(no subject)
i just wanted to post this. i apologize in advance for all the scientific jargon...
HIV Infection Control
Investigated Immune Mechanisms Involving CTL’s
In Vitro Mechanisms and Evidence
The replication of HIV-1 in CD4+ T lymphocyte can be suppressed by the addition of autologous CD8+ lymphocytes from HIV-1 infected patients7. Monocyte derived dendritic cells (DC) pulsed with inactivated virus were able to increase the numbers of virus-specific CD8+ T cells which subsequently eradicated the virus from cultured cells8. As CTL’s destroy the cells infected with HIV, the virus is left without cells that allow attachment and so is not able to replicate. CTL’s may have the ability to control the early stages of viral replication.
In Vivo Mechanisms and Evidence
In patients showing the early symptoms of HIV infection, HIV-specific CTL responses were detected prior to the viral replication peak1. The appearance of CTL’s was a major factor in the subsequent decline of the viral load, as neutralizing antibodies have not yet had time to appear7. In macaques infected with simian immunodeficiency virus (SIV), CTL’s have shown the ability to control infected cells7. In vivo experiments confirm what in vitro experiments have indicated.
Epidemiological Evidence
Virus-specific CTL responses play a vital role in controlling viral load, an important part of slowing the progression of the disease7. The decline of peripheral blood CD4+ lymphocytes, which are the cells that HIV targets, has been observed in HIV-1 infected patients with high virus-specific CTL activity7,9. The level of CTL activity has also been shown to parallel the control of viremia2. Some sex workers in Kenya that are routinely exposed to HIV remain uninfected; these individuals had HIV-1 specific CTL activity to a variety of HIV epitopes5. Invoking a virus-specific CTL response has a significant impact upon the infection with HIV and subsequent progression to AIDS.
Investigated Immune Mechanisms Involving Antibodies
In Vitro Mechanisms and Evidence
The generation of HIV-1 cell envelope protein-specific CD4+ T cells, leading to Th2 cells and the production of antibody, has been obtained from a HIV negative patient’s serum through stimulation in vitro with HIV cell envelope proteins11. Serum IgA that is HIV-specific is capable of inhibiting infection as well as replication in vitro3,4. Despite this evidence, many mechanisms that work in vitro have not been shown to be effective in vivo, possibly due to the isolation of mechanisms that often occurs during in vitro experiments.
In Vivo Mechanisms and Evidence
Passive protection in the form of neutralizing antibodies has been conferred to rhesus monkeys against a pathogenic chimeric simian-human immunodeficiency virus (SHIV)7. Antibodies that neutralize the virus (often by not allowing attachment) stop the spread of the virus. In HIV-1 infected individuals at the early stages of the immune response, the classical complement pathway is active and inactivates both autologous and heterologous HIV strains1. Antibodies that induce complement also stop virus replication by killing the cells that contain the virus. Both neutralizing and complement inducing antibodies may contribute to the control of HIV.
Epidemiological Evidence
In a small number of HIV-1 infected individuals, the antibodies produced are capable of neutralizing several HIV variants7. This is important (and rare) because of the high rate of antigenic variation in HIV, which causes many of the antibodies developed to become ineffective when the virus changes9. A vaccine specific for a HIV envelope protein (gp120) was given to human volunteers but not protection was conferred; some subsequently contracted HIV7. Antibodies that confer protection are difficult to produce because HIV is highly variable and many epitopes on the surface are masked7.
Vaccine Design
Traditional vaccine approaches are not proving effective against HIV. Any virus that is capable of inducing protection against a wild-type virus is likely to induce the disease itself (especially when administered to immunocompromised individuals which is likely) while non-live vaccines do not elicit a response that is capable of containing the infection of HIV variants7. A vaccine that induces a CTL response would be the most efficacious as T cell mediated immunity seems to be more efficient at conferring protection than humoral.
Genes that encode for surface proteins of HIV-1 can be inserted into a live viral vector5. Once the vector is within the subject, the vector would produce the HIV-1 proteins and stimulate a CTL response10. A good candidate for the vector would be the avian poxviruses. They produce the proteins in enough quantity to stimulate an immune response while only undergoing partial replication within the human, limiting the chances of the vector itself causing disease as well as stimulating Th1 responses due to the low level of antigen produced6,7,10. Because the proteins are processed through the MHC I pathway, they stimulate a CTL response, while proteins conjugated to an adjuvant do not7. The genes must be chosen carefully to insure that the proteins are those that will stimulate protection against many variants of HIV-1. Gag, Pol, and Env are virion structural proteins and are common to all HIV-1 strains, which makes them good candidates for this vector9.
