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Proof of principle for epitope-focused vaccine design

Nature volume 507, pages 201–206 (2014)Cite this article

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Abstract

Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

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Figure 1: A new computational method to design epitope-focused vaccines, illustrated with a neutralization epitope from RSV.
Figure 2: Biophysical and structural characterization of scaffold FFL_001.
Figure 3: Serological analysis of immunized macaques.
Figure 4: Analysis of monoclonal antibodies isolated from an immunized macaque.

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Accession codes

Accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for FFL_005, FFL_001–mota, and FFL_001–17-HD9 structures have been deposited in the Protein Data Bank with accession codes 4L8I, 4JLR and 4N9G, respectively.

References

  1. Plotkin, S. A., Orenstein, W. A. & Offit, P. A. Vaccines 6th edn (Elsevier, 2012)

    Google Scholar 

  2. Rappuoli, R., Mandl, C. W., Black, S. & De Gregorio, E. Vaccines for the twenty-first century society. Nature Rev. Immunol. 11, 865–872 (2011)

    Article  CAS  Google Scholar 

  3. Lozano, R. et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380, 2095–2128 (2012)

    Article  Google Scholar 

  4. Murray, C. J. et al. Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380, 2197–2223 (2012)

    Article  Google Scholar 

  5. Rerks-Ngarm, S. et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N. Engl. J. Med. 361, 2209–2220 (2009)

    Article  CAS  Google Scholar 

  6. The RTS,S Clinical Trials Partnership. A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants. N. Engl. J. Med. 367, 2284–2295 (2012)

  7. Tameris, M. D. et al. Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet 381, 1021–1028 (2013)

    Article  CAS  Google Scholar 

  8. Osterholm, M. T., Kelley, N. S., Sommer, A. & Belongia, E. A. Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis. Lancet Infect. Dis. 12, 36–44 (2012)

    Article  Google Scholar 

  9. Bhatt, S. et al. The global distribution and burden of dengue. Nature 496, 504–507 (2013)

    Article  CAS  ADS  Google Scholar 

  10. Anderson, L. J. et al. Strategic priorities for respiratory syncytial virus (RSV) vaccine development. Vaccine 31 (Suppl. 2). B209–B215 (2013)

    Article  Google Scholar 

  11. U.S. Food and Drug Administration. Vaccines, Blood & Biologics http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/

  12. Burton, D. R. Antibodies, viruses and vaccines. Nature Rev. Immunol. 2, 706–713 (2002)

    Article  CAS  Google Scholar 

  13. Burton, D. R., Poignard, P., Stanfield, R. L. & Wilson, I. A. Broadly neutralizing antibodies present new prospects to counter highly antigenically diverse viruses. Science 337, 183–186 (2012)

    Article  CAS  ADS  Google Scholar 

  14. Wu, H. et al. Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract. J. Mol. Biol. 368, 652–665 (2007)

    Article  CAS  Google Scholar 

  15. McLellan, J. S. et al. Structural basis of respiratory syncytial virus neutralization by motavizumab. Nature Struct. Mol. Biol. 17, 248–250 (2010)

    Article  CAS  Google Scholar 

  16. Correia, B. E. et al. Computational design of epitope-scaffolds allows induction of antibodies specific for a poorly immunogenic HIV vaccine epitope. Structure 18, 1116–1126 (2010)

    Article  CAS  Google Scholar 

  17. Ofek, G. et al. Elicitation of structure-specific antibodies by epitope scaffolds. Proc. Natl Acad. Sci. USA 107, 17880–17887 (2010)

    Article  CAS  ADS  Google Scholar 

  18. McLellan, J. S. et al. Design and characterization of epitope-scaffold immunogens that present the motavizumab epitope from respiratory syncytial virus. J. Mol. Biol. 409, 853–866 (2011)

    Article  CAS  Google Scholar 

  19. Azoitei, M. L. et al. Computation-guided backbone grafting of a discontinuous motif onto a protein scaffold. Science 334, 373–376 (2011)

    Article  CAS  ADS  Google Scholar 

  20. Azoitei, M. L. et al. Computational design of high-affinity epitope scaffolds by backbone grafting of a linear epitope. J. Mol. Biol. 415, 175–192 (2012)

