Two-Component Ferritin Nanoparticles for ... - ACS Publications

Article Cite This: ACS Infect. Dis. 2018, 4, 788−796

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Two-Component Ferritin Nanoparticles for Multimerization of Diverse Trimeric Antigens Ivelin S. Georgiev,†,∥ Michael Gordon Joyce,†,∥ Rita E. Chen,† Kwanyee Leung,† Krisha McKee,† Aliaksandr Druz,† Joseph G. Van Galen,† Masaru Kanekiyo,† Yaroslav Tsybovsky,‡ Eun Sung Yang,† Yongping Yang,† Priyamvada Acharya,† Marie Pancera,† Paul V. Thomas,† Timothy Wanninger,† Hadi M. Yassine,† Ulrich Baxa,‡ Nicole A. Doria-Rose,† Cheng Cheng,† Barney S. Graham,† John R. Mascola,*,† and Peter D. Kwong*,† †

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Bethesda, Maryland 20892, United States ‡ Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, 8560 Progress Drive, Frederick, Maryland 21702, United States S Supporting Information *

ABSTRACT: Antigen multimerization on a nanoparticle can result in improved neutralizing antibody responses. A platform that has been successfully used for displaying antigens from a number of different viruses is ferritin, a self-assembling protein nanoparticle that allows the attachment of multiple copies (24 monomers or 8 trimers) of a single antigen. Here, we design two-component ferritin variants that allow the attachment of two different antigens on a single particle in a defined ratio and geometric pattern. The two-component ferritin was specifically designed for trimeric antigens, accepting four trimers per particle for each antigen, and was tested with antigens derived from HIV-1 envelope (Env) and influenza hemagglutinin (HA). Particle formation and the presence of native-like antigen conformation were confirmed through negative-stain electron microscopy and antibody−antigen binding analysis. Immunizations in guinea pigs with two-component ferritin particles, displaying diverse Env, HA, or both antigens, elicited neutralizing antibody responses against the respective viruses. The results provide proof-of-principle for the self-assembly of a two-component nanoparticle as a general technology for multimeric presentation of trimeric antigens. KEYWORDS: antibody, HIV-1, immunogenicity, influenza, vaccine

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In the standard ferritin−antigen formulation, an antigen is genetically fused to the N terminus of each of the 24 copies of the ferritin protein, allowing for the formation of outward-facing spike-like structures, which in the case of influenza HA assemble into eight trimer spikes (Figure 1a). The standard ferritin technology, however, can only permit the random coassembly of diverse antigens (by, e.g., coexpressing multiple ferritin−antigen genes) and cannot guarantee the pattern nor the ratio of each antigen on a single particle. Here, we design a two-component ferritin, that allow the attachment of two different antigens in a regular geometric pattern and at an equal (1:1) ratio. We specifically tailor our designs for the presentation of trimeric antigens, which makes this technology especially applicable to viruses such as HIV-1, where antigens in a native-like trimer, rather than monomer, form are believed to be more optimal as immunogens.14 These two-component ferritin particles allow for the presentation of four trimers, each for two distinct antigens. We show that two-component ferritin can form with two diverse

he presentation of viral antigens in a regular repetitive pattern on the surface of virus particles facilitates B cell activation.1−3 Multimerization of antigens on engineered particles that mimic the geometric patterns observed for native viral proteins can lead to improved antibody responses.4−6 Recently, ferritin, a self-assembling sphere-like nanoparticle consisting of 24 copies of a single protein, was used for the multimerization of influenza hemagglutinin (HA) antigens, resulting in the elicitation of antibodies with substantially improved neutralization breadth and potency in immunized animals.7 The ferritin technology has since been extended to allow for the multimerization of other antigens such as the HIV-1 envelope (Env) glycoprotein8 and Epstein−Barr virus gp350 glycoprotein9 and can thus be viewed as a general platform for immunogen design. In addition to ferritin, other multimeric selfassembling platforms such as lumazine synthase have been assessed in immunogenicity studies,10 and multicomponent nanoparticles have been developed through computational design11,12 that may also be capable of multimeric antigen presentation.13 © 2018 American Chemical Society

