International Journal of Molecular Sciences

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3 pages, 162 KiB

Open AccessEditorial

Virus Engineering and Applications

by Laura Menotti

Int. J. Mol. Sci. 2023, 24(23), 16788; https://doi.org/10.3390/ijms242316788 - 27 Nov 2023

Cited by 1 | Viewed by 1248

Abstract

This Special Issue highlights multiple facets of virus engineering, ranging from the dissection of the biological properties of individual viral functions in the context of safe genomic backbones, virus genetic modification for applications in gene therapy, oncolytic virotherapy and vaccine production, to the [...] Read more.

This Special Issue highlights multiple facets of virus engineering, ranging from the dissection of the biological properties of individual viral functions in the context of safe genomic backbones, virus genetic modification for applications in gene therapy, oncolytic virotherapy and vaccine production, to the hurdles presented by quality control and the delivery of viruses for their final applications and finally to the simulation, prediction and validation of virus evolution [...] Full article

(This article belongs to the Special Issue Virus Engineering and Applications)

Research

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15 pages, 2051 KiB

Open AccessArticle

Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses

by Mariana V. Ferreira, Sofia Fernandes, Ana Isabel Almeida, Salomé Neto, João P. Mendes, Ricardo J. S. Silva, Cristina Peixoto and Ana Sofia Coroadinha

Int. J. Mol. Sci. 2023, 24(13), 10524; https://doi.org/10.3390/ijms241310524 - 23 Jun 2023

Cited by 1 | Viewed by 2673

Abstract

Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for diseases requiring large-gene delivery. To overcome this, dual-AAV vector systems that rely on protein trans-splicing were developed, with the split-intein Npu [...] Read more.

Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for diseases requiring large-gene delivery. To overcome this, dual-AAV vector systems that rely on protein trans-splicing were developed, with the split-intein Npu DnaE among the most-used. However, the reconstitution efficiency of Npu DnaE is still insufficient, requiring higher vector doses. In this work, two split-inteins, Cfa and Gp41-1, with reportedly superior trans-splicing were evaluated in comparison with Npu DnaE by transient transfections and dual-AAV in vitro co-transductions. Both Cfa and Gp41-1 split-inteins enabled reconstitution rates that were over two-fold higher than Npu DnaE and 100% of protein reconstitution. The impact of different vector preparation qualities in split-intein performances was also evaluated in co-transduction assays. Higher-quality preparations increased split-inteins’ performances by three-fold when compared to low-quality preparations (60–75% vs. 20–30% full particles, respectively). Low-quality vector preparations were observed to limit split-gene reconstitutions by inhibiting co-transduction. We show that combining superior split-inteins with higher-quality vector preparations allowed vector doses to be decreased while maintaining high trans-splicing rates. These results show the potential of more-efficient protein-trans-splicing strategies in dual-AAV vector co-transduction, allowing the extension of its use to the delivery of larger therapeutic genes. Full article

(This article belongs to the Special Issue Virus Engineering and Applications)

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19 pages, 8915 KiB

Open AccessArticle

Human Monocytes Are Suitable Carriers for the Delivery of Oncolytic Herpes Simplex Virus Type 1 In Vitro and in a Chicken Embryo Chorioallantoic Membrane Model of Cancer

by Alberto Reale, Lea Krutzke, Massimiliano Cadamuro, Adriana Vitiello, Jens von Einem, Stefan Kochanek, Giorgio Palù, Cristina Parolin and Arianna Calistri

Int. J. Mol. Sci. 2023, 24(11), 9255; https://doi.org/10.3390/ijms24119255 - 25 May 2023

Cited by 7 | Viewed by 2152

Abstract

Oncolytic viruses (OVs) are promising therapeutics for tumors with a poor prognosis. An OV based on herpes simplex virus type 1 (oHSV-1), talimogene laherparepvec (T-VEC), has been recently approved by the Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) [...] Read more.

