Vaccination with mRNA-encoded membrane-bound HIV Envelope trimer induces neutralizing antibodies in animal models

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.

Fig. 1.. Rabbit serum antibody responses.

(A) Illustration of MD39.3 mRNA immunogen expression. The cartoon illustrates the in vivo translation of MD39.3 mRNA in transfected cells, producing either soluble or membrane-bound MD39.3 proteins. All MD39.3 variants are designed with furin cleavage independence provided by a Link14 linker and a filled glycan hole achieved through N241/N289 glycans. (B) AUC ELISA response for serum antibodies binding to soluble BG505 MD39.3 gp140. (C) AUC ELISA response for serum antibodies binding to soluble BG505 MD39.3 gp140 in the presence of the base binding mAb RM19R. (D) Ratio of AUC for BG505 MD39.3 gp140 + RM19R over AUC for BG505 MD39.3 gp140 alone. Lower values indicate the presence of more base binding antibodies in the sera. (E) Serum neutralization against BG505 T332N pseudovirus. Bars indicate median values for AUC measurements and neutralization data. Each point indicates a single animal (n=6/group). Statistical significance was assessed using the Kruskal-Wallis test, followed by Dunn’s multiple comparisons test. Significance levels were indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.. Electron microscopy polyclonal epitope mapping of rabbit serum.

(A) Composite 3D map representing the epitopes observed in negative stain EMPEM analysis. (B) Bar graphs showing the number of animals per with detectable antibodies directed to each specific epitope at 10 and 24 weeks post first immunization. Colored to match (A).

Fig. 3.. NHP serum antibody responses.

(A) Three doses of BG505 MD39.3 immunogens were administered at weeks 0, 8 and 24. All doses were administered bilaterally and intramuscularly into the deltoid muscle. PBMCs were isolated from whole blood and LN FNA from axillary LNs. Five groups of six animals per group were immunized with BG505 MD39.3 immunogens. Groups 1–4 received BG505 MD39.3 mRNA immunogens while G5 received BG505 MD39.3 protein plus SMNP adjuvant. The mRNA groups included: soluble MD39.3 (G1), membrane-bound MD39.3 (G2 & G4) and membrane-bound MD39.3 CD4KO (G3). (B) Longitudinal AUC ELISA responses for serum antibodies binding to soluble BG505 MD39.3 gp140. (C) Ratio of AUC for BG505 MD39.3 gp140 BaseKO over AUC for BG505 MD39.3 gp140 WT. Lower values indicate the presence of more base binding antibodies in the sera. (D) Week 26 serum neutralization against BG505 T332N pseudovirus. Bars indicate median values for AUC measurements and neutralization data. Each point indicates a single animal (n=6/group). Groups 1 and 2 were compared for statistical significance using the Mann-Whitney test. Significance levels were indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.. Electron microscopy polyclonal epitope mapping of NHP serum.

(A) Composite 3D map representing the epitopes observed in negative stain EMPEM analysis. (B) Bar graphs showing the number of animals per group with detectable antibodies against each specific epitope at 24 weeks post first immunization. Colored to match (A).

Fig. 5.. MD39.3-binding B cell responses in NHPs.

(A) Representative flow plots show CD20⁺IgD⁻IgM⁻Env^+/+ B_Mem cells (left) and BaseKO⁺ within Env^+/+ (right). (B) Frequency of Env^+/+ B_Mem cells in total B cells in PBMCs at different time points are shown. Responses that are lower than the LOD (0.001375) were set at the LOD. (C) Fold-change of Env^+/+ B_Mem cells per group and time points over baseline Env^+/+ B_Mem frequencies (pre-immunization week −2). (D) Median of Env^+/+ B_Mem cells shown in (B) graphed over time including one additional time point at week 24 (G2 & G5 only). (E) Percentage of BaseKO⁺ within total Env^+/+ B_Mem cells in all groups post-boosts. G2 values were compared with corresponding G1 values for statistical significance. (F) Amount of Env-specific IgG⁺ B_PC in BM aspirates at week 119 (G1 & G5 only). Bars indicate median for B cell frequencies and each point indicates a single animal (n=6/group) except (D) shows median of the respective group. Animals with missing baseline samples were excluded from the fold-change analysis in (C). Groups 1 and 2 were compared for statistical significance using the Mann-Whitney test, followed by the Holm-Šídák multiple comparisons test. Significance levels were indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 6.. MD39.3-specific T cell responses in NHPs.

(A) Env-specific AIM⁺ CD4 T cell responses of G2 and G5 at week 2 (post-prime) and week 26 (post-third). (B) Env-specific AIM⁺ CD8 T cell responses of G2 and G5 at week 2 (post-prime) and week 26 (post-third). Data are shown as background subtracted. Non-responder samples are set at baseline. The dotted black line indicates the limit of quantification (LOQ). Bars represent geometric mean and each point indicate a single animal (n=6/group). Animals with missing or poor-viability samples were excluded from the analysis. Statistical significance was assessed using the Kruskal-Wallis test, followed by Dunn’s multiple comparisons test. Significance levels were indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 7.. Single cell analysis of MD39.3-binding B_Mem cells in NHPs.

(A) Heavy chain mutations in total Env-binding B_Mem cells were assessed for G2 and G5 at week 8, 10, 24, 26 and 32. (B) Heavy chain mutations on non-base-Env-binding B_Mem cells (BaseKO⁺) for the same groups and timepoints as in (A). (C) Total Env-binding B_Mem clonal families were analyzed for groups 2 and 5 across timepoints. Numbers within the Donut plot indicate total number of clonal families detected for each measurement. (D) Non-base-Env-binding B_Mem clonal families were analyzed for the groups and timepoints shown in (C). (E) Uniform manifold approximation and projection (UMAP) visualization of single-cell gene expression profiles identifying clusters among Env-binding B_Mem cells sorted from PBMCs for G2 and G5. Bars indicate median for mutational analysis and each point indicates a single B cell sequence.