key: cord-0966096-zf8fpyee
authors: Xiang, Qi; Li, Linhao; Wu, Jie; Tian, Miao; Fu, Yang
title: Application of pseudovirus system in the development of vaccine, antiviral-drugs, and neutralizing antibodies
date: 2022-02-16
journal: Microbiol Res
DOI: 10.1016/j.micres.2022.126993
sha: 4a59339ceb0f5b5585a78141321b0972424c3b14
doc_id: 966096
cord_uid: zf8fpyee

Pseudoviruses are viral particles coated with a heterologous envelope protein, which mediates the entry of pseudoviruses as efficiently as that of the live viruses possessing high pathogenicity and infectivity. Due to the deletion of the envelope protein gene and the absence of pathogenic genes, pseudoviruses have no autonomous replication ability and can infect host cells for only a single cycle. In addition, pseudoviruses have the desired characteristics of high safety, strong operability, and can be easily used to perform rapid throughput detection. Therefore, pseudoviruses are widely employed in the mechanistic investigation of viral infection, the screening and evaluation of monoclonal antibodies and antiviral drugs, and the detection of neutralizing antibody titers in serum after vaccination. In this review, we will discuss the construction of pseudoviruses based on different packaging systems, their current applications especially in the research of SARS-CoV-2, limitations, and further directions.

As a commonly used viral tool, pseudoviruses facilitate the study of high-risk and highly pathogenic enveloped viruses that require biosafety level (BSL)-3 or higher laboratories in a biosafety level (BSL)-2 environment. Pseudoviruses are usually based on the genome of virus with low biological risk (e.g., murine leukemia virus, MLV and vesicular stomatitis virus, VSV) or modified virus (human immunodeficiency virus, HIV), in which the envelope protein genes required for infecting host cells are replaced by reporter genes that can be used for in vitro detection, such as GFP and luciferase genes. At the same time, plasmids or stably expressed cell lines are used to express the envelope protein of the high-risk virus to be studied. The core genome and envelope proteins derived from two different viruses are assembled in vitro to form a complete pseudovirus particle, which could be secreted into the cell culture supernatant. At this time, the supernatant is collected and can be used to infect the target cells. Thus, the pseudoviruses can simulate the process of live virus infection by using the envelope protein of highly infectious virus (Li et al., could be secreted to extracellular environment before harvesting. (B) HEK 293T cells were firstly transfected with an envelope protein expression plasmid, twenty-four hours post-transfection, the cells were infected with VSV*∆G encoding firefly luciferase or GFP. Pseudotyped particles were harvested 20 h post-inoculation. (C) HEK 293T cells were co-transfected with an envelope protein encoding-plasmid, an MLV Gag-Pol packaging plasmid and the MLV transfer vector encoding a luciferase reporter. The transfected cells produced pseudotyped MLV particles like the HIV systems. Red bar in plasmid represents packaging elements such as gag and pol; green bar in plasmid represents reporter genes, such as GFP and Luciferase; orange bar in plasmid represents envelope protein gene; purple bar in plasmid represents packaging signals, 3'LTR and 5'LTR. The HIV pseudoviruses system is the most widely used method. The HIV genome contains three structural genes (gag, pol, and env), three regulatory genes (tat, rev, and nef) and four helper genes (vif, vpr, vpu, and vpx) (Wain-Hobson et al., 1985) ,

In order to reduce the chance of homologous recombination in vitro to form complete HIV, the different parts of HIV genome are cloned into different DNA expression vectors, and some dispensable elements of HIV genome, such as nef, are deleted.

Meanwhile, the envelope protein gene env will be mutated by frame shift mutation or deletion in order to load the envelope proteins of other viruses. Hence, another plasmid heterologously expressing the envelope protein is required to form pseudoviruses based on HIV. Depending on the number of plasmids used in the system, the HIV pseudoviruses system can be classified into two-plasmid, threeplasmid, and four-plasmid systems. The preferred one is the two-plasmid system, which includes an expression plasmid and a packaging plasmid. The commonly used packaging plasmid is pSG3Δenv and pNL4-3(Bosch and Pawlita, 1990) . The threeplasmid system divides the HIV skeleton into packaging plasmid and transfer plasmid based on the two-plasmid system. The packaging plasmid expresses Gag and Pol. The transfer plasmid contains HIV reverse transcriptase gene, cis regulatory elements required for integration, and reporter genes driven by CMV promoter (Dull et al., 1998) . The commonly used packaging plasmid and transfer plasmid are psPAX2 and pLenti-GFP (Kretschmer et al., 2020; Ou et al., 2020) . Noteworthily, the third generations of packing system not only evaluated the transactors but also improved J o u r n a l P r e -p r o o f cis elements. For better safety, the transferred plasmids need to be replication incompetent and render "self-inactivating" after integration by shortened 3'LTR.

