Myomedin replicas of gp120 V3 loop glycan epitopes recognized by PGT121 and PGT126 antibodies as non-cognate antigens for stimulation of HIV-1 broadly neutralizing antibodies

Abstract

IntroductionImprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified.MethodsIn this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes.ResultsIn the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro.DiscussionOur data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.

Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging

“…In our previous work, we demonstrated that the human myomesin-1 domain 10 structure exhibited sufficient stability to be used as a scaffold for the generation of a highly complex combinatorial library using randomization of 12 mutable amino acid residues located in the three-domain loops, named Myomedin scaffold [ 34 , 39 ]. Based on the available crystal structure of mouse/human PD-1/PD-L1 complex (PDB ID 3bik), it seems to be evident that critical interaction residues of human PD-L1 required for the recognition of PD-1 receptor are located on β-sheet surface (Additional file 1 : Fig.…”

“…The injected substances were well tolerated for several days after application as the mice used for the imaging study were sacrificed approximately 2 weeks after the imaging without clinical and neurological marks of alterations. In support, we have previously published experiments on Myomedin-derived binders (each containing 10 µg) for immunization (in Freund adjuvant) through intradermal application of experimental BALB/c mice [ 34 , 39 ]. We did not record any marks of somatic or behavioral alterations, with exception of local irritation due to adjuvant co-administered with individual Myomedins.…”

“…Collectively, we present here a concept of highly complex β-sheet combinatorial library developed on Myomedin scaffold as a valuable alternative for the generation of small protein binders for biomedical applications. Randomization of amino acid residues on a flat β-sheet surface provides a proof-of-concept for the selection of high-affinity binders and, thus, completes a portfolio of loop-type scaffold libraries [ 34 , 39 , 51 ] and three-helix bundle-based libraries [ 68 – 70 ]. Herein, we provide evidence that β-sheet-derived MBA variants exhibit PD-1 specificity and stability in human serum.…”