Research ArticleHIV

3M-052, a synthetic TLR-7/8 agonist, induces durable HIV-1 envelope–specific plasma cells and humoral immunity in nonhuman primates

See allHide authors and affiliations

Science Immunology  19 Jun 2020:
Vol. 5, Issue 48, eabb1025
DOI: 10.1126/sciimmunol.abb1025
  • Fig. 1 Study design.

    (A) Six groups of RMs (n = 8 per group) were immunized with the HIV-1 clade C–derived 1086.C gp140 immunogen admixed with different adjuvants in groups 1 to 6 (referred to as durability arm of the study). Additional (n = 6) animals were allocated to groups 1 and 4 (also referred to as mechanistic arm of the study). (B) All animals were vaccinated four times with 6-week intervals as shown in the experimental timeline schematic. Blood and BM aspirates were collected from animals at time points indicated for various assays described in the study. n = 6 animals in groups 1 and 4 were euthanized at ~week 22 or 1 month after the final vaccination. Immune responses in all other animals were monitored for close to a year after the final vaccination before sacrifice at ~week 70 in the study. Adjuvant names are color coded for clarity and consistency in all data plots described in the study.

  • Fig. 2 3M-052 adjuvant formulated in PLGA NPs with or without GLA induces high magnitude of Env-specific plasmablasts in peripheral blood and BM-resident LLPCs that are durable for ~1 year.

    Env-specific ASCs were enumerated using a B cell ELISPOT assay. (A) Line graphs indicate frequencies of plasmablast responses in peripheral blood at pre-boost, days 4 and 7 after the second, third, and fourth vaccinations in the study. y axis represents the numbers of Env-specific IgG+ plasmablasts per million peripheral blood mononuclear cells. (B) Line graphs indicate frequencies of Env-specific LLPCs per million BM mononuclear cells (BMMCs) quantified in aspirates collected from iliac crest at indicated time points. (C) Graph highlights frequencies of Env-specific LLPCs per million BMMCs assayed in femur tissue scoop harvested at termination (~week 70) of the study. Statistical significance of differences in magnitude of immune responses was tested with a one-tailed nonparametric Mann-Whitney test for comparing groups 2, 3, and 4 with group 1 as per the primary study objective. All other comparisons were performed using a two-tailed nonparametric Mann-Whitney test using the GraphPad Prism version 8.0 software. Asterisks are color coded to highlight the difference between two distinct treatment groups. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

  • Fig. 3 3M-052 adjuvant formulated in PLGA NPs with or without GLA improves rapidity and durability, respectively, of tier 1A HIV nAb responses in serum up to ~1 year in comparison with the clinically used alum adjuvant.

    Tier 1A HIV-1 pseudovirus neutralizing activity was assayed using a standardized TZM-bl assay. (A) Line graphs indicate the kinetics of tier 1A MW965.26 HIV-1 pseudovirus neutralizing activity in serum. (B) Scatter plots display peak tier 1A MW965.26 HIV-1 pseudovirus neutralization at 2 weeks after the third vaccination or week 14 in the study. (C) Scatter plots display durable tier 1A MW965.26 pseudovirus neutralization at week 70 or close to a year after the fourth vaccination. (D) MB curve of tier 1A HIV-1 pseudovirus nAb responses in treatment groups 1 to 4 is shown. (E) MB curve of tier 1A HIV-1 pseudovirus nAb responses in groups 3 to 6 is displayed to highlight differences between adjuvanting with 3M-052 with or without GLA in comparison with R848 with or without MPL. (F) Graph shows a Spearman correlation of all combined tier 1A MW965.26 HIV-1 pseudovirus nAb response (all treatment groups) versus corresponding frequencies of 1086.C Env gp140–specific LLPCs in the BM. Statistical significance of differences in magnitude of immune responses was tested using a nonparametric Wilcoxon rank sum test (also known as Mann-Whitney U test) using the R software version 3.6.1. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. ID50, 50% inhibitory dose.

  • Fig. 4 3M-052 adjuvant formulated in PLGA NPs with or without GLA significantly improves the magnitude and breadth of binding Ab responses against HIV-1 Env gp140, gp120, and V1V2 epitope–bearing antigens in comparison with other adjuvants in the study.

