Neutralization Beginner Guide

---

Viral neutralization refers to the ability to stop virus from infecting cells. Take SARS-CoV-2 virus as an example, the virus that causes COVID-19 pandemic. The virus infects cells through viral surface envelope Spike protein binding to cell surface ACE2 receptor (see picture [1]), followed by protein fusion and virus replication, resulting in people sickness. An effective medicine would directly block this binding between virus and cell, or immune serum containing neutralizing antibodies elicited by vaccines would also block this binding. Once the binding is blocked, the virus won’t be able to replicate and virus amount would be controlled. The immune system would clear them from the body. 

The neutralizing antibodies (nAbs) either man-made (such as bebtelovimab) or elicited by vaccines are considered as the functional antibodies, different from the IgG/IgM measured with serological tests. The whole level of IgG does not always represent neutralizing antibody, especially in HIV vaccine field where the current HIV vaccine candidates are capable to elicit robust IgG but not neutralizing antibody response [2]. The vaccines’ protective immunity has been widely found to strongly correlates with the level of virus-neutralizing antibodies (nAb) [3, 4].

Basically, there are two methods of neutralization assays to measure the neutralizing power of samples. The traditional way is plaque-reduction neutralization test (PRNT), performed as the addition of laboratory-adapted live virus to the cultured virus-susceptible cells and subsequent counting of the virus-induced plaques in the cell monolayer. This PRNT method usually takes few working days and labor consuming [5]. When this assay involves the live virus requiring high biosafety level (BSL) laboratory containment, such as BSL-3 lab required for SARS-CoV-2 or BSL-4 for Ebola/Marburg/High Pathogenic Avian Influenza viruses, it is impossible to use this assay for any mass screenings [6]. The second method is to use pseudovirus reporters, which are bioengineered products with viral replication limited/restricted. Therefore, a BSL-2 laboratory containment is sufficient for this assay. Besides, the reporter (such as GFP, luciferase) improves the reading with the better sensitivity and higher throughput, enabling the mass screening. More conveniently, pseudovirus is easily manipulated to keep the peace of virus mutation, enabling the vaccine advancement. For example, when SARS-CoV-2 virus mutates from wild type to Omicron strains, a pseudovirus for Omicron was rapidly produced to validate the protection efficacy of vaccine at that time point, facilitating vaccine advancement to protect people from Omicron strain. 

Through either method mentioned above, the half maximal inhibitory concentration (IC50) is obtained as an absolute number that directly relates to the neutralizing power of a therapy [7]. When this therapy is a neutralizing antibody or compound, the unit for IC50could be µg/ml or µMol or similar. A potent therapy would have a low IC50, indicating that less medicine is needed to neutralize the 50% of the virus. When measure vaccine’s efficacy, the similar number called as half-maximal inhibitory dilution (ID50) that represents the plasma dilution to inhibit 50% of the infection. A robust vaccine would have a high ID50, indicating that a greater level of neutralizing antibodies was induced by this vaccine, suggesting the vaccine-induced protection likely lasts longer.  Either IC50 or ID50 might be different when the same sample was tested using live or pseudovirus. It has been reported that one sample that neutralizes Ebola pseudovirus but not live virus, possibly due to the glycosylation variation of two different viruses [8]. 

Pseudovirus Reporter is bioengineered with packaging system, reporter and pseudotyped heterogenous protein (such as Spike protein of SARS-CoV-2). The packaging system includes HIV-1 lentiviral, vesicular stomatitis virus (VSV), retrovirus (RV) or murine leukemia virus (MLV), with lentiviral or VSV backbone commonly used. The recent generation of lentivirus backbone has greatly reduced the safety concern [9]. However, the potency of pseudovirus on lentivirus is relatively lower than that produced on VSV backbone. The potency comparison is based on same condition (without concentration) -yielded signal. Pseudovirus on VSV backbone is safe as wild VSV classified as a BSL-2 pathogen and barely infecting human [10]. Even so, VSV pseudovirus is unable to pack large particles, unlike lentiviral vector with capability of packaging several kb genetic cargo. 

1. Brown, E.E.F., et al., Characterization of Critical Determinants of ACE2-SARS CoV-2 RBD Interaction. Int J Mol Sci, 2021. 22(5).

2. Kim, J., et al., Current approaches to HIV vaccine development: a narrative review. J Int AIDS Soc, 2021. 24 Suppl 7(Suppl 7): p. e25793.

3. Gao, Q., et al., Development of an inactivated vaccine candidate for SARS-CoV-2. Science, 2020. 369(6499): p. 77-81.

4. Gu, M., et al., One dose of COVID-19 nanoparticle vaccine REVC-128 protects against SARS-CoV-2 challenge at two weeks post-immunization. Emerg Microbes Infect, 2021. 10(1): p. 2016-2029.

5. Kolesov, D.E., et al., Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD. Diagnostics (Basel), 2022. 12(2).

6. Bewley, K.R., et al., Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays. Nat Protoc, 2021. 16(6): p. 3114-3140.

7. Chang, S., et al., The Antiviral Activity of Approved and Novel Drugs against HIV-1 Mutations Evaluated under the Consideration of Dose-Response Curve Slope. PLoS One, 2016. 11(3): p. e0149467.

8. Wang, Y., et al., Prominent Neutralizing Antibody Response Targeting the Ebolavirus Glycoprotein Subunit Interface Elicited by Immunization. J Virol, 2021. 95(8).

9. Leander Johansen, J., et al., A new versatile and compact lentiviral vector. Mol Biotechnol, 2005. 29(1): p. 47-56.

10. Abdelmageed, A.A. and M.C. Ferran, The Propagation, Quantification, and Storage of Vesicular Stomatitis Virus. Curr Protoc Microbiol, 2020. 58(1): p. e110.