Two-Component Ferritin Nanoparticles for Multimerization of Diverse

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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 YASSINE, Ulrich Baxa, Nicole Doria-Rose, Cheng Cheng, Barney S. Graham, John R. Mascola, and Peter D. Kwong ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00192 • Publication Date (Web): 16 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

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ACS Infectious Diseases

Two-Component Ferritin Nanoparticles for Multimerization of Diverse Trimeric Antigens

Ivelin S. Georgiev1*, Michael Gordon Joyce1*, Rita E. Chen1, Kwanyee Leung1, Krisha McKee1, Aliaksandr Druz1, Joseph G. Van Galen1, Masaru Kanekiyo1, Yaroslav Tsybovsky2, Eun Sung Yang1, Yongping Yang1, Priyamvada Acharya1, Marie Pancera1, Paul V. Thomas1, Timothy Wanninger1, Hadi M. Yassine1, Ulrich Baxa2, Nicole A. Doria-Rose1, Cheng Cheng1, Barney S. Graham1, John R. Mascola1# and Peter D. Kwong1#

1

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Bethesda, MD 20892, USA 2 Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, 8560 Progress Drive Frederick, MD 21702, USA

*

Equal contribution

#

Corresponding authors: John R. Mascola [email protected] [email protected]

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& Peter D.

Kwong,

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 (twenty-four monomers or eight trimers) of a single antigen. Here we design two-component ferritin variants that allow the attachment of two diverse 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 proofof-principle for self-assembly of a two-component nanoparticle as a general technology for multimeric immunogen presentation of trimeric antigens.

Keywords: vaccine, HIV-1, influenza, antibody, immunogenicity

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The 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 the multimerization of other antigens such as the HIV-1 envelope (Env) glycoprotein 8 and Epstein-Barr virus gp350 glycoprotein 9, and can thus be viewed as a general platform for immunogen design. In addition to ferritin, other multimeric self-assembling platforms such as lumazine synthase have been assessed in immunogenicity studies 10, while multi-component nanoparticles have been developed through computational design 11-12 that may also be capable of antigen presentation 13. In the standard ferritin-antigen formulation, an antigen is genetically fused to the Nterminus of each of the 24 copies of the ferritin protein, allowing the formation of outwardfacing spike-like structures, which in the case of influenza HA assemble into eight trimer spikes (Fig. 1a). The standard ferritin technology, however, can only permit the random co-assembly of diverse antigens (by, e.g., co-expressing multiple ferritin-antigen genes) and cannot guarantee the pattern and ratio of each antigen on a single particle. Here we design ferritin variants, twocomponent 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 3 ACS Paragon Plus Environment


optimal as immunogens 14. These two-component ferritin particles allow the attachment of four trimers each for two distinct antigens. We show that two-component ferritin can form with two diverse 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 addition 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 selfassembles naturally as a 24-mer with twelve 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 is available, enabling structure-based design 15. The location of the N-termini of the wildtype iFerr, however, is not optimal for attachment of trimeric antigens (Fig. 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) (Fig. 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 and affect the formation of the 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-term deletions in both chains (Fig. 2a). Next, we sought to determine whether antigens can be added properly 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 (Fig. 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 were linked to the N-term of the respective iFerr 4 ACS Paragon Plus Environment

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sequences using flexible 2 or 5 amino acid Gly-Ser linkers for attachment to iFerr HC and 5 for 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 the particles in all three cases (Fig. 2c); importantly, the EM images show that aggregates, where present, were formed from irregular, disorganized proteins, whereas correctly assembled nanoparticles primarily existed in a non-clustered 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 (Fig. 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 (dual-HIV iFerr): soluble gp140 based on strain CNE58 (on iFerr LC) and another cladeC strain, ZM106.9 19 with the SOSIP and 201C-433C stabilizing mutations and a C-terminal strep-tag (on iFerr HC) were used (Fig. 3a). For the dual-HIV iFerr particle, strep-based negative selection was also performed prior to size-exclusion 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 (non-particulated) protein (Fig. 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 5 ACS Paragon Plus Environment


