Vaccines | Free Full-Text | Antigen-Heterologous Vaccination Regimen Triggers Alternate Antibody Targeting in SARS-CoV-2-DNA-Vaccinated Mice
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1. Introduction
In December 2019, the SARS-CoV-2 Index strain emerged in Wuhan, China. Since then, the virus has evolved substantially, acquiring consecutively more amino acid changes in the major surface glycoprotein, the spike protein, which is the primary target for vaccine- and infection-induced neutralizing antibodies. Following the introduction of SARS-CoV-2 into Europe, the ancestral strain with the spike D614G substitution became dominant, followed by the emergence of a myriad of variants that include Alpha, Beta, Gamma, Delta, and several Omicron variants and recombinants. Immune pressure likely contributed to virus evolution that altered antigenicity, leading to escape of vaccine-induced immunity, natural immunity, and hybrid immunity. The pandemic has seen to an unprecedented and ongoing race to develop novel and updated vaccines that generate effective broadly neutralizing antibodies that are able to protect from severe disease against an everchanging virus.
SARS-CoV-2 vaccine development focused primarily on the spike protein due to its essential functions in virus entry, which makes it an ideal target for protective antibody responses. The spike protein is a trimer, and each monomer is built up by two non-covalently associated subunits S1 and S2. SARS-CoV-2 enters host cells by binding of the viral receptor-binding motif (RBM), located in the receptor-binding domain (RBD) of the spike protein, to the host cell receptor, angiotensin-converting enzyme 2 (ACE2). The RBD embedded in the S1 subunit shifts between a shielded ‘down’ conformation and an “open” receptor-binding conformation. Pre-cleavage at the S1/S2 cleavage site at the junction between S1 and S2 by furin or other cellular proteases promotes the RBD ‘up’ conformation, priming ACE2 binding [1,2,3]. In the ‘up’ position, the RBD can bind to the ACE2 receptor, leading to conformational changes in the spike and exposure of the S2’ cleavage site. To facilitate membrane fusion, S2’ is cleaved either by cell surface proteases such as TMPRSS2 or furin, or by cathepsin in the endosomal pathway, leading to a destabilization of the pre-fusion trimer and detachment of S1. Mediated by the S2 domain of the spike protein, the viral envelope fuses with the cell membrane through the insertion of the fusion peptide and the formation of a six-helix bundle by heptad repeat 1 and 2, pulling the two membranes together and resulting in fusion.
Rational vaccine design or monoclonal antibody therapy relies on knowledge about B-cell epitopes generating effective broadly neutralizing antibodies. As a host receptor engaging viral protein domain, the RBD is the primary target for neutralizing antibodies and a clear target for intervention strategies [4,5,6]. RBD-specific antibodies directed against ACE2 binding epitopes are classified as class 1 or 2 antibodies [7]. Class 1 antibodies bind only RBD ‘up’ epitopes and class 2 antibodies bind both RBD ‘up’ and ‘down’ epitopes. Conversely, RBD-specific antibodies of classes 3 and 4 are non-ACE2 blocking antibodies that bind ‘up’ and ‘down’ (class 3) or only ‘up’ (class 4). Multiple factors contribute to an enhanced frequency of mutations in the RBD, thereby influencing vaccine immunogenicity and monoclonal antibody recognition [8]. The N-terminal domain (NTD) upstream of the RBD in the S1 subunit, similarly known to elicit potent neutralizing antibodies [9,10,11], is also a region of highly mutated residues primarily centered around a supersite located in the N3 loop (residues 141–156) [9,11]. Deletions compromising neutralizing antibody efficiency are known to exclusively affect the S1 regions outside the RBD [12,13]. The key to effective broadly neutralizing antibodies is therefore to target epitopes that are conserved across variants. Protein function and structural constraints are believed to have a strong impact on the likeliness that a gene sequence will mutate [14]. Targeting conformationally “locked” regions could be crucial for effective neutralization.
Statens Serum Institut, Denmark, developed two candidate SARS-CoV-2 vaccines that encode either the unmodified spike protein of the Index strain or the spike protein of the Beta VOC (PANGO lineage B.1.351) [15,16]. Both vaccines are immunogenic in rabbits and mice, producing robust binding and neutralizing antibody responses. In studies conducted by Lassauniere et al. [15], where CB6F1 mice received a homologous regimen of three immunizations of either of these vaccines or a combination of both, the authors observed an increased breadth of neutralization responses when the vaccines were mixed. In this study, we aim to determine if this increased breadth is the result of different epitopes being targeted. To this end, we study serum from mice that received homologous or heterologous vaccine regimens. We use microarray technology with circular constrained peptides of the entire Index strain spike ectodomain to identify targeted B-cell epitopes.
