Research ArticleHIV

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

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Science Immunology  19 Jun 2020:
Vol. 5, Issue 48, eabb1025
DOI: 10.1126/sciimmunol.abb1025

Live long and protect

While vaccines elicit robust short-term responses, maintaining sustained and durable antibody responses to vaccines remains a challenge. Durability of antibody responses not only depends on the antigen but also on the adjuvants used in the vaccine. Here, Kasturi et al. have used a nanoparticle and a clinically applicable alum adsorbed formulation of 3M-052, a TLR7/8 agonist to promote sustained antibody responses to an HIV-1 envelope (Env) protein vaccine in rhesus macaques. They report that 3M-052–adjuvanted Env vaccination led to the development of durable antibody responses that were readily detectable a year after immunization, and they attribute this to the ability of 3M-052 to prime and sustain long-lived plasma cell responses. 3M-052 now joins an elite set of adjuvants that could help shape vaccination in the 21st century.

Abstract

A fundamental challenge in vaccinology is learning how to induce durable antibody responses. Live viral vaccines induce antibody responses that last a lifetime, but those induced with subunit vaccines wane rapidly. Studies in mice and humans have established that long-lived plasma cells (LLPCs) in the bone marrow (BM) are critical mediators of durable antibody responses. Here, we present data that adjuvanting an HIV-1 clade C 1086.C–derived gp140 immunogen (Env) with a novel synthetic Toll-like receptor (TLR)–7/8 agonist named 3M-052 formulated in poly(lactic-co-glycolic)acid or PLGA nanoparticles (NPs) or with alum, either alone or in combination with a TLR-4 agonist GLA, induces notably high and persistent (up to ~1 year) frequencies of Env-specific LLPCs in the BM and serum antibody responses in rhesus macaques. Up to 36 and 18% of Env-specific cells among total IgG-secreting BM-resident plasma cells were detected at peak and termination, respectively. In contrast, adjuvanting Env with alum or GLA in NP induced significantly lower (~<100-fold) LLPC and antibody responses. Immune responses induced by 3M-052 were also significantly higher than those induced by a combination of TLR-7/8 (R848) and TLR-4 (MPL) agonists. Adjuvanting Env with 3M-052 also induced robust activation of blood monocytes, strong plasmablast responses in blood, germinal center B cells, T follicular helper (TFH) cells, and persistent Env-specific plasma cells in draining lymph nodes. Overall, these results demonstrate efficacy of 3M-052 in promoting high magnitude and durability of antibody responses via robust stimulation of innate immunity and BM-resident LLPCs.

INTRODUCTION

Vaccination has had a profound impact on preventing infectious diseases, but one major challenge remains in learning how to develop durable immunity against global pandemics such as HIV, malaria, and tuberculosis (1). Although live attenuated virus-based smallpox or yellow fever vaccines induce durable antibody (Ab) responses that can last a lifetime, waning immunity has been documented with inactivated and subunit vaccines against HIV, malaria, influenza, Bordetella pertussis, Salmonella enterica serovar Typhi, and Neisseria meningitidis, which represents a major shortcoming in inducing long-lasting protective immunity (2, 3). Specifically, in a phase 3 HIV vaccine clinical trial (the RV144 trial), vaccine efficacy of ~60% during the first year rapidly declined to ~31% by 42 months, again highlighting the lack of durable immunity (46). Therefore, a great challenge in vaccinology is learning how to program the immune system to yield long-lived Ab response and plasma cells (PCs) that last a lifetime (7).

During an immune response, activated antigen-specific B cells either differentiate into short-lived PCs, producing low-affinity Abs, or initiate germinal center (GC) reactions (8), which produce high-affinity memory B cells (9) and long-lived PCs (LLPCs) that secrete high-affinity Abs (1012). LLPCs migrate to the bone marrow (BM) where they can reside for extended periods and produce Abs, there maintaining the levels of Abs in serum (1314). Hence, understanding immunological mechanisms that regulate the generation of LLPCs is key to developing vaccines that elicit durable protective humoral immunity.

Adjuvants are an important component of subunit vaccines and can substantially improve the magnitude and durability of Ab responses (15). It is well established that adjuvants activate the innate immune system, which, in turn, enhances magnitude and durability of antigen-specific T and B cell responses induced by vaccines (3, 15). In particular, recent research has highlighted a new generation of molecular adjuvants targeted to pathogen recognition receptors on dendritic cells (DCs). Specific agonists of various Toll-like receptors (TLRs) hold much promise as vaccine adjuvants (3, 15, 16). In this regard, the live attenuated yellow fever vaccine YF-17D induces innate activation by signaling via multiple TLRs on distinct subsets of DCs, and this appears to be essential for its immunogenicity, highlighting the importance of TLR signaling in inducing effective immune responses (17). Our previous studies in mice have demonstrated that multiple TLR agonists delivered in poly(lactic-co-glycolic)acid (PLGA) nanoparticles (NPs) could induce notably enhanced GC and Ab responses (18, 19). In addition, we and others have reported the potency of TLR agonists as vaccine adjuvants in rhesus macaques (RMs), a clinically relevant nonhuman primate (NHP) model (1826). The TLR-4–targeted monophosphoryl lipid A (MPL) formulated with alum has been licensed for clinical use with subunit vaccines against the human papillomavirus (HPV) (27) and herpes zoster (Shingrix) (28). TLR agonists are also being evaluated for use in cancers (15, 29). However, there is currently a paucity of data on the longevity of the Ab responses induced by these adjuvants and, in particular, in their capacity to induce LLPCs in humans or in NHPs.

