Announcements

“Zoonotic Animal Influenza Virus and Potential Mixing Vessel Hosts”
by Elsayed M. Abdelwhab and Thomas C. Mettenleiter
Viruses 2023, 15(4), 980; https://doi.org/10.3390/v15040980
Available online: https://www.mdpi.com/1999-4915/15/4/980


“The Molecular Epidemiology of Clade 2.3.4.4B H5N1 High Pathogenicity Avian Influenza in Southern Africa, 2021–2022”
by Celia Abolnik, Thandeka Phiri, Belinda Peyrot, Renee de Beer, Albert Snyman, David Roberts, Katrin Ludynia, Frances Jordaan, Michele Maartens, Zehaad Ismail et al.
Viruses 2023, 15(6), 1383; https://doi.org/10.3390/v15061383
Available online: https://www.mdpi.com/1999-4915/15/6/1383

“The Molecular Epidemiology of Clade 2.3.4.4B H5N1 High Pathogenicity Avian Influenza in Southern Africa, 2021–2022”
by Celia Abolnik, Thandeka Phiri, Belinda Peyrot, Renee de Beer, Albert Snyman, David Roberts, Katrin Ludynia, Frances Jordaan, Michele Maartens, Zehaad Ismail et al.
Viruses 2023, 15(6), 1383; https://doi.org/10.3390/v15061383
Available online: https://www.mdpi.com/1999-4915/15/6/1383

“Disappearance and Re-Emergence of Influenza during the COVID-19 Pandemic: Association with Infection Control Measures”
by Hikaru Takeuchi and Ryuta Kawashima
Viruses 2023, 15(1), 223; https://doi.org/10.3390/v15010223
Available online: https://www.mdpi.com/1999-4915/15/1/223

“Spreading of the High-Pathogenicity Avian Influenza (H5N1) Virus of Clade 2.3.4.4b into Uruguay”
by Ana Marandino, Gonzalo Tomás, Yanina Panzera, Carmen Leizagoyen, Ramiro Pérez, Lucía Bassetti, Raúl Negro, Sirley Rodríguez and Ruben Pérez
Viruses 2023, 15(9), 1906; https://doi.org/10.3390/v15091906
Available online: https://www.mdpi.com/1999-4915/15/9/1906

“Different Infectivity and Transmissibility of H5N8 and H5N1 High Pathogenicity Avian Influenza Viruses Isolated from Chickens in Japan in the 2021/2022 Season”
by Yoshihiro Takadate, Ryota Tsunekuni, Asuka Kumagai, Junki Mine, Yuto Kikutani, Saki Sakuma, Kohtaro Miyazawa and Yuko Uchida
Viruses 2023, 15(2), 265; https://doi.org/10.3390/v15020265
Available online: https://www.mdpi.com/1999-4915/15/2/265

“Avian Influenza Virus Tropism in Humans”
by Umarqayum AbuBakar, Lina Amrani, Farah Ayuni Kamarulzaman, Saiful Anuar Karsani, Pouya Hassandarvish and Jasmine Elanie Khairat
Viruses 2023, 15(4), 833; https://doi.org/10.3390/v15040833
Available online: https://www.mdpi.com/1999-4915/15/4/833

“Multi-Influenza HA Subtype Protection of Ferrets Vaccinated with an N1 COBRA-Based Neuraminidase”
by Amanda L. Skarlupka, Xiaojian Zhang, Uriel Blas-Machado, Spencer F. Sumner and Ted M. Ross
Viruses 2023, 15(1), 184; https://doi.org/10.3390/v15010184
Available online: https://www.mdpi.com/1999-4915/15/1/184

“Determining the Protective Efficacy of Toll-Like Receptor Ligands to Minimize H9N2 Avian Influenza Virus Transmission in Chickens”
by Sugandha Raj, Mohammadali Alizadeh, Bahram Shoojadoost, Douglas Hodgins, Éva Nagy, Samira Mubareka, Khalil Karimi, Shahriar Behboudi and Shayan Sharif
Viruses 2023, 15(1), 238; https://doi.org/10.3390/v15010238
Available online: https://www.mdpi.com/1999-4915/15/1/238

