Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2001-07-16
2004-02-17
Wehbe', Anne M. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S325000, C435S455000, C514S04400A
Reexamination Certificate
active
06692955
ABSTRACT:
The invention relates to a method of virus manipulation; means therefor and products thereof which have particular, but not exclusive, application in gene therapy/vaccine development.
A virus-derived vector capable of efficient gene delivery to human T lymphocytes would have a wide range of uses in human gene therapy. An obvious disease target would be human immunodeficiency virus (HIV) infection, where such a vector could have use both in the prevention and therapy of infection. Two virus vector systems which do not cause a cytopathic effect in vitro, retroviruses and adeno-associated virus, both target dividing cells only, and are thus inappropriate for use with a CD4 T cell population which are mainly non-dividing.
Virus based gene therapy systems currently in clinical trial or in development include vectors based on adenoviruses, retroviruses and human herpesviruses (1, 2, 3). All these systems have inherent disadvantages. Adenovirus vectors have constraints on the size of heterologous DNA incorporated, can cause toxic side effects and induce a vigorous immune response resulting in rapid clearance of infected and therefore gene targeted cells (4, 5). A major drawback in retroviral systems based on murine leukemia viruses is their inability to infect non-dividing cells. Thus cells must be removed, activated, infected in vitro, and then delivered back to the patient. A further disadvantage is the high inherent mutation rate caused by reverse transcription (6).
Herpesvirus vector systems offer the potential of delivering >50 kb of heterologous DNA, the infection of non-dividing cells and maintenance of their genome episomally in a non-replicative form (7). However, nearly all vector systems in development to date are based on herpes simplex virus and are likely to be ineffective in many individuals due to an immune response present in >80% of the population, already induced by the wild type virus (8). A herpesvirus of non-human origin, capable of infecting human cells, therefore represents an attractive candidate as a gene therapy vector, as there will be no innate immune response in the recipient to prevent infection in vitro.
Herpesvirus saimiri (HVS) is a lymphotropic rhadinovirus (&ggr;2 herpesvirus) of squirrel monkeys (
Saimiri sciureus
). The virus genome may be detected in an episomal form in T cells and causes no apparent disease in the natural host. Whereas type A and B strains similarly do not cause apparent disease in other monkey species, C type strains of this virus are oncogenic in certain New World primates (9). C strains also have the ability to transform human T cells in vitro. The gene product responsible for cell transformation has been identified as the STP gene (ORF1) (10). STP is non-essential for virus replication in vitro and in vivo; natural deletion mutants exist in C strains which are non-oncogenic. Therefore a virus of strain A, which has the STP gene deleted is unable to transform any type of cell. Virus strains lacking this gene and carrying several heterologous genes have been constructed and studies carried out in vitro have demonstrated high efficiency and long term expression of the heterologous gene product (11). The virus DNA remains episomal with no detectable expression of virus genes, but with stable heterologous expression in the absence of selection. Furthermore, the virus genome segregates efficiently between dividing cells, presumably in a manner similar to the human &ggr;2 herpesvirus, Epstein-Barr Virus. Advantageously, HVS which naturally infects non-human primates has also been found to infect human T lymphocytes. As previously mentioned, this feature of HVS can be used to advantage for providing in man a wide range of gene therapies. For example, it is of note that a major target cell type for HIV infection in man is T lymphocytes. We therefore speculated that the expression of HIV protein in the correct oligomeric configuration in vivo on the surface of a T lymphocyte should induce an effective humoral and cellular anti-HIV immune response. HVS can be used to advantage in the delivery of a gene encoding large HIV proteins because, as previously mentioned, it has the potential of delivering >50 kb of heterologous DNA and is further capable of infecting T lymphocytes.
In our studies we chose an envelope gene from a primary isolate of HIV as the gene to be delivered by HVS for the development of the potential vaccine.
Studies have shown that immunogens based on the envelope protein or defined epitopes thereof induce both virus neutralizing antibodies and HIV-specific cytotoxic T lymphocytes (12, 13, 14, 15). Furthermore, chimpanzees have been successfully protected against HIV challenge, with protection correlated with the presence of high titre neutralizing antibodies (12). However, recent studies have shown that antibodies raised against these immunogens encoding envelope sequences from laboratory adapted isolates are ineffective at neutralizing primary isolates from HIV infected individuals (16). Envelope proteins from primary isolates confer different characteristics to the virus as compared to laboratory adapted isolates. These include the ability to infect macrophages and the inability to induce syncytia, the cytopathic effect seen with laboratory adapted isolates. Furthermore, anti-envelope antibodies from HIV positive individuals show cross isolate neutralizing ability (17, 18). Analysis of HIV neutralizing antibodies have also suggested that the envelope immunogen must be presented in the authentic, oligomeric, non-denatured form to induce heterologous neutralizing antibodies (15, 19). Recent reports also indicate the need to elicit a polyclonal antibody response as human monoclonal antibodies isolated from patients fail to neutralize certain primary isolates (18, 20). Further evidence for the feasibility of a vaccine strategy utilizing a primary macrophage-tropic envelope is provided by a study using the simian immunodeficiency model (SIV) model (21). Macaques immunized with an attenuated macrophage-tropic SIV developed cross-neutralizing antibodies and were successfully protected against heterologous isolate challenge.
The studies outlined above indicate that the induction of a vigorous immune response against the HIV envelope protein may protect against virus challenge or constrain the virus if infection does occur. Long term survivors who remain health); 12-15 years after infection have high levels of neutralizing antibodies (17). Delivery of a primary isolate envelope gene by a virus based vector to the correct cell population offers the potential to induce an effective anti-HIV cross-isolate immune response, an essential requirement of any candidate HIV vaccine. This gene therapy approach may also have therapeutic value to individuals who have progressed to AIDS. It has been shown that expression of envelope protein in T cells induces partial resistance to infection, and significantly induces total resistance to cytopathic effects (22). T cells infected with the recombinant HVS may therefore be protected in vivo.
We believe that vectors derived from HVS offer a wide range of opportunities to target both protective and therapeutic genes to human T cells, offering the unique opportunity to deliver a stably-expressing, extrachromosmal element, to a non-dividing cell population. Moreover we have demonstrated efficient infection of the T-cell line Jurkat, by a recombinant HVS vector expressing HIV gp160.
It is therefore a first object of the invention to provide a gene delivery system/vaccine to deliver at least a part of at least one preselected gene to a specific cell population in which the encoded heterologous protein is presented as an antigen.
In its broadest aspect the invention concerns the use of HVS to deliver heterologous genetic material to a specific cell population and ideally to T lymphocytes and/or macrophages.
According to a first aspect of the invention there is therefore provided a herpesvirus saimiri vector which has inserted therein at least a part of a gene encoding an envelope protein of
Markham Alexander Fred
Meredith David Mark
Li Q. J.
Myers Bigel Sibley & Sajovec P.A.
University of Leeds
Wehbe' Anne M.
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