Viral Vectors Research Articles

A Special Insight on the Causal Agents and Mode of Occurrence of Japanese Encephalitis (JE) Infections in Rural Regions of Assam, India.

Japanese Encephalitis (JE) is a life-threatening disease, especially in the Indian subcontinent. Knowledge about the nature and ecology of the dispersal of JE virus (JEV) vectors needs to be increased. This study mechanistically explores the ecology of JEV vectors and the mode and frequency of occurrence of Acute Encephalitis Syndrome (AES) and JEV infections. We established a linear relationship between environmental variables and JEV infection by JEV vectors (mosquitoes)-Culex tritaeniorhynchus, Culex vishnui, and Culex pseudovishnui. The relative abundance of three mosquito species was evaluated, and the JE Sample Positivity Rate (SPR) and JE Case Fatality Rate (CFR) were computed. Culex vishnui had a high abundance in residential areas during the transition from hot-dry (77.34%) to hot-wet period (78.66%) at temperatures between 31°C and 34°C and relative humidity of 80%-85.3%; this period also coincided with occurrence of AES (39 cases in hot-dry and 88 cases in hot-wet period) and JE (8 instances in hot-dry and 31 cases in hot-wet period). JE infection dominated near rainfed rice fields (rainfall: R = 0.67 at p < 0.05; rainy days: R = 0.74, p < 0.01). SPR was up to 32.28%, and CFR was as high as 42.86%; JEV infection was concentrated in adult male humans near rice fields (15.66%). Climate modulation over the years can influence the distribution of Culex vishnui and, hence, AES and JE cases. JEV infections tend to rise in JE hotspot regions, especially near rice fields. Thus, comprehensive epidemiological investigations will help prevent a silent outbreak of JE.

Janus Kinase 2/Signal Transducer and Activator of Transcription 3/Cyclooxygenase 2 Signaling Pathway Mediates the Effect of Central Angiotensin II on the Elevation of Rostral Ventrolateral Medulla Prostaglandin E2-Induced Oxidative Stress in Hypertension.

Prostaglandin E2 (PGE2) in the rostral ventrolateral medulla (RVLM) has been recognized as a pivotal pressor substance in hypertension, yet understanding of its effects and origins in the RVLM remains largely elusive. This study aimed to elucidate the pivotal enzymes and molecular mechanisms underlying PGE2 synthesis induced by central Ang II (angiotensin II) and its implications in the heightened oxidative stress and sympathetic outflow in hypertension. RVLM microinjections of PGE2 and Tempol were administered in Wistar-Kyoto rats. Intracisternal drug delivery and adeno-associated viral vectors microinjection were used in both Wistar-Kyoto rats and spontaneous hypertensive rats to modulate the function of Ang II, PGE2 receptor 3, and expression of COX2 (cyclooxygenase 2). Microinjection of PGE2 into the RVLM significantly augmented sympathetic activity (25.380±1.566%) and oxidative stress level, whereas intracisternal infusion of a prostaglandin E receptor 3 antagonist attenuated sympathetic activity in both spontaneous hypertensive rats and Ang II-induced hypertensive rats. Furthermore, Ang II treatment upregulated COX2 expression in RVLM neurons (1.000±0.112 versus 1.506±0.370 fold change), with no significant effect on other enzymes involved in PGE2 synthesis. Additionally, inhibition of the JAK2/STAT3 (Janus kinase 2/signal transducer and activator of transcription 3) signaling pathway nullified Ang II-mediated elevation of COX2 expression, as evidenced by phosphorylated STAT3 binding to the COX2 sequence in PC12 cells. Central Ang II induces the accumulation of RVLM PGE2 through the neuronal AT1R (angiotensin type 1 receptor)/JAK2/STAT3/COX2 pathway, thereby promoting oxidative stress, augmenting sympathetic outflow, and contributing to essential hypertension.

