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Recombinant antibodies have increased in prominence as therapeutics and diagnostic tools since their introduction to the market in the mid-1980s. They are used to treat diverse conditions from Crohn's disease to cancer. Since the Anthrax letter attacks of 2001, a great deal of work has been carried out to develop therapeutics to this disease, and antibodies that neutralize the toxic action of Bacillus anthracis are prominent among them. This dissertation describes the elucidation of the structure of the 14B7 family of neutralizing antibodies directed at protective antigen (PA) of B. anthracis and the complex of PA domain 4 (PAD4) with an ultra-high affinity neutralizing antibody (M18), and then utilizes this information to aid in the engineering of the antibody to various ends. Chapter 2 presents the structure of the M18-PAD4 complex and of the 14B7 family of antibodies, which aids in the understanding of the affinity maturation process for this antibody family. Chapter 3 describes the affinity maturation of M18 to a PA variant by applying the knowledge gained from the complex structure. This previously intractable challenge was met by employing saturation mutagenesis in highly focused libraries to M18 directed by the complex structure to the area of variation on PA. These results indicate that this could be a generalizable method for the engineering of M18 to natural and deliberate variation of PA. Chapter 4 reports work toward the development of a reversible, photoresponsive antibody using small molecule and polymer-protein conjugates. The results indicate that a probable site on M18 was located for placement of the polymer appendage, although further work is necessary to empirically refine the properties of the photoresponsive polymer. Chapter 5 presents an unrelated project, which was to confirm the existence of a proposed RNA thermosensor in the 5' untranslated region of LcrF from the pathogenic bacterium Yersinia pestis, the causative agent of plague. Overall, these studies reveal the power of structure-based engineering in this antibody-antigen system. In addition, the structural elucidation of the M18-PAD4 complex and the 14B7 family of antibodies furthers our basic understanding of protein-protein interactions and the process of affinity maturation of antibodies. / text

Foot-and-mouth disease (FMD) is one of the most contagious infectious diseases of domestic and wild cloven-hoofed animals, particular in cattle, sheep, pigs, goats and domestic buffalo, as well as wild ruminants such as deer. In Taiwan, there was a severe outbreak of FMD after more than 60 years freedom from the disease. The virus strain, O Taiwan 97 from the March 1997 outbreak of FMD in Taiwan, however, has been shown to have a species-specific adaptation to pigs. Although there are 7 distinct serotypes of FMD found in different regions of the world, this study focuses on the pig-adapted type O strain of FMD. After the FMD outbreak commenced in Taiwan, the spread of disease was very rapid and the whole of the western parts of Taiwan was affected within a few days after the diagnosis of FMD was confirmed. In some situations airborne transmission of FMD virus was suspected and it was speculated that this was the explanation for such rapid spread in Taiwan. Therefore, studies were conducted to investigate the transmissibility of O Taiwan/97 FMDV to susceptible pigs by direct and indirect spread including airborne spread in an enclosed animal house. This study showed that pigs in direct contact with challenged pigs became infected but none of the close-contact pigs became infected. These experiments clearly demonstrated that the pig adapted strain O Taiwan/97 was only efficiently transmitted by direct contact. This indicates that effective control against future outbreaks of pig adapted FMDV strains could be achieved by restriction of pig movement and stamping out if the outbreak has been detected in the early stages and prior to the movements of pigs from the infected premises. The measures used to control the Taiwanese FMD outbreak in 1997 were initially the slaughter of whole herds in the infected premises. However, with the rapid spread and large numbers of cases, the decision was taken to use universal compulsory vaccination of pig herds to control the outbreak when sufficient supply of vaccines was organized. Type O FMD vaccines were imported from a number of major FMD vaccine manufactures from around the world. Initially, vaccine efficacy for the imported vaccines was tested by measurement of neutralizing antibody titers in vaccinated pigs. To establish the relationship between serum neutralizing titers and protection from foot and mouth disease in pigs after vaccination, challenge studies were conducted with O/Taiwan/97 FMD in vaccinated pigs. Additionally, antibody responses to structural (neutralizing antibody) and non-structural proteins (NSP) were evaluated in vaccinated pig herds after primary and secondary vaccination in herds infected before and after vaccination. In order to be able to monitor the circulation of virus in vaccinated pig populations, valid diagnostic kits based on the detection of antibody against NSP were required. These tests needed to be evaluated against pig sera derived from challenge studies and natural FMD outbreaks. Three commercially available ELISAs (Cedi, UBI and Checkit), which were available to differentiate infected from vaccinated pigs, were tested and results showed that the Cedi test had the optimal sensitivity and specificity for pig adapted type O FMD testing. This test was used to retrospectively evaluate the sera collected from infected and non-infected pig herds collected sequentially in the year after the 1997 FMD outbreak in Taiwan. These studies also showed that the early vaccines used, stimulated NSP antibody production in swine herds that were vaccinated but not infected. This resulted in the requirement for purified FMD vaccines to be used when monitoring programs for FMD infection by NSP testing were in place. In these studies, it was also demonstrated that the purified FMD vaccines used later in the control program did not induce NSP antibody after multiple double dosage to pigs. Although clinical FMD appeared to be successfully controlled with vaccination program in Taiwan it was essential for the eradication plan to maintain active surveillance for NSP reactors in the pig population. The UBI and Cedi NSP kits were applied as screening and confirmatory tests, respectively, to pig sera collected in auction markets distributed around Taiwan to monitor for evidence of the circulation of FMD virus. Herds with positive reactors were followed-up by clinical inspection and 15 sera from suspected herds were further sampled. Negative results were obtained from all these investigation. With the absence of clinical outbreaks and the lack of evidence of FMDV circulation in the field from the NSP reactor surveillance, the Taiwanese government has progressed the eradication plan to a progressive cessation of vaccination, commencing with banning of vaccination on one isolated island in December 2006. The absence of outbreaks on that island, paved the way for further cessation of FMD vaccination in Taiwan from July 2008.

