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 virus-sera mixture. By centrifuging the LLC-MK2 cells and removing the bovine trypsin, an even adherent, monolayer resulted despite the presence of a low concentration of porcine trypsin in the hMPV growth media.
With minor modifications of the RSV MNA methodology, we found that MNA for hMPV is technically easy to perform and results in reproducible values over a wide range of antibody titers. Because the readout does not require laborious manual plaque counting with a microscope, the MNA can be used for studies which require rapid screening of large numbers of sera for hMPV neutralizing activity. Selection of susceptible volunteers for human challenge studies, screening donors to develop a plasma pool of high hMPV titer immunoglobulin, and clinical trials of hMPV vaccine candidates would be facilitated by a rapid and accurate assay such as the MNA.
Another potential advantage of the MNA compared to the standard plaque reduction assay may be better prediction of in-vivo protection from hMPV infection similar to the RSV MNA.10
Although hMPV neutralizing titers measured by plaque reduction correlated with MNA titers, some differences were apparent. In the MNA, antibody remains in the culture media throughout the assay, whereas, after the initial period of neutralization antibody is removed in the plaque reduction assay. The continued presence of antibody throughout the course of the assay neutralizes virus released from infected cells as well as prevents viral spread by inhibiting cell to cell fusion. Functionally distinct antibodies may be measured by MNA (i.e., anti-binding + anti-fusion) compared to plaque reduction which may primarily measure anti-binding antibody. In addition, the plaque assay measures only visible plaques, in contrast to the MNA, which quantitates total viral antigen production in the monolayer. These attributes of the MNA may best mimic in-vivo virus neutralization and thus, correlate better than plaque reduction assays with protection from hMPV infection.
Genetic analyses of hMPV indicate two major genotypes (Group A & B) can be defined and that the greatest diversity is found in the G protein.11–13
However, it is not clear if genotypes translate into antigenically distinct serotypes. Most, but not all investigators, have demonstrated cross protection from infection with heterologous strains during animal challenge studies.11, 14
Using reverse genetics, the individual contributions of the F, G and SH proteins of hMPV have been evaluated in the hamster model.11, 15
The F protein was found to be highly immunogenic and protective, whereas the SH and G proteins were not. Thus, the neutralizing activity of sera may be driven primarily by antibody to the conserved F protein without a significant role of the antigenically more diverse G protein.14
Thus, it is not unexpected we found a high degree of correlation between neutralizing antibody titers to group A and group B strains of hMPV in naturally infected adults. Although our diagnostic RT-PCR does not identify the infecting strain of hMPV, subjects had similar responses to both strains. Comparable observations have been noted with RSV infected adults, although neutralizing activity of antibodies directed against the RSV G protein have been demonstrated.16
Because our results may not be applicable to children with primary infection, studies of strain specific immune responses are needed in children.