Validated Pseudovirus

Our pseudoviruses are based on a 3rd-generation of an HIV-based lentiviral vector. The 3rd-generation is well known and well characterized by the scientific community because the technology has been used for more than 20 years.

The 3rd-generation of lentiviral vectors resulted from several safety modifications from the original viral genome:

• It contains only 10% of the viral genome sequence (notably excluding the viral tat gene that is essential for the wild-type replication) [1–2],
• It consists of a split-genome packaging system to reduce the risk of recombination during plasmid amplification and viral vector manufacture,
• A chimeric 5’ LTRs have been constructed in the transfer vector to make the lentiviral promoter Tat-independent. This has been achieved by replacing the U3 region of the 5’LTR with a strong tat-independent constitutive promoter [3],
• An altered 3′ LTR renders the vector “self-inactivating” to prevent integrated genes from being repackaged and so, strongly limiting both genome mobility and possibilities of recombination in the host cell [4].

Finally, the modifications characterizing the 3rd generation of lentiviral vectors make them safe enough to be used in approved gene therapies [5]. As explained in this publication, “the third-generation SIN lentiviral vector system makes the generation of replication-competent lentiviruses very unlikely because the viral genome is split into separate plasmids”. For this reason, the scientific community considers the 3rd-generation of lentiviral vectors as replication-incompetent and self-inactivating vectors.

Hence, we did not perform any additional experiments to demonstrate the safety of our pseudoviruses and we rely on the numerous publications about this topic. However, our pseudoviruses are infectious viruses that integrate their genome into the host cell genome. They must be manipulated under the hood in BSL-2 containment, according to the Good Laboratory Practices, and with a decontamination procedure involving virucide or bleach.

A pseudovirus, defined as ‘a defective nonpathogenic enveloped virus that displays a heterologous viral envelope protein of interest,’ can enter a cell, and mimic the initial stages of infection, but cannot complete a full replication cycle to produce new infectious particles. To make them replication-defective and to facilitate the incorporation of the heterologous viral envelope protein, the native virus envelope gene is deleted, and other genes involved in pathogenicity are also removed. What remains are the structural and enzymatic proteins and genomic elements required for virion assembly, host cell entry, and gene expression. Pseudoviruses can be produced at large scale in vitro, by co-transfecting producer cells with plasmids expressing the envelope protein from the viral pathogen of interest, viral packaging plasmids to express structural proteins, and a defective viral genome that often includes a reporter gene for ease of quantifying infection. The incorporation of the heterologous viral envelope protein allows the pseudovirus to utilize the same pathway that its native pathogenic virus uses to enter cells (Xiang Q et al., 2022), and through selective expression of envelop proteins, these pseudoviruses can facilitate systematic studies on the mechanisms of viral entry into host cells and allow research into entry inhibitors as antivirals (Syamala R. Thimmiraju et al., 2024).

NAbs are a type of antibodies produced by B lymphocytes when pathogenic microorganisms invade the human body. NAbs can inhibit viral infection during the whole virus replication cycle, especially in the process of viral attachment and the entry to host cells (Shanmugaraj B et al., 2020). NAbs can facilitate virus particle aggregation, reduce the number of host cells that virions attach to; degrade the virus or inhibit virus internalization through endocytosis; inhibit viral metabolic events (replication or transcription) (Klasse PJ et al. 2002), among which the most important inhibitory effect is that NAbs can block the infection by interfering with the virus-receptor binding site when the virus is attaching to the target cell (Klasse PJ et al. 2002; Tong PB et al. 2020).The level of total antibodies as well as the level of NAbs, should be measured in clinical trials of vaccines (Yang J et al. 2020; Corbett KS et al. 2020). In short, NAbs can reduce the infectivity of the virus by blocking the binding of the virus to the cell receptor or interfering during the virus fusion with the cell.NAbs are promising candidates for providing prophylactic and therapeutic protection against infectious diseases

Neutralization assays determine the ability of an antibody to inhibit a virus infection of cultured cells and the resulting cytopathic effects of viral replication, which are important for drug discovery and vaccine development. Virus neutralization is a specialized type of immunoassay since it detects antibodies that can block virus replication. Neutralizing antibodies generally recognize proteins or glycoproteins on the virus surface. Those antibodies have several mechanisms, including blocking the virus attachment to the host cell, inhibiting its penetration into the host cell membrane, or interfering with uncoating of the virus within the cell.

In the live virus neutralization assay, the viruses and antibodies are mixed and incubated under appropriate conditions and then, inoculated to sensitive hosts (including animals, chicken embryos and cells). Virus infection, that is the infectivity of the remaining virus to the host, is observed. Neutralization assays involving cell culture are mainly used to identify the antibodies that can inhibit cytopathic effect or the formation of virus plaques.

Live virus neutralization assays mainly include Focus-Reduction Neutralization Test (FRNT) (Case JB et al. 2020), Plaque Reduction Neutralization Test (PRNT) (Perera RA et al. 2020) and live virus microneutralization (MN) assay (Bond K et al. 2020; Amanat F et al. 2020).

PRNT is a traditional method and gold standard for measuring the neutralizing ability of antibodies against viruses. This method quantifies the number of infectious virus particles by amplifying the cytopathic effect (CPE) caused by a single virus into ‘plaque’ by staining. PRNT is reliable and requires few specific reagents, but it is usually carried out in low-throughput 6 or 24 well plates (in a few cases also can be carried out in 48- and 96-well format) (Lau EHY et al. 2020) which leads to a long sample analysis time (about 4 days). Moreover, the final read-out may be affected by the subjective judgment of the operator. Although PRNT is highly sensitive, it is not efficient to be suitable for large-scale detection of NAbs.

