Innate immune recognition of glycans targets HIV nanoparticle immunogens to germinal centers

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Science  08 Feb 2019:
Vol. 363, Issue 6427, pp. 649-654
DOI: 10.1126/science.aat9120

HIV glycans and nanoparticle vaccines

Synthetic nanoparticles have attracted widespread interest for vaccine design, but how the immune system generates a response to multimeric nanoparticles remains unclear. Tokatlian et al. studied immunity generated by HIV envelope antigens arranged in either multivalent nanoparticle forms or as single monomers (see the Perspective by Wilson). The nanoparticle HIV immunogens triggered greater antibody responses compared with the monomeric forms. Glycosylation appeared key for enhanced humoral immunity because it spurred binding to mannose-binding lectin, complement fixation, and antigen trafficking to follicular dendritic cells. The findings highlight how the innate immune system recognizes HIV nanoparticles and the importance of antigen glycosylation in the design of next-generation nano-based vaccines.

Science, this issue p. 649; see also p. 584


In vaccine design, antigens are often arrayed in a multivalent nanoparticle form, but in vivo mechanisms underlying the enhanced immunity elicited by such vaccines remain poorly understood. We compared the fates of two different heavily glycosylated HIV antigens, a gp120-derived mini-protein and a large, stabilized envelope trimer, in protein nanoparticle or “free” forms after primary immunization. Unlike monomeric antigens, nanoparticles were rapidly shuttled to the follicular dendritic cell (FDC) network and then concentrated in germinal centers in a complement-, mannose-binding lectin (MBL)–, and immunogen glycan–dependent manner. Loss of FDC localization in MBL-deficient mice or via immunogen deglycosylation significantly affected antibody responses. These findings identify an innate immune–mediated recognition pathway promoting antibody responses to particulate antigens, with broad implications for humoral immunity and vaccine design.

The immune system has evolved to recognize and respond to nano- and micrometer-sized particles such as viruses and bacteria. Nanoparticles are trafficked to lymphoid tissues through afferent lymph, are internalized and processed for antigen presentation by dendritic cells, and activate B cells via cross-linking of B cell receptors (1). These features of immune recognition have motivated the use of nanoparticulate antigens in licensed vaccines, such as vaccines for human papilloma virus and hepatitis B virus (2, 3), and have motivated the design of nanoparticle forms of immunogens in the development of new vaccines (46). With respect to HIV, evidence from preclinical animal models indicates that nanoparticulate HIV immunogens can more effectively activate low-affinity germline precursor B cells than monomeric antigens (69), promote enhanced follicular helper T (Tfh) cell induction and germinal center (GC) responses (911), and enhance the induction of neutralizing antibody responses (9, 12, 13). However, the mechanisms by which such adaptive immunity is influenced by the physical form of immunogens remain poorly understood.

To define pathways regulating the HIV immune response to multivalent particulate antigens in vivo, we examined the fates of two distinct HIV envelope antigens as soluble monomers or as protein nanoparticles. We compared a germ line–targeting engineered outer domain of gp120 (eOD-GT8, herein referred to as eOD) and a gp140 envelope trimer (MD39) (6, 1416). This trimer is an improved version of BG505 SOSIP gp140 with enhanced thermal stability and expression level and reduced exposure of the V3 loop (8, 17). We selected these two antigens as representatives of “reductionist” antigens designed to elicit an immune response against a particular neutralizing epitope and of whole-envelope protein immunogens bearing multiple neutralizing sites, respectively.

