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. 2021 Jun 10;95(13):e0000521.
doi: 10.1128/JVI.00005-21. Epub 2021 Jun 10.

Membrane Env Liposomes Facilitate Immunization with Multivalent Full-Length HIV Spikes

Daniel P Leaman  1 , Armando Stano  1 , Yajing Chen  1 , Lei Zhang  1 , Michael B Zwick  1
Affiliations

Membrane Env Liposomes Facilitate Immunization with Multivalent Full-Length HIV Spikes

Daniel P Leaman et al. J Virol. .

Abstract

A major goal of HIV vaccine design is to elicit broadly neutralizing antibodies (bNAbs). Such bNAbs target HIV's trimeric, membrane-embedded envelope glycoprotein spikes (mEnv). Soluble Env (sEnv) trimers have been used as vaccines, but engineering sEnvs for stability, multivalency, and desired antigenicity is problematic and deletes key neutralizing epitopes on glycoprotein 41 (gp41) while creating neoepitopes that elicit unwanted antibodies. Meanwhile, multivalent mEnv vaccines are challenging to develop due to trimer instability and low mEnv copy number amid other extraneous proteins on virus-like particles. Here, we describe a multivalent mEnv vaccine platform that does not require protein engineering or extraneous proteins. mEnv trimers were fixed, purified, and combined with naked liposomes in mild detergent. On removal of detergent, mEnv spikes were observed embedded in liposome particles (mean diameter, 133 nm) in correct orientation. These particles were recognized by HIV bNAbs and not non-NAbs and are designated mEnv liposomes (MELs). Following a sequential immunization scheme in rabbits, MELs elicited antibodies that neutralized tier 2 HIV isolates. Analysis of serum antibody specificities, including those to epitopes involving a missing conserved N-glycosylation site at position 197 near the CD4 binding site on two of the immunogens, provides clues on how NAb responses can be improved with modified immunogens. In sum, MELs are a biochemically defined platform that enables rational immunization strategies to elicit HIV bNAbs using multimerized mEnv. IMPORTANCE A vaccine that induced broadly neutralizing antibodies against HIV would likely end the AIDS pandemic. Such antibodies target membrane-embedded envelope glycoprotein spikes (mEnv) that HIV uses to enter cells. Due to HIV Env's low expression and instability, soluble stabilized Env trimers have been used as vaccine candidates, but these have an altered base that disrupts targets of HIV broadly neutralizing antibodies that bind near the membrane and are not available for all HIV isolates. Here, we describe membrane Env liposomes (MELs) that display a multivalent array of stable mEnvs on liposome particles. MELs showed the expected antibody recognition properties, including targeting parts of mEnv missing on soluble Envs. Immunization with MELs elicited antibodies that neutralized diverse HIV isolates. The MEL platform facilitates vaccine development with potentially any HIV Env at high valency, and a similar approach may be useful for eliciting antibodies to membrane-embedded targets of therapeutic interest.

Keywords: HIV; envelope; gp120; gp41; immunogen; liposome; vaccine.