    Article  CAS  Google Scholar 

  21. Kuhlman, B. et al. Design of a novel globular protein fold with atomic-level accuracy. Science 302, 1364–1368 (2003)

    Article  CAS  ADS  Google Scholar 

  22. Koga, N. et al. Principles for designing ideal protein structures. Nature 491, 222–227 (2012)

    Article  CAS  ADS  Google Scholar 

  23. Correia, B. E. et al. Computational protein design using flexible backbone remodeling and resurfacing: case studies in structure-based antigen design. J. Mol. Biol. 405, 284–297 (2011)

    Article  CAS  Google Scholar 

  24. Zhu, Q. et al. Analysis of respiratory syncytial virus preclinical and clinical variants resistant to neutralization by monoclonal antibodies palivizumab and/or motavizumab. J. Infect. Dis. 203, 674–682 (2011)

    Article  CAS  Google Scholar 

  25. Clarke, B. E. et al. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature 330, 381–384 (1987)

    Article  CAS  ADS  Google Scholar 

  26. Jegerlehner, A. et al. A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses. Vaccine 20, 3104–3112 (2002)

    Article  CAS  Google Scholar 

  27. Jafri, H. S., Wu, X., Makari, D. & Henrickson, K. J. Distribution of respiratory syncytial virus subtypes A and B among infants presenting to the emergency department with lower respiratory tract infection or apnea. Pediatr. Infect. Dis. J. 32, 335–340 (2013)

    Article  Google Scholar 

  28. Sundling, C. et al. High-resolution definition of vaccine-elicited B cell responses against the HIV primary receptor binding site. Sci. Transl. Med. 4, 142ra196 (2012)

    Article  Google Scholar 

  29. Wen, X. et al. Structure of the human metapneumovirus fusion protein with neutralizing antibody identifies a pneumovirus antigenic site. Nature Struct. Mol. Biol. 19, 461–463 (2012)

    Article  CAS  Google Scholar 

  30. Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 487, 545–574 (2011)

    Article  CAS  Google Scholar 

  31. Rohl, C. A., Strauss, C. E., Misura, K. M. & Baker, D. Protein structure prediction using Rosetta. Methods Enzymol. 383, 66–93 (2004)

    Article  CAS  Google Scholar 

  32. Sheffler, W. & Baker, D. RosettaHoles2: a volumetric packing measure for protein structure refinement and validation. Protein Sci. 19, 1991–1995 (2010)

    Article  CAS  Google Scholar 

  33. Bansal, M., Kumar, S. & Velavan, R. HELANAL: a program to characterize helix geometry in proteins. J. Biomol. Struct. Dyn. 17, 811–819 (2000)

    Article  CAS  Google Scholar 

  34. Greenfield, N. J. Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nature Protocols 1, 2527–2535 (2007)

    Article  Google Scholar 

  35. Greenfield, N. J. Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism. Nature Protocols 1, 2733–2741 (2007)

    Article  Google Scholar 

  36. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995)

    Article  CAS  Google Scholar 

  37. Johnson, B. A. & Blevins, R. A. NMR View: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994)

    Article  CAS  Google Scholar 

  38. Murphy, B. R., Sotnikov, A. V., Lawrence, L. A., Banks, S. M. & Prince, G. A. Enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (RSV) or purified F glycoprotein and challenged with RSV 3-6 months after immunization. Vaccine 8, 497–502 (1990)

    Article  CAS  Google Scholar 

  39. Chen, M. et al. A flow cytometry-based assay to assess RSV-specific neutralizing antibody is reproducible, efficient and accurate. J. Immunol. Methods 362, 180–184 (2010)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Menis, D. Kulp and D. Burton for comments on the manuscript, Y.-E. Ban, D. Alonso and K. E. Laidig for computing assistance, E. Gribben and R. Carnahan for assistance with mouse immunizations and C. Slaughter for assistance in statistical analysis. Adjuplex adjuvant was a gift from Advanced BioAdjuvants. The University of Washington has filed patents relating to immunogens in this manuscript. Materials and information will be provided under Materials Transfer Agreement (MTA). Support for this work was provided by Fundação para a Ciência e a Tecnologia fellowship SFRH/BD/32958/2006 (B.E.C.), National Institutes of Health NRSA Training Grant fellowship T32CA080416 (J.G.J.), The Children’s Hospital of Philadelphia (P.R.J.), a Bill and Melinda Gates Foundation CAVD award (W.R.S., R.K.S. and D.B.), the International AIDS Vaccine Initiative Neutralizing Antibody Consortium (W.R.S. and D.B.), the International AIDS Vaccine Initiative Neutralizing Antibody Center (W.R.S. and R.T.W.), a grant from the March of Dimes (J.E.C.), National Institutes of Health grants 2T32 GM007270 (V.V.) and U54 AI 005714 (R.E.K.), National Institute of Allergy and Infectious Diseases grants P01AI094419 (W.R.S. and R.K.S.), 5R21AI088554 (W.R.S.), 1UM1AI100663 (W.R.S. and R.T.W.), 1R01AI102766-01A1 (Y.L. and R.T.W.), P30AI36214 (from the Center for AIDS Research, University of California, San Diego, to Y.L.), and the National Institute of Allergy and Infectious Diseases Intramural Research Program (B.S.G.). This is manuscript 26069 from The Scripps Research Institute.

