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More Than Just Gene Therapy Vectors: Lentiviral Vector Pseudotypes for Serological Investigation

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More Than Just Gene Therapy Vectors: Lentiviral Vector Pseudotypes for Serological Investigation viruses Review More Than Just Gene Therapy Vectors: Lentiviral Vector Pseudotypes for Serological Investigation Kamilla Toon 1,2, Emma M. Bentley 1,* and Giada Mattiuzzo 1,* 1 Division of Virology, National Institute for Biological Standards and Control-MHRA, Blanche Lane, South Mimms EN6 3QG, UK; [email protected] 2 Division of Infection and Immunity, University College London, London WC1E 6BT, UK * Correspondence: [email protected] (E.M.B.); [email protected] (G.M.); Tel.: +44-17-0764-1158 (E.M.B.); +44-17-0764-1283 (G.M.) Abstract: Serological assays detecting neutralising antibodies are important for determining the immune responses following infection or vaccination and are also often considered a correlate of protection. The target of neutralising antibodies is usually located in the Envelope protein on the viral surface, which mediates cell entry. As such, presentation of the Envelope protein on a lentiviral particle represents a convenient alternative to handling of a potentially high containment virus or for those viruses with no established cell culture system. The flexibility, relative safety and, in most cases, ease of production of lentiviral pseudotypes, have led to their use in serological assays for many applications such as the evaluation of candidate vaccines, screening and characterization of anti-viral therapeutics, and sero-surveillance. Above all, the speed of production of the lentiviral pseudotypes, once the envelope sequence is published, makes them important tools in the response to viral outbreaks, as shown during the COVID-19 pandemic in 2020. In this review, we provide an overview of the landscape of the serological applications of pseudotyped lentiviral vectors, with a brief discussion on their production and batch quality analysis. Finally, we evaluate their role as surrogates for the real virus and possible alternatives. Citation: Toon, K.; Bentley, E.M.; Mattiuzzo, G. More Than Just Gene Keywords: pseudotype; lentivirus; serology; vector Therapy Vectors: Lentiviral Vector Pseudotypes for Serological Investigation. Viruses 2021, 13, 217. https://doi.org/10.3390/v13020217 1. At a Glance: History of the Evolution of the Lentiviral Pseudotype System The first naturally observed pseudotype was a replication-defective Rous sarcoma Academic Editor: Birke Bartosch virus (RSV) carrying on the particle surface the envelope protein (Env) of avian leukosis Received: 22 December 2020 virus (ALV) [1]. The RSV genome had the envelope gene, env, replaced with an oncolytic Accepted: 26 January 2021 Published: 31 January 2021 gene, src. Co-infection of the same cells with ALV allowed the envelope-less RSV to acquire the ALV Env and infect cells, leading to malignancy. The pseudotyped viruses’ tropism Publisher’s Note: MDPI stays neutral at entry level and neutralisation properties are therefore determined by the Env on their with regard to jurisdictional claims in surface, and not by the genetic information within the virus particles. Nowadays, this published maps and institutional affil- naturally occurring phenomenon has been engineered to serve many purposes such as gene iations. therapy, virus–host interaction and serological studies. Many viruses have been exploited as a basis for a pseudotype [2,3], with lentiviral vectors (LVs) at the forefront [4–6]. LVs are mainly derived from human immunodeficiency virus type 1 (HIV-1), a single-stranded RNA virus from the Retroviridae family, genus Lentivirus. The relatively simple genome structure (less than 10,000 bases) is convenient for genetic manipulation [7]. Initially, the Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. use of HIV-1 as a vector to deliver genes to a host cell, known as transgenes, was carried out This article is an open access article with a replication-competent virus that had its own genes swapped for those of interest [8]. distributed under the terms and However, as HIV-1 is classified as a hazard group 3 pathogen, many modifications to the conditions of the Creative Commons system have been made since to improve the safety of these vectors [9]. The most commonly Attribution (CC BY) license (https:// used HIV-1 system has the viral genome split in three plasmids: the packaging plasmid creativecommons.org/licenses/by/ containing the structural and essential accessory genes, the transfer plasmid containing the 4.0/). packaging signal (Y) for inclusion into the viral particles with the transgene to be integrated Viruses 2021, 13, 217. https://doi.org/10.3390/v13020217 https://www.mdpi.com/journal/viruses Viruses 2021, 13, x FOR PEER REVIEW 2 of 19 the packaging plasmid containing the structural