Myomedin scaffold variants targeted to 10E8 HIV-1 broadly neutralizing antibody mimic gp41 epitope and elicit HIV-1 virus-neutralizing sera in mice

Abstract

One of the proposed strategies for the development of a more efficient HIV-1 vaccine is based on the identification of proteins binding to a paratope of chosen broadly neutralizing antibody (bNAb) that will mimic cognate HIV-1 Env (glyco)protein epitope and could be used as potent immunogens for induction of protective virus-neutralizing antibodies in the immunized individuals. To verify this "non-cognate ligand" concept, we developed a highly complex combinatorial library designed on a scaffold of human myomesin-1 protein domain and selected proteins called Myomedins specifically binding to variable regions of HIV-1 broadly neutralizing antibody 10E8. Immunization of mice with these Myomedin variants elicited the production of HIV-1 Env-specific antibodies. Hyperimmune sera bound to Env pseudotyped viruses and weakly/moderately neutralized 54% of tested clade A, B, C, and AE pseudotyped viruses variants in vitro. These results demonstrate that Myomedin variants have the potential to mimic Env epitopes and could be used as potential HIV-1 vaccine components.

Keywords: Env glycoprotein; HIV vaccine; broadly neutralizing antibody; combinatorial library; protein mimetics; protein scaffold.

Figure 1.

Human myomesin-1 Domain 10, a Loop-type Myomedin library concept. A) The superposition of the crystal structure of the myomesin-1 domain 10 used for randomization (PDB ID 6t3o) to the structures of longer myomesin fragments. The domain 10 is shown in green and the randomized residues indicated as red spheres. The myomesin fragment 10–11 (PDB ID 3rbs) is shown in gray and the 9–10-11 fragment (PDB ID 2y23) is shown in light blue. The Cα RMSD values for the 6t3o domain 10 compared to 2y23 and 3rbs structures are 0.81 Å and 0.70 Å, respectively, showing a high structural similarity of the isolated domain. B) A rotated view on domain 10 is shown in green cartoon representation with the randomized residues indicated as red spheres. C) Diagram of the primers used for the assembly of the Myomedin loop-type combinatorial library via multiple PCR steps

Figure 2.

Binding of MLA protein variants to 10E8 bNAb, IgG isotype control and BSA. Myomedin variants of eight selected MLA clones in the form of purified fusion proteins with N-terminal polyhistidine tag and C-terminal V5 tag produced in E.coli BL21 (DE3) were assayed in ELISA, the parental non-randomized Myomedin was used as a negative control. Binding to immobilized 10E8 bNAb (labeled as 10E8 IgG), IgG1λ isotype (labeled as IgG lambda) and BSA was detected by anti-V5 Ab-HRP conjugate. Each point is shown as the mean value of triplicates with standard deviation

Figure 3.

Modeling of MLA-10E8 interaction by docking. A), A crystal structure of the scaffolded HIV-1 Env gp41 MPER/10E8 complex (PDB ID 5sy8) showing the 10E8 antibody heavy chain in yellow cartoon representation, the light chain in magenta, and the gp41/Env MPER as a green surface embedded in a gray helper protein construct. The 10E8 interaction interface is shown as cyan sticks and the positions of the key 10E8 interaction sites are indicated by arrows, Site 1 residues are highlighted as red sticks and Site 2 as orange sticks. B), Predicted binding mode of the MLA092 (orange) to the 10E8 antibody; the upper row of figures shows the whole complex (left) and a detail of the interacting interface (right) in the antibody orientation corresponding to the left-hand side of the panel A (focusing on the interaction site 2). The lower row corresponds to the second antibody orientation (focusing on the interaction site 1). C), Predicted binding mode of the MLA158 (pickle) in the same orientation as in panel B. D), Predicted binding mode of the MLA025 (violet) in the same orientation as in panel B. MLA interface residues within 4 Å of the 10E8 residues are: MLA016 A6, E8, I9, L10, R15, W17, K21, A22, Q23, Q24, H47, I48, D49, I50, M51, F52, E56. MLA024 L21, S22, V23, F24, G25, N26, R50, N51, F76, M77, L78, M79. MLA025 S24, N26, I32, N34, T69, T71, Q73, Q75, I77, L78, L80, H82, T84. MLA092 E7, K11, R15, W17, I22, M23, W24, G25, H47, I48, D49, P50, S51, W52, E56, F58. MLA093 S24, N26, I32, T71, Q73, Q75, F77, I78, W80, H82. MLA132 L22, L24, H47, D49, Y50, F51, W52, E56, M76, W77, S78. MLA158 E3, M18, Q19, A20, W21, M22, W24, N26, L74, L76, Y77, Y78, A79, N81. MLA159 Y24, N26, I32, E35, T71, Q73, M77, I78, E79, Y80, H82. The randomized residues are in bold. The source PyMOL session containing the results for all identified MLA variants is available on 10.5281/zenodo.4650182

Figure 4.

