Generation of SARS CoV 2 and ZIKV-specific monoclonal antibodies by in vitro immunization using cell-permeable virus-like particles as antigen carrier

SARS-CoV-2 und ZIKV sind zwei neu auftretende Krankheitserreger. Ein wesentliches Werkzeug für die Charakterisierung des viralen Lebenszyklus sind Antikörper. Monoklonale Antikörper (MAbs) sind ein immunologisches Instrument mit vielfältigen Anwendungen in Forschung, Diagnose und Therapie. Die konventionelle Herstellung von MAbs ist jedoch zeitaufwändig, kostspielig und erfordert die Verwendung vieler Tiere und geht außerdem mit einer Belastung der Versuchstiere einher. Alle tierexperimentell arbeitenden Wissenschaftler sind sich der großen Verantwortung bewusst, die sie für das Wohlergehen der Versuchstiere tragen. Obwohl Tierversuche in der Forschung unerlässlich sind, besteht Einigkeit darüber, sie auf ein notwendiges Minimum zu beschränken. Als Richtlinie gilt dabei das ethische Prinzip der „3R“: Replace (Vermeiden), Reduce (Verringern) und Refine (Verbessern). Nach dem 3R-Prinzip zielte dieses Projekt darauf ab, SARS-CoV-2 und ZIKV-spezifische monoklonale Antikörper durch In-Vitro Immunisierung zu erzeugen. In diesem Projekt wurden zellpermeable Carrier-Capside benutzt, die einerseits das Zellpermeabilität vermittelnde TLM-Peptid beinhalten und im Bereich des Spike tips einen insertierten Strep-tag aufweisen. Dies ermöglicht die flexible Beladung mit Antigenen, die an Streptavidin fusioniert sind, so dass zellpermeable VLPs entstehen, die an ihrer Oberfläche mit Antigen beladen sind. Durch die Verwendung dieser Plattformtechnologie, die einen effizienteren Antigentransfer und eine robustere Immunantwort induziert als eine konventionelle Immunisierung mit dem freien Antigen, soll die geringere Effizienz der In-Vitro Immunisierung kompensiert werden. Rekombinante Antigene und VLPs konnten in E. coli hergestellt und durch Affinitätschromatographie gereinigt werden. Dichtegradientenzentrifugation und Elektronenmikroskopie zeigten den Aufbau der vlps und die Beladung mit den Antigenen. Diese Antigen-beladenen Partikel wurden zur In-Vitro Immunisierung von Milz-abgeleiteten Lymphozyten verwendet. Bei der In-Vvitro Immunisierung erfolgt die Immunisierung nicht im Tier, sondern in Kultur. Nach 4 Tagen war die Immunisierung abgeschlossen und die Zellen bereit für die Fusion mit Myeloma Zellen, um Hybridoma herzustellen. Wir haben ein Elektrofusionsprotokoll zur Fusion von B-Zellen mit Myelomzellen erstellt. B-Zellen und Myelomzellen werden über Komplexe aus Biotin- und Streptavidin–gekoppeltem Antigen verknüpft. Dazu wurde eine Biotinylierung von Oberflächenproteinen der Sp2/0-Ag14 Zellen durchgeführt. Fusionsproteine bestehend aus monomerem Streptavidin und ZIKV.E oder spike RBD können an das Biotin auf der Zelloberfläche binden. Auf der anderen Seite kann das Antigen an Oberflächen von B-Zellen binden. Nach Inkubation der B-Zellen mit Myeloma Zellen entstehen Verbindungen zwischen beiden Zellen, was zu einer effizienten Fusion führt. Anschließend erfolgt die Elektrofusion der Zellsuspension in einem Puffer niedriger Ionenstärke. Die monoklonalen Antikörper werden von den Zellen an das Medium abgegeben und können daraus in großen Mengen in vitro gewonnen werden. Durch diesen Ansatz wurden SARS-CoV2-Spike-RBD- und ZIKV.E-spezifische Antikörper-Mabs in vitro erhalten. Die Antikörperbindung wurde durch die Oberflächenplasmonresonanz auf einem Biacore-System charakterisiert. Somit können diese Antikörper zum Nachweis von SARS-CoV2-Spike bzw. ZIKV.E durch Western-Blot- oder Immunfluoreszenzmikroskopie und zur Quantifizierung von SARS-CoV2 S oder ZIKV.E durch spezifische ELISAs verwendet werden. In Übereinstimmung mit dem 3R-Prinzip haben wir ein Protokoll zur In-Vitro Immunisierung und Erzeugung monoklonaler Antikörper entwickelt. Es wurden hochspezifische Antikörper zum Nachweis und zur Quantifizierung von entweder SARS-CoV2-Spike oder ZIKV-Hüllprotein erhalten.ZIKV and SARS-CoV-2 are two emerging pathogens. For detailed characterization of the viral life cycle, specific monoclonal antibodies for viral proteins are required. Monoclonal antibodies (MAbs) are an established immunological tool with multiple applications in research, diagnosis and therapy. However, the conventional production of MAbs is time-consuming, costly and requires the use of many animals. The 3R principle aims to avoid animal experiments altogether (replacement), to limit the number of animals (reduction) and their suffering (refinement). According to the 3R principle, this project aimed to generate SARS-CoV-2 and ZIKV-specific monoclonal antibodies by in vitro immunization. The HBV capsid is highly immunogenic and helps to trigger a strong B-cell response against the antigens. In this project, TLM-HBV core fused to Streptavidin serves as membrane permeable antigen carrier and monomeric streptavidin fused to RBD of SARS-CoV-2 or ZIKV envelope (E) is used as cargo. TLM-HBV core served as antigen carrier. Recombinant antigens and vlps could be produced in E. coli and purified by affinity chromatography. Density gradient centrifugation and electron microscopy revealed the assembly of the vlps and the loading with the respective antigens. These antigen-loaded particles were used for immunization of spleen-derived lymphocytes. We established an electrofusion protocol for fusing B-cells with myeloma partners. This was achieved by Streptavidin-mediated coupling of B-cells to biotinylated myeloma cells to generate hybridomas secreting functional monoclonal antibodies. By this approach SARS-CoV-2 spike-RBD and ZIKV.E specific antibodies mabs were obtained in vitro. Antibody binding was characterized by surface plasmon resonance on a Biacore system. Thus, these antibodies can be used for detection of SARS-CoV-2 spike or ZIKV.E, respectively by western blot or immunofluorescence microscopy and for quantification of SARS-CoV-2 or ZIKV.E by specific ELISAs. In line with the 3R principle we developed a protocol for in vitro immunization and generation of monoclonal antibodies. Highly specific antibodies for detection and quantification of either SARS-CoV-2 spike or ZIKV envelope protein were obtained