“Experimental Pathogenicity of H9N2 Avian Influenza Viruses Harboring a Tri-Basic Hemagglutinin Cleavage Site in Sonali and Broiler Chickens”
by Jahan Ara Begum, Ismail Hossain, Mohammed Nooruzzaman, Jacqueline King, Emdadul Haque Chowdhury, Timm C. Harder and Rokshana Parvin
Viruses 2023, 15(2), 461; https://doi.org/10.3390/v15020461
Available online: https://www.mdpi.com/1999-4915/15/2/461

“Association between Temperature and Influenza Activity across Different Regions of China during 2010–2017”
by Dina Wang, Hao Lei, Dayan Wang, Yuelong Shu and Shenglan Xiao
Viruses 2023, 15(3), 594; https://doi.org/10.3390/v15030594
Available online: https://www.mdpi.com/1999-4915/15/3/594

1. “Topoisomerase II as a Novel Antiviral Target against Panarenaviral Diseases”
by Tosin Oladipo Afowowe, Yasuteru Sakurai, Shuzo Urata, Vahid Rajabali Zadeh and Jiro Yasuda
Viruses 2023, 15(1), 105; https://doi.org/10.3390/v15010105
Available online: https://www.mdpi.com/1999-4915/15/1/105

2. “Differential Cellular Sensing of Fusion from within and Fusion from without during Virus Infection”
by David N. Hare, Tetyana Murdza, Susan Collins, Katharina Schulz, Subhendu Mukherjee, Roberto de Antueno, Luke Janssen, Roy Duncan and Karen L. Mossman
Viruses 2023, 15(2), 301; https://doi.org/10.3390/v15020301
Available online: https://www.mdpi.com/1999-4915/15/2/301

3. “SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128”
by Steven Kopcho, Marina McDew-White, Wasifa Naushad, Mahesh Mohan and Chioma M. Okeoma
Viruses 2023, 15(3), 622; https://doi.org/10.3390/v15030622
Available online: https://www.mdpi.com/1999-4915/15/3/622

4. “Antiviral Activity of Acetylsalicylic Acid against Bunyamwera Virus in Cell Culture”
by Sara Yolanda Fernández-Sánchez, José P. Cerón-Carrasco, Cristina Risco and Isabel Fernández de Castro
Viruses 2023, 15(4), 948; https://doi.org/10.3390/v15040948
Available online: https://www.mdpi.com/1999-4915/15/4/948

5. “Immune Prophylaxis Targeting the Respiratory Syncytial Virus (RSV) G Protein”
by Harrison C. Bergeron, Jackelyn Murray, Aakash Arora, Ana M. Nuñez Castrejon, Rebecca M. DuBois, Larry J. Anderson, Lawrence M. Kauvar and Ralph A. Tripp
Viruses 2023, 15(5), 1067; https://doi.org/10.3390/v15051067
Available online: https://www.mdpi.com/1999-4915/15/5/1067

6. “Novel Mode of nanoLuciferase Packaging in SARS-CoV-2 Virions and VLPs Provides Versatile Reporters for Virus Production”
by Rebekah C. Gullberg and Judith Frydman
Viruses 2023, 15(6), 1335; https://doi.org/10.3390/v15061335
Available online: https://www.mdpi.com/1999-4915/15/6/1335

7. “Structure and Function of Hoc—A Novel Environment Sensing Device Encoded by T4 and Other Bacteriophages”
by Andrei Fokine, Mohammad Zahidul Islam, Qianglin Fang, Zhenguo Chen, Lei Sun and Venigalla B. Rao
Viruses 2023, 15(7), 1517; https://doi.org/10.3390/v15071517
Available online: https://www.mdpi.com/1999-4915/15/7/1517

8. “A Reverse-Transcription Loop-Mediated Isothermal Amplification Technique to Detect Tomato Mottle Mosaic Virus, an Emerging Tobamovirus”
by Kan Kimura, Akio Miyazaki, Takumi Suzuki, Toya Yamamoto, Yugo Kitazawa, Kensaku Maejima, Shigetou Namba and Yasuyuki Yamaji
Viruses 2023, 15(8), 1688; https://doi.org/10.3390/v15081688
Available online: https://www.mdpi.com/1999-4915/15/8/1688

