African swine fever (ASF) continues to hamper pork production worldwide. In addition to vaccines, there is a need to develop curative therapeutics, such as highly-specific antibodies or nanobodies (NB), against immunogenic proteins with important functions such as the viral CD2 (CD2v)-like protein. However, further characterization of CD2v, such as its binding with cellular partners, is needed for the rational development of such biologics.We aimed to analyze the binding interaction of CD2v from pandemic genotypes I and II and swine CD58 using molecular simulations and optimize the NB to target the conserved epitope around the identified binding site using a computational pipeline.CD2v-CD58 complexes and equivalents from swine and humans were generated and simulated to characterize their binding. Mapping of residue contributions to the binding, variability, epitope propensity, and epitope multiplicity allowed us to select a conserved region near the site. Optimized NB were developed from sequences deposited in the NanoLAS repository, following screening for physicochemical and reactogenic parameters, docking, iterative optimization, and molecular dynamics simulations compared to controls.Simulations report poor affinity of CD2v to CD58 in the "canonical" pose and present a novel pose backed by docking, simulation data, and recent observations. The new site might have developed due to immunological considerations and may reproduce observed lymphocyte non-proliferation by hijacking CD58-CD2 ruler functions in immunological synapse formation. Two NB were also developed targeting genotype I and II CD2vs with stable binding and better affinity, solubility, allergenicity, and antigenicity than the controls NB22 and 18a3.Herein, we showcase the power of computational biology techniques to elucidate ASF virus (ASFV)-host interactions and simultaneously design therapeutics to target the identified novel binding site. Further validation is needed to confirm these findings; however, future work on other relevant ASFV proteins may lead to potent cocktails with therapeutic potential.