Microneutralization assay for the measurement of neutralizing antibodies to human metapneumovirus
Human metapneumovirus (hMPV) is an RNA virus in the pneumovirus subfamily of Paramyxoviridae first described in young children with bronchiolitis from the Netherlands.1 Since its discovery in 2001, the virus has been shown to have world wide circulation and to be a common cause of respiratory illness in persons of all ages.2 Similar to a related virus, respiratory syncytial virus (RSV), immunity to hMPV appears to be incomplete and re-infections occur throughout adulthood.3 Correlates of
In this report, we describe the development of a MNA assay for use with trypsin dependent strains of hMPV as a simple and accurate method for the measurement of hMPV neutralizing antibody titers.
LLC-MK2 cells and Vero cells were maintained in MEM, 5% fetal calf serum (FCS). The CAN 97-83 (group A) and CAN 98-75 (group B) strains of hMPV were kindly provided by Dr. Guy Boivin at Laval University Quebec, Canada. Viruses were grown in LLC-MK2 cells with viral growth media (VGM) consisting of 1% bovine serum albumin, 1% HEPES, 0.5% glucose and porcine pancreatic trypsin (2 units/ml). The titer of the viral stock was quantified at 1.3 × 106 using a plaque assay described below.
Plaque reduction neutralization assay
Serum neutralizing antibody was measured using a modification of a plaque assay as described by Williams et al.5 Twenty-four well plates were set with 2 × 105 cells/ml Vero cells to achieve ∼80% confluence the next day. Ten serial 2-fold dilutions of sera starting at 1:50 were incubated at RT for 60 min with an equal volume hMPV to provide approximately 50 plaque forming units (pfu)/200 μL. Two hundred microliters of the serum–virus mixtures were added in duplicate to cell monolayers and allowed to
Twenty acute and convalescent serum pairs were analyzed using the microneutralization assay using CAN 97-83 (MNA-A) and CAN 98-75 (MNA-B) strains of hMPV. Twenty samples (10 acute and convalescent pairs) were run in three assays using CAN 97-83 hMPV performed on different days with excellent inter-assay variability (coefficient of variance = 7%). The mean log 2 titers for MNA-A and MNA-B values were 11.53 ± 1.96 and 11.90 ± 2.10, respectively. The correlation of MNA-A and MNA-B values was high with a
Adapting the MNA methodology used for RSV to hMPV was complicated by the nature of LLC-MK2 cells and the need for trypsin in the growth media of hMPV. In the RSV assay, EDTA is used to disperse Hep-2 cells but was not effective for LLC-MK2 cells and resulted in an poorly adherent irregular monolayer. While the use of bovine pancrease trypsin produced a homogenous single cell suspension of LLC-MK2 cells, a monolayer would not set in the 96 well plates with the trypsin present when added to the
In summary, MNA is a simple and reproducible method for the measurement of serum neutralizing antibody against hMPV. This assay should be useful in studies of disease pathogenesis and facilitate clinical studies of hMPV vaccine candidates.
Conflicts of interest
Dr Falsey consults for Medimmune, GSK, Sanofipasteur, Astrazeneca, and ADMA Biologics and has received honoraria. She also serves on the advisory board of Quidel Inc. Dr Walsh consults for Astrazeneca and ADMA Biologics and has received honoraria. Ms. Formica has no conflicts of interest.
The authors wish to thank Patricia A. Hennessey and Mary Criddle for collection of serum samples and Jamie Biear for technical assistance.
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