Although the system has been designed with many safety measures, the system still has potential threat to initiate tumor genes because the transgene sequence will insert into host genome by 2 long terminal repeat (LTR) via viral transduction (Mautino, 2002) . The four-plasmid system further separates rev into an independent expression plasmid based on the three plasmid system to reduce the chance of HIV recombination in vitro and further improve the safety (Dull et al., 1998) . Through transfecting the combined plasmids into 293T cells, the HIV genome elements transcribed from the packaging plasmid and transfer plasmid are assembled into incomplete and safe HIV pseudoviruses particles, and the viral particles will be secreted into the extracellular area by exocytosis. During the secretion process, the pseudovirus particles will take the opportunity to integrate the heterologously expressed envelope proteins into the viral membrane, which is derived from cell membrane. Finally, collecting the cell supernatant and obtaining the pseudovirus through centrifugation, purification, and concentration will yield pseudovirus particles for subsequent research.

Pseudoviruses with VSV genome as the core are also widely used. VSV is an enveloped RNA virus which mainly infects animals. VSV genome is a non-segmented, negative strand, single strand RNA (ssRNA), with a size of about 11 kb. Five nonoverlapping genes are arranged from the 3′ end to the 5′ end, encoding five different main proteins, including nuclear (N) protein, phosphate (P) protein, matrix (M) protein, sugar (G) protein and RNA polymerase (L) protein. VSV is also regarded as an ideal virus tool, because it has simple genome structure and can easily infect a reporter genes, VSV can also complete the process of budding from cells if G protein or other virus-derived surface glycoproteins are expressed by plasmids (Whitt, 2010; Tani et al., 2011) . Therefore, we can utilize G protein deficient VSV to load a variety of surface glycoproteins from others virus to assemble recombinant VSV pseudoviruses. In the preparation of recombinant VSV, we should firstly construct a ΔG transfer plasmid which can transcribe the negative strand RNA of VSV genome, and its transcript will be served as an RNA template for RNA-dependent RNA polymerase (RdRp) to synthesize viral RNA. The vector is co-transfected with four auxiliary vectors, which expressing VSV N, P, G and L proteins respectively, into packaging cells expressing phage T7 RNA polymerase inoculated with cowpox virus.

The T7 RNA polymerase acts on the T7 promoter of the transfer plasmid to drive the transcription of the negative RNA VSV-ΔG genome. Recombinant VSV is assembled in and released from packaging cells. The cell supernatant is collected to obtain Gdeficient VSV virus. Subsequently, we use it to re-infect with packaging cells expressing G glycoprotein to obtain high-yield VSV-ΔG/G* stock and we can use VSV-∆G virion coated with G protein to infect cells expressing other viral glycoproteins to generate the target pseudo virus. But the G protein of initial viral particles can be recycled on the newly produced particles which may cause super high background noise. So using an antibody against the G protein is critical for this type of pseudo virus (Almahboub et al., 2020) .

Like HIV, MLV is also a retrovirus, which mainly infects mice and causes leukemia. MLV is an encapsulated positive strand RNA virus with a genome about 8000 nucleotides, including three structural genes (gag, pol and env), encoding viral capsid proteins [matrix (MA), capsid (CA) and nucleocapsid (NC)], protease [reverse transcriptase (RT), integrase (IN) and protease (PR)] and envelope protein (Fan, 1997) .

The early work of Witte' team showed that when vesicular stomatitis virus (VSV) infected cells that had integrated MLV genomes, the harvested VSV pseudoviruses, although containing VSV genome, were resistant to VSV antiserum, which suggested that MLV also had the potential to package pseudoviruses(Witte and Baltimore, 1977).

In subsequent studies, it was found that the G protein of VSV can also be independently integrated into MLV virus particles transcribed by plasmid when other VSV related genes did not exist (Emi et al., 1991) . Since then, the genome of MLV had been split into 2 parts: one encoding gag-pol and the other containing the reporter gene. The 2 gene sets were further cloned into plasmids to generate highly efficient MLV packaging systems (Soneoka et al., 1995) . The packaging process of MLV pseudoviruses is similar to that of HIV. MLV structural genes gag and pol and a gene encoding heterologous virus envelope protein are transfected into cells by plasmid transfection. After intracellular recombination, the pseudoviruses particles carrying heterologous virus envelope protein will be secreted into the cell culture medium (Millet and Whittaker, 2014; Millet and Whittaker, 2016) .