Three-dimensional linkage analysis with digital PCR for genome integrity and identity of recombinant adeno-associated virus

Recombinant adeno-associated virus (rAAV) has emerged as the vector of choice for in vivo gene delivery, with numerous clinical trials underway for the treatment of various human diseases. Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer and further establish the optimal therapeutic dosage for a rAAV product. The conventional single-channel droplet digital PCR (1D ddPCR) method offers only partial information regarding the viral vector genome titer, lacking insights into its integrity. In our pursuit of further advancing rAAV analysis, we have developed a novel 3D ddPCR assay with advanced 3D linkage analysis. We have designed the three amplicon sites targeting both ends of the viral genome, as well as the center of key therapeutic gene of interest (GOI). This study aims to offer a more comprehensive and insightful assessment of rAAV products which includes not only quantity of viral genome titer but also the quality, distinguishing between partial ones and intact full-length viral genomes with the right GOI. Importantly, due to the random partitioning property of a digital PCR system, the 3D linkage analysis of rAAV viral genome requires a proper mathematical model to identify the true linked DNA molecules (full-length/intact DNA) from the population of false/unlinked DNA molecules (fragmented/partial DNA). We therefore have developed an AAV 3D linkage analysis workflow to characterize genomic integrity and intact titer for rAAV gene therapy products. In this study, we focus on evaluating our 3D linkage mathematical model by performing DNA mixing experiments and a case study using multiple rAAV samples. Particularly, we rigorously tested our algorithms by conducting experiments involving the mixing of seven DNA fragments to represent various AAV viral genome populations, including 3 single partials, 3 double partials, and 1 full-length genomes. Across all 37 tested scenarios, we validated the accuracy of our workflow’s output for the percentages of 3D linkage by comparing to the known percentages of input DNA. Consequently, our comprehensive AAV analytical package not only offers insights into viral genome titer but also provides valuable information on its integrity and identity. This cost-effective approach, akin to the setup of traditional 1D or 2D dPCR, holds the potential to advance the application of rAAV in cell and gene therapy for the treatment of human diseases.

Single Dose of Attenuated Vaccinia Viruses Expressing H5 Hemagglutinin Affords Rapid and Long-Term Protection Against Lethal Infection with Highly Pathogenic Avian Influenza A H5N1 Virus in Mice and Monkeys

Background/Objectives: In preparation for a potential pandemic caused by the H5N1 highly pathogenic avian influenza (HPAI) virus, pre-pandemic vaccines against several viral clades have been developed and stocked worldwide. Although these vaccines are well tolerated, their immunogenicity and cross-reactivity with viruses of different clades can be improved. Methods: To address this aspect, we generated recombinant influenza vaccines against H5-subtype viruses using two different strains of highly attenuated vaccinia virus (VACV) vectors. Results: rLC16m8-mcl2.2 hemagglutinin (HA) and rLC16m8-mcl2.3.4 HA consisted of a recombinant LC16m8 vector encoding the HA protein from clade 2.2 or clade 2.3.4 viruses (respectively); rDIs-mcl2.2 HA consisted of a recombinant DIs vector encoding the HA protein from clade 2.2. A single dose of rLC16m8-mcl2.2 HA showed rapid (1 week after vaccination) and long-term protection (20 months post-vaccination) in mice against the HPAI H5N1 virus. Moreover, cynomolgus macaques immunized with rLC16m8-mcl2.2 HA exhibited long-term protection when challenged with a heterologous clade of the HPAI H5N1 virus. Although the DIs strain is unable to grow in most mammalian cells, rDIs-mcl2.2 HA also showed rapid and long-lasting effects against HPAI H5N1 virus infection. Notably, the protective efficacy of rDIs-mcl2.2 HA was comparable to that of rLC16m8-mcl2.2 HA. Furthermore, these vaccines protected animals previously immunized with VACVs from a lethal challenge with the HPAI H5N1 virus. Conclusions: These results suggest that both rLC16m8-mcl2.2 HA and rDIs-mcl2.2 HA are effective in preventing HPAI H5N1 virus infection, and rDIs-mcl2.2 HA is a promising vaccine candidate against H5 HA-subtype viruses.

