Turnover of Antigen Specific Lymphocytes After Immunization With the 17D Yellow Fever Vaccine

University of California, Berkeley, National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID)

The yellow fever vaccine is a live, attenuated virus that results in a robust immune response, especially in the T cell compartment. We have been studying immune responses to live viral infections using the yellow fever vaccine as a model for a live viral infection. In this study, we are interested in looking at the processing and lifespan of yellow fever specific CD8 T cells. We plan to accomplish this by measuring DNA replication and cell proliferation in humans using a naturally occurring stable isotope called deuterium (D20). This technique has been used to track the turnover of a number of human cell types in vivo. We plan to use D20 labeling to track YFV specific CD8+ T cells in human vaccinees who are positive for a specific HLA type, HLA A202. Deuterium labeled water (D2O), also known as heavy water is physically and chemically very similar to ordinary drinking water. In water, two hydrogen atoms bond to an oxygen and create H20. However in deuterated water, deuterium atoms replace the hydrogen atoms. Deuterium is a form of hydrogen that has an extra neutron. This neutron gives the atom extra weight, hence the name "heavy water." This extra weight can be detected in the lab with very sensitive instruments. Scientists have been using heavy water as a tracer to gain a better understanding of animal and human metabolic rates. Deuterium is in fact already in the water we drink daily. It is not radioactive, and it occurs naturally at a concentration of about 1 part per 5,000. Researchers have used heavy water since 1934 as a safe and effective tool in clinical trials.

In general, T cell responses to viral infections occur in three distinct stages: expansion, contraction and memory. During expansion, antigen-specific CD8+ T cells proliferate to increase in numbers as well as acquire the ability to kill virus infected cells, i.e become 'effectors'. Once the virus is cleared, 90-95% of the effectors die. The small pool of remaining cells differentiates gradually to become memory cells and provide life-long protection. The rate of cell division is distinct in the generation of effectors and memory cell maintenance. These concepts have been primarily derived from mouse studies. Human CD8+ T cell turnover is not completely understood because of the lack of appropriate tools and techniques suitable for human studies. In this protocol, the researchers want to understand the lifespan and decay curve of effector CD8+ T cells and the rate of homeostatic turnover of memory CD8+ (CD28+/- subsets) T cells after 17D YFV immunization using an innovative method developed by Dr. Marc Hellerstein's group (at the University of California, Berkeley) for measuring DNA replication and cell proliferation in humans using a naturally occurring stable isotope called deuterium (2H). This technique has been used to track the turnover of a number of human cell types in vivo. The researchers of this study plan to use 2H labeling to track YFV specific CD8+ T cells in human vaccinees (HLA-A2 positive participants only). The availability of a T cell epitope (A2-NS4B214), a major component of the human YFV specific CD8+ T cell response, allows for the longitudinal analysis of virus specific CD8+ T cells. The unique feature of this study is that it allows for tracking of differentiation of YFV specific CD8+ T cells in humans. Thus, the researchers can overcome the inherent limitations due to heterogeneity in cross-sectional studies that involve bulk CD8+ T cells. We plan to enroll 50 healthy, adult volunteers (18-45 of age) for Projects 1 and 2 to study memory CD8 T cell responses. All subjects will receive the FDA approved YFV-17D vaccine (at the FDA approved dose and route of administration). CD28 expression will be determined to see if it is coupled to the generation and/or maintenance of YFV-specific memory CD8 T cells. Phenotypic differences between CD28+ and CD28-_YFV-specific CD8 T cells will be studied to determine differences in longevity. Gene expression profiles of CD28+ and CD28 YFV-specific CD8 T cells will be determined. The influence of IL-7 and IL-15 on survival of YFV-specific CD8 T cells will be studied. This information will be gained from YFV specific cells isolated at Day 0, 14, 28 and 3 to 6 months, 1-2 years and >5 years after vaccination. The study will be conducted at the Hope Clinic of Emory Vaccine Center. Individuals who are planning to travel to yellow fever endemic areas and who require the vaccine for travel purposes as well as non-travelers will be recruited into the study. Potential participants may be recruited from the Emory Travel Well Clinic or the CDC employee health or the Emory University campus or the general population of metro Atlanta using radio and print advertisements, electronic mailings, Internet postings, flyers, and posters. IRB approval from Emory University will be obtained prior to the initiation of the study. There will be a screening visit to determine the eligibility for the study. Signed informed consent forms will be obtained prior to the initiation of study procedures. The inclusion and exclusion criteria and a brief history and physical examination will be performed. In addition, participants will be asked to undergo phlebotomy to determine the HLA type. Participants who are positive for the HLA A202 allele, will be asked to participate in this study. It is estimated that 25% of the Caucasian US population will be positive for this allele. Participants who test negative for this allele will be offered other vaccine trials that are being conducted at the Emory Hope clinic and their participation in this protocol will end. Prior to the administration of the yellow fever vaccine, the risk-benefit ratio of the vaccine-related adverse events versus the risk of contracting yellow fever is determined. Healthy individuals who have not been previously vaccinated with YFV vaccine will be included in the study. Both female and male volunteers will be recruited. We plan to enroll 50 healthy volunteers between 18 to 45 years of age. In order to meet these enrollment goals, we may need to screen 200 subjects. All subjects will receive the FDA approved YFV-17D vaccine (at the FDA approved dose and route of administration) once on day 0 of the study visit. Blood specimens will be obtained from the volunteers on Day 0, and on scheduled time points post-vaccination. The total volume of blood drawn will not exceed the Red Cross limit of 473mls in 56 days. The study volunteers will be maintained on a 100ml - 150ml daily intake of 2H2O, with a goal of maintaining 1.5-2% body water enrichment (assuming total body water turnover of ~3.5 liters per day in healthy, ambulatory subjects). Plasma and saliva samples will be obtained during the 2H2O administration protocol for measurement of body 2H2O enrichment.

