Staphylococcus aureus has a large set of finely-tuned virulence-associated genes that has endowed this bacterium with highly adaptive and versatile strategies to survive in beneficial as well as in hostile environments (1–6). Two major classes of virulence factors belong to Cell Wall-Anchored (CWA) adhesins (2) and a group of secreted proteins called Secretable Expanded Repertoire Adhesive Molecules (SERAMs) (7). The most represented activity in both groups of virulence factors is their ability to bind fibrinogen (Fg), a host blood glycoprotein. For instance, SERAMs coagulase (Coa), von Willebrand factor binding protein (vWbp), Extracellular fibrinogen-binding protein (Efb), Extracellular adhesive protein (Eap), Extracellular matrix binding protein (Emp) all bind Fg (8). Amongst them, prothrombin-activating proteins Coa and vWbp engage Fg independently from prothrombin (9–12). The Fg binding activity of SERAMs, especially well studied for Coa, vWbp and Efb, is mainly located in unordered regions of these proteins (8, 9, 11).

Fg is a large, fibrous plasma glycoprotein with three pairs of polypeptide chains, designated Aα, Bβ and γ. During haemostasis and clot formation, it self-assembles into an insoluble fibrous gel upon conversion to fibrin (13, 14). The role of Fg in bacterial infection has been mainly regarded as protective “haemostatic containment”, owing to the ability of Fg/fibrin to entrap bacteria, reducing their proliferation and dissemination, and fibrin-mediated recruitment of immune cells to clear invading bacteria (15–17). As mentioned so far, S. aureus harnesses an impressive array of virulence factors that can interact with Fg. Multiple recent evidence has demonstrated that the interaction with Fg may drive different host responses based on the tissual context (8). In peritonitis mouse infection models, binding of Fg is fundamental to elicit an antibacterial response and contain infection (18–21). However, the picture is completely reversed in bloodstream infections, where Fg instead promotes spreading of S. aureus (22). Therefore, understanding the interactions between S. aureus virulence factors and Fg is crucial to understand how new therapeutic opportunities should be designed against the multiple antibiotic-resistant strains of this pathogen.

Efb and Coa are the best characterized SERAM proteins. The Efb::Fg interaction is located in the N-terminal half of Efb (23), whereas Coa can bind Fg all throughout its length, with the more potent interactions located in the C-terminal domain (9, 11, 24). Furthermore, both Coa and Efb mediate the formation of a Fg/fibrin shield around staphylococcal cells, thereby protecting bacteria from host immune responses (23, 25–27). Coa also mediates allosteric activation of prothrombin through its N-terminal D1D2 domains promoting fibrin polymerization (28–30). In respect to therapeutic potential of Coa- and Efb-targeted antibodies, polyclonal rabbit sera raised against Coa (10, 25) or Efb-specific antibodies derived from patients with S. aureus infection (31) could reduce Fg binding in vitro and protected mice from lethal S. aureus sepsis.

Figure 1A shows the domain organization of Coa protein. The full length protein can be divided into N-terminal and C-terminal halves. The N-terminal half of the protein contains D1D2 domains. The C–terminal part of Coa can be divided into two portions: the repeat region of Coa, located at the most C-terminal of the protein, and a linker that connects the D1D2 domain and the repetitive region of Coa. As mentioned earlier, both N-terminal and C-terminal halves can bind Fg but the more potent binding region is located at the C-terminal half. Different recombinant constructs used in the study are also depicted in Figure 1A . CoaF contains the linker region and harbours a first, slightly divergent and longer repeat termed CoaR0 ( Figures 1A, C ). The remaining repeats are covered in recombinant construct CoaR, which harbours relatively conserved tandem repeats I-V of 27-residue each. CoaR, together with CoaF, constitutes the C-terminal domain of Coa, expressed as recombinant protein named CoaC. The number of repeats present in Coa protein varies from 1 to 9 copies depending on the S. aureus strain, 5 or more being the most common amount (33). These repeats are shorter than CoaR0, which is the longest repeat able to bind Fg and is present in CoaF, spanning residues from 474 to 505. Therefore, Coa can be divided into several functional domains that have different affinities for Fg (9, 11).

As mentioned, S. aureus Efb also interacts with Fg and belongs to SERAMs (9, 23, 27, 34). It is reported to inhibit complement activation by engaging C3b (35–38), block platelet aggregation and their interaction with leukocytes (39, 40) and interact with immune cells blocking cellular-mediated immunity (23, 27, 41, 42). Furthermore, Efb can also bind to Complement Receptor 2 on B cells, further tackling adaptive responses of the host (43). The Fg-binding activity is located at the N-terminal of Efb and has been mapped to relatively long amino acid stretches termed EfbO and EfbA ( Figure 1B ). The affinity of EfbO for Fg is 200 times higher than that of EfbA, indicating that EfbO is the primary Fg binding site in Efb (23). Coa repeats and Efb N-terminal share homology in their Fg binding mechanisms and likely target the same or overlapping sites in Fg, given that EfbO, CoaR0 and CoaRI peptides are able to inhibit Fg binding of both EfbN and CoaC (9).

