...WRPRC Interim Director Joseph Kemnitz's April Congressional testimony
in favor of the Regional Primate Research Centers Program.
The WRPRC Immunology and Virology Core Laboratory is at the heart of AIDS-related research at our center and is also serving companies and investigators across the United States. Our lab contracts with clients to conduct basic, time-saving experiments and maintains a widely used data base of "virus knowledge" for investigators studying infectious diseases, primarily the various strains of simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV). Lab personnel offer standardized techniques, reagents and virus stocks. They ensure purity and consistency among the virus strains and concentrations provided. Scientists can obtain and share accurate information, avoid duplicating research, and minimize the number of animals to be infected. Having a centralized, experienced staff conducting blood draws and other lab work also minimizes health risks to all workers.
David Pauza, Ph.D., Immunology and Virology Research Group chair, developed the lab in 1991. A professor of pathology and laboratory medicine, Pauza came to UW-Madison in 1990 to organize the AIDS research program at the WRPRC and to integrate this research into clinical studies of AIDS and other infectious diseases. He has a $1 million, four-year grant to study the selection and spread of SIV after mucosal infection in rhesus monkeys. He is also involved in clinical AIDS drug therapy trials at University Hospital and Clinics. One of the advantages of having a centralized core lab is that we can accumulate enough examples of how long a particular SIV strain takes to cause disease," Pauza explains.
"With several virus strains, we don't have to do infection studies anymore. We can concentrate on health maintenance and palliative care therapies in monkeys, with the ultimate goal of finding a cure for people with AIDS."
Pauza adds that the lab has also documented treatment trials that did not really alter the course of disease in monkeys, or that had too many side effects. "These studies are now in a centralized data base and do not have to be repeated," he said.
Scientists can examine a monkey's initial response to SIV at the time of infection, unlike the situation with HIV in humans. They can measure early immune responses to various strains and concentrations of viruses. They can look for patterns between how a monkey's antibody levels at the time of infection correlate to how long it will stay healthy. They may even be able to link these patterns to slight genetic or other differences in monkeys, and eventually in people, to further drive society's efforts toward finding a vaccine, better therapy, or a cure.
There is a lot to learn. "These invaders, these retroviruses, are so similar to the biochemistry of our own cells that our greatest challenge is to learn more about how they work in order to develop effective therapies, those that will kill the virus without killing the host," Pauza explains. He adds that the "triple therapy" being used with many AIDS patients today does keep the virus at bay, but when it's stopped, the virus immediately rebounds. "And the longer patients are on the drugs, the more negative side effects occur," he says. "Many people can't tolerate the drugs at all."
Because of this, our investigators do not work on standard, anti-viral drugs, "First, those drugs are rarely tested in macaques, so we would make no contribution to their development and, second, there are plenty of people doing this type of work already," Pauza explains. "So we look at drugs that require animal models, the immuno-modulating approaches or immunologic therapies, drugs with so many complexities you can't test them in people."
Tissue and cell cultures are also grown from single cells of infected animals whenever practical, Pauza adds. By supplying cultured samples, the WRPRC has saved hundreds of thousands in research dollars and has reduced the number of animals involved in its SIV research by 50 percent in the last six years. About 50 rhesus monkeys are now involved in core experiments.
HL9001 is showing promise as a supportive treatment and as a way to reverse the wasting that occurs with AIDS," Pauza says. He adds that a recombinant Salmonella bacterium is also being studied here as a potential vaccine approach. These are just a few of the unique immunology and therapy studies being conducted at our center.
"There is also a great deal of collaboration in everyone's search to better understand these retroviruses," Pauza says. SIV investigators here, for instance, have collaborated with scientists at the California Regional Primate Research Center on virus transmission studies.
Pauza points out that the core team informs all clients of its full range of activities. "We want investigators to think about how their intervention fits into the broader picture, we want them to understand how their project affects all aspects of an animal's health and welfare, not only the simple experimental objective they're looking at. Our objective is not to become just a service and execute what others ask us to do; it is to make individual projects much stronger, to make science much better." As the core lab expands its services, Pauza continues to lead the Immunology and Virology Research Group, while Leonid Iakoubov, Ph.D., assistant scientist, has been taking over day-to-day direction of the core lab, working closely with Jack Mitchen, M.S., to maintain protocol compliance. Iakoubov, previously an immunologist at Massachusetts General Hospital and Harvard Medical School, has broad experience in molecular biology, neuromorphology and immunology. Because of this experience, he can interact with researchers in various fields to assess their needs and how the lab can help them.
"I foresee more collaboration with AIDS researchers to complement our own SIV studies," Iakoubov says. "There is a huge variability among monkeys concerning individual survival rates. We are trying to predict the dynamics of the disease in individual animals, which has a significant theoretical and practical impact."
David Watkins, Ph.D., chair of our Immunogenetics Group, is conducting studies to address these differences. The core lab is supporting his work to generate a genetically homogenous group of monkeys for conducting SIV research under more controlled circumstances. "If we can predict, from the earliest stages of infection, in which monkeys the disease will progress more quickly and in which ones it will progress more slowly, we can ultimately determine those factors that might be important in resistance to the AIDS virus," he says.