One potential approach to vaccine development against HIV involves generating an immunogen that can elicit the production of broadly neutralizing monoclonal antibodies (bnmAbs), which target specific sites on the HIV-1 envelope. Using site-directed mutagenesis and ELISA assays, this thesis investigates the idea of a secondary binding site of one of the bnmAbs, 2F5, as suggested by previous studies that identified residues Asp64, Thr65, and Arg82B on 2F5 that are recognized by its anti-idiotypic antibody 3H6. Results show that 2F5 binds only very weakly to the gp41 ectodomain in its post-fusion conformation. However, a small but significant difference was observed between the binding of the mutants and the T-20 peptide, a fusion inhibiting drug. Due to the limited effect, the results need to be confirmed using more quantitative techniques and more optimal conformations of the antigen, but raise the prospect that design of immunogens to elicit HIV-specific antibodies might have to incorporate this novel interaction site.

One potential approach to vaccine development against HIV involves generating an immunogen that can elicit the production of broadly neutralizing monoclonal antibodies (bnmAbs), which target specific sites on the HIV-1 envelope. Using site-directed mutagenesis and ELISA assays, this thesis investigates the idea of a secondary binding site of one of the bnmAbs, 2F5, as suggested by previous studies that identified residues Asp64, Thr65, and Arg82B on 2F5 that are recognized by its anti-idiotypic antibody 3H6. Results show that 2F5 binds only very weakly to the gp41 ectodomain in its post-fusion conformation. However, a small but significant difference was observed between the binding of the mutants and the T-20 peptide, a fusion inhibiting drug. Due to the limited effect, the results need to be confirmed using more quantitative techniques and more optimal conformations of the antigen, but raise the prospect that design of immunogens to elicit HIV-specific antibodies might have to incorporate this novel interaction site.

T follicular helper cells (T-FH) are a specialized subset of CD4 T cells that reside in B cell follicles and promote B cell maturation into plasma cells and long-lived memory B cells. During chronic infection prior to the development of AIDS, HIV-1 (HIV) replication is largely concentrated in T-FH. Paradoxically, T-FH numbers are increased in early and midstages of disease, thereby promoting HIV replication and disease progression. Despite increased T-FH numbers, numerous defects in humoral immunity are detected in HIV-infected individuals, including dysregulation of B cell maturation, impaired somatic hypermutation, and low quality of antibody production despite hypergammaglobulinemia. Clinically, these defects are manifested by increased vulnerability to bacterial infections and impaired vaccine responses, neither of which is fully reversed by antiretroviral therapy (ART). Deficits in T-FH function, including reduced HIV-specific IL-21 production and low levels of co-stimulatory receptor expression, have been linked to these immune impairments. Impairments in T-FH likely contribute as well to the ability of HIV to persist and evade humoral immunity, particularly the inability to develop broadly neutralizing antibodies. In addition to direct infection of T-FH, other mechanisms that have been linked to T-FH deficits in HIV infection include upregulation of PD-L1 on germinal center B cells and augmented follicular regulatory T cell responses. Challenges to development of strategies to enhance T-FH function in HIV infection include lack of an established phenotype for memory T-FH as well as limited understanding of the relationship between peripheral T-FH and lymphoid tissue T-FH. Interventions to augment T-FH function in HIV-infected individuals could enhance immune reconstitution during ART and potentially augment cure strategies.

