The lack of HBsAg secretion does neither facilitate induction of antiviral T cell responses nor Hepatitis B Virus clearance in mice.

Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts is regarded as a key contributor. HBsAg is expressed in HBV-infected hepatocytes and those carrying an HBV integration. Whether either HBsAg secretion or the high antigen amount expressed in the liver determines its immunomodulatory properties, however, remains unclear. We, therefore, developed a novel HBV animal model that allowed us to study the role of secreted HBsAg. We introduced a previously described HBs mutation, C65S, abolishing HBsAg secretion into a replication-competent 1.3-overlength HBV genome and used adeno-associated virus vectors to deliver it to the mouse liver. The AAV-HBV established a carrier state of wildtype and C65S mutant HBV, respectively. We investigated antiviral B- and T-cell immunity in the HBV-carrier mice after therapeutic vaccination. Moreover, we compared the effect of a lacking HBsAg secretion with that of an antiviral siRNA. While missing HBsAg secretion allowed for higher levels of detectable anti-HBs antibodies after therapeutic vaccination, it did neither affect antiviral T-cell responses nor intrahepatic HBV gene expression, irrespective of the starting level. A treatment with HBV siRNA restricting viral antigen expression within hepatocytes, however, improved the antiviral efficacy of therapeutic vaccination, irrespective of the ability of HBV to secrete HBsAg. Our data indicate that clearing HBsAg from blood cannot significantly impact HBV persistence or T-cell immunity. This indicates that a restriction of hepatic viral antigen expression will be required to break HBV immunotolerance.

Introducing adjuvant-loaded particulate hepatitis B core antigen as an alternative therapeutic hepatitis B vaccine component.

Background & Aims: Particulate hepatitis B core antigen (HBcoreAg) is a potent immunogen used as a vaccine carrier platform. HBcoreAg produced in E. coli encapsidates random bacterial RNA (bRNA). Using the heterologous protein-prime, viral-vector-boost therapeutic hepatitis B vaccine TherVacB, we compared the properties of different HBcoreAg forms. We explored how the content of HBcoreAg modulates antigen stability, immunogenicity, and antiviral efficacy. Methods: bRNA was removed from HBcoreAg by capsid disassembly, followed by reassembly in the absence or presence of specific nucleic acid-based adjuvants poly I:C or CpG. The morphology and structure of empty, bRNA-containing and adjuvant-loaded HBcoreAg were monitored by electron microscopy and nuclear magnetic resonance spectroscopy. Empty, bRNA-containing or adjuvant-loaded HBcoreAg were applied together with HBsAg and with or without nucleic acid-based external adjuvants within the TherVacB regimen in both wild-type and HBV-carrier mice. Results: While HBcoreAg retained its structure upon bRNA removal, its stability and immunogenicity decreased significantly. Loading HBcoreAg with nucleic acid-based adjuvants re-established stability of the capsid-like antigen. Immunization with poly I:C- or CpG-loaded HBcoreAg induced high antibody titers against co-administered HBsAg. When applied within the TherVacB regimen, they activated vigorous HBcoreAg- and HBsAg-specific T-cell responses in wild-type and HBV-carrier mice, requiring a significantly lower dose of adjuvant compared to externally added adjuvant. Finally, immunization with adjuvant-loaded HBcoreAg mixed with HBsAg led to long-term control of persistent HBV replication in the HBV-carrier mice. Conclusion: Adjuvant-loaded HBcoreAg retained capsid integrity and stability, was as immunogenic in vivo as externally adjuvanted HBcoreAg, requiring lower adjuvant levels, and supported immunity against co-administered, non-adjuvanted HBsAg. Thus, adjuvant-loaded HBcoreAg represents a promising novel platform for vaccine development. Impact and implications: Hepatitis B core antigen (HBcoreAg) recapitulates the capsid of the HBV that hosts the viral genome. Produced recombinantly, it is not infectious but emerges as a potent immunogen in vaccine development. In this preclinical study, we show that loading HBcoreAg with defined nucleic-acid-based adjuvants on the one hand stabilizes the HBcoreAg with standardized capsid content and, on the other hand, efficiently promotes the immunity of HBcoreAg and a co-administered antigen, allowing for reduced adjuvant doses. Therefore, adjuvant-loaded HBcoreAg not only serves as an encouraging option for therapeutic hepatitis B vaccines, but could also act as an efficient adjuvant delivery system for other types of vaccine.

High-Throughput Screening for the Prevalence of Neutralizing Antibodies against Human Adenovirus Serotype 5.

Adenoviral vectors based on the human adenovirus species C serotype 5 (HAdV-C5) are commonly used for vector-based gene therapies and vaccines. In the preclinical stages of development, their safety and efficacy are often validated in suitable animal models. However, pre-existing neutralizing antibodies may severely influence study outcomes. Here, we generated a new HAdV-C5-based reporter vector and established a high-throughput screening assay for the multivalent detection of HAdV-C5-neutralizing antibodies in serum. We screened the sera of rhesus macaques at different primate centers, and of rabbits, horses, cats, and dogs, showing that HAdV-C5-neutralizing antibodies can be found in all species, albeit at different frequencies. Our results emphasize the need to prescreen model animals in HAdV-C5-based studies.

