AIDScasestudy41307 (2)

Finding a Cure: Which HIV vaccine would you choose? How does the Immune System fight invaders? How does the Immune System fight invaders? How does the Immune System fight invaders? How does the Immune System fight invaders? How do vaccines ‘help’ the immune system to fight off viruses? Vaccines Currently Available What is HIV? Four Potential Vaccines Which genes are in the vaccines? Gel electrophoresis of digested vaccines Loading your gel Loading your gel Gel electrophoresis of digested vaccines Which genes are in the vaccines? Which genes are in the vaccines? Which genes are in the vaccines? Which genes are in the vaccines? Which genes are in the vaccines? Which genes are in the vaccines? Which one of these would you use in a human trial? Vaccine study in an animal model Vaccine study in an animal model More about HIV/AIDS… What is in the vaccines? Which ones would you use in human trials? Clinical Trials: Testing in Humans You will each be given one serum sample from one volunteer – follow the instructions in your handout to determine if the volunteer made the appropriate antibodies… Which vaccine worked the best in the trial? Why do we need to test immunogenicity? Diversity, diversity, diversity… People have different MHC alleles MHC Polymorphism Ramil Sapinoro Life Sciences Learning Center University of Rochester Medical Center AIDSVax Inc. Department of Vaccine Research 1979 Cure Drive Rochester, NY 14620 Dear Research Technician, We are excited to collaborate with your organization in developing a AIDS vaccine. I am sending you 4 possible DNA vaccines that you could use in your vaccine trials. I’m not sure which combinations of the three HIV genes are in each one. You will have to check this. Along with the DNA, I have included some data collected from preliminary animal studies. We injected animals with each of the three HIV genes alone: the gag gene codes for HIV structural proteins; the env gene codes for the virus envelope proteins; and the pol gene codes for viral enzymes. We used mice as an experimental animal. Sincerely, Clarke Kent, Ph.D Director Center for Vaccine Research University of Rochester APC Antigen Presenting Cells (ex: macrophages) Capture invaders (pathogens), destroy them, and display parts of them (antigens) on proteins called MHC’s Virus Antigen MHC APC T Helper T Cells See antigens presented by APC’s and does two things: Turn on Cytotoxic T Cell – these T Cells can go on to kill other cells that present the antigen (infected cells as well as macrophages) Activate B Cells APC T T Cytotoxic T Cell B B Cell T Infected Cell B Cytotoxic T cell produces signaling proteins that tell infected cells to kill themselves Helper T cell produces signaling proteins that tell B Cells that have antibodies against the antigen to make more B Cells and free floating antibodies B B B What would a Successful Vaccine Do? Stimulate the production of antibodies Stimulate the production of Cytotoxic T Cells Remember the pathogen (Memory cells) Not cause disease B B Vaccine Approaches What parts of a pathogen could we use to stimulate the immune response and not cause disease? Live-attenuated Virus Inactivated Virus DNA Protein subunit Synthetic peptide Virus-like particles Cervical Cancer Inactivated virus Influenza Recombinant protein Hepatitis B Live vaccinia virus Smallpox Live-attenuated Chicken Pox Live-attenuated Measles/Mumps/Rubella Inactivated virus Polio Type of Vaccine Disease/Virus HIV = Human Immunodeficiency Virus Small Replicates within cells of living hosts Composed of RNA Pathogenic: Has the ability to cause disease (AIDS) HIV causes disease because it kills helper T cells APC T HIV Genome and Structure env proteins Viral envelope proteins gag proteins Capsid Matrix pol proteins Viral enzymes gag gene pol gene env gene The HIV genome has three main genes… V1 V2 V3 V4 gag gene pol gene env gene gag gene pol gene gag gene pol gene env gene pol gene env gene Which of these possible pieces of DNA are in each vaccine? How will you find out? V1 DNA Vaccine V2 DNA Vaccine V3 DNA Vaccine V4 DNA Vaccine Cut with Restriction Enzyme ? ? ? ? V1 V2 V3 V4 You have been given vaccines that have been pre-digested with restriction enzyme. You’ll need to run the DNA fragments out on a gel to see what genes are in each vaccine. Load the gel Carry the gel to a power source Fill the gelbox with TAE buffer (fill the box so that the top of the gel is just covered) Put the lid on the gel Run the gel for about 10 minutes at 120 volts V1 V2 V3 V4 gag gene pol gene env gene Restriction Enzyme gag gene pol gene env gene Gel electrophoresis Well gag gene pol gene Restriction Enzyme gag gene pol gene Gel electrophoresis Well pol gene env gene Restriction Enzyme pol gene env gene Gel electrophoresis Well gag gene Restriction Enzyme gag gene Gel electrophoresis Well pol gene Restriction Enzyme pol gene Gel electrophoresis Well env gene Restriction Enzyme env gene Gel electrophoresis Well gag gene pol gene env gene gag gene pol gene gag gene pol gene env gene pol gene env gene Which ones do you KNOW you would NOT want to use? Of the others, how will you decide which ones to use in human trials? B B Which one(s) would you use to make a vaccine? DNA injected into mice Amount of Antibodies B B Which one(s) would you use to make a vaccine? DNA injected into mice Amount of cytotoxic T-cell Activity HIV Lifecycle Helper T Cell CD4 Global HIV infections, Dec 2006 HIV Treatment: Anti-virals X X Reverse Transcription Inhibitors Protease Blockers Which genes are in each of the four potential vaccines? Which of the potential vaccines would you NOT use? What other information might you need to make a decision on which one to use? V1 V2 V3 V4 Phase Objective Number of Volunteers I Safety and dose 20–50 / trial Additional safety and immunogenicity 100’s / trial Efficacy and protection 1000’s / trial II III Phase Objective Number of Volunteers I Safety and dose 20–50 / trial Additional safety and immunogenicity 100’s / trial Efficacy and protection 1000’s / trial II III V2 and V4 You will be testing the serum of a group of people who have either been injected with V2, V4 or a placebo to see if they respond to the vaccine by making antibodies against the appropriate proteins V2 Serum Each volunteer is injected with one vaccine. After a time, serum is collected from the volunteer, and analyzed for the presence of antibodies using an ELISA assay… Obtain three absorbent squares and label “G” (gag) “P” (pol) and “E” (env)… Gag Pol Env Spot the gag, pol and env proteins on their appropriate spots… Gag Pol Env Place serum on each of the squares – the serum contains antibodies… Gag Pol Env If the volunteer has antibodies against the HIV proteins, those antibodies will bind to the proteins on the paper. Excess antibodies will wash off… Gag Pol Env Add labeled “Secondary Antibody” This secondary antibody binds to all human antibodies, and has a colored tag… Gag Pol Env Upon the addition of “developing solution,” the colored tag will become visible… Env Pol Gag Volunteer # Vaccine Given APC Antigen MHC allele #1 APC MHC allele #2 Person 2 No immune response Person 1 Immune response Challenges in HIV Vaccine Research Viral Genetic Diversity: HIV is not just one specific virus. Immune Protection: Scientists don’t know what immune responses are needed, or how strong they need to be. Neutralizing Antibody: Difficult to generate broadly neutralizing antibodies. Vaccine Testing: Slow process, very expensive …but on the Brightside… Precedent from other systems: Success against other viral infections Precedent from animal studies: Long-term control of infection in vaccinated monkeys Immune control of HIV-1: Infected individuals control infection Vaccine Trials: In progress