The principle of FRNT is similar to PRNT, but in FRNT, virus is detected using specific antibodies that are conjugated to horseradish peroxidase (HRP), and the results are presented by observing the foci of virus-infected cells with microanalyser. Compared with PRNT, FRNT can be performed in a 96-well plate, takes less time and requires fewer reagents and cells (Vanderheiden A et al. 2020).

For those indistinct CPE plaques caused by some viruses, MN can be selected. The purpose of MN is similar to that of PRNT, which aims to directly quantify the virus neutralization in the initial inoculation of virus, but the result of MN is more sensitive than PRNT (Khoury DS et al. 2020).

The live virus neutralization assay is an effective method to evaluate the level of NAbs (Wang K et al. 2021). Although the live virus neutralization assay can measure the level of NAbs blocking viral infection, it is expensive and requires well-trained professionals to work with highly pathogenic viruses in a biosafety level-3 or -4 (BSL-3, BSL-4) laboratory. It is time-consuming, labor-intensive, and usually takes 4-10 days to complete. Therefore, this method is not suitable for large-scale and high throughput serological diagnosis and vaccine evaluation (Xi Y. 2020).

Understand the NAbs immune responses of infected people.

In some recent studies, the rapid decline of NAbs within a few weeks has been a concern, which is obvious in individuals with asymptomatic infection or mild symptoms (Seow J et al. 2020; Long QX et al. 2020). There have also been reports of some cases of re-infection, increasing concerns about susceptibility to re-infection (An J et al. 2020; Lee JS et al. 2020). However, in general, antibody levels will always decrease after the acute infection period. Most of the responses of effector B cells induced by infection are short-lived, while the antibody level needs to be maintained by longer-lived plasma cells and memory B cells (Stephens DS et al. 2020; Ripperger TJ et al. 2020).In the early stage, the decline of NAbs level should not cause concern. Neutralization assays are useful tools to determine the level of antibody produced as well as the duration of the antibody titer stability after a natural infection or a vaccination.

Evaluation of convalescent plasma (CP) therapy, mAbs and vaccines

Before effective drugs and vaccines are developed, convalescent plasma could represent an immediate stopgap treatment against viruses. Passive immunity driven by convalescent plasma can provide NAbs that inhibit infection. Other antibody-mediated pathways, such as complement activation, antibody-dependent cytotoxicity and/or phagocytosis, may also promote the therapeutic effect of convalescent plasma (Zhou G et al. 2020). Patients with high NAbs titers who have recovered from a virus may be a valuable source of donors for convalescent plasma. However, the titer of NAbs in the donor’s plasma, the efficiency of the plasma therapy, and the capacity of the recipient to produce efficient specific NAbs after the plasma administration are part of the data that need to be evaluated.
Passive antibody protection conferred by convalescent plasma can provide rapid but short-term immunity for susceptible individuals, while vaccines induce long-lasting protective antiviral immunity (Bloch EM et al. 2020). In many vaccine trials, the titer of NAbs is usually regarded as an important evaluation endpoint (Sahin U et al. 2020; Zhu FC et al. 2020). There are still some key questions to be answered in the development of vaccines, such as which antigen (epitope) will trigger powerful NAbs in most people.

In addition, neutralization assays can be used to do some valuable research on controlling infectious diseases. Studies could, for example, be performed to describe the NAbs response of recovered patients or normal individuals after immunization, including the average titers of asymptomatic, mild and severe patients and the titers of different age groups; to study the kinetics and lifespan of NAbs; to determine the titers of protective NAbs and the level of NAbs required to observe an antiviral effects.

“Vaccine developers should assess immunogenicity of modified vaccines against targeted variants using a neutralization assay with SARS-CoV-2 wildtype or pseudoviruses.” Learn more

“Antibody developers should report the neutralization activity of their antibody against circulating variants.” Learn more

In neutralization assays, the selection of cell lines and virus models will have an impact on the neutralizing activity of antibodies. In most pseudovirus neutralization assays, cell lines of human and animal origin have shown high sensitivity to pseudovirus binding and entry (Hoffman M, 2020), especially Vero and Huh7 cell lines (Nie J, 2020 et Yang R, 2020). However, it is worth noting that some viruses such as SARS-CoV-2 have a poor infection of some common cell lines used in some virus neutralization assays (Manenti A, 2020 and Harcourt J, 2020). To overcome this issue in pseudovirus assays, cell lines such as HEK293T can be transfected to overexpress the specific receptor to the targeted virus.

Most sVNTs are based on the principle of blocking the interaction between the receptor binding site (RBD) and the cellular receptors (Abe KT et al. 2020; Tan CW et al. 2020). The selected specific antigen, which can be biotinylated, is coated on a plate and incubated with test serum. Then the soluble cellular receptor conjugated with HRP is added in the plate, followed by its colorimetric substrate 3,3′,5,5′-Tetramethylbenzidine. Or the plate is coated with the cellular receptor, and soluble antigen is used to compete with antibodies. The antibody that blocks the receptor-antigen interaction can be detected by the reduction of the HRP luminescence signal or surface plasmon resonance. Unfortunately, this detection method still has some limitations: it can only detect designated antibodies, such as blocking antibodies, but not all NAbs (Liu L et al. 2020). Therefore, the synergy between NAbs is difficult to assess. The sensitivity and specificity of sVNTs are lower than cell-based neutralization assays (Sholukh AM et al. 2020). These shortcomings make sVNTs currently only moderately used as supplementary tests for cell-based neutralization assays.