particles were observed for fraction 33 from the second SEC peak (Fig. 3c). Importantly, the EM images showed the correctly assembled nanoparticles to exist primarily in non-clustered 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 (Fig. 3d,e). The full ELISA curves (Fig. 3d) are shown in addition to OD value at 1 mg/ml (Fig. 3e) for comparison. Importantly, strong binding was observed for the quaternary-specific antibodies VRC26.09 20 and PGT145 21, indicating the formation of the closed conformation 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 non-multimerized soluble gp140 before negative selection against species binding to 447-52D 23 and other ineffective antibodies targeting the V3 variable region of Env 18. Binding was observed for a number of other antibodies targeting various sites on Env, including the CD4 receptor binding site (antibodies VRC01 24 and b12 25), V3-glycan site (PGT128 26) and gp120-gp41 composite epitope sites (35O22 27 and PGT151 28). 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 flu HA antigen and an HIV Env antigen (flu/HIV iFerr). The dual-flu iFerr particle (Fig. 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 6 ACS Paragon Plus Environment

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B/Phuket/3073/2013 (B/Yamagata lineage) strain on iFerr LC. Negative-stain EM confirmed the formation of the dual-HA constructs as non-clustered, well-formed particles (Fig. 4a). The dualflu construct bound 5J8, an H1N1-neutralizing antibody that targets the HA head region, and three of the four tested stem antibodies (Fig. 4b), in agreement with the expected reactivity of the tested antibodies 29. The combined flu/HIV particle (Fig. 5) incorporated the A/California/7/2009 (H1N1) flu 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 non-clustered well-formed particles (Fig. 5a). The combined flu/HIV construct showed binding to both flu and HIV-1 antibodies (Fig. 5b,c). The antigenicity profiles were similar to those observed with the dual-HIV and dual-flu particles, with the exception of the observed lack of binding to HIV-1 antibodies 8ANC195 and b12, both of which do not neutralize the wildtype CNE58 virus 30. Taken together, these results underscore the ability of iFerr particles to display HIV-1 and flu 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 previously described for HIV-1 Env trimer immunizations 31. The three immunization groups comprised of (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 flu; the dual-flu iFerr elicited neutralizing antibodies against flu, but not HIV-1, and the flu/HIV iFerr elicited neutralizing antibodies against both flu and HIV-1 (Fig. 6). While in the case of HIV-1, only marginal and sporadic autologous neutralization could be observed ( Table S1), 7 ACS Paragon Plus Environment


strong autologous influenza neutralization was observed for the iFerr particles incorporating flu strains (Fig. 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 flu. 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 ncomponent (where n>2) particles through structure-based design, thus enabling the display of an even larger number of diverse antigens on the same particle. The multi-component 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.

METHODS Design of iFerr particles for multimerization of trimeric antigens. The iFerr structure was obtained from PDB ID 1Z6O 15. 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 8 ACS Paragon Plus Environment

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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 strains 17 were used in the analysis: a SOSIP-type molecule based on strain CNE58 16 and strain ZM106.9 19. Both gp140 constructs had a disulfide mutation (201C 433C) for stabilizing the closed trimer conformation 18

. Flu: HA antigens from strains A/California/7/2009(H1N1) and B/Phuket/3073/2013. The C-