Immunization of experimental mice with a panel of MLA variants induced serum antibodies specifically recognizing HIV-1 Env on the virus surface. A) Mice were immunized by the administration of four doses of individual MLA variants including wild-type (MyoWT). Following immunization, sera were collected and tested in their reactivity with non-replicative HIV-1 viruses pseudotyped with B) Clade C Env (ZM109F) or C) Clade B Env (AC10.0, QH0692, RHPA) coated on ELISA plates. Antibody titers of IgG isotype were measured in ELISA. Statistical comparison was performed by ANOVA Kruskal-Wallis test with Dunn’s posttest (* P < 0.05, ** P < 0.01). The red asterisk indicates a comparison with MyoWT. D) Sera from mice immunized with MLA092 and MLA025 Myomedins were tested for reactivity with RHPA-pseudotyped virus coated on ELISA panel in competition with 10E8 or VRC01. 10E8 serially diluted to achieve final concentrations 2; 1; 0.5; 0.25; 0.125; 0.0625; and 0 μg/mL in blocking buffer was applied with individual MLA092- or MLA025-immunized mouse sera diluted 1:400. After washing, the plates were incubated with rabbit anti-mouse IgG HRP-conjugated antibody, developed with a substrate and O.D. 492 nm was measured. VRC01 antibody was applied in control reaction, as irrelevant antibody, at final 3.33; 1; 0.33; 0.1; 0.033; and 0 μg/mL analogously to 10E8. In separate experiment 10E8 at indicated concentration was applied with individual MyoWT-immunized or naive mouse sera diluted 1:400 as the control (two right panels). All experiments were performed in triplicates. Mean values are indicated by horizontal lines

Figure 5.

Neutralization activity of MLA-immunized mice sera. A) Values represent the reciprocal serum dilution which resulted in 50% virus neutralization. Relative mean neutralization dilution represents mean values of detected titers among all tested pseudoviruses. No. of neutralized PVs indicates the sum of pseudoviruses neutralized at reciprocal titer higher than 30. Murine leukemia virus (MULV) was used in neutralization assay as a control. Pseudotyped viruses used for analyses were chosen to cover the phylogenetic tree of HIV-1 Env. B) An unrooted phylogenetic tree of 194 representative sequences from HIV-1 2019 Compendium of HIV-1 ENV genes (https://www.hiv.lanl.gov) enriched for 22 HIV-1 ENV sequences used in this analysis. Sequences were processed at EMBL-EBI Simple Phylogeny using neighbor-joining clustering method [68,69]. HIV-1 strains are classified into four groups M, N, O and P. The major group M is further divided into nine genetically distinct subtypes A, B, C, D, AE, G, H, J, F and K. Each subtype contains hybrid viruses – circulating recombinant forms as a result of genetic material combination. For better orientation, only the 22 HIV-1 ENV sequences used in the analysis are visualized. The complete and high-resolution tree is available in figure S6

Figure 6.

Model-based interpretation of neutralizing potential of all MLA proteins. A) Insufficient mimicking of MPER F673 (green sticks) by MLA variants 024 and 093, B) Partial mimicking of MPER F673 (green sticks) by MLA variants 092 and 159, C) Larger interacting surface within site 2 evolved in MLA132 variant, D) Partial mimicking of MPER W672 (green sticks) by variants MLA025 and MLA158 (left), Comparison of interaction surfaces within site 1 between MLA025 and MLA158, demonstrating the formation of a deep cavity for binding of 10E8 F100a residue (right), E) Potential of variants MLA016 and MLA092 for mimicking of site 3 interaction. As an example, Fig.S11 is summarizing interacting residues of MLA092. For more detailed representation we included the source PyMOL session containing the results for all identified MLA variants available on 10.5281/zenodo.4650182