Here, we describe data from two RM studies involving a total of 90 animals, highlighting the adjuvanticity of 3M-052, a novel synthetic TLR-7/8 agonist used by itself or in combination with a synthetic TLR-4–targeted agonist glucopyranosyl lipid adjuvant (GLA). The synthetic imidazoquinolinone 3M-052 is structurally similar to resiquimod (R848), a small molecule TLR-7/8 agonist; however, 3M-052 has an 18-C fatty acyl chain that confers enhanced hydrophobicity, reduced systemic dissemination, and hence, improved bioavailability at the site of immunization and in draining lymphoid organs (30). Specifically, in the first study, we report notably superior immune responses with the 3M-052 adjuvant with or without GLA formulated in PLGA-based NPs when used with 1086.C, an HIV-1 clade C–derived gp140 immunogen (Env) in comparison with immune responses induced by GLA alone in NPs or by alum, an adjuvant approved for human use. The Env immunogen used here is currently a component as gp120 of the HVTN 702 ALVAC-bivalent gp120 efficacy trial (NCT02968849) (31). We also report a successful induction and maintenance of Env-specific LLPCs for 1 year after vaccination with the 3M-052 adjuvant. Furthermore, these responses were also significantly superior to those induced by R848 or a combination of R848 and TLR-4 agonist MPL. Last, we report reproducible innate and Env-specific B cellular and serological responses in an independent RM study not only with PLGA NP–formulated 3M-052 but also with a novel alum-adsorbed 3M-052 formulation that is currently being developed for use in human clinical trials.

RESULTS

Study design

Our first preclinical study involved a total of 60 RMs distributed among six cohorts (n = 8 per cohort for longitudinal analyses and n = 6 each in groups 1 and 4 allocated for necropsy at 4 weeks after the last vaccination). All RMs were immunized with Env and adjuvants as detailed in Fig. 1A. Male and female RMs (n = 30 each) were equally distributed among the experimental groups. Animals in group 1 were immunized with Env and alum, a benchmark adjuvant used in many licensed vaccines. Animals in groups 2, 3, and 4 were immunized with soluble Env and GLA, 3M-052, and a combination of GLA+3M-052, respectively, wherein all adjuvants were formulated in PLGA NPs. Animals in groups 5 and 6 were immunized with soluble Env and R848 and a combination of MPL and R848, respectively, also formulated in PLGA NPs. All treatment groups with TLR agonists in NPs will be referenced with the suffix NP for brevity. All animals received four immunizations at weeks 0, 6, 12, and 18. Blood and BM samples were analyzed for immune responses at various time points for up to 70 weeks after the final immunization (Fig. 1B).

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.

The primary objective of this study was to investigate whether immune responses stimulated by GLA NP, 3M-052 NP, or the combination of GLA and 3M-052 in NP were superior to those induced by alum and to identify the best candidate adjuvant that promotes high magnitude and durable Ab responses to the Env immunogen. The secondary objective of the study was to investigate whether immune responses stimulated by 3M-052 or the combination of GLA and 3M-052 were superior to those induced by the first generation of TLR-4 and TLR-7/8 agonists or MPL and R848, respectively. Hence, the statistical tests used to establish significance of differences in immune responses were designed to address these two study objectives as detailed in Methods.

Alum was used in the study at a dose of 500 μg as recommended and used previously in RMs (19, 32). All TLR-targeted agonists were formulated in PLGA-based NPs (table S1) (18, 19). 3M-052, an 18-C fatty acyl chain–bearing hydrophobic imidazoquinoline, was designed to facilitate improved incorporation of the molecule in emulsion or liposome-based formulations or synthetic particles (30). Hence, we observed increased loading efficiency of 3M-052 in NPs (up to 100% of target loading) in comparison with previously reported ~50% loading of the R848 molecule (table S1) (18, 19). Similar ~100% loading efficiency of hydrophobic purified or synthetic MPL in PLGA particles has been reported previously and used accordingly (table S1) (33). Experimental dose of R848 (750 μg) was similar to that used previously in RMs (21, 25). MPL dose (50 μg) was chosen on the basis of the dose used with the HPV vaccine (34). The initial dose of 3M-052 and GLA with primary immunization was also chosen to match the dose of R848 and MPL at 750 and 50 μg, respectively. However, because of localized reactions at the injection site several weeks after the primary vaccination (swelling, redness, and ulceration) in 2 of 8 and 4 of 14 total animals in groups 3 and 4, respectively, the doses of 3M-052 and GLA were scaled down 10-fold to 75 and 5 μg, respectively, for the second, third, and fourth immunizations. Because these reactions were observed during the second immunization, a small group of animals in groups 2 (n = 4), 3 (n = 2), and 4 (n = 2) received the original high dose before the 10-fold reduction of the GLA and 3M-052 doses for all subsequent doses. One animal included in the listed number above displayed a reaction only after this second high-dose vaccination. Also, for the second immunization alone, vaccinations were administered in the contralateral flanks for animals receiving the 3M-052 adjuvant to avoid potential aggravation or induction of localized reaction. All animals were included in statistical analyses because no impact was observed in long-term immune responses between animals within a single treatment regimen with these dose reductions.

