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Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Pharmaceutical Science." Includes bibliographical references (p. 167-189).

Thesis (Ph. D.)--University of Texas at Austin, 1997. / Vita. Includes bibliographical references (leaves 232-244). Available also in a digital version from Dissertation Abstracts.

M.Sc. / It is generally believed that the development of a completely effective vaccine for the human immunodeficiency virus (HIV) will likely require neutralizing antibodies that react with the diverse strains of cell-free forms of this virus, as well as induce cellular responses in the form of cytotoxic T-lymphocytes (CTL), to eliminate cell-associated virus. Vaccines based on viral envelope proteins attempt to induce the former response, whilst DNA/vector based approaches aim to induce CTL. The membrane proximal external region (MPER) of HIV-1 gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is an important lead for vaccine design. It is conserved among several strains of HIV-1, except for subtype C where restricted mutations are found, especially in the epitopes of 2F5 and 4E10. Mono- and polyvalent (homologous and heterologous) synthetic peptide constructs of the epitopes recognised by 2F5 and 4E10, based on HIV-1 subtype C, have been designed and their immunogenicity compared in this study. The peptide constructs, designated MPER 1 / 2, a / b, induced humoral immune responses in mice and rabbits with the use of adjuvants. The homologous constructs (designated a) induced better humoral immune responses than the heterologous versions (designated b) in small animals. However the antibodies generated in rabbits were not potent enough to neutralize isolates of HIV- 1. The induction of neutralizing antibodies may be addressed by further conformational considerations, as conjugation to an octameric lysine core was insufficient. The peptide constructs did induce proliferative and inflammatory immune responses in a murine model. Additionally, the peptide constructs were highly antigenic as neutralizing anti-HIV antibodies present in naturally infected sera were able to recognise and bind to the MPER peptides as antigen in ELISAs. This suggests that the peptide constructs may be of value for characterizing anti-MPER antibody responses in infected individuals. The constructs were further able to mimic the true representation of these regions in vivo, as human monoclonal antibodies 2F5 and 4E10 were able to recognize and bind 3 of the 4 constructs. The human anti-MPER antibodies as well as the recombinant monoclonal antibodies had a higher binding affinity for the heterologous constructs. The MPER constructs exhibited many beneficial characteristics and may therefore hold application as a component in HIV-1 preventative and therapeutic vaccination following further modification.

The development of an effective HIV vaccine is hindered by several obstacles. One of the leading challenges is the antigenic variability of HIV-1 that is exhibited throughout all viral gene products but to greatest extent in the viral envelope proteins. This phenomenon is the result of continuous mutations in the HIV genome and is responsible for the immune escape of viral mutants. Many studies have suggested that a multivalent vaccine that elicits broadly cross-reactive antibodies is required to efficiently target antigenic variability. To this end, we have designed and analyzed a synthetic peptide construct that mimicked the major variability exhibited in the V3 loops of HIV-1 subtype C isolates. The peptide construct, described as a multiple epitope immunogen of the V3 loop with 8 branches and termed MEIV3b8, was shown to be non-toxic but highly immunogenic in experimental animals (mice and rabbits) and produced antibodies that were reactive to V3 loop peptides of various subtypes, variant envelope proteins and whole viral isolates [at antibody titers 1000 in enzyme-linked immunosorbent assays (ELISAs)]. Furthermore, functional antibodies were generated in rabbits that mediated neutralization of a neutralization-sensitive HIV-1 isolate and two distinct primary HIV-1 isolates in several different neutralization assays (at antibody titres 1213). Additionally, the MEIV3b8 induced both proliferative and inflammatory immune responses in a murine model.Finally, antibodies in the plasma of individuals (n = 148) infected with HIV-1 subtype C, subtype B and HIV-2 were found to bind to the MEIV3b8 as antigen in ELISAs. Through these findings, this study demonstrated that the variable MEIV3b8 effectively addressed antigenic variability and provided evidence that this peptide construct may hold application in HIV-1 preventative and therapeutic vaccination as well as HIV immunodiagnosis. / Dr. D. Meyer

A thesis submitted to Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Doctor of Philosophy Johannesburg, March 2014 / The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine targeting the human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the surface of the virion, thereby preventing cell entry. Previous work in our laboratory focused on the generation of a 2dCD4S60C molecule (a variant of the CD4 primary Env receptor) with higher affinity for HIV-1 Env through targeted disulphide exchange. This study reports on the design and construction of an HIV-1 subtype C founder virus consensus Env immunogen derived from newly transmitted/founder virus sequences, and the ability of the purified recombinant Env proteins (2dCD4S60C-liganded and unliganded) to induce a broadly neutralizing antibody response in small animals. A total of 1894 founder sequences from 80 HIV-1 subtype C infected patients were available and downloaded from the databases. A consensus sequence was generated for each of the patients, and this alignment was subsequently used to generate a founder virus consensus env sequence. The env sequence was used to create codon-optimized constructs encoding monomeric (gp120FVCm), dimeric (gp120FVCGCN4d) and trimeric (gp140FVCGCN4t(+) and gp140FVCGCN4t(-) founder virus conformations cloned into the pcDNA3.1(-) mammalian expression vector. All four Env constructs were successfully expressed in HEK293T mammalian cell culture. The 2dCD4S60C was expressed in E. coli BL21 (DE3) and purified by nickel affinity chromatography. Large scale expression and purification of the gp120, gp120GCN4 and gp140GCN4 +/- in the unliganded or 2dCD4S60C liganded state were purified by lectin affinity chromatography, followed by conformation and complex purification using size exclusion chromatography. Immunogens/immune complexes were evaluated by ELISA, SDS-PAGE, native PAGE and surface plasmon resonance, and confirmed they were functional and conformationally intact. Immunogenicity of each conformation alone or complexed to 2dCD4S60C was evaluated in rabbits. Breadth and potency of the rabbit sera was tested against 12 pseudoviruses (Tiers 1-3), derived from HIV-1 subtype B and C Env, using the PhenoSense Neutralizing antibody assay (Monogram Bioscience, Inc.). Minimal neutralizing breadth was obtained from animals immunized exclusively with Env conformations. However, animals that received the Env/2dCD4S60C complex showed extensive neutralizing capacity against all 12 viruses tested, including the tier 2 and 3 virus strains. End-point ELISA titer results revealed that the rabbits that were immunized with Env/2dCD4S60C produced both Env and 2dCD4S60C specific titers, but those directed towards 2dCD4 were on average 10x lower than the 2dCD4S60C control group. This implies a proportion of the NAb activity is directed towards conserved epitopes exposed on the Env/2dCD4S60C immunogens. Overall, these results show that the use of founder Env/2dCD4S60C complexes as vaccine immunogens dramatically improves the antibody neutralization breadth and magnitude as compared to founder Env or 2dCD4S60C alone. This level of broad neutralization has not been previously reported in the literature, and these results provide encouraging data to inform us of the best envelope vaccine immunogen to include in a preventative vaccine.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

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.

There is currently no protective vaccine against HIV. It is known that a high mutation rate and the existence of many subspecies or clades generated by point mutations or recombination events, are at least partly responsible for the ability of the virus to escape immune responses elicited by classical vaccines. Protein subunit vaccines may not be effective due to this pronounced viral mutability. An immune evasion mechanism has been postulated in which variable domains occlude conserved epitopes crucial for infectivity. The use of DNA vaccines appeared as a favorable approach. Here, a DNA vaccine approach is presented in which the DNA constructs have been engineered to circumvent the aforementioned problems by 1) introducing elements to enhance expression, such as a heterologous promoter, a heterologous signal sequence and intron sequences, 2) by optimizing codon usage, and 3) by vaccinating with antigens that have a modified glycosylation pattern which will make them more immunogenic. The results indicated that deglycosylation of different clades of gp120 did not affect the protein conformation, and 'in vitro' expression levels were good. Antigen codon optimization dramatically increased antibody production. In the animals vaccinated with non-codon-optimized constructs, the presence of an intron and a heterologous signal sequence was required to achieve a good antibody response. Therefore, antigen engineering is required to obtain a powerful immune response against HIV-1 gp120.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.