Received: October 24, 2017 Published: February 16, 2018 788

DOI: 10.1021/acsinfecdis.7b00192 ACS Infect. Dis. 2018, 4, 788−796

ACS Infectious Diseases

Article

Figure 1. Design of two-component ferritin nanoparticles for attachment of diverse trimeric antigens. (a) Schematic of (upper) single-component ferritin (light blue) with eight copies of trimeric antigen A (black) and (lower) two-component ferritin (light blue and light green) with four copies each of trimeric antigens A (black) and B (gray). (b) Design of insect ferritin (left) heavy chain (HC, light blue) and (right) light chain (LC, light green) in monomer (upper) and particle (lower) form to allow attachment of trimeric antigens.

nonclustered state. Of note, the particles with antigen on both HC and LC had visibly more spikes than the particles with antigens on only one of the two chains, highlighting the importance of utilizing both iFerr chains for the assembly of particles with a full assortment of (eight) spikes (Figure 2c). To determine whether iFerr particles could form when two different antigens were attached to, respectively, iFerr HC and LC, we first tested particles with two diverse HIV-1 strains (dualHIV iFerr): soluble gp140 based on strain CNE58 (on iFerr LC) and another clade C strain, ZM106.9,19 with the SOSIP and 201C-433C stabilizing mutations and a C-terminal strep-tag (on iFerr HC) were used (Figure 3a). For the dual-HIV iFerr particle, strep-based negative selection was also performed prior to sizeexclusion chromatography (SEC). Two SEC peaks were observed, with the first peak attributed to higher-order particle formation and the second peak possibly attributed to free (nonparticulated) protein (Figure 3b). Protein yield after lectin and strep-tag purification was ∼1 mg/L, with ∼1/3 of that amount obtained for fractions 16−26 belonging to the first SEC peak. To determine particle formation for the dual-HIV iFerr, we carried out negative-stain EM for several pooled fractions of the dual-HIV iFerr SEC profile: three fraction groups were taken from the first peak (fractions 16−22, 23−25, and 26) and one from the second peak (fraction 33). Particles were observed in all three fraction sets from the first SEC peak, with nanoparticle amount decreasing with latter fractions, while virtually no particles were observed for fraction 33 from the second SEC peak (Figure 3c). Importantly, EM images showed the correctly assembled nanoparticles to exist primarily in nonclustered states. To assess the formation of the trimeric form of the HIV-1 gp140 antigens, we analyzed the antigenicity profiles for the three fraction sets from the first SEC peak of the dual-HIV iFerr using antibody binding by Galanthus nivalis lectin-based ELISA and found that the three sets exhibited similar antibody binding profiles (Figure 3d,e). The full ELISA curves (Figure 3d) are shown in addition to OD value at 1 μg/mL (Figure 3e) for comparison. Importantly, strong binding was observed for the quaternary-specific antibodies CAP256-VRC26.09 20 and PGT145,21 indicating the formation of a closed conformation

HIV-1 Env antigens or two diverse influenza HA antigens, as well as both HIV-1 Env and influenza HA antigens displayed on a single two-component ferritin particle. To allow the presentation of two different antigens on the same particle, we used as a platform a ferritin molecule derived from the insect Trichoplusia ni (iFerr) since it self-assembles naturally as a 24-mer with 12 copies each of a heavy and light chain (we term these iFerr HC and iFerr LC, respectively) and an atomic-level structure of the iFerr particle was available, enabling structure-based design.15 The location of the N-termini of the wild-type iFerr, however, was not optimal for attachment of trimeric antigens (Figure 1b). Hence, we modified the iFerr particle by deleting N-term residues from both iFerr HC and iFerr LC. This resulted in antigen attachment points on ferritin that formed an equilateral triangle with distances of ∼34 Å (HC) and ∼31 Å (LC) (Figure 1b), in line with the close to 30 Å distance between the C-term attachment points for influenza HA and HIV-1 Env. To determine whether these residue deletions would destabilize or affect the formation of ferritin particles, we performed negative-stain electron microscopy (EM). We observed spherical particles with a diameter of 145 ± 11 Å, indicating that iFerr particles could successfully form even with N-terminal deletions in both chains (Figure 2a). Next, we sought to determine whether antigens can be added correctly to each of the iFerr chains. To that end, we tested particles where antigen was attached to iFerr HC only (with no antigen on iFerr LC), to iFerr LC only (with no antigen on iFerr HC), or to both iFerr HC and LC (Figure 2b). As antigen, we used a soluble SOSIP-type gp140 trimer based on the HIV-1 clade C strain CNE58.16,17 The C-term of the antigens was linked to the N-terminus of the respective iFerr sequences using 2 or 5 amino-acid Gly-Ser linkers for attachment to iFerr HC and 5 amino-acid linkers for attachment to iFerr LC. All HIV-1 gp140 constructs had a disulfide mutation (201C 433C) for stabilizing the closed trimer conformation.18 Negative-stain EM confirmed the formation of nanoparticles in all three cases (Figure 2c); importantly, the EM images showed that aggregates, where present, were formed from irregular, disorganized proteins, whereas correctly assembled nanoparticles primarily existed in a 789



