An effective HIV-1 vaccine will likely elicit broadly neutralizing antibodies (bnAbs). However, development of vaccine immunogens that induce bnAbs remains a challenging goal. Understanding the somatic maturation pathways of known broadly neutralizing HIV-1 human monoclonal antibodies (bnmAbs) may help vaccine immunogen design. All known HIV-1 bnmAbs are highly somatically matured, and the putative germline antibodies of the known HIV-1 bnmAbs lack measurable binding activity to HIV-1 envelope glycoprotein (Env). Based on these observations, we hypothesize that somatic maturation of known HIV-1 bnmAbs may be initiated by primary immunogens which may not be related to HIV-1 Env; such primary immunogens trigger the somatic maturation of the germline antibodies and induce intermediate antibodies that bind to HIV-1 Env and further mature to bnAbs upon HIV-1 infection or Env vaccination. The main objective of my study is to identify intermediate antibodies of bnmAb b12 in uninfected and infected human individuals, as well as in uninfected rhesus macaques, the model animals for vaccine development. I constructed two nonimmune cDNA antibody VH1 scFv libraries using the mRNAs isolated from pooled PBMCs of 200 uninfected healthy human individuals and one uninfected rhesus macaque, respectively, and identified 5 and 10 possible b12 intermediate immunoglobulin heavy chain V-segments (IGHVs) from the human and macaque nonimmune libraries, respectively. 454 deep sequencing of the VHs and VLs in the nonimmune and two immune human cDNA Fab libraries confirmed the existence of b12 intermediate IGHVs, but we did not find further maturation of the b12 intermediate IGHVs in HIV-1-infected human individuals. Further sequence analysis revealed the extremely low frequency of the VHs with exactly the same V(D)J recombination as b12, which may explain the lack of further maturation of the intermediate IGHVs of b12 in HIV-1-infected humans. Characterization of HIV-1 Env trimer may aid in Env-based vaccine immunogen design. Therefore, I investigated the importance of Env variable loops in Env-mediated viral function. A panel of gp160JRFL loop deletion/replacement mutants were constructed and tested. The results indicate that, besides the CD4 binding loop and V3, V1V2 and loop D are also critical for virus entry into permissive cells. Deletion of variable V4 or V5 loop or replacement of V4 or V5 loop with a flexible linker of the same length abolish Env cell surface display, which may result from the conformational changes of the V4 or V5 loop deletion or replacement Env proteins. V4 or V5 deletion or replacement knocks out the CD4 binding site and CD4-induced site on Env, but enhances the exposure of the membrane-proximal external region (MPER) and N-trimer structure. In summary, my study demonstrated the existence of intermediate b12 IGHVs in uninfected and HIV-1-infected humans and rhesus macaques. These intermediate antibody fragments may be used to identify primary immunogens that initiate b12 somatic maturation. My study also showed the importance of Env variable loops for Env structural integrity and Env-mediated viral function. The enhanced exposure of the MPER in gp160JRFL ΔV4 or ΔV5 may be further explored for vaccine development to induce MPER-specific bnAbs. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Lupus nephritis is characterized by the production of anti-dsDNA antibodies, deposition of immune complexes within the kidney parenchyma, proliferation of resident renal cells and induction of inflammatory and fibrotic processes. Approximately 70% of patients with lupus nephritis show immune aggregates along the tubular basement membrane, which is accompanied by an influx of infiltrating cells and increased intra-renal expression of IL-6. Much attention has focused on the inflammatory processes in the kidney during pathogenesis of lupus nephritis whereas mechanisms of fibrogenesis are less well characterized. Tubulo-interstitial injury is a key indicator of poor prognosis of renal function. Given that the tubulo-interstitium occupies over 80% of the kidney volume, injury to this compartment will have a major impact on renal function. There is evidence to show that proximal tubular epithelial cells (PTEC) undergo epithelial-to-mesenchymal transition (EMT) during pathological disorders and adopt a fibroblastic morphology with increased fibrogenic potential. We have previously demonstrated that anti-dsDNA antibodies bound directly to the surface of PTEC through cross-reactive proteins, which were subsequently internalized and translocated to the nucleus where they induced functional changes. Using a proteomic approach, this study identified the cross-reactive antigens that mediated anti-dsDNA antibody binding and intracellular localization in PTEC and the functional consequences thereafter, focusing on EMT and fibrogenic events. Human polyclonal anti-dsDNA antibodies isolated from patients with lupus nephritis bound to Ku70 in plasma membrane extracts isolated from PTEC, and to Ku70, Ku80 and major vault protein in cytosolic and nuclear fractions. Anti-dsDNA antibodies increased synthesis of Ku70, Ku80 and major vault protein in PTEC in a time-dependent manner. Expression of these proteins was localized to proximal tubules especially those undergoing atrophy, and staining was more prominent in renal biopsies from patients with lupus nephritis compared to non-lupus renal disease or control specimens. Binding of anti-dsDNA antibodies to PTEC increased phosphorylation of MAPK and PKC signaling pathways that was accompanied by a concomitant increase in IL-6, IL-8 and TGF-1 secretion and synthesis of β-catenin, fibroblast specific protein-1, fibronectin and laminin. Inhibition of MAPK and PKC signaling pathways with specific inhibitors revealed differential regulation of inflammatory and fibrotic processes by these signaling pathways. In this respect, increased ERK, p38 MAPK, JNK and PKC phosphorylation in PTEC following anti-dsDNA antibody stimulation enhanced IL-6, IL-8 and fibronectin synthesis, whereas increased ERK and JNK phosphorylation upregulated TGF-β1 secretion. Increased β-catenin synthesis was mediated through JNK and PKC phosphorylation. Taken together, our data suggest that PTEC contribute to the pathogenesis of renal inflammation and fibrosis in lupus nephritis. We hypothesize that anti-dsDNA antibodies bind to Ku70 on the plasma membrane of PTEC to mediate inflammation, cell activation and increased fibrogenesis. Although synthesis of EMT markers was increased in PTEC after anti-dsDNA antibody stimulation, transition to a fibroblastic morphology was not observed under our experimental setting suggesting that induction of the EMT cascade is an early event before phenotypic alterations. / published_or_final_version / Medicine / Master / Master of Philosophy

