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Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cells
Thesis (MSc)--Stellenbosch University, 2000.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2012
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| _version_ | 1867614141159571456 |
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| access_status_str | Open Access |
| author | De Villiers, Tania |
| author2 | Janse van Rensburg, Estrelita |
| author_browse | De Villiers, Tania Janse van Rensburg, Estrelita |
| author_facet | Janse van Rensburg, Estrelita De Villiers, Tania |
| author_sort | De Villiers, Tania |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2000. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/53329 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:47:19.123Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2012 |
| publishDateRange | 2012 |
| publishDateSort | 2012 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/53329 Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cells De Villiers, Tania Janse van Rensburg, Estrelita Engelbrecht, S. Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Pathology. HIV (Viruses) HIV (Viruses) -- Research HIV infections -- Epidemiology AIDS vaccines Dissertations -- Medicine Dissertations -- Medical virology Theses -- Medical virology Thesis (MSc)--Stellenbosch University, 2000. ENGLISH ABSTRACT: At the end of 2002, human immunodeficiency virus (HIV) had infected 42 million people worldwide. The morbidity and mortality rate, as well as the epidemic proportions of the disease have led to concentrated scientific efforts to reveal the disease's pathogenesis and develop effective preventative and treatment measures. Advances have been made to inhibit viral replication by suppressing the virus' ability to replicate by developing antiretroviral treatments, although development of a save and effective vaccine is the only way to stem the pandemic. Advances in vaccine design, animal models and clinical research have led to the creation of promising candidate vaccines to counter this rampage, but most of these vaccines entering phase I-III clinical trials are based mainly only subtype B genomes. HIV-1 subtype C is the most commonly transmitted subtype worldwide, and is the predominant subtype in India, China, East and Southern Africa. A subtype C vaccine is critical for the developing nations such as South Africa, where antiretroviral therapies are largely unaffordable. The envelope gene (env) is an attractive target as immunogen to be included in a HIV vaccine. The envelope protein (Env) elicits neutralising antibodies and cytotoxic T-Iymphocyte (CTl) responses. This protein will therefore be useful in creating a humoral and cellular immune response in the host. A shortage in characterised subtype C env gene sequences from South Africa was recognised, and this study focussed on the characterisation of generated sequences, as well as the expression of selected env genes. These immunogens were created for possible use in a prime-boost vaccine modality. The env genes from recent circulating strains in South Africa were amplified by polymerase chain reaction (PCR). The genes were then cloned for sequencing and expression purposes. Phylogenetic relationships were determined by comparing the sequences to reference subtype strains and subtype C strains. Expression of the genes was assessed by Western Blot in 293 cells with HIV- 1 positive patient sera. Sequence analysis showed a more conserved third variable (V3) loop in South African subtype C sequences, with a more variable region downstream from the loop. The crown sequence (GPGQ) and positions of uncharged or negatively charged residues in the V3 loop indicated a non-syncytium-inducing (NSI) phenotype for the isolates. Phylogenetic analysis showed the sequences to all belong to the C subtype, and further that the sequences were not recombinant, which was confirmed by recombination analysis. The intersample diversity observed for strains from South Africa was significantly higher than distances observed to the subtype C consensus sequence. The South African sequences were distributed across several subclusters in a subtype C phylogenetic tree, highlighting the concept that these infections represent a more longstanding epidemic with multiple introductions from different geographic areas. Western Blot with HIV-1 positive patient sera showed the expression of uncleaved gp160 Env proteins, which were Rev dependent. This study has generated much needed subtype C South African env gene sequences that can be used as basis for modification for use as immunogens in a South African vaccine. AFRIKAANSE OPSOMMING: Teen die einde van 2002 was 42 miljoen mense wêreldwyd geïnfekteer met die menslike immuniteitsgebrekvirus (MIV). Die dode- en sterfte syfers, asook die skaal van die epidemie, het gelei tot 'n wetenskaplike poging om die siekte se patogenese te openbaar en om effektiewe voorkomende en terapeutiese middels te ontwikkel. Vordering is reeds gemaak om die virus se replikasie te hinder deur die ontwerp van antivirale middels, alhoewel die ontwikkeling van 'n doeltreffende en veilige entstof die enigste manier is om die pandemie te stuit. As gevolg van die vordering in entstof ontwerp, diere modelle en kliniese navorsing is belowende kandidaat entstowwe wat die infeksie kan teenwerk ontwikkel, maar die meeste van hierdie enstowwe wat vir fase I-III kliniese proewe gebruik word is gebaseer op subtipe B genome. MIV-subtipe C is wêreldwide die algemeenste subtipe wat oorgedra word en is die oorheersende subtipe in lande soos Indië, China, oostelike en suidelike Afrika. 'n Subtipe C entstof word dringend benodig in ontwikkelende lande soos Suid-Afrika waar antivirale middels onbekostigbaar is. Die membraangeen is 'n aanloklike teiken om as immunogeen in 'n MIV entstof te dien. Die membraanproteïen lok neutraliserende teenliggame en sitotoksiese T-limfosiet reaksies uit. Die proteïen sal dus 'n humorale en sellulêre immuunrespons in die gasheer ontlok. 'n Tekort aan gekarakteriseerde subtipe C membraangeen volgordes van Suid-Afrika is opgemerk, en dus fokus hierdie studie op die karakterisering van gegenereerde volgordes, asook die uitdrukking van geselekteerde membraangene. Die immunogene is geskep om moontlik gebruik te word in 'n stimuleer-versterkingsenstof toedieningstrategie. Die membraangene van onlangs sirkulerende virusstamme in Suid-Afrika was geamplifiseer deur polimerase kettingreaksie (PKR). Die gene is daarna gekloneer vir beide volgordebepalings en uitdrukkingdoeleindes. Filogenetiese verhoudings is uitgewerk deur die volgordes met verwysingsstamme en subtipe C stamme te vergelyk. Uitdrukking van die gene is waargeneem in 293 selle deur die Westerse kladtegniek te gebruik met MIV-1 positiewe pasiëntsera as teenliggaam. Volgorde-analise het aangetoon dat die derde varieerbare (V3) lus meer gekonserveer is, en dat die gedeelte wat op die lus volg meer varieerbaar is. Die kroonvolgorde (GPGQ) asook posisies van ongelaaide of negatief gelaaide aminosure in die V3 lus het aangedui dat die isolate 'n nie-syncytia induserende fenotipe het. Filogenetiese analise het aangedui dat al die volgordes subtipe C is en dat die volgordes nie rekombinant is nie. Dit is ook deur rekombinasie analise bewys. Die inter-monster diversiteit van die Suid-Afrikaanse volgordes was hoër as die waargenome afstand vanaf die subtipe C konsensus volgorde. Die Suid-Afrikaanse volgordes is versprei oor verskeie subgroepe in 'n subtipe C boom, wat die konsep dat hierdie infeksies 'n meer gevestigde epidemie voorstel waar veelvuldige infeksies met verskillende geografiese oorspronge plaasgevind het beklemtoon. Die Westerse klad het ongeprosesseerde gp160 membraanproteïne aangetoon wat Rev afhanklik was. Hierdie studie het hoogs benodigde subtipe C Suid-Afrikaanse volgordes van membraangene geproduseer. Die volgordes kan as basis dien om die gene te modifiseer sodat dit gebruik kan word as immunogene in 'n entstof vir Suid-Afrika. 2012-08-27T11:35:25Z 2012-08-27T11:35:25Z 2003-03 Thesis http://hdl.handle.net/10019.1/53329 en_ZA Stellenbosch University 152 p. : ill. application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | HIV (Viruses) HIV (Viruses) -- Research HIV infections -- Epidemiology AIDS vaccines Dissertations -- Medicine Dissertations -- Medical virology Theses -- Medical virology De Villiers, Tania Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cells |
| title | Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cells |
| title_full | Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cells |
| title_fullStr | Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cells |
| title_full_unstemmed | Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cells |
| title_short | Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cells |
| title_sort | characterisation of the hiv 1 subtype c env gene and the expression of the env protein from selected isolates in mammalian cells |
| topic | HIV (Viruses) HIV (Viruses) -- Research HIV infections -- Epidemiology AIDS vaccines Dissertations -- Medicine Dissertations -- Medical virology Theses -- Medical virology |
| url | http://hdl.handle.net/10019.1/53329 |
| work_keys_str_mv | AT devillierstania characterisationofthehiv1subtypecenvgeneandtheexpressionoftheenvproteinfromselectedisolatesinmammaliancells |