Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function. First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens. Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells. Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies. Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.

Human immunodeficiency type 1 (HIV-1) is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years’ infection and as a result, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with antibody dependent cellular cytotoxicity (ADCC) activity. Previous studies in the Lu lab demonstrated that in an HIV-1 vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities along with multiple Fc mediated effector functions. I developed a protocol for the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from these DP6-001 vaccinees. By utilizing a labeled gp120 bait to isolate Env specific B cells, paired heavy and light chain immunoglobulin (Ig) genes were cloned and allowed for the production of monoclonal antibodies with specificity for gp120. By using this protocol, 13 isolated mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens, with mostly conformational epitopes and a few V3 and C5 specific mAbs. Equally cross-reactive Fc-mediated functional activities, including ADCC and antibody dependent cellular phagocytosis (ADCP), were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional antibodies by the DNA prime- protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV-1 vaccine design.

Thesis (MPhil)-- University of Stellenbosch, 2000 / ENGLISH ABSTRACT: It is a well-known fact that the sub-Saharan Africa is a continent most affected by HIV/AIDS. The HIV/AIDS pandemic has in other words become our disease. For many of us, this fact may be difficult to fully accept. There are elements of prejudice in our reactions. Ignorance and intolerance can be found around the world. Therefore, by presenting the facts about HIV/AIDS, this assignment challenges the misconceptions and focuses on the profound dilemmas confronting society. I think the success in combating the HIV/AIDS pandemic could be found in President Thabo Mbeki's terminology "Partnership against HIV/AIDS". In his speech, the President appealed to both the private and public sectors and all South Africans to work together with greater determination than before to fight against HIV infection and AIDS. Arguably, this was the best speech President Thabo Mbeki ever made on HIV/AIDS on October 9, 1998. Back then, the government seems to have had a direction and led from the front in the battle against HIV/AIDS. The title of this thesis reads: "Ethical perspectives on surveillance and preventive strategies for HIV/AIDS in South Africa". Presently, the South African Government through the Ministry of Health is seriously considering making AIDS a notifiable medical condition. This is a serious and a controversial move that has serious ethical and legal implications that will be discussed. Should partners of HIV-infected individuals be informed? If the answer is on the affirmative, who should inform them? I am also looking at the ethical obligation of health care workers to treat HIV/AIDS patients despite the fear of being accidentally infected. Tough questions need to be asked. Should health workers be informed of the HIV status of every patients they treat? On the other hand, some patients have some fears too that HIV-infected health professionals may infect them. Again, the fundamental ethical concerns related to confidentiality, privacy, the right to treatment will also be discussed. The country is divided on this issue. Ethical principles are directly involved in such a decision, for instance, the principle of confidentiality, respect for autonomy and informed consent. How can the government go about implementing this without disregarding these fundamental ethical requirements?Another ethical issue that comes to mind regarding HIV/AIDS concerns AIDS vaccine trials, which are so far dominantly manufactured in 'developed countries' while subjects of these trials are from 'third world' or 'developing countries '. The ethical concerns here are: How will informed consent be protected, especially where subjects of the trials are not educated and do not understand the terms used? What are the cost-effects or benefits of such trials? What are the risks involved? Together with this, other issues include ethical debates concerning market prices of drugs, which are too expensive for poorer countries and affordable for richer countries. Finally, this work does not treat everything that