Comparative analysis of the neutralizing activity against SARS-CoV-2 Wuhan-Hu-1 strain and variants of concern: Performance evaluation of a pseudovirus-based neutralization assay

Abstract

ObjectivesEmergence of new variants of SARS-CoV-2 might affect vaccine efficacy. Therefore, assessing the capacity of sera to neutralize variants of concern (VOCs) in BSL-2 conditions will help evaluating the immune status of population following vaccination or infection.MethodsPseudotyped viruses bearing SARS-CoV-2 spike protein from Wuhan-Hu-1/D614G strains (wild type, WT), B.1.617.2 (Delta), or B.1.1.529 (Omicron) VOCs were generated to assess the neutralizing antibodies (nAbs) activity by a pseudovirus-based neutralization assay (PVNA). PVNA performance was assessed in comparison to the micro-neutralization test (MNT) based on live viruses. Sera collected from COVID-19 convalescents and vaccinees receiving mRNA (BNT16b2 or mRNA-1273) or viral vector (AZD1222 or Ad26.COV2.S) vaccines were used to measure nAbs elicited by two-dose BNT16b2, mRNA-1273, AZD1222 or one-dose Ad26.CO2.S, at different times from completed vaccination, ~ 1.5 month and ~ 4-6 months. Sera from pre-pandemic and unvaccinated individuals were analyzed as controls. Neutralizing activity following booster vaccinations against VOCs was also determined.ResultsPVNA titers correlated with the gold standard MNT assay, validating the reliability of PVNA. Sera analyzed late from the second dose showed a reduced neutralization activity compared to sera collected earlier. Ad26.CO2.S vaccination led to very low or absent nAbs. Neutralization of Delta and Omicron BA.1 VOCs showed significant reduction of nAbs respect to WT strain. Importantly, booster doses enhanced Omicron BA.1 nAbs, with persistent levels at 3 months from boosting.ConclusionsPVNA is a reliable tool for assessing anti-SARS-CoV-2 nAbs helping the establishment of a correlate of protection and the management of vaccination strategies.

Comparative analysis of serological assays and sero-surveillance for SARS-CoV-2 exposure in US cattle

“…However, the sample was negative in SARS-CoV-2 live virus neutralization assay. Although pVNTs yield comparable neutralization titer as that of live virus neutralization assays for detecting the SARS-CoV-2 antibodies [44][45][46], their use as a diagnostic tool could be limited due to the highly sensitive luciferase reporter system. pVNTs are widely used to determine the variant specific neutralization titer and generate antigen cartography to assess the relationship between the variants and serum antibodies [26].…”

Importance, Applications and Features of Assays Measuring SARS-CoV-2 Neutralizing Antibodies

“…Whichever assay format or readout, the assay must be optimized for a particular SARS-CoV-2 isolate or variant. For example, the Omicron BA.1 variant of concern (VOC) replicates slower in Vero cells than the ancestral (D614G) SARS-CoV-2 strain, leading to the prolongation of the MNT turnaround time by 1–2 days [ 70 , 80 , 81 ].…”

“…They provide a relatively safe model for studying virus entry because of their inability to produce infectious progeny virus. In the pseudotyped virus-neutralization test (pVNT) ( Figure 1 B) ( Table 1 ), a single-cycle, replication-defective virus, generally a lentivirus [ 81 , 82 , 83 ] or a glycoprotein (G) protein-deficient vesicular stomatitis virus (VSV-ΔG) [ 82 , 84 , 85 , 86 ], bearing a reporter gene, is pseudotyped with the SARS-CoV-2 S protein expressed from a separate plasmid. The resulting pseudotyped virions are mixed with the test sample containing NAbs, which reduce the infection rate and thus reporter gene readout, allowing the measurement of anti-SARS-CoV-2 NAbs under BSL-2 conditions [ 81 , 82 , 87 ].…”

“…In the pseudotyped virus-neutralization test (pVNT) ( Figure 1 B) ( Table 1 ), a single-cycle, replication-defective virus, generally a lentivirus [ 81 , 82 , 83 ] or a glycoprotein (G) protein-deficient vesicular stomatitis virus (VSV-ΔG) [ 82 , 84 , 85 , 86 ], bearing a reporter gene, is pseudotyped with the SARS-CoV-2 S protein expressed from a separate plasmid. The resulting pseudotyped virions are mixed with the test sample containing NAbs, which reduce the infection rate and thus reporter gene readout, allowing the measurement of anti-SARS-CoV-2 NAbs under BSL-2 conditions [ 81 , 82 , 87 ]. Luminescent (NanoLuc, firefly luciferase, renilla luciferase) [ 66 , 81 , 85 , 87 ], fluorescent (enhanced green fluorescent protein (eGFP), mNeonGreen) [ 88 ] or dual (NanoLuc-eGFP, NeonGreen-NanoLuc) [ 82 , 83 , 84 ] reporters are effectively used in both lenti- and VSV-based pseudoviruses.…”

“…The resulting pseudotyped virions are mixed with the test sample containing NAbs, which reduce the infection rate and thus reporter gene readout, allowing the measurement of anti-SARS-CoV-2 NAbs under BSL-2 conditions [ 81 , 82 , 87 ]. Luminescent (NanoLuc, firefly luciferase, renilla luciferase) [ 66 , 81 , 85 , 87 ], fluorescent (enhanced green fluorescent protein (eGFP), mNeonGreen) [ 88 ] or dual (NanoLuc-eGFP, NeonGreen-NanoLuc) [ 82 , 83 , 84 ] reporters are effectively used in both lenti- and VSV-based pseudoviruses. However, VSV pseudotypes are advantageous over their lentiviral counterparts because rapid single-round intracellular replication of the VSV genome enables robust reporter gene expression that can be detected within a few hours of infection [ 82 ].…”