term of the antigens were linked to the N-term of the respective iFerr sequences using 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 co-transfected with Furinencoded DNA. The culture supernatant was harvested at 5 days post-transfection and centrifuged at 8,000 × g 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-dmanno-pyranoside–phosphate-buffered saline (PBS), pH 7.4. After concentration (concentrators of size 10K-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 mis-folded molecules. Antigenic characterization. 96-well MaxiSorp plates (Thermo Fisher Scientific) were coated overnight at 4°C with 100µl/well of snowdrop lectin from Galanthus nivalis (Sigma – Aldrich) at 2µg/ml, diluted in 1X PBS. The plates were then blocked at room temperature for 1 hr using 200µl/well of 5% skim milk, 1.5% bovine serum albumin (BSA) in 0.05% Tween-20 + 1X PBS, followed by washing (wash buffer: 0.05% Tween-20 + 1X PBS). iFerr nanoparticles at 2µg/ml, diluted in 10% fetal bovine serum (FBS) 1X PBS were added to the plates and incubated for two hours followed by washing. Antibodies were 5-fold serially diluted in 0.2% Tween-20 + 1X PBS starting at 5µg/ml and transferred to the plate and incubated for 1 hr followed by washing. Plates were then incubated for one hour with horseradish peroxidase (HRP) –conjugated anti-human IgG (1:5000) diluted in 0.2% Tween-20 + 1X PBS, washed and incubated with SureBlue TMB Peroxidase Substrate (KPL) for 10 minutes. The reaction was stopped with 1 N H2SO4 and then the absorbance was measured at 450 nm. All incubations were at 100µl/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 iFerrr particles or HA or Env proteins. All the assays were performed at 30 °C with agitation set to 1,000 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 10 ACS Paragon Plus Environment

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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 15s, and stained with 0.7% uranyl formate. Images were collected semi-automatically using SerialEM (48) on a FEI Tecnai T20 with a 2k x 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 13-413. Female Hartley guinea pigs with body weights of 300 grams 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 retro-orbital 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 co-transfected by using the following plasmids: 17.5 µg of pCMV-R8.2, 17.5 µg of pHR’CMV-Luc, 1 µg CMV/R H1 A/California/04/2009-Mut, or B/Phuket/3073/2013; and 0.125 µg of the corresponding NA (18 million cells in a 15cm 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. Forty-eight hours later,

supernatants were harvested, filtered through a 0.45-um 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 2h later. Three days after infection, cells were lysed in 20 µl of cell culture lysis buffer (Promega, Madison, WI). Luciferase assay reagent (50 µl; Promega) was added to the cell lysate prior to measuring luciferase activity. Animal sera is 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. Supporting Information. Figure S1 and Table S1.

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


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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.

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., A.D., J.V.G., M.K., Y.T., Y.Y., P.A., H.M.Y., and U.B. performed experiments; I.S.G., M.G.J., R.E.C., M.K., Y.T., M.P., P.V.T., C.C., U.B., 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.