Vaccination with Env and 3M-052 or GLA+3M-052 NP induces robust plasmablast responses and durable BM LLPCs in RMs

Recall responses upon secondary vaccinations lead to rapid generation of plasmablasts in peripheral blood both in humans and RMs (19, 35). Here, we evaluated the kinetics of Env-specific plasmablast responses induced by immunization with GLA and/or 3M-052, using an Env-specific B cell enzyme linked immune absorbent spot (ELISPOT) assay (fig. S1A). We observed a rapid and notable increase in numbers of Env-specific plasmablasts 4 days after each of the booster vaccinations relative to preimmunization time points in all treatment groups (Fig. 2A). Although no significant difference in the magnitude of these plasmablast responses was observed at day 4 after the second vaccination in animals receiving the 3M-052 adjuvant in comparison with alum or GLA adjuvants, significantly higher frequencies of Env-specific plasmablasts were observed after the third and the fourth vaccinations (Mann-Whitney test at 4 days after fourth vaccination, P = 0.0001 and 0.0003 for 3M-052 NP and P = <0.0001 and 0.0002 for GLA+3M-052 NP versus alum and GLA NP, respectively). These responses were similar at day 4 time points across all animals receiving either the R848 or the 3M-052 adjuvants. However, plasmablast responses contracted rapidly by 7 days to less than 10 spots per million mononuclear cells in animals vaccinated with alum or GLA NP and also substantially decreased in animals vaccinated with R848 NP or MPL+R848 NP (Fig. 2A) (~8-fold change in median frequencies after the second and third vaccinations versus 10- to 23-fold change after the fourth vaccination) confirming previous observations (19). In contrast, noticeably persistent Env-specific plasmablast frequencies at day 7 (less than threefold contraction after the third and fourth vaccinations) were observed selectively in animals treated with 3M-052 NP or GLA+3M-052 NP adjuvants, respectively (Fig. 2A). Responses at day 7 in animals were significantly higher once again, selectively only in animals vaccinated with 3M-052 NP (P = <0.0001 versus alum, P = 0.0002 versus GLA NP and P = 0.0281 versus R848 NP after the fourth vaccination; Mann-Whitney test) and GLA+3M-052 NP (P = <0.0001 versus alum and GLA-NP and P = 0.0001 versus MPL+R848 NP). We have recently reported identical kinetics with changes in frequencies of Env-specific immunoglobulin A (IgA)–secreting plasmablasts (~twofold lower in absolute magnitude) and very low levels of Env-specific IgM induced in RMs when using the MPL+R848 NP adjuvant (19), and hence, no Env-specific IgM+ plasmablast responses were assayed in the current 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.

LLPCs in the BM are critical for sustained Ab responses (10, 11, 36). We assayed for Env-specific LLPCs in BM aspirates collected over the duration of the study (Fig. 2B). While a transient yet significant increase in Env-specific plasmablasts was observed in peripheral blood in RMs vaccinated with Env and alum or GLA-NP at 4 days after boost vaccinations, very minimal accumulation of Env-specific LLPCs in the BM was observed when vaccinating with these adjuvants (Fig. 2, B and C). In contrast, a notably higher frequency of Env-specific LLPCs was observed in animals vaccinated with 3M-052 NP or GLA+3M-052 NP, with median frequencies per million mononuclear cells at 472.5 (min, max: 27, 729) and 513 (min, max: 63, 1782), respectively, observed 5 weeks (at week 23) after the final vaccination. These responses were significantly higher in comparison with those induced by alum and GLA NP, respectively (P = <0.0001 for 3M-052 NP versus alum and P = 0.0002 versus GLA NP and P = 0.0002 for GLA+3M-052 NP versus alum and P = 0.0003 versus GLA NP]. Env-specific LLPCs remarkably persisted in these 3M-052 NP– or GLA+3M-052 NP–vaccinated animals at median frequencies per million mononuclear cells at 445.5 (min, max: 87, 729) and 702 (min, max: 162 and 2025), respectively, at termination at week 70. These responses were again significantly higher in comparison with those induced by alum and GLA NP (P = <0.0001 for both 3M-052 NP and GLA+3M-052 NP versus alum and P = 0.0002 versus GLA NP, respectively). Furthermore, we observed an additive to synergistic impact on enrichment of Env-specific IgG+ LLPCs within all IgG-secreting BM PCs (fig. S1, B and C). In particular, vaccination with GLA+3M-052 NP constituted a median frequency of 20.24% (min, max: 5.6 and 36.70%) of all total IgG+ BM PCs at week 23 (peak after the final vaccination). This persisted at 6.7% (min, max: 1.7 and 18.12%) at ~1 year after the final vaccination. The LLPC response observed when vaccinating with GLA+3M-052 NP was only slightly improved than that seen when vaccinating with 3M-052 NP both at peak and termination time points. Last, in addition to LLPCs in BM, we also detected persistent PC responses in the primary draining iliac lymph nodes (LNs) close to ~1 year after the final vaccination (fig. S1, D and E). In summary, these data demonstrate the potent adjuvanticity of 3M-052 in inducing superior magnitude and durability of blood plasmablasts and BM-resident LLPCs in comparison with other adjuvants used in the study.