Author information

Author notes

  1. Margaret A. Holmes: Deceased.

Authors and Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, 98195, Washington, USA

    Bruno E. Correia, Gretchen Baneyx, Joseph G. Jardine, Oleksandr Kalyuzhniy, Vinayak Vittal, Eric Stevens, Alexandria Schroeter, Skye MacPherson, Andreia M. Serra, Yumiko Adachi, Rachel E. Klevit, David Baker & William R. Schief

  2. PhD Program in Computational Biology, Instituto Gulbenkian Ciência and Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras 2780-157, Portugal,

    Bruno E. Correia

  3. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Bruno E. Correia

  4. The Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, 37232, Tennessee, USA

    John T. Bates & James E. Crowe

  5. The Children’s Hospital of Philadelphia Research Institute, Philadelphia, 19104, Pennsylvania, USA

    Rebecca J. Loomis, Mary J. Connell & Philip R. Johnson

  6. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, 98109-1024, Washington, USA

    Chris Carrico, Peter Rupert, Colin Correnti, Margaret A. Holmes & Roland K. Strong

  7. Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, 92037, California, USA

    Joseph G. Jardine, Skye MacPherson, Yuxing Li, Richard T. Wyatt & William R. Schief

  8. IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, 92037, California, USA

    Joseph G. Jardine, Oleksandr Kalyuzhniy, Skye MacPherson, Andreia M. Serra, Yumiko Adachi, Yuxing Li, Richard T. Wyatt & William R. Schief

  9. Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, 92037, California, USA

    Joseph G. Jardine, Oleksandr Kalyuzhniy, Skye MacPherson, Andreia M. Serra, Yumiko Adachi, Yuxing Li, Richard T. Wyatt & William R. Schief

  10. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Man Chen & Barney S. Graham

  11. Department of Pathology, Microbiology and Immunology, Vanderbilt Medical Center, Nashville, 37232, Tennessee, USA

    James E. Crowe

  12. Department of Pediatrics, Vanderbilt Medical Center, Nashville, 37232, Tennessee, USA

    James E. Crowe

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  1. Bruno E. Correia
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Contributions

B.E.C., J.T.B., R.J.L., R.E.K., B.S.G., R.T.W., D.B., R.K.S., J.E.C, P.R.J. and W.R.S. designed research. B.E.C. wrote the code for FFL, designed proteins and performed biophysical characterization. J.T.B., R.J.L., M.C. and M.J.C. performed serological analysis. G.B. and E.S. prepared and characterized particle immunogens. C. Carrico, J.G.J., P.R., C. Correnti and M.A.H. performed X-ray crystallography. O.K. performed biophysical characterization. V.V. performed NMR studies. M.J.C. performed NHP immunizations. O.K., A.S., S.M., A.M.S., Y.A. and E.S. performed protein expression and purification. Y.L. performed B-cell sorting and RT–PCR. B.E.C. and W.R.S. wrote, and all co-authors edited, the manuscript.

Corresponding author

Correspondence to William R. Schief.

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Competing interests

W.R.S. owns shares in and is co-founder of a company (CompuVax, Inc.) that intends to develop vaccines for RSV and other pathogens based on epitope-focused immunogens pursuant to a research and license agreement in negotiation with The Scripps Research Institute.

Extended data figures and tables

Extended Data Figure 1 Overview of the Fold From Loops (FFL) computational procedure.

Initially large conformational spaces are sampled by low-resolution folding, and subsequently iterative sequence design and small structural optimizations are performed to accommodate the target functional motif.

