Comparison of the immunogenicity of mRNA-encoded and protein HIV-1 Env-ferritin nanoparticle designs

Abstract

Nucleoside-modified mRNA technology has revolutionized vaccine development with the success of mRNA COVID-19 vaccines. We used modified mRNA technology for the design of envelopes (Env) to induce HIV-1 broadly neutralizing antibodies (bnAbs). However, unlike SARS-CoV-2 neutralizing antibodies that are readily made, HIV-1 bnAb induction is disfavored by the immune system because of the rarity of bnAb B cell precursors and the cross-reactivity of bnAbs targeting certain Env epitopes with host molecules, thus requiring optimized immunogen design. The use of protein nanoparticles (NPs) has been reported to enhance B cell germinal center responses to HIV-1 Env. Here, we report our experience with the expression of Env-ferritin NPs compared with membrane-bound Env gp160 when encoded by modified mRNA. We found that well-folded Env-ferritin NPs were a minority of the protein expressed by an mRNA design and were immunogenic at 20 µg but minimally immunogenic in mice at 1 µg dose in vivo and were not expressed well in draining lymph nodes (LNs) following intramuscular immunization. In contrast, mRNA encoding gp160 was more immunogenic than mRNA encoding Env-NP at 1 µg dose and was expressed well in draining LN following intramuscular immunization. Thus, analysis of mRNA expression in vitro and immunogenicity at low doses in vivo are critical for the evaluation of mRNA designs for optimal immunogenicity of HIV-1 immunogens.IMPORTANCEAn effective HIV-1 vaccine that induces protective antibody responses remains elusive. We have used mRNA technology for designs of HIV-1 immunogens in the forms of membrane-bound full-length envelope gp160 and envelope ferritin nanoparticle. Here, we demonstrated in a mouse model that the membrane-bound form induced a better response than envelope ferritin nanoparticle because of higher in vivo protein expression. The significance of our research is in highlighting the importance of analysis of mRNA design expression and low-dose immunogenicity studies for HIV-1 immunogens before moving to vaccine clinical trials.

Keywords: HIV-1; bnAb; ferritin NP; gp160; mRNA vaccine.

Fig 1

Protein Env–NP induced higher binding and neutralizing antibody responses than mRNA–LNP in rhesus macaques. (A) Protein and mRNA–LNP prime-boost regimens in rhesus macaques (n = 4). One group of rhesus macaques was primed intramuscularly with 50 µg per dose of CH848 10.17DTe Env–NP mRNA and were boosted with 50 µg per dose of CH848 10.17e Env–NP mRNA at indicated time points. The other group of rhesus macaques was immunized with 100 µg per dose of the same immunogens in the form of purified recombinant protein adjuvanted with 3M-052 and alum following the same regimen. (B) Quantification of serum-binding IgG levels to CH848 10.17 gp120, CH848 10.17DT SOSIP, CH848 10.17 SOSIP, H. pylori ferritin, and human ferritin by ELISA. Each thin line represents an individual rhesus macaque; thick lines represent means in each group. Mann-Whitney U test was performed on the Log-transformed area-under-curve of each group. (C) Quantification of serum autologous tier-2 neutralizing antibody titers 2 weeks after prime or boost by TZM-bl pseudovirus neutralization assay. Data shown are 50% inhibition dilutions (ID50s). Murine leukemia virus Senecavirus A (MLV-SVA) is a negative control virus. LLOD, lower limit of detection; GMT, geometric mean titer of ID50s. (D) Lack of V3-glycan bnAb epitope N332-dependent neutralization activities in mRNA–LNP- and recombinant protein-vaccinated rhesus macaques. Data are from one experiment. *P < 0.05; ns, not significant; Mann-Whitney U test.

Fig 2

CH848 10.17e SOSIP mRNA boost induced similar responses to CH848 10.17e Env–NP boost. (A) Protein and mRNA–LNP prime-boost regimens in rhesus macaques (n = 4 each group). One group of rhesus macaques was primed with 50 µg per dose of CH848 10.17DTe Env–NP mRNA–LNP at indicated time points; the other group of rhesus macaques was primed with 100 µg per dose of the same immunogen in the form of purified recombinant protein adjuvanted with 3M-052 and alum. Both groups of rhesus macaques were boosted with CH848 10.17e SOSIP trimer mRNA–LNP. (B) Serum-binding IgG levels to CH848 10.17 gp120, CH848 10.17DT SOSIP, CH848 10.17 SOSIP, H. pylori ferritin, and human ferritin measured in ELISA. Each thin line represents an individual rhesus macaque; thick lines represent the means in each group. (C) Serum autologous tier-2 neutralizing antibody titers 2 weeks after prime or boost measured in TZM-bl pseudovirus neutralization assay. Murine leukemia virus Senecavirus A (MLV-SVA) is negative control. Data shown are ID50s. LLOD, lower limit of detection; GMT, geometric mean titer. (D) Lack of V3-glycan N332-dependent neutralization activities in mRNA–LNP- and recombinant protein-vaccinated rhesus macaques. (E) ELISA for serum IgG or IgM for anti-dsRNA antibodies. No anti-dsRNA antibodies were induced by mRNA–LNP or by protein. (F) ELISA for serum IgG or IgM for anti-PEG antibodies. Low levels of anti-PEG antibodies were induced by mRNA–LNP but not by protein. *P < 0.05; area-under-curve (AUC) of each group were compared in Mann-Whitney U test. Data are from one experiment.

