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. 2023 May 27;20(1):9.
doi: 10.1186/s12977-023-00624-9.

Conformational antigenic heterogeneity as a cause of the persistent fraction in HIV-1 neutralization

Philippe Colin  1   2 , Rajesh P Ringe  1   3 , Anila Yasmeen  1 , Gabriel Ozorowski  4 , Thomas J Ketas  1 , Wen-Hsin Lee  4 , Andrew B Ward  4 , John P Moore  1 , P J Klasse  5
Affiliations

Conformational antigenic heterogeneity as a cause of the persistent fraction in HIV-1 neutralization

Philippe Colin et al. Retrovirology. .

Abstract

Background: Neutralizing antibodies (NAbs) protect against HIV-1 acquisition in animal models and show promise in treatment of infection. They act by binding to the viral envelope glycoprotein (Env), thereby blocking its receptor interactions and fusogenic function. The potency of neutralization is largely determined by affinity. Less well explained is the persistent fraction, the plateau of remaining infectivity at the highest antibody concentrations.

Results: We observed different persistent fractions for neutralization of pseudovirus derived from two Tier-2 isolates of HIV-1, BG505 (Clade A) and B41 (Clade B): it was pronounced for B41 but not BG505 neutralization by NAb PGT151, directed to the interface between the outer and transmembrane subunits of Env, and negligible for either virus by NAb PGT145 to an apical epitope. Autologous neutralization by poly- and monoclonal NAbs from rabbits immunized with soluble native-like B41 trimer also left substantial persistent fractions. These NAbs largely target a cluster of epitopes lining a hole in the dense glycan shield of Env around residue 289. We partially depleted B41-virion populations by incubating them with PGT145- or PGT151-conjugated beads. Each depletion reduced the sensitivity to the depleting NAb and enhanced it to the other. Autologous neutralization by the rabbit NAbs was decreased for PGT145-depleted and enhanced for PGT151-depleted B41 pseudovirus. Those changes in sensitivity encompassed both potency and the persistent fraction. We then compared soluble native-like BG505 and B41 Env trimers affinity-purified by each of three NAbs: 2G12, PGT145, or PGT151. Surface plasmon resonance showed differences among the fractions in antigenicity, including kinetics and stoichiometry, congruently with the differential neutralization. The large persistent fraction after PGT151 neutralization of B41 was attributable to low stoichiometry, which we explained structurally by clashes that the conformational plasticity of B41 Env causes.

Conclusion: Distinct antigenic forms even of clonal HIV-1 Env, detectable among soluble native-like trimer molecules, are distributed over virions and may profoundly mold neutralization of certain isolates by certain NAbs. Affinity purifications with some antibodies may yield immunogens that preferentially expose epitopes for broadly active NAbs, shielding less cross-reactive ones. NAbs reactive with multiple conformers will together reduce the persistent fraction after passive and active immunization.