Mecp2: An Important Role in the Pathogenesis of RETT Syndrome

RETT syndrome is an X-chromosome-linked neurodevelopmental disorder mainly affecting female children, and its pathogenesis is mainly related to mutations in the Mecp2. In recent years, the role of Mecp2 in neurodevelopment and its relationship with RETT syndrome has become a hot research topic. Current research is focused on the effects of Mecp2 mutations on neuronal function and neural circuits, as well as exploring potential therapeutic approaches in animal models. Despite the results achieved, there are still gaps in the understanding of the precise molecular mechanisms of the Mecp2, gender specificity and the role of non-coding RNAs. This paper analyzed the role of Mecp2 in neurodevelopment and its relationship with Rett syndrome, and explored the molecular mechanisms and potential therapeutic strategies for Mecp2 mutations. It was found that Mecp2 mutations lead to neurological dysfunction by affecting neuronal differentiation and synaptic plasticity. In addition, this paper evaluates the current prospects for gene therapy, noting that adeno-associated viral vectors (AAV) have shown favorable therapeutic effects in animal experiments. This paper provides new insights into understanding the pathogenic mechanism of the Mecp2 in RETT syndrome and provides an important reference for future therapeutic strategies. However, the precise molecular mechanism of the Mecp2 and its gender-specific differences still need further investigation. Future studies could focus on developing safer and more effective gene therapies and exploring the role of non-coding RNAs in RETT syndrome with a view to achieving more comprehensive therapeutic strategies

Targeting Brain Drug Delivery with Macromolecules Through Receptor-Mediated Transcytosis

Brain diseases pose significant treatment challenges due to the restrictive nature of the blood–brain barrier (BBB). Recent advances in targeting macromolecules offer promising avenues for overcoming these obstacles through receptor-mediated transcytosis (RMT). We summarize the current progress in targeting brain drug delivery with macromolecules for brain diseases. This exploration details the transport mechanisms across the BBB, focusing on RMT and its use of natural ligands for drug delivery. Furthermore, the review examines macromolecular ligands such as antibodies, peptides, and aptamers that leverage RMT for effective BBB traversal. Advancements in macromolecules-based delivery systems for brain diseases are summarized, emphasizing their therapeutic potential and limitations. Finally, emerging RMT strategies, including viral vectors, exosomes, and boron neutron capture therapy, are discussed for their precision in brain-targeted treatments. This comprehensive overview underscores the potential of RMT-based approaches to revolutionize brain disease therapy.

Strategic and Technical Considerations in Manufacturing Viral Vector Vaccines for the Biomedical Advanced Research and Development Authority Threats

Over the past few decades, the world has seen a considerable uptick in the number of new and emerging infectious disease outbreaks. The development of new vaccines, vaccine technologies, and platforms are critical to enhance our preparedness for biological threats and prevent future pandemics. Viral vectors can be an important tool in the repertoire of technologies available to develop effective vaccines against new and emerging infectious diseases. In many instances, vaccines may be needed in a reactive scenario, requiring technologies than can elicit rapid and robust immune responses with a single dose. Here, we discuss how viral vector vaccines are utilized in a vaccine portfolio for priority biological threats, some of the challenges in manufacturing viral vector vaccines, the need to strengthen live virus manufacturing capabilities, and future opportunities to capitalize on the use of viral vectors to improve the sustainability of the Biomedical Advanced Research and Development Authority’s vaccine portfolio.

Susceptibility to organophosphate insecticides in Aedes aegypti (Diptera: Culicidae) from northern Colombia and associated resistance mechanisms

BackgroundAedes aegypti is the primary vector of dengue, chikungunya, and Zika viruses in Colombia. Various insecticides, including pyrethroid, organophosphate, and carbamate insecticides; growth regulators; and biological insecticides, such as Bacillus thuringiensis var. israelensis, have been used to control Ae. aegypti populations. However, organophosphates such as malathion, pirimiphos-methyl, and temephos have been used over the last decade owing to the high resistance to pyrethroids.MethodsThis study assessed the susceptibility to organophosphates in 14 Ae. aegypti populations from the Córdoba department in northern Colombia. Moreover, possible resistance mechanisms were investigated by determining the activity levels of α-esterases, β-esterases, mixed function oxidases (MFOs), glutathione S-transferases (GSTs), and insensitive acetylcholinesterase (iAChE). Additionally, the Ace-1 gene was sequenced to identify mutations at the target site of action.ResultsThe populations were susceptible to temephos and malathion but resistant to fenitrothion, and in three of them, to pirimiphos-methyl. Alterations in the enzyme activity levels of α-esterases and β-esterases, GST, and iAChE were observed among the populations, with high enzyme activity levels of α and β esterases associated with resistance to fenitrothion. No mutations were identified in the Ace-1 gene.ConclusionsThese findings are highly relevant for vector control programs in the region, as they allow for adjustments in resistance management strategies and improve the effectiveness of interventions against these arboviruses.Graphical

