AIDSVaxTeacher040907

TEACHER GUIDE All the materials presented at this workshop can be downloaded from the Life Sciences Learning Center Website _http://lifesciences.envmed.rochester.edu/_ in the curriculum section _http://lifesciences.envmed.rochester.edu/curriculum.html_ AIDS Vaccine Case Study In this case study, students will learn about how the immune system responds to subunits of the HIV virus genome. They will then learn about using immunoassays to determine the effectiveness of potential AIDS vaccines. Part 1 of the case involves an agarose gel electrophoresis activity using dyes to represent DNA vaccines composed of HIV genes. In Part 2 of the case, students perform a mock ELISA (enzyme linked immunosorbant assay) to determine which potential AIDS vaccine works best in a test population. Part 1: DNA Vaccines Learning Objectives Students will learn about how the immune system responds to pathogens Students will learn about how a vaccine stimulates the immune system Students will perform gel electrophoresis to determine what DNA fragments are in four potential vaccines Students will use data from the gel electrophoresis activity and data from animal models to determine which vaccines would be most useful for a human clinical trial Gel electrophoresis Lab Set-Up Prepare the four “vaccine” samples: V1 (20mL) 4mL 1% Bromophenol Blue 4mL 1% Indigo Carmine 4mL 1% Xylene Cyanole 2mL Glycerol 0.4 ml 50X TAE electrophoresis buffer V2 (20mL) 4mL 1% Indigo Carmine 4mL 1% Bromophenol Blue 2mL Glycerol 0.4 ml 50X TAE electrophoresis buffer V3 (20mL) 4mL 1% Xylene Cyanole 2mL Glycerol 0.4 ml 50X TAE electrophoresis buffer V4 (20mL) (NOTE: Indigo Carmine diluted in TAE has a shelf life of about 2 months) 4mL 1% Indigo Carmine 2mL Glycerol 0.4 ml 50X TAE electrophoresis buffer Each group of 2-4 students should have: 1 tube of V1 (Each student will only run 10uL of V1) 1 tube of V2 (Each student will only run 10uL of V2) 1 tube of V3 (Each student will only run 10uL of V3) 1 tube of V4 (Each student will only run 10uL of V4) 1 micropipettor capable of dispensing 10uL 4 disposable micropipettor tips Enough 1.5% agarose for one gel (agarose gel should be made with 1X TAE electrophoresis buffer) 1X TAE electrophoresis buffer One gel box Access to a powersource Procedures Give each student a copy of part 1. Choose a student to read the AIDSVax letter out loud. Use PowerPoint slides 3-10 to go over how the immune system works and how vaccines can be used to boost the immune system. This will prepare students for how to interpret the animal model data. Also go over the structure of the AIDS virus and its genome to prepare students for interpreting the gel electrophoresis data (slides 11-14) Have students load the “digested DNA fragments” onto their gels, using slides 15-18 to illustrate how to load a gel. Students will work in groups of 2-4 to complete the agarose gel electrophoresis activity. They will load 10mL of each of the “DNA vaccines” into the well of an agarose gel and run the gel for about 15 minutes at 120 Volts. While the gels are running, use slides 19-25 to reinforce how students should interpret the gel data. Use slides 26-27 to analyze the animal model data in figures 1 and 2. In Figure 1, students should recognize that immunizing with the pol gene does not stimulate the production of antibodies, and is therefore not a good candidate for a vaccine when given on its own. In Figure 2, students should recognize that immunizing with the pol gene does not stimulate the production of cytotoxic T-Cells, and is therefore not a good candidate for a vaccine when given on its own Have students draw the bands that they observe on their gel and complete the questions. The gel observations must be done immediately, and cannot be saved until the next day (the dyes will diffuse out of the gel). 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 an 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 HIV DNA, I have included some data collected from preliminary animal studies. We injected mice 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. Sincerely, Clarke Kent, Ph.D Director Center for Vaccine Research University of Rochester Part 1: Gel Electrophoresis You have been given 4 potential AIDS vaccines (V1, V2, V3, V4). These are DNA vaccines that contain different parts of the HIV genome, which is shown below. The DNA vaccines have already been cut with a restriction enzyme. The restriction enzyme cut sites are shown below. The entire HIV genome is cut into three pieces (gag, pol and env). HIV Genome: You will need to identify which of the 4 DNA vaccines have parts of the HIV genome that you might be interested in using in an AIDS vaccine clinical trial. For example, you will not want to use a vaccine that contains all three genes (why not?) You will use agarose gel electrophoresis to determine which parts of the HIV genome are in each of the 4 DNA vaccines. Load 10mL of each of the pre-cut DNA vaccines into the wells of an agarose gel and run the gel for about 15 minutes at 120 Volts Part 1: Observations/Conclusions Draw what you see on your gel: What does each band represent on the gel? Each band represents a different HIV gene. The largest band is pol. The middle band is env. The smallest band is gag. Part 1: Studies in mouse models Figure 1. Comparison of antibody titers collected from 10 mice injected with different DNA HIV vaccines encoding pol, env, gag, or control DNA. Serum from each animal was collected 8 weeks after injection. Each filled circle represents one mouse. Figure 2. Cytotoxic T cell activity