    Env antigen–specific IgG Ab responses were measured using a BAMA assay. Ab responses are represented as area under the serum titration curve (AUTC) detailed in Methods. Scatter plots represent percentage change in binding Ab responses from peak (week 20) to termination (~week 70) time points against (A) autologous 1086.C gp140 Env antigen, (B) 1086.C gp120 Env antigen, (C) gp41 antigen, and (D) 1086.C V1V2 tag antigen. (E) Binding Ab responses to various HIV-1 Env antigens (n = 24) are summarized by clade groups. (F) Line graphs show group average MB curves highlighting breadth of binding activity against Env gp140 (n = 7) and Env gp120 (n = 8). Statistical differences are color coded to indicate the respective group comparisons. Statistical significance of differences in magnitude of immune responses was tested using a nonparametric Wilcoxon rank sum test (also known as Mann-Whitney U test) using the R software version 3.6.1. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

  • Fig. 5 3M-052 adjuvant formulated in PLGA NPs with or without GLA significantly improves ADCC as well as avidity of Ab responses durable in serum up to ~1 year in comparison with the clinically used alum adjuvant.

    ADCC activity was assayed using an established GTL assay. Avidity of polyclonal sera was assayed on purified serum IgG using a BIAcore SPR assay. (A) Line graphs show kinetics of ADCC titers observed in the study. (B) Scatter plots display an early peak ADCC activity observed after the second vaccination in the study at week 8. (C) Scatter plots show persistent ADCC activity at termination of the study at week 70. (D) Scatter plots display avidity analysis at termination (week 70) against homologous 1086.C Env gp140, 120, and V1V2 antigens. Avidity is calculated by dividing BRUs by inverse of the dissociation rate constant or kOFF values. (E) Scatter plots display kOFF or dissociation rates against homologous, consensus, and heterologous antigens. (F) Scatter plots display avidity scores against homologous, consensus, and heterologous antigens. Statistical significance of differences in magnitude of immune responses was tested using a nonparametric Wilcoxon rank sum test (also known as Mann-Whitney U test) using the R software version 3.6.1. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. ND, not detected.

  • Fig. 6 3M-052 adjuvant formulated in PLGA NPs with GLA induces significantly higher GC B cells, TFH, and Env-specific PC responses in RM draining LNs in comparison with those induced by alum adjuvant.

    Data shown in the figure summarize immune responses at ~week 22 or 4 weeks after the final vaccination in n = 6 animals in the mechanistic arm, assigned to treatment groups 1 and 4 in the study (see study design in Fig. 1). (A) Representative flow cytometry plots are shown, highlighting GC B cells (live, CD3CD20+Ki67+Bcl6+) and GC TFH cells (live, CD3+CD4+ PD-1 high and CXCR5 high). PD-1, programmed death 1. Bar graphs summarize (B) frequencies of GC B cells and (C) frequencies of GC TFH cells. (D) Immunohistochemistry was performed on draining iliac LNs in n = 6 animals from the alum- and GLA+3M-052 NP adjuvant-treated animals, respectively. Green, magenta, and black colors were used to represent staining for IgD, Ki67, and CD3 staining, respectively. Figure represents low-magnification and high-magnification images from alum and GLA+3M-052 NP–adjuvanted animals (see Methods for details). (E) Graph summarizes percent of B cell follicles staining positive for GCs with immunohistochemistry. (F) Graph shows relative increase in CXCL13 chemokine in RM plasma 1 week before and after the final vaccination. (G) Graph summarizes Env-specific PCs (B cell ELISPOT assay) per million live mononuclear cells in various draining and nondraining LNs. Statistical significance of differences in magnitude of immune responses was tested with a two-tailed nonparametric Mann-Whitney test using the GraphPad Prism version 8.0 software. **P < 0.01, *P < 0.05.

  • Fig. 7 3M-052 adjuvant formulated with alum induces robust and durable Env-specific LLPCs in BM comparable with those induced when formulating in PLGA NPs and also significantly higher than alum adjuvant.