Student Number : 9000987E - PhD thesis - School of Medicine - Faculty of Health Sciences / The generation of human monoclonal antibodies (mAbs) that are able to block HIV-1 infection in vitro would be useful reagents for studying virus neutralization, and assist in identifying neutralizing antibody (NAb) epitopes of HIV-1 envelope glycoprotein. This may provide important information for designing HIV-1 vaccine that aim to induce NAbs. HIV-1 subtype C individuals with high levels of NAb titres were identified, and peripheral blood mononuclear cells (PBMC) from these individuals were isolated and B-cells transformed with Epstein-Barr virus (EBV). Clones specific to HIV-1 gp120 using cell lysate preparations derived from HIV-1 subtype C infected cell lines were generated by performing limiting dilutions. Transformation efficiencies were estimated at over 80% by evaluating EBV-transformation cultures by microscopic visualization. Of these approximately 5% were HIV-1-specific. Five clones derived from the Du23 (1) sample secreting anti-HIV-1 antibodies were generated: 2.3C, 2.9D, 3.2C, 4.12E, and 1.5D. The 1.5D mAb could not be confirmed as anti-HIV-1 clone and it was probably lost during the process of subculturing. The remaining four Du23 mAbs were determined to be of IgG1 isotype lambda (λ) light chain. These mAbs bind to gp120, and 2.9D is probably a polyreactive clone. Clones 2.3C, 3.2C and 4.12E appear to be A32-like, but do not share the same epitope. We have determined that the binding sites for all four Du23 mAbs require at least the C1 region, and they also showed binding sites overlapping with F91 and 1.5E. All four Du23 mAbs required intact gp120 proteins for their binding, and soluble CD4 enhance their binding. Thus, their binding site is discontinuous and conformational. These mAbs are non-neutralizing as they showed limited activity of 30-59% when tested using T-cell line grown viruses or 0-30% when tested against pseudovirions. This activity is rather low when compared to over 80% shown by broadly neutralizing mAbs that have been described in the literature. The challenge in generating mAbs, in particular subtype C-derived, is to find those antibodies capable of suppressing viral replication in vivo and be capable of preventing infection. These reagents could be used to identify epitopes to guiding the design of HIV-1 subtype C envelope immunogens or vaccines. It is also envisaged that neutralizing antibodies used in therapeutic setting or in combination with antiviral drug therapy could reduce viral load and retard disease progression in infected people.