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Figure 2. Structural characterization of modified iFerr particles. (a) Negative-stain EM of designed insect ferritin particles with no antigen attached (scale bars indicate 50 and 15 nm (inset), respectively). (b) SDS-PAGE of iFerr particles with HIV-1 Env attached to iFerr heavy (HC) or light chains (LC), nonreduced (NR), or reduced (R). Lanes are as follows: M, molecular weight marker; lanes 1 and 2, antigen on HC only; lanes 3 and 4, antigen on LC only; lanes 5 and 6, antigen on both HC and LC. (c) (upper) Particle schematic and construct design and expression components and (lower) negative-stain EM of HIV-1 Env gp140 from strain CNE58 attached to iFerr HC only (left), iFerr LC only (middle), and both iFerr HC and LC (right). Scale bars indicate 50 and 15 nm (inset), respectively.

of the HIV-1 gp140 trimer.22 Low or no binding was observed for ineffective HIV-1 antibodies F105 and 17b, whereas higher levels of binding were observed for antibody 447-52D, similar to what is seen for nonmultimerized soluble gp140 before negative selection against species binding to 447-52D23 and other ineffective antibodies targeting the third variable region (V3) of Env.18 Binding was observed for a number of other antibodies targeting various sites on Env, including the CD4 receptor binding site (antibodies VRC0124 and b1225), glycan-V3 site (PGT12826), and gp120−gp41 interface sites (35O2227 and PGT15128). Taken together, the antigenicity results indicate that HIV-1 Env trimers in a prefusion-closed conformation structurally compatible with broadly neutralizing antibodies can be successfully displayed on iFerr particles. To demonstrate the generality of the iFerr technology, we further tested particles with two diverse influenza strain HAs (dual-flu iFerr), as well as particles that incorporated both a influenza HA antigen and an HIV-1 Env antigen (flu/HIV iFerr). The dual-flu iFerr particle (Figure 4) incorporated two HAs from

viral strains that were part of the 2015−2016 trivalent or quadrivalent vaccine recommendations: an A/California/7/ 2009 (H1N1) strain on iFerr HC and a B/Phuket/3073/2013 (B/Yamagata lineage) strain on iFerr LC. Negative-stain EM confirmed the formation of the dual-HA constructs as nonclustered, well-formed particles (Figure 4a). The dual-flu construct bound 5J8, an H1N1-neutralizing antibody that targets the HA head region, and three of the four tested stem antibodies (Figure 4b), in agreement with the expected reactivity of the tested antibodies.29 The combined flu/HIV particle (Figure 5) incorporated the A/California/7/2009 (H1N1) influenza HA on iFerr HC and the CNE58 Env on iFerr LC. Although sample purity appeared to be lower than the dual-HIV and dual-flu cases, negative-stain EM confirmed the formation of combined flu/HIV iFerr as nonclustered well-formed particles (Figure 5a). The combined flu/HIV construct showed binding to both influenza and HIV-1 antibodies (Figure 5b,c). The antigenicity profiles were similar to those observed with the dual-HIV and dual-flu particles, with the 790



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Figure 3. Characterization of iFerr particles with attached antigens from two HIV-1 strains (CNE58 and ZM106.9). (a) Schematic representation of the particle construct components. (b) Size-exclusion chromatography and fractionation and (c) corresponding negative-stain EM (scale bar is equal to 20 and 100 nm (inset)). (d) Antigenic characterization of the two-component HIV iFerr by lectin-capture ELISA following size-exclusion chromatography. (e) A summary of the antigenicity data for the HIV-1 antibodies (represented as a heatmap for OD450 values at 1 μg/mL antibody concentration) is shown.

