Researchers in the Cheloha Group at the National Institutes of Health, published in the Journal of the American Chemical Society, have developed nanobody-fusion inhibitor peptide conjugates that block HIV infection with potencies up to 10,000-fold greater than standalone peptides and broad activity across diverse viral strains.
HIV continues to affect more than 37 million people worldwide. Drug resistance and toxicity remain persistent obstacles to treatment, driving interest in new approaches that engage multiple mechanisms of inhibition. HIV enters host cells through the trimeric surface protein Env, whose gp120 subunit binds the host receptor CD4 and a coreceptor, while gp41 mediates membrane fusion. Fusion inhibitor peptides derived from the HR2 region of gp41 block viral entry by preventing the conformational rearrangement of gp41 into the six-helix bundle required for membrane fusion. The clinically approved peptide enfuvirtide validates this mechanism but requires high doses due to modest potency, resulting in unwanted side effects. Previous strategies attached cholesterol moieties to fusion inhibitor peptides, concentrating them at host cell membranes and improving activity. However, lipid-based delivery lacks specificity for the CD4-positive cells that HIV actually targets for infection.
The team combined chemical peptide synthesis with nanobody engineering to build a modular conjugation platform. They linked the fusion inhibitor peptide C34 to nanobodies targeting proteins on HIV-susceptible host cells, including the primary receptor CD4 and the coreceptor CXCR4. Sortase-mediated enzymatic ligation connected the C-terminus of each nanobody to the N-terminus of C34. Click chemistry generated C-to-C terminal conjugates as well, a linkage topology inaccessible through conventional genetic fusion. This semisynthetic strategy allowed the team to vary nanobody identity, target specificity and linkage geometry across a broad set of conjugates.
The lead conjugate Nb3F11-C34, targeting CD4, achieved an IC50 of 0.005 nM against HIV pseudovirus, compared to 30 nM for C34 alone. Simply mixing the nanobody and peptide together without covalent linkage produced no meaningful improvement, confirming that physical conjugation drives the enhancement. A panel of ten HIV strains spanning four viral clades and both CCR5-tropic and CXCR4-tropic variants revealed broad-spectrum activity, with CD4-targeting conjugates reaching IC50 values as low as 0.001 nM. These conjugates outperformed both enfuvirtide and the broadly neutralizing antibody VRC01 by nearly 1,000-fold in several comparisons. Notably, the semisynthetic conjugates also surpassed analogous genetic fusion proteins produced in mammalian cells, with the enzymatically ligated version showing substantially greater potency regardless of linker design. The team further demonstrated that conjugates delivered to cells expressing the target antigen resisted depletion by off-target cell populations, while conjugates targeting broadly expressed markers like MHC-I lost activity when exposed to non-susceptible cells acting as unproductive reservoirs.
These results establish nanobody-mediated delivery as a powerful strategy for concentrating antiviral peptides at their site of action on the host cell surface. The modularity of the platform enables rapid exploration of targeting strategies and conjugate architectures, including topologies that genetic methods cannot access. The exceptional potency and breadth of activity across diverse HIV strains position these conjugates as promising candidates for therapeutic or prophylactic development. Future work will address pharmacokinetic challenges related to the small size of nanobody constructs, potentially through PEGylation or albumin-binding modifications to extend circulation time. The broader principle of delivering bioactive peptides through antibody fragment targeting may also extend to other viral infections and disease contexts.