Measurement of Response
MHC:peptide tetramers (some specific for each protein that will be expressed by the vector) can be synthesized and labeled with a fluorochrome to allow for detection of binding5. A sample of blood serum will be taken from each subject, repeated at certain intervals to determine the rapidity and longevity of the response. The MHC:peptide tetramer will then be mixed with the serum and will identify those T cells that are specific for that MHC:peptide complex. The binding will be monitored by flow cytometry which (when also stained with antibodies specific for CD4 and CD8) will allow the quantification of the percentage of the total population of T cells that are specific for that complex5.
Since a CTL response is preferred over a CD4+ T cell response, an assay to determine that cytotoxic T cells were generated would be desirable. Target cells (cells that express one of the proteins encoded within the vector) will be grown up in a culture containing 51Cr. The radioactive Cr is incorporated into the live cells10. The subject’s blood serum will be then applied to the culture. Any CD8+ T cells present in the serum will kill target cells which causes the release of the radioactive Cr into the supernatant, which can then be quantified5.
Both assays determine whether there was a T cell response generated and the relative immunogenicity of each protein encoded for in the vector. Before there is a chance of the vaccine conferring protection, it needs to be demonstrated that it generates a CTL response. Knowing which specific proteins generate the immune system will be important to determine whether to include them in the next version of the vector or to attempt to find other HIV surface proteins that would generate a response.
Measurement of Protection
Detection of protection in humans is hampered by the inability to induce HIV in the subjects through deliberate infection. To produce the most data possible, the study will be limited to subjects that are routinely exposed to HIV, as with the sex workers previously mentioned. The immune response must be challenged because the ability to stimulate an immune response does not confer protection10. Without being infected with HIV, there is no way to determine the protective effects of the vaccine.
Traditional tests used to diagnose HIV would be sufficient to measure protective immunity. If the vaccine confers protection, no HIV infection will be successful in causing disease. ELISA’s can be used to test for HIV-specific antibodies in the patient10. An indirect ELISA in which HIV antigen is fixed, the patient’s serum is added (the primary antibody), and an antibody against human IgG conjugated to alkaline phosphatase (the secondary antibody) would be used. Some commonly used antigens are gp120, gp160, gp41, and p2410. It would be important to avoid using the proteins that were included in the vaccine as the antibodies could have been caused by the vaccine or by HIV infection.
Works Cited
1. Aasa-Chapman, M. M, I., S. Holuigue, K. Aubin, M. Wong, N. A. Jones, D. Cornforth, P. Pellegrino, P. Newton, I. Williams, P. Borrow, & Á. McKnight. 2005. Detection of antibody-dependent complement-mediated inactivation of both autologous and heterologous virus in primary human immunodeficiency virus type 1 infection. J. Virol. 79:2823-2830. [online.]
2. Borrow, P., H. Lewicki, B.H. Hahn, G.M.Shaw, & M.B. Oldstone. 1994. Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of primary viremia in primary human immunodeficiency virus type 1 infection. J. Virol. 68:6103-6110. [online.]
3. Burnett, P.R., T.C. VanCott, V.R. Polonis, & D.L. Birx. 1994. Serum IgA-mediated neutralization of HIV type 1. J. Immunol. 152:4642-4648. [online.]
4. Huang, Y. T., A.Wright, X. Gao, L. Kulick, H. Yan, & M. E. Lamm. 2005. Intraepithelial cell neutralization of HIV-1 replication by IgA. J. Immunol. 174: 4828-4835. [online.]
5. Janeway Jr., C. A, P. Travers, M. Walport, & M. J. Schomchik. 2005. Immunobiology: the immune system in health and disease, p. 491-507, 651, 711-721. Garland Science Publishing, New York, N.Y.
6. Kent, S.J., A. Zhao, S. J. Best, J. D. Chandler, D. B. Boyle, & I. A. Ramshaw. 1998. Enhanced T cell immunogenicity and protective efficacy of a human immunodeficiency virus type 1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowlpox virus. J. Immunol. 72:10180-10188.
7. Letvin, N. L, D. H. Barouch, & D. C. Montefiori. 2002. Prospects for vaccine protection against HIV-1 infection and AIDS. Ann. Rev. Immunol. 20:73-99.
8. Lu, W. & J.M. Andrieu. 2001. In vitro human immunodeficiency virus eradication by autologous CD8+ T cells expanded with inactivated-virus-pulsed dendritic cells. J. Virol. 75:8949-8956. [online.]
9. Sharon, J. 1998. Basic Immunology, p. 151. Williams & Wilkins, Baltimore, MD.
10. Taylor, B. Personal communication.
11. Venturini, S., D. E. Mosier, D. R. Burton, & P. Poignard. 2002. Characterization of human immunodeficiency virus type 1 (HIV-1) Gag- and Gag peptide-specific CD4+ T-cell clones from an HIV-1 seronegative donor following in vitro immunization. J. Virol. 76:6987-6999. [online.]