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16. Shang, H.; Han, X.; Shi, X.; Zuo, T.; Goldin, M.; Chen, D.; Han, B.; Sun, W.; Wu, H.; Wang, X.; Zhang, L., Genetic and neutralization sensitivity of diverse HIV-1 env clones from chronically infected patients in China. The Journal of biological chemistry 2011, 286 (16), 14531-41. 17. Joyce, M. G.; Georgiev, I. S.; Yang, Y.; Druz, A.; Geng, H.; Chuang, G. Y.; Kwon, Y. D.; Pancera, M.; Rawi, R.; Sastry, M.; Stewart-Jones, G. B. E.; Zheng, A.; Zhou, T.; Choe, M.; Van Galen, J. G.; Chen, R. E.; Lees, C. R.; Narpala, S.; Chambers, M.; Tsybovsky, Y.; Baxa, U.; McDermott, A. B.; Mascola, J. R.; Kwong, P. D., Soluble Prefusion Closed DS-SOSIP.664-Env Trimers of Diverse HIV-1 Strains. Cell reports 2017, 21 (10), 2992-3002. 18. Do Kwon, Y.; Pancera, M.; Acharya, P.; Georgiev, I. S.; Crooks, E. T.; Gorman, J.; Joyce, M. G.; Guttman, M.; Ma, X.; Narpala, S.; Soto, C.; Terry, D. S.; Yang, Y.; Zhou, T.; Ahlsen, G.; Bailer, R. T.; Chambers, M.; Chuang, G. Y.; Doria-Rose, N. A.; Druz, A.; Hallen, M. A.; Harned, A.; Kirys, T.; Louder, M. K.; O'Dell, S.; Ofek, G.; Osawa, K.; Prabhakaran, M.; Sastry, M.; Stewart-Jones, G. B.; Stuckey, J.; Thomas, P. V.; Tittley, T.; Williams, C.; Zhang, B.; Zhao, H.; Zhou, Z.; Donald, B. R.; Lee, L. K.; Zolla-Pazner, S.; Baxa, U.; Schon, A.; Freire, E.; Shapiro, L.; Lee, K. K.; Arthos, J.; Munro, J. B.; Blanchard, S. C.; Mothes, W.; Binley, J. M.; McDermott, A. B.; Mascola, J. R.; Kwong, P. D., Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env. Nature structural & molecular biology 2015, 22 (7), 522-31. 19. Derdeyn, C. A.; Decker, J. M.; Bibollet-Ruche, F.; Mokili, J. L.; Muldoon, M.; Denham, S. A.; Heil, M. L.; Kasolo, F.; Musonda, R.; Hahn, B. H.; Shaw, G. M.; Korber, B. T.; Allen, S.; Hunter, E., Envelopeconstrained neutralization-sensitive HIV-1 after heterosexual transmission. Science 2004, 303 (5666), 2019-22. 20. Doria-Rose, N. A.; Schramm, C. A.; Gorman, J.; Moore, P. L.; Bhiman, J. N.; DeKosky, B. J.; Ernandes, M. J.; Georgiev, I. S.; Kim, H. J.; Pancera, M.; Staupe, R. P.; Altae-Tran, H. R.; Bailer, R. T.; Crooks, E. T.; Cupo, A.; Druz, A.; Garrett, N. J.; Hoi, K. H.; Kong, R.; Louder, M. K.; Longo, N. S.; McKee, K.; Nonyane, M.; O'Dell, S.; Roark, R. S.; Rudicell, R. S.; Schmidt, S. D.; Sheward, D. J.; Soto, C.; Wibmer, C. K.; Yang, Y.; Zhang, Z.; Mullikin, J. C.; Binley, J. M.; Sanders, R. W.; Wilson, I. A.; Moore, J. P.; Ward, A. B.; Georgiou, G.; Williamson, C.; Abdool