Presenting native-like HIV-1 envelope trimers on ferritin nanoparticles improves their immunogenicity
Retrovirology volume 12, Article number: 82 (2015)
Presenting vaccine antigens in particulate form can improve their immunogenicity by enhancing B cell activation.
We describe ferritin-based protein nanoparticles that display multiple copies of native-like HIV-1 envelope glycoprotein trimers (BG505 SOSIP.664). Trimer-bearing nanoparticles were significantly more immunogenic than trimers in both mice and rabbits. Furthermore, rabbits immunized with the trimer-bearing nanoparticles induced significantly higher neutralizing antibody responses against most tier 1A viruses, and higher responses (but not significantly), to several tier 1B viruses and the autologous tier 2 virus than when the same trimers were delivered as soluble proteins.
This or other nanoparticle designs may be practical ways to improve the immunogenicity of envelope glycoprotein trimers.
An HIV-1 subunit vaccine should induce a broad and potent neutralizing antibody (NAb) response against the envelope glycoprotein spike (Env) . Soluble, stable mimics of the native spike, such as the BG505 SOSIP.664 gp140 trimer, might be good starting points for such a vaccine [2–5]. These trimers bind virtually all known broadly neutralizing antibodies (bNAbs) but almost no non-neutralizing antibodies (non-NAbs), and adopt a native-like conformation with a well-defined structure [2, 6–8]. Furthermore, unlike other gp140 proteins, soluble, adjuvanted BG505 SOSIP.664 trimers induce NAbs against the autologous, neutralization-resistant (tier 2) virus efficiently in animals . Licensed subunit vaccines against viral pathogens, such as hepatitis B virus and human papillomavirus, are however particulate antigens . The greater size and the capacity for multivalent antigen presentation and B cell receptor cross-linking provide such particulate vaccines with advantages over soluble proteins for inducing antibody responses . For example, fusing eight influenza hemagglutinin (HA) trimers or engineered HA stem antigens to Helicobacter pylori ferritin greatly improved NAb responses against influenza in animals [12, 13].
Modeling showed that H. Pylori ferritin (GenBank accession no. NP_223316) could potentially present eight BG505 SOSIP.664 trimers. Therefore we fused the ferritin N-terminus, starting from Asp5, to the SOSIP.664 C-terminus, separated by a Gly-Ser-Gly (GSG) linker (Fig. 1a). The SOSIP.664-ferritin plasmid was co-transfected into 293F cells with a furin plasmid to maximize trimer cleavage and ensure it adopts a native conformation . To select for antigenically and structurally well-folded Env proteins, the secreted nanoparticles and control trimers were purified using PGT145 bNAb-affinity chromatography . Judged by BN-PAGE and SDS-PAGE analysis followed by Coomassie staining this purification method yielded highly pure (>95 % purity) SOSIP.664 trimer and SOSIP.664-ferritin protein preparations (Fig. 1b). SDS-PAGE also confirmed that the SOSIP.664 component of the nanoparticles was cleaved efficiently between gp120 and gp41 (Fig. 1b, left panel).
The antigenic structure of SOSIP.664 trimers and SOSIP.664-ferritin was compared using ELISA. Proteins were captured using Galanthus nivalis lectin and probed with bNAbs and non-NAbs (Fig. 1c). Several bNAbs that bind to distinct Env epitopes (VRC01, PGT121, PG9) showed similar binding to SOSIP.664 and SOSIP.664-ferritin, moreover non-NAbs (F105 and F240) displayed similarly poor reactivity with both proteins (Fig. 1c). We did observe lower affinity of gp120/gp41 interface (8ANC195, 35O22 and PGT151) and gp41 (3BC315) bNAbs for SOSIP.664-ferritin, which might be explained by steric hindrance of neighboring trimers on the nanoparticle (Fig. 1c).
The purified nanoparticles were analyzed by negative stain electron microscopy (NS-EM). More than 70 % of the particles on the EM grid resembled ferritin cages with protruding spikes that were 30–40 nm in diameter (Fig. 1d). When single particles were automatically picked and processed as described elsewhere , 2D class averages representing views along the three- and fourfold symmetry axes suggested that 65–80 % of the SOSIP.664-ferritin particles were fully decorated with Env trimers (three and four spikes visible, respectively) (Fig. 1e). The lack of views along the twofold symmetry axis (i.e. six spikes visible) may be a result of the immobilization on the EM grid or flexibility of the GSG-linker that affects the alignment of the particles and visualization of each Env trimer.
