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The E2DISP Antigen Display System: A Novel HIV Vaccine Approach
Dina Lauman, Antonella Caivano, Gonzalo Domingo, Luca Meoli, Sunil Thomas, William F. Sutton, Murali-Krishna Kaja, Nancy L. Haigwood, Piergiuseppe De Berardinis, Nicole Doria-Rose Seattle Biomedical Research Institute University of Washington Institute for Protein Biochemistry August 14, 2006
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Virus-Like Particles as Vaccine Approaches
Inactivated virions Complex composition Potential safety risks Virus-like particles (VLPs) made of viral structural proteins HIV Gag or Gag/Env particles Hepatitis B Virus Human Papillomavirus VLPs as display scaffolds Regions or epitopes of heterologous proteins Size constraints on inserts
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The E2 Protein serves as a scaffold for a Multienzyme Complex
PSBD Catalytic Domain (CD) Multiple copies of E1 and E3 associate tightly but non-covalently with E2 N-terminal domains Adapted from Domingo et al., J Mol Biol 305:259.
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The E2 Antigen Display System (E2DISP)
Heterologous Protein HP PSBD Catalytic Domain (CD) Multiple copies of E1 and E3 associate tightly but non-covalently with E2 N-terminal domains
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The E2DISP Antigen Display System as a Vaccine Candidate
Advantages Particles self-assemble in vitro Elicit antibodies, T cell help and CTL to peptides Up to 60 different antigens on VLPs Polypeptides of up to 25 kDa expressed Up to 3 mg protein per 1 L E. coli culture Limitations Prokaryotic system: No glycosylations Unknown whether large antigens will be effectively processed for T cell responses
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Generation of E2DISP-HIV Particles
Express E2DISP-HIV molecules in large-volume E. coli BL21 cultures Purification of 60mers Ammonium Sulfate Fractionation Ion Exchange Chromatography Gel Filtration Mix “Pure” particles in varying ratios, denature, renature “Hybrid” particles
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E2DISP Effectively Displays HIV Peptides
E2 with two HIV peptides elicited: Antibody CD4+ T cells CTL In mice Domingo et al., Vaccine 21:1502
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HIV Peptides (3) and Proteins (17) as E2DISP-HIV Fusions
* Gag-p17 Gag-p24 Gag-p17/24 Gag-ep24 Gag-CTL-p17 Rev * Tat Nef NefLL174/5AA rev TM gag pol vif vpr env SU tat nef vpu LTR RT2 Pep23 RTp51 RTp66 Pro-RTD26E Env-gp41EC EnvC2 EnvC3 EnvV3 His6EnvV3 Env-gp120
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Western blots of E2DISP-HIV Constructs
E2Env-V3 34 kDa a-HIV MW U I 52 33 98 21 a-E2 MW U I 51 kDa & 41 kDa a-E2 E2Nef & E2Rev MW U I U I 52 33 98 21 Here are some examples of protein expression in E. coli. These are westerns etc etc. We have tested several of these constructs as immunogens in mice.
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E2Gag-p17 forms 60-mer Particles
ABS 280nm Fraction Trimers, other 60-mers Here are some examples of protein expression in E. coli. These are westerns etc etc. We have tested several of these constructs as immunogens in mice. Sizing Column Gag-p17 E2
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E2Gag-p17 Particles are Immunogenic in HLA-A2 Transgenic Mice
We first tested E2-Gag-p17 in HLA-A2 transgenic mice. We detected killing of cells loaded with a gag peptide in a classic chromium release assay. Gag-p17 E2 One dose in Incomplete Freund’s Adjuvant Specific lysis of target cells in 51Cr release assay
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E2DISP-HIV Particles Effectively Boost Antibody Responses in Non-Transgenic Mice
Antibodies to E2 Antibodies to Gag Dose 1 Dose 2 10 1 2 3 4 5 6 E2 alone E2-p17 E2-p17/23 Immunizations Titer We then looked at the effect of giving multiple doses. Gag-p17 E2
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CTL elicited to ova embedded in E2Gag-p17
Constructs: ova-E2 (peptide “SIINFEKL”) p17-ova-E2 Immunized mice had CTL for ova Positive by JAM CTL and by tetramer Both constructs Conclusion: known epitopes are immunogenic when embedded in E2DISP-HIV fusion protein E2 ova Gag-p17 E2 ova
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Summary E2 fusion proteins can display many different HIV peptides and proteins E2DISP-HIV particles elicit antibodies in the absence of adjuvant Multiple doses boost antibody responses There is evidence for weak CTL responses in mice
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Future Directions E2Env-V3 immunogenicity studies
Test boosting potential of 3rd dose and use of E2 in heterologous prime-boost Design new peptide-based E2 constructs to target cellular responses Compare immunogenicity of “Pure” versus “Hybrid” particles
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Acknowledgements Nancy Haigwood Nicole Doria-Rose William Sutton
Piergiuseppe De Berardinis Antontella Caivano Luca Meoli Gonzalo Domingo Murali-Krishna Kaja Sunil Thomas Benjamin Buelow Support Provided by: amfAR NIH
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