To generate the protein nanoparticles, eOD was formulated as a ~32-nm-diameter nanoparticle (eOD-60mer) by fusion to a bacterial protein, lumazine synthase, which self-assembles into a 60-mer as previously described (Fig. 1A) (1416). By contrast, a ~40-nm-diameter nanoparticle form of MD39 (MD39-8mer) was generated by fusing the MD39 gp140 chain to an archaeal ferritin; 24 subunits of ferritin self-assemble to form a nanoparticle (ferritin core outer diameter, ~12 nm) displaying eight copies of gp140 trimer (Fig. 1B) (13, 18). MD39-8mer eluted as a relatively uniform peak in size exclusion chromatography, showed an enzyme-linked immunosorbent assay (ELISA) binding profile to neutralizing and nonneutralizing monoclonal antibodies consistent with expectations for the MD39 trimer (13), and was observed to have a reasonably homogeneous morphology by transmission electron microscopy (TEM) (Fig. 1B and fig. S1, A to C). In vitro, both eOD-60mer and MD39-8mer nanoparticles stimulated stronger calcium signaling in VRC01-expressing B cells than their monomer counterparts (6) (fig. S1D). In immunized mice, the nanoparticle forms of eOD and MD39 elicited higher immunoglobulin G (IgG) titers (up to 90 times as high) than the soluble immunogens (Fig. 1, C to E). Analysis of responding cells in lymph nodes (LNs) revealed that Tfh cell responses were not altered by nanoparticle immunization (Fig. 1, F and G) but that GC B cells were substantially increased (Fig. 1, H and I). A deeper analysis of eOD-immunized animals further showed that polyclonal IgGs isolated from eOD-60mer–immunized sera exhibited lower off rates than IgG from monomer-immunized animals, indicative of enhanced affinity maturation (Fig. 1J and fig. S2, A and B). Thus, the nanoparticle forms of either eOD or MD39 elicited substantially enhanced humoral responses in vivo compared with their monomer forms.

Fig. 1 Nanoparticle forms of gp120 and envelope (Env) trimer immunogens elicit enhanced humoral immune responses.

(A and B) Model representations and TEM images of (A) eOD-60mer and (B) MD39-8mer nanoparticles. eOD or MD39 is shown in green, glycans are shown in blue, and the lumazine synthase or ferritin core is in red. (C and D) BALB/c mice (n = 4 mice per group) were immunized with 2 μg of the eOD monomer (blue) or 3.7 μg of eOD-60mer (red), with each preparation containing the same number of moles of eOD, together with saponin adjuvant. Shown are (C) serum eOD-specific IgG titers analyzed over time by ELISA and (D) individual titers 1 month postimmunization. Data represent the means with 95% confidence intervals (CIs) from one of five independent experiments. (E) BALB/c mice (n = 10 per group) were immunized with 1 μg of MD39 or ~1.3 μg of MD39-8mer, with each preparation containing the same number of moles of trimer, together with saponin adjuvant and received a booster immunization at 6 weeks; individual gp120-specific IgG titers were analyzed 3 weeks post–booster immunization by ELISA. Data show the means with 95% CIs from one of three independent experiments. (F to I) Mice were immunized with eOD or MD39 as described for (C) and (E); [(F) and (G)] absolute (abs.) counts of antigen-specific Tfh cells and [(H) and (I)] GC B cells in lymph nodes from individual mice were determined by flow cytometry on day 7. Shown are the means with SD from one of three independent experiments. (J) Dissociation rates (koff) of day-21 purified polyclonal IgG bound to immobilized eOD analyzed via biolayer interferometry for mice immunized with eOD or eOD-60mer. Shown are the means and SD from one of three independent experiments. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, determined by a Mann-Whitney test (serum titers), one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test (for GC Tfh cells), or an unpaired t test.

To understand the differences in immune responses induced by HIV antigen nanoparticles versus soluble monomers, we examined the lymphatic trafficking and tissue localization of each immunogen. Whole-tissue fluorescence measurements of infrared (IR) dye–labeled immunogens in draining LNs (dLNs) showed higher total accumulation of both nanoparticle formulations than of monomeric forms in dLNs at 3 days postimmunization (Fig. 2A). However, confocal imaging of cleared whole dLNs revealed that soluble MD39 accumulated primarily in the subcapsular sinus and medullary areas, whereas MD39-8mer was observed to begin concentrating within follicles by day 3 and was strongly colocalized with follicular dendritic cells (FDCs) by day 7 (Fig. 2, B and C). Liposomes (~95 nm in diameter) surface-conjugated with densely packed MD39 also exhibited FDC accumulation over 7 days postimmunization, though with a lower efficiency than the smaller ferritin-based nanoparticles, suggesting that FDC targeting is independent of the nature of the nanoparticle core (Fig. 2C and fig. S3A) (8). Trafficking of the eOD monomer versus eOD-60mer was even more distinct: Whereas the eOD monomer showed low levels of accumulation in dLNs over a 14-day time course and colocalized primarily with SIGN-R1+ macrophages, as reported previously for other gp120 antigens (19), eOD-60mer was already beginning to colocalize with FDCs after 24 hours (Fig. 2D and fig. S3, B and C). By day 7, eOD nanoparticles were almost exclusively localized within the FDC network and persisted there for ~4 weeks (Fig. 2, D and E, and fig. S3, D and E); FDC localization occurred in the presence or absence of coadministered adjuvant, albeit with lower overall accumulation in the absence of adjuvant (fig. S3F). Targeting of FDCs required high antigen valency, as eOD trimers failed to show follicular localization similar to that of the eOD monomer (Fig. 2E and fig. S3G). Bare lumazine synthase nanoparticles lacking eOD also did not traffic to FDC networks (fig. S3H). Costaining to identify GCs showed that both the monomer and eOD-60mer initiated GCs (Fig. 2F), but much higher levels of eOD-60mer were localized in GCs, aligning with FDCs in the light zone (Fig. 2, D and F). MD39-8mers and MD39 conjugated to liposomes exhibited a similar pattern of concentration within GCs (fig. S3, I to K).