Karim, S. S.; Morris, L.; Kwong, P. D.; Shapiro, L.; Mascola, J. R.; Becker, J.; Benjamin, B.; Blakesley, R.; Bouffard, G.; Brooks, S.; Coleman, H.; Dekhtyar, M.; Gregory, M.; Guan, X.; Gupta, J.; Han, J.; Hargrove, A.; Ho, S. L.; Johnson, T.; Legaspi, R.; Lovett, S.; Maduro, Q.; Masiello, C.; Maskeri, B.; McDowell, J.; Montemayor, C.; Mullikin, J.; Park, M.; Riebow, N.; Schandler, K.; Schmidt, B.; Sison, C.; Stantripop, M.; Thomas, J.; Thomas, P.; Vemulapalli, M.; Young, A., Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies. Nature 2014, 509 (7498), 55-62. 21. McLellan, J. S.; Pancera, M.; Carrico, C.; Gorman, J.; Julien, J. P.; Khayat, R.; Louder, R.; Pejchal, R.; Sastry, M.; Dai, K.; O'Dell, S.; Patel, N.; Shahzad-ul-Hussan, S.; Yang, Y.; Zhang, B.; Zhou, T.; Zhu, J.; Boyington, J. C.; Chuang, G. Y.; Diwanji, D.; Georgiev, I.; Kwon, Y. D.; Lee, D.; Louder, M. K.; Moquin, S.; Schmidt, S. D.; Yang, Z. Y.; Bonsignori, M.; Crump, J. A.; Kapiga, S. H.; Sam, N. E.; Haynes, B. F.; Burton, D. R.; Koff, W. C.; Walker, L. M.; Phogat, S.; Wyatt, R.; Orwenyo, J.; Wang, L. X.; Arthos, J.; Bewley, C. A.; Mascola, J. R.; Nabel, G. J.; Schief, W. R.; Ward, A. B.; Wilson, I. A.; Kwong, P. D., Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9. Nature 2011, 480 (7377), 336-43. 22. Julien, J. P.; Cupo, A.; Sok, D.; Stanfield, R. L.; Lyumkis, D.; Deller, M. C.; Klasse, P. J.; Burton, D. R.; Sanders, R. W.; Moore, J. P.; Ward, A. B.; Wilson, I. A., Crystal structure of a soluble cleaved HIV-1 envelope trimer. Science 2013, 342 (6165), 1477-83. 23. Gorny, M. K.; Conley, A. J.; Karwowska, S.; Buchbinder, A.; Xu, J. Y.; Emini, E. A.; Koenig, S.; ZollaPazner, S., Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody. Journal of virology 1992, 66 (12), 7538-42. 24. Wu, X.; Yang, Z. Y.; Li, Y.; Hogerkorp, C. M.; Schief, W. R.; Seaman, M. S.; Zhou, T.; Schmidt, S. D.; Wu, L.; Xu, L.; Longo, N. S.; McKee, K.; O'Dell, S.; Louder, M. K.; Wycuff, D. L.; Feng, Y.; Nason, M.; DoriaRose, N.; Connors, M.; Kwong, P. D.; Roederer, M.; Wyatt, R. T.; Nabel, G. J.; Mascola, J. R., Rational 15 ACS Paragon Plus Environment


