A Reciprocal Cross-Reactivity between Monoclonal Antibodies to SARS-CoV-2 Spike Glycoprotein S1 and Human CXCR2—An Implication of a Viral Mimic of Human CXCR2

Some viruses contain mimics of host chemokine receptors that influence host immunity; however, such viral mimics have not yet been reported for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, I focused on C-X-C motif chemokine receptor 2 (CXCR2) as a candidate chemokine receptor exploited by SARS-CoV-2. Similarities between the extracellular domain (ECD) of human CXCR2 and the SARS-CoV-2 spike glycoprotein S1 (CoV2S1) were investigated. Flow cytometric analysis of healthy donor-derived peripheral leukocytes was performed to examine the cross-reactivity between specific monoclonal antibodies against these two proteins. The results showed that CR3022, a monoclonal antibody to the receptor binding domain of CoV2S1, recognized the CXCR2 ECD, and a murine monoclonal antibody to human CXCR2 recognized recombinant CoV2S1. This reciprocal cross-reactivity suggests that CoV2S1 harbors a mimic of the


Introduction
Some viruses reportedly evade host immunity by acquiring mimics of host chemokine receptors during the genetic process of virus-host coevolution [1]. Because chemokine receptors possess multiple chemokines as their own cognate ligands [2], such viral mimics can lead to global alterations in host chemokine-mediated functions, such as leukocyte chemotaxis, angiogenesis, and neuronal maintenance.
The severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia has been associated with the overexpression of ligands that activate the C-X-C motif chemokine receptor 2 (CXCR2) pathway in the nasopharynx [3]. The serum levels of CXCL2 and CXCL8 (interleukin (IL)8), both of which are chemokine ligands for CXCR2, are also significantly elevated in the early phase of infection [4]. Given that the CXCR2-mediated axis plays an important role in controlling peripheral granulocyte migration, extravasation, and endothelial homeostasis [5], prolonged dysregulation of this axis may induce aberrant recruitment of activated granulocytes and embolic pathology, as observed in advanced cases of SARS-CoV-2 infection [6][7][8][9]. As coronaviruses possess an exceptionally high potential for genetic recombination ability relative to other RNA viruses [10,11], SARS-CoV-2 may have implemented host gene fragments into its own genome during evolution [12]. Thus, SARS-CoV-2 may harbor a mimic of human CXCR2 that induces pathological consequences by altering the CXCR2-mediated axis in peripheral granulocytes.
CXCR2 is a chemokine G-protein-coupled receptor (GPCR). Its extracellular domain (ECD) is composed of an N-terminal domain (NTD) and three extracellular loops (I-III) [5]. CXCR2 can recognize not only IL8 but also a broader variety of C-X-C chemokines through discontinuous amino acid sequences in the NTD [13][14][15]. The IL8-binding amino acid sequences of the CXCR2 NTD wrap around the IL8 globular core mainly through hydrophobic interactions to form an IL8-CXCR2-NTD complex. This complex attaches to the CXCR2 extracellular loops by bending and positioning through a disulfide bond between the NTD and extracellular loop III, leading to the activation of intracellular signaling pathways for granulocyte chemotactic migration [5,14,16].
IL8 binding to the CXCR2 NTD induces internalization of the IL8-CXCR2 complex to desensitize the receptor in an IL8 concentration-dependent manner. This binding also contributes to the termination of granulocyte chemotactic migration at the inflammatory site, where the local IL8 concentration is the highest [17,18]. Therefore, aberrant recruitment of activated peripheral granulocytes during SARS-CoV-2 infection may be a secondary event due to the perturbed formation of the CXCR2 chemokine-concentration gradient at the site of viral infection.
To elucidate the accumulation of activated granulocytes observed in SARS-CoV-2 infection, I investigated whether it is possible that SARS-CoV-2 harbors a mimic of the ECD in human CXCR2. To identify the location of such a mimic, the SARS-CoV-2 spike glycoprotein S1 domain (CoV2S1) of the original Wuhan-Hu strain was examined.

Materials and Methods
This study was performed from July to November in 2021.  Table S1 for healthy donor information). Whole blood (2 mL) in a collecting tube coated with EDTA-2K (453533; Sekisui Medical, Tokyo, Japan) was centrifuged at 400× g for 10 min. After plasma removal, the remaining sample was mixed with 5 mL of 1× red blood cell (RBC) lysis buffer diluted from a 10× stock (420301; BioLegend, San Diego, CA, USA) and incubated at room temperature for 10 min. To stop the lysis reaction, 30 mL of 1× phosphate-buffered saline (PBS) was added to the mixture, which was then centrifuged at 400× g for 10 min, and the supernatant was aspirated. RBC lysis was repeated twice. The pellet was resuspended in 1000 µL of flow cytometry buffer (FACS buffer, 1× PBS containing 1% bovine serum albumin and 0.2% sodium azide). Approximately 140 µL aliquots of the suspension containing 10 5 -10 6 leukocytes were transferred into FACS tubes (Falcon 352235; Corning Inc., Corning, NY, USA). The cells were washed with 1 mL of FACS buffer and centrifuged at 400× g for 5 min, and the supernatant was discarded.

