Thesis (MScMedSc) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The gag gene of HIV-1 encodes for one of the major structural proteins, which
contains several conserved cytotoxic T cell (CTL) epitopes. Gag specific CTL
responses are important in controlling viral load during acute infection and
asymptomatic stages of the infection. Currently, only one complete South African
HIV-1 subtype C gag sequence has been published. The first aim of this study
was to characterise the complete gag gene of 15 HIV-1 subtype C isolates, to be
used as a set of reference sequences in the design of a South African HIV-1
subtype C vaccine.
Fifteen HIV-1 subtype C isolates selected for this study, were isolated during 1998
and 1999 from the HIV-1 positive patients attending the Infectious Disease Clinic at
Tygerberg Hospital. The gag gene of these isolates was amplified by PCR, cloned
into mammalian expression vectors and sequenced. Restriction digest analyses as
well as phylogenetic analyses were performed on the sequencing data. Previously
published mutational analyses and CTL epitopes were compared to the predicted
amino acid sequences of the gag clones.
Sequences of 23 complete gag genes representing the 15 HIV-1 subtype C
isolates as well as one complete sequence of an HIV-1 subtype B isolate were
compiled. Subtyping by restriction fragment length polymorphism (RFLP) would
have correctly identified 14 of the 15 subtype C isolates as subtype C and one as
unidentifiable. The subtype B isolate would have also been correctly identified.
Phylogenetic analyses showed that our subtype C isolates clustered with reference
subtype C strains from various countries, including Botswana, India, Israel,
Tanzania and Zambia. Strains from Ethiopia and Brazil formed a separate subtype
C cluster. The diversity between our isolates was comparable to the diversity seen
between all the HIV-1 subtype C strains. Comparisons of previously published
mutational analyses and CTL epitopes to the predicted amino acid sequences of the gag clones, showed conservation in most of the clones throughout the
sequence.
A second aim was to establish transfection and Western Blot techniques in our
laboratory for use in future studies. An in vitro transcription! translation assay was
performed on the gag clones and the protein producing clones were used to
transfect mammalian cells using electroporation. A Western blot was then used to
screen for Gag protein expression in the transfected cell Iysates.
The in vitro transcription! translation assay showed that seven of the 23 clones
could produce a protein of -55 kDa in size. Four out of the seven of these clones
gave a weak expression of a-55 kDa protein after transfection in a mammalian
cell line. Since the completion of the experimental work of this study, other cloned
HIV-1 genes have successfully been transfected into mammalian cells using the
electroporation technique and the proteins produced were screened for by Western
blot.
To conclude with; the native form of the gag gene does not elicit strong expression
of the protein, but studies have shown that expression can be improved by
sequence-modification of the gag nucleotide sequence. Due to the conservation of
gag, the sequence of any subtype C strain can be used for the development of a
Southern African vaccine. / AFRIKAANSE OPSOMMING: Die HIV-1 gag geen kodeer vir een van die hoof strukturele proterene en bevat
verskeie sitotoksiese T-limfosiet epitope. Gag spesifieke sellulere immuun respons
is belangrik vir die beheer van virale lading tydens akute infeksies en tydens
asimptomatiese fases van die infeksie. Tans is slegs een volledige Suid
Afrikaanse HIV-1 subtipe C nuklerensuur volgorde gepubliseer. Die eerste doel
van hierdie studie was om die volledige gag geen van 15 HIV-1 subtipe C isolate te
karakteriseer, om gebruik te word as In stel verwysings nukleiensuur volgordes, vir
die ontwerp van In Suid Afrikaanse HIV-1 subtipe C entstof.
Die 15 HIV-1 subtipe C isolate wat vir hierdie studie geselekteer is, is tydens 1998
en 1999 ge·lsoleer vanaf HIV-1 positiewe pasiente wat die Infeksiesiekte Kliniek,
Tygerberg Hospitaal bygewoon het. Die gag geen van hierdie isolate is
geamplifiseer deur PKR, gekloneer in soogdier ekspressie vektore en die
nukleiensuur volgorde is bepaal. Die nuklerensuur volgorde is gebruik in restriksie
ensiem analises asook filogenetiese analises. Reeds gepubliseerde mutasie
analises en limfosiet epitope is met die voorspelde aminosuur volgorde van die gag
klone vergelyk.
Die nukleiensuur volgordes van die 23 volledige gag gene wat die 15 HIV-1
subtipe C isolate verteenwoordig, asook een volledige nukleiensuur volgorde van
een HIV-1 subtipe B isolaat, is saamgestel. Subtipering deur middel van restriksie
fragment lengte polimorfisme (RFLP) sou 14 uit die 15 subtipe C isolate korrek
qerdentifiseer het, maar sou een nie kon identifiseer nie. RFLP sou ook die
subtipe B isolaat korrek qerdentifiseer het. Filogenetiese analises het gewys dat
ons subtipe C isolate met die verwysings subtipe C stamme van verskeie lande,
insluitend Botswana, lndie, Israel, Tanzania en Zambie groepeer. Stamme van
Ethiopie en Brasilie het In aparte subtipe C groep gevorm. Die diversiteit tussen
ons isolate was vergelykbaar met die diversiteit tussen al die subtipe C stamme.
Vergelykings van gepubliseerde mutasie analises en limfosiet epitope met die voorspelde aminosuur volgorde van die gag klone, het konservasie in meeste van
die klone, deur die hele nukleiensuur volgorde, getoon.
Die tweede doel was om die metodes van transfeksie en Westerse klad in ons
laboratorium tot stand te bring. In vitro transkripsie/ translasie toetse is gedoen op
die gag klone en die proteten produserende klone is gebruik om soogdierselle te
transfekteer deur gebruik te maak van elektroporasie. In Westerse klad is toe
gebruik om vir Gag proterenuitdrukkinq in die sellisate te toets.
Die in vitro transkripsie/ translasie toets het getoon dat sewe uit 23 klone, In
proteren van -55 kDa kon produseer. Vier uit die sewe van hierdie klone het In
-55 kDa proteren swak uitgedruk na transfektering van soogdier selle. Sedert die
voltooiing van die eksperimentele werk van hierdie stud ie, is ander gekloneerde
HIV-1 gene suksesvol in soogdierselle getransfekteer met die gebruik van
elektroporasie en die proterene is met In Westerse klad aangetoon.
Ten slotte: die natuurlike vorm van die gag geen ontlok nie In sterk ekspressie van
die proteren nie, maar ander studies het wei aangetoon dat die ekspressie verbeter
kan word met modifikasie van die gag nukleiensuur volgorde. As gevolg van die
konservasie van gag, kan die nuklerensuur volgorde van enige subtipe C stam
gebruik word vir die ontwikkeling van In Suider Afrikaanse entstof. / The Poliomyelitis Research Foundation / The South African AIDS Vaccine Initiative / Harry Crossley Foundation
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53091 |
| Date | January 2002 |
| Creators | Sampson, Candice Corene |
| Contributors | Van Rensburg, E.J., Engelbrecht, S., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | viii, 254 leaves : ill. |
| Rights | Stellenbosch University |
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