The SARS-CoV-2 Variant Omicron Is Able to Escape Vaccine-Induced Humoral Immune Responses, but Is Counteracted by Booster Vaccination

The SARS-CoV-2 variant Omicron has spread world-wide and is responsible for rapid increases in infections, including in populations with high vaccination rates. Here, we analysed in the sera of vaccinated individuals the antibody binding to the receptor-binding domain (RBD) of the spike protein and the neutralization of wild-type (WT), Delta (B.1.617.2), and Omicron (B.1.1.529; BA.1) pseudotyped vectors. Although sera from individuals immunized with vector vaccines (Vaxzevria; AZ and COVID-19 Janssen, Ad26.COV2.S; J&J) were able to bind and neutralize WT and Delta, they showed only background levels towards Omicron. In contrast, mRNA (Comirnaty; BNT) or heterologous (AZ/BNT) vaccines induced weak, but detectable responses against Omicron. While RBD-binding antibody levels decreased significantly six months after full vaccination, the SARS-CoV-2 RBD-directed avidity remained constant. However, this still coincided with a significant decrease in neutralization activity against all variants. A third booster vaccination with BNT significantly increased the humoral immune responses against all tested variants, including Omicron. In conclusion, only vaccination schedules that included at least one dose of mRNA vaccine and especially an mRNA booster vaccination induced sufficient antibody levels with neutralization capacity against multiple variants, including Omicron

Quantitative and Qualitative Difference in Antibody Response against Omicron and Ancestral SARS-CoV-2 after Third and Fourth Vaccination

Waning immunity against SARS-CoV-2 and the emergence of variants, especially of the most distant variant, Omicron, affect titers of neutralizing antibodies in the sera of vaccinated individuals. Thus, two vaccinations with the mRNA vaccine BNT162b fail to induce neutralizing antibodies against the Omicron variant. A first booster vaccination increases Omicron-RBD-binding IgG and IgA and neutralizing capacity. In comparison, the Wuhan isolate titers of the Omicron variant binding antibodies are 8.5 lower. After a third vaccination, induction of Omicron-RBD- and Wuhan-RBD-binding antibodies follows the same kinetic. Five to six months after the third vaccination, there are still Omicron-RBD-binding antibodies detectable, but 35.9 percent of the analyzed sera fail to neutralize the Omicron variant, while all sera efficiently neutralize the Delta isolate. In the case of the Wuhan-RBD, a significantly larger number of stable antigen–antibody complexes is formed than in Omicron-RBD. A fourth vaccination with mRNA-1273 temporarily restores levels of Omicron-, Delta- and Wuhan-specific antibodies. Comparing different booster strategies revealed that the breadth of the immune response is not affected by the vaccination regimen. Taken together, these data indicate that booster vaccinations (third and fourth dose) increase the breadth of the immune response, but there is a qualitative difference of antibodies with respect to the stability of antigen–antibody complexes and persistence of antibody titers

Persistence of infectious SARS-CoV-2 particles for up to 37 days in patients with mild COVID-19

BACKGROUND: People suffering from COVID-19 are typically considered non-infectious 14 days after diagnosis if symptoms have disappeared for at least 48 h. We describe three patients who independently acquired their infection. These three patients experienced mild COVID-19 and completely recovered symptomatically within 10 days, but remained PCR-positive in deep pharyngeal samples for at least 38 days. We attempted to isolate virus from pharyngeal swabs to investigate whether these patients still carried infectious virus. METHODS: Infectious virus was amplified in Vero E6 cells and characterized by electron microscopy and WGS. The immune response was investigated by ELISA and peptide arrays. RESULTS: In all three cases, infectious and replication-competent virus was isolated and amplified in Vero E6 cells. Virus replication was detected by RT-PCR and immunofluorescence microscopy. Electron microscopy confirmed the formation of intact SARS-CoV-2 particles. For a more detailed analysis, all three isolates were characterized by whole-genome sequencing (WGS). The sequence data revealed that the isolates belonged to the 20A or 20C clade, and two mutations in ORF8 were identified among other mutations that could be relevant for establishing a long-term infection. Characterization of the humoral immune response in comparison to patients that had fully recovered from mild COVID-19 revealed a lack of antibodies binding to sequential epitopes of the receptor-binding domain (RBD) for the long-term infected patients. CONCLUSION: Thus, a small portion of COVID-19 patients displays long-term infectivity and termination of quarantine periods after 14 days, without PCR-based testing, should be reconsidered critically

Comparative Investigation of Methods for Analysis of SARS-CoV-2-Spike-Specific Antisera

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen–antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera—as in cases of non-ARDS sera—combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen–antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility