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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53329 |
| Date | 03 1900 |
| Creators | De Villiers, Tania |
| Contributors | Janse van Rensburg, E., Engelbrecht, S., Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Pathology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 152 p. : ill. |
| Rights | Stellenbosch University |
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