Vaccination with Env and 3M-052 or GLA+3M-052 NPs induces enhanced and durable tier 1A HIV-1 virus neutralizing Ab responses in RMs

We next evaluated Env-specific neutralizing Ab (nAb) responses in serum. We observed a robust tier 1A MW965.26 clade C HIV-1 nAb response in animals immunized with 3M-052 NP or GLA+3M-052 NP that was durable for 1 year after the final vaccination (Fig. 3A). Specifically, we observed an early and significantly higher nAb response in animals immunized with Env + GLA+3M-052 NP at week 14, in comparison with animals immunized with Env + 3M-052 NP (sixfold higher, P = 0.017) or GLA NP (47-fold higher, P < 0.001) (Fig. 3B). These results are consistent with our work and other studies previously reported in mice and mini pigs, highlighting an advantage of combining TLR-4 and TLR-7/8 adjuvants to induce higher Ab responses (18, 37, 38). However, at week 70, the magnitude of nAb response was highest and comparable in animals immunized with Env + GLA+3M-052 NP or 3M-052 NP (Fig. 3C). Breadth of tier 1 HIV-1 virus neutralizing activity against MW965.26, MN.3, and SF162.LS was calculated as the average of the log10 nAb titer over the panel of isolates and plotted as magnitude-breadth (MB) plots as described previously (39) and shown in Fig. 3 (D and E). These combined analyses continued to highlight higher nAb responses in animals receiving 3M-052 or the combination of GLA+3M-052 NP adjuvants in comparison with alum, GLA NP, R848, and MPL+R848 NP adjuvants, respectively. Last, a significant correlation (Spearman r = 0.8064, P = 0.0001) overall was observed between the frequencies of Env-specific BM LLPCs and tier 1 MW965.26-specific nAb titers (Fig. 3F). These results highlight the adjuvanticity of 3M-052 to induce a high magnitude and durable nAb response in RMs.

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.

Vaccination with Env and 3M-052 or GLA+3M-052 NPs induced enhanced and durable HIV-1 Env binding Ab responses in RMs

An optimal panel of antigens composed of Env gp140, gp120, gp41, and V1V2 antigenic domains have recently been downselected to guide mapping of Ab responses with HIV Env vaccinations (40). We assayed for HIV-1–specific IgG Ab responses against this panel of antigens using the binding Ab multiplex assay (BAMA assay) (Fig. 4, A to D). Vaccination with Env + 3M-052 NP or GLA+3M-052 NP induced significantly higher magnitude of binding Ab titers against the whole homologous 1086.C gp140 immunogen (Fig. 4A), the gp120 and gp41 antigenic domains (Fig. 4, B and C), and against the V1/V2 epitope (Fig. 4D) at 2 weeks after the final vaccination (week 20) (P values between 0.01 and <0.0001 for all antigens assayed between groups 3 and 4 versus 5 and 6 and 1 and 2, respectively, as shown in the figures). A notable aspect of these data was that the magnitude of persistent Env Ab responses in 3M-052 NP– and GLA+3M-052 NP–adjuvanted animals at week 70 was significantly higher than those induced by alum and GLA NP at week 20, the peak time point in the study (Fig. 4, A to D). One such example worth highlighting is the magnitude of Ab response against the homologous 1086.C V1V2 antigen (Fig. 4D). At week 70, Ab titers in animals vaccinated with 3M-052 and GLA+3M-052 NP were 30,681 (min, max: 15,564, 43,804) and 34,813 (min, max: 17,543, 57,919) higher than the median magnitude of Ab responses in animals receiving alum and GLA NP at week 20, which were 15,653 (min, max: 5279, 36,679) and 8043 (min, max; 1065, 16,825), respectively. Enhanced responses in 3M-052–adjuvanted animals were observed not only against the whole Env or its antigenic domains but also against a panel of HIV-1 antigens representing most circulating clades of HIV-1 (Fig. 4E). Group average MB curves were also used to display the breadth of binding Ab activity against various gp140 (n = 7) and gp120 (n = 8) antigens (Fig. 4F). These data demonstrate that 3M-052 adjuvant by itself or in combination with GLA+3M-052 in PLGA NPs induced enhanced and long-lived IgG binding Ab responses against a large panel of HIV-1 Env antigens.

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.

Vaccination with Env and 3M-052 or GLA+3M-052 NPs induces enhanced and durable ADCC activity in RMs

Emerging evidence in both preclinical and clinical studies points to a role for non-neutralizing Fc receptor–mediated effector functions of HIV-specific Abs as a complementary mechanism of protection to neutralization responses (4144). We thus measured serum Ab-dependent cellular cytotoxicity (ADCC) activity against the whole 1086.C gp120 immunogen–coated target cells using a standardized Granzyme Toxi Lux (GTL) assay (45). Env-specific ADCC activity was induced in RMs immunized with Env + 3M-052 NP (Fig. 5, A to C). Similar to rapid induction of nAb titers observed in animals immunized with GLA+3M-052 NP, we noted that enhanced ADCC responses were detected as early as week 8 in those animals that were immunized with GLA+3M-052 NP in comparison with responses in animals receiving 3M-052 NP (2.9-fold higher, P = 0.13) or GLA NP (180-fold higher, P = 0.049) alone (Fig. 5, A and B). These responses were also higher than those induced by alum (~21-fold higher) and the combination of MPL+R848 in NPs (~6-fold higher). Notable durability and enhanced ADCC titers (100% response rates) were observed for the entire duration of the study (Fig. 5, A and C) only when vaccinating with Env + 3M-052 NP or GLA+3M-052 NP. In contrast, a substantial contraction in responses was observed as early as 3 months after the final vaccination in animals immunized with alum or with GLA NP, with only 37.5 and 0% animals responding at termination, respectively (Fig. 5C).

