The E2DISP Antigen Display System: A Novel HIV Vaccine Approach

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The E2DISP Antigen Display SystemA 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
Adapted from Domingo et al., 2001. J Mol Biol
305259.
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The E2 Antigen Display System (E2DISP)
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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

Domingo et al., 2003. Vaccine 211502
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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
Nef NefLL174/5AA
Tat
RT2 Pep23 RTp51 RTp66 Pro-RTD26E
Env-gp41EC
EnvC2 EnvC3 EnvV3 His6EnvV3 Env-gp120
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Western blots of E2DISP-HIV Constructs
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E2Gag-p17 forms 60-mer Particles
Sizing Column
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E2Gag-p17 Particles are Immunogenic in HLA-A2
Transgenic Mice
One dose in Incomplete Freunds 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
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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

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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