Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-05-12
2003-02-25
Bui, Phuong T. (Department: 1638)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C424S208100, C424S188100, C424S192100, C435S007100, C435S069100, C435S069300, C435S070100, C435S071100, C435S071200, C435S235100, C435S320100, C514S04400A, C536S023720
Reexamination Certificate
active
06525173
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention refers to the general field of the technology of the DNA recombinant proteins, for the production of a recombinant hybrid protein (p24/p17) derived from human immunodeficiency virus (HIV-1) to be used in diagnosis of acquired immunodeficiency disease virus (AIDS), vaccination, antibody production or in research.
BACKGROUND TO THE INVENTION
The epidemic provoked by the human immunodeficiency virus(HIV), today global, continues without bamers, and has as a consequence the syndrome of the acquired immunodeficiency(AlDS), recognized 12 years ago. The studies of the World Organization of Health estimated that more than 18 million people are infected and by the year 2000, 40 to 100 million people worldwide would have been infected by HIV-1 or HIV-2, from which 10 million will be children.
The hope for an effective therapy and, mainly, the prevention resides in the acquisition of new information on the virus, the mechanisms of the pathogenesis and in the search of experimental models (COOPER. Immunol. 4: 461, 1992). The current knowledge is that blocking the dissemination will be very important and therefore the early detection is needed for the treatment with the new drugs.
The virus concentration can greatly influenced the virus transmission in a body fluid. In the beginning of the epidemic the major routes of AIDS transmission were sexual contact and through transfusion with contaminated blood (Levis in: HIV and pathogenesis of AIDS p.26,1989). The syndrome was initially described in homosexual and bisexual men and in intravenous drug users (Mansur et al.,N. Engl. J. Med.305:1431,1989, but its occurrence from heterosexual activity were soon recognized (Harris, C et al.,N.Engl. J. Med.308:1181,1983). Today the principal means of transmission are the sexual, maternal-child, drug users and still blood in undeveloped countries. All of them can be explained to a great extent by the relative concentration of HIV in various body fluids.
HIV entry in the body and developed acute infection as observed by other virus. The HIV pathogenesis reflects several properties of the virus and the host immune response. AIDS final outcome is the differential expression of those major components of HIV infection
The first step of HIV infection is the interaction between the major lymphocyte receptor CD4 molecule, a member of immunoglobulin superfamily with the envelope gp120 protein. According to some reports described gp 120 is displaced, leading to the uncovering of domain on the envelope gp41 needed for virus cell fusion. Once in the cytoplasm several intracellular events take place ending with integration of a proviral form into cellular genome.
Besides entering cells via the direct interaction of the virus envelope with cell surface receptors, HIV can infect cells by other mechanisms. For example, during the course of studies on the humoral response to HIV-1 (infection, the phenomenon of antibody dependent enhancement (ADE), of HIV infection was found to occur (Homsy, J. et al. Science 244: 1357, 1989; Homsy, J. et al. Lancet i: 1285, 1988). The transfer of the virus into a cell through the complement or Fc receptor involves the binding of the Fab portion of nonneutralizing antibodies to the virus surface (Levy in: HIV and the pathogenesis.p.53,1994)
Clinical manifestations of acute HIV infection were recognized in the very early studies, and have been described in various articles (Thindall and Cooper, AIDS:5:1, 1991; NIV, MT et al. J. Infect. Dis. 168:1490, 1993). A newly infected host present within 1 to 3 weeks symptoms as headache, pain, muscle aches, sore throat, fever, swollen lymph nodes, a non pruritic macular erythematous rash involving the trunk and later the extremities.
It is estimated that 50 to 70% of patients primary infected with HIV will developed a syndrome of acute mononucleosis-like illness. This period is associated with high viremia levels, and the immune humoral response against the virus is detected between one week and three months (DAAR et al. New Engl. J. Med 324: 961,1991).
This specific immunity that initiates in this period is associated to a dramatic decline of the viremia, but the level of this immunity inadequate to suppress the viral multiplication. The expression of the virus persists in the lymph nodes, even when the presence of the virus in the plasma is difficult to be detected, and the mRNA is not detectable in the mononuclear cells of blood stream (MICHAEL et al. J. Virol. 66: 310-316, 1992).
HIV is classified, based on its morphology, genomic organization and pathogenic properties, as a member of the sub-family lentivirus of the family Retrovindae.
HIV-1 and HIV-2 as a lentivirus have the characteristic cone-shaped core composed of the viral p24 GAG protein. Inside this capsid are two identical RNA strands with the reverse transcriptase (RNA dependent DNA polymerase) and the nucleocapsid proteins (p9, p6) are closely associated. The inner portion of the viral membrane is surrounded by a myristylated p17 core protein (GAG) that provides the matrix (MA) for the viral structure which is important for virion integrity (McCune, J. M. et al. Cell 53: 55, 1988; Shulz, T. F. et al. AIDS Res. Human Retrov 8:1584, 1992). The virus surface is made by envelope glycoproteins derived from a precursors of Mw 160.000 which is inserted inside the cell into a gp120 and a gp41 transmembrane protein (TM). The central region of TM protein binds to the external viral gp120 in a non covalent ligation at two hydrophobic regions in the amino and carboxi termini of gp120 (Helseth, E. et al. J.Virol. 65:2119-2123, 1991.
Two other genes tat and rev are positive regulators for the replication of HIV, besides other proteins with accessory function, as the vpu, vif, vpr and nef (ROSEN, TIG 7: 9-14. 1991.).
One of the most notable properties of the genoma of the HIV-1 is its genetic variation (DESAI et al Proc. Natl. Acad. Sci. 83: 8380, 1986). The diversity can be important for many aspects of the biology of the virus, among them the tissue and cell specificity, clinical-pathological picture of the disease, geographical and temporary virus distribution, difference in the susceptibility of immune response, virulence and, especially, development of a vaccine of wide crossed reactivity. The mistake range esteemed for the variation of HIV is of a substitution in 10
4
synthesized nucleotides. Besides the substitution, deletions and inserts, whose frequency is more difficult of being evaluated can happen.
The gene env shows, along its structure, variable (V) and constant (C) regions. The principal neutralizing domain of the HIV-1 is placed in the third variable region (it raises V3) of the envelop glycoprotein (gp120). The loop V3 is an important neutralization epitop for, viral tropism and syncytium formation. In the V3 loop is the neutralyzing epitop for type-specific antibodies.
The laboratory diagnosis of any infectious agent is an important aspect for the control of infectious diseases. The precise diagnosis wins importance in the blood derivatives, whose use depends on the capacity of the tests in the detection of infectious agents' or its antigens. During the last decade, the technological progresses developed precise tests for the diagnosis of AIDS. Most of the researches concentrated efforts for the development of tests to be used in development countries were are the economic limitation. In those countries the HIV detection is not a routine.
The HIV-1 diagnosis is made with the indirect detection of the presence of the virus, indicated by the patient's immune response, evidenced by the presence of specific antibodies against the HIV-1 or the detection of the virus or its components.
The direct methods identify the virus multiplication in culture, detection of virus or antigens in immunoassays (ELISA), molecular hybridization or amplification of nucleic acids (PCR). However, some of these tests demand qualified personnel and equipped laboratories, what hinders its widespread use. An exception is the test of ELISA for t
Ferreira Paulo Cesar Peregrino
Geessien Kroon Erna
Bui Phuong T.
Universidade Federal de Minas Gerais
Young & Thompson
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