9. “The Human Cytomegalovirus Latency-Associated Gene Product Latency Unique Natural Antigen Regulates Latent Gene Expression”
by Emma Poole, Jonathan Lau, Ian Groves, Kate Roche, Eain Murphy, Maria Carlan da Silva, Matthew Reeves and John Sinclair
Viruses 2023, 15(9), 1875; https://doi.org/10.3390/v15091875
Available online: https://www.mdpi.com/1999-4915/15/9/1875

10. “Effect of Interferon Gamma on Ebola Virus Infection of Primary Kupffer Cells and a Kupffer Cell Line”
by José A. Aguilar-Briseño, Jonah M. Elliff, Justin J. Patten, Lindsay R. Wilson, Robert A. Davey, Adam L. Bailey and Wendy J. Maury
Viruses 2023, 15(10), 2077; https://doi.org/10.3390/v15102077
Available online: https://www.mdpi.com/1999-4915/15/10/2077

11. “ZBP1 Drives IAV-Induced NLRP3 Inflammasome Activation and Lytic Cell Death, PANoptosis, Independent of the Necroptosis Executioner MLKL”
by R. K. Subbarao Malireddi, Bhesh Raj Sharma, Ratnakar R. Bynigeri, Yaqiu Wang, Jianlin Lu and Thirumala-Devi Kanneganti
Viruses 2023, 15(11), 2141; https://doi.org/10.3390/v15112141
Available online: https://www.mdpi.com/1999-4915/15/11/2141

12. “Tetherin Restricts SARS-CoV-2 despite the Presence of Multiple Viral Antagonists”
by Elena Hagelauer, Rishikesh Lotke, Dorota Kmiec, Dan Hu, Mirjam Hohner, Sophie Stopper, Rayhane Nchioua, Frank Kirchhoff, Daniel Sauter and Michael Schindler
Viruses 2023, 15(12), 2364; https://doi.org/10.3390/v15122364
Available online: https://www.mdpi.com/1999-4915/15/12/2364

More than 100 German universities and research institutions have entered into a national agreement with MDPI. The publication agreement negotiated by ZB MED comes into effect on 1 January 2025 and is valid until the end of 2026. Joining the consortium is still possible until the beginning of 2025. We are delighted by the high level of interest this agreement has already garnered, reflecting the strong demand for accessible and cost-effective open access publishing solutions among German research institutions.

This new cooperation aims to make scientific gold open access publishing more affordable and less administratively burdensome for researchers in Germany. The agreement includes substantial discounts on article processing charges (APCs) for corresponding authors from participating institutions. It offers flexible payment options, including centralized invoicing or individual payment of fees by researchers or their institutions. Additionally, the agreement features a flat-fee model that enables institutions to precisely plan expenses and optimize their library budgets.

"MDPI can look back on over a decade of successful partnerships with German research institutions," says Peter Roth, MDPI Head of Publishing. "The new agreement marks another milestone in the long-standing co-operation between MDPI and the German scientific community. It emphasizes our commitment to developing up-to-date and inventive solutions for the diverse needs of scientific institutions to promote open research for the benefit of researchers."

Petra Labriga, Head of Strategic License Management at ZB MED, highlighted the agreement's significance: "As one of the world's leading Gold OA publishers, MDPI plays a central role in the German publishing landscape. We are particularly pleased that we were able to achieve considerable potential cost savings for scientific institutions and their authors at a national level through our negotiations."

The partnership reflects a common goal of advancing the idea of open access and supporting researchers in making their scientific excellence internationally visible.

"We would like to thank the ZB MED consortium team for their excellent collaboration," added Adrian Stefan Zamfir, MDPI Institutional Partnership Manager for the DACH region. "We are delighted that this agreement will give even more researchers in Germany access to our tried-and-tested and reliable publication platform."

Franziska Fischer (right), Commercial Director at ZB MED, an Peter Roth (left), Head of Publishing at MDPI, celebrate the signing of the new national open access agreement between MDPI and the ZB MED Consortium.

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About ZB MED

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More information at www.zbmed.de.

About MDPI

Headquartered in Basel, Switzerland, MDPI is one of the world's leading open access publishers with a current portfolio of more than 440 journals in all scientific disciplines. MDPI‘s goal is to advance open science worldwide through greater transparency, efficiency and collaboration. To date, more than 3.7 million researchers have published their results in MDPI journals. The editorial process is overseen by a large network of dedicated reviewers and editors and supported by more than 6500 MDPI employees. MDPI currently works with over 800 academic institutions and 180 scientific societies worldwide, which benefit from a wide range of MDPI services and products.