Keywords: Antigenic heterogeneity; Binding kinetics; Broadly active neutralizing antibodies (bNAbs); Efficacy; HIV-1 neutralization; Persistent fraction; Stoichiometry.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Neutralization efficacy of BG505 and B41 pseudovirus. The extent of neutralization (%) of PV in a TZM-bl assay is plotted as a function of NAb concentration (μg/ml); means ± s.e.m. of 2–4 replicate titrations are shown. Neutralization by the three bNAbs 2G12 (N332 glycan), PGT145 (V2-apex), and PGT151 (gp41-gp120 interface) of BG505 PV (A) and B41 (B) gave different potencies and efficacies. C The log10 of the relative remaining infectivity after neutralization by bNAb at a fixed concentration (50 μg/ml) is plotted as a function of the log10 of varied viral inoculum dilution. D The schematic shows the procedure for partial depletion of a PV preparation by the absorption of virions binding most avidly to bNAbs on beads
Fig. 2
Fig. 2
Neutralization of bNAb-depleted B41 PV by bNAbs. The extent of neutralization (%) is plotted as a function of bNAb concentration (μg/ml). PV was first incubated with Sepharose beads covalently conjugated with bNAbs PGT145 or PGT151 or mock-conjugated (see color-coded legend). The unbound virions were then tested for neutralization by the NAbs indicated in each diagram in a TZM-bl neutralization assay. The extent of neutralization (%) is plotted as a function of bNAb concentration (μg/ml), so that potencies rise as curves are shifted from right to left
Fig. 3
Fig. 3
Neutralization of bNAb-depleted B41 PV by sera from immunized rabbits. The extent of neutralization (%) of PV depleted as in Figs. 1D and 2 is plotted as a function of serum dilution factor. Thus, in contrast to diagrams for monoclonal Abs, the potency rises as curves are shifted from left to right
Fig. 4
Fig. 4
Neutralization of bNAb-depleted B41 PV by mNAbs from immunized rabbits. The extent of neutralization (%) of PV depleted as in Figs. 1D, 2, and 3 is plotted as a function of autologous mNAb concentration (μg/ml). In contrast to the plots for the sera in Fig. 3, the potency rises as curves are shifted from right to left
Fig. 5
Fig. 5
B41 SOSIP.664 trimer. A A surface-rendered model of the B41 SOSIP.664 trimer (PDB 6MCO [52]) oriented with the apex up (top model) or viewed from above (bottom model). The peptidic surface is gray on one protomer and light blue on the other two. Man9 glycans were added to the published model [52] and are depicted as sticks and colored green unless they are directly involved in the epitopes of the three bNAbs used for affinity purification, in which case they are colored as the rest of the epitope. Contacts with PGT145 are colored blue, with 2G12 yellow, and with PGT151 purple. N289, which is not part of a PNGS in B41 SOSIP.664, is colored magenta. B Purified Env proteins were analyzed by electrophoresis in 4–12% Bis–Tris BN-PAGE gels with Coomassie-blue staining. 2 μg protein per well was loaded from each purification. C NS-EM analyses of unliganded the B41 SOSIP.664 trimer purified by PGT151-affinity chromatography. The propeller-like, triangular particles show 100% native-like trimer molecules
Fig. 6
Fig. 6
SPR analysis of bNAb binding to bNAb-purified and -depleted BG505 and B41 SOSIP.664. Sensorgrams for individual bNAbs are shown. A BG505 (top) and B41 (bottom) SOSIP.664 trimers were affinity-purified the bNAbs 2G12, PGT151, or PGT145 in a first step and thereafter by SEC. To the right in the B41 row is the sensorgam for the autologously neutralizing rabbit mNAb 16D. Each sensorgram shows the response after background subtraction on the y axis (response units, RU) over time after start of injection on the axis (s). Association was monitored for 300 s and dissociation for 600 s. B B41 SOSIP trimer was first 2G12-affinity and then SEC-purified and thereafter depleted by passage through Sepharose columns with the bNAbs PGT151 or PGT145 or no antibody on the beads. The flowthrough trimer was immobilized and analyzed by SPR as in A. The sensorgrams represent 2–3 highly similar replicates
Fig. 7
Fig. 7
bNAb-Fab binding to BG505 and B41 SOSIP.664 SOSIP,664. Fabs of PTG145 or PGT151 were injected over trimer immobilized freshly in each cycle at concentrations as indicated in the color-coded legend. BG505 SOSIP.664 trimer was 2G12-purifed and B41 SOSIP.664 trimer 2G12- or PGT145-purified, as indicated. Each sensorgram shows the response after background subtraction on the y axis (response units, RU) over time after start of injection on the axis (s). Association was monitored for 300 s and dissociation for 600 s. The model-fitted functions (heterogeneous-ligand for BG505 and Langmuir for B41) are shown as thin black curves. The sensorgram for PGT151 Fab binding to PGT145-purified B41 trimer has no model curves because no model fitted to the weak binding. The number of replicate titrations is given in Table 1
Fig. 8
Fig. 8
Structural constraints of PGT151 binding to B41 SOSIP.664. A PGT151 (PDB 5FUU; in complex with JRFL Env [65]) aligned to a cryo-EM structure of B41 SOSIP.664 (PDB 6U59 [33]). The fusion peptide is depicted in red and derived from the PGT151 + JRFL SOSIP.664 [41] model for reference. B PGT151 contact residues in the epitope (as defined by < 4 Å distance from paratope atoms in the PGT151 + JRFL SOSIP.664 model) and equivalent positions in BG505 and B41 SOSIP.664 reveal high conservation. Amino acids different between B41 and BG505 are highlighted in light blue. C Alignment of PGT151 Fab from the PGT151 + JRFL SOSIP.664 structure onto the b12-bound (top, PDB 5VN8 [27]) or 8ANC195- and sCD4-bound (bottom, PDB 6EDU [29]) conformation using one of three reference chains [27, 29]. Note that for clarity, the b12, 8ANC195, and sCD4 structures have been removed from the reference model. Steric clashes are depicted with translucent buff-colored stars. The clashing gp120 subunit is depicted in transparent blue. For the rest, antibody and Env colors follow those in panel A
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