Use of lentiviral pseudotypes as an alternative to reassortant or Triton X‐100‐treated wild‐type Influenza viruses in the neuraminidase inhibition enzyme‐linked lectin assay

Abstract

Background Formulation of neuraminidase (NA) within influenza vaccines is gaining importance in light of recent human studies. The enzyme‐linked lectin assay (ELLA) is considered a reliable assay to evaluate human anti‐NA antibodies. Objectives To overcome interference by hemagglutinin (HA)‐specific antibodies and detect neuraminidase inhibitory (NI) antibodies only, two different sources of antigen have been studied in ELLA: reassortant viruses with a mismatched avian origin‐HA or Triton X‐100 (Tx)‐treated wild‐type viruses. Pseudotypes or pseudovirus (PV), characterized by a lentivirus core bearing human influenza NA and avian influenza HA, were investigated as an alternative source of antigen and compared to HA‐mismatched and Tx‐treated viruses, since represent a safer product to be handled. Methods Two independent panels of sera were analyzed by ELLA to evaluate the anti‐NA response against N1 (A/California/07/2009 (H1N1pdm)) and N2 (A/Hong Kong/4801/2014 (H3N2)). The NA inhibition (NI) antibody titers measured as either the 50% end point or 50% inhibitory concentration (IC 50 ) were compared for every source of antigen. Results The ELLA assay performed well with all three sources of antigen. NI titers measured using each antigen type correlated well when reported either as end point titers or as the IC 50 . Conclusions This study suggests that HA‐mismatched whole virus, Triton‐treated wild‐type virus or PV can be used to measure NI antibody titers of human sera, but further comparability/validation assays should be performed to assess statistical differences. The data support the use of PV as an attractive alternative source of antigen and justify further investigation to improve stability of this antigen source.

Influenza A (N1-N9) and Influenza B (B/Vic and B/Yam) Neuraminidase Pseudotypes as Tools for Pandemic Preparedness and Improved Influenza Vaccine design

“…All values were normalized against the highest PV value obtained (100% OD 450 ) and the sample diluent only control (0% OD 450 ). Traditionally, 90% activity is optimal for use in the inhibition assay (33,39), however, we also set the additional criteria of the OD 450 to be a minimum of 2.0 for use in pELLA inhibition. All human and avian PV produced demonstrated NA enzymatic activity and met the additional criteria we set for use in pELLA inhibition ( Figure 2C ) apart from bat N10 and N11.…”

“…Additionally, pseudotype virus (PV) can also be used as a substitute to wild type virus in these assays. Neuraminidase pseudotyped viruses have already been successfully used in place of reassortant virus or Triton X-treated wild type virus in the ELLA assay for N1 and N2 subtypes (38,39) As NA has the potential to be included in a more broadly protective vaccine approach, a toolbox of assays capable of assessing NA inhibition will be required. To this end, we have produced an NA PV library encompassing IAV N1-N11 and IBV from the Victoria-like (B/Vic) and Yamagata-like (B/Yam) lineages for use in the pELLA assay and potentially the NA-Fluor™ assay.…”

Tetrameric Neuraminidase of Influenza A Virus Is Required to Induce Protective Antibody Responses in Mice

“…The enzymatic activity of recombinant NA proteins was determined by the cleavage of two specific substrates of NA, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) ( Job et al, 2018 ; Ju et al, 2018 ) and fetuin ( Prevato et al, 2015 ; Biuso et al, 2019 ) as previously described, with minor modifications.…”

More Than Just Gene Therapy Vectors: Lentiviral Vector Pseudotypes for Serological Investigation

“…Although the MN assay is commonly used in the evaluation of influenza virus vaccines, it has demonstrated significant inter-laboratory variability [ 95 ], in addition to the risk of working with highly pathogenic strains; as such, studies have shown pseudotyped LV neutralisation assays as a reliable alternative [ 96 ]. Further, a pseudotyped LV has been used as an alternative source of antigen for enzyme-linked lectin assays (ELLA), measuring antibodies raised to neuraminidase and alleviating complications of working with reassortant or detergent-treated viruses [ 97 , 98 ].…”