We first immunized mice (approved by the AMC animal ethics committee: DMB-102836; n = 8 mice per group) to compare the antibody response of SOSIP.664-ferritin nanoparticles with soluble (i.e. monovalent) SOSIP.664 trimers. The anti-trimer binding responses were eightfold higher in mice vaccinated with nanoparticle-displayed trimers compared to soluble trimers (medians: 86 vs. 686; P = 0.015) (Fig. 2a). We next immunized rabbits (approved by the Covance Institutional Animal Care and Use Committee (IACUC): 0082-14; n = 5 rabbits per group), using a triple DNA-prime, protein-boost regimen (Fig. 2b). Given the limited group sizes and the large spread in neutralization titers generally observed in other HIV-1 vaccination studies , we included historic control sera from four rabbits to increase the statistical power of this study. These rabbits were immunized with the soluble trimers in an independent experiment using the same DNA prime + protein boost protocol (approved by the Covance IACUC: 0001-14; n = 4 rabbits per group; unpublished results). As expected, the anti-trimer binding antibody responses rose and fell between immunizations, and were boosted by the protein-only immunization [9, 16]. The titers were two- to threefold higher at several time points for the rabbits given SOSIP.664-ferritin nanoparticles compared to the soluble trimers. Although the improved immunogenicity was less pronounced in rabbits compared to mice, it is consistent with other observations showing the benefits of particulate antigen presentation [12, 17, 18] (Fig. 2b).
We used the TZM-bl cell neutralization assay and viruses from different clades to assess the serum NAb titers 2 weeks after the protein boost in rabbits . Sera from 4/5 rabbits given the SOSIP.664-ferritin nanoparticles neutralized the autologous BG505.T332 N tier 2 virus, and the median titer in this group was higher than in the soluble trimer group (603 vs. 186). However, because of the small group sizes, the difference was not statistically significant (P = 0.34) (Fig. 2c). The NAb titers against heterologous tier 1 viruses were also higher in the rabbits that received SOSIP.664-ferritin nanoparticles (Fig. 2d). Median NAb titers against tier 1A viruses were 10- to 90-fold higher in the nanoparticle group: MN.3 (4,857 vs. 282; P = 0.019); SF162 (2,799 vs. 31; P = 0.004); MW.965 (18,563 vs. 1,127; P = 0.019). For the more resistant tier 1B viruses the titers were also higher, although this did not reach statistical significance in all cases: 6535.3 (472 vs. 82; P = 0.029); BaL (171 vs. 71; P = 0.083); DJ286.3 (195 vs. 64; P = 0.11). The tier 1B viruses HxB2, Q23env17, ZM109F and ZM197M and the tier 2 viruses 94UG103, 92RW020, Q259.d2.17, Q769.d22, Q842.d12 (all clade A), YU2 (clade B) and Ce1176_A3 (clade C) were not neutralized by any rabbit sera (data not shown).
We conclude from this exploratory study that the nanoparticle display of SOSIP.664 trimers improves the magnitude of the overall antibody response and neutralization breadth at the tier 1 level. We are seeking to solve the substantial problem of inducing a bNAb response (at the tier 2 level) by improving the design of native-like trimers such as BG505 SOSIP.664 and/or how they are used as immunogens. If and when this goal is achieved, the superior immunogenicity of a particulate antigen presentation should be valuable.
Van Gils MJ, Sanders RW. In vivo protection by broadly neutralizing HIV antibodies. Trends Microbiol. 2014;22:550–1.
Sanders RW, Derking R, Cupo A, Julien JP, Yasmeen A, de Val N, Kim HJ, Blattner C, de la Peña AT, Korzun J, Golabek M, de los Reyes K, Ketas TJ, Vangils MJ, King CR, King CR, Wilson IA, Ward AB, Klasse PJ, Moore JP. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS Pathog. 2013;9:e1003618.
Lyumkis D, Julien J-P, de Val N, Cupo A, Potter CS, Klasse P-J, Burton DR, Sanders RW, Moore JP, Carragher B, Wilson IA, Ward AB. Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer. Science. 2013;342:1484–90.