from 10 mice injected with different DNA HIV vaccines encoding pol, env, gag, or control DNA. Immune cells from each animal were collected 8 weeks after injection. Use the information from your gel and the animal studies to answer the following questions: Vaccine Name What genes are in this vaccine? Would you use this in a human trial? If no, why not? V1 pol, env, gag No It contains all the HIV genes and could cause disease V2 env, gag Yes V3 pol No It only contains the pol gene, which was shown in the animal studies to not stimulate the production of antibodies or of active cytotoxic T cells V4 gag Yes Part 2: Enzyme Linked ImmunoSorbant Assay (ELISA) Learning Objectives: Students will learn about variability in alleles that lead different people to respond differently to antigens in a vaccine Students will learn about experimental design – why are placebos used in a clinical trial Students will learn about Enzyme Linked ImmunoSorbant Assay (ELISA) Students will learn how to interpret data from a group to determine which vaccine works the best in the test population Mock ELISA Lab Activity Set-Up: Prepare 34 tubes of “serum” from people vaccinated with V2, V4 or placebo. These represent people participating in a clinical trial of an AIDS vaccine. Each student will receive 1 of these serum samples to test: Serum from V2 infected people: Patients #’s 1-14 (Some of these people will make antibodies to gag; others will not make antibodies to gag) Tubes 1-6 serum = 5mL pH 3 buffer (people who do NOT make gag antibodies) Tubes 7-14 serum = 5mL water (people who DO make gag antibodies) Serum from V4 infected people: Patient #’s 15-28 (all of these people will make antibodies to gag) Serum = 5mL water Serum from Placebo infected people: Patient #’s 29-34 Serum = 5mL pH 3 buffer Prepare the other reagents for the mock ELISA. Each group of 2-4 students will get the following: Pol Proteins: 1mL water Env Proteins: 1mL water Gag Proteins: 1mL pH 11 buffer Labeled Antibodies: 5mL water Developing Solution: 1mL 1% _Phenolphthalein_  Water: 5mL water Give each student A tube of patient “serum” for one vaccine trial participant Three absorbent squares (1-3mm filter paper cut into ¾ inch squares) 6 transfer pipettes 3 small plastic weigh boats Procedures Give each student a copy of part 2. Use the powerpoint slides 34-38 to explain the phases of clinical trials, and to prepare students for the “study” they will be performing. Slides 39-44 can be used to explain how an ELISA assay works. Students should use the protocols in part 2 to perform their own ELISA, using the patient’s serum sample they are given. They should assemble class data in the chart to determine which vaccines elicited the most consistent immune response in the clinical trial volunteer patients. Use powerpoint slides 47-48 to briefly discuss why different people might respond differently to the same vaccine. Slides 49-50 can be used to discuss current challenges and advances in AIDS vaccine research. Part 2: Testing for an Immune Response using ELISA (Enzyme Linked ImmunoSorbant Assay) Which vaccine results in the best immune response, V2 or V4? You have tested V2 and V4 vaccines by injecting volunteer patients with V2 or V4 vaccine, or with a placebo. 14 people were injected with V2 14 people were injected with V4 6 people were injected with placebo Several months later, you collect serum from these patients. You will test their serum samples to see if these patients have made antibodies against the proteins that are coded for by the DNA vaccine. This is done using an Enzyme Linked ImmunoSorbant Assay, or ELISA. ELISA: Part 2: Clinical Trial: Individual ELISA Data PROCEDURE Label three absorbent squares for each HIV protein using a pencil. Place one labeled absorbent square in each of the six small plastic trays Use a transfer pipette to: drop 1 drop of env proteins onto the square labeled “env” drop 1 drop of gag proteins onto the square labeled “gag” drop 1 drop of pol proteins onto the square labeled “pol” Let the squares dry for 1 minute Use a transfer pipette to put 0.5mL of your patient serum onto each square Let soak for 5 seconds Pour off the excess serum Transfer 0.5mL of labeled antibodies on the square Let soak for 5 seconds Transfer 0.5mL of water onto each square to quickly rinse the card Pour off the water Drop 1 drop of Developing Solution onto each square Record your results Patient # Patient was given vaccine Part 2: Clinical Trial Group Data Patient # Vaccine given pol antibodies? env antibodies? gag antibodies? 1 V2 - - - 2 V2 - - - 3 V2 - - - 4 V2 - - - 5 V2 - - - 6 V2 - - - 7 V2 - - yes 8 V2 - - yes 9 V2 - - yes 10 V2 - - yes 11 V2 - - yes 12 V2 - - yes 13 V2 - - yes 14 V2 - - yes 15 V4 - - yes 16 V4 - - yes 17 V4 - - yes 18 V4 - - yes 19 V4 - - yes 20 V4 - - yes 21 V4 - - yes 22 V4 - - yes 23 V4 - - yes 24 V4 - - yes 25 V4 - - yes 26 V4 - - yes 27 V4 - - yes 28 V4 - - yes 29 Placebo - - - 30 Placebo - - - 31 Placebo - - - 32 Placebo - - - 33 Placebo - - - 34 Placebo - - - What antibodies did you expect a person injected with V2 would have? env and gag antibodies What antibodies did you expect a person injected with V4 would have? gag antibodies What antibodies did you expect a person injected with Placebo would have? None Which vaccine do you think works the best? Why? V4 – everyone who was injected with V4 made the right antibodies (gag) Only some of the people who were injected with V2 made the gag antibodies. None of the people injected with V2 made env antibodies. 1 Copyright © 2007, University of Rochester May be copied for classroom use