Cell Staining and Flow Cytometric Analysis
Cells in the FACS tube were incubated with Fc block, which was diluted with 30 µL of FACS buffer for 10 min on ice. Subsequently, 40 µL of FACS buffer containing a mixture of primary antibodies against murine monoclonal PE/anti-human CXCR2, murine monoclonal FITC/anti-human CXCR1, murine monoclonal BV421/anti-human CD66b, and rabbit monoclonal CR3022 was incubated for 20 min on ice. The same dose of isotype IgG was incubated instead of CR3022 as a negative control. The cells were washed once with FACS buffer, resuspended in FACS buffer containing AF 647/anti-rabbit IgG as the secondary antibody against CR3022, and incubated for 20 min on ice. The cells were then washed twice with FACS buffer, and 1 µL of 7-AAD (559925; BD Biosciences) was added to exclude nonviable cells. The fluorescence intensity of the sample was measured using an FACS Aria II (BD Biosciences). The number of electrical events for the measurements per sample was set at 30,000 in the live-cell population. The data were analyzed using FlowJo (v10.0; FlowJo LLC, Ashland, OR, USA).

Study 2 Flow Cytometric Analysis for Samples with rCoV2S1
rCoV2S1 (273068; Abcam, Cambridge, UK) contains an amino acid sequence that covers Val16 to Arg685 of the original Wuhan-Hu strain. rCoV2S1 is cytometrically detectable by incubation with a fluorochrome-conjugated anti-spike S1 antibody in cells overexpressing angiotensin-converting enzyme 2 (ACE2) [19]. The experimental procedures in Study 1 were repeated by adding rCoV2S1 to a mixture of primary antibodies.
The protocols of studies 1 and 2 were repeated three times on different days to confirm the within-subject repeatability of the 1st subject. To confirm the between-subject repeatability, the protocols of studies 1 and 2 were performed for the 2nd subject.

Results
The same results were obtained in studies 1 and 2 three times for the 1st and 2nd subjects.

Study 1
Monoclonal Anti-SARS-CoV-2 Spike S1 Antibody (CR3022) Recognizes the ECD of Human CXCR2 The similarities between the ECD of human CXCR2 and CoV2S1 were evaluated using multicolor flow cytometric analysis of fresh leukocytes obtained from two healthy donors. Whether CR3022, a monoclonal anti-SARS-CoV-2 spike S1 antibody against non-ACE2-binding sites in the receptor binding domain of CoV2S1 [20], cross-reacts with the ECD of human CXCR2 was examined. Murine monoclonal antibodies against the ECD of human CXCR2 and CXCR1, which is homologous to human CXCR2, were used. A murine monoclonal antibody against CD66b, a specific surface marker of the granulocytic lineage, was also used. The entire leukocyte population of interest was defined using the gating strategy shown in Supplementary Figure S1. The intensity of CR3022 (red) showed a significant rightward shift compared with that of the negative control (blue), indicating that CR3022 recognized granulocytes. However, in the nongranulocyte population (Figure 1a-c, *), the intensity of CR3022 completely overlapped with that of the negative control, indicating that CR3022 did not recognize the nongranulocyte population. The signal intensity of CR3022 in the granulocyte population was positively correlated with the anti-human CXCR2 antibody (Figure 1a). In contrast, the cross-reactivity of CR3022 with the granulocyte population did not correlate with CXCR1 and CD66b (Figure 1b,c). These results suggest that CR3022 recognizes the ECD of human CXCR2 based on the observed specific cross-reactivity of CR3022 against the ECD of human CXCR2.