    (A) Study design details treatment groups with various adjuvants and Env vaccinations in the second study. (B) Experimental timeline details blood and BM collections as well the sacrifice time point in the study. (C) Env-specific LLPC responses were enumerated using a B cell ELISPOT assay. Line graphs indicate frequencies of Env-specific LLPCs per million BMMCs quantified in aspirates collected from iliac crest at indicated time points. (D) Graph highlights frequencies of Env-specific LLPCs per million BMMCs assayed in femur tissue scoop harvested at termination (~week 65). (E) Enrichment of Env-specific IgG+ LLPCs in BM within total IgG+ ASCs is highlighted for the peak and termination time points. Statistical significance of differences in magnitude of immune responses was tested with a one-tailed nonparametric Mann-Whitney test comparing groups 2 and 3 versus 1 and two-tailed nonparametric Mann-Whitney test comparing the rest of the groups using the GraphPad Prism version 8.0 software. Asterisks are color coded to highlight the difference between two distinct treatment groups. **P < 0.01, *P < 0.05. n.s, not significant.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/48/eabb1025/DC1

    Methods

    Fig. S1. Representative ELISPOTs, frequencies of Env-specific ASCs/total IgG+ ASCs and LN LLPCs.

    Fig. S2. Avidity scores and dissociation rate comparison across all groups at termination.

    Fig. S3. Flow cytometry gating strategy in identifying LN GC B cells and TFH in Fig. 6.

    Fig. S4. Whole LN images (at low magnification) for all animals euthanized ~4 weeks after the final vaccination in study #1.

    Fig. S5. Correlation of LN GC B cells, TFH, LN, and BM PCs with nAbs at ~4 weeks after the final vaccination in study #1.

    Fig. S6. Env-specific CD4+ T cell responses in study #1 by intracellular cytokine staining.

    Fig. S7. Change in frequencies of CCR5+CD4+ T cells in rectal mucosa after vaccinations in study #1.

    Fig. S8. Flow cytometry gating strategy in identifying monocyte subsets in fig. S9.

    Fig. S9. Change in frequencies of monocyte subsets and activation markers after primary vaccination in study #1.

    Fig. S10. Antibody responses (nAb, ADCC, and avidity) in study #2.

    Fig. S11. Binding Ab responses against Env gp140, g120, V1V2, and gp41 by BAMA assay in study #2.

    Fig. S12. Change in frequencies of monocyte subsets and activation markers after primary vaccination in study #2.

    Table S1. Adjuvant characterization.

    Data file S1. Primary data.

    References (6675)

  • Supplementary Materials

    The PDF file includes:

    • Methods
    • Fig. S1. Representative ELISPOTs, frequencies of Env-specific ASCs/total IgG+ ASCs and LN LLPCs.
    • Fig. S2. Avidity scores and dissociation rate comparison across all groups at termination.
    • Fig. S3. Flow cytometry gating strategy in identifying LN GC B cells and TFH in Fig. 6.
    • Fig. S4. Whole LN images (at low magnification) for all animals euthanized ~4 weeks after the final vaccination in study #1.
    • Fig. S5. Correlation of LN GC B cells, TFH, LN, and BM PCs with nAbs at ~4 weeks after the final vaccination in study #1.
    • Fig. S6. Env-specific CD4+ T cell responses in study #1 by intracellular cytokine staining.
    • Fig. S7. Change in frequencies of CCR5+CD4+ T cells in rectal mucosa after vaccinations in study #1.
    • Fig. S8. Flow cytometry gating strategy in identifying monocyte subsets in fig. S9.
    • Fig. S9. Change in frequencies of monocyte subsets and activation markers after primary vaccination in study #1.
    • Fig. S10. Antibody responses (nAb, ADCC, and avidity) in study #2.
    • Fig. S11. Binding Ab responses against Env gp140, g120, V1V2, and gp41 by BAMA assay in study #2.
    • Fig. S12. Change in frequencies of monocyte subsets and activation markers after primary vaccination in study #2.
    • Table S1. Adjuvant characterization.
    • References (6675)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article