In group 1, participants will receive the Yellow fever vaccine or YFV-17D and will be asked to drink Deuterium (70% enriched 2H2O) labeled water for 2 weeks (Days 14 to 28 post-vaccination). They will undergo phlebotomy on Day 0, 14, 28 and 3 to 6 months, 1-2 years and >5 years after vaccination.

In group 2, participants will receive the Yellow fever vaccine or YFV-17D and will be asked to drink Deuterium (70% enriched 2H2O) labeled water for 3 weeks. They will undergo phlebotomy on Day 0, 14, 28 and 3 to 6 months, 1-2 years and >5 years after vaccination. In group 2a, phlebotomies will occur at weekly at 8, 9, 10, 11,12, 14 and 20 weeks post vaccination Participants in Group 2a will begin drinking deuterated water at month 2 Participants will drink 2H2O 50ml TID for the first week, then 50ml BID for 3 weeks. In group 2b, phlebotomies will occur weekly at 24, 25, 26, 27, and 28 post vaccination.

In group 2, participants will receive the Yellow fever vaccine or YFV-17D and will be asked to drink Deuterium (70% enriched 2H2O) labeled water for 3 weeks. They will undergo phlebotomy on Day 0, 14, 28 and 3 to 6 months, 1-2 years and >5 years after vaccination. Participants in Group 2b will begin drinking deuterated water at month 6 Participants will drink 2H2O 50ml TID for the first week, then 50ml BID for 3 weeks.

The yellow fever vaccine (YFV-17D) manufactured by Sanofi Pasteur as a one-dose vial will be purchased from the manufacturer. Vaccine will be stored at the Emory Investigational Drug Service (IDS) between 2 to 8 degrees Celsius as per the manufacturer's instructions. It will be transported to the Hope Clinic per the Standard Operating Procedures.

70% enriched 2H2O will be obtained from Cambridge Isotope Laboratories (Andover, MA) in sterile 1 Liter containers. An Emory Investigational Drug Service (IDS) pharmacist will prepare sterile 50 milliliter aliquots with a tamper seal which will be transported to the Hope Clinic at room temperature.

CD8 T cells will be measured on Day 0, 14, 28 and 3 to 6 months, 1-2 years and >5 years after vaccination.

CD8 T cells will be measured on Day 0, 14, 28 and 3 to 6 months, 1-2 years and >5 years after vaccination.

Inclusion Criteria: Able to understand and give informed consent Age 18-45 years Participants agree not to take any live vaccines 30 days before or after (14 days for inactivated) yellow fever vaccination Women of child bearing potential must agree to use effective birth control for the entire duration of the study. A negative urine pregnancy test must be documented prior to vaccination and prior to the initiation of drinking deuterium labeled water. Must be positive for the HLA A202 allele Must agree not to receive any other vaccination during the labeling period with heavy water and in the first 28 days after receipt of yellow fever vaccination Exclusion Criteria: Travel to or having lived in a country/area which is endemic for yellow fever History of previous yellow fever, West Nile, Dengue, St. Louis encephalitis, Japanese encephalitis vaccination or infection Any history of allergy to eggs, chicken or gelatin or to any previous vaccine A history of a medical condition resulting in impaired immunity (such as HIV infection, cancer, particularly leukemia, lymphoma, use of immunosuppressive or antineoplastic drugs or X-ray treatment). Persons with previous skin cancers or cured non-lymphatic tumors are not excluded from the study. History of HIV infection, Hepatitis B or Hepatitis C infection History of any chronic medical conditions that are considered progressive (ex, diabetes, heart disease, lung disease, liver disease, kidney disease, gastrointestinal diseases and uncontrolled hypertension). Use of systemic immunosuppressive medications (ex, prednisone) for 2 weeks or more in the past 3 months History of excessive alcohol consumption, drug abuse, psychiatric conditions, social conditions or occupational conditions that in the opinion of the investigator would preclude compliance with the trial Thymus gland problems (such as myasthenia gravis, DiGeorge syndrome, thymoma) or removal of thymus gland or history of autoimmune disorder Recipient of a blood products or immune globulin product within 42 days of the vaccination visit Pregnant women and nursing mothers or women who are planning to become pregnant within 2 months after receiving the yellow fever vaccination Any condition in the opinion of the investigator that would interfere with the proper conduct of the trial