Study 2
Monoclonal Anti-ECD of the Human CXCR2 Antibody Recognizes rCoV2S1 The cross-reactivity of the anti-human CXCR2 antibody against CoV2S1 was examined by incubating leukocytes with a mixture of primary antibodies and rCoV2S1. rCoV2S1 is not directly detectable using flow cytometry; thus, cross-reactivity was estimated indirectly based on the reduction in the signal intensity of the anti-human CXCR2 antibody in the leukocyte population caused by the competitive effect of rCoV2S1. Different concentrations of rCoV2S1 (0, 0.5, 1.5, and 3 μg per sample) were prepared to examine the dose-dependency of the reduction caused by anti-human CXCR2 antibody binding to rCoV2S1.
A competitive effect of rCoV2S1 on CR3022 in the granulocyte population due to the binding of CR3022 to rCoV2S1 was clearly observed at rCoV2S1 concentrations of 0-3 μg (Figure 2a). The signal intensity of anti-human CXCR2 antibodies in the same granulocyte population decreased with increasing concentrations of rCoV2S1 (Figure 2b). This result indicates that rCoV2S1 competed with the anti-human CXCR2 antibody, suggesting crossreactivity of the anti-human CXCR2 antibody with rCoV2S1. These results, along with those described in Section 3.1, showed that CR3022, a specific monoclonal antibody against CoV2S1 and rCoV2S1, recognized the ECD of human CXCR2. Additionally, a murine monoclonal antibody against the ECD of human CXCR2 recognized rCoV2S1. This reciprocal cross-reactivity suggests that CoV2S1 harbors a mimic of the ECD of human CXCR2.

Study 2 Monoclonal Anti-ECD of the Human CXCR2 Antibody Recognizes rCoV2S1
The cross-reactivity of the anti-human CXCR2 antibody against CoV2S1 was examined by incubating leukocytes with a mixture of primary antibodies and rCoV2S1. rCoV2S1 is not directly detectable using flow cytometry; thus, cross-reactivity was estimated indirectly based on the reduction in the signal intensity of the anti-human CXCR2 antibody in the leukocyte population caused by the competitive effect of rCoV2S1. Different concentrations of rCoV2S1 (0, 0.5, 1.5, and 3 µg per sample) were prepared to examine the dose-dependency of the reduction caused by anti-human CXCR2 antibody binding to rCoV2S1.
A competitive effect of rCoV2S1 on CR3022 in the granulocyte population due to the binding of CR3022 to rCoV2S1 was clearly observed at rCoV2S1 concentrations of 0-3 µg (Figure 2a). The signal intensity of anti-human CXCR2 antibodies in the same granulocyte population decreased with increasing concentrations of rCoV2S1 (Figure 2b). This result indicates that rCoV2S1 competed with the anti-human CXCR2 antibody, suggesting cross-reactivity of the anti-human CXCR2 antibody with rCoV2S1. These results, along with those described in Section 3.1, showed that CR3022, a specific monoclonal antibody against CoV2S1 and rCoV2S1, recognized the ECD of human CXCR2. Additionally, a murine monoclonal antibody against the ECD of human CXCR2 recognized rCoV2S1. This reciprocal cross-reactivity suggests that CoV2S1 harbors a mimic of the ECD of human CXCR2.

Discussion
This study showed that CoV2S1 may harbor a viral mimic of human CXCR2, which is the IL8 receptor. Increased levels of IL8 reportedly correlate with the severity of SARS-CoV-2 infection [21,22]. Viruses and viral proteins can upregulate IL8 transcription, which is translated and secreted by endothelial cells, epithelial cells, and peripheral leukocytes [23]. IL8 in the bloodstream is captured by upregulated heparan sulfate on endothelial cells at the infection site to form an IL8 concentration gradient and attract peripheral granulocytes to undergo chemotactic migration and extravasation [24]. The chemotactic migration of peripheral granulocytes is regulated by CXCR2 [25]. Rose et al. [17] showed that CXCR2 signaling driving granulocyte migration is counter-regulated by receptor