needs to be dealt with insofar as HIV/AIDS is concerned. However, I hope that this thesis will contribute (in a small way) in making people appreciate the ethical dilemmas that are presented by HIV/AIDS. / AFRIKAANSE OPSOMMING: Dit is algemeen bekend dat Afrika suid van die Sahara die gebied is met die hoogste voorkoms van MIV/vIGS. Die MIV/VIGS-pandemie het dus ons siekte geword. Dit is vir baie van ons moeilik om hierdie feit te aanvaar, en ons reaksies is dikwels bevooroordeeld. Onkunde en onverdraagsaamheid oor MIV/vIGS word trouens wereldwyd aangetref. Hierdie verhandeling Ie klem op die feite van MIV/VIGS, en konfronteer sodoende hierdie wanopvattings terwyl daar gefokus word op die diepgaande dilemmas waarmee die samelewing gekonfronteer word. President Thabo Mbeki se woorde "Vennootskap teen MIV/VIGS" verwoord myns insiens die enigste oplossing vir die MIV/VIGS-pandemie. Die President doen in sy toespraak 'n beroep op al1e Suid-Afrikaners, in private en openbare sektore, om met groter determinasie saam te veg teen MIV-infeksie en VIGS; Hierdie toespraak, gelewer op 9 Oktober 1998, toe die regering klaarblyklik nog rigting gehad het en op die voorfront was in die styd teen MIV/VIGS, was moontlik President Thabo Mbeki se beste ooit oor die onderwerp MIV/VIGS. Die titel van hierdie verhandeling is "Etiese perspektiewe ten opsigte van waarnemende en voorkomende strategiee vir MIV/VIGS in Suid-Afrika". Die Suid-Afrikaanse regering, by monde van die Ministerie van Gesondheid, oorweeg dit tans sterk om VIGS 'n aanmeldbare mediese kondisie te verklaar. Die ernstige etiese en regsimplikasies van so 'n daadwerklike en kontroversiele stap sal in die verhandeling bespreek word. Behoort die rnetgesel1e van MIV-positiewe persone ingelig te word? Indien wei, wie moet hulle in kennis stel? Daar sal ook gekyk word na die etiese verpligting van gesondheidsorgwerkers om MIV/VIGS-pasiente te behandel ten spyte van hul1e vrees om per ongeluk besmet te word. Indringende vrae moet gevra word. Behoort gesondheidsorgwerkers ingelig te word oor die MIV-status van elke pasient wat hul1e behandel? Aan die ander kant vrees sornmige pasiente dat hul1e deur MIV-positiewe gesondheisorgwerkers besmet kan word. Die fundamentele etiese aangeleenthede rakende vertroulikheid, privaatheid en die reg tot mediese behandeling sal ook bespreek word. Suid-Afrika is verdeeld oor hierdie kwessies. Etiese waardes, soos die beginsel van vertroulikheid, respek vir outonomie en ingeligte goedkeuring is direk betrokke by besluite oor etiese kwessies. Die regering kan nie hierdie aangeleenthede implementeer sonder om die fundamentele etiese vereistes in ag te neem nie. VIGS-entstofproefnemings is'n verdere etiese kwessie wat ter sprake kom. Hierdie proefnemings word grotendeels deur "ontwikkelde" lande uitgevoer, tewyl die proefpersone van "derdewereldse" of "ontwikkelende" lande afkomstig is. Die etiese kwessies hierby betrokke is: hoe sal ingeligte goedkeuring beskerm word, veral wanneer proefpersone onopgevoed is en nie die tersaaklike terme verstaan nie? Wat is die kosteeffektiwiteit of voordele van hierdie proefnemings? Watter risiko's is betrokke? Die etiese debat oor die markprys van medisyne, wat heel bekostigbaar vir ryk lande, maar duur vir armer lande is, word ook aangeraak. Hierdie verhandeling dek nie alle relevante kwessies wat betref MIV/VIGS nie. Tog hoop ek dat dit 'n bydrae sal lewer tot mense se bewuswording van die etiese dilemmas wat MIV/VIGS inhou.

A formulação HIVBr18, previamente desenvolvida e testada, é uma vacina de DNA que codifica 18 epítopos CD4, promíscuos e conservados do HIV-1, e que após imunização de camundongos transgênicos para diversas moléculas de HLA de classe II humanas, observou-se proliferação de linfócitos T CD4+ e CD8+ e produção de IFN-? direcionadas a múltiplos epítopos codificados pela vacina. Abordamos aqui estratégias baseadas na fusão ou combinação dos epítopos codificados pela vacina HIVBr18 à glicoproteína D (gD) do HSV-1, e também na coadministração de plasmídeos que codificam citocinas (IL-2, -12, -15 e GM-CSF) visando aumentar a imunogenicidade de HIVBr18. A sequencia de DNA que codifica os 18 peptídeos da vacina HIVBr18 foi amplificada por PCR e clonada em um plasmídeo que abrigava a sequencia da gD do HSV-1. dando origem ao plasmídeo pVAX-gDh-HIVBr18. Animais imunizados com gDh-HIVBr18 apresentaram resposta imunológica similar ao grupo que recebeu somente HIVBr18, não sendo diferente também daqueles que receberam plasmídeos gDh-HIVBr18 que sofreram alterações nas sequências para melhorar o padrão de