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

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

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., 2001. J Mol Biol 305:259.

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

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

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

E2DISP Effectively Displays HIV Peptides E2 with two HIV peptides elicited: Antibody CD4+ T cells CTL In mice Domingo et al., 2003. Vaccine 21:1502

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

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.

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

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

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

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

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

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

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