Julien J-P, Cupo A, Sok D, Stanfield RL, Lyumkis D, Deller MC, Klasse P-J, Burton DR, Sanders RW, Moore JP, Ward AB, Wilson IA. Crystal structure of a soluble cleaved HIV-1 envelope trimer. Science. 2013;342:1477–83.
Pancera M, Zhou T, Druz A, Georgiev IS, Soto C, Gorman J, Huang J, Acharya P, Chuang G-Y, Ofek G, Stewart-Jones GBE, Stuckey J, Bailer RT, Joyce MG, Louder MK, Tumba N, Yang Y, Zhang B, Cohen MS, Haynes BF, Mascola JR, Morris L, Munro JB, Blanchard SC, Mothes W, Connors M, Kwong PD. Structure and immune recognition of trimeric pre-fusion HIV-1 Env. Nature. 2014;514:455–61.
Blattner C, Lee JH, Sliepen K, Derking R, Falkowska E, de la Peña AT, Cupo A, Julien J-P, van Gils M, Lee PS, Peng W, Paulson JC, Poignard P, Burton DR, Moore JP, Sanders RW, Wilson IA, Ward AB. Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 Interface on intact HIV-1 Env trimers. Immunity. 2014;40:669–80.
Falkowska E, Le KM, Ramos A, Doores KJ, Lee J, Blattner C, Ramirez A, Derking R, VanGils MJ, Liang CH, Mcbride R, von Bredow B, Shivatare SS, Wu CY, Chan-Hui PY, Liu Y, Feizi T, Zwick MB, Koff WC, Seaman MS, Swiderek K, Moore JP, Evans D, Paulson JC, Wong CH, Ward AB, Wilson IA, Sanders RW, Poignard P, Burton DR. Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers. Immunity. 2014;40:657–68.
Huang J, Kang BH, Pancera M, Lee JH, Tong T, Feng Y, Imamichi H, Georgiev IS, Chuang G-Y, Druz A, Doria-Rose NA, Laub L, Sliepen K, van Gils MJ, de la Peña AT, Derking R, Klasse PJ, Migueles SA, Bailer RT, Alam M, Pugach P, Haynes BF, Wyatt RT, Sanders RW, Binley JM, Ward AB, Mascola JR, Kwong PD, Connors M. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface. Nature. 2014;515:138–42.
Sanders RW, van Gils MJ, Derking R, Sok D, Ketas TJ, Burger JA, Ozorowski G, Cupo A, Simonich C, Goo L, Arendt H, Kim HJ, Lee JH, Pugach P, Williams M, Debnath G, Moldt B, van Breemen MJ, Isik G, Medina-Ramirez M, Back JW, Koff WC, Julien J-P, Rakasz EG, Seaman MS, Guttman M, Lee KK, Klasse PJ, LaBranche C, Schief WR, et al. HIV-1 neutralizing antibodies induced by native-like envelope trimers. Science. 2015;349:aac4223.
Schiller J, Chackerian B. Why HIV virions have low numbers of envelope spikes: implications for vaccine development. PLoS Pathog. 2014;10:e1004254.
Bachmann MF, Jennings GT. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev Immunol. 2010;10:787–96.
Kanekiyo M, Wei C-J, Yassine HM, McTamney PM, Boyington JC, Whittle JRR, Rao SS, Kong W-P, Wang L, Nabel GJ. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature. 2013;499:102–6.
Yassine HM, Boyington JC, McTamney PM, Wei C, Kanekiyo M, Kong W, Gallagher JR, Wang L, Zhang Y, Joyce MG, Lingwood D, Moin SM, Andersen H, Okuno Y, Rao SS, Harris AK, Kwong PD, Mascola JR, Nabel GJ, Graham BS: Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection. Nat Med. 2015;21:1065–70.
Ringe RP, Sanders RW, Yasmeen A, Kim HJ, Lee JH, Cupo A, Korzun J, Derking R, van Montfort T, Julien J-P, Wilson IA, Klasse PJ, Ward AB, Moore JP. Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation. Proc Natl Acad Sci USA. 2013;110:18256–61.