The possibility to interfere with Fg binding is thus crucial to understand and block one S. aureus pathogenic mechanism. Here, antibodies are not only a tool for blocking Coa and Efb interaction with Fg for research but also potential therapeutic molecules. We used antibody phage display to select several fully human, sequence-defined antibodies against CoaC and characterized them in vitro. We found that the anti-CoaC antibodies showed crossreactivity with Efb and were able to discriminate between CoaR0 and CoaRI peptides. In addition, we identified four antibodies that were able to inhibit Coa and Efb Fg-binding.

S. aureus has for a long time been a critical global healthcare threat owing to increase in spread and virulence of antibiotic-resistant strains (47, 48). The discovery and introduction of radically new antibiotic classes into the market has been lagging for two decades (49). Therefore, new approaches to tackle S. aureus infections are of foremost interest. A first crucial aspect in S. aureus pathogenesis is the attachment to host tissues. Among these interactions, Fg seems to play a dominant role (8). Indeed, S. aureus has evolved a vast arsenal of proteins to interact with this soluble plasma protein: first, Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are cell wall-bound proteins primarily involved in extracellular matrix components binding to secure bacterial adhesion to host tissues (2). A second class is collectively referred to as SERAMs proteins, which are secreted and still interact with several extracellular matrix molecules displaying also an immune evasion and dissemination function (7). Among MSCRAMMs, ClfA and ClfB, FnbpA and FnbpB and SdrE/Bbp bind different segments of Fg molecule. Fg-binding activity is also prominent in SERAMs, where unordered regions of Coa, vWbp, Efb, Eap and Emp present Fg binding as a common feature. The comprehensive picture of these proteins seems not yet fully disclosed, as the recent initial characterization of vhp shows (50).

Also the role of Fg is at the crossroad between its well described role in haemostasis and its importance in mediating immune responses (14, 51). Much preclinical evidence also showed that mutated versions of Fg cannot efficiently clear infections mediated by S. aureus thus compromising immune response towards the pathogen (19–21, 52). Furthermore, preclinical studies together with vaccine candidates have shown that ClfA-mediated Fg interaction is a viable alternative for possible therapeutic strategies (53–55). Therefore, all these presented interactions show how intricate the interplay between S. aureus and Fg is and thus its extremely high potential as a therapeutic target for alternative treatment strategies.

To this end, anti-Coa antibodies have been generated by antibody phage display, using human naïve libraries (44), providing sequence-defined mAbs: 11 mAbs with unique sequences recognized CoaF and CoaR0 fragments upon panning against CoaC (FBE5 mAbs) and 2 mAbs were directed against CoaR (LIG40 mAbs). Of note, to obtain the latter antibodies, 4 times our usual number of clones had to be screened, to obtain only 10 hits and in the end 2 unique clones. In comparison, selection of FBE 5 mAbs had a higher hit rate (45 positive hits/95 colonies screened). This may be a consequence of the unstructured organization of CoaR (11, 30), even if selection was performed on ELISA plates which should “immobilize” antigens in a fixed position.

Coagulase protein and expecially its gene have been extensively harnessed over the years to classify S. aureus strains based on the antigenicity of the protein or through restriction fragment length polymorphism analysis, respectively (56, 57). The former classification allowed the identification of twelve different serotypes of Coa and the N- terminal D1D2 domains have been found to be major contributor to this difference in antigenicity, despite conserved prothrombin binding and activation (33). On the other hand, the C-terminal repeats are highly conserved with an average of 92% homology and are only polymorphic for the number of tandem repeats present in each strain (58, 59). Since both FBE5 and LIG40 antibodies target the repeated region of Coa ( Figures 6 , 7 ), they are expected to target most if not all Coa proteins belonging to different Coa serotypes.Since Coa shares sequence and functional homology to Efb, cross-recognition of the generated antibodies was investigated. All FBE5 antibodies showed binding to both Coa and Efb fragments ( Figure 3 , Supplementary Figures 3, 4 ). In particular, all FBE5 mAbs bound to different extents CoaF, CoaR0, EfbN, EfbA and EfbO fragments, except FBE5-C8 and FBE5-E5 that showed low-affinity binding to Coa and substantially no binding to Efb. Among them, FBE5-F11 had the highest apparent affinity ( Table 1 ) and showed the greatest inhibitory effect on Fg binding to CoaF, CoaR0, EfbN, EfbA and EfbO constructs ( Figure 5 ). FBE5-A12, FBE5-D10 and FBE5-F9 could also efficiently inhibit all Efb and Coa fragments tested, although to a lower extent than FBE5-F11 ( Figure 5 ). This activity correlated with their apparent affinities determined in ELISA ( Table 1 ). FBE5-A5, FBE5-A6, FBE5-B9, FBE5-C1 and FBE5-D9, instead, showed inhibition of CoaF, CoaR0 and EfbA ( Supplementary Figure 5 ). Essentially, these antibodies were able to inhibit CoaR0-mediated Fg binding, since EfbA is unlikely to be the most functionally relevant Fg-binding region in Efb, given its low affinity for Fg (1µM) (23). Finally, FBE5-C1, FBE5-C8 and FBE5-E5 displayed only minor blocking activity on EfbA, matching the apparent affinity measurement of these antibodies.