Oncolytic viruses (OVs) are promising therapeutics for tumors with a poor prognosis. An OV based on herpes simplex virus type 1 (oHSV-1), talimogene laherparepvec (T-VEC), has been recently approved by the Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for the treatment of unresectable melanoma. T-VEC, like most OVs, is administered via intratumoral injection, underlining the unresolved problem of the systemic delivery of the oncolytic agent for the treatment of metastases and deep-seated tumors. To address this drawback, cells with a tropism for tumors can be loaded ex vivo with OVs and used as carriers for systemic oncolytic virotherapy. Here, we evaluated human monocytes as carrier cells for a prototype oHSV-1 with a similar genetic backbone as T-VEC. Many tumors specifically recruit monocytes from the bloodstream, and autologous monocytes can be obtained from peripheral blood. We demonstrate here that oHSV-1-loaded primary human monocytes migrated in vitro towards epithelial cancer cells of different origin. Moreover, human monocytic leukemia cells selectively delivered oHSV-1 to human head-and-neck xenograft tumors grown on the chorioallantoic membrane (CAM) of fertilized chicken eggs after intravascular injection. Thus, our work shows that monocytes are promising carriers for the delivery of oHSV-1s in vivo, deserving further investigation in animal models. Full article

(This article belongs to the Special Issue Virus Engineering and Applications)

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22 pages, 3752 KiB

Open AccessArticle

Exploring FeLV-Gag-Based VLPs as a New Vaccine Platform—Analysis of Production and Immunogenicity

by Raquel Ortiz, Ana Barajas, Anna Pons-Grífols, Benjamin Trinité, Ferran Tarrés-Freixas, Carla Rovirosa, Victor Urrea, Antonio Barreiro, Anna Gonzalez-Tendero, Maria Cardona, Laura Ferrer, Bonaventura Clotet, Jorge Carrillo, Carmen Aguilar-Gurrieri and Julià Blanco

Int. J. Mol. Sci. 2023, 24(10), 9025; https://doi.org/10.3390/ijms24109025 - 19 May 2023

Cited by 4 | Viewed by 2402

Abstract

Feline leukemia virus (FeLV) is one of the most prevalent infectious diseases in domestic cats. Although different commercial vaccines are available, none of them provides full protection. Thus, efforts to design a more efficient vaccine are needed. Our group has successfully engineered HIV-1 [...] Read more.

Feline leukemia virus (FeLV) is one of the most prevalent infectious diseases in domestic cats. Although different commercial vaccines are available, none of them provides full protection. Thus, efforts to design a more efficient vaccine are needed. Our group has successfully engineered HIV-1 Gag-based VLPs that induce a potent and functional immune response against the HIV-1 transmembrane protein gp41. Here, we propose to use this concept to generate FeLV-Gag-based VLPs as a novel vaccine strategy against this retrovirus. By analogy to our HIV-1 platform, a fragment of the FeLV transmembrane p15E protein was exposed on FeLV-Gag-based VLPs. After optimization of Gag sequences, the immunogenicity of the selected candidates was evaluated in C57BL/6 and BALB/c mice, showing strong cellular and humoral responses to Gag but failing to generate anti-p15E antibodies. Altogether, this study not only tests the versatility of the enveloped VLP-based vaccine platform but also sheds light on FeLV vaccine research. Full article

(This article belongs to the Special Issue Virus Engineering and Applications)

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17 pages, 2241 KiB

Open AccessArticle

Engineering an Optimal Y280-Lineage H9N2 Vaccine Strain by Tuning PB2 Activity

by Se-Hee An, Seung-Min Hong, Jin-Ha Song, Seung-Eun Son, Chung-Young Lee, Kang-Seuk Choi and Hyuk-Joon Kwon

Int. J. Mol. Sci. 2023, 24(10), 8840; https://doi.org/10.3390/ijms24108840 - 16 May 2023

Cited by 1 | Viewed by 2162

Abstract

H9N2 avian influenza A viruses (AIVs) cause economic losses in the poultry industry and provide internal genomic segments for the evolution of H5N1 and H7N9 AIVs into more detrimental strains for poultry and humans. In addition to the endemic Y439/Korea-lineage H9N2 viruses, the [...] Read more.