Discussion
This study showed that CoV2S1 may harbor a viral mimic of human CXCR2, which is the IL8 receptor. Increased levels of IL8 reportedly correlate with the severity of SARS-CoV-2 infection [21,22]. Viruses and viral proteins can upregulate IL8 transcription, which is translated and secreted by endothelial cells, epithelial cells, and peripheral leukocytes [23]. IL8 in the bloodstream is captured by upregulated heparan sulfate on endothelial cells at the infection site to form an IL8 concentration gradient and attract peripheral granulocytes to undergo chemotactic migration and extravasation [24]. The chemotactic migration of peripheral granulocytes is regulated by CXCR2 [25]. Rose et al. [17] showed that CXCR2 signaling driving granulocyte migration is counterregulated by receptor desensitization following receptor-IL8 complex internalization in an IL8 concentration-dependent manner. They suggested that receptor endocytosis acts as a terminal stop signal when granulocytes reach the infection site, where the IL8 concentration is the highest.
Viral mimics of human GPCR chemokine receptors are exploited by some large DNA viruses to escape host immunity [1]. One important example is the viral mimic of human C-X 3 -C and C-C chemokine receptors, which is referred to as US28 and encoded by human cytomegalovirus. US28 can bind to a broad variety of chemokines, including CX 3 CL1, and ligands for C-C chemokine receptor 5 (CCR5), such as CCL5 (also known as RANTES) [26]. Human CCR5 plays an important role in host defense mechanisms through T cell migration and adaptive immune functions [27,28]. Bodaghi et al. [29] reported that US28 sequesters C-C chemokines such as RANTES in virus-infected cells, and that the sequestration of host RANTES by the viral mimic leads to a decrease in the extracellular RANTES concentration. Additionally, Billstrom et al. [30] demonstrated that RANTES concentration is decreased by internalization of the membrane-bound RANTES-US28 complex in virus-infected cells and suggested that CCR5-mediated chemotactic activity is perturbed in host cells. The structural similarities between human proteins and SARS-CoV-2 have been studied using computational approaches [31,32]. I hypothesized that a viral mimic of human CXCR2 is embedded in CoV2S1. The flowcytometric analysis using healthy donor-derived leukocytes revealed reciprocal cross-reactivity between specific monoclonal antibodies against the ECD of human CXCR2 and rCoV2S1 (Figures 1a and 2b), suggesting similarities between the ECD of human CXCR2 and CoV2S1.
CR3022, a monoclonal antibody to the receptor binding domain of CoV2S1 [20], recognizes the ECD of human CXCR2 (Figure 1a). A monoclonal anti-NTD of the human CXCR2 antibody (abN48) reportedly induces receptor internalization after binding to the NTD of human CXCR2 [15]. As CR3022 cross-reacts with the ECD of CXCR2, human antibodies produced during CoV2 infection may recognize this domain, leading to antibody-dependent desensitization. Moreover, anti-CoV2S1 antibodies that recognize the human CXCR2 ECD may cause antibody-dependent cytotoxicity. A previous study suggested that CoV2S1 activates autoreactive T cells by cross-reacting with the T cell receptor (TCR) [33]. Cheng et al. [34] demonstrated that this TCR cross-reactivity is induced through a similar motif as bacterial superantigen, which is located adjacent to the C-terminus of CoV2S1. In the present study, CoV2S1 was found to be similar to human CXCR2. This finding suggests that molecular mimicry, which is defined as a viral mimic of host protein [35], contributes to the TCR cross-reactivity of CoV2S1. In some viruses, molecular mimicry in a viral protein reportedly mediates autoimmune reactions related to viral infection and vaccination [35,36]. It is of clinical significance to elucidate whether the human CXCR2 mimic leads to autoimmune pathologies, as observed in the case of SARS-CoV-2 [37,38].
The flowcytometric analysis showed that a murine monoclonal anti-ECD of human CXCR2 antibody recognized rCoV2S1 (Figure 2b), indicating that CoV2S1 harbors a mimic of the CXCR2 ECD. Notably, epitopes of murine monoclonal antibodies to human CXCR2 closely overlap with the IL8-binding sites of the CXCR2 NTD [15,[39][40][41]. Thus, CoV2S1 may attract IL8 through the mimic of the CXCR2 ECD and induce its unavailability around granulocytes.
Because membrane-bound IL8 in endothelial cells plays a major role in forming a local IL8 concentration gradient [24], its unavailability may affect CXCR2-mediated chemotactic granulocyte migration. A previous study reported that CXCR2 signaling is counter-regulated through receptor desensitization in an IL8-concentration-dependent manner [17]. This counter-regulation is important for terminating the chemotactic migration of granulocytes at the infection site, where the local IL8 concentration is the highest [17]. If the mimic of CXCR2 in CoV2S1 attracts IL8 and contributes to its unavailability, the local concentration gradient of IL8 at the infection site may be perturbed.
This study had some limitations. First, only the rCoV2S1 of the Wuhan-Hu strain was used in this study. Second, the binding affinity of rCoV2S1 to IL8 and chemotactic ability were not measured. Third, structural analyses were not performed. Finally, a small number of healthy donors was examined.

Conclusions
A reciprocal cross-reactivity occurs between monoclonal antibodies to SARS-CoV-2 spike glycoprotein S1 and human CXCR2. Further studies of the viral characteristics may contribute to the development of new therapeutic compounds.
Supplementary Materials: The following information can be downloaded at https://www.mdpi. com/article/10.3390/covid2050042/s1: Figure S1: Gating strategy to define the leukocyte population of interest; Figure S2: Representative results of flow cytometric analysis for samples without recombinant CoV2S1 (rCoV2S1) presented as a dot plot; Table S1: Healthy donor information; Table S2: Overview of reagents used for flow cytometric analysis. Ref. [42] is cited in Supplementary Materials.