Concerning LIG40 antibodies raised against CoaR, they did not show any cross-reaction to Efb. Surprisingly, both of them could not inhibit Fg-binding of both CoaC and CoaR ( Supplementary Figure 6 ), despite their high affinity binding to functional Fg-binding sequences CoaR0 and CoaRI ( Figure 4 , Table 2 ). When binding of LIG40-A11 and LIG40-D8 to CoaC and CoaR was challenged with synthetic CoaR0 and CoaRI peptides ( Figure 7 ), high concentrations of peptides were necessary for competition. This could indicate, on one side, that the epitope is not properly represented in the peptide. On the other hand, it is very plausible that given the repetitive nature of CoaR, multiple binding sites for these antibodies are available within the same construct, thus higher concentrations of peptide are needed in order to exert a competitive effect. It is also highly unlikely that a single mAb’s paratope could span the entire linear 27 amino acid-long Fg binding repeat. These considerations hint that the inability of these antibodies to block Fg binding might be due to the repetitive nature of CoaR.

Furthermore, CoaR0 peptide could inhibit all FBE5 and, surprisingly, LIG40-D8 mAbs binding to CoaC, instead had no effect on LIG40-A11 binding to both CoaR and CoaC. Conversely, CoaRI peptide did inhibit LIG40-A11 binding, leaving unaffected the binding of all FBE5 and LIG40-D8 mAbs. The latter antibody showed indeed binding, albeit weaker, to CoaF and CoaR0 constructs ( Figure 4 , Table 2 ), even though its selection was performed on CoaR, which does not contain CoaR0 repeat. These results together suggest that these antibodies are targeting different epitopes in CoaR and that Fg binding repeats may assume similar conformations, however representing two functionally distinct epitopes in Coa. It could be speculated that LIG40-D8 is targeting conserved residues present both in CoaR0 and CoaRs repeats. Their role needs further clarification since it is clear that the binding site of CoaR0 and CoaRI in the Fg molecule is similar or overlapping. Both peptides are indeed able to inhibit Coa binding to Fg (9). Recent evidence has validated these previous results, highlighting the role of the CoaR0 repeat in Fg binding, further confirming that both CoaR0 and CoaRI are indeed the functional Fg binding repeats. It was also shown that increasing the number of Fg binding repeats does not lead to a cooperative effect and the stoichiometry remains 1:1 (number of repeats: Fg D molecules) (60). This is in further support of the possibility that more than one antibody molecule is necessary to efficiently inhibit Fg binding by all CoaRI-similar repeats, thereby giving a possible explanation why no efficient inhibition could be seen by antibodies directed to CoaR.

Other antibodies that bind either Coa or Efb have been reported. Thomer and colleagues (24) generated 13 mouse monoclonal antibodies by hybridoma technology targeting Coa and investigated two of them (5D5 and 3B3) in vivo in a mouse model of S. aureus bacteraemia. 5D5 mAb was assessed to bind the D1 domain of Coa and 3B3 bound the domain containing R repeats. No analysis of their crossreaction with Efb was provided. The only information available about crossreactivity is that no binding to vWbp and IsdA was detected. MAb 3B3 proved its clear efficacy in the bacteraemia mouse model, further highlighting clinical relevance of the repeated region of Coa (24). A detailed biochemical analysis of these antibodies would provide orthogonal confirmation to our results, also in respect to the hypothesis of two classes of motives by CoaR0 and CoaRI repeats. It is also a possibility that the efficacy of mAb 3B3 could be due to the parallel targeting of Coa and Efb. A clear obstacle for 5D5 and 3B3 clinical translation is their fully murine origin.

Shannon and colleagues found that antibodies against Efb from patient sera could be neutralizing in vitro and also crossreacting to Coa (61). A further peculiar class of antibodies against Efb, named catalytic antibodies, were isolated (62). This discovery led to the hypothesis that Efb could also act as a B cell superantigen. Another group instead focused on the selection and characterization of recombinant divalent (Fab)2 mAbs from a synthetic phage display library against Efb C-terminal domain (63). The latter work showed both the presence of antibodies specific to Efb C-terminal in patient sera and also that blocking Efb interaction with C3b with the selected divalent mAbs improved mice survival in an infection model.

This present work and research from other groups show how pivotal may be blocking the multiple activities of proteins engaging Fg, further strengthening a possible therapeutic strategy involving Coa and Efb (64). To the best of our knowledge, this is the only report that investigates these two proteins as potential targets for generation of monoclonal antibodies. None has provided sequenced-defined human mAbs to the Fg-binding domain of Efb and Coa. The use of combination(s) of antibodies directed against either N- or C-terminal of both these proteins most presumably will result in additive effect in inhibiting S. aureus pathology. Selection of such antibodies is already underway.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

FB, Y-PK, SA, MHu, MHö and LV contributed to conception and design of the study. FB, ST, LG, FRS, DM, SHP, MB, SA designed and performed research. All authors analyzed data. FB wrote the manuscript, with inputs from Y-PK and SA. All authors contributed to the article and approved the submitted version.