Different COVID-19 vaccines and their respective technological platforms: systematic review of clinical trials

Introduction: The COVID-19 pandemic has highlighted the importance of vaccination in reducing severe cases and deaths. However, the emergence of new SARS-CoV-2 variants raises concerns about the efficacy and safety of available vaccines, mainly due to the diversity of technological platforms used in their development. Objectives: To elucidate the current knowledge about the main vaccine against COVID-19 and their respective technological platforms, licensed and in the licensing phase. Methods: Systematic review according to the PRISMA statement of articles published from 2020, through the PubMed database. Results: The search resulted in fifty studies, of which twenty-one were included. Clinical trials addressed vaccines that use mRNA platforms (n=4), non-replicant viral vector (n=3), attenuated virus (n=2), inactivated vaccine (n=1), recombinant protein nanoparticles (n=3), vaccine using virus-like particles (n=1), subunit vaccine (n=1), DNA vaccine (n=1). Most of the vaccines showed good tolerability and induced neutralizing antibodies against various variants of SARS-CoV-2. Conclusions: Except for the non-replicant viral vector vaccine, intranasal ChAdOx1 nCoV-19 that did not induce mucosal and systemic immune responses, and the DNA vaccine, which induced only a good cellular response, the other vaccines demonstrated good tolerability and efficacy in inducing immune responses.

Genome-wide association studies to assess genetic factors controlling cucumber resistance to CABYV and CMV in crop fields and the attractiveness for their Aphis gossypii vector”

Abstract Cucumber crops face high pressure from pathogens, including various viral species. Mapping quantitative trait loci (QTL) for vegetable resistance to viruses has primarily been conducted after mechanical inoculation in controlled environments, but not in crop field conditions. Moreover, viruses which cannot be mechanically inoculated, e.g. the cucurbit aphid-borne yellows virus (CABYV), have been overlooked in resistance studies. Here we aimed to identify QTLs reducing epidemics of two prevalent cucumber viruses: CABYV and the cucumber mosaic virus (CMV). We evaluated the resistance of 256 elite cucumber lines and landraces in crop field conditions by screening for the presence of both viruses six-times during the season. We mapped twelve QTLs reducing CABYV epidemics and seven QTLs reducing CMV epidemics by combining multi-loci genome-wide association studies (GWAS) and local score approach analyses. We also examined the attractiveness of this cucumber panel for Aphis gossypii, a major cucumber virus vector. We identified five QTLs that reduced the attractiveness, including one co-localizing with a QTL reducing CABYV epidemics. Interestingly, some accessions deemed CMV-resistant after mechanical inoculation in controlled environments showed high infection rates in crop field conditions. Only one QTL for CMV resistance was detected in both conditions, indicating that these phenotypes are regulated by independent QTLs. Local linkage disequilibrium study findings suggested that certain QTLs reducing epidemics were introduced early into elite lines through serendipity or selection. QTLs could be pyramided with other low effect QTLs through genomic selection to obtain cucumber cultivars with enhanced field resistance to viruses.

Strong immune responses and robust protection following a novel protein in adjuvant tuberculosis vaccine candidate

BCG remains the only licensed vaccine for tuberculosis (TB), but its efficacy wanes over time. Subunit vaccines, aim to improve BCG immunity and protection, by inducing responses to a few mycobacterial antigens delivered with a specific platform. Since the platform shapes the immune response induced, selecting the right platform has been challenging due to the lack of immune correlates of protection. Recently, the protein-adjuvated subunit vaccine. M72/AS01E, demonstrated 49.7% efficacy in preventing active TB in latently infected adults, indicating that protective immunity through subunit vaccines is possible. In this study we evaluated the immunogenicity and efficacy of the promising mycobacterial antigen PPE15, formulated with five adjuvants developed by the Vaccine Formulation Institute. While all adjuvants were immunogenic, PPE15 with LMQ protected vaccinated mice against an in vivo Mycobacterium tuberculosis challenge, both as a standalone vaccine and as a boost to BCG. Vaccinated mice had enriched lung parenchymal antigen-specific CD4 + CXCR3 + KLRG1− T cells previously associated with TB protection. Heterologous vaccination strategies were also explored by combining intranasal ChAdOx1.PPE15 viral vector, with intramuscular PPE15-LMQ resulting in improved protection compared to individual vaccines. These findings support the progression of this vaccine candidate to the next stages of development.