previously described for HIV-1 Env trimer immunizations.31 The three immunization groups comprised (i) a dual-flu iFerr particle, (ii) a flu/HIV iFerr particle, and (iii) a dual-HIV iFerr particle. The dual-antigen iFerr particles elicited neutralizing antibody responses against the respective antigens: the dual-HIV iFerr elicited neutralizing antibodies against HIV-1 but not against influenza; the dual-flu iFerr elicited neutralizing antibod-

exception of the observed lack of binding to HIV-1 antibodies 8ANC195 and b12, both of which do not neutralize the wild-type CNE58 virus.30 Taken together, these results underscore the ability of iFerr particles to display HIV-1 and influenza antigens. To test the immunogenicity of the dual-antigen iFerr particles, we immunized guinea pigs (5 animals/group) with 25 μg of iFerr immunogen in the presence of Adjuplex at weeks 0, 4, and 16 as 791



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Figure 4. Characterization of iFerr particles with attached antigens from two influenza strains (A/California/7/2009 (H1N1) and B/Phuket/3073/ 2013). (a) For the two-component particle, a schematic representation of the respective construct, a size-exclusion chromatography profile with highlighted fractions, and corresponding negative-stain EM, with close-ups of selected particle structures, are shown (scale bar is equal to 20 and 100 nm (inset)). (b) Antigenic characterization by lectin-capture ELISA following size-exclusion chromatography. iFerr nanoparticles were assessed against a panel of both HA head-specific (5J8, CH67, and f045-092) and HA stem-specific (CR9114, CR6261, FI6, and CR8020) antibodies. (c) Summary of the antigenicity data for the influenza antibodies (represented as a heatmap for OD450 values at 1 μg/mL antibody concentration).

ies against influenza but not against HIV-1, and the flu/HIV iFerr elicited neutralizing antibodies against both influenza and HIV-1 (Figure 6). While in the case of HIV-1 only marginal and sporadic autologous neutralization could be observed (Table S1), strong autologous influenza neutralization was observed for the iFerr particles incorporating influenza HA (Figure 6). These results suggest that some antigens may be more readily amenable to the iFerr technology, while in other cases, such as with HIV-1, additional antigen optimization may be required to achieve improved immune responses. The sera binding responses to the antigens and insect ferritin were also observed with a similar pattern (Figure S1). The iFerr technology presented here could in principle be extended to trimeric or monomeric antigens other than those derived from HIV-1 and influenza. While the combined flu/HIV particles are likely not of value for clinical purposes, they serve as a proof-of-concept that diverse antigens can be placed on a single particle, in a regular repetitive pattern. By exploiting the geometric symmetry within ferritin particles, it should further be possible to obtain 3, 4, or 6 component particles through structure-based design, thus enabling the display of an even larger number of diverse antigens on the same particle. The

multicomponent design concept should also be generalizable to other types of protein nanoparticles and can thus be useful as a general platform for multimerized immunogen presentation and vaccine design.

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METHODS

Design of iFerr Particles for Multimerization of Trimeric Antigens. The iFerr structure was obtained from PDB ID 1Z6O.15 By using structure-based design, several iFerr HC and iFerr LC variants were selected: 16-residue, 18-residue, and 19-residue N-term deletions for iFerr HC and 29-residue and 35-residue N-term deletions for iFerr LC. The iFerr HC variants were tested with and without a L113Y mutation; the iFerr LC variants were tested with and without a L123E/I189K double mutation. Expression levels (data not shown) were best for the 18-residue N-term deletion in iFerr HC, effectively placing the new N-term at residue position 19, and the 29-residue N-term deletion for iFerr LC, effectively placing the new N-term at residue position 30. Antigen Constructs. HIV-1. gp140 variants from two diverse clade C strains17 were used in the analysis: a SOSIP-type molecule based on strain CNE5816 and strain ZM106.9.19 Both 792



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Figure 5. Characterization of iFerr particles with attached antigens from a particle made with one influenza (A/California/7/2009 (H1N1)) and one HIV-1 strain (CNE58). (a) Schematic representation of the respective construct, a size-exclusion chromatography profile with highlighted fractions, and corresponding negative-stain EM, with close-ups of selected particle structures, are shown (scale bar is equal to 20 and 100 nm (inset)). (b, c) Antigenic characterization against (b) HIV-1-specific antibodies and (c) influenza-specific antibodies.

