Vaccination with mRNA-encoded membrane-bound HIV Envelope trimer induces neutralizing antibodies in animal models
Parham Ramezani-RadiD, Christopher A. CottrelliD, Ester Marina-ZárateiD, Alessia LiguoriiD, Elise LandaisiD, Jonathan L. TorresiD, Amber MyersiD, Jeong Hyun LeeiD, Sabyasachi BabooiD, Claudia T. FlynniD, Katherine R. McKenneyiD, Eugenia SalcedoiD, Xiaoya ZhouiD, Oleksandr KalyuzhniyiD, Erik GeorgesoniD, Nicole PhelpsiD, Danny LuiD, Saman EskandarzadehiD, Sergey MenisiD, Michael KubitziD, Bettina GröscheliD, Nushin B. AlaviiD, Abigail M. JacksoniD, Wen-Hsin LeeiD, Andy S. TraniD, , William R. SchiefiD
Abstract
A protective vaccine against HIV will likely need to induce broadly neutralizing antibodies (bnAbs) that engage relatively conserved epitopes on the HIV envelope glycoprotein (Env) trimer. Nearly all vaccine strategies to induce bnAbs require the use of relatively complex immunization regimens involving a series of different immunogens, most of which are Env trimers. Producing protein-based clinical material to evaluate such relatively complex regimens in humans presents major challenges in cost and time. Furthermore, immunization with HIV trimers as soluble proteins induces strong non-neutralizing responses to the trimer base, which is normally occluded on the virion. These base responses could potentially detract from the induction of nAbs and the eventual induction of bnAbs. mRNA vaccine platforms offer potential advantages over protein delivery for HIV vaccine development, including increased production speed, reduced cost, and the ability to deliver membrane-bound trimers that might facilitate improved immuno-focusing to non-base epitopes. We report the design of mRNA-delivered soluble and membrane-bound forms of a stabilized native-like Env trimer (BG505 MD39.3), initial immunogenicity evaluation in rabbits that triggered clinical evaluation, and more comprehensive evaluation of B cell, T cell, and antibody responses in non-human primates. mRNA-encoded membrane-bound Env immunization elicited reduced off-target base-directed Env responses and stronger neutralizing antibody responses, compared with mRNA-encoded soluble Env. Overall, mRNA delivery of membrane-bound Env appears promising for enhancing B cell responses to subdominant epitopes and facilitating rapid translation to clinical testing, which should assist HIV vaccine development. One Sentence SummaryHIV envelope trimer mRNA enables membrane-bound expression and represents a functional immunogen in pre-clinical mammalian models.
02 /Specification & Classification
Click any tag to browse matching papers- Citations
- 42
- FWCI
- 20.93
- Field percentile
- top 1%
- Organism
- Assay
- Research Focus
- Approach
In-depth summary
Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (25 Jan 2025) · CC BY 4.0
Membrane-bound BG505 MD39.3 Env trimer delivered as mRNA-LNP reduced base-directed antibody responses in rabbits and macaques and elicited neutralizing antibodies, with memory B cell responses documented in macaques.
mRNA vaccines make the antigen in vivo, which allows membrane-bound display but requires trimers that fold correctly without purification. Soluble trimers expose an immunodominant, non-neutralizing base, so this work compares soluble and membrane-bound mRNA-delivered MD39.3 against adjuvanted protein in rabbits and macaques.
Key results
ShownIn rabbits, mRNA-delivered membrane-bound trimers shifted serum IgG away from the trimer base compared with soluble trimers.
Fraction of IgG binding AUC not blocked by base-binding mAb RM19R: 90 to 93% (membrane-bound) vs 65 to 69% (soluble). Kruskal-Wallis P = 0.0008 (MD39.2 gp140 vs gp151) and P = 0.0399 (MD39.3); n=6/group. · Membrane-bound MD39.3 elicited neutralizing and…; Figure legends
ShownIn macaques, membrane-bound MD39.3 mRNA groups showed less base-directed antibody and detectable autologous neutralization in some animals, while soluble mRNA gave none. Against a historical control, filling glycan holes at 241 and 289 appeared to lower neutralization.
30 NHPs, 5 groups of 6. At week 26 no neutralization for soluble gp140 mRNA (G1); all other groups detectable. Historical glycan-hole MD39 protein median 353 vs 19 for glycan-filled MD39.3 protein. · mRNA-delivered membrane-bound trimers elicited…; Figure legends
ShownEnv-binding memory B cells expanded with each dose in macaques and carried extensive somatic hypermutation. Membrane-bound antigen yielded a higher share of non-base-binding memory cells.
Over 95% of Env-binding BMem mutated at week 8 and >99% at week 10 (groups 2 and 5). Median SHM higher at week 24 than week 10 (Mann-Whitney P < 0.0001 for both groups). Fold change over baseline surpassed 100-fold in some animals. · MD39.3 mRNA induced B cell memory responses in…; Sequencing of antigen-binding BMem cells…
ShownEMPEM found base-directed antibodies in every group analyzed. At week 26, trimer-degrading antibodies were seen in both rabbit mRNA groups but not the protein group; in macaques only the membrane-bound mRNA group showed them.