Targeting genomic SARS-CoV-2 RNA with siRNAs allows efficient inhibition of viral replication and spread.

A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5' and 3' end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy.

[Comparison of Long-term Results with Small Incision Refractive Lenticule Extraction (ReLEX SMILE) vs. Femto-LASIK]. / Vergleich der Langzeitergebnisse bei Small Incision Refractive Lenticule Extraction (ReLEx SMILE) und Femto-LASIK.

AIM: Our aim was to retrospectively compare ReLEx Smile to femtosecondlaser-assisted LASIK (FsLASIK, femto-LASIK) in terms of safety, efficacy, stability as well as intraoperative complications. Comparable studies only show the results over the course of 3 years, making our data the first to examine longer term results. MATERIALS/METHODS: To accomplish this, we compared 404 eyes after FsLASIK (Mel 80, Carl Zeiss Meditec) and 1192 eyes after ReLEx SMILE (VisuMax, Carl Zeiss Meditec). We collected patients' data at 6 months, 1 year, 2 years and 5 years after refractive surgery. RESULTS: Five years postoperatively, the 2 methods showed equally good results in all investigated parameters. Over the course of these 5 years, the ReLEx SMILE achieved significantly better results for safety (p < 0.05) after 6 months, 1 year and 2 years. The results for visual acuity were significantly better for ReLEx SMILE after 1, 2 and 3 years. The predictability of both methods was consistently good over the entire period of time and intraoperative complications were equally low. CONCLUSION: After retrospective analysis of the visual outcomes of our patients up to 5 years after surgery, the ReLEx SMILE method turned out to be at least as safe and efficient as the FsLASIK. The stability of the refractive outcome was equally good with the 2 methods. Due to the high level of satisfaction experienced by the patients, high patient comfort intra- and postoperatively, absence of dry eyes, and the absence of flap complications, ReLEx SMILE has replaced the FsLASIK in our daily practice and become our method of choice for corneal refractive surgery when it comes to the correction of myopia and myopic astigmatism.

[Clinical Evaluation of a Novel Intraocular Lens with Enhanced Depth of Focus (EDOF) to Increase Visual Acuity for Intermediate Distances]. / Klinische Auswertung einer neuartigen Intraokularlinse mit erweiterter Tiefenschärfe (EDOF) zur Verbesserung der Sehkraft im Intermediärbereich.

BACKGROUND: Modern intraocular lens surgery has made great progress over the last few years towards creating independency of spectacles in daily life. Especially in the areas of distant and near visual acuity, optimisation has been possible. Nevertheless, with new media and requirements in professional life, there is an increasing need for optimisation of the intermediate range. In a prospective, non-randomised clinical study, the functional and refractive results after implantation of a novel intraocular lens with enhanced depth of focus were analysed. PATIENTS/MATERIAL AND METHODS: We have evaluated eleven patients after binocular implantation of the AT LARA 829MP (Carl Zeiss Meditec AG, Germany) 3 months after surgery. We examined refraction, corrected and uncorrected monocular and binocular distant visual acuity (CDVA, UDVA) as well as distance-corrected monocular and binocular visual acuity at different intermediate distances (with DCIVA 90, 80 and 60 cm) and 40 cm (DCNVA). We also performed defocus curve analysis. RESULTS: We found a mean increase of monocular CDVA from 0.35 to - 0.01 logMAR. Binocular DCIVA at 90, 80 and 60 cm was - 0.07, 0.04 and 0.07 logMAR, respectively. Even with a principle focus on intermediate distances, we found a functional DCNVA of 0.33 logMAR. Defocus curve analysis showed a visual acuity of 0.3 logMAR in a range of - 2.5 D to + 1.0 D. CONCLUSION: Binocular implantation of the AT LARA 829MP targeting emmetropia gives stable visual acuity from the distant to the near intermediate range, still with functional vision at the near distance of 40 cm.

Multifocal Intraocular Lenses and Extended Depth of Focus Intraocular Lenses.

Presbyopia and cataract patients' desire for increased spectacle independence after surgery is one of the main drivers for the development of multifocal intraocular lenses (MIOLs) and extended depth of focus (EDOF) intraocular lenses (IOLs). As education, biometry, diagnostics, surgical techniques, and MIOL/EDOF IOL designs have improved over the past decade, an increasing number of cataract surgeons have become cataract-refractive surgeons to help address this need. There is not 1 single MIOL/EDOF IOL, however, that suits all patients' needs. The wide variety of MIOLs and EDOF IOLs, their optics, and their respective impact on our patients' quality of vision have to be fully understood to choose the appropriate IOL for each individual; MIOL/EDOF IOL surgery has to be customized. This review article looks at the different optical aspects and clinical consequences of MIOLs/EDOF IOLs to help surgeons find an appropriate solution for each of their individual patients.