Non-Viral Delivery Systems to Transport Nucleic Acids for Inherited Retinal Disorders

Inherited retinal disorders (IRDs) represent a group of challenging genetic conditions that often lead to severe visual impairment or blindness. The complexity of these disorders, arising from their diverse genetic causes and the unique structural and functional aspects of retinal cells, has made developing effective treatments particularly challenging. Recent advancements in gene therapy, especially non-viral nucleic acid delivery systems like liposomes, solid lipid nanoparticles, dendrimers, and polymersomes, offer promising solutions. These systems provide advantages over viral vectors, including reduced immunogenicity and enhanced targeting capabilities. This review delves into introduction of common IRDs such as Leber congenital amaurosis, retinitis pigmentosa, Usher syndrome, macular dystrophies, and choroideremia and critically assesses current treatments including neuroprotective agents, cellular therapy, and gene therapy along with their limitations. The focus is on the emerging role of non-viral delivery systems, which promise to address the current limitations of specificity, untoward effects, and immunogenicity in existing gene therapies. Additionally, this review covers recent clinical trial developments in gene therapy for retinal disorders.

Development of Lentiviral Packaging Cells and Scale Up of Production to Meet the Growing Demand in Cell and Gene Therapy.

Gamma-Retroviral (RVVs) and lentiviral vectors (LVVs) represent indispensable tools in somatic gene therapy, mediating the efficient, stable transfer of therapeutic genes into a variety of human target cells. LVVs, in contrast to RVVs, are capable of stably genetically modifying non-proliferating target cells, making them the superior instrument in cell and gene therapy. To date, the LVV manufacturing process employs human embryonic kidney cells (HEK293) and derivatives thereof transiently transfected with multiple plasmids encoding the required viral vector components. Alternatively, stable packaging cell lines were developed and engineered to express all vector components in trans. Currently, these cells are mostly cultured in cell stacks, where they grow adherently in 2D layers, limiting the scale-up of vector production. The production of viral vectors using stable suspension cell lines enables larger-scale production and higher yields under controlled conditions. Here, we review the improvements made to enhance vector safety and production yield. Current advancements in the establishment of stable packaging cell lines enabling inducible and constitutive LVV production are summarized and discussed. Manufacturing processes for lentiviral vectors using bioreactors with perfusion systems are required to meet the growing demand in cell and gene therapy and to reduce production and therapy costs.

Novel Immune Response Evasion Strategy to Redose Adeno-associated Viral Vectors and Prolong Survival in Surfactant Protein-B Deficient Mice.

Surfactant protein-B (SP-B) deficiency is a lethal neonatal respiratory disease with few therapeutic options. Gene therapy using adeno-associated viruses (AAV) to deliver human SFTPB cDNA (AAV-hSPB) can improve survival in a mouse model of SP-B deficiency. However, the effect of this gene therapy wanes. Gene therapy efficacy could be prolonged if AAV vectors were able to be redosed, but readministering vectors is hindered by an immune response which includes toll like receptor 9 (TLR9) recognition of unmethylated CpG DNA motifs in the AAV genome. One strategy to mitigate TLR9 recognition of AAV is to incorporate decoy nucleotide sequences within the AAV genome. This work examined if AAV containing these TLR9 inhibitory oligonucleotide sequences (AAV-hSPBTLR9i) could mitigate the immune response sufficiently to redose AAV in the lungs and prolong the survival of SP-B deficient mice. Indeed, AAV-hSPBTLR9i was able to be redosed multiple times which significantly improved survival in our mouse model. This was partially a result of long-term increased SFTPB RNA and SP-B protein expression. Conversely, redosing AAV-hSPB resulted in the rapid death of SP-B deficient mice after the second AAV dose. TLR9 inhibition enabled readministration by avoiding the broad stimulation of genes belonging to multiple pathways in the host immune and inflammatory responses, including components of the interferon pathways. Thus, redosing of AAV vectors in the lungs using TLR9 inhibitory sequences is a promising strategy for prolonging gene therapy efficacy, with a proof-of-concept for AAV readministration in a clinically relevant mouse model of SP-B deficiency.

Innovations in RNA therapeutics: a review of recent advances and emerging technologies.