design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 2010, 329 (5993), 856-61. 25. Roben, P.; Moore, J. P.; Thali, M.; Sodroski, J.; Barbas, C. F., 3rd; Burton, D. R., Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. Journal of virology 1994, 68 (8), 4821-8. 26. Walker, L. M.; Huber, M.; Doores, K. J.; Falkowska, E.; Pejchal, R.; Julien, J. P.; Wang, S. K.; Ramos, A.; Chan-Hui, P. Y.; Moyle, M.; Mitcham, J. L.; Hammond, P. W.; Olsen, O. A.; Phung, P.; Fling, S.; Wong, C. H.; Phogat, S.; Wrin, T.; Simek, M. D.; Koff, W. C.; Wilson, I. A.; Burton, D. R.; Poignard, P., Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 2011, 477 (7365), 466-70. 27. Huang, J.; Kang, B. H.; Pancera, M.; Lee, J. H.; Tong, T.; Feng, Y.; Imamichi, H.; Georgiev, I. S.; Chuang, G. Y.; Druz, A.; Doria-Rose, N. A.; Laub, L.; Sliepen, K.; van Gils, M. J.; de la Pena, A. T.; Derking, R.; Klasse, P. J.; Migueles, S. A.; Bailer, R. T.; Alam, M.; Pugach, P.; Haynes, B. F.; Wyatt, R. T.; Sanders, R. W.; Binley, J. M.; Ward, A. B.; Mascola, J. R.; Kwong, P. D.; Connors, M., Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface. Nature 2014, 515 (7525), 13842. 28. Blattner, C.; Lee, J. H.; Sliepen, K.; Derking, R.; Falkowska, E.; de la Pena, A. T.; Cupo, A.; Julien, J. P.; van Gils, M.; Lee, P. S.; Peng, W.; Paulson, J. C.; Poignard, P.; Burton, D. R.; Moore, J. P.; Sanders, R. W.; Wilson, I. A.; Ward, A. B., Structural Delineation of a Quaternary, Cleavage-Dependent Epitope at the gp41-gp120 Interface on Intact HIV-1 Env Trimers. Immunity 2014, 40 (5), 669-80. 29. Lee, P. S.; Ohshima, N.; Stanfield, R. L.; Yu, W.; Iba, Y.; Okuno, Y.; Kurosawa, Y.; Wilson, I. A., Receptor mimicry by antibody F045-092 facilitates universal binding to the H3 subtype of influenza virus. Nature communications 2014, 5, 3614. 30. Chuang, G. Y.; Acharya, P.; Schmidt, S. D.; Yang, Y.; Louder, M. K.; Zhou, T.; Kwon, Y. D.; Pancera, M.; Bailer, R. T.; Doria-Rose, N. A.; Nussenzweig, M. C.; Mascola, J. R.; Kwong, P. D.; Georgiev, I. S., Residue-level prediction of HIV-1 antibody epitopes based on neutralization of diverse viral strains. Journal of virology 2013, 87 (18), 10047-58. 31. Zhou, T.; Doria-Rose, N. A.; Cheng, C.; Stewart-Jones, G. B. E.; Chuang, G. Y.; Chambers, M.; Druz, A.; Geng, H.; McKee, K.; Kwon, Y. D.; O'Dell, S.; Sastry, M.; Schmidt, S. D.; Xu, K.; Chen, L.; Chen, R. E.; Louder, M. K.; Pancera, M.; Wanninger, T. G.; Zhang, B.; Zheng, A.; Farney, S. K.; Foulds, K. E.; Georgiev, I. S.; Joyce, M. G.; Lemmin, T.; Narpala, S.; Rawi, R.; Soto, C.; Todd, J. P.; Shen, C. H.; Tsybovsky, Y.; Yang, Y.; Zhao, P.; Haynes, B. F.; Stamatatos, L.; Tiemeyer, M.; Wells, L.; Scorpio, D. G.; Shapiro, L.; McDermott, A. B.; Mascola, J. R.; Kwong, P. D., Quantification of the Impact of the HIV-1-Glycan Shield on Antibody Elicitation. Cell reports 2017, 19 (4), 719-732. 32. Montefiori, D. C., Measuring HIV neutralization in a luciferase reporter gene assay. Methods in molecular biology 2009, 485, 395-405. 33. Shu, Y.; Winfrey, S.; Yang, Z. Y.; Xu, L.; Rao, S. S.; Srivastava, I.; Barnett, S. W.; Nabel, G. J.; Mascola, J. R., Efficient protein boosting after plasmid DNA or recombinant adenovirus immunization with HIV-1 vaccine constructs. Vaccine 2007, 25 (8), 1398-408. 34. Seaman, M. S.; Janes, H.; Hawkins, N.; Grandpre, L. E.; Devoy, C.; Giri, A.; Coffey, R. T.; Harris, L.; Wood, B.; Daniels, M. G.; Bhattacharya, T.; Lapedes, A.; Polonis, V. R.; McCutchan, F. E.; Gilbert, P. B.; Self, S. G.; Korber, B. T.; Montefiori, D. C.; Mascola, J. R., Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. Journal of virology 2010, 84 (3), 1439-52. 35. Bottcher, E.; Matrosovich, T.; Beyerle, M.; Klenk, H. D.; Garten, W.; Matrosovich, M., Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium. Journal of virology 2006, 80 (19), 9896-8.


