High-dimensional analysis of 16 SARS-CoV-2 vaccine combinations reveals lymphocyte signatures correlating with immunogenicity.

The range of vaccines developed against severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) provides a unique opportunity to study immunization across different platforms. In a single-center cohort, we analyzed the humoral and cellular immune compartments following five coronavirus disease 2019 (COVID-19) vaccines spanning three technologies (adenoviral, mRNA and inactivated virus) administered in 16 combinations. For adenoviral and inactivated-virus vaccines, heterologous combinations were generally more immunogenic compared to homologous regimens. The mRNA vaccine as the second dose resulted in the strongest antibody response and induced the highest frequency of spike-binding memory B cells irrespective of the priming vaccine. Priming with the inactivated-virus vaccine increased the SARS-CoV-2-specific T cell response, whereas boosting did not. Distinct immune signatures were elicited by the different vaccine combinations, demonstrating that the immune response is shaped by the type of vaccines applied and the order in which they are delivered. These data provide a framework for improving future vaccine strategies against pathogens and cancer.

C-reactive protein (CRP) is essential for efficient systemic transduction of recombinant adeno-associated virus vector 1 (rAAV-1) and rAAV-6 in mice.

The clinical relevance of gene therapy using the recombinant adeno-associated virus (rAAV) vectors often requires widespread distribution of the vector, and in this case, systemic delivery is the optimal route of administration. Humoral blood factors, such as antibodies or complement, are the first barriers met by the vectors administered systemically. We have found that other blood proteins, galectin 3 binding protein (G3BP) and C-reactive protein (CRP), can interact with different AAV serotypes in a species-specific manner. While interactions of rAAV vectors with G3BP, antibodies, or complement lead to a decrease in vector efficacy, systemic transduction of the CRP-deficient mouse and its respective control clearly established that binding to mouse CRP (mCRP) boosts rAAV vector 1 (rAAV-1) and rAAV-6 transduction efficiency in skeletal muscles over 10 times. Notably, the high efficacy of rAAV-6 in CRP-deficient mice can be restored by reconstitution of the CRP-deficient mouse with mCRP. Human CRP (hCRP) does not interact with either rAAV-1 or rAAV-6, and, consequently, the high efficiency of mCRP-mediated muscle transduction by these serotypes in mice cannot be translated to humans. No interaction of mCRP or hCRP was observed with rAAV-8 and rAAV-9. We show, for the first time, that serum components can significantly enhance rAAV-mediated tissue transduction in a serotype- and species-specific manner. Bioprocessing in body fluids should be considered when transfer of a preclinical proof of concept for AAV-based gene therapy to humans is planned.

Adaptive Hybrid Surgery Experiences in Benign Skull Base Tumors.

BACKGROUND: The treatment of benign skull base tumors remains challenging. These tumors are often located in close relationship to critical structures. Therefore, radical resection of these tumors can be associated with high morbidity. Multimodal treatment concepts, including controlled partial tumor resection followed by radiosurgery, should be considered. METHODS: Adaptive hybrid surgery analysis (AHSA) is an intraoperative tool that has been introduced for the automatic assessment of tumor properties, and virtual real-time radiosurgical treatment simulation and continuous feasibility analysis of adjuvant radiosurgery. The AHSA method (Brainlab®, Munich, Germany) was applied to five patients who underwent partial resection of a benign skull base tumor. Tumor volumetry was obtained on pre- and postoperative MR scans. Organs at risk were, preoperative, automatically delineated with atlas mapping software (Elements® Segmentation Cranial), and adaptations were made if necessary. RESULTS: Five patients with benign skull base lesions underwent planned partial tumor resection in a multimodal therapeutic surgery followed by radiosurgery. The preoperative tumor volumes ranged between 8.52 and 25.2 cm3. The intraoperative residual tumor volume measured with the AHSA® software ranged between 2.13-12.17 cm3 (25-52% of the preoperative tumor volume). The intraoperative automatic AHSA plans of the remaining tumor volume suggested, in all five patients, that safe hypofractionated radiation was feasible. Patients were followed for 69.6 ± 1.04 months, and no complications occurred after the patients were treated with radiation. CONCLUSIONS: Intraoperative SRS planning based on volumetric assessments during resection of skull base tumors using AHSA® is feasible and safe. The AHSA method allows the neurosurgeon to continuously evaluate the feasibility of adjuvant radiosurgery while planning and performing a surgical resection. This method supports the treatment strategy of a complementary approach during surgical resection of complex skull base tumors and might contribute to preventing surgical and radiosurgical complications.