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.

In addition to ADCC activity, avidity of Env-specific Ab responses has previously been correlated with their ability to protect against intrarectal simian immunodeficiency virus or simian-human immunodeficiency virus challenges in macaques (46, 47). We performed a surface plasmon resonance (SPR)–based comparison of avidity of Env-specific Ab responses. Avidity was assayed against three homologous (1086.C gp120, gp140, or V1V2) antigens, consensus gp120 and gp140, and two heterologous gp120 antigens at week 20 (peak) and at week 70 in the study. We report both kd or dissociation rate, which is a parameter independent of the magnitude of the response, and the avidity score, which is dependent on the magnitude of the response at a given time point, calculated as a ratio of binding response units (BRUs) and kd. Consistent with significant differences observed in the magnitude of binding Ab responses in Fig. 4, we observed differences with avidity scores of Ab responses in animals receiving 3M-052 or GLA+3M-052 adjuvants in comparison with alum or GLA NPs at week 70, where an Ab response is expected to have matured over time (Fig. 5D and fig. S2A). Specifically, avidity score with responses against the homologous 1086.C gp120 was ~100-fold higher in animals receiving the GLA+3M-052 NPs in comparison with those receiving alum (Fig. 5D). Similar responses in these animals were observed against both the 1086.C gp140 and the 1086.C V1V2 antigens. Avidity scores against the 1086.C V1V2 antigen in animals immunized with the 3M-052 adjuvants were also significantly higher than those achieved by R848 or MPL+R848 NP, respectively (P = 0.0283, 3M-052 versus R848, and P = 0.0007, GLA+3M-052 versus MPL+R848), whereas no binding responses were detected when vaccinating with alum or GLA NP highlighting rapid decay. Furthermore, this notable difference in avidity score was predominantly due to differences in kd values highlighting increased avidity independent of the magnitude of the response (fig. S2, C and D). Both avidity score and kd responses were highest when stratifying responses against homologous antigens followed by consensus and heterologous antigens (Fig. 5, E and F). Overall, these data highlight the ability of 3M-052 to promote persistent high-avidity responses against Env immunogens in RMs.

Vaccination with Env + GLA+3M-052 NP induces an enhanced GC response in RM draining LNs in comparison with that induced by alum

Combination of TLR-4 and TLR-7 agonists significantly enhances GC responses in mouse draining LNs (18). Here, we investigated GC B cell and T follicular helper (TFH) responses in draining and nondraining LNs of animals immunized with Env + GLA+3M-052 NP or alum at ~4 weeks after the fourth immunization (Fig. 1B). GC B and TFH cells were identified by flow cytometry as described previously (48, 49) (fig. S3). A high frequency of GC B cells was induced in both the draining iliac and proximal popliteal LNs of animals vaccinated with the GLA+3M-052 NP, and this was significantly higher (P = 0.0022 and P = 0.0260 for right iliac and popliteal LNs, respectively) than that induced by alum (Fig. 6B). A corresponding and significant increase in GC TFH (P = 0.0043, GLA+3M-052 versus alum) was also observed in the draining iliac LN (Fig. 6C). Visualization of these responses by immunohistochemistry revealed a remarkably high proportion of follicles with GCs in animals vaccinated with the GLA+3M-052 NP (Fig. 6D and fig. S4). These GC frequencies were significantly higher (P = 0.0043) than those induced by alum (Fig. 6E).

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.

Because LNs after immunizations were only available from animals in groups 1 and 4, it was unclear whether GC responses were also induced in other groups in the study. CXCL13 in plasma can be indicative of GC activity in LNs (50). This biomarker provided us an opportunity to assess GC activity without having to sacrifice animals. We assayed the concentration of CXCL13 in serum in all animals before and after the final vaccination. Significant increases in CXCL13 1 week after the final vaccination in plasma were observed in animals immunized with Env and 3M-052 NP or GLA+3M-052 NP or in animals immunized with Env and R848 or MPL+R848 NP (Fig. 6F). No appreciable increase in CXCL13 was observed in animals receiving alum or GLA NP, consistent with the weaker Ab response in these animals.

A significantly higher frequency of Env-specific PCs was observed in draining iliac and popliteal LNs in animals vaccinated with Env + GLA+3M-052 NP (Fig. 6G), highlighting differential drainage of the vaccine and priming of Env-specific responses. Few Env-specific PCs were observed in the spleen, highlighting a more localized response when vaccinating subcutaneously. Last, a notable correlation was observed between GC B cells, TFH cells, and BM PC responses with the corresponding tier 1A clade C and heterologous clade B neutralizing responses (fig. S5).

In addition to inducing a strong GC TFH response in draining LNs, both 3M-052 and GLA+3M-052 NP adjuvants induced enhanced Env-specific CD4+ T cell responses in peripheral blood both after primary and booster vaccinations in comparison with other groups in the study (fig. S6). We have previously reported very low or no appreciable induction of CD8+ T cells in RMs when vaccinating with soluble protein immunogens and TLR agonists (19). We also investigated any potential increases in frequencies of CCR5+CD4+ T cells in the rectal mucosa after vaccinations with Env and adjuvants in the study (fig. S7). In contrast with increased Env-specific immune responses observed when vaccinating with Env and 3M-052 NP, no significant changes in frequencies of CCR5+CD4+ T cells were observed after each round of vaccination in comparison with baseline frequencies. Overall, these data suggest that the 3M-052 adjuvant with or without GLA in NPs is capable of orchestrating strong B cellular and serological responses in RMs without enhancing the frequency of target CD4+ T cells in the rectal mucosa.