Pugach P, Ozorowski G, Cupo A, Ringe R, Yasmeen A, de Val N, Derking R, Kim HJ, Korzun J, Golabek M, de Los Reyes K, Ketas TJ, Julien J-P, Burton DR, Wilson IA, Sanders RW, Klasse PJ, Ward AB, Moore JP. A native-like SOSIP.664 trimer based on an HIV-1 subtype B env gene. J Virol. 2015;89:3380–95.
Anderson KP, Lucas C, Hanson CV, Londe HF, Izu A, Gregory T, Ammann A, Berman PW, Eichberg JW. Effect of dose and immunization schedule on immune response of baboons to recombinant glycoprotein 120 of HIV-1. J Infect Dis. 1989;160:960–9.
Moon JJ, Suh H, Li AV, Ockenhouse CF, Yadava A, Irvine DJ. Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction. Proc Natl Acad Sci USA. 2012;109:1080–5.
Bachmann MF, Zinkernagel RM. Neutralizing antiviral B cell responses. Annu Rev Immunol. 1997;15:235–70.
Seaman MS, Janes H, Hawkins N, Grandpre LE, Devoy C, Giri A, Coffey RT, Harris L, Wood B, Daniels MG, Bhattacharya T, Lapedes A, Polonis VR, McCutchan FE, Gilbert PB, Self SG, Korber BT, Montefiori DC, Mascola JR. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J Virol. 2010;84:1439–52.
Schrodinger LLC. The PyMOL molecular graphics system, Version 1.3r1. 2010.
Binley JM, Sanders RW, Master A, Cayanan CS, Wiley CL, Schiffner L, Travis B, Kuhmann S, Burton DR, Hu S, Olson WC, Moore JP. Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins. J Virol. 2002;76:2606–16.
Binley JM, Sanders RW, Clas B, Schuelke N, Master A, Guo Y, Kajumo F, Anselma DJ, Maddon PJ, Olson WC, Moore JP. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J Virol. 2000;74:627–43.
Sanders RW, Vesanen M, Schuelke N, Master A, Schiffner L, Kalyanaraman R, Paluch M, Berkhout B, Maddon PJ, Olson WC, Lu M, Moore JP. Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. J Virol. 2002;76:8875–89.
Maraskovsky E, Schnurr M, Wilson NS, Robson NC, Boyle J, Drane D. Development of prophylactic and therapeutic vaccines using the ISCOMATRIX adjuvant. Immunol Cell Biol. 2009;87:371–6.
Derking R, Ozorowski G, Sliepen K, Yasmeen A, Cupo A, Torres JL, Julien J-P, Lee JH, van Montfort T, de Taeye SW, Connors M, Burton DR, Wilson IA, Klasse P-J, Ward AB, Moore JP, Sanders RW. Comprehensive antigenic map of a cleaved soluble HIV-1 envelope trimer. PLOS Pathog. 2015;11:e1004767.
KS conceived the project and carried out experiments, analyzed the data and wrote the manuscript. GO and ABW performed the electron microscopy experiments, analyzed the data and co-wrote the manuscript. JB, CLB and DCM carried out and interpreted neutralization experiments. TvM and MS purified proteins, performed mice vaccinations and performed ELISA experiments; JPM co-wrote the manuscript. RWS conceived the project, participated in the experimental design and wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by the International AIDS Vaccine Initiative (IAVI), The Bill and Melinda Gates Foundation, National Institutes of Health Grant P01 AI082362, NIAID-NIH Contract HHSN27201100016C, and Scripps CHAVI-ID UM1 AI100663. RWS is a recipient of a Vidi grant from the Netherlands Organization for Scientific Research (NWO) and a Starting Investigator Grant from the European Research Council (ERC-StG-2011–280829-SHEV). We thank Ronald Derking for providing the D7324-tagged BG505 SOSIP.664 trimer and Hansi Dean for the historic control rabbit sera. We thank Nicola Janus for her help with mouse immunizations and Mitch Hartog for technical assistance.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
About this article
Cite this article
Sliepen, K., Ozorowski, G., Burger, J.A. et al. Presenting native-like HIV-1 envelope trimers on ferritin nanoparticles improves their immunogenicity. Retrovirology 12, 82 (2015). https://doi.org/10.1186/s12977-015-0210-4