We are pleased to announce a series of updates to our template, aimed at improving the readability and overall aesthetics of our publications. These changes have been meticulously designed to enhance the user experience and ensure consistency across all our publications. The updated template will be available for download from the Instructions for Authors page.

The following updates will be applied to articles published in the 2025 volumes, starting on 24 December 2024:

• Main text: The line spacing has been increased to improve the readability of publications;
• Header and footer: The link to the journal website will be removed, as a hyperlink has been integrated into the journal logo. Additionally, the DOI link will be moved from the left-hand side to the right-hand side, and both the header and footer will be slightly raised to achieve a better balance;
• Left information bar: The information provided here has been rearranged for better organization; the CC-BY logo will be removed;
• Font size: The font size used for the abstract, keywords, and first-level headings will be increased.

Furthermore, MDPI journals will continue to use article numbers. This approach enables us to maintain a rapid and efficient production process by being able to define pagination as soon as a paper is accepted.

We hope that the new version of the template will provide users with a better experience and make the process more convenient.

Please contact production@mdpi.com if you have any questions or suggestions.

1. “SARS-CoV-2 T Cell Responses Elicited by COVID-19 Vaccines or Infection Are Expected to Remain Robust against Omicron”
by Syed Faraz Ahmed, Ahmed Abdul Quadeer and Matthew R. McKay
Viruses 2022, 14(1), 79; https://doi.org/10.3390/v14010079
Available online: https://www.mdpi.com/1999-4915/14/1/79

2. “Structural Assembly of Qβ Virion and Its Diverse Forms of Virus-like Particles”
by Jeng-Yih Chang, Karl V. Gorzelnik, Jirapat Thongchol and Junjie Zhang
Viruses 2022, 14(2), 225; https://doi.org/10.3390/v14020225
Available online: https://www.mdpi.com/1999-4915/14/2/225

3. “Mapping of Antibody Epitopes on the Crimean-Congo Hemorrhagic Fever Virus Nucleoprotein”
by Boniface Pongombo Lombe, Takeshi Saito, Hiroko Miyamoto, Akina Mori-Kajihara, Masahiro Kajihara, Masayuki Saijo, Justin Masumu, Takanari Hattori, Manabu Igarashi and Ayato Takada
Viruses 2022, 14(3), 544; https://doi.org/10.3390/v14030544
Available online: https://www.mdpi.com/1999-4915/14/3/544

4. “Structural Insight into KsBcl-2 Mediated Apoptosis Inhibition by Kaposi Sarcoma Associated Herpes Virus”
by Chathura D. Suraweera, Mark G. Hinds and Marc Kvansakul
Viruses 2022, 14(4), 738; https://doi.org/10.3390/v14040738
Available online: https://www.mdpi.com/1999-4915/14/4/738

5. “SARS-CoV-2 Causes Lung Inflammation through Metabolic Reprogramming and RAGE”
by Charles N. S. Allen, Maryline Santerre, Sterling P. Arjona, Lea J. Ghaleb, Muna Herzi, Megan D. Llewellyn, Natalia Shcherbik and Bassel E. Sawaya
Viruses 2022, 14(5), 983; https://doi.org/10.3390/v14050983
Available online: https://www.mdpi.com/1999-4915/14/5/983

6. “Evidence of RedOX Imbalance during Zika Virus Infection Promoting the Formation of Disulfide-Bond-Dependent Oligomers of the Envelope Protein”
by Grégorie Lebeau, Jonathan Turpin, Etienne Frumence, Daed El Safadi, Wissal Harrabi, Philippe Desprès, Pascale Krejbich-Trotot and Wildriss Viranaïcken
Viruses 2022, 14(6), 1131; https://doi.org/10.3390/v14061131
Available online: https://www.mdpi.com/1999-4915/14/6/1131

7. “Phosphomimetic S207D Lysyl–tRNA Synthetase Binds HIV-1 5′UTR in an Open Conformation and Increases RNA Dynamics”
by William A. Cantara, Chathuri Pathirage, Joshua Hatterschide, Erik D. Olson and Karin Musier-Forsyth
Viruses 2022, 14(7), 1556; https://doi.org/10.3390/v14071556
Available online: https://www.mdpi.com/1999-4915/14/7/1556

