Rasmussen, Taalin
Development of an Animal Model in Which to Study HIV-2
Faculty Mentor: Berges, Bradford
Microbiology and Molecular Biology
Introduction
Human Immunodeficiency Virus (HIV) attacks and destroys the cells of the immune system necessary to mounting an effective immune response to successfully fight off infection. Unchecked, an HIV infection leads to a gradual deterioration of the immune system, giving rise to many opportunistic infections not normally seen in people with a functioning immune system. There are two subtypes of HIV: HIV-1, which accounts for the most HIV infections worldwide, and HIV-2, which is most commonly seen in West Africa and countries with strong socioeconomic ties to West Africa, such as France, Portugal, Spain, Angola, Mozambique, and Brazil (1). There is, however, an increasing number of cases of HIV-2 in the United States, Europe, and India. In 2011, there was an estimated 1-2 million people infected with HIV-2; it has been estimated that HIV-2 now accounts for 4.5% of HIV infections (2).
Clinically, HIV-2 is important because the virus is intrinsically resistant to many of the anti-retroviral treatments (ART) that are used when treating HIV-1 (1). There are cases of concomitant infection with HIV-1 and HIV-2, and only the HIV-1 infection is treated, still allowing the HIV-2 infection to progress (3). Because of its lower incidence rate among the HIV-positive population, there has not been much research done in developing novel treatments for HIV-2. Even among treatments that may be useful in preventing Acquired Immunodeficiency Syndrome (AIDS), these treatments have demonstrated varying success rates due to naturally-occurring genetic polymorphisms in the viral genome (1). Additionally, due to a lack of large observational studies and randomized treatments, there is not a generalized consensus about when to begin treatment, or which treatments to use for an infection of HIV-2 before it progresses to causing advanced immunodeficiency (6). So little research has been done on HIV-2 that there is not even an accepted animal model aside from non-human primates in which to conduct HIV-2 studies. Developing an animal model that is not prohibitively expensive, as doing research on monkeys is, on which researchers could study HIV-2 would be useful in determining the differences in how HIV-2 infection progresses to AIDS, assist in drug development, allow for identification of which coreceptors HIV-2 uses to infect cells of the immune system, and help researchers develop the most useful treatment combinations with which to treat HIV-2 infection.
Previous research in our lab has indicated humanized mice to be an excellent animal model in which to study HIV-1 infection and progression to AIDS. We hypothesize that humanized mice infected with HIV-2 will also show symptoms of AIDS, allowing for more research on the pathogenesis and development of AIDS from HIV-2.
Our project will develop an animal model that can successfully maintain an HIV-2 infection that will be predicative of HIV-2 pathogenesis and treatment options in humans.
Methodology
We will have four groups of humanized mice, of which three groups will be infected with different strains of HIV.
1. The first control group of 3 mice will serve as a negative control, and will be uninfected with HIV.
2. The second group of 6 control mice, serving as a positive control, will be infected with a strain of HIV-1 called NL4-3 that has been proven to cause AIDS in humanized mice.
3. The two experimental groups consisting of 8 mice each will be infected with two different strains of HIV-2. The first strain is HIV-2 ROD-10 and is considered less virulent than the second strain. This second strain is known as HIV-2 287 and considered to be the more virulent of the two strains. Information about the virulence of the ROD-10 vs 287 strains was obtained in non-human primate studies. ROD-10 did not produce AIDS in monkeys, while the 287 strain did lead to immunodeficiency.
4. To prepare the mice for infection, Rag2-/γc-/- mice will be irradiated and injected with human hematopoietic stem cells. After 8 weeks, the mice will be screened for human cells. After a sufficient number of humanized mice have been produced, they will be screened for the number of peripheral blood engraftment of human cells. Those with at least 40% peripheral blood engraftment of human cells expressing receptor CD4 will be injected in the intraperitoneal cavity with our virus stocks. The titer of these stocks will be matched by diluting the more concentrated stocks to match the stock with the lowest titer.
Proof of Infection:
1. Quantitative PCR will be performed on blood sample to determine whether the virus is detectable in the blood. DNA PCR will be performed to test for latency of infection.
2. Histology samples from the organs will be taken and analyzed for evidence of the virus in tissues associated with the immune system.
3. Whether or not the mice get AIDS will be determined by FACS staining. With no infection, there should be a higher level of CD4+ cells than CD8+ cells in the blood. However, HIV attacks the CD4+ cells, leading to a CD4 inversion. With the CD4+ cells being destroyed by the virus, CD4+ levels will drop and the relative number of other immune cells, such as CD8+ cells, with rise. AIDS is defined as a CD4+ count below 200 cells/mm.
Discussion and Conclusion:
HIV belongs to the Lentiviral family, and as such replicates very slowly. It is often difficult to determine whether the virus is replicating properly in the cell culture. We have been using a cancerous, non-adherent T-cell line to grow the virus, relying on the presence of cytopathic effects to determine whether our virus has been replicating satisfactorily. After allowing the virus to propagate for about three weeks in continually replenished cells, we harvested the virus. However, titering the virus has proven problematic. We have been using ghost cells to determine the titer of our HIV-2 strains, but the cells have consistently died either before infection with the virus can occur or soon after infection. Doing the gold-standard plaque assay to determine our viral titer is not possible, since the cell lines with the necessary HIV coreceptors are non-adherent cells. We then tried to use PCR to determine our viral load, but there are currently no primers specific for any HIV-2 strains. Currently, we are on the verge of determining our viral titer using PCR and non-specific HIV-2 primers, but still need to determine the percentage of human cells in our mouse models. Once there are accomplished, we will be able to infect the mice and get the project moving again.