Vaccination with Env + GLA+3M-052 NP induces persistent activation of monocytes (up to 2 weeks) in peripheral blood

Immunization of RMs with TLR agonists has previously highlighted activation of DCs and monocytes in the blood (19, 21). Immunization with Env + 3M-052 NP or GLA+3M-052 NP induced a rapid increase in the proportion of CD14+CD16+ intermediate monocytes (figs. S8 and S9, A and B). In contrast, frequencies of classical and intermediate monocytes were minimally changed after vaccination with Env + alum or GLA NP. In addition, vaccination with 3M-052 NP or GLA+3M-052 NP induced enhanced expression of CD86 and/or CCR7 on all monocyte subsets that persisted for at least 2 weeks after primary vaccination (fig. S9C). We have also recently reported transient increase in frequencies of plasmacytoid DCs (pDCs) and conventional DC subset #1 (BDCA-1 or CD1c expressing DC) at day 1 followed by increased and peak activation (CD86 and CCR7) of these DC subsets at day 2 after primary vaccination when vaccinating with TLR agonists in NP (19). Overall, these data highlight a critical role for TLR-7/8 agonists in activating innate immunity.

Vaccination with Env + alum (3M-052), an adjuvant designed for clinical use, recapitulates induction of BM LLPCs and durability of Ab responses in RMs

Currently, there are no approved vaccine formulations with PLGA micro or NPs with challenges foreseen in scale-up (51, 52). Hence, we compared the immune responses stimulated by the 3M-052 or GLA+3M-052 NP with 3M-052 or GLA+3M-052 formulated in alum, which offers an easier path toward translation (53). Also, the first NHP study [described in Fig. 1 (A and B)] used a dose of 750 μg of the 3M-052 adjuvant (harmonized initially with the dose of R848) for the primary vaccination, followed by a reduced dose of 75 μg in all boost vaccinations. Hence, we wanted to evaluate whether the long-lived cellular and serological responses were dependent on the higher priming dose of the adjuvant used. We performed a second experiment with five groups of n = 6 RMs, each as detailed in Fig. 7A. (We have left out reporting data from a sixth group of n = 6 animals due to an error with vaccinations encountered during the study.) Animals in group 1 received vaccinations with 1086.C Env gp140 immunogen with alum. Animals in groups 2 and 3 received vaccinations with Env + alum–formulated 3M-052 or GLA+3M-052 adjuvants, respectively. Animals in groups 4 and 5 received vaccinations with Env + 3M-052 and GLA+3M-052 NPs, respectively. The primary objective of this study was to compare immune responses induced by the alum (3M-052) and 3M-052 NP formulations with those induced by alum-only clinical adjuvant. The secondary objective of this study was to compare immune responses induced by combinations of GLA+3M-052 in alum and NP formulations, respectively, with those induced by 3M-052 adjuvant itself. Hence, the statistical tests used to establish significance of differences in immune responses were designed to address these two study objectives as detailed in Methods.

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.

Consistent with our observations in the first study, we observed a robust induction and persistence (up to ~1 year) of BM-resident Env-specific LLPCs in RMs when vaccinating with Env and 3M-052 adjuvant with or without GLA in both NPs and the novel alum-adsorbed formulations (Fig. 7, C and D). No differences were noted between frequencies of Env-specific LLPCs in groups 2 to 5 (Fig. 7, C and D). Notably, similar to study 1 (fig. S1C), enrichment of Env-specific LLPCs among all IgG+ PCs was highest in animals vaccinated with GLA+3M-052 NP [P = 0.041 versus 3M-052 NP and P = 0.0043 versus alum (3M-052)] (Fig. 7E). We observed a reproducible induction and persistence of high magnitude and durability of nAb responses against MW965.26, an HIV-1 tier-1 pseudovirus (fig. S10A). A significant correlation (Spearman r = 0.7840, P < 0.0001) was observed between the presence of a range of frequencies with Env-specific BM LLPCs (upon combining all treatment groups) and corresponding clade C MW965.26 pseudovirus–specific nAb responses (fig. S10B). Ab responses with higher ADCC activity and avidity were observed in animals in groups 2 to 5 in comparison with group 1 (fig. S10, C and D). Furthermore, we observed high magnitude and durability of binding Ab responses against an optimal panel of HIV-1 Env antigens (fig. S11) when vaccinating with all 3M-052 adjuvant formulations with or without GLA. Last, we demonstrate reproducible changes in monocyte subsets and persistent activation with enhanced CD86 and CCR7 expression (in comparison with alum) when vaccinating with the novel alum (3M-052) adjuvant identical to those induced by the 3M-052 NP adjuvant (fig. S12, A to C). These data are consistent with those described in fig. S9. Overall, these data highlight much promise for rapid testing of the 3M-052–adsorbed alum formulation with HIV-1 Env protein immunogens in human clinical trials.

