Characterization of HIV-1 Nucleoside-Modified mRNA Vaccines in Rabbits and Rhesus Macaques

Abstract

Despite the enormous effort in the development of effective vaccines against HIV-1, no vaccine candidate has elicited broadly neutralizing antibodies in humans. Thus, generation of more effective anti-HIV vaccines is critically needed. Here we characterize the immune responses induced by nucleoside-modified and purified mRNA-lipid nanoparticle (mRNA-LNP) vaccines encoding the clade C transmitted/founder HIV-1 envelope (Env) 1086C. Intradermal vaccination with nucleoside-modified 1086C Env mRNA-LNPs elicited high levels of gp120-specific antibodies in rabbits and rhesus macaques. Antibodies generated in rabbits neutralized a tier 1 virus, but no tier 2 neutralization activity could be measured. Importantly, three of six non-human primates developed antibodies that neutralized the autologous tier 2 strain. Despite stable anti-gp120 immunoglobulin G (IgG) levels, tier 2 neutralization titers started to drop 4 weeks after booster immunizations. Serum from both immunized rabbits and non-human primates demonstrated antibody-dependent cellular cytotoxicity activity. Collectively, these results are supportive of continued development of nucleoside-modified and purified mRNA-LNP vaccines for HIV. Optimization of Env immunogens and vaccination protocols are needed to increase antibody neutralization breadth and durability.

Keywords: ADCC; HIV-1; mRNA vaccine; neutralizing antibody; nucleoside modification; rhesus macaque.

Figure 1

Antibody Responses after Nucleoside-Modified 1086C Env mRNA-LNP Immunization in Rabbits (A) Schematic of experimental design. Rabbits were immunized intradermally with 50 μg HIV-1 1086C B2 ecto Env or influenza virus A/California/07/2009 HA-encoding nucleoside-modified mRNA-LNPs or poly(C) RNA-LNPs at weeks 0, 6, 18, and 30. (B) Sera were collected at weeks 0, 6, 18, 30, 34, and 38, and the kinetics of 1086C anti-gp120 IgG titers were determined by endpoint dilution ELISA. Vertical black arrows indicate dates of immunizations. Shaded area indicates the limit of detection. n = 5 rabbits and each symbol represents one animal. (C) Neutralization titers (expressed as the reciprocal serum dilution resulting in 50% inhibition of infection) from sera were determined against the MW965.26 (tier 1A) virus. Each symbol represents one animal. Means of values with SEM are displayed. Statistical significance of differences (denoted by asterisks) was determined using two-way ANOVA with Bonferroni multiple comparisons after log transformation of values; p < 0.0001. ** denotes significant difference to *p < 0.005.

Figure 2

Neutralizing Antibody Titers in Rabbits Neutralizing antibodies generated by 1086C B2 ecto Env mRNA-LNP immunizations in rabbits were measured in week 0, 18, and 34 sera by the TZM-bl assay against HIV-1 isolates MW965.26 (tier 1A), Ce1086_B2 (autologous tier 2), and Ce1176_A3 (tier 2). Data are reported as dilution of sera required to inhibit 50% of viral infectivity (ID₅₀) and labeled in blue (21–100), yellow (101–1,000), orange (1,001–5,000), and red (>5,001).

Figure 3

ADCC Activity after Nucleoside-Modified 1086C Env mRNA-LNP Immunization in Rabbits ADCC activity in rabbit sera obtained at weeks 0, 18, and 34 was assessed. (A and B) ADCC responses against the 1086C recombinant gp120-coated target cells (GTL assay). Granzyme B activity (A) and ADCC antibody titers (B) were determined. The cut-off for positivity (indicated with a horizontal dotted line) was ≥8% of granzyme B activity after subtracting the background measured in the uncoated cells. Antibody titers are expressed as the dilution at which the dilution curve interpolate with the 15% cut-off value. Each symbol represents one animal. (C and D) ADCC responses against HIV-infected cells (luciferase-based assay). Specific killing activity (C) and ADCC antibody titers (D) were determined. The analysis of the results was conducted after subtracting the background detected with the pre-immunization (week 0) samples. The horizontal dotted line indicates the cut-off for positivity. n = 5 rabbits and each symbol represents one animal. Means of values with SEM are displayed. Statistical significance of differences (denoted by asterisks) was determined using two-way ANOVA with Bonferroni multiple comparisons after log transformation of values; p < 0.0001. ** denotes significant difference to *p < 0.005.