Foi investigada a existência de proteção cruzada entre bacterinas bivalentes formuladas com um de três representantes do Sorogrupo Sejroe: hardjo (bacterina A), wolffi (bacterina B) e guaricura (bacterina C), e uma estirpe do sorovar pomona, empregada por ser patogênica para hamsters (Mesocricetus auratus) e possibilitar a realização do teste de potência com desafio. Os ensaios foram efetuados em hamsters machos, comparando-se os níveis de anticorpos aglutinantes e neutralizantes, respectivamente obtidos nos testes de soroaglutinação microscópica (SAM) e inibição de leptospiras in vitro (ICL). Os animais receberam duas doses de vacinas via subcutânea; aos dez dias da segunda dose, foram inoculados com culturas não inativadas dos respectivos sorovares do Sorogrupo Sejroe. Aos 21 dias pós-infecção (d.p.i.), os animais foram sangrados, os soros (n=8) foram agrupados em pools e submetidos aos testes de SAM e ICL. O teste de potência com desafio para o sorovar pomona foi adaptado do protocolo preconizado pelo United States Department of Agriculture, mas as vacinas não foram diluídas e o esquema de imunização empregou duas aplicações de 1,0mL pela via subcutânea em intervalo de dez dias; o desafio foi efetuado aos dez dias da segunda aplicação; os óbitos por leptospirose foram registrados e, aos 21 d.p.i., os sobreviventes foram sacrificados e a condição de portadores renais foi investigada através de cultivos de tecido renal para isolamento de leptospiras. No teste de potência com o sorovar pomona, o número de doses infectantes empregado para desafio (100) situou-se dentro da faixa preconizada (10 a 100); respectivamente para as bacterinas A, B e C, as proporções de mortes por leptospirose entre os animais vacinados foram de 1/10, 0/10 e 3/10, e as de portadores renais de leptospiras entre os sobreviventes foram 2/9, 1/10 e 2/7. Os resultados do teste SAM revelaram que a bacterina A induziu reações para os sorovares hardjo e wolffi; a bacterina B, para hardjo, wolffi e guaricura, e a bacterina C, apenas para a guaricura, e do teste de ICL, que animais vacinados com as bacterinas B ou C apresentaram proteção para hardjo, wolffi e guaricura; entretanto, a bacterina A conferiu proteção apenas para wolffi. Apesar das variações no poder imunogênico segundo a estirpe de leptospira empregada para a produção das bacterinas, houve proteção cruzada entre os sorovares hardjo, wolffi e guaricura. / The existence of cross-protection among bivalent bacterins, produced with one of three leptospires belonging to Serogroup Sejroe: hardjo (bacterin A), wolffi (bacterin B) and guaricura (bacterin C), and a strain of serovar pomona (included because of its pathogenicity to hamsters and the possibility of performing potency assay with challenge), was investigated in male hamsters (Mesocricetus auratus) by comparison of agglutinating and neutralizing antibodies titers, respectively measured by microscopic agglutination (MAT) and in vitro growth inhibition (GIT) tests. All animals received two doses of bacterins by subcutaneous route; after ten days from the second dose, they were inoculated with non-inactivated cultures of respective serovars of Serogroup Sejroe. At 21 post-challenge day (p.c.d.), all animals were bled and their sera (n=8) were joined in pools and tested by MAT and GIT. The potency assay with challenge performed only with serovar pomona was modified from protocol of USDA, but vaccines were not diluted and the immunization schedule employed two 1.0 mL vaccine doses by subcutaneous route with 10?day interval; the challenge was performed after ten days from the second dose; the number of deaths due to leptospirosis was registered; at 21 p.c.d., the survivors were sacrificed and their renal carrier state was investigated by culture of renal tissue for leptospires isolation. In the potency assay with serovar pomona, the number of infectious doses employed for challenge (100 infective units) was in accordance with the recommended range (10-1,000 infective units); respectively to bacterins A, B and C, proportions of deaths due to leptospirosis among vaccinated animals were 1/10, 0/10 and 3/10, and proportions of leptospires renal carrier among survivors were 2/9, 1/10 and 2/7. Results of MAT showed that bacterin A induced reactions against serovars hardjo and wolffi; bacterin B, against hardjo, wolffi and guaricura, and bacterin C, against guaricura; results of GIT revealed that vaccinated animals with bacterins B or C presented protection against serovar hardjo, wolffi and guaricura; however, bacterin A induced protection only against wolffi. A cross?protection was observed among serovars hardjo, wolffi and guaricura, although variations exist in the immunogenic capacity according to the strain of leptospires used for the bacterins production.

The influenza virus hemagglutinin (HA) surface protein is a primary antigenic target for neutralization of viral infection. HA also mediates membrane fusion between the virus and a cell, which is the first critical step during infection. Traditional techniques to study infection neutralization by antibodies or the membrane fusion process rely on ensemble measurements, confounding the precise mechanism of infection neutralization and obscuring transient conformational intermediates. This dissertation describes advances made in a fluorescence microscopy-based single-particle fusion assay to overcome the limitations of ensemble measurements in these types of studies. Virus particles are labeled to visualize lipid mixing between a virus and a target membrane formed upon a glass or polymer support. Optionally, the viral lumen can be labeled to visualize the subsequent release of viral contents.

An effective vaccine to protect against HIV-1/AIDS remains elusive due to the extensive mechanisms employed by the HIV-1 virus to evade immune attack. Highly potent broadly neutralizing antibodies isolated from chronically infected individuals, however, show that such relevant antibodies can be naturally produced, implying that their elicitation through vaccination is a realistic possibility. These broadly neutralizing antibodies target different regions on the trimeric spikes formed by three protomers of the envelope (Env) protein. Each Env protein is comprised of the gp120 surface subunit in non-covalent association with the gp41 transmembrane subunit. Four regions have been identified: the CD4 binding site, the V1/V2 segment and the V3/glycan area all on the gp120 subunit as well as the MPER segment on the gp41 subunit. This dissertation focuses on the gp41 MPER segment given its highly conserved amino acid sequence among all HIV-1 clades and viral strain isolates and essential function in Env-mediated fusion and HIV entry. Of note, the MPER segment contains several adjacent epitopes targeted by broadly neutralizing antibodies, suggesting that the immune system is capable of producing neutralizing antibodies against this specific region. Analysis of both clade B and C MPER segments shows them to be L-shaped, consisting of two  helices separated by a hinge. We have found that the hinge region of the MPER segment provides the conformational flexibility necessary for the Env-mediated hemifusion and fusion processes. A significant reduction in virus infectivity is observed when the hinge region is disrupted by introduction of two amino acid mutations that eliminate -helical capping residues and the tandem hinge joints. The importance of the hinge region of the MPER segment is further supported by the action of four MPER-specific neutralizing antibodies 2F5, 4E10, 10E8 and Z13E1. These neutralizing antibodies block virus infection by disrupting MPER hinge-related function.