2. Materials and Methods
2.1. Study Population
The DNA vaccines, vaccination strategies, and animal experiments are described in detail elsewhere [15,16]. In brief, eight-week-old female CB6F1 mice (Envigo, Horst, The Netherlands) were immunized with 50 µg of a DNA plasmid vector, encoding either the complete and unaltered spike protein of the SARS-CoV-2 Index strain (pNTC-Spike), derived from the Wuhan-Hu-1 strain (MN908947), or of the Beta (B.1.351) strain (pNTC-Spike.351). Nature Technology Corporation (Lincoln, NE, USA) subcloned human codon-optimized SARS-CoV-2 spike sequences synthesized by GeneArt (Thermo Fisher Scientific, Dreieich, Germany) into the NTC8685-eRNA41H vector backbone and produced the vaccines in NTC4862 E. coli cells (DH5α attλ::P5/6 6/6-RNA-IN-SacV, Cmr) using their antibiotic-free RNA-OUT selection procedure. The vaccine stocks were provided in a concentration of 10 mg/mL in phosphate-buffered saline (PBS) [15]. Immunizations were performed in weeks 0, 2, and 4. Groups of five mice were immunized intradermally with needle injection at the base of the tail according to three different vaccine regimens (Figure 1). The first group received only the pNTC-Spike vaccine at weeks 0, 2, and 4, while the second group received only the pNTC-Spike.351 vaccine at the same time points. These antigen-homologous groups are hereafter referred to as the Index and Beta groups, respectively. The third group received a combination of the two vaccines as the pNTC-Spike vaccine administered at weeks 0 and 2, and the pNTC-Spike.351 vaccine administered at week 4. The latter antigen-heterologous group is hereafter referred to as the mixed group. Blood samples were taken two weeks after the final vaccination. The animals were housed in facilities at Statens Serum Institut, Copenhagen, Denmark. All procedures were supervised by the laboratory animal veterinarians and complied with the Danish legislation, which is based on EU Directive 2010/63/EU on the protection of animals used for scientific purposes. The experiments received ethical approval from The Animal Experimentation Council, the National Competent Authority within this field (approval number 2017-15-0201-01322).
2.2. Microarray Method
Epitope mapping was performed on pooled sera from each group of mice, according to vaccination regimen, using a custom-made microarray with 10′mer cyclic overlapping peptides spanning the full Index strain spike protein and 35 additional mutation sequences representing key mutations throughout the spike protein. To perform the epitope mapping, the peptide microarray obtained from PEPperPRINT (Heidelberg, Germany) was adjusted to room temperature and assembled in accordance with the manufacturer’s instructions. Subsequently, a washing buffer comprising Dulbecco’s phosphate-buffered saline (DPBS) with 0.05% Tween20, at a pH of 7.4, was added to each subarray. After incubation at room temperature for 15 min, the washing buffer was removed using a pipette and each subarray was blocked in 400 µL of Rockland Blocking Buffer (MB-070, Rockland Immunochemicals, Pottstown, PA, USA) at room temperature for 30 min. To evaluate non-specific reactions, we then pre-treated the peptide array with a secondary antibody/streptavidin solution diluted in staining buffer (DPBS with 0.005% Tween20 and 10% blocking buffer, pH 7.4) as described below and scanned. The microarray was then reinserted into the PEPperPRINT microarray cassette and incubated with staining buffer for 15 min to equilibrate. The staining buffer was then removed with a pipette. Diluted in staining buffer, 400 µL of each serum pool was pipetted into separate subarrays, and the microarray was left overnight at 2 to 8 °C on an orbital shaker at 140 rpm and in the dark. The following day, sample dilutions were removed by pipetting, followed by a two-times wash with 400 µL wash buffer for 10 s on an orbital shaker at 140 rpm, protected from light. Moreover, 400 µL of biotinylated goat anti-mouse IgG F(c) (cat. #31805, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was added to each subarray. This secondary antibody was diluted 1:500 in staining buffer and the microarray was subsequently incubated for 45 min at room temperature. The secondary antibody was removed by pipetting, followed by 2× wash with 400 µL wash buffer as previously described, and 400 µL streptavidin Alexa Fluor 647 conjugate (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:750 in staining buffer was added, and the peptide microarray was incubated at room temperature for 45 min. Each subarray was then washed two times with 400 µL wash buffer as previously described, and the entire array slide was submerged two times into dipping buffer (1 mM Tris buffer, pH 7.4), followed by a 1 min centrifugation at 1000 rpm to completely remove all dipping buffer. Microarray was scanned on a microarray scanner (SureScan from Agilent, Santa Clara, CA, USA) and analyzed using MAPIX Analyzer Software v. 9.1.0. The microarray was then re-inserted into the microarray cassette and equilibrated as previously described. After removal of the staining buffer using a pipette, the microarray was finally incubated with an anti-HA PEPperCHIP® control antibody from PEPperPRINT diluted 1:2000 in staining buffer and incubated at room temperature for 45 min. Subarrays were washed twice with 400 µL wash buffer, and, after disassembly, the entire array slide was dipped twice into dipping buffer, centrifuged, scanned, and analyzed as previously described. All RT incubations were carried out in the dark using an orbital shaker at 140 rpm.
