Towards the definition of HIV-related antibody signatures and new immunogens for vaccine design

Ziel meiner Dissertation war es, HIV-1 Subtyp C-spezifische Antikörper-Reaktivitätsprofile in HIV-infizierten Patienten zu analysieren. Die Charakterisierung der polyklonalen Antikörperantworten erfasste die Identifizierung der Virus-Komponenten wogegen sich Antikörper richteten, die Bestimmung der Antikörper Spiegel, Isotypen und Subklassen, und deren möglichen Veränderungen während des Verlaufes der Erkrankung und der Therapie. Strukturelle, funktionelle und akzessorische HIV-1 Subtyp C Proteine wurden rekombinant exprimiert, gereinigt und bezüglich ihrer biophysikalischen Eigenschaften charakterisiert. Gp120 und gp41-Peptide wurden durch Festphasen-Peptidsynthese produziert. Die hergestellten HIV Komponenten wurden für ELISA Experimente verwendet: IgG, IgG-Subklassen, IgA und IgM Serumspiegel wurden in HIV-infizierten Patienten bestimmt, die aus HIV-1 Subtyp C und B endemischen Regionen stammten. Anschließend, wurden auf Basis der ISAC-Technologie HIV-Microarrays entwickelt, die miniaturisierte Multiplex Messungen von HIV-spezifischen Antikörperprofilen ermöglichten. Wir zeigten, dass polyklonale Antikörperantworten von HIV-infizierten Patienten gp120 und gp41 Proteine und deren Peptide erkennen, sowie strukturelle und funktionelle aber nur selten akzessorische Proteine binden. Die wichtigsten Peptid-Epitope waren zwei Regionen im gp120 Protein, wobei beide Epitope nicht mit der CD4-Bindungsstelle überlappen. Antikörperantworten bestanden aus gemischten IgG Subklassen und Isotypprofilen, und IgG1 stellte die überwiegende Subklasse dar. In der Mehrzahl der Patienten waren die Antikörper-Reaktivitätsprofile während des Verlaufes der Erkrankung und der Therapie konstant, selbst wenn die Patienten an starken Verlusten der CD4-Zellen litten. Die entwickelten HIV-Microarrays wurden erfolgreich für die Bestimmung von IgG Subklassen und Antikörper-Isotypen validiert und stellten eine Weiterentwicklung der aktuellen verfügbaren Antikörperteste dar. Die aus den Antikörperreaktionsprofilen gewonnenen Erkenntnisse können nun zum Entwurf neuer Immunogene beitragen, die schützende Immunantworten gegen HIV hervorrufen sollen.Aim of my thesis was the dissection of HIV-1 clade C specific antibody reactivity profiles in HIV-infected individuals. The characterization of patients' polyclonal antibody responses included identification of antibodies' molecular targets, determination of antibody levels, isotypes and subclasses, and their monitoring during the course of infection and antiretroviral-treatment. Recombinant HIV-1 clade C structural, functional and accessory proteins were expressed, purified and characterized regarding their biophysical properties. Gp120 and gp41 synthetic peptides were produced by solid-phase-synthesis. The produced HIV components were used in ELISA experiments for determinations of IgG, IgG subclass, IgA and IgM reactivities in serum samples from HIV-infected patients from HIV-1 subtype C and subtype B endemic regions. Subsequently, ISAC technology was used to develop an HIV-microarray for miniaturized-multiplexed measurements of HIV-specific antibody profiles. We demonstrated that polyclonal antibody responses of HIV-infected individuals were directed towards gp120 and gp41 proteins and peptides, structural and pol-derived proteins, and rarely to accessory proteins. Two major peptide epitopes were identified in gp120, both residing outside the CD4-binding site. A mixed IgG subclass and isotype distribution was observed with a predominance of IgG1. In the majority of patients antibody profiles were constant during infection and treatment, even when patients suffered from severe drops in CD4 cell counts. The developed HIV-microarrays were successfully validated for determination of IgG subclasses and antibody isotypes and may be used for routine testing. The retrieved information may be further used for the design of new immunogens able to elicit protective immune responses against HIV.submitted by Daniela GalleranoZsfassung in dt. SpracheWien, Med. Univ., Diss., 2015OeBB(VLID)488582

HIV-specific antibody responses in HIV-infected patients: From a monoclonal to a polyclonal view