8. “TRIM7 Restricts Coxsackievirus and Norovirus Infection by Detecting the C-Terminal Glutamine Generated by 3C Protease Processing”
by Jakub Luptak, Donna L. Mallery, Aminu S. Jahun, Anna Albecka, Dean Clift, Osaid Ather, Greg Slodkowicz, Ian Goodfellow and Leo C. James
Viruses 2022, 14(8), 1610; https://doi.org/10.3390/v14081610
Available online: https://www.mdpi.com/1999-4915/14/8/1610

9. “Evaluation of Virulence in Cynomolgus Macaques Using a Virus Preparation Enriched for the Extracellular Form of Monkeypox Virus”
by Eric M. Mucker, Josh D. Shamblin, Arthur J. Goff, Todd M. Bell, Christopher Reed, Nancy A. Twenhafel, Jennifer Chapman, Marc Mattix, Derron Alves, Robert F. Garry and Lisa E. Hensley
Viruses 2022, 14(9), 1993; https://doi.org/10.3390/v14091993
Available online: https://www.mdpi.com/1999-4915/14/9/1993

10. “Biophysical Modeling of SARS-CoV-2 Assembly: Genome Condensation and Budding”
by Siyu Li and Roya Zandi
Viruses 2022, 14(10), 2089; https://doi.org/10.3390/v14102089
Available online: https://www.mdpi.com/1999-4915/14/10/2089

11. “Integrated Omics Reveal Time-Resolved Insights into T4 Phage Infection of E. coli on Proteome and Transcriptome Levels”
by Maik Wolfram-Schauerte, Nadiia Pozhydaieva, Madita Viering, Timo Glatter and Katharina Höfer
Viruses 2022, 14(11), 2502; https://doi.org/10.3390/v14112502
Available online: https://www.mdpi.com/1999-4915/14/11/2502

12. “Novel 3′ Proximal Replication Elements in Umbravirus Genomes”
by Philip Z. Johnson, Hannah M. Reuning, Sayanta Bera, Feng Gao, Zhiyou Du and Anne E. Simon
Viruses 2022, 14(12), 2615; https://doi.org/10.3390/v14122615
Available online: https://www.mdpi.com/1999-4915/14/12/2615

We are thrilled to announce a successful series of webinars organized by a Viruses-affiliated society—the Global Virus Network (GVN). The Global Virus Network is an essential and critical defense against viral disease. It is a coalition comprised of leading virologists around the world working to advance knowledge about how viruses cause us to be ill and to develop drugs and vaccines to lower the likelihood and severity of illness, as well as to prevent death.

On 26 November 2024, the GVN collaborated with Dr. Tony Schountz to present a webinar titled “Jamaican Fruit Bats as an Experimental Model for Virology and Immunology”. Dr. Tony Schountz, Ph.D., is a professor at the Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, at Colorado State University, USA. He is an accomplished virologist and immunologist with expertise in the study of virus–host interactions in reservoir species.

Following the mentioned collaboration, on 10 December 2024, the GVN welcomed Dr. Esteban Domingo for a fascinating exploration of SARS-CoV-2 genomes. The webinar, titled “Quasispecies and error catastrophe: the “be or not to be” of SARS-CoV-2 genomes”, delved into the complexity and variability of SARS-CoV-2 genomes, providing valuable knowledge and insights to the attendees. Keep an eye on their webpage for their next webinars!

Viruses (ISSN: 1999-4915) would like to congratulate the Global Virus Network for the success of their webinars and their great impact on as well as contribution to science. Viruses also cooperates with other societies, such as the American Society for Virology (ASV), the Spanish Society for Virology (SEV), the Canadian Society for Virology (CSV), the Italian Society for Virology (SIV-ISV), the Australasian Virology Society (AVS), and others affiliated with Viruses. For details, please visit https://www.mdpi.com/journal/viruses/societies.

Authors: Kentaro Yamada¹, Di Cao¹, Ning Li¹, Roland W. Herzog¹, Dongsheng Duan², Sandeep R.P. Kumar¹ 

¹Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University, Indianapolis, IN, USA;
²Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, USA. 