2.3. Statistical Analysis and Calculations
Fluorescence (relative fluorescence units, RFU) was determined as fluorescence intensity at 635 nm with a subtraction of background determined at 635 nm from an area manually set within each subarray in MAPIX. Microarray fluorescence data were imported into Excel. Fluorescence was calculated as the mean of Δ-fluorescence from two identical peptides and normalized according to the mean of a positive HA control (n = 48) included on each subarray. Targeted epitopes were defined as the fluorescence intensities above the cut off. The cut off was defined as
where is the mean of n negative control determinations (n = 96) included on each subarray, SD is the standard deviation, and f is the standard deviation multiplier with a confidence interval of 99.0% as provided by Frey et al. [17].
4. Discussion
This study applied peptide microarray technology to map B-cell epitopes targeted by SARS-CoV-2 spike-specific antibodies induced by three different regimens of DNA vaccines to aid rational vaccine development in the future. It is a follow-up study of the development and pre-clinical immunogenicity studies of the SARS-CoV-2 DNA vaccines described in Lassauniere et al. [15,16]. These studies found an enhanced breadth in heterologous-vaccinated mice that received a combination of a DNA plasmid encoding the full Index strain spike protein sequence and an identical plasmid vector encoding the full Beta VOC spike protein. Being one of the first variants bearing immune escape capabilities such as the E484K and K417N/T substitutions, the Beta VOC represented a highly antibody-resistant variant compared to the ancestral Index strain. To determine if the observed improved ability to cross-neutralize different VOCs in the heterologous-vaccinated mice is the result of different antibody targeting, we established B-cell epitope profiles from polyclonal serum from groups of mice representing each vaccination regimen. The animals following the antigen-heterologous vaccination regimen, combining both of the two used DNA vaccine constructs leading to a slightly improved cross-neutralizing capacity, showed the most distinct binding profile.
The ideal vaccine should induce a broad neutralizing antibody response, conferring protection against present and emerging virus strains. Our results indicate that using an antigen-homologous vaccine strategy directs the immune system into a focused strain-specific response. In contrast, applying an antigen-heterologous vaccination regime resulted in broader cross-neutralization [15]. We observed an antigen-heterologous vaccine regimen antibody profile targeting common regions in the NTD, RBD, and SD3 equally targeted by the antigen homologous regimens, with the addition, however, of a high level of antibody binding to an SD2 region spanning residues 616–627 and an HR2 region spanning residues 1160–1169. This group displayed a more focused antibody response with markedly lower omnipresent binding, and lower binding to mutational spots, although with a potent antibody response at the targeted regions. This diverging antibody binding profile was associated with a slightly improved ability to cross-neutralize variants (Supplementary Figure S1).
