Influenza microneutralization assay

The microneutralization assay is another technique used by the Centers for Disease Control and Prevention to determine that some adults have serum cross-reactive antibodies to the new influenza H1N1 virus. Let’s explore how this assay works.

Viral replication is often studied in the laboratory by infecting cells that are grown in plastic dishes or flasks, commonly called cell cultures. Many viruses kill such cells. Here is an example of HeLa cells being killed by poliovirus:

cpe

The upper left panel shows uninfected cells, and the other panels show the cells at the indicated times after infection. As the virus replicates, infected cells round up and detach from the cell culture plate. These visible changes are called cytopathic effects.

There is another way to visualize viral cell killing without using a microscope: by staining the cells with a dye. In the example shown below, cells have been plated in the small wells of a 96 well plate. One well was infected with virus, the other was not. After a period of incubation, the cells were stained with the dye crystal violet, which stains only living cells. It is obvious which cells were infected with virus and which were not.

cpe-stain

We can use this visual assay to determine whether a serum sample contains antibodies that block virus infection. A serum sample is mixed with virus before infecting the cells. If the serum contains antibodies that block viral infection, then the cells will survive, as determined by staining with crystal violet. If no antiviral antibodies are present in the serum, the cells will die.

In its present form, this assay tells us only whether or not there are antiviral antibodies in a serum sample. To make the assay quantitative, two-fold dilutions of the serum are prepared, and each is mixed with virus and used to infect cells. At the lower dilutions, antibodies will block infection, but at higher dilutions, there will be too few antibodies to have an effect. The simple process of dilution provides a way to compare the virus-neutralizing abilities of different sera. The neutralization titer is expressed as the reciprocal of the highest dilution at which virus infection is blocked.

neutralizationIn the example shown here, the serum blocks virus infection at the 1:2 and 1:4 dilutions, but less at 1:8 and not at all at 1:16. Each serum dilution was tested in triplicate, which allows for more accuracy. In this sample, the neutralization titer would be 4, the reciprocal of the last dilution at which infection was completely blocked.

This explanation should clarify how the neutralization titers were obtained that are reported in the CDC study cited below. By the way, microneutralization simply means that the neutralization assay is done in a small format, such as a 96 well plate, instead of larger cell culture dishes.

The authors of the CDC study note that “although serum hemagglutination inhibition (HI) antibody titers of 40 are associated with at least a 50% reduction in risk for influenza infection or disease in populations, no such correlate of protection exists for microneutralization antibody titers”. They used mathematical analysis to determine the relationship between HI and microneutralization titers. They found that in sera from children, an HI titer of 40 corresponded to a microneutralization titer of 40. However, in adults, an HI titer of 40 corresponded to a microneutralization titer of 160 or more. I don’t know the reason for this difference, but one possibility is that not all neutralizing antibodies in adult sera are able to inhibit hemagglutination. Understanding why this situation might occur will require a discussion of how antibodies block viral infection.

J Katz, PhD, K Hancock, PhD, V Veguilla, MPH, W Zhong, PhD, XH Lu, MD, H Sun, MD, E Butler, MPH, L Dong, MD, PhD, F Liu, MD, PhD, ZN Li, MD, PhD, J DeVos, MPH, P Gargiullo, PhD, N Cox, PhD (2009). Serum Cross-Reactive Antibody Response to a Novel Influenza A (H1N1) Virus After Vaccination with Seasonal Influenza Vaccine Morbid. Mortal. Weekly Rep., 58 (19), 521-524

Adults have cross-reactive antibodies to A/California/04/2009 (H1N1)

hemagglutinationDoes previous exposure to influenza H1N1 viruses, either by infection or vaccination, provide any protection against infection with the new H1N1 influenza virus strains? The answer to this question might provide insight as to why over 60% of confirmed cases of influenza caused by the swine-like H1N1 viruses in the US are in 5- to 24-year-olds, as reported at a CDC press conference.

To answer this question, CDC has analyzed serum specimens that were collected during previous vaccine studies. These sera were collected from children and adults before and after they received influenza vaccine in the 2005-06, 2006-07, 2007-08, or 2008-09 influenza seasons. Virus neutralization and hemagglutination-inhibition assays were done to determine whether these sera contain antibodies that cross-react with the new H1N1 strain. The authors of the study used the A/California/04/2009 as a representative of the new H1N1 virus isolates.

The results show that previous immunization of children (age 6 months to 9 years, total of 79 specimens) with either seasonal trivalent inactivated vaccine or infectious, attenuated influenza vaccine of the previous four years did not induce cross-reactive antibody to the new influenza A H1N1 strain. Previous immunization did induce a low cross-reactive antibody response to A/California/04/2009 in adults. Among 18-64 year olds, there was a twofold increase in cross reactivity antibody to the virus, compared with a 12-19 fold increase in antibody titers against the seasonal strains. There was no increase in cross reactive antibodies in adults over 60 years of age. These data indicate that immunization with seasonal influenza vaccines containing previous H1N1 strains (years 2005-2009) is not likely to confer protection against infection with the new H1N1 strains.

An important question is whether the sera obtained before administration of vaccine contain cross-reactive antibody titers against A/California/04/2009. Such analyses would indicate whether natural infection with H1N1 strains confers some protection agains the new isolates. There were no pre-vaccination cross-reactive antibodies to A/California/04/2009 in sera of any of the 79 children of ages 6 months to 9 years. However, 6% of adults 18-40 years old, 9% of adults 18-64 years old, and 33% of adults over 60 years of age had pre-vaccination neutralizing antibody titers to A/California/04/2009 greater than or equal to 160. These antibodies were likely acquired by infection with an H1N1 virus that is antigenically more similar to the A/California/04/2009 than other seasonal H1N1 strains. Whether such antibodies would confer protection against infection is unknown, but they could reduce the severity of disease symptoms.

I suspect that not all readers of virology blog are familiar with the microneutralization and hemagglutination-inhibition assays used in this study. In a separate post, I will explain how the assays work, and the significance of the test results.

J Katz, PhD, K Hancock, PhD, V Veguilla, MPH, W Zhong, PhD, XH Lu, MD, H Sun, MD, E Butler, MPH, L Dong, MD, PhD, F Liu, MD, PhD, ZN Li, MD, PhD, J DeVos, MPH, P Gargiullo, PhD, N Cox, PhD (2009). Serum Cross-Reactive Antibody Response to a Novel Influenza A (H1N1) Virus After Vaccination with Seasonal Influenza Vaccine Morbid. Mortal. Weekly Rep., 58 (19), 521-524