Extended Data Figure 2 Properties of designed proteins in this study.

a, Sequence alignment of the FFL designs. 3LHP_S is the protein used as the template topology. b, Sequence alignment for the FFL_surf series designed on the basis of the FFL_001 design model. c, Parameters and filtering criteria and results in the design process for FFL designs. a, standard deviation allowed on the constraints derived from the target topology; b, design epitope segment design of residues within the epitope segment that were not part of the epitope–antibody interface; c, filtering criteria based on the helix bend angle; d, Rosetta energy after human-guided optimization. d, Structural diversity in the FFL design models. Values give the backbone r.m.s.d. in Å between two designs or between the template (3LHP_S) and the designs. e, Mutational diversity in the FFL designs. Values give the number of mutations between two designs or between the template (3LHP_S) and the designs.

Extended Data Figure 3 Structural properties of FFL designs in solution.

a, Characterization of the oligomeric state by size exclusion chromatography-coupled inline to multi-angle light scattering (SEC-MALS). All molecules that showed a single monodisperse species by SEC had molecular weights computed from MALS that were consistent with expectation for a monomer (approximately 15 kDa). b, Secondary structure and thermal stability of FFL designs assessed by circular dichroism. Wavelength scans at T = 25 ° (left row) show the double minima typical for helical proteins. Thermal denaturation curves (right row) reveal high thermostability. c, HSQC spectra of several 15N-labelled FFL designs. The spectra exhibit features typical for properly folded proteins with high α-helical content, particularly FFL_006. FFL_005 and FFL_007 exhibited reduced dispersion possibly due to self-association at higher concentrations.

Extended Data Figure 4 SPR data for FFL designs binding to mota or pali.

a, Binding of FFL_005 and FFL_007 to mota, in which the epitope scaffolds were amine-coupled to the sensor chip and mota Fab was used as analyte. The concentrations of mota Fab ranged from 950 nM to 436.5 pM and were used in serial dilutions with a dilution factor of three. b, Binding of FFL_001 and FFL_005 to mota. Mota IgG was captured on the sensor chip by anti-human IgG and epitope scaffolds were used as analytes. The concentrations of scaffold ranged from 6.9 nM to 255.6 pM and were used in serial dilutions with a dilution factor of three. Kinetic fits are shown in red for both panels. c, Binding of FFL_001 and FFL_007 to pali assessed by SPR. FFL_001 was amine-coupled to the sensor chip and pali fab was analyte (left), or pali IgG was captured by anti-human IgG and FFL_007 was analyte (right). d, Mota-binding specificity of FFL_001 assessed by SPR. Mota IgG was the ligand, captured by anti-human IgG on the sensor chip, and FFL_001 (blue) and an epitope point mutant of FFL_001 (FFL_001_K82E, black) were analytes at a concentration of 22 nM. The interaction between FFL_001 and mota was eliminated by the point mutation.

Extended Data Figure 5 Crystallographic statistics for crystal structures determined.

Values in parentheses refer to the highest resolution shell.

Extended Data Figure 6 Immunological evaluation of FFL scaffolds by different means.

a, Evaluation of scaffolds as probes to detect the presence of epitope-specific antibodies in human sera. Sera from six healthy seropositive individuals were tested by ELISA for reactivity to FFL_001, FFL_001 with two different epitope point mutants (FFL_001_K82E and FFL_001_N72Y), and to recombinant RSV F glycoprotein. b, ELISA end point titres from mice immunized with immunogens shown on the x axis. Autologous titres were measured against 001, 005, 007 or HBcAg particles without conjugated scaffold (triangles), and titres were also measured against RSV F protein (red). Titres after two immunizations are on the left, titres after four immunizations are on the right. c, ELISA end point titres for binding to recombinant RSV F protein, from non-human primates (NHPs) immunized with 001, 005, 007 and HBcAg–FFL_001. d, RSV microneutralization assay results for NHPs immunized with 001, 005, 007 and HBcAg–FFL_001. In c and d, values at each time point are mean ± standard deviation computed for the four animals per group at that time point.

Extended Data Figure 7 Neutralization of RSV by week 20, post-5 immunization NHP sera assessed by a flow cytometry-based assay.

a, The neutralization curves for several vaccinated animals are shown. 07C0012 was immunized with FFL_001; 07C004 and 07D039 were immunized with HBcAg–FFL_001; 07C0010 and 07D087 were immunized with FFL_007. b, Table showing 50% neutralization titres measured in two independent assays. c, Flow cytometry assay results for RSV subtypes A and B.