Influenza. HA antigens from strains A/California/7/2009(H1N1) and B/Phuket/3073/2013 were used. The C-term of

gp140 constructs had a disulfide mutation (201C 433C) for stabilizing the closed trimer conformation.18 793



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absorbance was measured at 450 nm. All incubations were at 100 μL/well at room temperature, except where noted otherwise. His-tagged iFerr particles with no antigens were immobilized onto ForteBio HIS1K probes. Typical capture levels were between 0.8 and 1 nm, and variability within a row of eight tips did not exceed 0.1 nm. A fortéBio Octet Red384 instrument was used to measure diluted sera (1:100) binding to the iFerr particles or HA or Env proteins. All the assays were performed at 30 °C with agitation set to 1000 rpm in PBS supplemented with 1% BSA to minimize nonspecific interactions. The final volume for all the solutions was 100 μL/well. Assays were performed at 30 °C in solid black 96-well plates (Geiger Bio-One). Biosensor tips were equilibrated for 60 s in PBS−1% BSA buffer before binding measurements. Upon antibody addition, association was allowed to proceed for 300 s. Negative-Stain Electron Microscopy. Samples were diluted to ∼0.03 mg/mL, adsorbed to a freshly glow-discharged carbon-film grid for 15 s, and stained with 0.7% uranyl formate. Images were collected semiautomatically using SerialEM (48) on a FEI Tecnai T20 with a 2k × 2k Eagle CCD camera at a pixel size of 0.22 nm/px. Animal Immunizations. For immunization studies, animals were housed and cared for in accordance with local, state, federal, and institute policies in an American Association for Accreditation of Laboratory Animal Care-accredited facility at VRC, NIAID, NIH. All animal experiments were reviewed and approved by the Animal Care and Use Committee of the Vaccine Research Center, NIAID, NIH under protocol VRC 13413. Female Hartley guinea pigs with body weights of 300 g were purchased from Charles River Laboratories (MA). For each immunization, muscles of the two hind legs were injected with 400 μL of immunogen mix, containing 25 μg of specified, filter sterilized protein immunogen and 80 μL of Adjuplex (SigmaAldrich Inc., MO) in PBS. Blood was collected through retroorbital bleeding under anesthesia for serological analyses. HIV-1 Neutralization Assays. HIV-1 Env pseudoviruses that perform a single round of replication were prepared; titers were determined, and the pseudoviruses were used to infect TZM-bl target cells as described previously.32,33 Neutralization curves were fit by nonlinear regression using a 5-parameter hill slope equation as previously described.34 The 50% and 80% inhibitory dilutions (ID50 and ID80) are reported as the serum concentrations required to inhibit infection by 50% and 80%, respectively. Influenza Neutralization Assays. Influenza HA-pseudotyped lentiviral vectors expressing a luciferase reporter gene were produced as described.31 Briefly, 293T cells were cotransfected by using the following plasmids: 17.5 μg of pCMV-R8.2, 17.5 μg of pHR’CMV-Luc, 1 μg of CMV/R H1 A/California/04/2009Mut or B/Phuket/3073/2013, and 0.125 μg of the corresponding NA (18 million cells in a 15 cm dish). For the production of H1N1 and influenza B pseudovirus, a human type II transmembrane serine protease TMPRSS2 gene was included in transfection for the proteolytic activation of HA.35 Cells were transfected overnight and replenished with fresh medium. Fortyeight hours later, supernatants were harvested, filtered through a 0.45 μm syringe filter, aliquoted, and frozen at −80 °C before use. Neutralization assays were carried out as follows: monoclonal Abs at various dilutions were mixed with pseudoviruses for 45 min and then added to 293A cells in 96-well dishes (10 000 cells per well). Additional fresh medium was added 2 h later. Three days after infection, cells were lysed in 20 μL of cell culture lysis buffer (Promega, Madison, WI). Luciferase assay reagent (50 μL;

Figure 6. Immunogenicity of dual-antigen iFerr particles. Shown are the neutralization titers (y-axis) for two influenza and two HIV-1 strains with animal sera (points) elicited against the dual-flu, flu/HIV, and dualHIV iFerr particles (x-axis).