Correspondence:
Sandeep R. P. Kumar, Ph.D.
Herman B Wells Center for Pediatric Research
Department of Pediatrics
Indiana University
1044 W. Walnut Street
Indianapolis, IN 46202, USA
E-mail: sankuma@iu.edu 

In the context of muscle gene delivery, we recently demonstrated in a murine model that the innate sensing of adeno-associated virus (AAV) vectors and their encoded transgenes in muscle tissue is quite complex and involves multiple innate sensors and associated pathways¹. Innate immune sensors recognize unique molecular structures associated with invading microbes and trigger a rapid cascade of events, which enables communication with the key players involved in adaptive immunity to mount an effective anti-microbial response. Though critical to fighting off invading microbes, innate immune responses are one of the major impediments to the success of viral vector-based gene therapies to treat genetic diseases. Among the various viral vectors, AAV-based vectors are preferred for in vivo gene delivery due to their high degree of tissue tropism and comparatively low immunogenic profile². However, the immunotoxicity observed in multiple clinical trials clearly indicates that AAV vectors are capable of activating the host immune system and rendering gene therapy ineffective. Despite the fact that multiple AAV-based gene therapy products have received regulatory approval, the immunogenicity of AAV and its derivative vectors remains incompletely understood. Over the past 15 years, multiple innate immune sensors such as toll-like receptor (TLR) 2 (AAV capsid sensing), TLR9 (AAV genome sensing), and MDA5-MAVS (dsRNA sensing) have been implicated in AAV immunogenicity³. Among these, TLR9 and the downstream adaptor molecule, myeloid differentiation primary response 88 (MyD88), are most firmly established as the drivers of anti-AAV responses. To prevent TLR9-mediated immune responses, strategies such as the removal of unmethylated CpG motifs (which are potential TLR9 agonists) or the incorporation of TLR9 inhibitory DNA sequences (such as inflammation-inhibiting oligonucleotide 2 (io2)) in the therapeutic gene expression cassette are being employed^4,5. Recently, we have uncovered a TLR9-independent innate sensing pathway that activates cellular immune responses to AAV-encoded transgene products in the liver⁶. This pathway instead utilizes cytokines IL-1a and IL-1b for the induction of IL-1R1–MyD88 signaling but does not rely on the inflammasome machinery (Fig. 1a). 

Figure 1. Innate immune sensors implicated in cellular response to AAV encoded transgene product following hepatic (a) and muscle (b) directed gene delivery. Strategies to prevent TLR9 activation such as depletion of unmethylated CpG motifs from AAV expression cassette are already in clinical use. TLR9 inhibitory sequences such as io2 can be incorporated in the expression cassette to further prevent TLR9 activation due to unmethylated CpG motifs in AAV ITRs. Anakinra, a recombinant version of naturally occurring IL-1 receptor antagonist (IL-1Ra) and Rilonacept, a fusion protein to neutralize IL-1a and IL-1b are already in clinics to treat other disease indications and can be repurposed for AAV gene therapy application. TLR3 antagonists are only available experimentally but not as approved medication. 

Skeletal muscle is one of the target tissues for AAV-mediated gene therapy to treat neuromuscular diseases⁷. Therefore, it is prudent to understand the mechanisms of AAV-driven immune responses following intramuscular gene delivery. Compared to hepatic gene transfer, the intramuscular route of vector administration is typically more prone to immune responses against the transgene product, including CD8⁺ T cell responses through TLR9>MyD88-mediated activation. In our study¹, we asked two questions. First, whether IL-1 signaling is important for CD8⁺ T cell activation against the transgene product expressed in the skeletal muscle, and second, whether additional innate sensing pathways are involved¹. To address these questions, we utilized a variety of strategies to dampen TLR9 activation (either by reducing the CpG content or the incorporation of io2 in the expression cassette) or to inhibit both TLR9 activation and IL-1R1 signaling. 