Fig. 2 Nanoparticle eOD and MD39 trimer immunogens are targeted to the FDC network and concentrated in GCs.

(A) BALB/c mice (n = 4 to 5 mice per group) were immunized with IR dye–labeled MD39, MD39-8mer, eOD, or eOD-60mer, and total fluorescence integrated (Int.) intensity in dLNs was recorded over time. Shown are the means and SD from one of two independent experiments. k counts, 1000 counts. (B and C) BALB/c mice (n = 5 per group, 10 dLNs) were immunized with fluorescent MD39 or MD39-8mer (with the equivalent of 5 μg of trimer in each group) and adjuvant. FDCs were labeled in situ with anti-CD35 antibody, excised dLNs were cleared and (B) imaged by confocal microscopy, and (C) colocalization of antigen with follicles was quantified by the percentage of MD39 signal within follicles and by the percentage of follicular area that contained MD39. Shown are the means and SD from one of three independent experiments. Lipos, liposomes. (D to F) BALB/c mice (n = 5 to 9 per group, 10 to 18 dLNs) were immunized with fluorescent eOD monomer or eOD-60mer (with the equivalent of 2 μg of eOD in each group) and adjuvant. (D) FDCs or GCs were labeled in situ with anti-CD35 or anti-CD157, respectively, and excised dLNs were cleared and imaged by confocal microscopy. In images from eOD-immunized mice, eOD brightness was increased to allow for visualization. (E) Colocalization of antigen with follicles was quantified by the percentage of eOD signal within follicles and by the percentage of follicular area that contained eOD. Shown are the means and SD from one of three independent experiments. (F) dLNs were cut into 100-μm-thick slices and stained with anti-B220 and anti-Ki67, and individual follicles were imaged by confocal microscopy. *P < 0.05, **P < 0.01, determined by a one-way ANOVA followed by Tukey’s multiple comparisons test. IR dye–tagged antigen–trafficking analysis included comparisons to unimmunized LN controls at each time point.

Targeting to FDCs and GCs is not a generic property of nanoparticles in naïve animals, as many vaccine studies have shown particles of diverse sizes and material compositions localizing in a manner suggesting exclusion rather than enrichment in B cell follicles (2023). By contrast, immune complexes (ICs) have been reported to elicit a similar type of antigen delivery to FDCs (2426). IC trafficking to FDCs is mediated by the relay of complexes from subcapsular sinus macrophages to migrating B cells, which in turn transfer antigen to FDCs, in a complement- and complement receptor–dependent manner (2426). To determine whether complement is also involved in the recognition of envelope nanoparticles and to test whether nanoparticle trafficking is mediated by interactions with complement receptors, we immunized mice lacking the C3 component of the complement system [C3 knockout (KO) mice] and mice lacking complement receptors (Cr1/2 KO mice) with eOD-60mer or MD39-8mer. As shown in Fig. 3, A and B, both nanoparticles were strongly localized to the FDC network in wild-type (WT) mice at day 7, but only low levels of antigen were detected in C3 KO dLNs, with a diffuse distribution. Nanoparticle trafficking to FDCs was also abrogated in Cr1/2 KO animals (Fig. 3, C and D). Consistent with these findings, in vitro addition of normal serum to plate-immobilized eOD-60mer, but not the eOD monomer or trimer, led to substantial deposition of C3 as detected by ELISA (Fig. 3E). These data suggest that opsonization of nanoparticle antigens by complement is required for rapid trafficking to FDCs and that this trafficking is complement receptor dependent.

Fig. 3 Complement and complement receptor are required for follicular targeting of nanoparticle immunogens.