distribuição hidrofóbica e permitir a migração da proteína de fusão para o meio extracelular. Construímos e testamos um plasmídeo bicistrônico que expressa gDh e HIVBr18 isoladamente, mas também não observamos aumento na resposta imune induzida. A coadministração com o plasmídeo HIVBr18 e plasmídeos que codificam as citocinas IL-12, IL-15 e GM-CSF, proporciona um aumento na magnitude da resposta imunológica induzida contra o pool de peptídeos codificados pela vacina, entretanto sem alteração da amplitude da resposta. Além disso, o plasmídeo de GM-CSF induziu maior número de células T CD4+ polifuncionais. Demonstramos também que a coadministração do plasmídeo que codifica GM-CSF, induz uma resposta imune celular de maior magnitude mesmo em uma condição de dose reduzida. Entretanto, observamos que esta citocina não é um bom adjuvante quando utilizamos como vetor de imunização um adenovírus que expressa os 18 epítopos / The formulation HIVBr18, previously developed and tested, is based on a DNA vaccine encoding 18 conserved and promiscuous HIV-1 CD4 epitopes and after immunization of transgenic mice for many human HLA class II molecules using this DNA vaccine, could be observed proliferation of CD4+ and CD8+ T cells and IFN-y production directed to multiple epitopes encoded by the vaccine. We intend to explore here, strategies based on fusion or combination of epitopes encoded by HIVBr18 vaccine with glycoprotein D (gD) of HSV- 1 and also the coadministration of cytokine-encoding plasmids (pIL-2, -12, -15 and pGM -CSF) aiming to enhance immunogenicity of HIVBr18. The DNA sequence of epitopes encoded by HIVBr18 vaccine was amplified by PCR and cloned into a plasmid that contained the sequence of gD, giving rise to plasmid pVAX-gDh-HIVBr18. After mice immunization, animals immunized with this construct showed similar immune response to the group that received HIVBr18, and also the group of animals that received gDh-HIVBr18 plasmid that had been modified by exchange in peptides order to assure to the molecule a better hydrophobic distribution and allow translocation to the extracellular face of cell membrane. We constructed and injected mice with a bicistronic plasmid expressing gDh and HIVBr18, simultaneously and isolated, but no increase in the magnitude of the immune response was observed. HIVBr18 coadministration with cytokine-encoding plasmids pIL-12, pIL-15 and pGM-CSF, provides an increase in the magnitude of immune response induced against the peptides encoded by the vaccine, and similar breadth. In addition, co-immunization with pGM-CSF induced greater number of polyfunctional CD4 + T cells. We also demonstrate that, even in a low dose approach coadministration of pGM-CSF induced a higher immune response than HIVBr18 alone in the same dose. However, we observed that this cytokine is not a good adjuvant when used in combination with an adenovirus that expresses the 18 HIV-1 epitopes.

A formulação HIVBr18, previamente desenvolvida e testada, é uma vacina de DNA que codifica 18 epítopos CD4, promíscuos e conservados do HIV-1, e que após imunização de camundongos transgênicos para diversas moléculas de HLA de classe II humanas, observou-se proliferação de linfócitos T CD4+ e CD8+ e produção de IFN-? direcionadas a múltiplos epítopos codificados pela vacina. Abordamos aqui estratégias baseadas na fusão ou combinação dos epítopos codificados pela vacina HIVBr18 à glicoproteína D (gD) do HSV-1, e também na coadministração de plasmídeos que codificam citocinas (IL-2, -12, -15 e GM-CSF) visando aumentar a imunogenicidade de HIVBr18. A sequencia de DNA que codifica os 18 peptídeos da vacina HIVBr18 foi amplificada por PCR e clonada em um plasmídeo que abrigava a sequencia da gD do HSV-1. dando origem ao plasmídeo pVAX-gDh-HIVBr18. Animais imunizados com gDh-HIVBr18 apresentaram resposta imunológica similar ao grupo que recebeu somente HIVBr18, não sendo diferente também daqueles que receberam plasmídeos gDh-HIVBr18 que sofreram alterações nas sequências para melhorar o padrão de distribuição hidrofóbica e permitir a migração da proteína de fusão para o meio extracelular. Construímos e testamos um plasmídeo bicistrônico que expressa gDh e HIVBr18 isoladamente, mas também não observamos aumento na resposta imune induzida. A coadministração com o plasmídeo HIVBr18 e plasmídeos que codificam as citocinas IL-12, IL-15 e GM-CSF, proporciona um aumento na magnitude da resposta imunológica induzida contra o pool de peptídeos codificados pela vacina, entretanto sem alteração da amplitude da resposta. Além disso, o plasmídeo de GM-CSF