and essential accessory genes, the trans- Viruses 2021, 13, 217 fer plasmid containing the packaging signal (Ψ) for inclusion into the viral particles with2 of 18 the transgene to be integrated in the target cell and the third plasmid, which is for the expression of the Env which will determine the tropism of the viral particles (Figure 1a). A morein the detailed target description cell and the of third the molecular plasmid, which basis isand for evolution the expression of the oflentiviral the Env vectors which will is provideddetermine in the tropismreview by of theDuvergé viral particles and Negroni (Figure in1 a).this A Special more detailed Issue [5]. description The other of the advantagemolecular of LVs basis is the and lack evolution of interaction of the lentiviral between vectors the core is providedand Env for in theviral review egress, by with Duverg é the capabilityand Negroni to bud inthis in the Special absence Issue of [Env5]. The [10,11] other or advantagemore valuably of LVs incorporate is the lack a of heterol- interaction ogousbetween envelope the [12]. core The and ability Env forof an viral LV egress, to have with multiple the capability different viral to bud Envs in the“plugged absence of in” toEnv the [surface10,11] or makes more it valuably a flexible incorporate tool for gene a heterologous delivery, basic envelope viral research [12]. The (e.g., ability viral of an entry,LV receptor to have studies), multiple drug different screening viral Envs and “pluggedserology (Figure in” to the 1b). surface This review makes itfocuses a flexible on tool the usefor of gene LVs delivery, in serological basic viral assays. research LVs have (e.g., proven viral entry, to be receptor a safe and studies), efficient drug tool screening for screeningand serology for neutralising (Figure1 b).antibodies This review to a focuseswide range on the of useviral of pathogens, LVs in serological especially assays. as a LVs substitutehave provenfor pathogens to be a safedesignated and efficient hazard tool groups for screening 3 and 4, forwhere neutralising accessibility antibodies for re- to a searchwide is restricted, range ofviral especially pathogens, in resource-limited especially as a areas substitute where for high-containment pathogens designated facilities hazard are notgroups available. 3 and 4,Additionally, where accessibility pseudotyped for research LVs play is restricted, an important especially role inin resource-limitedserological researchareas for where viruses high-containment that cannot be propagated facilities are in not cell available. culture, such Additionally, as most strains pseudotyped of hep- LVs atitisplay C virus an important or emerging role viruses in serological that do research not have for cell viruses culture that systems cannot be established propagated yet in cell [13,14].culture, such as most strains of hepatitis C virus or emerging viruses that do not have cell culture systems established yet [13,14]. Figure 1. Figure 1. ApplicationApplication of pseudotyped of pseudotyped lentiviral lentiviral vectors vectors (LVs) (LVs) to neutralisation to neutralisation assays. assays. Pseudo- Pseudotyped typedlentiviral lentiviral particles particles are are generated generated by by transfection transfection of of a a permissive permissive cell cell line line (e.g., (e.g., HEK293T) HEK293T) with a mix with aof mix plasmids of plasmids coding coding for HIV-1 for HIV-1 structural structur andal enzymatic and enzymatic proteins proteins (HIV gagpol),(HIV gagpol), the lentiviral the len- vector tiviralwith vector a transgene with a transgene of interest, of interest, e.g., luciferase, e.g., luciferase, GFP, contained GFP, contained within within the HIV the long HIV terminal long ter- repeats minal(LTR) repeats and (LTR) an expression and an expression plasmid coding plasmid the codi Envng of the the Env virus of of the interest. virus of Particles interest. are Particles collected in the are collectedsupernatant in the of supernatan the transfectedt of the cells transfected usually 48–72 cells usually h post-transfection 48–72 hours (post-transfectionA). In a neutralisation (A). assay, In a neutralisationthe pseudotyped assay, LV the particles pseudotyped are incubated LV particles with the are serological incubated samplewith the to serological test before sample adding to the to test before adding to the target cells. If no neutralising antibody is present, the pseudotyped LV target cells. If no neutralising antibody is present, the pseudotyped LV will enter the target cells, and will enter the target cells, and the transgene will be expressed, and infection can be monitored by the transgene will be expressed, and infection can be monitored by the signal produced. Neutralising the signal produced. Neutralising antibody in the sample will inhibit LV entry and no signal will B be visibleantibody
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