DISCUSSION

It is now well established that adjuvants substantially improve immunogenicity of protein-based vaccines (15). On the basis of data in mice and more recent report in humans, induction of LLPCs is a critical objective in vaccine and/or adjuvant design (10, 11, 14). However, no studies with adjuvants so far have demonstrated long-term (>1 year) persistence of BM-resident HIV-1 vaccine–specific LLPCs in RMs, given that NHPs are used as the most relevant model for HIV vaccine testing. A key goal of our study here was to carefully downselect a single or combination of TLR-4 and TLR-7/8–targeted adjuvants and independent formulation approaches that could promote high magnitude and durable HIV-1 Env–specific Ab responses and, most critically, LLPCs in RMs. Our results highlight a significant and superior ability of the 3M-052 adjuvant in stimulating HIV-1 Env–specific Ab responses that are not only substantially higher at peak (~ 2 weeks) after four vaccinations but also remarkably persistent for a year at levels higher than that achieved by alum at peak (Figs. 2 to 7). Moreover, broad immune responses were notably durable against multiple antigenic components of the Env protein (gp41 and V1V2) as shown in Figs. 4 and 5, including against the gp120 component previously reported to be immunosuppressive when interacting with both DCs and B cells (54, 55). Both with alum, a benchmark adjuvant used in clinic, and GLA, a TLR-4 agonist formulated in PLGA NPs, we did observe robust Env-specific plasmablast responses in blood (Fig. 2B) after each round of boost vaccination but failed to detect appreciable Env-specific LLPCs in the BM.

Several immunological outcomes in groups 3 to 6 in comparison with groups 1 and 2 could have contributed to successful induction of LLPCs: (i) robust and more persistent (day 7 versus day 4) plasmablast responses, which suggests a threshold of frequencies above which the surviving emigrating cells could traffick to the BM; (ii) the direct targeting of TLR-7 by 3M-052 or R848 in B cells, resulting in distinct programming of antibody secreting cell (ASCs) helping them home to the BM; (iii) successful induction of GC B and GC TFH cells in addition to T helper 1 (TH1)–polarized CD4+ T cells; and last, (iv) robust differentiation and activation of monocyte subsets both contributing to PC differentiation. Although the requirement and impact of direct targeting of TLRs on B cells for vaccine efficacy are unclear from work in mice (56, 57), we find consistent and robust induction of Env-specific BM-resident PCs only when vaccinating with TLR-7/8 agonists, suggesting a critical role for B cell–intrinsic TLR-7 in PC differentiation. Future studies investigating the direct impact of distinct adjuvants on B cells could identify mechanisms, if any, that improve both memory and PC differentiation. We would also like to hypothesize that use of an adjuvant capable of directly targeting memory B cells may be able to minimize the dependency on CD4+ T cells and hence reduce the generation of target cells for HIV-1 (fig. S7) (58). Recent work also highlights a synergy between TLR-7/8 and interferon-γ (IFN-γ) signaling in B cells in promoting PC differentiation (59, 60). As a consequence, it was also reported that IFN-γ signaling increased responsiveness of B cells to interleukin-21 cytokine, predominantly secreted by TFH cells. In line with these observations, we report induction of significantly higher IFN-γ–responding CD4+ T cells when vaccinating with 3M-052 NP (fig. S5) and notably high GC TFH responses when vaccinating with both 3M-052 and GLA+3M-052 NPs (Fig. 6, E and F), both of which could have supported PC differentiation. Last, we have previously reported robust plasmablast differentiation of B cells supported by the CD14+CD16++ intermediate monocytes that are largely expanded and activated in RMs when vaccinating with TLR-7/8 agonists (21, 61). Again, it is highly likely that robust differentiation and activation of intermediate monocytes induced by 3M-052 (fig. S8) could have contributed to programming of LLPCs. Collectively, all these data strongly suggest a critical role for TH1 immune response–polarizing TLR-7/8 agonists in supporting and augmenting PC differentiation.

Many new adjuvants either in clinical testing or recently approved for use in humans are combination adjuvants (15, 29). Combination adjuvants such as with AS01 and AS04 (MPL and QS-21 in liposomes and alum + TLR-4 agonist, MPL) include components that perform additively or synergistically better than when used individually (15, 62). Here, we report that the combination of GLA+3M-052 adjuvants improves the rapidity of neutralizing and ADCC activity as observed in Figs. 3B and 5B at early time points after vaccinations. However, no such advantage was observed with the combination of GLA and 3M-052 on the magnitude of responses at termination (Figs. 2 to 7). One possible explanation is that higher responses induced early by the combination of GLA and 3M-052 or preexisting vaccine-induced immunity could have perhaps limited the boostability of the final vaccination. Studies with longer intervals in between vaccinations in general (63) can improve boostability when using such potent adjuvants. An alternate explanation is the use of NP- or alum-based formulations with GLA in this study. Squalene-based stable emulsion has been reported to be superior in enhancing adjuvanticity of GLA (64), and it could very well enhance the efficacy of GLA in combination with 3M-052 as well. Here, we conclude that 3M-052, even in the absence of GLA as an adjuvant, demonstrates potential to improve both peak and durability of key non-neutralizing effector functions desired in HIV-specific Ab responses.