Therapeutic antibodies have achieved exceptional clinical success in the treatment of cancer and other human diseases. Now, new approaches are required to enhance the potency of antibodies to further increase the number of patients responding to therapy. By engineering the antibody Fc domain through mutation of the amino acid sequence, binding affinity to activating or inhibitory Fc receptors on effector cells can be increased to modulate the cellular immune response. However, attaining selectivity for closely related Fc receptors has proved challenging and the technique has not been applied to access the function of antibody isotypes other than IgG. Here we present new methods for enhancing antibody potency using both hybrid IgA/G and aglycosylated Fc domains. In the first instance, a chimeric antibody Fc domain has been created by combining residues from IgA with those from IgG. The new variant, MutD, introduces binding to FcαRI while retaining affinity for certain members of the FcγR family. ADCC assays show MutD, when part of a full length trastuzumab antibody against Her2 antigen, can kill Her2-overexpressing tumor cell lines as effectively as IgA antibodies. Moreover, MutD shows improved assembly compared to IgA and thus provides access to potent FcαRI function while overcoming the expression and purification barriers that have limited the use of IgA as a therapeutic. Alternatively, aglycosylated antibodies may be engineered for exceptional effector function. Glycans anchored to residue N297 of the antibody IgG Fc domain are typically critical in mediating binding toward the FcγRs. Yet, using a full length bacterial IgG display system, we have isolated aglycosylated Fc1004 with mutations that confer a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous inhibitory receptor, FcγRIIb. Incorporation of this engineered Fc into trastuzumab resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with medium Her2-expressing cancer cells. In vivo testing of Fc1004 using NOD/SCID mouse model, reconstituted by adoptive transfer of leukocytes from FcγRIIa-R131 homozygous donors, showed a promising reduction in tumor burden in SkBr-3 Her2+ xenografts. / text

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

No description available.

Aim: The purpose of this study was to describe the experiences of people living with the human immunodeficiency virus who underwent a physiotherapy rehabilitation programme, with the aim of informing and improving future physiotherapy rehabilitation interventions. Methodology: Design: A qualitative research design was adopted, using the International Classification of Function, Disability and Health as a guiding framework. Population: All adult HIV positive patients who were referred for physiotherapy rehabilitation at a public-funded South African hospital during the course of a five week clinical block. Sample: Fourteen participants were eligible for the study. Eight of these participants, who were able to attend the post-rehabilitation interview, were considered for final analysis in the study. Study setting: A public-funded hospital within the eThekwini district of KwaZulu-Natal, South Africa. Research procedure: All eligible participants were requested to complete a questionnaire, the World Health Organisation Disability Assessment Schedule, prior to commencing a physiotherapy rehabilitation programme. After the period of rehabilitation, participants were interviewed using 14 open-ended questions designed to explore their experiences of this programme. Results: The questionnaire data described the participants’ demographics and illustrated the varying cognitive and physical challenges faced by these eight individuals. Content analysis of the eight interviews revealed the following themes: the participants’ knowledge of their health conditions and their prescribed medication, the impact of their illness on their impairments, activities and participation in their daily lives, the context in which these factors exist, the participants’ experience of physiotherapy rehabilitation and the barriers they faced in accessing continued rehabilitation. Conclusion: While participants reported mostly positive experiences related to physiotherapy rehabilitation, they face a number of barriers that limit their access to continued rehabilitation. It is hoped that the results of this study will assist in informing the development of future physiotherapy interventions, which are better designed to suit the needs of PLHIV in a South African public health context. / Thesis (M.Physio.)-University of KwaZulu-Natal, Westville, 2012.