HIV Infection Control
Investigated Immune Mechanisms Involving CTL’s
In Vitro Mechanisms and Evidence
The replication of HIV-1 in CD4+ T lymphocyte can be suppressed by the addition of autologous CD8+ lymphocytes from HIV-1 infected patients7. Monocyte derived dendritic cells (DC) pulsed with inactivated virus were able to increase the numbers of virus-specific CD8+ T cells which subsequently eradicated the virus from cultured cells8. As CTL’s destroy the cells infected with HIV, the virus is left without cells that allow attachment and so is not able to replicate. CTL’s may have the ability to control the early stages of viral replication.
In Vivo Mechanisms and Evidence
In patients showing the early symptoms of HIV infection, HIV-specific CTL responses were detected prior to the viral replication peak1. The appearance of CTL’s was a major factor in the subsequent decline of the viral load, as neutralizing antibodies have not yet had time to appear7. In macaques infected with simian immunodeficiency virus (SIV), CTL’s have shown the ability to control infected cells7. In vivo experiments confirm what in vitro experiments have indicated.
Epidemiological Evidence
Virus-specific CTL responses play a vital role in controlling viral load, an important part of slowing the progression of the disease7. The decline of peripheral blood CD4+ lymphocytes, which are the cells that HIV targets, has been observed in HIV-1 infected patients with high virus-specific CTL activity7,9. The level of CTL activity has also been shown to parallel the control of viremia2. Some sex workers in Kenya that are routinely exposed to HIV remain uninfected; these individuals had HIV-1 specific CTL activity to a variety of HIV epitopes5. Invoking a virus-specific CTL response has a significant impact upon the infection with HIV and subsequent progression to AIDS.
Investigated Immune Mechanisms Involving Antibodies
In Vitro Mechanisms and Evidence
The generation of HIV-1 cell envelope protein-specific CD4+ T cells, leading to Th2 cells and the production of antibody, has been obtained from a HIV negative patient’s serum through stimulation in vitro with HIV cell envelope proteins11. Serum IgA that is HIV-specific is capable of inhibiting infection as well as replication in vitro3,4. Despite this evidence, many mechanisms that work in vitro have not been shown to be effective in vivo, possibly due to the isolation of mechanisms that often occurs during in vitro experiments.
In Vivo Mechanisms and Evidence
Passive protection in the form of neutralizing antibodies has been conferred to rhesus monkeys against a pathogenic chimeric simian-human immunodeficiency virus (SHIV)7. Antibodies that neutralize the virus (often by not allowing attachment) stop the spread of the virus. In HIV-1 infected individuals at the early stages of the immune response, the classical complement pathway is active and inactivates both autologous and heterologous HIV strains1. Antibodies that induce complement also stop virus replication by killing the cells that contain the virus. Both neutralizing and complement inducing antibodies may contribute to the control of HIV.
Epidemiological Evidence
In a small number of HIV-1 infected individuals, the antibodies produced are capable of neutralizing several HIV variants7. This is important (and rare) because of the high rate of antigenic variation in HIV, which causes many of the antibodies developed to become ineffective when the virus changes9. A vaccine specific for a HIV envelope protein (gp120) was given to human volunteers but not protection was conferred; some subsequently contracted HIV7. Antibodies that confer protection are difficult to produce because HIV is highly variable and many epitopes on the surface are masked7.
Vaccine Design
Traditional vaccine approaches are not proving effective against HIV. Any virus that is capable of inducing protection against a wild-type virus is likely to induce the disease itself (especially when administered to immunocompromised individuals which is likely) while non-live vaccines do not elicit a response that is capable of containing the infection of HIV variants7. A vaccine that induces a CTL response would be the most efficacious as T cell mediated immunity seems to be more efficient at conferring protection than humoral.
Genes that encode for surface proteins of HIV-1 can be inserted into a live viral vector5. Once the vector is within the subject, the vector would produce the HIV-1 proteins and stimulate a CTL response10. A good candidate for the vector would be the avian poxviruses. They produce the proteins in enough quantity to stimulate an immune response while only undergoing partial replication within the human, limiting the chances of the vector itself causing disease as well as stimulating Th1 responses due to the low level of antigen produced6,7,10. Because the proteins are processed through the MHC I pathway, they stimulate a CTL response, while proteins conjugated to an adjuvant do not7. The genes must be chosen carefully to insure that the proteins are those that will stimulate protection against many variants of HIV-1. Gag, Pol, and Env are virion structural proteins and are common to all HIV-1 strains, which makes them good candidates for this vector9.