Rabbit week 10: all three groups had trimer-degrading antibodies, most consistently the gp151 mRNA group. NHP week 26: only G2 showed degradation (G1 and G5 did not); non-base/non-degrading antibodies were seen in G2 and G5. EMPEM is qualitative. · EMPEM revealed base-directed and…; mRNA-delivered membrane-bound trimer elicited…
SuggestedMembrane-bound MD39.3 mRNA at the 100-µg dose induced Env-specific CD4 T cell responses comparable to adjuvanted protein, plus detectable CD8 responses in most mRNA animals.
G2 vs G5 at week 2 and week 26, n=6/group: similarly robust AIM+ CD4 responses; cTFH cells at comparable levels; CD8 responses in the majority of G2 animals. Animals with poor-viability PBMCs were excluded. · MD39.3 mRNA induced antigen-specific T cell…; Discussion
Methods, data and reproducibility
Rabbits and rhesus macaques received three intramuscular doses of soluble or membrane-bound MD39.3 mRNA-LNP or adjuvanted protein. Responses were assessed by ELISA with base-knockout probes, neutralization, EMPEM, flow cytometry, T cell assays and single-cell BCR sequencing.
- Rabbit immunization study comparing mRNA-LNP soluble/membrane-bound MD39.2 and MD39.3 with adjuvanted protein controls (weeks 0, 8, 24) · New Zealand white rabbit · n = 6 per group
- NHP study: soluble gp140 mRNA (G1), membrane-bound gp151 mRNA (G2, G4), CD4KO gp151 mRNA (G3), protein plus SMNP (G5) · rhesus macaque (Macaca mulatta) · n = 30 animals, 5 groups of 6
- Historical NHP control group given glycan-hole-containing MD39 protein plus SMNP, used for neutralization comparison · rhesus macaque (Macaca mulatta)
- Bone marrow aspirates for Env-specific plasma cells, G1 and G5 only, more than 1 year after last immunization · rhesus macaque (Macaca mulatta) · n = 3 animals per group with detectable cells (G1, G5)
- Single-cell BCR and gene expression profiling of sorted Env-binding BMem cells (G2 and G5, five timepoints) · rhesus macaque (Macaca mulatta) · GSE00000000; SRX00000000 (as printed)
- Methods & tools
- EMPEM (negative stain), Carterra LSA SPR, Biolayer interferometry, Spectral flow cytometry (Cytek Aurora), AIM/ICS T cell assay, 10x Genomics Chromium 5' v2 or v3, CellRanger, IgBLAST, Change-O (Immcantation), SHazaM, Seurat v5.0.1, Relion 4.0
- Reagents & environment
- Seurat v5.0.1; Prism v10.4.1 (GraphPad); FlowJo v10.8; Relion 4.0; CellRanger de novo option
- Reproducibility gaps
- scRNA-seq accessions appear as placeholders (GSE00000000, SRX00000000); materials on request with possible MTA; no analysis code link; EMDB map IDs not given.
Limitations (5)
- EMPEM gives qualitative epitope information and does not quantify the share of the response aimed at a given site. · stated by the authors
- MD39.3 alone, as a native-like immunogen, is insufficient to elicit broad neutralization. · stated by the authors
- Axillary LN FNAs apparently missed draining nodes, so germinal center B cells were not reliably sampled; germinal center induction is inferred indirectly from somatic hypermutation in circulating memory cells. · stated by the authors
- The glycan-hole comparison uses a historical control with a different route (subcutaneous) and schedule, so it is not a randomized contrast. · noted in this summary
- Groups of six unblinded animals, with T cell and BCR analyses restricted to G2 vs G5 at one mRNA dose, leave mRNA vs protein comparisons with limited statistical power. · noted in this summary
Who should read:Read this if you design mRNA-delivered HIV Env trimer immunogens and want rabbit and macaque data on membrane-bound versus soluble formats.
Reproducibility ledger
Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.
Sign in to file a reproducibility report.
Related papers
- Ability of nucleoside-modified mRNA to encode HIV-1 envelope trimer nanoparticles
Z. Mu, K. Wiehe, … B. F. Haynes·bioRxiv·Aug 9, 2021
- Membrane Env liposomes for immunization with HIV spikes
D. P. Leaman, A. Stano, … M. B. Zwick·bioRxiv·Nov 29, 2020
- CD4 binding-site antibodies induced by a subtype B HIV-1 envelope trimer
A. Schorcht, T. L. G. M. van den Kerkhof, … R. W. Sanders·bioRxiv·Mar 23, 2022
- HIV Env trimers elicit NHP apex cross-neutralizing antibodies mimicking human bNAbs
Javier Guenaga, Mónika Ádori, … Richard T. Wyatt·bioRxiv·Sep 19, 2025
- Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens
J. M. Binley, E. Crooks, … L. Verkoczy·bioRxiv·Jul 20, 2021
- Immunogenicity and Structure of a Stabilized HIV -1 clade C Env from a Pediatric Elite neutralizer in Complex with an Autologous bnAb
Swarandeep Singh, Sanjeev Kumar, … Kalpana Luthra·bioRxiv·Apr 26, 2025