Extended Data Figure 8 Properties of NHP monoclonal antibodies isolated by B-cell sorting from an animal immunized with HBcAg–FFL_001.

a, ELISA binding of recombinant NHP monoclonal antibodies to FFL_001 (left) and recombinant RSV F glycoprotein (right). b, Sequence alignment of heavy (left) and light (right) chains of the Fv domains of NHP monoclonal antibodies 17-HD9 and 31-HG7 along with mota and pali. c, SPR data for monoclonal antibodies 17-HD9 and 31HG7 binding to FFL_001. Monoclonal antibodies IgGs were captured by anti-human IgG on the sensor chip (monoclonal antibodies were expressed with human Fc) and FFL_001 was flowed as analyte. d, Head-to-head comparison of the neutralization potency of NHP monoclonal antibodies, mota and pali in the plaque reduction assay. The data values are shown as mean ± standard deviation from two assays. The data were fit by the equation for one site specific binding with Hill slope, implemented in GraphPadPrism. According to the fits, the IC50s were 0.21 μg ml−1 (pali), 0.046 μg ml−1 (mota), 0.031 μg ml−1 (17-HD9) and 0.049 μg ml−1 (31-HG7). e, EC50 values for neutralization of RSV subtypes A and B by 17-HD9 and 31-HG7 as reported by the flow cytometry assay.

Extended Data Figure 9 SPR data for the binding of NHP monoclonal antibodies to FFL_001 variants.

a–c, FFL_001_surf1 (a), FFL_001_K82E (b), FFL_001_R33C_N72Y_K82E (c). Monoclonal antibodies were captured by anti-human IgG on the sensor chip (antibodies were expressed with human Fc) and FFL_001 variants were flowed as analytes.

Extended Data Figure 10 Four complex structures of 17-HD9 plus peptide in the asymmetric unit, from PDB 4N9G.

The four complexes in the asymmetric unit consisted of two pairs of nearly identical structures (r.m.s.d. within each pair was 0.3 Å), with the pairs differing from each other primarily in the Fv angle of approach to the epitope (angle difference ∼9°) and in the Fab elbow angle (angle difference ∼10°); differences within the peptide between pairs were small (r.m.s.d. over peptide between pairs was 0.7 Å). a, Chains A+B+C. b, Chains E+F+D. c, Chains H+L+Y. d, Chains M+N+Z. e, View of crystal packing interaction, in which the ‘backside’ of one peptide interacts with the backside of another. Partial scaffolds (peptides) are packed against each other at crystal contacts between complexes through an interface outside of the epitope, with perfect dyad symmetry broken by a translation along the non-crystallographic symmetry (NCS) dyad axis to accommodate complementary packing of apolar side chains. The crystal packing is incompatible with the scaffold being present as a three-helix bundle as in the mota or 31-HG7 complex structures. Clear density was lacking for the scaffold outside the helix-turn-helix peptide. Scaffold missing density is possibly due to partial proteolysis or unfolding of the scaffold that may have occurred while purified Fab–scaffold complexes incubated at high concentration (∼10 mg ml−1) in crystallization liquor for 3 months before crystal formation (see Supplementary Methods). The location and size of solvent channels in the crystal could accommodate the disordered region of the scaffold as an extended, flexible peptide unfolded under the conditions of crystallization, but it is also plausible that limited proteolysis has reduced the scaffold to a minimal structure protected by contacts with the antibody.

Supplementary information

Supplementary Information

This file contains Supplementary Methods and Supplementary Tables 1-5. Supplementary Methods include command lines and input files used to perform protein design with Rosetta and extended methods for crystallography and RSV microneutralization assay. Supplementary Table 1 shows raw data for NHP sera binding to RSVF and viral lysates, Supplementary Table 2 shows raw data for RSV neutralization titers and Supplementary Tables 3-5 contain structural analysis of the crystal structures of 17-HD9 and Mota in complex with RSV peptide epitope. (PDF 422 kb)

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Correia, B., Bates, J., Loomis, R. et al. Proof of principle for epitope-focused vaccine design. Nature 507, 201–206 (2014). https://doi.org/10.1038/nature12966

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