H9N2 avian influenza A viruses (AIVs) cause economic losses in the poultry industry and provide internal genomic segments for the evolution of H5N1 and H7N9 AIVs into more detrimental strains for poultry and humans. In addition to the endemic Y439/Korea-lineage H9N2 viruses, the Y280-lineage spread to Korea since 2020. Conventional recombinant H9N2 vaccine strains, which bear mammalian pathogenic internal genomes of the PR8 strain, are pathogenic in BALB/c mice. To reduce the mammalian pathogenicity of the vaccine strains, the PR8 PB2 was replaced with the non-pathogenic and highly productive PB2 of the H9N2 vaccine strain 01310CE20. However, the 01310CE20 PB2 did not coordinate well with the hemagglutinin (HA) and neuraminidase (NA) of the Korean Y280-lineage strain, resulting in a 10-fold lower virus titer compared to the PR8 PB2. To increase the virus titer, the 01310CE20 PB2 was mutated (I66M-I109V-I133V) to enhance the polymerase trimer integrity with PB1 and PA, which restored the decreased virus titer without causing mouse pathogenicity. The reverse mutation (L226Q) of HA, which was believed to decrease mammalian pathogenicity by reducing mammalian receptor affinity, was verified to increase mouse pathogenicity and change antigenicity. The monovalent Y280-lineage oil emulsion vaccine produced high antibody titers for homologous antigens but undetectable titers for heterologous (Y439/Korea-lineage) antigens. However, this defect was corrected by the bivalent vaccine. Therefore, the balance of polymerase and HA/NA activities can be achieved by fine-tuning PB2 activity, and a bivalent vaccine may be more effective in controlling concurrent H9N2 viruses with different antigenicities. Full article

(This article belongs to the Special Issue Virus Engineering and Applications)

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15 pages, 2006 KiB

Open AccessArticle

Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches

by Megane K. Aebischer, Thomas Bouvarel, Emmalyn Barrozo, Dominik Kochardt, Carsten Elger, Markus Haindl, Raphael Ruppert, Davy Guillarme and Valentina D’Atri

Int. J. Mol. Sci. 2023, 24(10), 8503; https://doi.org/10.3390/ijms24108503 - 9 May 2023

Cited by 5 | Viewed by 3367

Abstract

The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins [...] Read more.

The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this study, the potential benefits and limitations of different electrophoretic and chromatographic methods were evaluated, including capillary electrophoresis–sodium dodecyl sulfate (CE-SDS), reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), for the analysis of VPs obtained from different serotypes (i.e., AAV2, AAV5, AAV8, and AAV9). CE-SDS is considered to be the reference method and provides a suitable separation of VP1-3 proteins using generic conditions and laser induced fluorescence detection. However, the characterization of post-translational modifications (i.e., phosphorylation, oxidation) remains difficult, and species identification is almost impossible due to the lack of compatibility between CE-SDS and mass spectrometry (MS). In contrast, RPLC and HILIC were found to be less generic than CE-SDS and require tedious optimization of the gradient conditions for each AAV serotype. However, these two chromatographic approaches are inherently compatible with MS, and were shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications. Finally, despite being non-denaturing, HIC offers disappointing performance for viral capsid proteins characterization. Full article

(This article belongs to the Special Issue Virus Engineering and Applications)

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13 pages, 3974 KiB

Open AccessArticle

Analysis of SARS-CoV-2 Spike Protein Variants with Recombinant Reporter Viruses Created from a Bacmid System

by Arne Cordsmeier, Doris Jungnickl, Alexandra Herrmann, Klaus Korn and Armin Ensser

Int. J. Mol. Sci. 2023, 24(9), 8156; https://doi.org/10.3390/ijms24098156 - 2 May 2023

Cited by 2 | Viewed by 1796

Abstract

SARS-CoV-2, the causative agent of COVID-19, has spread around the world with more than 700 million cases and 6.8 million deaths. Various variants of concern (VoC) have emerged due to mutations and recombination and concurrent selection for increased viral fitness and immune evasion. [...] Read more.

