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Presented by Sarah Mohn and Mari Hammerquist

Killing Kinetics of Simian Immunodeficiency Virus-Specific CD8+ T Cells: Implications for HIV Vaccine Strategies. Erik Rollman, Miranda Z. Smith, Andrew G. Brooks, Damian F.J. Purcell, Bartek Zuber, Ian A. Ramshaw, and Stephen J. Kent. Presented by Sarah Mohn and Mari Hammerquist. Goals.

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Presented by Sarah Mohn and Mari Hammerquist

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  1. Killing Kinetics of Simian Immunodeficiency Virus-Specific CD8+ T Cells: Implications for HIV Vaccine Strategies Erik Rollman, Miranda Z. Smith, Andrew G. Brooks, Damian F.J. Purcell, Bartek Zuber, Ian A. Ramshaw, and Stephen J. Kent Presented by Sarah Mohn and Mari Hammerquist

  2. Goals • To show that magnitude and rapid response of vaccine induced CTLs are important in the control of HIV/SIV (Simian Immunodeficiency Virus) • SIV is studied in this paper for implications in HIV vaccines • To determine which method of vaccination induces a better immune response • Prime-boost vaccination: First vaccination primes the immune system and second vaccination boosts the immune response • Live attenuated virus (LAV) vaccine: vaccination with a weakened form of the live virus

  3. Prime-Boost Vaccine • Recombinant DNA vectors contain the target gene for wild-type SIV Gag • Viruses used to incorporate target DNA into host cell • Vaccinia virus (VV) (prime) • Fowlpoxvirus (FPV) (boost) • Host cells produce SIV Gag • Antibodies are formed against SIV Gag

  4. Experiment 1: RT-PCR • Determined number of KP9-specific CD8+ CD3+ T cells present after prime boost vaccination and LAV inoculation • KP9 is immunodominant SIV Gag epitope and is targeted by a MHC class I tetramer (Mane-A* 10+) • Examined amount of virus RNA present in blood plasma (viremia)

  5. Prime/boost macaques had a significant increase in KP9-specific T cells post challenge • LAV inoculated macaques had a much lower percent of KP9

  6. RT-PCR • CD4+ T cell levels drop and then recover with the prime/boost vaccine, but remain fairly constant with the LAV vaccine • Viral loads are similar regardless of vaccination type

  7. Flow Cytometer Flow Cytometry Tutorial

  8. CD8+ T cell • Granzyme B: a serine protease that initiates apoptosis, present in cytolytic vesicles • Perforin: forms transmembrane pores to kill target cell • IFN-γ: a cytokine released during T cell degranulation, increases immune response • CD107α: a degranulation marker • KP9 Gag: SIV epitope targeted by CD8+ T cell

  9. Kinetic Tetramer Degranulation Assay • CD8+ T cells from a vaccinated animal were restimulated in vitro 5 weeks post challenge • Showed an increase in levels of IFN-γ, granzyme B, perforin and CD 107 as time progressed

  10. Prime/Boost • Challenge occurred at 18 weeks after vaccination • At challenge, there are no signs of degranulation • Hours post challenge IFN-γ is increased • Restimulation at 5 weeks post challenge • Showed high levels of granzyme B • Hours after restimulation show increasing levels of CD107 and IFN-γ • Restimulation at 28 weeks post challenge • Low levels of CD107, granzyme B, and IFN-γ • Gradual increase in IFN-γ and greater increase in CD107

  11. LAV • Restimulation at 8 weeks post inoculation • Initially low levels of CD107, granzyme B and IFN-γ • CD107 gradually increases and IFN-γ follows later

  12. Prime/boost vs. LAV • Top: prime/boost 2 weeks post challenge, week 20 post vaccination • Bottom: LAV week 8 post challenge • IFN-γ and CD107 increase in both cases • Greater increase in IFN-γ for prime/boost • Greater increase in CD107 in LAV

  13. Ex Vivo Levels of Granzyme B • Intracellular Granzyme B levels in SIV specific CD8+ T cells in vaccinated animals rise after challenge and eventually return to normal levels • Intracellular Granzyme B levels in other CD8+ T cells remain relatively constant throughout exposures

  14. Further Experiments • Phenotypes of memeory CD8+ T cells were investigated • The Gag-specific memory T cell phenotype is clearly different early on in LAV animals compared to prime-boost vaccinated animals • As time progresses, these phenotypes become more similar

  15. Conclusions • The response of the CTLs were different • LAV had a much more rapid cytolytic phenotype and less IFN-γ expression • Prime-boost showed a less rapid response phenotype and much greater levels of IFN-γ • Showed that IFN-γ is not always representative of CTL response • Important implications for HIV vaccine research, which commonly use IFN-γ as an indicator • LAV was shown to be a better candidate for HIV vaccination

  16. Questions?

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