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Retrovirus-Based Pseudotyped Virus Neutralisation Assays Overestimate Neutralising Activity in Sera from Participants Receiving Integrase Inhibitors
27 Pages Posted: 21 Feb 2025
More...Abstract
Background: Retroviral pseudotype-based virus neutralisation assays (PVNAs), including human immunodeficiency virus (HIV) and murine leukaemia virus (MLV)-based systems, are widely used in estimating functional immunity, but may be unsuitable when testing HIV-infected participants receiving integrase inhibitors. We assessed these assays for quantifying neutralisation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and hepatitis C virus (HCV) in sera from people living with HIV.
Methods: SARS-CoV-2 neutralisation was assessed using HIV(SARS-CoV-2) pseudoviruses in sera from a longitudinal population-based cohort in Malawi (n=1,876). Sera from HIV-infected participants (n=96) were re-tested using vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudoviruses (VSV(SARS-CoV-2)) and HIV-based pseudoviruses bearing the VSV-G protein (HIV(VSV-G)), confirming neutralisation specificity. MLV(HCV) pseudoviruses assessed HCV neutralisation in UK-based hepatitis patient samples (n=100, n=90 HIV-infected). Sera were re-tested using VSV(HCV) and MLV(VSV-G) pseudoviruses, identifying possible interference. The HIV(SARS-CoV-2) and MLV(HCV) pseudoviruses were tested against antiretroviral drugs to determine the interference source.
Findings: Using the HIV(SARS-CoV-2)-based assay, SARS-CoV-2 neutralisation was detected in 10·5-54·5% of HIV-uninfected participants,compared with 85·5-93·9% of HIV-infected participants (Malawi cohort). The VSV(SARS-CoV-2)-based assay estimated seroprevalence at 5·6-65·2% in HIV-infected participants, suggesting overestimation by the HIV(SARS-CoV-2)-based assay. 75·0-87·9% of HIV-infected samples inhibited HIV(VSV-G) pseudotypes, indicating SARS-CoV-2 spike independent inhibition. Using MLV(VSV-G) pseudotypes, non-HCV-specific inhibition was identified in 12·2% of UK-based HIV-infected participants (all receiving integrase inhibitors). Median percent neutralisation of MLV(HCV) pseudotypes was substantially higher in those on integrase inhibitors (71·8%, IQR 37·3-82·4%) than those not (21·3%, IQR 0·0-45·0%) Testing HIV(SARS-CoV-2) and MLV(HCV) pseudotypes against antiretroviral drugs showed that dolutegravir (integrase inhibitor) interferes with assay measures.
Interpretation: Sera from HIV-infected participants interfere with retrovirus-based neutralisation assays, due to residual integrase inhibitor activity, making these assays unsuitable for testing samples from participants receiving these drugs. Protective immunity to many viruses has likely been over-reported in HIV prevalent populations.
Keywords: pseudovirus, assay, interference, integrase inhibitors
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