SARS-CoV-2, the causative agent of COVID-19, has spread around the world with more than 700 million cases and 6.8 million deaths. Various variants of concern (VoC) have emerged due to mutations and recombination and concurrent selection for increased viral fitness and immune evasion. The viral protein that primarily determines the pathogenicity, infectivity, and transmissibility is the Spike protein. To analyze the specific impact of variant Spike proteins on infection dynamics, we constructed SARS-CoV-2 with a uniform B.1 backbone but with alternative Spike proteins. In addition, ORF6 was replaced by EYFP as a biological safety measure, and for use of this well-established reporter. We show that namely the delta variant Spike proteins cause a distinct phenotype from the wild type (B.1, D614G) and other variants of concern. Furthermore, we demonstrate that the omicron BA.1 Spike results in lower viral loads and a less efficient spread in vitro. Finally, we utilized viruses with the two different reporters EYFP and mCherry to establish a competitive growth assay, demonstrating that most but not all Spike variant viruses were able to outcompete wild type SARS-CoV-2 B.1. Full article

(This article belongs to the Special Issue Virus Engineering and Applications)

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16 pages, 587 KiB

Open AccessArticle

Quantifying In-Host Quasispecies Evolution

by Josep Gregori, Marta Ibañez-Lligoña and Josep Quer

Int. J. Mol. Sci. 2023, 24(2), 1301; https://doi.org/10.3390/ijms24021301 - 9 Jan 2023

Cited by 2 | Viewed by 1767

Abstract

What takes decades, centuries or millennia to happen with a natural ecosystem, it takes only days, weeks or months with a replicating viral quasispecies in a host, especially when under treatment. Some methods to quantify the evolution of a quasispecies are introduced and [...] Read more.

What takes decades, centuries or millennia to happen with a natural ecosystem, it takes only days, weeks or months with a replicating viral quasispecies in a host, especially when under treatment. Some methods to quantify the evolution of a quasispecies are introduced and discussed, along with simple simulated examples to help in the interpretation and understanding of the results. The proposed methods treat the molecules in a quasispecies as individuals of competing species in an ecosystem, where the haplotypes are the competing species, and the ecosystem is the quasispecies in a host, and the evolution of the system is quantified by monitoring changes in haplotype frequencies. The correlation between the proposed indices is also discussed, and the R code used to generate the simulations, the data and the plots is provided. The virtues of the proposed indices are finally shown on a clinical case. Full article

(This article belongs to the Special Issue Virus Engineering and Applications)

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Review

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21 pages, 1297 KiB

Open AccessReview

Viral Vector-Based Gene Therapy

by Xuedan Li, Yang Le, Zhegang Zhang, Xuanxuan Nian, Bo Liu and Xiaoming Yang

Int. J. Mol. Sci. 2023, 24(9), 7736; https://doi.org/10.3390/ijms24097736 - 23 Apr 2023

Cited by 47 | Viewed by 8990

Abstract

Gene therapy is a technique involving the modification of an individual’s genes for treating a particular disease. The key to effective gene therapy is an efficient carrier delivery system. Viral vectors that have been artificially modified to lose their pathogenicity are used widely [...] Read more.

Gene therapy is a technique involving the modification of an individual’s genes for treating a particular disease. The key to effective gene therapy is an efficient carrier delivery system. Viral vectors that have been artificially modified to lose their pathogenicity are used widely as a delivery system, with the key advantages of their natural high transduction efficiency and stable expression. With decades of development, viral vector-based gene therapies have achieved promising clinical outcomes. Currently, the three key vector strategies are based on adeno-associated viruses, adenoviruses, and lentiviruses. However, certain challenges, such as immunotoxicity and “off-target”, continue to exist. In the present review, the above three viral vectors are discussed along with their respective therapeutic applications. In addition, the major translational challenges encountered in viral vector-based gene therapies are summarized, and the possible strategies to address these challenges are also discussed. Full article

(This article belongs to the Special Issue Virus Engineering and Applications)

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