Please use this identifier to cite or link to this item: http://dl.umsu.ac.ir/handle/10616/44415
Title: Methods for detection of HIV-2/SIV infections
Authors: Walther Jallow, Lilian
Year: 5-May-2000
26-May-2000
Publisher: Mikrobiologiskt och Tumörbiologiskt Centrum (MTC) / Microbiology and Tumor Biology Center (MTC)
Description: The main objective of this thesis was to develop and improve laboratory methods for detection of HIV-2 in infected humans and detection of HIV-2/SIV in experimentally infected cynomolgus monkeys. Early in the HIV epidemic no assays were commercially available for detection of HIV-2/SIV infections and laboratory testing often relied on cross-reactivity in the assays. Indirect, serological methods are commonly used to demonstrate HIV infection. The screening for anti-HIV antibodies by ELISA is usually followed by confirmation of positive screening results by immunoblot. The sensitivity of different immunoblots for confirmation of HIV-2 infection was evaluated and the presence and form of the envelope proteins in the different WB assays were investigated. One of three commercial WB kits tested had a lower sensitivity for detection of HIV-2 seroconversion, due to lack of gp125, when applying WHO criteria for interpretation of results. There was no difference in sensitivity for detection of HIV-2 seroconversion dependent on the form of the transmembranous glycoprotein. An HIV-2 PCR assay was optimized with regard to primers, other reagents and sample preparation method. The assay was used together with an HIV-1 PCR assay to evaluate the accuracy of antibody assays designed to discriminate between HIV- 1 and HIV-2. We found a high correlation between dual serological reactivity and dual HIV-1 and HIV-2 infection as demonstrated by PCR. An antigen capture ELISA for direct detection of HIV-2/SIV antigen was developed and a PCR assay to discriminate between HIV-2 and SIV was established. These methods were applied to monitoring infection in monkeys experimentally infected with HIV-2 and SIV. We were able to demonstrate protection against infection or disease progression in HIV-2 preinfected cynomolgus monkeys challenged either intravenously or intrarectally with pathogenic SIV. By applying a method for quantification of plasma viral load, we demonstrated that a viral RNA threshold value can be used to predict disease progression not only in infected monkeys but also in monkeys immunized with live HIV-2 SBL-6669 or with MVA expressing HIV-2 env, gag-pol and challenged with pathogenic SIV. A plasma virus load greater than 105 RNA equivalents/ml of plasma at 6 to 12 weeks after inoculation was predictive of a rapid disease progression while less than 104 RNA equivalents/ml of plasma at 6 to 12 weeks was indicative of slow progression to AIDS. The tests presented in this thesis are necessary tools in our research regarding HIV-2 infections in humans and HIV-2/SIV vaccine studies in monkeys. Both the antigen test and the WB assay are less expensive than the current commercially available tests. The discriminatory PCR assays are crucial for investigation of dual HIV- 1 and HIV-2 infection in humans as well as for vaccine research involving coinfection with HIV-2 and SIV in monkeys. Furthermore, the establishment of a threshold SIV RNA value predictive of disease outcome in the vaccinated monkeys allows shorter follow-up periods in the vaccine experiments. The main objective of this thesis was to develop and improve laboratory methods for detection of HIV-2 in infected humans and detection of HIV-2/SIV in experimentally infected cynomolgus monkeys. Early in the HIV epidemic no assays were commercially available for detection of HIV-2/SIV infections and laboratory testing often relied on cross-reactivity in the assays. Indirect, serological methods are commonly used to demonstrate HIV infection. The screening for anti-HIV antibodies by ELISA is usually followed by confirmation of positive screening results by immunoblot. The sensitivity of different immunoblots for confirmation of HIV-2 infection was evaluated and the presence and form of the envelope proteins in the different WB assays were investigated. One of three commercial WB kits tested had a lower sensitivity for detection of HIV-2 seroconversion, due to lack of gp125, when applying WHO criteria for interpretation of results. There was no difference in sensitivity for detection of HIV-2 seroconversion dependent on the form of the transmembranous glycoprotein. An HIV-2 PCR assay was optimized with regard to primers, other reagents and sample preparation method. The assay was used together with an HIV-1 PCR assay to evaluate the accuracy of antibody assays designed to discriminate between HIV- 1 and HIV-2. We found a high correlation between dual serological reactivity and dual HIV-1 and HIV-2 infection as demonstrated by PCR. An antigen capture ELISA for direct detection of HIV-2/SIV antigen was developed and a PCR assay to discriminate between HIV-2 and SIV was established. These methods were applied to monitoring infection in monkeys experimentally infected with HIV-2 and SIV. We were able to demonstrate protection against infection or disease progression in HIV-2 preinfected cynomolgus monkeys challenged either intravenously or intrarectally with pathogenic SIV. By applying a method for quantification of plasma viral load, we demonstrated that a viral RNA threshold value can be used to predict disease progression not only in infected monkeys but also in monkeys immunized with live HIV-2 SBL-6669 or with MVA expressing HIV-2 env, gag-pol and challenged with pathogenic SIV. A plasma virus load greater than 105 RNA equivalents/ml of plasma at 6 to 12 weeks after inoculation was predictive of a rapid disease progression while less than 104 RNA equivalents/ml of plasma at 6 to 12 weeks was indicative of slow progression to AIDS. The tests presented in this thesis are necessary tools in our research regarding HIV-2 infections in humans and HIV-2/SIV vaccine studies in monkeys. Both the antigen test and the WB assay are less expensive than the current commercially available tests. The discriminatory PCR assays are crucial for investigation of dual HIV- 1 and HIV-2 infection in humans as well as for vaccine research involving coinfection with HIV-2 and SIV in monkeys. Furthermore, the establishment of a threshold SIV RNA value predictive of disease outcome in the vaccinated monkeys allows shorter follow-up periods in the vaccine experiments.
URI: https://openarchive.ki.se/xmlui/handle/10616/44415
Standard no: 91-628-4214-5
20000526walt
Appears in Collections:Dept of Microbiology, Tumor and Cell Biology

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