Among the complications of current treatments for hemophilia A, the development of anti-FVIII antibodies including “FVIII inhibitors” remains the major clinical problem in treating hemophiliacs. Factor VIII inhibitors work through neutralizing the coagulation cofactor activity of the infused FVIII and preventing the restoration of normal hemostasis. This thesis explains the influence of genetic background on the generation of FVIII inhibitors, introduces a new pre-clinical approach that reduces the immunological response towards FVIII and predicts the in vivo behavior of recombinant and plasma-derived FVIII products in hemophilic patients. First, we studied the influence of the genetic background on the formation of FVIII antibodies by treating hemophilia A Balb/c and C57BL/6 mice with repetitive FVIII infusions. We observed that the C57BL/6 mice developed higher FVIII antibody titers than the Balb/c mice. Our results suggest that differences in the cytokine immune responses due to FVIII in Balb/c and C57BL/6 mice are responsible for the different FVIII antibody titers in each of these strains. Second, we investigated the use of FVIII-pulsed immature dendritic cells in inducing immune tolerance against FVIII prior to the FVIII treatment. We showed that in vivo, FVIII does not induce the activation and proliferation of hemophilic T cells. Furthermore, infusing FVIII-pulsed immature dendritic cells into hemophilic mice resulted in a long-term reduction in immune reactivity towards FVIII. Also, we have proposed methods on how to improve the tolerogenic abilities of dendritic cells. Our results indicate that the immature dendritic cells induced the formation of T regulatory cells and that these T regulatory cells were responsible for the observed reduction in immune reactivity. Finally, we were able to identify the mechanisms behind the immune system activation in mice treated with either recombinant or plasma-derived FVIII products. We showed that plasma-derived FVIII results in reduced FVIII antibody titer formation in hemophilic mice. Our results demonstrate that the differences in antibody formation in hemophilic mice treated with either recombinant or plasma- derived FVIII products are due to the distinct cytokine micro-environment induced by each product. This thesis contributes to the current knowledge on FVIII immunology and the in vivo behavior of FVIII in hemophilic mice. The results generated from this thesis can be used to modify the available FVIII treatments in order to minimize the immunological complications of FVIII and improve the quality of life of hemophilic patients. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-08-14 18:22:51.56

No description available.

Antiphospholipid syndrome (APS) is an autoimmune disorder defined as the persistent presence of antiphospholipid antibodies (aPL) associated with recurrent thrombotic events and/or fetal loss. The mechanisms for fetal loss in this syndrome have not yet been clearly explained, although several hypotheses based on experimental data have been put forward. It has been shown that proliferation of human umbilical vein endothelial cells (HUVECs) decreased significantly in cultures that contained sera positive for anticardiolipin antibody activity collected from patients with recurrent fetal loss. We explored the effect of ACAs on Vascular Endothelial Growth factor- A (VEGF-A) secretion of cultured HUVECs. VEGF appears to be the most endothelial cell-specific and unequivocal angiogenic factor. In vitro, VEGF causes endothelial cell proliferation and migration; in vivo, it is potently angiogenic and causes vascular permeability. We determined the effect of ACA IgG 40μg/ml and 80μg/ml on VEGF secretion using ELISA. The results were measured in mean ± SD and expressed in pg of VEGF/5 x 10⁴ cells. P ≤ 0.05 considered significant as compared to control. The results showed that ACA increases VEGF-A secretion and/or may bind at VEGF-A binding sites. This finding may be useful for finding therapies for patients with recurrent miscarriages. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science

Two monoclonal antibodies (mabs), the newly generated mab anti-type I and the previously documented mab anti-zebrin II, were reacted with brainstem sections of two ostariophysan siluriforms, the gymnotoid Rhamphichthys rostratus and the siluroid Ictalurus punctatus. Mab anti-type I recognizes a 47 kD polypeptide present in the dendrites and soma of projection neurons. Mab anti-zebrin II recognizes a 36 kD polypeptide present throughout the neuronal cytoplasm, including the axon. Strongly type I immunopositive cells include all cerebellar Purkinje cells, pyramidal cells of the nucleus medialis, electrosensory lateral line lobe, and tectum, pacemaker relay cells, Mauthner neurons, lateral line ganglion cells, and cells of the reticular formation, lateral reticular nucleus, and inferior olive. Weakly reactive type I cells include neurons in the torus semicircularis, medial and efferent octavolateralis nuclei, magnocellular and lateral tegmentum, and motor neurons of the Vth, V I Ith, and Xth cranial nerves. All type I positive cells are projection neurons. Zebrin II expression is restricted to subsets of two cell types which also express the type I antigen -- Purkinje cells and developing acousticolateralis pyramidal cells. Both of these neurons develop from the region of the rhombic lip. Thus, the mutual expression of the type I antigen can be explained by the shared function of projection neurons, while the common expression of the zebrin II antigen may be due to a shared embryological lineage. / Department of Physiology and Health Science