain are necessary to promote a humoral response that is both cross-neutralizing or more potent. Additionally, our study only used Addavax as the adjuvant, which elicits both a cellular and a humoral response. It is also possible that mixing different adjuvants could promote a more robust cross-neutralizing response. Future studies involving B domain immunogens may need to consider different nanoparticles and different adjuvants.

We show that presenting the CHIKV E2-B on the aaLS nanoparticle elicits homotypic neutralizing antibody response against CHIKV, but not MAYV. We also demonstrate that displaying MAYV E2-B elicits non-neutralizing titers. Our study enhances our understanding of antibody-mediated neutralization of heterologous arthritogenic alphaviruses and lays the groundwork for continued refinement of E2-B based immunogens. Future work is needed to map the epitopes that are targeted by E2-B immunization to identify the regions that elicit a homotypic neutralizing response. These regions could then be masked using resurfacing techniques, such as hyperglycosylation, to focus the immune response on epitopes that would better induce broadly neutralizing titers. This investigation would aid the design of future E2-B based immunogens guided by rational protein design approaches, in pursuit of next-generation vaccines against CHIKV, MAYV, and even additional emerging arthritogenic alphaviruses.

Materials and methods

Ethics Statement:

All mouse immunization studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee at the Albert Einstein College of Medicine.

Cell lines and viruses:

Vero cells (CCL-81) were passaged in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 100 U/ml penicillin/streptomycin. CHIKV 181/25 was obtained from Dr. Robert B. Test (University of Texas Medical Branch). MAYV TRVL-4674 was obtained from Dr. Margaret Kielian (Albert Einstein College of Medicine).

Expression and purification of proteins:

To express recombinant CHIKV-B-SpyT (GenBank Accession No. ABD95938.1, residues 495-561) and MAYV-B-SpyT (GenBank Accession No. AF339482.1, residues 495-562), sequences were codon-optimized for E. coli expression, synthesized as a gene fragment, and inserted into a pET21a expression vector. An N-terminal SpyTag was introduced via Q5 mutagenesis and confirmed by Sanger Sequencing. The constructs were transformed into One-Shot^™ BL21 (DE3) E. coli cells. Cells were grown to an OD6₀₀ of 0.6-0.8 at 37°C and induced with .2 mM IPTG overnight at 28°C. Cells were then pelleted and resuspended in 50 mM Tris-HCl pH 8.0, 25% Sucrose, 1 mM EDTA, 0.01% sodium azide, and 10 mM DTT. They were then lysed in 50 mM Tris-HCl pH 8.0, 1% Triton X-100, 1% sodium deoxycholate, 100 mM NaCl, 0.01% sodium azide, and 10 mM DTT. Inclusion bodies were washed 4 times with 50 mM Tris-HCl pH 8.0, 0.5% Triton X-100, 100 mM NaCl, 1 mM EDTA, 0.01% sodium azide, 1 mM DTT, followed by 2 washes with 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.01% sodium azide, and 1 mM DTT. Inclusion bodies were resuspended in TE buffer via sonication, denatured in 7M guanidinium hydrochloride and 40 mM β-mercaptoethanol, and diluted in 2M guanidinium hydrochloride, 50 mM sodium acetate. This mixture was filtered and denatured protein was added dropwise 1ml/hour and oxidatively refolded in 100 mM Tris-Hcl, 400 mM L-arginine, .5 mM oxidized glutathione, 5 mM reduced glutathione, 2 mM EDTA, supplemented with Pierce^™ Protease Inhibitor Tablets (Thermo Scientific^™A32963), according to manufacturer’s instructions. Protein was concentrated using a 3 kDa molecular weight cut-off ultra-centrifugal filter (EMD Millipore) and purified by HiLoad 16/600 Superdex 75 size exclusion chromatography (GE Healthcare).

To express aaLS-SpyC, the Aquifex aeolicus lumazine synthase with a C-terminal SpyCatcher moiety was cloned into a pET-28a vector with an N-terminal His tag for expression in BL21(DE3) cells (50, 51). Cells were grown to an OD₆₀₀ of 0.6–0.8 at 37°C, followed by induction with .2mM IPTG for 18 hours at 28°C. Cells were pelleted and then frozen at −20°C. Following a freeze-thaw, the cells were lysed by sonication in 50 mM Tris pH 8.0, 250 mM NaCl, and purified by Ni-NTA chromatography. To display E2-B on aaLS, SpyTagged E2-B were coupled to aaLS-SpyC overnight at room temperature in a 2:1 molar ratio, followed by size exclusion chromatography with a Sephacryl 16/60 S400 HR size exclusion chromatography column (GE Healthcare).