To this end, Watkins is exploring the role of cytotoxic T lymphocytes (CTLs) in fighting SIV. In addition to helper T-cells, CTLs are generated during the immune system's initial response to infection. The CTLs recognize peptides presented by major histocompatibility (MHC) class I molecules. So Watkins is studying infected animals with genetic differences in terms of their MHC class I allele and discovering that animals with certain types of MHC class I alleles mount a similar, strong offense to the virus. His goal is to identify those parts of the SIV molecule that are being presented to the CTLs during infection, then use those regions to immunize uninfected animals and see if protection occurs.
The core team members ensure that Watkins' study animals are infected with the same amount of the same virus. They monitor lymphocyte levels and antibody responses. Watkins gets the blood draws and can then concentrate on conducting CTL experiments, his lab's specialty.
"I have benefited immensely from the well organized core lab," she testifies. "I have been able to study cell-mediated immunity in infected animals that have been extensively characterized by the core lab so that I do not have to do all the characterizations myself." She cited the accurate, computerized records on the hematology, flow cytometry, virological status and appearance of the monkeys involved in core experiments. She has used data generated by the core lab in six publications over the last two years.
"One of the best examples of efficient data coordination is the flow cytometry data," Salvato details. "My lab discovered that not only the SIV-infected monkeys, but also uninfected monkeys can have vigorous cell-mediated immune responses to SIV. We found that some monkeys have a higher response than others and that the effector cells of this immune response are CD4+ cells. It was then a simple matter to go back into the core lab flow cytometry records to find out that monkeys with better responses have higher absolute numbers of CD4+ cells in their blood."
Core laboratory staff members have also carefully selected animals for Mirek Malkovsky's studies, which address the role of gamma/delta T-cells during SIV infection. Malkovsky explains that the lab has prepared protocols for immunizing and tolerizing rhesus monkeys with nonpeptidic phosphoantigens and is performing blood drawings for the in vitro studies associated with this project.
"Simply put, without the intensive and extensive support from the lab, it is difficult to imagine that these gamma/delta studies could ever happen," Malkovsky attests. Moreover, he adds that the lab's future flow analyses of (including the analyses of SIV-, SHIV- or HIV-infected cells) from Macaca mulatta or Homo sapiens will be vital for a better understanding of the role of gamma/delta T cells in the pathogenesis of SIV/SHIV/HIV-induced disorders.
For more information on the WRPRC Immunology and Virology Core Laboratory
services, contact Jack Mitchen at (608) 265-4072 (jmitchen@primate.wisc.edu).
The core staff includes Jack Mitchen, M.S., Paul Hinds, M.T. (ASCP), Marta Dykhuizen, B.A., and Leonard Acker. Mitchen, who joined us in 1995 and has 24 years of experience managing large laboratories, develops and ensures compliance to protocols. He spends half his time managing the multi-use Biological Safety Level 3 Lab and the other half scheduling and overseeing experiments. He is also responsible for staff safety.
Paul Hinds, senior research specialist, has over 20 years of pathology,
toxicology and medical technology experience with nonhuman primates. He
has been with the core lab since 1993, performing the physical examinations
and blood draws on rhesus macaques involved in SIV studies. He maintains
virus stocks and performs the biopsies and necropsies. Hinds also does
virus titer assays, in which he incubates infected cells with regular cell
lines to determine whether an animal is infected and to measure the degree
of infection. Marta Dykhuizen, research specialist, was hired in 1995 to conduct flow
cytometry, a procedure that allows the detailed study of white blood cells.
She is also responsible for testing for cell-free SIV and the antibody
responses to that virus. She is finalizing a paper correlating core staff
laboratory data that show early antibody responses can statistically help
researchers predict rhesus life spans after SIV infection. She previously
worked in a basic immunology lab at the University of Chicago, where she
gained her flow lab experience.
"A peripheral blood sample has many different types of cells, including
different subsets of lymphocytes, each with a specialized function," Dykhuizen
explains. "You can find out a lot about the general health of an animal
or how a disease is progressing by studying how those subsets are changing."
Cells tagged with fluorescent antibodies run through a flow cytometer
in single file and are hit by two different lasers. The lasers illuminate
their stained surfaces to reveal which cell surface molecules are binding
to which subsets. The machine records the numbers of cells in each subset
and outputs electronic images and data to a computer screen.
One such image, at right, depicts forward scatter (X axis), which is
the change in the laser beam due to cell size, and side scatter (Y axis),
the change in the beam due to the complexity, or density of the cell. Molecular
biologists using flow cytometry data can study the various parameters of
cell groups, or even individual cells, to unlock the secrets of viral infection
and progression.
"Flow cytometry existed before the AIDS epidemic, but AIDS spurred its
development," Pauza says. "It's important for the experimenter to know
how the virus is progressing. It's also important for the veterinarian
to follow the animal's clinical progression before it shows signs of disease,
so that we can give the best supportive care we can for each monkey while
seeking effective treatments for the disease."
Copyright 1997. Wisconsin Regional Primate Research Center.