ANIMAL LENTIVIRUSES--STRATEGIES FOR HIV VACCINATION

The human imunodeficiency virus (HIV) is a member of the lentivirus
subfamily, a taxonomic group of nononcogenic retroviruses that
persistently infect cells of the host immune system. These are
enveloped viruses that have a highly glycosylated envelope glycoprotein
(env gp) that stimulates strong immune responses in the infected host.
However, immune responses elicited during natural infection fail to
control virus replication resulting in a persistent infection that
eventually leads to chronic multisystemic disease and death. Many of
the functional domains that mediate cell tropism, cytopathicity, and
neutralization of virus by antibody have been mapped to the env gp of
HIV. However, the properties of the lentivirus envelope that underlie
ineffective anti-lentivirus immune responses are poorly understood and
difficult to evaluate with HIV itself. Insight into these areas are
essential to developing effective immune based strategies against HIV.
Caprine arthritis encephalitis virus (CAEV) of goats and visna virus of
sheep are two closely related, well-characterized lentiviruses that
mirror HIV in many of their biological and structural characteristics.
Infectious molecular clones of both CAEV and visna that have been
completely sequenced are available for study and provide a system in
which to address questions of lentivirus immunogenicity and pathogenesis
in the natural host. This proposal focuses on mapping and
characterizing the functional domains of the ovine/caprine lentivirus
env gp. A set of truncated and mutated env gp genes will be constructed
using the polymerase chain reaction with synthetic oligonucleotides as
primers and the subcloned into a baculovirus expression vector.
Recombinant baculovirus expressing the various forms of the env gp will
be used to infect spinner flasks of insect cells to obtain milligram
quantities of purified env gp. The various purified glycoprotein
constructs will be compared for their ability to block in various
functional assays. In years 4-5 these mapping studies will be extended
by using PCR to construct infectious molecular clones that are envelope
chimeras of CAEV and visna. Switching specific domains between the env
genes of the two viruses will map determinants of cell tropism, antibody
neutralization, immunogenicity and pathogenicity for the ovine/caprine
lentiviruses in their natural host. These biological properties are
mediated at least in part by env gp and differ markedly between the well
characterized parental viruses, CAEV and visna virus. The candidate has limited experience in applying techniques of molecular
biology to the study of disease pathogenesis. The supervisor is a
recognized expert in the field of lentivirus molecular biology with a
large active lab. The candidate has been provided with independent
laboratory space at the Bayview Research Campus of the Johns Hopkins
University, School of Medicine along with equipment and support from the
Division of Comparative Medicine and the university. This combination
of factors should provide an excellent environment for completing the
proposed studies and greatly enhance the candidate's development as an
independent researcher.