The field of biomedical science has witnessed another milestone with the advent of RNA-based therapeutics. This review explores three major RNA molecules, namely: messenger RNA (mRNA), RNA interference technology (RNAi), and Antisense Oligonucleotide (ASO), and analyses U.S. Food and Drug Administration drugs from 14 RNA-based pharmaceutical companies in terms of targeted genes, diseases and types, clinical trials and status, the mode of delivery, and the year of production. Many of such drugs are clinically approved or pending approval by the U.S. Food and Drug Administration (FDA) alongside other leading drugs agencies. Regarding diseases, this article emphasizes cancer therapy, genetic diseases, viral infections, and two categories of drug delivery systems include viral vectors and nanoparticles. Despite the tremendous progress made, key issues associated with these delivery systems are stability, off-target activities of RNA payloads, efficiency in cellular uptake, and the innovative need for engineering techniques for modifications. This review emphasizes the transformative potential of RNA therapeutics and the role of innovative technologies in addressing clinical needs, paving the way for a new era in precision medicine.

Trophectoderm-specific gene manipulation using adeno-associated viral vectors.

In mammals, blastocyst-stage trophectoderm (TE) contacts the maternal body at the time of implantation and forms the placenta after implantation, which supports the development of the fetus. Studying gene function in TE and placenta is important to understand normal implantation and pregnancy processes and their dysfunction. However, genetically modified mice are commonly generated by manipulating pronuclear-stage zygotes, which modify both the genome of the fetus and the placenta. Therefore, we previously developed TE/placenta-specific gene expression technology by transducing blastocysts with lentiviral vectors. However, the zona pellucida (ZP) needed to be removed before transduction. In this study, we examined various adeno-associated viral (AAV) vectors to develop a new TE/placenta-specific gene transduction method. As AAV1 can path through ZP, we succeeded in trophoblast-specific gene expression without ZP removal. Furthermore, TE cells genetically modified by AAV1-Cre contributed uniformly to the placenta. Our new technology contributes to advances in implantation and placenta research and leads to the development of new assisted reproductive technology.

Insights into eye genetics and recent advances in ocular gene therapy.

The rapid advancements in the field of genetics have significantly propelled the development of gene therapies, paving the way for innovative treatments of various hereditary disorders. This review focuses on the genetics of ophthalmologic conditions, highlighting the currently approved ophthalmic gene therapy and exploring emerging therapeutic strategies under development. Inherited retinal dystrophies represent a heterogeneous group of genetic disorders that manifest across a broad spectrum from infancy to late middle age. Key clinical features include nyctalopia (night blindness), constriction of the visual field, impairments in color perception, reduced central visual acuity, and rapid eye movements. Recent technological advancements, such as multimodal imaging, psychophysical assessments, and electrophysiological testing, have greatly enhanced our ability to understand disease progression and establish genotype-phenotype correlations. Additionally, the integration of molecular diagnostics into clinical practice is revolutionizing patient stratification and the design of targeted interventions, underscoring the transformative potential of personalized medicine in ophthalmology. The review also covers the challenges and opportunities in developing gene therapies for other ophthalmic conditions, such as age-related macular degeneration and optic neuropathies. We discuss the viral and non-viral vector systems used in ocular gene therapy, highlighting their advantages and limitations. Additionally, we explore the potential of emerging technologies like CRISPR/Cas9 in treating genetic eye diseases. We briefly address the regulatory landscape, concerns, challenges, and future directions of gene therapy in ophthalmology. We emphasize the need for long-term safety and efficacy data as these innovative treatments move from bench to bedside.

Heparan sulfate proteoglycan affinity of adeno-associated virus vectors: Implications for retinal gene delivery.

Adeno-associated virus (AAV)-based vectors have emerged as an effective and widely used technology for somatic gene therapy approaches, including those targeting the retina. A major advantage of the AAV technology is the availability of a large number of serotypes that have either been isolated from nature or produced in the laboratory. These serotypes have different properties in terms of sensitivity to neutralizing antibodies, cellular transduction profile and efficiency. The infectivity of AAV vectors depends on the affinity to certain molecules on the cell surface, in particular to cellular glycosaminoglycans (GAGs) such as heparan sulfate proteoglycans (HSPGs). Here, we tested how altering HSPG affinity in AAV vectors affects cellular tropism and transduction efficiency. The previously developed AAV2.GL variant was used as a starting variant to alter or disrupt HSPG affinity. The HSPG-independent AAV9 serotype was used to introduce different HSPG-binding sites. As an indicator of HSPG affinity, we measured the binding strength of the vector variant on a heparin chromatography column. We show that modification of capsid-exposed residues has a strong impact on HSPG affinity, cellular tropism and transduction efficiency in HeLa cells and in vivo in mouse retina. Our study shows that key properties of AAV vectors can be tailored in different directions and used to improve tropism and efficiency.