Figure 4

Antibody Responses after Nucleoside-Modified 1086C Env mRNA-LNP Immunization in Rhesus Macaques (A) Schematic of experimental design. Rhesus macaques were immunized intradermally with 50 μg HIV-1 1086C.DRss Env mRNA-LNPs at weeks 0, 4, 20, 32, and 48. (B) Sera were collected at weeks 0, 4, 8, 24, 34, 40, 50, and 52, and the kinetics of 1086C anti-gp120 IgG titers were determined by endpoint dilution ELISA. Vertical black arrows indicate dates of immunizations. Shaded area indicates the limit of detection. Each symbol represents one animal. (C–E) Neutralization titers (expressed as the reciprocal serum dilution resulting in 50% inhibition of infection) from sera were determined against MW965.26 (C, tier 1A), 92BR025.9 (D, tier 1B), and Ce1086_B2 (E, autologous tier 2). n = 6 monkeys and each symbol represents one animal. Means of values with SEM are displayed. Statistical significance of differences (denoted by asterisk) was determined using one-way ANOVA with Bonferroni multiple comparisons to week 0 samples after log transformation of values; p < 0.0001.

Figure 5

Neutralizing Antibody Titers in Rhesus Macaques Neutralizing antibodies generated by 1086C.DRss Env mRNA-LNP immunizations in rhesus macaques were measured in week 0, 8, 24, 34, 40, 50, and 52 sera by the TZM-bl assay against HIV-1 isolates MW965.26 (tier 1A), 92BR025.9 (tier 1B), Ce1086_B2 (autologous tier 2), 25710-2.43 (tier 2), and Ce1176_A3 (tier 2). Data are reported as dilution of sera required to inhibit 50% of viral infectivity (ID₅₀) and labeled in blue (21–100), yellow (101–1,000), orange (1,001–5,000), and red (>5,001).

Figure 6

Neutralizing Antibody Epitope Mapping in Rhesus Monkey A16334 Epitope-mapping experiments were performed with week 34 serum from rhesus macaque A16334 using pseudoviruses with resistance mutations for known bnAbs targeting various regions of the HIV-1 envelope. Concentration (μg/mL) of bnAbs required to inhibit 50% of viral infectivity (IC₅₀) is shown. Serum neutralization titers are reported as dilution of serum required to inhibit 50% of viral infectivity (ID₅₀). *Positive deflection (44%) was detected for Ce1086_B2.N611A.

Figure 7

ADCC Activity after Nucleoside-Modified 1086C Env mRNA-LNP Immunization in Rhesus Macaques ADCC activity in monkey sera obtained at weeks 0 and 34 was assessed. (A and B) ADCC responses against HIV-1 TV-1 and 1086C recombinant gp120-coated target cells (GTL assay). Granzyme B activity (A) and ADCC antibody titers (B) were determined. The cut-off for positivity (indicated with a horizontal dotted line) was ≥8% of granzyme B activity after subtracting the background measured in the uncoated cells. Antibody titers are expressed as the dilution at which the dilution curve interpolate with the 15% cut-off value. n = 6 monkeys and each symbol represents one animal. Means of values with SEM are displayed. Statistical significance of differences (denoted by asterisk) was determined using two-way ANOVA with Bonferroni multiple comparisons (after log transformation of values in the case of B); p < 0.01.