There were two goals in the second study. First, we wanted to investigate whether we could induce and sustain high frequencies of Env-specific LLPCs with a reduced dose of 3M-052 (75 versus 750 μg) with a primary vaccination. Also, we aimed to address whether formulation (NP versus alum) affected the adjuvanticity of 3M-052. We hypothesized that an alum-adsorbed 3M-052 formulation has an accelerated path for testing in humans due to the previously reported challenges with the PLGA platform (53). Innate and Env-specific Ab responses induced by alum-formulated 3M-052 (Fig. 7 and figs. S10 and S11) were not only identical to results from the first study but also demonstrated notable similarity to that induced by distinct 3M-052 NP formulations. Hence, these data suggest a minimal dependency of the 3M-052 molecule on the delivery vehicles tested so far. Furthermore, now with reduced priming doses in comparison with the first study, we continued to observe a lack of impact on immune response longevity when combining GLA and 3M-052 in comparison with the 3M-052 adjuvant alone. One caveat worth highlighting is the route of vaccination used in our studies. We reasoned that subcutaneous vaccinations could recapitulate our findings of improved vaccine efficacy in mice when using protein immunogens and particulate adjuvants (18). However, most approved subunit vaccines are administered via the intramuscular route of vaccination in humans. Recent studies comparing subcutaneous versus intramuscular routes of vaccinations with HIV-1 immunogens and adjuvants have reported either improved immunogenicity or no impact on innate or adaptive immunity with subcutaneous versus intramuscular vaccinations (49, 65). Future studies in macaques comparing (i) impact of subcutaneous versus intramuscular routes of vaccinations with 3M-052 adjuvant formulations and (ii) dose titration of the agonist in adjuvanting protein immunogens could significantly inform the choice of route and dose for human use. In summary, these results highlight the potential for 3M-052 use as an adjuvant in vaccines against a variety of infections such as with HIV, malaria, and influenza. Furthermore, these results have prompted a phase 1 clinical trial to assess the adjuvant potential of 3M-052 in the context of HIV Env antigens (http://clinicaltrials.gov NCT04177355).

METHODS

Materials and methods are available as supplementary materials.

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.

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REFERENCES AND NOTES

Acknowledgments: We thank A. Weiner at the BMGF for continuous support, encouragement, and insights. We would like to thank all animal staff at the Yerkes National Primate Research Center at Emory University and specifically C. Souder, R. Sheffield, J. Wambua, and S. Ehnert in assisting with the RM study. We would also like to thank E. Strobert, J. Wood, and S. Jean for exceptional assistance with animal health assessment with these adjuvant studies. We would like to acknowledge the NIH AIDS reagent program for consensus peptide pool for clade C envelope procured to run the T cell ICS assay reported in fig. S6. Funding: This work was supported by a Center for AIDS Vaccine Discovery (CAVD) grant from the Bill and Melinda Gates Foundation (BMGF) to B.P. and a grant to B.F.H. for Env protein production. S.P.K. was additionally supported by an NIH K01 award OD023039-03. S.C. and C.H.-D. were supported by NIH NIAID R01 AI125068. Adjuvant formulation work at IDRI was supported by the Bill and Melinda Gates Foundation (BMGF) grant #OPP1055855. The CFAR Immunology/Emory Vaccine Center Flow Cytometry Core is supported by the NIH grant (P30 A050509). We also acknowledge the support from grants NIH P51-RR000165 and P51-OD011132 to the Yerkes National Primate Research Center. Author contributions: B.P., S.P.K., R.A., and M.A.U.R. designed the research. S.P.K. and B.P. were responsible for the overall study execution, compiled all data, and wrote the manuscript. R.A. and M.A.U.R. helped edit and finalize the manuscript. M.T. and J.V. provided the 3M-052 compound. S.P.K. synthesized and characterized the NP adjuvants, received and processed samples with help from M.P., T.L., and Z.J.S., executed all innate and T cell assays, and analyzed data. C.B.F. and S.G.R. provided the alum-formulated 3M-052 adjuvants and provided the GLA raw material as well. B.F.H. provided the 1086.C immunogen. M.A.U.R., Y.K., J.W., and J.H. performed the B cell ELISPOT assays. C.H.-D. and S.C. performed the GC B cell and TFH experiments. C.L. and D.M. performed the nAb assays. N.Y., S.S., and G.T. performed the BAMA assays. S.S.-O. and G.F. performed the ADCC assay. S.M.A. performed the SPR assay. F.V. along with the Yerkes animal staff helped vaccinate animals and oversaw sample collections and freezing. S.G. performed the immunohistochemistry. R.G., W.F., and A.S. provided overall guidance in statistical tests to use with the study objectives and analyzed nAb, BAMA, ADCC, and SPR data from both studies. All authors helped edit the manuscript. Competing interests: M.T. and J.V. are employees of 3M Drug Delivery Systems and are co-inventors of the synthetic TLR-7/8 agonist. C.B.F. is a member of the Scientific Advisory Board of MaxHealth Biotechnology LLC. C.B.F. is an inventor on patent application (US 2019/0142935) held/submitted by IDRI that covers formulations of TLR ligands with lipid excepients and aluminum salts. R.G. has received consulting income from Juno Therapeutics, Takeda, Infotech Soft, Celgene, and Merck, has received research support from Janssen Pharmaceuticals and Juno Therapeutics, and declares ownership in CellSpace Biosciences. All other authors declare that they have no competing interests. Data and materials availability: All raw data used in graphs in the manuscript are included in master Excel file included in the Supplementary Materials. 3M-052 and GLA are available from C.B.F. and M.T. under a material transfer agreement with IDRI and 3M Drug Delivery Systems. 1086.C Env is available from B.F.H. under a material agreement with Duke.

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