Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations

The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide/N-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome’s physical integrity and the immunogen’s conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles

Anwendung von HPLC, DLS und LDA zur Formulierungsentwicklung eines neuartigen liposomalen HIV-Impfstoffs

Liposomen haben sich in den letzten Jahren und Jahrzehnten als bewährtes Trägersystem (engl. drug delivery system, DDS) für verschiedenste Substanzen erwiesen. Sie finden nicht nur in der therapeutischen, sondern darüber hinaus beispielsweise auch in der Lebensmittel- und Kosmetikindustrie Anwendung. Nachdem für Hepatitis A und Influenza bereits liposomale Impfstoffe zugelassen wurden, soll nun in einem neuen Ansatz ein liposomenbasierter Impfstoff gegen das humane Immundefizienz-Virus (HIV) entwickelt werden. Aufgrund der gegenwärtigen Pandemie scheint dieser Ansatz die geeignetste Vorgehensweise zu sein, um weiteren Infektionen vorzubeugen. Dabei enthalten die per Impfung verabreichten Liposomen im Inneren Antigene (AG), mit denen das Immunsystem der geimpften Personen bekannt ist. Auf der äußeren Oberfläche befinden sich HIV-Hüllproteine. Im Körper stoßen diese Liposomen auf AG-präsentierende Zellen, werden von diesen aufgenommen, prozessiert und via dem Haupthistokompatibilitätskomplex II (engl. major histocompatibility complex, MHC) präsentiert. Ziel ist es, die starke Immunantwort gegen das eingeschlossene Peptid auf das HIV-Antigen zu übertragen und so einen ausreichenden Immunschutz zu erreichen. Diese Methode wird als intrastrukturelle Hilfe bezeichnet. Für die Entwicklung des Impfstoffs wurde OVA 323-339 als Modellpeptid gewählt. Da der passive Einschluss von Peptiden in der Regel mit einer geringen Effizienz einhergeht, wurden Modifikationen am OVA 323-339 vorgenommen, indem es durch ein bis drei Lysine am C und N- Terminus erweitert wurde und dadurch eine positive Ladung erhielt. Durch den Einsatz der negativ geladenen Liposomenkomponente 1,2-Distearoyl-sn-glycero-3-phosphoglycerol (DSPG) soll es zur Ausbildung von elektrostatischen Bindungen zwischen Liposomen und Peptiden kommen und dadurch eine erhöhte Einschlusseffizienz erreicht werden. Die vorliegenden Ergebnisse bestätigten diese These und stellen auch unter Beweis, dass ein kontrollierbarer Einschluss möglich ist. Die Einschlusseffizienz von modifizierten Peptiden im Vergleich zu der nicht modifizierten OVA-Variante ist um bis 42% höher.In the past, it has been proven that liposomes have great potential as carriers for different substances. They are not only used for pharmaceutical applications but also in the food and cosmetic industry for example. Currently, two liposomal vaccines namely against Hepatitis A and Influenza are approved for market. In a European consortium a new approach for the development of a liposomal vaccine against the Human Immunodeficiency Virus (HIV) is aimed. Due to the current pandemic, vaccination against HIV seems to be the best attempt to prevent new infections. Therefore, peptides recognized by the immune system of the vaccinated individual are being encapsulated into liposomes. On the outer surface of the liposomes HIV antigens will be presented. Once the liposomes are administered, the antigens are taken up, processed and presented by antigen presenting cells to other cells of the immune system. Thats why the Immune response addresses against both antigens. The goal is to delegate the strong immunological response against the encapsulated antigen to the HIV antigen and to achieve a sufficient immune protection by that. The term for this method is intrastructural help. For the proof of principle, OVA 323-339 has been used as a model peptide. Since passive encapsulation is known to be inefficient, the peptide was modified by adding one to three additional lysine residues on the C- and N terminus to make them positively charged. The use of negatively charged 1,2-dioctadecanoyl-sn-glycero-3-phosphoglycerol (DSPG) in the liposomal composition should facilitate electrostatic interactions and result in an increased encapsulation efficiency. The results show that a controllable encapsulation is possible. Comparing the encapsulation of the original and the modified OVA peptides an increase up to 42% has been shown.vorgelegt von:Mirjam BatzoniArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftWien, FH Campus Wien, Masterarb., 2018(VLID)289599

Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations

The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide/N-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome’s physical integrity and the immunogen’s conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles

Design and Functional Characterization of HIV-1 Envelope Protein-Coupled T Helper Liposomes

Functionalization of experimental HIV-1 virus-like particle vaccines with heterologous T helper epitopes (T helper VLPs) can modulate the humoral immune response via intrastructural help (ISH). Current advances in the conjugation of native-like HIV-1 envelope trimers (Env) onto liposomes and encapsulation of peptide epitopes into these nanoparticles renders this GMP-scalable liposomal platform a feasible alternative to VLP-based vaccines. In this study, we designed and analyzed customizable Env-conjugated T helper liposomes. First, we passively encapsulated T helper peptides into a well-characterized liposome formulation displaying a dense array of Env trimers on the surface. We confirmed the closed pre-fusion state of the coupled Env trimers by immunogold staining with conformation-specific antibodies. These peptide-loaded Env-liposome conjugates efficiently activated Env-specific B cells, which further induced proliferation of CD4+ T cells by presentation of liposome-derived peptides on MHC-II molecules. The peptide encapsulation process was then quantitatively improved by an electrostatically driven approach using an overall anionic lipid formulation. We demonstrated that peptides delivered by liposomes were presented by DCs in secondary lymphoid organs after intramuscular immunization of mice. UFO (uncleaved prefusion optimized) Env trimers were covalently coupled to peptide-loaded anionic liposomes by His-tag/NTA(Ni) interactions and EDC/Sulfo-NHS crosslinking. EM imaging revealed a moderately dense array of well-folded Env trimers on the liposomal surface. The conformation was verified by liposomal surface FACS. Furthermore, anionic Env-coupled T helper liposomes effectively induced Env-specific B cell activation and proliferation in a comparable range to T helper VLPs. Taken together, we demonstrated that T helper VLPs can be substituted with customizable and GMP-scalable liposomal nanoparticles as a perspective for future preclinical and clinical HIV vaccine applications. The functional nanoparticle characterization assays shown in this study can be applied to other systems of synthetic nanoparticles delivering antigens derived from various pathogens