the antigens were linked to the N-term of the respective iFerr sequences using a flexible Gly-Ser linker of size 2 or 5 for attachment to iFerr HC and 5 for attachment to iFerr LC. In the case of the HIV-iFerr constructs, a C-terminal strep-tag was added. Expression and Purification. All iFerr proteins were expressed by transient transfection in 293F cells using 293fectin (Invitrogen). HIV-1-based constructs were cotransfected with Furin-encoded DNA. The culture supernatant was harvested at 5 days post-transfection and centrifuged at 8000g for 45 min to remove cell debris. The culture supernatants were sterile filtered prior to protein purification. Proteins were purified using snowdrop lectin from Galanthus nivalis (EY Laboratories, San Mateo, CA) affinity chromatography at 4 °C and eluted using 1 M methyl-α-D-manno-pyranoside−phosphate-buffered saline (PBS), pH 7.4. After concentration (concentrators of size 10− 150K MWCO were used (Millipore)), size-exclusion chromatography was performed using a HiPrep 16/60 Sephacryl S-500 HR column (GE Healthcare Bio-Sciences AB). Fractions of interest were pooled and concentrated. For the dual-HIV iFerr particle, strep-based negative selection was also performed prior to size-exclusion chromatography. In brief, the concentrated lectin chromatography eluant was applied to a StreptactinII affinity column, and the flowthrough and wash fractions were pooled and concentrated prior to size-exclusion chromatography, with the C-terminal strep-tag allowing removal of misfolded molecules. Antigenic Characterization. 96-well MaxiSorp plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 100 μL/well of snowdrop lectin from Galanthus nivalis (Sigma− Aldrich) at 2 μg/mL, diluted in 1× PBS. The plates were then blocked at room temperature for 1 h using 200 μL/well of 5% skim milk and 1.5% bovine serum albumin (BSA) in 0.05% Tween-20 + 1× PBS, followed by washing (wash buffer: 0.05% Tween-20 + 1× PBS). iFerr nanoparticles at 2 μg/mL, diluted in 10% fetal bovine serum (FBS) and 1× PBS, were added to the plates and incubated for 2 h followed by washing. Antibodies were 5-fold serially diluted in 0.2% Tween-20 + 1× PBS starting at 5 μg/mL and transferred to the plate and incubated for 1 h followed by washing. Plates were then incubated for 1 h with horseradish peroxidase (HRP)-conjugated antihuman IgG (1:5000) diluted in 0.2% Tween-20 + 1× PBS, washed, and incubated with SureBlue TMB Peroxidase Substrate (KPL) for 10 min. The reaction was stopped with 1 N H2SO4, and then, the 794



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Promega) was added to the cell lysate prior to measuring luciferase activity. Animal sera was initially pretreated with receptor destroying enzyme II (Denka Seiken, Japan) to eliminate serum nonspecific inhibitors in accordance with the manufacturer’s protocol prior to beginning the neutralization assays.36

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.7b00192. iFerr particles eliciting neutralizing antibody responses against HIV-1; iFerr antigen particles eliciting antibody responses (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.R.M.). *E-mail: [email protected] (P.D.K.). ORCID

Peter D. Kwong: 0000-0003-3560-232X Author Contributions ∥

I.S.G. and M.G.J. contributed equally.

Author Contributions

I.S.G. and M.K. conceived the two-component iFerr model. I.S.G. and M.G.J. designed experiments. I.S.G., M.G.J., R.E.C., K.L., K.M., A.D., J.G.V.G., M.K., Y.T., E.S.Y., Y.Y., P.A., T.W., H.M.Y., U.B., N.A.D.-R., and C.C. performed experiments. I.S.G., M.G.J., R.E.C., M.K., Y.T., M.P., P.V.T., C.C., U.B., N.A.D.-R., C.C., B.S.G., J.R.M., and P.D.K. analyzed and interpreted data. I.S.G. and M.G.J. wrote the manuscript, on which all authors commented. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Zhiping Ye (FDA-CBER) kindly provided influenza viruses used in the microneutralization assay. We thank the members of the Structural Biology Section and Structural Bioinformatics Core, Vaccine Research Center, for discussions and comments on the manuscript. Support for this study was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, and by Federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

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