A SARS-CoV-2 vaccine design using a native spike protein, as used for this study cohort, may induce a different RBD-binding antibody repertoire compared to that induced by a pre-fusion-stabilized spike protein. The pre-fusion-stabilized S-2P spike used in the mRNA-1273 vaccine [25] is believed to adopt a more open conformation with one or more monomers in the ‘up’ state in comparison with the native spike protein [3,26,27]. With a maximum RFU of 8529 (Beta-vaccinated group), none of the vaccinated groups tend to focus their antibody repertoire against the RBD. The antibodies in all three groups were mainly targeting class 4 and class 1 epitopes, both of which target RBD ‘up’ conformations. These epitopes are not accessible in the closed formation. Surprisingly, all three vaccinated groups targeted an epitope containing the glycan-shielded N343 with considerable intensity (Figure 2). The N343 RBD glycan is, besides shielding, suspected to interact with the ACE2 backbone and stabilize ACE2-RBD binding [4,28]. The mAb S309 (classified as a class 3 antibody) does not bind this exact epitope; however, N343 is sandwiched between the mAb’s binding sites, and the binding of this antibody possibly compromises ACE2-RBD binding stability, thereby inhibiting infectivity [28]. The observed antibody binding to the peptide containing N343 in all the groups of vaccinated mice may behave similarly. Despite the usage of a native spike, supposedly adopting a more ‘closed’ spike conformation compared to the pre-fusion-stabilized spike, the main antibody targeting is directed against epitopes only accessible in the ‘open’ conformation. Antibodies directed against the down state are regarded as being more potent, and, because of a more conserved area of the exposed surface of the closed conformation, they are also believed to have a higher level of cross-neutralization [10,27]. The observed weak antibody binding to closed conformation epitopes, combined with overall moderate RBD targeting, point towards effective antibody repertoires targeting regions outside the receptor-binding domain.
As opposed to moderate RBD antibody targeting, we observed high antibody binding just outside the RBD, in the SD1 region, most noticeably observed in the Index-vaccinated group. The non-mutated sequence of peptide 579 displayed high antibody binding even at a 1:100 dilution compared to the remaining targeted peptides in this group, indicating a higher proportion of high-affinity antibodies targeting this peptide (Supplementary Figure S2). For serum diluted 1:20, this peptide had an RFU of 12,733. However, a single substitution, A570D, significantly increased the antibody binding to the neighboring mutated peptide spanning residues 569–578 (IDDTTDAVRD) to an RFU of 36,706 (3-fold). The A570D substitution has been reported to increase viral entry into target cells [29]. The targeted peptide is overlapping with a known broadly neutralizing epitope in the SD1 [8,30,31,32,33] that presumably functions as a hinge of the open/closed state of RBD. Vaccination with the native spike, used for this study cohort [16], is known to elicit antibodies targeting this region [34]. In contrast, this SD1 region is occluded when using a vaccine design with a pre-fusion-stabilized spike protein. Being immunized with the Index strain without the A570D substitution, it is surprising that the elicited antibodies bind the peptide with the substitution with 3-fold greater intensity. However, despite high-resolution amino acid coverage on the microarray, an amino acid shift of two amino acids could have some limitations. Thus, an exact comparison of an identical peptide frame differentiating only in the substitution was not possible. The enhanced antibody fit could therefore partly be explained by the slight frame shift varying two terminal amino acids. It is unlikely, however, to be the sole reason for the increased signal as the terminal amino acids are positioned close to the linker attaching the 10′mer circular peptide to the glass plate; hence, the minimal epitope is likely unaffected by these amino acid changes.
The S1 subdomain, and in particular the RBD, has been the primary target for research within neutralizing antibodies. However, the more conserved regions of S1 and S2 are speculated to have a higher contribution to cross-reactivity [35,36]. Residues 616–627 of the S2 region are parts of the 630 loop stretching from residue 617 to residue 644 [37]. The 630 loop is, along with the fusion peptide, believed to play a critical role in stabilizing the spike protein and serves a function in retaining the RBD in the ‘down’ mode [37,38,39]. Targeting this region with antibodies could possibly disturb the structural rearrangements necessary for opening up the RBD, leaving it in a locked ‘down’ position. The stability of the 630 loop is affected by the neighboring H655Y substitution; however, all three vaccinated groups share enhanced binding to the mutated form (Figure 3) [37]. HR2, which interacts with HR1 in the formation of the 6-helix bundle during membrane fusion [39,40], similarly reveals a broadly neutralizing epitope that becomes accessible after ACE2 binding [40,41,42]. Heavily targeting of this epitope, as observed in the mixed-vaccinated group, is likely to interfere with membrane fusion. Binding to HR2 has been reported to correlate with disease severity of SARS-CoV-2 [43].
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