In this study we report the successful production of anti-CD81 mAbs using DNA immunization which potently inhibit cell-free HCV infection from different genotypes in a dose-dependent manner and block cell-cell transmission and dissemination. Production of mAbs using DNA immunization has been reported to induce higher avidity antibodies than protein immunization 
, which may be advantageous for the development of antibodies efficiently inhibiting HCV infection. Indeed, among the four anti-CD81 mAbs generated in this study, anti-CD81 mAb QV-6A8-F2-C4 showed very effective inhibition of HCVcc infection and HCVpp entry during HCV-CD81 post-binding interaction(s).
The CD81 LEL has been shown to play an important role in the entry process as soluble recombinant forms of CD81 LEL are able to inhibit HCV infection 
. The amino acid residues within the CD81 LEL and HCV E2 involved in E2-CD81 binding have been extensively characterized 
. Interestingly, our studies identify anti-CD81 mAbs with different inhibition profiles on HCV infection. Anti-CD81 mAb QV-6A8-F2-C4 which most efficiently inhibited HCV infection was characterized by binding to cell surface-expressed human CD81 and mutual cross-competition between QV-6A8-F2-C4 and the well-characterized commercially available anti-CD81 antibody JS81 suggests that they recognize similar epitopes on CD81.
These novel anti-CD81 antibodies may be very useful for investigators studying the HCV entry process. Indeed, a panel of antibodies inhibiting HCV entry with different efficacy and recognizing different epitopes is of interest as it may be used to (i) further decipher structural and functional domains in CD81 which are crucial for inhibition and (ii) to more deeply dissect its mechanistic role in the entry process. This will allow a better understanding of CD81 regions binding to envelope glycoprotein E2 or domains involved in the formation of the CD81-CLDN1 complex 
. Furthermore, the antibodies are useful to study CD81 expression by flow cytometry.
The identification of novel anti-CD81 antibodies may also be relevant for the development of novel antiviral antibodies for prevention and treatment of HCV infection. CD81 may be an attractive therapeutic target for the development of HCV entry inhibitors as it is a key player in the HCV entry process. Small molecules and mAbs targeting CD81 and interfering with HCV infection have previously been described 
. So far, the effect of the majority of these compounds has been solely assessed on cell-free HCV entry 
. While cell-free viral entry is undoubtedly essential for initiation of infection, direct cell-cell transmission probably constitutes the dominant mechanism of viral spread and thus persistence of infection 
. Direct cell-cell transfer has an important impact for the development of antivirals as this process allows viral spreading by escaping extracellular neutralizing antibodies as well as defined antibodies interfering with host cell entry factors 
. Most of the known HCV entry factors are involved in this process 
. In addition to CD81-dependent HCV cell-cell transmission, a fraction of viral spread appears to be independent of CD81 
. Noteworthy, the anti-CD81 mAb QV-6A8-F2-C4 described in our study not only inhibited cell-free HCV entry but also efficiently and dose-dependently blocked cell-cell transmission and viral spread, providing novel options for the development of efficient anti-HCV therapeutics interfering with this process.
Entry inhibitors, such as anti-CD81 mAbs, are ideal to be applied for the prevention of HCV re-infection in the transplantation setting where currently no clinical option exists to protect HCV-negative transplanted livers from re-infection 
. An anti-CD81 antibody inhibiting HCV infection in vitro
has already been demonstrated to prevent HCV infection in the human liver-chimeric Alb-uPA/SCID mouse model 
. This suggests that targeting CD81 may be an efficient strategy to prevent HCV infection e. g. in transplant recipients where entry has been shown to be a key determinant for infection of the liver graft 
. In this study, we demonstrate that anti-CD81 mAbs efficiently inhibited the entry of highly infectious HCV escape variants that are resistant to autologous host responses and re-infect the liver graft. Interestingly, combination of HCV envelope-specific antibodies with a CD81-specific mAb resulted in a synergistic activity on the inhibition of HCVcc infection and HCVpp escape variant entry. The combination decreased the concentration needed to achieve a 50% antiviral activity of the individual compounds up to 100-fold. The ability of anti-CD81 mAbs to block entry of HCV escape variants and the marked synergy with anti-envelope antibodies on inhibiting HCV entry indicate that the novel CD81-specific mAbs are prime candidates for prevention of liver graft infection. Furthermore, entry inhibitors may also be efficient antivirals for treatment of HCV infection 
. Indeed, the ability of anti-CD81 mAb QV-6A8-F2-C4 to block cell-cell transmission and dissemination post-infection without any detectable toxicity suggests that targeting CD81 may also hold promise for the treatment of chronic infection in combination with other antivirals. A potential challenge for the clinical development of anti-CD81 antibodies could be adverse effects. Indeed, CD81 is ubiquitously expressed on the surface of various cell types. Antibodies binding to CD81 may alter the function, expression or signaling of the receptor resulting in side effects. Interestingly, using anti-CD81 mAb QV-6A8-F2-C4, no toxic effects were detected in MTT-based cellular assays (). However, further in vivo
studies are needed to address toxicity in hepatic and extrahepatic tissues.
In conclusion, we identified and functionally characterized a novel panel of anti-CD81 mAbs generated by DNA immunization which efficiently inhibit HCV infection and dissemination. These antibodies will be useful for the molecular investigations of virus-host interactions during the HCV entry process and the characterization of CD81 expression in cell lines, primary cells and tissues. Furthermore, one antibody is an interesting and relevant candidate for the development of novel preventive and improved therapeutic antiviral strategies against HCV infection.