Negative stain electron microscopy:

400 mesh, carbon only grids were cleaned using a Tergeo-EM Plasma Cleaner (PIE Scientific, USA). Purified LS-CHIKV-B and LS-MAYV-B nanoparticles were adsorbed onto grids for 3 min followed by washing with dH₂O. Samples were negatively stained with 1% uranyl acetate and viewed on a Tecnai 20 transmission electron microscope (ThermoFisher) at 120 kV.

Mouse immunizations and sera collection:

Female BALB/c mice (6-8 week old) were purchased from Charles River Laboratories and housed in vented cages. Mice were immunized with 200 ul of either 15 ug LS-CHIKV-B nanoparticles, LS-MAYV-B or unconjugated LS by intraperitoneal injection using a 1 ml syringe. Addavax adjuvant (InvivoGen) was mixed in a 1:1 ratio in all immunizations as per the manufacturer’s instructions. The immunization schedule included a prime dose followed by two boosts spaced 21 days apart. Blood was collected by submandibular bleeds, 1 week prior to prime immunization. Blood was also collected on day 56 via cardiac puncture. Samples were incubated at room temperature for one hour for the blood to coagulate, centrifuged for 2 min at 13,000 g, and the sera was collected.

ELISA binding assay:

To assess sera and antibody reactivity by ELISA, half-area 96-well high binding plates (Costar) were coated with either CHIKV-B-SpyT, MAYV-B-SpyT, LS-CHIKV-B, or LS-MAYV-B at 100 ng/well overnight at 4°C. Wells were blocked with 1% BSA at room temperature for 2 hours and washed 5 times with PBS-T (PBS pH 7.4, 0.05% Tween-20). Mouse sera or mAbs were diluted in PB-T (PBS pH 7.4, 0.2% BSA, 0.05% Tween) and incubated for 1 hour at room temperature. Plates were washed and HRP-conjugated anti mouse IgG (Sigma) or HRP-conjugated protein A (Life Technologies) was added for mouse sera or mAb, respectively. After 1 hour incubation at room temperature, plates were washed and developed using TMB (ThermoFisher) and absorbance at 450 nm was measured on Synergy H4 Hybrid reader (BioTek). Data were fit to a standard four-parameter logistic equation, the EC₅₀ was determined by the inflection point of the curve.

Neutralization assay:

All viruses were titered to 100-150 focus forming units (FFUs). Mouse sera was serially diluted in DMEM high glucose medium (Gibco), supplemented with 2% FBS (heat-inactivated, Atlanta Biologicals), 1% P/S (Gibco), and 10 mM HEPES (Gibco), and incubated at 37°C, 5% CO2 for 1 hour. The sera mixture was added in triplicate to 96-well plates (Costar) with confluent Vero cells seeded the day before and incubated for 1 hour, followed by addition of 1% (w/v) methylcellulose in MEM. After an 18-hour incubation, cells were fixed with 1% paraformaldehyde, permeabilized by .1% saponin, and stained for infection foci with 5E11 (1 ug/ml). Finally True-Blue reagent (Seracare) was added the plates developed for up to 20 min. Plates were imaged and the foci were quantified using a BioSpot Reader (immunospot). Antibody-dose response curves were analyzed using non-linear regression analysis with a variable slope (GraphPad Software).

Highlights:

• CHIKV E2-B and MAYV E2-B nanoparticle immunogens were designed and characterized.

• A CHIKV E2-B nanoparticle immunogen elicited homotypic neutralizing antibody.

• A MAYV E2-B nanoparticle immunogen elicited non-neutralizing antibody.

Abbreviations:

E2-B

E2 glycoprotein B domain

CHIKV

Chikungunya virus

MAYV

Mayaro virus

aaLS

Aquifex aeolicus lumazine synthase

SpyT

Spytag

SpyC

Spycatcher

CHIKV-B-SpyT

Spytagged E2 glycoprotein B from CHIKV

MAYV-B-SpyT

Spytagged E2 glycoprotein B from MAYV

LS-CHIKV-B

E2 glycoprotein B from CHIKV on lumazine synthase nanoparticles

LS-MAYV-B

E2 glycoprotein B from MAYV on lumazine synthase nanoparticles