Truncated NS1 Influenza A Virus Induces a Robust Antigen-Specific Tissue-Resident T-Cell Response and Promotes Inducible Bronchus-Associated Lymphoid Tissue Formation in Mice

Background: Influenza viruses with truncated NS1 proteins show promise as viral vectors and candidates for mucosal universal influenza vaccines. These mutant NS1 viruses, which lack the N-terminal half of the NS1 protein (124 a.a.), are unable to antagonise the innate immune response. This creates a self-adjuvant effect enhancing heterologous protection by inducing a robust CD8+ T-cell response together with immunoregulatory mechanisms. However, the effects of NS1 modifications on T-follicular helper (Tfh) and B-cell responses remain less understood. Methods: C57bl/6 mice were immunised intranasally with 10 μL of either an influenza virus containing a truncated NS1 protein (PR8/NS124), a cold-adapted influenza virus with a full-length NS1 (caPR8/NSfull), or a wild-type virus (PR8/NSfull). Immune responses were assessed on days 8 and 28 post-immunisation by flow cytometry, ELISA, and HAI assay. Results: In this study, we demonstrate that intranasal immunisation with PR8/NS124 significantly increases tissue-resident CD4+ and CD8+ T cells in the lungs and activates Tfh cells in regional lymph nodes as early as day 8 post-immunisation. These effects are not observed in mice immunised with caPR8/NSfull or PR8/NSfull. Notably, PR8/NS124 immunisation also leads to the development of inducible bronchus-associated lymphoid tissue (iBALT) in the lungs by day 28, characterised by the presence of antigen-specific Tfh cells and GL7+Fas+ germinal centre B cells. Conclusions: Our findings further underscore the potential of NS1-truncated influenza viruses to drive robust mucosal immune responses and enhance vaccine efficacy.

Safety and efficacy of valoctocogene roxaparvovec with prophylactic glucocorticoids: 1-year results from the phase 3b, single-arm, open-label GENEr8-3 study.

Valoctocogene roxaparvovec, an adeno-associated virus vector that transfers a human factor VIII (FVIII) coding sequence to hepatocytes, provides bleeding protection for people with severe hemophilia A (HA). Determine the efficacy and safety of valoctocogene roxaparvovec with concomitant prophylactic glucocorticoids in the open-label, single-arm, phase 3b GENEr8-3 trial. Participants with severe HA who were using HA prophylaxis received one 6x1013 vg/kg infusion of valoctocogene roxaparvovec concomitantly with daily prophylactic glucocorticoids (40 mg prednisolone equivalent/d weeks 0‒8; taper to 5 mg/d weeks 9‒19). The primary efficacy endpoint was change from baseline in FVIII activity (chromogenic substrate assay) at week 52. Secondary efficacy endpoints included annualized rate of FVIII use and annualized bleeding rate (ABR) for treated bleeds. Safety was assessed by adverse events (AEs). Analysis populations were intent-to-treat (ITT; received valoctocogene roxaparvovec) for safety analyses and modified ITT (mITT; ≥52 FVIII infusions in the year before dosing) for efficacy analyses. Overall, 22 participants with severe HA received valoctocogene roxaparvovec. In the mITT population (n = 21), mean week 52 FVIII activity increased from baseline (imputed as 1 IU/dL) to 16.1 IU/dL (standard deviation [SD], 22.4; P = 0.0057); post-HA prophylaxis, mean treated ABR and mean annualized FVIII use decreased 67.1% and 91.6% from baseline, respectively (P <0.05). The most common AE was alanine aminotransferase elevation (20/22 participants). Glucocorticoid-related AEs occurred in 19/22 participants. No participants discontinued the study. Based on cross-trial comparisons, prophylactic glucocorticoids do not confer safety or efficacy benefits compared with reactive glucocorticoid regimens.