(A to D) WT C57BL/6, C3 KO, or Cr1/2 KO mice (n = 4 mice per group, 8 dLNs) were immunized with 3.7 μg of fluorescent eOD-60mer (equivalent to 2 μg of eOD) or 6.4 μg of MD39-8mer (equivalent to 5 μg of trimer) and adjuvant. dLNs were recovered after 7 days, cleared, and imaged by confocal microscopy. Antigen localization was imaged in [(A) and (B)] WT and C3 KO or (C) Cr1/2 KO animal LNs for [(A) and (C)] eOD-60mer or (B) MD39-8mer, and (D) eOD localization in follicles in WT versus KO mice was quantified by the percentage of eOD signal within follicles and by the percentage of follicular area that contained eOD. Shown are the means and SD from one of two independent experiments. (E) WT mouse serum was added to ELISA plates coated with the eOD monomer, eOD-3mer, or eOD-60mer, and deposited C3 was detected by ELISA. Shown are the means and SD from one of four independent experiments. a.u., absorbance units. **P < 0.01, ****P < 0.0001, determined by a one-way ANOVA followed by Tukey’s multiple comparisons test.

We sought to define the mechanism of complement fixation by the nanoparticle immunogens. IgM from naïve animals did not bind eOD-60mer by ELISA (fig. S4), suggesting that natural IgM is not involved. The lectin-mediated pathway activates complement in bacterial immunity; here, mannose-binding lectin (MBL) binds to glycosylated microbes and activates complement via MBL-associated serine proteases (27). MBL is a large macromolecular complex composed of multimers of trimeric lectin stalks, which achieve high-avidity binding to pathogens through multivalent engagement with large patches of dense sugars (2729). Structural studies have shown that the three carbohydrate binding domains (CBDs) at the end of each stalk in the MBL multimer are arranged in a triangular configuration, separated by 4.5 nm (30); the distance between each stalk of MBL multimers is poorly defined but expected to be of similar order or larger (31). Each CBD recognizes mannose and other sugars with a very weak affinity of Kd (dissociation constant) ~10−3 M, but stable binding to larger patches of glycans is thought to be achieved by the avidity effect of engaging multiple trimeric stalks of MBL multimers. These considerations suggest that MBL will be unable to bind multiple stalks to an eOD monomer (diameter, 7.5 nm) and may be capable of engaging only a few stalks on MD39 trimers (diameter, 15 nm) (fig. S5). In agreement with these arguments, in an ELISA-type assay murine and human MBLs bound to immobilized eOD-60mer but exhibited only weak recognition of eOD-3mer and the eOD monomer (Fig. 4A). Biolayer interferometry measurements of eOD-60mer binding to immobilized MBL revealed an apparent affinity of ~4 pM, whereas binding by the eOD monomer was essentially undetectable; similarly, the nanoparticle MD39-8mer showed avid binding by MBL, whereas binding to the MD39 monomer was low (fig. S6, A and B).

Fig. 4 MBL-mediated innate recognition and follicular targeting amplifies humoral responses to envelope nanoparticle immunogens.