induziu maior número de células T CD4+ polifuncionais. Demonstramos também que a coadministração do plasmídeo que codifica GM-CSF, induz uma resposta imune celular de maior magnitude mesmo em uma condição de dose reduzida. Entretanto, observamos que esta citocina não é um bom adjuvante quando utilizamos como vetor de imunização um adenovírus que expressa os 18 epítopos / The formulation HIVBr18, previously developed and tested, is based on a DNA vaccine encoding 18 conserved and promiscuous HIV-1 CD4 epitopes and after immunization of transgenic mice for many human HLA class II molecules using this DNA vaccine, could be observed proliferation of CD4+ and CD8+ T cells and IFN-y production directed to multiple epitopes encoded by the vaccine. We intend to explore here, strategies based on fusion or combination of epitopes encoded by HIVBr18 vaccine with glycoprotein D (gD) of HSV- 1 and also the coadministration of cytokine-encoding plasmids (pIL-2, -12, -15 and pGM -CSF) aiming to enhance immunogenicity of HIVBr18. The DNA sequence of epitopes encoded by HIVBr18 vaccine was amplified by PCR and cloned into a plasmid that contained the sequence of gD, giving rise to plasmid pVAX-gDh-HIVBr18. After mice immunization, animals immunized with this construct showed similar immune response to the group that received HIVBr18, and also the group of animals that received gDh-HIVBr18 plasmid that had been modified by exchange in peptides order to assure to the molecule a better hydrophobic distribution and allow translocation to the extracellular face of cell membrane. We constructed and injected mice with a bicistronic plasmid expressing gDh and HIVBr18, simultaneously and isolated, but no increase in the magnitude of the immune response was observed. HIVBr18 coadministration with cytokine-encoding plasmids pIL-12, pIL-15 and pGM-CSF, provides an increase in the magnitude of immune response induced against the peptides encoded by the vaccine, and similar breadth. In addition, co-immunization with pGM-CSF induced greater number of polyfunctional CD4 + T cells. We also demonstrate that, even in a low dose approach coadministration of pGM-CSF induced a higher immune response than HIVBr18 alone in the same dose. However, we observed that this cytokine is not a good adjuvant when used in combination with an adenovirus that expresses the 18 HIV-1 epitopes.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcriptional repressor BCL6 has been shown to be essential for the differentiation of germinal center (GC) B cells and follicular T helper (TFH) cells. The interaction of TFH and GC B cells is necessary for the development of high affinity antibodies specific for an invading pathogen. Germline BCL6-deficient mouse models limit our ability to study BCL6 function in T cells due to the strong inflammatory responses seen in these mice. To overcome this, our lab has developed a new BCL6 conditional knockout (cKO) mouse using the cre/lox system, wherein the zinc finger region of the BCL6 gene is flanked by loxP sites. Mating to a CD4-Cre mouse allowed us to study the effects of BCL6 loss specifically in T cells, without the confounding effects seen in germline knockout models. Using this cKO model, we have reaffirmed the necessity of BCL6 for TFH differentiation, including its role in sustained CXCR5 surface expression, a signature marker for TFH cells. This model also allowed us to recognize the role of BCL6 in promoting the expression of PD-1, another key surface marker for TFH cells. Without BCL6, CD4+ T cells cannot express PD-1 at the high levels seen on TFH cells. Our discovery of DNMT3b as a target for BCL6 suggests BCL6-deficient T cells have increased DNA methyltransferase activity at the PD-1 promoter. This data establishes a novel pathway for explaining how BCL6, a transcriptional repressor, can activate genes. Experiments with the BCL6 cKO model have also established a role for BCL6 in naïve CD4+ T cell activation. Furthermore, we did not observe increased differentiation of other helper T cell subsets, in contrast to what has been reported elsewhere with germline BCL6-deficient models. Unexpectedly, we found decreased T helper type 2 (Th2) cells, whereas mouse models with a germline mutation of BCL6 have increased Th2 cells. These results indicate that BCL6 activity in non-T cells is critical for controlling T cell differentiation. Finally, using an HIV-1 gp120 immunization model, we have, for the first time, shown BCL6-dependent GCs to be limiting for antibody development and affinity maturation in a prime-boost vaccine scheme.