Efficient ex vivo expansion of cd4+ and cd8−...

Details Efficient ex vivo expansion of cd4+ and cd8−... Efficient ex vivo expansion of cd4+ and cd8−...

1999-04-23

2003-08-26

Fredman, Jeffrey (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S320100, C435S325000, C435S455000, C424S093210, C424S093200, C514S04400A, C530S350000, C530S351000

Reexamination Certificate

active

06610542

ABSTRACT:

BACKGROUND OF THE INVENTION
The human immunodeficiency virus (HIV) is a serious and growing health threat in virtually every part of the world. It has been estimated that over 22 million people are currently infected worldwide, and it is anticipated that over 40 million people will be infected by the end of this decade.
HIV infection typically leads to acquired immunodeficiency syndrome (AIDS) within 8 to 10 years after infection. Individuals with AIDS are subject to opportunistic infections and cancers, leading to severe illness and, ultimately, death. Although various treatments delaying the progression from HIV infection to AIDS are known, these treatments are of limited effectiveness and generally require the use of pharmaceuticals which have adverse side-effects. Moreover, the effectiveness of different drug treatments varies among individuals and no satisfactory system exists to screen different drug combinations for effectiveness in combating HIV infection in a particular subject.
HIV is a retrovirus, closely related to simian immunodeficiency virus (SIV). At least three variants of HIV, known as HIV-1, HIV-2, and HIV-0, are known. It is believed that HIV-1 is the predominant global form of human HIV infection at the present time. HIV-2 is believed to be common in West Africa, but rarer elsewhere in the world. HIV-2 appears to be less pathogenic that HIV-1. In addition to these three HIV variants, the high natural mutation rate of HIV DNA means that virtually every individual infected with HIV carries a slightly different virus. Differences between HIV isolates complicate efforts to devise effective anti-HIV approaches, including drugs and vaccines.
HIV enters target cells by the binding of gp120 (present on the HIV virion) to cellular receptors, followed by fusion of the viral envelope with the plasma membrane of the target cell. The major cellular receptor for the HIV gp120 is cluster of differentiation factor 4 (CD4). The highest levels of CD4 are generally found on T-helper (Th) cells; thus, the consequences of HIV infection are typically most obvious in the Th cell population. HIV can also infect other cells, including macrophages, monocytes, dendritic cells, Langerhans cells, and microglial cells. HIV-1 has a higher affinity for CD4 than does HIV-2, and it is thought that this may contribute to the greater pathogenicity of HIV-1 compared to HIV-2. HIV-1 also requires a chemokine coreceptor (e.g. CCR5 or CXCR4) to gain entry into susceptible cells.
There is evidence in the prior art suggesting that specific chemokines such as RANTES, MIP-1&agr; and MIP-1&bgr; may inhibit fusion between the HIV-1 virion and target cells by inhibiting the interaction between HIV surface proteins and cell surface receptors. This inhibits viral replication by reducing the rate of infection.
Fusion between the HIV virion and the plasma membrane of the target cell allows the HIV RNA to enter the target cell, where it is reverse transcribed into DNA by viral reverse transcriptase and integrated into the host cell's genome to form an HIV provirus. Once the HIV DNA is integrated into the host cell genome, it is replicated during cell division and is passed on to daughter cells.
The HIV provirus may remain inactive in the host cell for some time until it is activated. Upon activation, HIV structural genes are expressed, and single stranded HIV RNA (HIV ssRNA) is transcribed. The HIV structural proteins and HIV ssRNA assemble to form numerous virus particles which then exits from the host cell infects other cells.
At present, methods of inhibiting HIV replication in tissue samples have tended to focus on reducing the number of newly infected cells through the inhibition of infection by released virus particles. This has been effected through the use of compounds which inhibit fusion between the HIV virion and the plasma membrane, and inhibitors of viral reverse transcriptase (necessary to generate DNA from the viral RNA prior to integration into the host cell genome) to form the provirus. In addition, the production and release of viral particles from infected cells has been inhibited through the use of protease inhibitors which interfere with the post-translational processing of HIV gene products necessary for virus particle formation. The effectiveness of many current therapies is limited by the capacity of the HIV virus to mutate, resulting in the development of resistance. Methods for inhibiting the expression of HIV DNA in populations comprising CD8 and CD4 cells from infected subjects (thereby reducing the number of virus particles which can be formed) while greatly expanding CD4 and CD8 cells in these populations are not known in the art. Such methods might be less susceptible to circumvention by acquired resistance and therefore represent a potentially powerful form of HIV treatment.
It is desirable to have a means of inhibiting the expression of HIV DNA in infected cells. Individual infected cells are capable of producing a massive number of infectious HIV particles, and the release of such particles from a cell can cause the infection of numerous previously uninfected cells Levine et al. (
Science,
272:1939, Jun. 18, 1996) have reported that the interaction of CD4 cells with immobilized (but not soluble) CD28 monoclonal antibodies reduces the susceptibility of CD4 cells to HIV infection. However, it would be more efficient to inhibit the formation of HIV particles in infected cells, rather than to simply attempt to reduce the rate of infection by such particles following their formation and release from the infected cell.
HIV DNA sequences are flanked by long terminal repeats (LTRs). Promoter and enhancer sequences are located in the 5′ LTR, and polyadenylation sequences are contained in the 3′ LTR. The 5′ LTR sequence normally has only a low affinity for RNA polymerase, causing premature truncation of transcription products and preventing the formation of infectious viral particles. However, the viral protein Tat is capable of interacting specifically with a region (TAR) on the emerging RNA transcript and increasing the formation of full-length proviral transcripts.
One of the characteristic features of HIV infection is a reduction in the number of CD4
+
T-cells (“CD4 cells”) in the peripheral blood of infected subjects. Healthy uninfected individuals typically have approximately 1100 CD4 cells per microliter of whole blood. After an individual has been infected with HIV, CD4 cell levels generally drop gradually over a period of 8 to 10 years, but then drops more rapidly. In subjects with AIDS, CD4 cells levels below 200 cells per &mgr;l are common.
It is believed that in the early stages of HIV infection CD4 cells are destroyed at a high rate. However, at this stage the subject's immune system is able to replace many of the destroyed cells, resulting in only a gradual decline in observed CD4 cell numbers. Cells may be destroyed by various means following infection. One means for the destruction of infected cells is lysis resulting from the exit of large numbers of newly formed virs particles. A second means by which infected cells may be destroyed is by an immune response to HIV antigens expressed on the cell membrane. It is believed that enhanced levels of active anti-HIV specific CD4 cells in HIV infected patients allows the maintenance of low viral loads and non-progression into AIDS.
It appears that many uninfected CD4 cells lose their capacity to respond to foreign antigens and are also destroyed during HIV infection. The exact mechanism by which this occurs is not fully understood. However, it is suspected that free gp120 to CD4 molecules on the surface of uninfected cells. This binding may lead to the internalization of the gp120 by the uninfected CD4 cells. Proteolytic processing of the internalized gp120 in the endosome, followed by association of the processed peptides with class II MHC, may lead to the expression of an HIV peptide-MHC complex at the surface of uninfected cells. Such cells may thus be destroyed as a result of an immune

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