Fig 3

Characterization of modified mRNA-encoded CH848 10.17DTe Env–NPs. (A) Capillary gel electrophoresis of CH848 10.17DTe Env–NP mRNA showed a single peak at expected size. (B) Minimal levels of dsRNAs were detected in CH848 10.17DTe Env–NP mRNA materials. Exposure time was 6 min. (C) Antigenicity of modified mRNA-encoded CH848 10.17DT Env–NPs that were expressed in 293 F cells and purified by bnAb PGT145. Data are from one experiment.

Fig 4

CH848 10.17DTe Env–NP mRNA–LNP was immunogenic at high dose only in DH270 UCA KI mice. (A) Vaccination regimen. DH270 UCA KI mice were vaccinated with 20, 1, 0.5, or 0.25 µg CH848 10.17DTe Env–NP mRNA–LNP intramuscularly every 2 weeks for three times. (B) Quantification of serum-binding IgG levels by ELISA 1 week after the third vaccination. (C) Quantification of serum autologous tier-2 neutralizing antibody titers 1 week after the third vaccination by TZM-bl pseudovirus neutralization assay. Data shown are ID50s. Horizontal bar: GMT of ID50s. * P < 0.05; ** P < 0.01; ns, not significant; Mann-Whitney U test.

Fig 5

Quantification of CH848 10.17DTe Env–NPs expressed from mRNA in ExpiCHO cells. Modified mRNA-encoded CH848 10.17DTe Env–NPs were purified from transfected ExpiCHO cell supernantant using bnAb 2G12 affinity column. Well-folded Envs show the triangular forms attached to ferritin (central circle) in the 2D classification images above. NSEM analysis and quantification of mRNA-expressed Env–NPs showed that only 0.7% of observed particles were well-folded Env–NPs. Data are representative of two experiments.

Fig 6

CH848 10.17DTe Env–NP mRNA–LNP lacked significant dose-dependent response in rhesus macaques. (A) Vaccination regimen. Rhesus macaques were vaccinated intramuscularly with 50, 70, or 200 µg per dose of mRNA–LNP encoding CH848 10.17DTe Env–NP or 100 µg per dose of the same immunogen as protein adjuvanted in LNP at indicated time points. (B) Quantification of serum-binding IgG levels by ELISA 2 weeks after the third vaccination. (C) Quantification of serum autologous tier-2 neutralizing antibody titers 2 weeks after the third vaccination by TZM-bl pseudovirus neutralization assay. Data shown are ID50s. Horizontal bar: GMT of ID50s. (D) Lack of V3-glycan N332-dependent neutralizing activities in vaccinated rhesus macaques. Data are from one experiment. *P < 0.05; Mann-Whitney U test.

Fig 7

mRNA–LNP-encoded CH848 10.17DT gp160 was more immunogenic than Env–NP. (A) Comparison of CH848 10.17DT SOSIP- and 10.17 SOSIP-specific serum IgG levels induced by gp160 mRNA vs Env–NP mRNA. Sera from 1 week after the third vaccination were tested. (B) Comparison of autologous tier-2 neutralizing antibody titers against CH848 10.17DT pseudovirus induced by gp160 mRNA vs Env–NP mRNA. (C and D) NGS analysis of frequencies of key improbable mutations in DH270 antibody gene induced by 20 or 1 µg of Env–NP mRNA (C) or gp160 mRNA (D). Data shown are percentage of DH270 antibody genes with a specific mutation. Horizontal bar: median. Dotted line: expected mutation frequencies without antigen selection. (E) Standard curve for measuring Env expression by Western blot. Purified recombinant CH848 10.17DT Env–NP was ran in reduced SDS-page electrophoresis and blotted with anti-Env. Gray dotted line: 95% CI. (F) Quantification of Env–NP and gp160 from ExpiCHO cell mRNA transfection by Western blot. Values are interpolated from the standard curve in E. (G) Representative confocal images of mRNA–LNP-encoded gp160 and Env–NP expression in vivo. DH270 UCA KI mice were vaccinated intramuscularly with 20 µg of mRNA–LNP encoding gp160 or Env–NP; draining inguinal lymph nodes were collected 24 h after vaccination for IHC analysis. Blue, IgD; green, CD169; red, antigen stained by bnAb DH270.6. Images of whole LN sections are shown on the left; regions of interest (ROIs) are shown on the right. Scale bar in whole section image: 200 µm; in ROIs: 50 µm. (H) Percent area of lymph node sections covered by gp160 or Env–NP staining quantified by Fiji. Data shown are from 17 LN cryosections from three mice for each group. (I) Mean intensity of gp160 or Env–NP staining per µm² tissue quantified by Fiji. Data shown are from 17 LN cryosections from three mice for each group. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant; Mann-Whitney U test.