We utilized muscle-tropic AAV serotype 1 vectors that encoded chicken ovalbumin (OVA) under the control of the ubiquitous cytomegalovirus enhancer/human elongation factor-1α (CMV/EF1α) promoter or the muscle-specific promoter CK8. Additional AAV expression cassettes that either lacked CpG motifs or carried io2 alone or in combination with CpG depletion were also packaged in AAV1. In total, we evaluated five different AAV expression cassettes (Table 1). Two different doses of CMV/EF1a AAV1 vectors (2x10¹⁰ or 2x10¹¹ vector genomes/mouse) were injected into the quadriceps muscle of mice lacking individual innate sensors. In some experimental groups, IL-1 signaling was blocked by a combination of a-IL-1a and a-IL-1b antibodies. Flow cytometry was employed to monitor the kinetics of OVA-specific CD8⁺ T cells in peripheral blood, and an enzyme-linked immunosorbent assay (ELISA) was used to quantitate a-OVA IgGs in the plasma over 6 weeks. 

 Table 1: AAV constructs used in this study.

We first noticed a discrepancy in the need for TLR9 innate sensing between single-stranded (ss) and self-complimentary (sc) AAV vectors. The innate sensing of scAAV seemed to rely more strongly on TLR9 sensing. However, the innate sensing of ssAAV involved a number of different innate signaling pathways including TLR9, IL-1R1, and potentially TLR3 (Fig. 1b). The requirement for a single pathway was vector-dose-dependent. At a low dose, the inhibition of either of these signaling pathways significantly reduced the CD8⁺ T cell response against the transgene product. Interestingly, at a high vector dose, cellular responses were driven by a number of redundant pathways, including TLR9 and IL-1R1 signaling. Blocking one of these innate sensing pathways did not impact the response against the transgene product, whereas simultaneous blockade of both TLR9 and IL-1R1 signaling reduced the CD8⁺ T cell responses (Fig. 1b). Moreover, mice lacking downstream adaptor molecules such as MyD88 and TBK1 had minimal CD8⁺ T cell responses. We also found evidence for the involvement of RNA innate sensing via the TLR3-TRIF pathway. Taken together, these results clearly indicate that multiple innate pathways play a role in driving cellular responses during intramuscular gene transfer. Nevertheless, we did not find evidence of cytoplasmic DNA or RNA sensors being required, with the caveat that these results were entirely based on experiments performed in knockout mice (which may have skewed the results and therefore, they are not entirely conclusive). 

We examined whether genome engineering by CpG depletion or the incorporation of io2 in the AAV expression cassette would affect the immune response at a high dose. Interestingly, individually, these modifications had no impact. However, the presence of io2 in the AAV expression cassette in combination with the inhibition of IL-1R1 signaling was effective in reducing (but not preventing) the CD8⁺ T cell response against the transgene product. Surprisingly, the combined use of CpG depletion and IL-1R1 blockade failed to have a similar effect. Combining CpG depletion with io2 in a single AAV expression cassette reduced the CD8⁺ T cell response (which, however, was not further reduced when combined with IL-1R1 inhibition). These results illustrate the complexities and challenges of eliminating CD8⁺ T cell activation through innate immune blockade at high intramuscular vector doses. Even very low frequencies of CD8⁺ T cell responses in peripheral blood were associated with a loss of expression in the muscle. Nonetheless, we ultimately succeeded in achieving sustained transgene expression in skeletal muscle with minimal circulating or infiltrating CD8⁺ T cells when using a muscle-specific promoter along with CpG depletion and inclusion of the io2 sequence (raising the question of whether io2 inhibits additional pathways besides TLR9). 

In conclusion, we demonstrated the dose-dependent innate sensing of AAV-encoded transgene products following muscle gene transfer. Multiple innate signaling pathways (including TLR9, IL-1R1, and, potentially, TLR3) contribute to CD8⁺ T cell activation in AAV muscle gene transfer. Because of this redundancy, the blockade of multiple innate signaling pathways would be necessary to reduce CD8⁺ T cell responses against the transgene product. Further, we provided evidence that genome engineering (such as CpG motif removal or the incorporation of the TLR9 inhibitory sequence io2), could be beneficial in minimizing these cellular responses. Future work may reveal why the combination of TLR9 and IL-1R1 inhibition was effective in some but not other instances. It should be pointed out that blocking multiple innate signaling pathways, while helping to reduce CD8⁺ T cell responses, had no impact on antibody formation against the transgene product. Similarly, though the use of the muscle-specific promoter mitigated CD8⁺ T cell responses and provided long-lasting local transgene expression, this approach failed to prevent the antibody response. 

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