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Figures

FIG 1
FIG 1
Purified mEnvs are trimeric and retain native antigenicity. (A) mEnv, which was GA cross-linked and PGT151 affinity purified, was analyzed using size exclusion chromatography (SEC). (B) Two mEnv-containing fractions from SEC were analyzed by denaturing Coomassie SDS-PAGE. (C) MEnv (5 μg; from fraction 5 in panel B) was analyzed using BN-PAGE stained with Coomassie blue. (D) Purified mEnv was captured using lectin from Galanthus nivalis (GNL) and probed in an ELISA using a panel of antibodies. Antibodies are classified as NAbs if they neutralized cognate virus with an IC50 of <50 μg/ml. NAbs against all isolates include PGDM1400, PGT145, PGT128, PGT135, VRC01, CH103, PGT151, 35O22, 3BC176, and 10E8. Non-NAbs against all isolates include b6, F105, 7B2, and 98-6. VRC34 neutralizes CH505.N197D and JRFL.TD15 but not ADA.CM. PGT128, 2F5, and PGT135 neutralize ADA.CM and JRFL.TD15 but not CH505.N197D. HGN194 neutralizes ADA.CM but not CH505.N197D or JRFL.TD15. 19b neutralizes ADA.CM and CH505.N197D but not JRFL.TD15.
FIG 2
FIG 2
Production, properties, and antigenicity of mEnv liposomes (MELs). (A, left) Naked liposomes were treated with the mild detergent DDM, which destabilizes the membrane bilayer, and then combined with purified mEnv trimers. (Right) Detergent was depleted from the liposome-mEnv mixture by repeated incubations with Bio-beads, which yields MELs decorated with an array of spikes. Data shown in this and subsequent panels are from JRFL.TD15 MELs, but similar results were observed with ADA.CM and CH505.N197D. (B) Nanoparticle tracking analysis (NTA) reveals liposomes are monodispersed with a mean diameter of 143 nm; MELs containing mEnv remain monodispersed with a mean diameter of 133 nm. (C) Negative-stain EM shows MELs are embedded with mEnv trimeric spikes. (D) ELISA data show that MELs are bound by bNAbs but not by non-NAbs and the anti-CTT antibody Chessie8. MELs were labeled with a 2% molar ratio of biotin-DOPE and captured on streptavidin-coated microwells.
FIG 3
FIG 3
Stability of MELs in solution. (A) Negative-stain EM images of MELs following 0, 48, and 96 h of incubation periods at 37°C. (B) Antibody binding properties of MELs before and after incubation for 168 h at 37°C, as analyzed by streptavidin-capture ELISA as described for Fig. 2D.
FIG 4
FIG 4
Immunization with MELs in rabbits elicited HIV NAbs. (A) Schedule of immunization of NZW rabbits with MELs. Rabbits were immunized with MELs seven times, i.e., ADA.CM (4 times), CH505.N197D (2 times), and JRFL.TD15 (1 time), 6 weeks apart. (B) Kinetics of neutralization of autologous and heterologous tier 1 and tier 2 isolates by MEL immune sera. Mean IC50s of six rabbit sera from each bleed against the three immunizing or autologous isolates and JRFL.WT (top), heterologous tier 1 isolates (middle), and a panel of heterologous tier 2 isolates (bottom). The lowest serum dilution tested was 1:10, so any serum that did not reach an IC50 at a 1:10 dilution has been defined as <10. (C) Kinetics of neutralization of immunizing virus strains by individual rabbit sera.
FIG 5
FIG 5
HIV neutralization breadth and potency of MEL immunized rabbit sera. Sera taken from rabbits were tested in neutralization assays against a cross-clade panel of autologous and heterologous HIV isolates. Data shown are reciprocal serum dilutions at the IC50 from bleeds 4 (after ADA.CM), 6 (after CH505.N197D), and 7 (after JRFL.TD15) for each isolate.
FIG 6
FIG 6
Stability and neutralization sensitivity of HIV JRFL.TD15. (A) Stability of function of JRFL.TD15 and JRFL.WT mEnvs was studied by determining the relative infectivity of cognate pseudovirions incubated for an hour at different temperatures; the temperature at which infectivity is reduced by 90% (T90) is indicated to the left. (B) Neutralization of JRFL.TD15 by narrow neutralizing antibodies against V3 (447-52D, 19b, and HGN194) and CD4BS (F105 and b6), as well as by bNAbs to CD4BS (CH103) and to V2 (PGT145). (C) MEL-immunized rabbit sera were assayed for neutralization against JRFL.WT, JRFL.TD15, and mutants JRFL.TD15.R308H and JRFL.TD15.WTV2, which bear JRFL.WT V3 and V2 domains, respectively.
FIG 7
FIG 7
Antibody binding specificities in MEL antisera revealed by ELISA. (A) Sera from bleeds 4 (post-ADA.CM), 6 (post-CH505.N197D), and 7 (post-JRFL.TD15) were tested for binding to each of the three immunizing mEnvs as well as recombinant gp120 (JRCSF) and gp41 (JRFL) and various antigens and peptides of HIV Env. (B) Sera from the final bleed were tested for the ability to block biotinylated bNAb binding to JRFL.TD15 trimer. The biotinylated bNAbs include those against CD4BS (VRC01), N332 glycan supersite (PGT126), gp120-gp41 interface (3BC176, PGT151, and 35O22), and the MPER (10E8). (C) Alignment of the FP (aa 512 to 530) and MPER (aa 660 to 683) of mEnvs used in the sequential immunization. ADA.CM sequence is in blue, CH505.N197D sequence is in green, and JRFL.TD15 sequence is in red. Residues that differ in one isolate relative to the other two are highlighted in yellow.
FIG 8
FIG 8
ADA.CM neutralization activities in sera 5393 and 5396 target V1 and the gp120-gp41 interface. (A) Serum 5393 from bleed 3, following immunization with ADA.CM, was tested for neutralization of ADA.CM, parental ADA, and an ADA mutant containing an N139/I140 deletion and N142S substitution in V1 of gp120. (B) Four rabbit sera from bleed 4 that neutralized ADA.CM with an IC50 of >10 were tested in neutralization assays against ADA.CM with glycan knockout mutations (N88A, N241S, N611D, and N637K) or glycan hole-filling mutations (D230N/K232T and K289N) near the gp120-gp41 interface.
FIG 9
FIG 9
Serum neutralization of autologous isolates and some heterologous tier 2 isolates cannot be blocked by V3 peptide. Neutralization by monoclonal antibody F425-B4e8 that binds to the V3 crown (A) and sera from the final bleed (B) was tested in a neutralization assay against HIV isolates in the presence or absence of V3 peptide (JRFL sequence, NTRLSIHIGPGRAFYTTGEIIGDI).
FIG 10
FIG 10
MELs elicited potent NAbs to a glycan hole at position 197. (A) Neutralization by sera 5394 and 5396 against a panel of domain-swapped HIV Env chimeras between JRFL and the more resistant JRCSF as well as against N-glycosylation site mutants N197 and N332 of JRCSF. (B) N197D mutants of diverse HIV isolates were tested for neutralization by sera from the final bleed postsequential MEL immunization (left) as well as by a panel of bNAbs and non-NAbs (right). JRFL and JRCSF and their N197 mutants were tested for neutralization by V3 antibody F425-B4e8 and MPER antibody 2F5 rather than 447-52D and 4E10, respectively. (C) Rabbit sera from the final bleed were tested for neutralization against CH505.WT, CH505.N197D, CH505.N197D.N160A (knocks out V2 bNAb neutralization), and CH505.N197S.
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