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. 2022 Sep 30:12:100222.
doi: 10.1016/j.jvacx.2022.100222. eCollection 2022 Dec.

Immunization with HIV-1 trimeric SOSIP.664 BG505 or founder virus C (FVCEnv) covalently complexed to two-domain CD4S60C elicits cross-clade neutralizing antibodies in New Zealand white rabbits

Nancy L Tumba  1 , Gavin R Owen  1 , Mark A Killick  1 , Maria A Papathanasopoulos  1
Affiliations

Immunization with HIV-1 trimeric SOSIP.664 BG505 or founder virus C (FVCEnv) covalently complexed to two-domain CD4S60C elicits cross-clade neutralizing antibodies in New Zealand white rabbits

Nancy L Tumba et al. Vaccine X. .

Abstract

Background: An ongoing challenge in HIV-1 vaccine research is finding a novel HIV-1 envelope glycoprotein (Env)-based immunogen that elicits broadly cross-neutralizing antibodies (bnAbs) without requiring complex sequential immunization regimens to drive the required antibody affinity maturation. Previous vaccination studies have shown monomeric Env and Env trimers which contain the GCN4 leucine zipper trimerization domain and are covalently bound to the first two domains of CD4 (2dCD4S60C) generate potent bnAbs in small animals. Since SOSIP.664 trimers are considered the most accurate, conformationally intact representation of HIV-1 Env generated to date, this study further evaluated the immunogenicity of SOSIP.664 HIV Env trimers (the well characterized BG505 and FVCEnv) covalently complexed to 2dCD4S60C. Methods: Recombinant BG505 SOSIP.664 and FVCEnv SOSIP.664 were expressed in mammalian cells, purified, covalently coupled to 2dCD4S60C and antigenically characterized for their interaction with HIV-1 bnAbs. The immunogenicity of BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C was investigated in New Zealand white rabbits and compared to unliganded FVCEnv and 2dCD4S60C. Rabbit sera were tested for the presence of neutralizing antibodies against a panel of 17 pseudoviruses. Results: Both BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C elicited a potent, HIV-specific response in rabbits with antibodies having considerable potency and breadth (70.5% and 76%, respectively) when tested against a global panel of 17 pseudoviruses mainly composed of harder-to-neutralize multiple clade tier-2 pseudoviruses. Conclusion: BG505 SOSIP.664-2dCD4S60C and FVCEnvSOSIP.664-2dCD4S60C are highly immunogenic and elicit potent, broadly neutralizing antibodies, the extent of which has never been reported previously for SOSIP.664 trimers. Adding to our previous results, the ability to consistently elicit these types of potent, cross-neutralizing antibody responses is dependent on novel epitopes exposed following the covalent binding of Env (independent of sequence and conformation) to 2dCD4S60C. These findings justify further investment into research exploring modified open, CD4-bound Env conformations as novel vaccine immunogens.

Keywords: Broadly neutralizing antibodies; Covalent complexes; Envelope glycoprotein trimers; HIV-1 vaccine immunogens; Immunogen design; New Zealand White rabbits.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Biochemical characterization of FVCENV and BG505 SOSIP.664 trimers covalently complexed to 2dCD4S60C. FVCENV and BG505 SOSIP.664 trimers complexed to 2dCD4S60C were purified using size-exclusion chromatography and analysed by SDS-PAGE and BN-PAGE. (A) HiPrep Sephacryl S-300 HR SEC profile of lectin-purified FVCENV SOSIP.664 and BG505 SOSIP.664 gp140 expressed in HEK293T cells before and after complexing to 2dCD4S60C with peaks correlating to aggregates, trimers and monomers annotated. (B) Reducing (+DTT) and non-reducing (-DTT) SDS-PAGE analysis with 10% Tris-Glycine gels of lectin-purified FVCENV SOSIP.664 gp140 and BG505 SOSIP.664, following Coomassie blue staining. Subsequent SEC-purified FVCENV SOSIP.664 gp140 also shown. The shift of the gp140 band to gp120 under reducing conditions indicates furin-mediated cleavage to allow gp120-gp41 dissociation. The molecular weight marker (M), indicated on the left, is the broad-range, color prestained protein standard (NEB). (C) Coomassie-stained BN-PAGE analysis of FVCENV and BG505 pre- and post-SEC purification as well as the FVCENV:2dCD4S60C complex post- SEC pooled fractions. The molecular weight marker (M), indicated on the left, is the NativeMark™ protein standard (Thermo Fisher Scientific).
Fig. 2
Fig. 2
Antigenicity of SOSIP.664 trimers (FVCENV and BG505) and complexes. (A) Recognition of FVCENV and BG505 trimers by a selection of antibodies targeting the V1V2 apex (yellow curves- PG9 & PGT145), the CD4 binding site (green curves- IgG1b12 & VRC01), the V3 region (blue curve- 10–1074), the gp120/gp41 interface (purple curve- 35O22), the C1-C2 region (black curve- A32), and an MPER epitope (red curve- 10E8) used as a negative control. (B) Model of unliganded SOSIP.664 trimer in the closed, prefusion conformation (left) and the open, CD4-bound conformation (right). The trimer spike is represented with two of the three protomers shown in light grey and the third protomer shown in dark grey with broadly neutralizing antibodies epitopes indicated with arrows and colored in orange for the V1V2 apex, blue for the V3 glycan patch, green for the CD4 binding site, purple for the gp120/gp41 interface, and red for the MPER region. (C) Effect of complexing FVCENV to 2dCD4S60C on the high-binding antibodies PG16, VRC01, IgG1b12, and 10–1074 as well as the CD4-induced mAb 17b. Red curves represent FVCENV SOSIP.664 trimers and blue curves the FVCENV SOSIP.664 trimers complexed to 2dCD4S60C.
Fig. 3
Fig. 3
Rabbit sera neutralization of a Tier 2 pseudovirus panel. (A) Schematic of rabbit immunization schedule with FVCENV SOSIP.664, 2dCD4S60C, BG505 SOSIP.664-2dCD4S60C and FVCENV SOSIP.664-2dCD4S60C. The rabbits were immunized four times, monthly, with 50 μg of the soluble proteins adjuvanted with Adjuplex. Two weeks prior to the first immunization and two weeks following each immunization, blood samples were collected. Immunizations and bleeds time-points are indicated in weeks. (B) Neutralization titers of the rabbit sera in each of the four immunization groups. Sera obtained prior to immunizations (pre-bleed) and at week 14, following the final immunization, were tested in a TZM-bl reporter cell assay against the VSV-G specificity control pseudovirus and HIV-1 pseudoviruses QH0692.42, 398_F1, 246F3, TRO.11, X2278, CAP210.2.00.E8, Du422.12, 246F, 25710, CE0217, ZM53.12, CNE8, CNE55, CH119.10, BJOX2000, CE1176, and X1632. Monoclonal antibodies (mAbs) PG9 and VRC03 were included as neutralization controls for each pseudovirus except the non-HIV VSV-G pseudovirus. Numerical values represent ID50 (for sera) and IC50 (for mAbs) neutralization titers. ID50 < 40 and IC50 > 10 indicate no detectable neutralization for the rabbit sera or mAbs, respectively.
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