My laboratory focuses on the interactions between enveloped viruses and their cellular receptors, with particular emphasis on the human immunodeficiency virus (HIV). Our overall goals are to elucidate mechanisms by which virus-receptor interactions lead to fusion and entry, to understand how such interactions contribute to viral pathogenesis, and to use this knowledge to develop novel therapeutic strategies to treat viral infection. My initial studies concerned structural analysis of CD4, the primary HIV receptor. In collaboration with other lab members, I localized the region of CD4 involved in binding to gp120, the external subunit of the HIV envelope glycoprotein (env). My group then went on to characterize CD4-induced changes in the structure of env and the possible roles of discrete regions of CD4 in the fusion process.
On the basis of our structure-function analyses of CD4, we devised a novel therapeutic strategy for the targeted killing of HIV-infected cells. With NCI collaborators, we genetically engineered a chimeric toxin so that it contains a portion of CD4 linked to the active regions of Pseudomonas exotoxin A. This drug, CD4-PE40, kills HIV-infected cells with extremely high selectivity and potency in vitro. Unfortunately, clinical trials at NIH and several other U.S. centers revealed unexpectedly high liver toxicity in HIV-infected patients, and no benefit occurred at tolerated doses. However, other applications are very promising. For example, several investigators who are developing gene-therapy protocols are using CD4-PE40 ex vivo to eliminate HIV-infected cells before genetically altered cells are reintroduced to the patient.
Using recombinant vaccinia virus technology, my lab developed a highly versatile reporter gene assay system for quantitation of fusion between env-expressing and CD4-expressing cells. We are using this assay to probe mechanistic features of fusion mediated by the HIV env and have extended our studies to paramyxoviruses such as measles virus and respiratory syncytial virus. The assay is also a valuable tool for the rapid, quantitative screening of antiviral drugs and antibodies that block the fusion step of virus infection.
A major focus of our mechanistic work concerns the specificity of the fusion processes mediated by the interaction between HIV env and CD4. Studies from several groups, including mine, have indicated that CD4 must be expressed on a human cell in order to support fusion by the HIV-1 env. Working with NCI investigators, we demonstrated that this restriction is due to the requirement for an unidentified human-specific fusion accessory component of the CD4-expressing cells. A related problem concerns the marked tropism of different HIV-1 isolates for infection of human T-cell lines vs. primary macrophages. My group recently showed that this cytotropism is due primarily to the fusion specificities of the corresponding envs. Our subsequent results suggest that T-cell-line vs. primary macrophage tropism is due to the requirement of the corresponding envs for distinct fusion-accessory factors differentially expressed in various CD4-positive target-cell types.
A major goal of our work is to identify these accessory-fusion factors. To this end, we used the vaccinia-based reporter-gene assay system in functional screening of cDNA libraries. We isolated a cDNA encoding a seven-transmembrane segment G protein-coupled receptor, which has the properties expected for a fusion accessory-factor for T-cell-line-tropic HIV-1 isolates. The identification of a new molecular player in the fusion process opens major new directions for our mechanistic structure-function studies.
My laboratory investigates bacterial pathogens transmitted by ticks and fleas. We focus on the Lyme disease spirochete, Borrelia burgdorferi; a relapsing fever spirochete, Borrelia hermsii; and the plague bacillus, Yersinia pestis. My early work focused on developing rapid diagnostic techniques to detect these agents in their respective tick and flea vectors. We also applied recombinant techniques to clone and express genes of B. burgdorferi that would be useful in the serodiagnosis of Lyme disease. One recombinant antigen, P39, has been patented and developed commercially into several diagnostic test kits for testing human serum samples for specific antibodies associated with Lyme disease. We also produced the first recombinant-based vaccine for plague.
More recently, we have begun to examine how these bacteria adapt and change during their infections in their arthropod vectors. For this, we rear and maintain live colonies of several species of ticks, including Ixodes scapularis and Ornithodoros hermsi, the respective tick vectors of Lyme disease and relapsing fever spirochetes. We also maintain a colony of the Oriental rat flea, Xenopsylla cheopis, for our work on plague.
The relapsing fever spirochete, B. hermsii, contains at least 40 genes that encode variable major proteins (Vmps). At any given time, only one gene is expressed by the spirochete and each Vmp confers serotype specificity on the bacterium's surface. In humans and other animals, these genes form the basis of the spirochete's antigenic variation, allowing the organism to temporarily evade the mammalian host's humoral immune response to infection.
We are investigating the antigenic behavior of relapsing fever spirochetes during their infection in the tick vector and how the different serotypes affect transmission by ticks. By infecting different cohorts of ticks with different serotypes of B. hermsii, we have found that the spirochetes do not change antigenically while they are in ticks. Hence, ticks transmit the same serotype that was ingested during a previous blood meal. However, the serotype has a striking influence on the frequency at which spirochetes are transmitted when the infected ticks feed again. This means that one serotype is rarely transmitted, whereas another is transmitted during one out of every three tick feedings. We are now trying to understand the basis for such differences.
The closely related Lyme disease spirochete causes more human infections in the United States than all other vector-borne illnesses combined. Recently, we demonstrated that during the spirochete's residency in the tick midgut, its outer surface changes as the tick attaches to a mammalian host and ingests blood. An increase in temperature is also involved in the spirochete's synthesis of new proteins during the feeding, and it corresponds to the time at which these bacteria escape the tick's midgut, infect salivary glands, and are transmitted via the saliva. Such changes have important implications for both diagnostics and vaccine development. We hope that our studies with both species of spirochetes and their respective tick vectors will allow us to identify factors responsible for tick-spirochete specificity and critical events in the transmission of spirochetes to humans.
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