In general, each of the three methods discussed above have its own advantages and disadvantages. First, in terms of operational complexity, HIV and MLV packaging methods are simpler and less time-consuming than VSV packaging method. Second, in terms of virus yield, VSV and MLV packaging systems can achieve higher pseudovirus yields compared to HIV system (Li et al., 2018) . For experiments with lower titer requirements, subsequent concentration and purification procedures will be omitted. Another advantage of VSV pseudovirus over their HIV and MLV counterparts is that the rapid intracellular replication of the VSV genome enables robust reporter gene expression to be detected within a few hours after infection . Finally, from a safety point of view, VSV and MLV systems are safer than HIV system, and live VSV and MLV themselves are less toxic than HIV. (Organization, 2021) . Therefore, there is an urgent need for COVID-19 specific therapeutic drugs and preventive vaccines all over the world.

However, due to SARS-CoV-2's high pathogenicity and infectivity, all test procedures using live viruses to evaluate drug efficacy must be carried out in biosafety level (BSL) 3 facilities, which undoubtedly hinders the development of related products.

There is also an urgent need for standardized in vitro efficacy methods to evaluate preclinical and clinical antiviral products. In addition, the detection of neutralizing antibody against SARS-CoV-2 will help to understand the protective immune response status and the immune response of human body in COVID-19 patients and asymptomatic cases after vaccination. Because of the above reasons, the pseudoviruses system of SARS-CoV-2 plays an extremely important role during the outbreak of COVID-19, which greatly promotes the development of SARS-CoV-2 drug treatment, antibody screening, vaccine research, and receptor recognition, among others.

As mentioned above, pseudoviruses are composed of the core of one enveloped virus and the envelope glycoprotein of another enveloped virus. The envelope glycoprotein of the SARS-CoV-2 is the Spike protein on its surface. The Spike is responsible for recognizing the cell surface receptor ACE2 and mediating viruses' J o u r n a l P r e -p r o o f entry, in which the activation of Spike is a prerequisite for the entry of SARS-CoV-2. This S protein activation is enabled through two proteolytic cleavage steps following the engagement of ACE2. The first of these is located at the S1-S2 boundary and is cleaved by furin in virus-producing cells, and the second is located at the S2' site in the S2 subunit (Figure 2) , which is mainly cleaved by two major proteases, TMPRSS2 and cathepsin L. Since TMPRSS2 is present on the cell surface, TMPRSS2-mediated activation of the S protein occurs at the plasma membrane, whereas cathepsinmediated activation occurs in the endolysosome (Hoffmann et al., 2020a; Walls et al., 2020; Bayati et al., 2021; . Therefore, S protein is the preferred target of many SARS-CoV-2 antiviral drugs and small molecule inhibitors. In addition, because the spike protein is the major factor on SARS-CoV-2 virion function as both receptor recognition and membrane fusion, and it is poor on antigenicity because of the sugar shelter (Walls et al., 2016) , so S protein is also the preferred antigen of vaccine. For these reasons, pseudoviruses integrating SARS-CoV-2 S protein play an extremely important role and the research on these pseudoviruses is numerous. (Lontok et al., 2004; Stertz et al., 2007) . In this compartment, S protein interacts with another structural protein M to drive S protein to participate in virus particle assembly and infiltrate into virus envelope (McBride et al., 2007; Ujike et al., 2016) . In addition, some mature S proteins of SARS-CoV-2 reach the plasma membrane through secretory pathway, where they can mediate the fusion of infected cells and uninfected cells to fuse and form multinucleated giant cells (syncytia) (Malik, 2020) . What's more, the pseudovirus is packaged with S proteins located on the plasma membrane of the cell. Therefore, several studies have shown that the S protein of SARS-CoV-2 without ERRS sequence not only can increase the infectivity of single cycle VSV pseudovirues and VSV pseudoviruses with replication ability (Dieterle et al., 2020; Schmidt et al., 2020) , but also enhances the infectivity of HIV pseudoviruses or other retroviral pseudoviruses Yu et al., 2021) . This deletion will enhance the localization of the S glycoprotein of SARS-CoV-2 on the cell membrane surface and promote the integration of more S protein into pseudoviruses particles. Previously, our group have used the pNL4-3. Luc.R-E plasmid and pVAX-S-ΔC19 with 19 amino acids deleted at the C-terminal to successfully package HIV SARS-CoV-2 pseudoviruses and applied it to antibody screening targeting the RBD region of S protein of SARS-CoV-2 .