Intraoperative Spinal Angiography during Microsurgical Occlusion of Spinal Dural Arteriovenous Fistula within the Hybrid Operation Room.

BACKGROUND: Spinal dural arteriovenous fistula (SDAVF) is a rare cause of progressive myelopathy in predominantly middle-aged men. Treatment modalities include surgical obliteration and endovascular embolization. In surgically treated cases, failure of obliteration is reported in up to 5%. The aim of this technical note is to present a safe procedure with complete SDAVF occlusion, verified by intraoperative digital subtraction angiography (DSA). METHODS: We describe four patients with progressive leg weakness who underwent surgical obliteration of SDAVF with spinal intraoperative DSA in the prone position after cannulation of the popliteal artery. All surgeries took place in our hybrid operating room (OR) and were accompanied by electrophysiologic monitoring. Surgeries and cannulation of the popliteal artery were performed in the prone position. Ultrasound was used to guide the popliteal artery puncture. A 5-Fr sheath was inserted and the fistula was displayed using a 5-Fr spinal catheter. Spinal intraoperative DSA was performed prior to and after temporary clipping of the fistula point as well after the final SDAVF occlusion. RESULTS: The main feeder of the SDAVF fistula in the first patient arose from the right T11 segmental artery, which also supplied the artery of Adamkiewicz. The second patient initially underwent endovascular treatment and deteriorated 5 months later due to recanalization of the SDAVF via a small branch of the T12 segmental artery. The third and fourth cases were primarily scheduled for surgical occlusion. Access through the popliteal artery for spinal intraoperative DSA proved to be beneficial and safe in the hybrid OR setting, allowing the sheath to be left in place during the procedure. During exposure and after temporary and permanent occlusion of the fistulous point, intraoperative indocyanine green (ICG) video angiography was also performed. In one case, the addition of intraoperative DSA showed failure of fistula occlusion, which was not visible with ICG angiography, leading to repositioning of the clip. Complete fistula occlusion was documented in all cases. CONCLUSION: Spinal intraoperative DSA in the prone position is a feasible and safe intervention for rapid localization and confirmation of surgical SDAVF occlusion.

AAV-8 and AAV-9 Vectors Cooperate with Serum Proteins Differently Than AAV-1 and AAV-6.

Under intravenous delivery, recombinant adeno-associated vectors (rAAVs) interact with blood-borne components in ways that can critically alter their therapeutic efficiencies. We have previously shown that interaction with human galectin 3 binding protein dramatically reduces rAAV-6 efficacy, whereas binding of mouse C-reactive protein improves rAAV-1 and rAAV-6 transduction effectiveness. Herein we have assessed, through qualitative and quantitative studies, the proteins from mouse and human sera that bind with rAAV-8 and rAAV-9, two vectors that are being considered for clinical trials for patients with neuromuscular disorders. We show that, in contrast to rAAV-1 and rAAV-6, there was a substantial similarity in protein binding patterns between mouse and human sera for these vector serotypes. To establish an in vivo role for the vector binding of these sera proteins, we chose to study platelet factor 4 (PF4), which interacts with both vectors in both mouse and human sera. Experiments using PF4-knockout mice showed that a complete lack of PF4 did not alter skeletal muscle transduction for these vectors, whereas heart transduction was moderately improved. Our results strongly support our position that the impact of serum proteins on the transduction properties of rAAV-8 and rAAV-9, already observed in mouse models, should be similar in human preclinical trials.

Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates.

Vectors based on recombinant adeno-associated viruses (rAAV) attract a growing interest for human gene therapy. Recently, it was shown that many rAAV serotypes produced by transient transfection of human embryonic kidney 293 cell line (HEK293) are efficiently released into culture medium and functionally equivalent to those purified from cell lysates. Here, we report that HEK293 cells produce and secrete Galectin 3-binding protein (huG3BP), a protein that efficiently binds rAAV6 in vivo. Importantly, intracellular G3BP and secreted G3BP have different properties: while the secreted protein had the same electrophoretic mobility as serum huG3BP and interacted with rAAV6, intracellular protein migrated faster and did not bind rAAV6. Consequently, rAAV6 purified from culture medium (secreted, rAAV6-S) was physically associated with huG3BP while rAAV6 harvested from cell lysates (cellular, rAAV6-C) was huG3BP-free. After systemic injections, rAAV6-S bound to huG3BP was 3 times less efficient compared to rAAV6-C and induced an immune response against huG3BP protein. Our findings show that protein content of rAAVs purified from culture medium or from cell lysates can be different and these differences may impact vector efficacy and/or immune response.

Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.

From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.