Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: HIV has attained a global distribution and the number of infected people reached an
estimated 28.1 million in sub-Saharan Africa at the end of 2001. HIV-1 subtype C is
overwhelmingly prevalent in Botswana and South Africa and to date no interventions
have been successful enough to curb the rapid spread of the virus. A number of
HIV-1 vaccine strategies are being developed, however the breadth and efficacy of
such candidate vaccines, many of which are based on the HIV-1 structural genes pol,
gag and env, have mostly been found to be inadequate.
The HIV-1 accessory genes are attractive components of HIV vaccines due to their
role in viral pathogenesis, early expression and the high ratio of conserved CTl
epitopes. Yet, because of undesirable properties questions regarding their safety as
vaccine components are raised. In this study candidate tat, rev and nefmutants were
assessed for efficient expression and inactivation of undesirable functionality.
/
Plasmid constructs that encode the South African HIV-1 subtype C consensus Tat,
Rev and Nef proteins were constructed. The coding sequences of the genes were
codon-optimised for optimum protein expression and these synthetic genes were
constructed using overlapping 50-mer oligonucleotides. Furthermore, the proteins
were mutated at previously described sites by PCR-based site-directed mutagenesis
to render them inactive for their respective functions. Corresponding wild-type Tat,
Rev and Nef constructs were also made from viral isolates that were least dissimilar
to the respective consensus amino acid sequences. tn vitro expression of the
different constructs were assessed in 293 cells by Western blotting with polyclonal
mouse sera, which were generated by DNA immunisation with one of the Tat, Rev
and Nef constructs. The transactivation activity of Tat variants and Rev-mediated
nuclear export activity of RRE-containing transcripts were studied in cotransfection
experiments using reporter-gene-based assays while Nef functionality was assessed
in a cotransfection assay with subsequent flow cytometric analysis of surface CD4
and MHC-I expression on 293 cells.
Sequence analysis of the South African HIV-1 subtype C consensus sequences of
Tat, Rev and Nef revealed a high degree of similarity with a consensus sequence
that was drawn up from a large number of viruses from southern Africa. These
consensus sequences were also closer to individual viral isolate sequences than any
individual sequences were, indicating that the use of a consensus sequence may
serve to reduce genetic diversity between a vaccine and circulating viruses. Expression levels of the sequence-modified tat and nef gene constructs were not
significantly higher than the wild-type constructs, however, the codon-optimised rev
mutant exhibited markedly higher expression than the wild-type rev construct.
Immunoreactivity of the protein with the mouse sera demonstrates expression and
immunogenicity of the Tat, Rev and Nef immunogens in mice. In the background of
the subtype C Tat, a single C22 mutation was insufficient to inactivate l TRdependent
CAT expression in 293T and Hela cells. Yet, this activity was significantly
impaired using the single mutation, C3?, or the double mutation, C22C3? Compared
to the wild-type Rev, the function of the Rev with a double mutation, M5M10, was
completely abrogated. Similarly, while the wild-type Nef and native, codon-optimised
consensus Nef proteins mediated CD4 and MHC-I downregulation, CD4
downregulation was completely abrogated in one of the mutants, while both Nef
mutants were entirely deficient for MHC-I downregulation.
These data demonstrate the high expression levels and impaired functionality of
sequence-modified HIV-1 subtype C consensus Tat, Rev and Nef DNA immunogens
that may be used as single-standing vaccine components or form part of a multicomponent
HIV-1 vaccine. / AFRIKAANSE OPSOMMING: Sedert die eerste gevalle van MIV in die vroeë 1980's beskryf is het die virus
wêreldwyd versprei en 'n beraamde 28.1 miljoen mense in sub-Sahara Afrika was
teen die einde van 2001 geïnfekteer. MIV-1 subtipe C kom verreweg die meeste voor
in Botswana en Suid-Afrika en tans is daar geen suksesvolle tussenkoms wat die
vinnige verspreiding van die virus kan stuit nie. 'n Aantal MIV-1 subtipe C
entstofstrategieë word tans ontwikkel maar die spektrum en effektiwiteit van sulke
entstowwe, waarvan baie op die MIV strukturele gene gag, pol en env gebaseer is, is
tans onvoldoende.
Die MIV-1 bykomstige gene is aantreklike entstofkomponente omdat hulle vroeg
uitgedruk word, 'n belangrike rol in virale patogenese speel en omdat hulle 'n hoë
verhouding van gekonserveerde sitotoksiese T-limfosiet (STL) epitope tot grootte
besit. Vanweë hierdie gene se verskeie ongewenste eienskappe word vrae ten
opsigte van hul veilige insluiting in enstofstrategieë geopper. Hierdie studie omskryf
die evaluasie van kandidaat tat, reven nef mutante vir doeltreffende
proteïenuitdrukking en funksionele onaktiwiteit.
Plasmiedkonstrukte wat vir die Suid-Afrikaanse MIV-1 subtipe C konsensus Tat, Rev
en Nef proteïene kodeer is saamgestel. Die koderingsvolgordes van die gene is
geoptimiseer vir optimale uitdrukking en die sintetiese gene is van oorvleuelende 50-
mer oligonukleotiede vervaardig. Deur van PKR-gebaseerde site-directed
mutagenese gebruik te maak is hierdie proteïene gemuteer op posisies wat voorheen
geïdentifiseer is. Ooreenstemmende wilde-tipe Tat, Reven Nef konstrukte is gemaak
vanaf virale isolate waarvan die aminosuurvolgordes die meeste ooreenstem met dié
van die konsensusvolgorde. In vitro uitdrukking van die konstrukte in 293 selle is met
behulp van immunoklad met poliklonale muissera bepaal. Die serum is gegenereer
deur DNS immunisasie van muise met een elk van die Tat, Reven Nef konstrukte.
Die transaktiverings-aktiwiteit van Tat variante en Rev bemiddelde uitvoer van RREbesittende
transkripte uit die nukleus is in verklikkergeen kotransfeksie-eksperimente
bestudeer. Nef se funksionaliteit is deur kotransfeksie en die daaropvolgende
vloeisitometriese analise van 293 selle se oppervlak-CD4 en MHC-I uitdrukking
bestudeer.
Nukleotiedvolgorde-analise van die Suid-Afrikaanse MIV-1 subtipe C konsensus Tat,
Reven Nef proteiëne toon 'n hoë vlak van ooreenkoms met 'n konsensusvolgorde
wat afgelei is vanaf 'n groot aantal suider-Afrikaanse virusse. Hierdie konsensusvolgordes is ook meer soortgelyk aan individuele virale isolate as enige
individuele volgordes. Vanuit hierdie data kan afgelei word dat die gebruik van so 'n
konsensusvolgorde die genetiese diversiteit tussen 'n entstof en sirkuierende virusse
kan verminder.
Uitdrukkingsvlakke van die volgorde-geoptimiseerde tat en nef geenkonstrukte is nie
merkbaar hoër as die van die wilde-tipe konstrukte nie. In teenstelling het die
volgorde-geoptimiseerde rev mutant merkbaar hoër uitdrukkingsvlakke as die wildetipe
getoon. Immunoreaktiwiteit van die proteïene met die muissera demonstreer dat
die Tat, Reven Nef proteïene uitgedruk word en immunogenies in muise is. 'n
Enkele C22 mutasie in Tat is nie genoeg om lTR-afhanklike CAT uitdrukking in 293T
en Hela selle te inaktiveer nie. In teenstelling is hierdie aktiwiteit geïnhibeer vir Tat
proteïene met die enkel mutasie C37 en die dubbel mutasie C22C37. In vergelyking
met die funksionele aktiwiteit van die wilde-tipe Rev is dié van die Rev mutant
M5M10 heeltemal geïnhibeer. Die wilde-tipe en geoptimiseerde, konsensus Nef
proteïene het seloppervlak-CD4 en -MHC-I uitdrukking verlaag, maar hierdie effek
van afregulering van CD4 uitdrukking was heeltemaal opgehef in een Nef mutant en
van MHC-I uitdrukking in beide Nef mutante.
Hierdie data demonstreer die hoë uitdrukkingsvlakke en geïnhibeerde funksionaliteit
van volgorde-gemodifiseerde MIV-1 subtipe C konsensus Tat, Reven Nef DNS
immunogene wat as enkelstaande enstof kan optree of deel kan uitmaak van 'n
multi-komponent MIV-1 entstof.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52621 |
| Date | 12 1900 |
| Creators | Scriba, Thomas Jens |
| Contributors | Van Rensburg, E. Janse, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 162 p. : ill. |
| Rights | Stellenbosch University |
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