|
Novel viral vector systems for HIV/AIDS vaccine development
Herpesvirus vectors for HIV vaccine development
Infection of humans with members of the herpesvirus family results in long-term, potent immunologic responses,
including brisk CD8+ activity.
The potency of this response has been exploited to study the use of attenuated herpesviruses,
including HSV and varicella zoster virus (VZV) as vaccine vectors.
Herpes simplex virus type-1 (HSV-1) has the ability to infect a large number of cells, including dendritic cells (DC)
and other potential antigen presenting cells, and its size allows for incorporation of large amounts of foreign DNA.
Herpes vectors include both standard gene replacement vectors, as well as highly defective, replication-incompetent
plasmid DNAs which contain less than 1% of the viral genome.
The latter vectors use a helper virus-free HSV packaging system in which the DNA packaging signals are removed,
and the helper virus genome is contained within a bacterial artificial chromosome (bacmid).
Viral particles are produced by co-transfecting this bacmid with a plasmid vector that possesses the HSV origin of
replication, a viral packaging signal, and the desired gene(s) downstream of a potent promoter.
These particles, referred to as HSV amplicons, have multiple, concatameric copies of the desired gene product,
and induce both antibody and cellular immune responses in mice, including CD8+ T cell responses.
Based on promising preliminary data in the murine model, we will further evaluate the HSV amplicon
as a potential vaccine delivery vehicle for HIV-1 immmunization.
To do this, we will carefully construct a vector that has promise as a prophylactic vaccine.
In order to address the worldwide HIV-1 epidemic, we have chosen to use clade C gene products for our vaccine candidate.
The amplicon will express a codon-optimized, gag gene product, along with the env and other genes.
More details on this project can be found at the
HIV Vaccine Development Project website.
Targeted bacteriophage vectors for transduction of dendritic cells
Bacteriophage vectors are cheap to manufacture and purify, and have no inherent ability to infect mammalian cells.
As a consequence, suitably modified bacteriophage vectors may represent a cheap, safe and appealing alternative (or complement)
to existing mammalian virus vectors.
Coat-protein display of short antigenic peptides on bacteriophage particles can result in the elicitation of immune responses against
those peptides, and recent work by March and coworkers has established that unmodified phage vectors can elicit immune responses
against genomically-encoded antigens. We therefore hypothesize that: (i) bacteriophage vectors can be retargeted so as to allow
efficient transduction of DC, and (ii) that these modified bacteriophage vectors can be used to elicit specific and potent immune
responses to an encoded HIV-1 antigen. This hypothesis is presently being tested through an iterative series of experiments,
which involve (1) Development of modified bacteriophage vectors capable of transducing DC, using phage display technology;
(2) Insertion of a mammalian gene expression cassette encoding HIV-1 Env into these DC-targeted bacteriophage vectors; and
(3) Analysis of the immunogenicity of Env-encoding, DC-targeted bacteriophage vectors.
Interaction of HHV-7 with salivary gland (SG); SG gene transfer
Human herpesvirus (HHV)-7 infection is associated with persistence of viral genomes in salivary gland (SG) tissues,
and chronic expression of viral antigens in these sites. The virus is generally thought to be spread by a salivary route,
and there is lifelong shedding of large amounts of infectious virions in saliva. These observations suggest the following
hypothesis: that HHV-7 has evolved specific mechanisms to gain entry to SG cells and to evade host immune responses in SG tissues.
This may have important implications for the development of SG-targeted gene transfer applications.
We are interested in the interplay between the virus and its SG host cells. In collaboration with Dr. David Culp and others,
we have shown that HHV-7 antigens can be detected in salivary epithelial cells. We have also detected the expression of a
recently described, membrane-tethered delta-chemokine (fractalkine, CX3CL1) in salivary epithelial cells -
suggesting that SG chemokines may influence virus infection/replication in SG tissue.
We are therefore studying two putative 7-transmembrane (7-tm) receptors encoded by HHV-7, U51 and U12.
These receptors have previously been shown to interact with host chemokines, and may play a role in viral immune evasion
and/or salivary gland tropism, since homologous genes encoded by rat and mouse cytomegalovirus (CMV) are essential for efficient
viral replication in salivary glands.
We are presently examining the functional activity of U51 and U12 orthologs encoded by HHV-7 and by its close relative, HHV-6,
using both in vivo and in vitro model systems. It is expected that a greater understanding of the molecular pathways
exploited by HHV-7 will contribute to the future design of enhanced gene delivery vehicles for SG gene therapy;
such vector systems may incorporate components of HHV-7.
Regulation of neuronal survival by candidate HIV neurotoxins
A significant proportion of individuals infected with human immunodeficiency virus type-1 (HIV-1)
will develop HIV-associated dementia (HAD).
Neuronal injury and cell death are thought to contribute to the pathogenesis of this disorder,
and these events are believed to occur in response to the production and release of both viral and cellular gene products;
collectively, these molecules are refered to as candidate HIV neurotoxins.
We have shown that glycogen synthase kinase 3-beta (GSK-3b) may be an important contributor to HIV-1 induced neurotoxicity
and we are presently exploring the therapeutic implications of this finding, in collaboration with Dr. Howard Gendelman (U of Nebraska)
and others.
Related studies focus on mechanisms of microglial activation and monocyte recruitment to the CNS, in the context of HAD.
This is an important because microglia and brain-resident macrophages (which are derived from circulating monocytes that enter the brain)
are believed to be the principle source of candidate HIV neurotoxins; these cells are also the major site of HIV-1
replication within the brain.
Finally, collaborative studies being performed with Dr. Yuanan Lu (U of Hawaii) focus on the
development of novel gene transfer approaches, for delivery and expression of potentially therapeutic molecules
(such as neurotrophins) in the CNS. These studies involve the surface modification of mammalian virus vectors, in such a way as
to enhance their ability to cross the blood-brain barrier (BBB) and/or to transduce migratory cells than can traverse this interface
(such as circulating monocytes).
More details on this project can be found at the
HIV Neuroprotection Project website.
|