(A) Mouse MBL (200 ng/ml) or human MBL (7.5 μg/ml) was added to ELISA plates coated with the eOD monomer, eOD-3mer, or eOD-60mer, and bound MBL was detected by ELISA. Shown are the means and SD from one of four independent experiments. (B) MBL KO mice (n = 3 mice per group, 6 dLNs) were immunized with 3.7 μg of fluorescent eOD-60mer (equivalent to 2 μg of eOD) and adjuvant. Excised dLNs were cleared and imaged by confocal microscopy. eOD brightness was increased to allow for visualization. (C) Unmodified eOD-60mer (1) and eOD-60mer deglycosylated by PNGase F treatment (2) were analyzed via SDS–polyacrylamide gel electrophoresis for changes in subunit size and glycan content. (Left) Nonspecific protein stain. (Right) Glycoprotein stain. (D) Unmodified (red) and deglycosylated (blue) eOD-60mer hydrodynamic radii were evaluated by dynamic light scattering. (E) Cryo-TEM of deglycosylated eOD-60mer. (F) C57BL/6 mice (n = 5 per group, 10 dLNs) were immunized with 3.7 μg of fluorescent deglycosylated (degly) eOD-60mer and adjuvant. Excised dLNs were cleared and imaged by confocal microscopy. (G) C57BL/6 mice (n = 5 per group) were immunized with 3.7 μg of eOD-60mer (red) or deglycosylated eOD-60mer (blue) and adjuvant. Serum eOD-specific IgG titers from individual mice were analyzed over time by ELISA. Shown are the means with 95% CIs from one of two independent experiments. (H to L) C57BL/6 mice (red) or MBL KO mice (blue) (n = 5 per group) were immunized with 3.7 μg of eOD-60mer and adjuvant. Absolute (abs.) numbers of (H) GC Tfh cells and (I) GC B cells from individual mice were analyzed at day 7. Shown are the means and SD. (J) eOD-specific IgG titers from individual mice were analyzed over time by ELISA. Shown are the means and 95% CIs. (K) Isotype-specific midpoint titers were analyzed 1 month postimmunization. Shown are the means and SD. (L) Bone marrow eOD-specific antibody-secreting cells (ASCs) were quantified by enzyme-linked immunospot assay at 8 weeks postimmunization. Data show the means and SD. (M) C57BL/6 mice (n = 3 animals per group) were immunized with 5 μg of fluorescent bare ferritin nanoparticles lacking glycans or 5 μg of ferritin particles conjugated with trimannose moieties (~96 trimannose groups per particle), together with adjuvant. LNs were excised 3 days later, sectioned, and imaged by confocal microscopy. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, determined by either a one-way ANOVA followed by Tukey’s multiple comparisons test or a Mann-Whitney test (for serum titer analysis only).

To evaluate whether MBL is involved in trafficking of nanoparticles to FDCs in vivo, we immunized MBL KO mice with eOD-60mer or MD39-8mer. dLNs contained low levels of antigen with no accumulation on the FDC network (Fig. 4B and fig. S7, A to C). To evaluate whether FDC localization was immunogen glycan dependent, we deglycosylated eOD-60mer with peptide N-glycosidase F (PNGase F) and confirmed by light scattering, TEM, and ELISA analyses that the enzyme-treated protein retained its self-assembled particle structure and presentation of the key CD4 binding site epitope (Fig. 4, C to E, and fig. S8). MBL bound at only low levels to deglycosylated eOD-60mer in vitro (fig. S6A), and deglycosylated particles exhibited low accumulation in LNs in WT mice, with no FDC localization (Fig. 4F). These data imply that dense arrays of glycans trigger MBL-mediated innate immune recognition of nanoparticles. To assess the generality of this concept, we assessed trafficking of another highly glycosylated nanoparticle immunogen, influenza hemagglutinin-ferritin 8-mer particles (HA-8mers) (4), which were also targeted to the FDC network in WT mice but not MBL KO mice (fig. S9A).

To obtain insight into how the composition and density of glycans regulate MBL recognition of nanoparticles, we characterized the glycan profiles of each of the immunogens. eOD-60mer was typically prepared in the presence of kifunensine, such that its glycans were almost completely oligomannose (fig. S10). By contrast, the eOD monomer (typically prepared in 293F cells) contained predominantly complex-type glycans (fig. S10). To assess whether this difference in glycan composition affected in vivo antigen trafficking, we prepared eOD-60mer in 293F cells, which contained pedominantly complex glycans mirroring the eOD monomer (fig. S10). These eOD-60mer nanoparticles trafficked to FDCs in a manner identical to that of the eOD-60mer bearing only oligomannose glycans (fig. S9B), suggesting that small levels of oligomannose glycans are sufficient to trigger MBL-mediated trafficking to follicles. In support of this argument, MD39-8mers and HA-8mers exhibited an ~50/50 complex/oligomannose ratio and a predominantly complex glycan profile, respectively, but the two nanoparticle immunogens showed similar FDC localization patterns in vivo. Similar immunogen trafficking patterns elicited by these distinct glycan profiles are consistent with the ability of MBL to bind to both complex and simple sugars.

We then assessed the immunological effects of MBL recognition and FDC targeting. WT mice immunized with nanoparticle eOD-60mer showed an IgG response two times that elicited by deglycosylated eOD-60mer (Fig. 4G). Further, responses to eOD-60mer were stronger in WT mice than in MBL KO mice by several measures, with 53% greater Tfh cell responses, 43% greater GC B cell responses, and IgG titers about five times as high across multiple antibody isotypes, irrespective of the adjuvant used (Fig. 4, H to K, and fig. S11, A and B). IgG binding to plates coated with a low versus high density of antigen was also proportionally weaker for sera from MBL KO animals than for sera from WT mice, suggesting a lower mean avidity of IgG elicited in MBL KO animals (fig. S11C). Eight weeks postimmunization, WT mice also had approximately double the population of bone marrow–resident eOD-specific antibody-secreting cells compared with MBL KO animals (Fig. 4L). Notably, MBL binding to eOD did not obscure recognition of the CD4 binding site (fig. S12), suggesting that innate recognition of Env glycans would not inhibit generation of on-target antibody responses. Thus, despite preserving high multivalency for B cell receptor triggering, nanoparticles lacking MBL-mediated FDC targeting elicit weaker humoral responses.