Measurement of Response
MHC:peptide tetramers (some specific for each protein that will be expressed by the vector) can be synthesized and labeled with a fluorochrome to allow for detection of binding5. A sample of blood serum will be taken from each subject, repeated at certain intervals to determine the rapidity and longevity of the response. The MHC:peptide tetramer will then be mixed with the serum and will identify those T cells that are specific for that MHC:peptide complex. The binding will be monitored by flow cytometry which (when also stained with antibodies specific for CD4 and CD8) will allow the quantification of the percentage of the total population of T cells that are specific for that complex5.
Since a CTL response is preferred over a CD4+ T cell response, an assay to determine that cytotoxic T cells were generated would be desirable. Target cells (cells that express one of the proteins encoded within the vector) will be grown up in a culture containing 51Cr. The radioactive Cr is incorporated into the live cells10. The subject’s blood serum will be then applied to the culture. Any CD8+ T cells present in the serum will kill target cells which causes the release of the radioactive Cr into the supernatant, which can then be quantified5.
Both assays determine whether there was a T cell response generated and the relative immunogenicity of each protein encoded for in the vector. Before there is a chance of the vaccine conferring protection, it needs to be demonstrated that it generates a CTL response. Knowing which specific proteins generate the immune system will be important to determine whether to include them in the next version of the vector or to attempt to find other HIV surface proteins that would generate a response.
Measurement of Protection
Detection of protection in humans is hampered by the inability to induce HIV in the subjects through deliberate infection. To produce the most data possible, the study will be limited to subjects that are routinely exposed to HIV, as with the sex workers previously mentioned. The immune response must be challenged because the ability to stimulate an immune response does not confer protection10. Without being infected with HIV, there is no way to determine the protective effects of the vaccine.
Traditional tests used to diagnose HIV would be sufficient to measure protective immunity. If the vaccine confers protection, no HIV infection will be successful in causing disease. ELISA’s can be used to test for HIV-specific antibodies in the patient10. An indirect ELISA in which HIV antigen is fixed, the patient’s serum is added (the primary antibody), and an antibody against human IgG conjugated to alkaline phosphatase (the secondary antibody) would be used. Some commonly used antigens are gp120, gp160, gp41, and p2410. It would be important to avoid using the proteins that were included in the vaccine as the antibodies could have been caused by the vaccine or by HIV infection.
Works Cited
1. Aasa-Chapman, M. M, I., S. Holuigue, K. Aubin, M. Wong, N. A. Jones, D. Cornforth, P. Pellegrino, P. Newton, I. Williams, P. Borrow, & Á. McKnight. 2005. Detection of antibody-dependent complement-mediated inactivation of both autologous and heterologous virus in primary human immunodeficiency virus type 1 infection. J. Virol. 79:2823-2830. [online.]
2. Borrow, P., H. Lewicki, B.H. Hahn, G.M.Shaw, & M.B. Oldstone. 1994. Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of primary viremia in primary human immunodeficiency virus type 1 infection. J. Virol. 68:6103-6110. [online.]
3. Burnett, P.R., T.C. VanCott, V.R. Polonis, & D.L. Birx. 1994. Serum IgA-mediated neutralization of HIV type 1. J. Immunol. 152:4642-4648. [online.]
4. Huang, Y. T., A.Wright, X. Gao, L. Kulick, H. Yan, & M. E. Lamm. 2005. Intraepithelial cell neutralization of HIV-1 replication by IgA. J. Immunol. 174: 4828-4835. [online.]
5. Janeway Jr., C. A, P. Travers, M. Walport, & M. J. Schomchik. 2005. Immunobiology: the immune system in health and disease, p. 491-507, 651, 711-721. Garland Science Publishing, New York, N.Y.
6. Kent, S.J., A. Zhao, S. J. Best, J. D. Chandler, D. B. Boyle, & I. A. Ramshaw. 1998. Enhanced T cell immunogenicity and protective efficacy of a human immunodeficiency virus type 1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowlpox virus. J. Immunol. 72:10180-10188.
7. Letvin, N. L, D. H. Barouch, & D. C. Montefiori. 2002. Prospects for vaccine protection against HIV-1 infection and AIDS. Ann. Rev. Immunol. 20:73-99.
8. Lu, W. & J.M. Andrieu. 2001. In vitro human immunodeficiency virus eradication by autologous CD8+ T cells expanded with inactivated-virus-pulsed dendritic cells. J. Virol. 75:8949-8956. [online.]
9. Sharon, J. 1998. Basic Immunology, p. 151. Williams & Wilkins, Baltimore, MD.
10. Taylor, B. Personal communication.
11. Venturini, S., D. E. Mosier, D. R. Burton, & P. Poignard. 2002. Characterization of human immunodeficiency virus type 1 (HIV-1) Gag- and Gag peptide-specific CD4+ T-cell clones from an HIV-1 seronegative donor following in vitro immunization. J. Virol. 76:6987-6999. [online.]