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36. Talaat, K. R.; Karron, R. A.; Callahan, K. A.; Luke, C. J.; DiLorenzo, S. C.; Chen, G. L.; Lamirande, E. W.; Jin, H.; Coelingh, K. L.; Murphy, B. R.; Kemble, G.; Subbarao, K., A live attenuated H7N3 influenza virus vaccine is well tolerated and immunogenic in a Phase I trial in healthy adults. Vaccine 2009, 27 (28), 3744-53.



a

b

trimeric antigen A

iFerr HC

Onecomponent ferritin

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iFerr LC

ΔNTHC

ΔNTLC

Wildtype NT Engineered NT

trimeric antigen A trimeric antigen B

Twocomponent ferritin

34Å

31Å

ΔNTHC ΔNTLC

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.


Page 19 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a


b

iFerr with no antigens attached

M M

1

2

HCantigen 1 2 NR R

3

4

5

LCantigen 3 NR R4

6

HC-LCantigen 5 NR R6

198

gp140 on iFerr HC or iFerr LC gp120

98 62 49

gp41 on iFerr LC gp41 on iFerr HC

38 28

iFerr LC iFerr HC

17

c Antigen on iFerr LC only

Antigen on iFerr HC only

construct

gp120

gp41

iFerr HC iFerr LC

gp120

gp41

Antigen on iFerr HC and LC iFerr HC

gp120

gp41

iFerr HC

iFerr LC

gp120

gp41

iFerr LC

EM structure

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 nm and 15 nm (inset) respectively). (b) SDS-PAGE of iFerr particles with HIV-1 Env attached to iFerr heavy (HC) or light chains (LC), non-reduced (NR), or reduced (R). Lanes are as follows: M: molecular weight marker. (lanes 1-2) antigen on HC only; (lanes 3-4) antigen on LC only; (lanes 5-6) 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 nm and 15 nm (inset) respectively.



a

b

Diverse HIV-1 antigens on both iFerr HC and LC

Fractions

gp120

gp41

iFerr HC

gp120

gp41

iFerr LC

16-22 23-25 26

c

33

Fractions 16-22

Fractions 23-25

Fraction 26

Fraction 33

ml

d

F105

e

Fractions

VRC26.09 PGT145 F105 17b 447-52D 8ANC195 35O22 PGT151 VRC01 b12 2G12 PGT128

HIV-1 antibodies

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 24

16-22 23-25 3.95 3.95 3.95 3.95 1.01 0.73 0 0 3.06 3.3 0.55 0.37 3.95 3.95 3.43 2.73 3.95 3.95 3.95 3.95 3.88 2.81 2.74 2.35

26 3.94 3.51 0.56 0 2.82 0.25 3.19 1.99 3.94 3.4 2.02 1.77

OD450 at 1 µg/ml

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 nm and 100 nm (inset)). (d) Antigenic characterization of the two-component HIV iFerr by lectin-capture ELISA following sizeexclusion chromatography. (e) Summary of the antigenicity data for the HIV-1 antibodies (represented as a heatmap for OD450 values at 1 µg/ml antibody concentration) are shown.


Page 21 of 24

a

Fractions 20-21

Diverse flu antigens on both iFerr HC and LC HA1

HA2

iFerr HC

HA1

HA2

iFerr LC

c

b

Fractions

Flu antibodies

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5J8 CH67 f045-092 CR9114 CR6261 FI6 CR8020

20-21 3.95 0 0 3.95 3.95 3.95 0.4

OD450 at 1 µg/ml

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 nm 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).



a

Page 22 of 24

Fractions 20-21

Flu and HIV-1 antigens on both iFerr HC and LC

b

HA1

HA2

iFerr HC

gp120

gp41

iFerr LC

F105

Fractions 20-21 5J8 CH67 f045-092 CR9114 CR6261 FI6 CR8020

c

3.94 0 0 3.94 3.94 3.94 0.61

VRC26.09 PGT145 F105 17b 447-52D 8ANC195 35O22 PGT151 VRC01 b12 2G12 PGT128

3.95 1.95 0.76 0 2.03 0.07 1.32 3.11 1.98 0.17 0.4 1.45

OD450 at 1 µg/ml

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 nm and 100 nm (inset)). (b,c) Antigenic characterization against (b) HIV-1-specific antibodies and (c) Influenza-specific antibodies. ACS Paragon Plus Environment

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



80x40mm (300 x 300 DPI)


Page 24 of 24

Two-Component Ferritin Nanoparticles for Multimerization of Diverse

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