Lastly, we examined whether synthetic introduction of glycans could be used to engineer the delivery of nanoparticles to follicles, as a preliminary test of the utility of glycan engineering to alter the processing of nanoparticle vaccines in vivo. We expressed bare ferritin nanoparticles lacking any glycosylation; these particles showed low overall accumulation in LNs and no colocalization with FDCs after the immunization of WT mice (Fig. 4M). By contrast, conjugation of a synthetic trimannose moiety to these nanoparticles led to pronounced accumulation on FDCs within 3 days of immunization (Fig. 4M and fig. S13A); this FDC localization was glycan density dependent because ferritin nanoparticles functionalized with a lower density of trimannose groups (~25 versus ~96 trimannose groups per particle) did not localize to the FDC network (fig. S13B). To determine whether glycan-mediated delivery to FDCs could be achieved with synthetic nanoparticles and to assess the effect of particle size, we functionalized monodisperse polystyrene nanoparticles with the same trimannose groups at high density (fig. S14A). Polystyrene nanoparticles 40 nm in diameter accumulated on FDCs (albeit with lower efficiency than the protein nanoparticles, possibly because of some level of aggregation in vivo), whereas 100- or 200-nm-diameter particles were excluded from follicles (fig. S14B). Thus, synthetic introduction of even simple glycans through chemical or genetic approaches may provide a means to direct arbitrary vaccine nanoparticles of appropriate size to the FDC network.

Collectively, these data suggest that glycosylated nanoparticles trigger MBL-mediated innate immune recognition, leading to rapid complement-dependent transport to FDCs and subsequent concentration in GCs in vivo. This targeted trafficking was associated with enhanced antibody responses, suggesting that tuning immunogen glycosylation may be a key design criterion for future nanoparticulate vaccines or immunomodulators and providing an explanation for how FDC localization of immunogens can occur in the absence of preexisting antibody. These findings are especially interesting in the context of HIV vaccine development, where the dense envelope “glycan shield” is often viewed as a hurdle to achieving efficient antibody responses.

Supplementary Materials

Materials and Methods

Figs. S1 to S14

Table S1

References (3237)

References and Notes

Acknowledgments: We thank the Koch Institute Swanson Biotechnology Center for technical support, specifically the flow cytometry, microscopy, and nanotechnology materials core facilities. We also thank the Biophysical Instrumentation Facility for use of the Octet biolayer interferometry system. Funding: This work was supported in part by the NIAID under awards UM1AI100663 (to D.J.I, W.R.S., and M.C.), AI104715 (to D.J.I.), and AI048240 (to D.J.I.); the Koch Institute support (core) grant P30-CA14051 from the National Cancer Institute; the Ragon Institute of MGH, MIT, and Harvard; the International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Consortium (NAC) and Center (to W.R.S. and M.C.); and the Collaboration for AIDS Vaccine Discovery funding for the IAVI NAC Center (to W.R.S. and M.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. D.J.I. is an investigator of the Howard Hughes Medical Institute. Author contributions: T.T., B.J.R., C.A.J., S.K., J.Y.H.C., E.L.D., A.L., J.D.A., and M.C. designed, performed, and analyzed experiments. D.W.K., S.M., J.M.S., W.R.S., M.S., and D.L. designed and synthesized immunogens. T.T., B.J.R., D.W.K., W.R.S., and D.J.I. wrote the manuscript. Competing interests: The eOD and MD39 immunogens in this paper are included in patent filings from IAVI, the Scripps Research Institute, and MIT by inventors including D.W.K., S.M., J.M.S., T.T., B.J.R., D.J.I., and W.R.S. W.R.S. is a co-founder and stockholder in Compuvax, which has programs in non-HIV vaccine design that may benefit indirectly from this research. Data and materials availability: All data are available in the main text or the supplementary materials.

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