HIV infections represent a major global health threat, affecting more than 35 million individuals worldwide. High infection rates and problems associated with lifelong antiretroviral treatment emphasize the need for the development of prophylactic and therapeutic immune intervention strategies. It is conceivable that insights for the design of new immunogens capable of eliciting protective immune responses may come from the analysis of HIV-specific antibody responses in infected patients. Using sophisticated technologies, several monoclonal neutralizing antibodies were isolated from HIV-infected individuals. However, the majority of polyclonal antibody responses found in infected patients are nonneutralizing. Comprehensive analyses of the molecular targets of HIV-specific antibody responses identified that during natural infection antibodies are mainly misdirected towards gp120 epitopes outside of the CD4-binding site and against regions and proteins that are not exposed on the surface of the virus. We therefore argue that vaccines aiming to induce protective responses should include engineered immunogens, which are capable of focusing the immune response towards protective epitopes

Persistence of IgE-associated allergy and allergen-specific IgE despite CD4+ T cell loss in AIDS

The infection of CD4+ cells by HIV leads to the progressive destruction of CD4+ T lymphocytes and, after a severe reduction of CD4+ cells, to AIDS. The aim of the study was to investigate whether HIV-infected patients with CD4 cell counts <200 cells/µl can suffer from symptoms of IgE-mediated allergy, produce allergen-specific IgE antibody responses and show boosts of allergen-specific IgE production. HIV-infected patients with CD4 counts ≤ 200 cells/µl suffering from AIDS and from IgE-mediated allergy were studied. Allergy was diagnosed according to case history, physical examination, skin prick testing (SPT), and serological analyses including allergen microarrays. HIV infection was confirmed serologically and the disease was staged clinically. The predominant allergic symptoms in the studied patients were acute allergic rhinitis (73%) followed by asthma (27%) due to IgE-mediated mast cell activation whereas no late phase allergic symptoms such as atopic dermatitis, a mainly T cell-mediated skin manifestation, were found in patients suffering from AIDS. According to IgE serology allergies to house dust mites and grass pollen were most common besides IgE sensitizations to various food allergens. Interestingly, pollen allergen-specific IgE antibody levels in the patients with AIDS and in additional ten IgE-sensitized patients with HIV infections and low CD4 counts appeared to be boosted by seasonal allergen exposure and were not associated with CD4 counts. Our results indicate that secondary allergen-specific IgE production and IgE-mediated allergic inflammation do not require a fully functional CD4+ T lymphocyte repertoire

Rhinovirus-induced VP1-specific Antibodies are Group-specific and Associated With Severity of Respiratory Symptoms

Background: Rhinoviruses (RVs) are a major cause of common colds and induce exacerbations of asthma and chronic inflammatory lung diseases. Methods: We expressed and purified recombinant RV coat proteins VP1-4, non-structural proteins as well as N-terminal fragments of VP1 from four RV strains (RV14, 16, 89, C) covering the three known RV groups (RV-A, RV-B and RV-C) and measured specific IgG-subclass-, IgA- and IgM-responses by ELISA in subjects with different severities of asthma or without asthma before and after experimental infection with RV16. Findings: Before infection subjects showed IgG1 > IgA > IgM > IgG3 cross-reactivity with N-terminal fragments from the representative VP1 proteins of the three RV groups. Antibody levels were higher in the asthmatic group as compared to the non-asthmatic subjects. Six weeks after infection with RV16, IgG1 antibodies showed a group-specific increase towards the N-terminal VP1 fragment, but not towards other capsid and non-structural proteins, which was highest in subjects with severe upper and lower respiratory symptoms. Interpretation: Our results demonstrate that increases of antibodies towards the VP1 N-terminus are group-specific and associated with severity of respiratory symptoms and suggest that it may be possible to develop serological tests for identifying causative RV groups

Allergen-specific IgE antibody levels (kUA/L) and CD4 counts in HIV-positive patients with AIDS (#8–11) and in HIV-positive patients with CD4 counts below 200/µl (#12–13).

<p>CD4 counts (right y-axes) and allergen-specific IgE levels (left y-axes) measured in serum samples obtained at different points of time (x-axes) are shown. The usual periods of the grass and tree pollen season is indicated in green and brown, respectively.</p

Demographic, clinical and immunological characterization of ten HIV infected patients with low CD4 counts.

<p>Displayed are age, sex and ranges of CD4 counts for follow-up sera. Allergic symptoms and positive results obtained by skin prick testing and determination of allergen-specific IgE are summarized.</p><p>Abbreviations: F: female; M: male; R: rhinitis; RC: rhinoconjunctivitis; U: urticaria; AB: asthma bronchiale; AP: allergic pharyngitis; SPT: skin prick test; Der p: <i>Dermatophagoides pteronyssinus</i>; n.k.: not known;</p