At present, the pseudoviruses of SARS-CoV-2 has played an important role in the study of virus invasion mechanism, screening of antiviral drugs, evaluation of which the IC50 of multiple antibodies was less than 1ug/ml (Ju et al., 2020) . More examples of the applications of SARS-CoV-2 pseudoviruses are listed in Table 1 . (Hou et al., 2020; Korber et al., 2020) . In August 2020, a new mutant strain B.1.1.7 was identified in the UK, and eight important mutations occurred in its S glycoprotein (Davies et al., 2021; Galloway et al., 2021) . variant that was initially prevalent in India seems to be more worrying, because research indicated that Delta variant is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type bearing D614G (Mlcochova et al., 2021) . What's more, sera from Beta and Gamma variant show markedly reduced neutralizing abilities against Delta variant . It is reported that Alpha variant is resistant to most monoclonal antibodies targeting the NTD domain of S protein, while it is relatively sensitive to monoclonal antibodies targeting the RBD structure of S protein. In addition, it is not highly resistant to convalescent plasma or vaccinated sera.

The discovery of Beta viriant is more worrying, because this variant is neither neutralized by most NTD mAbs, nor by multiple individual mAbs of the RBD, mainly due to the E484K mutation. In addition, the neutralizing ability of convalescent plasma and vaccinated serum against Beta variant decreased by 9.4 times and 10.3-12.4 times, respectively (Wang et al., 2021b) . A latest research showed that in the serum of convalescence patients infected with the original SARS-CoV-2 strain, the mean neutralizing ED50 of their serum for Omicron decreased to 66, about 8.4 times that of the D614G reference strain (ED50=556), while the neutralizing activity of other variant pseudoviruses decreased only about 1.2 to 4.5 times (Zhang et al., 2021a) .

Despite the advantages of pseudoviruses listed above, the system also has many limitations. Firstly, pseudoviruses can only be used in the study of viruses with specific envelope protein, such as influenza virus (Ao et al., 2008; Wang et al., 2008; Guo et al., 2009; Ferrara et al., 2012) , coronavirus (Han et al., 2004; Simmons et al., 2004; Zhao et al., 2013; Wang et al., 2014) SARS-CoV-2 is a spherical virus, and the S protein covers two different states of before and after fusion (Yao et al., 2020) . However, among our commonly used pseudoviruses system, HIV-1 and MLV are spherical viruses, while VSV is a bullet virus, and the distribution of S protein on the surface of pseudoviruses is not clear.

Therefore, the distribution, conformation and density pattern of heterologous viral glycoproteins on pseudoviruses may not reflect their "natural" state on the surface of live viruses (Li et al., 2018) . In addition, it is inconvenient to calculate the titer of pseudoviruses quantitatively. When researchers use HIV to package pseudoviruses, J o u r n a l P r e -p r o o f they will choose to measure the level of HIV-1 p24 antigen by ELISA to quantify the titer of pseudoviruses (Deng et al., 2021; Motozono et al., 2021) . Alternatively, the copies of pseudoviruses were expressed as numbers of viral RNA genomes per mL of viral stock solution determined by using reverse transcription fluorescence quantitative PCR. Other titer quantification methods include the 50% tissue culture infectious dose (TCID50) calculated by the Reed-Muench method (Nie et al., 2020b; Garcia-Beltran et al., 2021) and using reverse transcription fluorescence quantitative PCR to determine the copy number of the sample and comparing it with the standard (e.g., The P protein gene of VSV virus was cloned into the vector pCDNA3.1(+) as a plasmid standard) (Li et al., 2020a) . However, among these methods, P24 Elisa assay or copy numbers seem to ignore a same problem, which is that what they measured is the amount of pseudoviruses core genome. Whereas the amount of envelope protein on pseudoviruses is not directly proportional to the copy number of core genome. 

In order to overcome the shortcomings of pseudoviruses, many research groups have developed an infectious cDNA clone technology of SARS-CoV-2 Ju et al., 2021; Rihn et al., 2021; Zhang et al., 2021b) . The infectious clone of J o u r n a l P r e -p r o o f SARS-COV-2 contains almost all its own genes, and the reporter gene is inserted for easy detection. With regard to both pathogenicity and replication capacity, the infectious clone is similar to the situation of wild-type live virus infection. Meanwhile, it can also be operated in a biosafety level-2 environment, and the entire SARS-CoV-2 genome can be edited in a site-specific manner in vitro. Recently, a research group used infectious clone of SARS-CoV-2 to find that many-but not all-of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2 . However, this method is technically difficult and relatively expensive, and experiments may not be able to obtain successful infectious clones. Therefore, this method still has some limitations in its use. In summary, pseudoviruses system is an effective tool for screening and evaluation of monoclonal antibodies or antiviral drugs, detection of serum neutralizing antibodies and evaluation of vaccine titer. It will still be widely used in the future. This review summarizes three main methods of packaging pseudoviruses, and focuses on the application of pseudovirus in COVID-19 research, which has proper guiding significance for the future development and application of pseudoviruses. 

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