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Semliki Forest virus (SFV) is an alphavirus that uses the very-low-density lipoprotein receptor (VLDLR) as a receptor during infection of its vertebrate hosts and insect vectors. Herein, we used cryoelectron microscopy to study the structure of SFV in complex with VLDLR. We found that VLDLR binds multiple E1-DIII sites of SFV through its membrane-distal LDLR class A (LA) repeats. Among the LA repeats of the VLDLR, LA3 has the best binding affinity to SFV. The high-resolution structure shows that LA3 binds SFV E1-DIII through a small surface area of 378 Å2, with the main interactions at the interface involving salt bridges. Compared with the binding of single LA3s, consecutive LA repeats around LA3 promote synergistic binding to SFV, during which the LAs undergo a rotation, allowing simultaneous key interactions at multiple E1-DIII sites on the virion and enabling the binding of VLDLRs from divergent host species to SFV. [Display omitted] • The receptor VLDLR binds multiple E1-DIII sites of SFV through its LA repeats • Single LA binds E1-DIII with a small contact surface and a low binding affinity • Multiple LAs promote synergistic binding and a high binding affinity to SFV Semliki Forest virus (SFV) enters host cells by binding to repeat regions of the host cell's very-low-density lipoprotein receptors. The repeat regions enable synergic, multimodal binding with SFV, which is highly tolerant of mutations, allowing SFV to bind efficiently to the receptors of highly divergent species. [ABSTRACT FROM AUTHOR]

Despite the success of immune checkpoint blockade for cancer therapy, many patients do not respond adequately. We aimed to improve this therapy by optimizing both the antibodies and their delivery route, using small monodomain antibodies (nanobodies) delivered locally with a self-amplifying RNA (saRNA) vector based on Semliki Forest virus (SFV). We generated nanobodies against PD-1 and PD-L1 able to inhibit both human and mouse interactions. Incorporation of a dimerization domain reduced PD-1/PD-L1 IC50 by 8- and 40-fold for anti-PD-L1 and anti-PD-1 nanobodies, respectively. SFV viral particles expressing dimeric nanobodies showed a potent antitumor response in the MC38 model, resulting in >50% complete regressions, and showed better therapeutic efficacy compared to vectors expressing conventional antibodies. These effects were also observed in the B16 melanoma model. Although a short-term expression of nanobodies was observed due to the cytopathic nature of the saRNA vector, it was enough to generate a strong proinflammatory response in tumors, increasing infiltration of NK and CD8+ T cells. Delivery of the SFV vector expressing dimeric nanobodies by local plasmid electroporation, which could be more easily translated to the clinic, also showed a potent antitumor effect. • Nanobodies able to block human and mouse PD-1/PD-L1 interactions have been developed. • Dimerization of nanobodies considerably decreased IC50 for PD-1/PD-L1 interactions. • SFV viral particles expressing dimeric nanobodies led to potent antitumor effects. • Delivery of SFV saRNA by plasmid electroporation showed a potent antitumor effect. [ABSTRACT FROM AUTHOR]