TWiV 332: Vanderbilt virology

On episode #332 of the science show This Week in Virology, Vincent visits Vanderbilt University and meets up with Seth, Jim, and Mark to talk about their work on a virus of Wolbachia, anti-viral antibodies, and coronaviruses.

You can find TWiV #332 at

TWiV 319: Breaking breakbone

On episode #319 of the science show This Week in Virology, the TWiVers review the outcomes of two recent phase 3 clinical trials of a quadrivalent dengue virus vaccine in Asia and Latin America.

You can find TWiV #319 at

Futures in Biotech 71: Genomics, Proteomics, Cellular Immunity, and Anti-Matter

I joined Marc Pelletier, Andre Nantel, and George Farr on futures-in-biotechepisode 71 of Futures in Biotech for a conversation about the 1000 genome project, the billion dollar human proteome, how antibodies block viral infection, and capturing anti-matter.

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Download audio FiB #71 (42 MB .mp3, 87 minutes)

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Antibodies neutralize viral infectivity inside cells

Antibodies are an important component of the host defense against viral infection. These molecules, produced 7-14 days after infection, neutralize viral infectivity, thereby limiting the spread of infection. Antibodies are thought to neutralize viral infectivity in several ways: by forming noninfectious aggregates that cannot enter cells, or by blocking virion attachment to cells or uncoating (figure). A new mechanism has just joined this list, in which antibody bound virions are degraded in the cell cytoplasm.

A cytoplasmic protein called TRIM21 (tripartite motif-containing 21) was recently found to bind with high affinity to the conserved regions of antibody molecules. The presence of this activity in many mammalian species suggested that there could be ways that antibodies operate within cells. This possibility was studied by using adenovirus infection of cultured cells. When adenovirions were mixed with neutralizing antibodies and added to cells, the antibody-coated particles entered the cytoplasm where they became associated with TRIM21. This behavior was observed when several different adenovirus antibodies were used, suggesting that it is not an unusual property of one type of antibody.

By definition, neutralizing antibodies reduce viral infectivity. When levels of cellular TRIM21 protein were depleted, neutralizing antibodies had little effect on adenovirus infectivity. This effect was found in several cell lines, using three different anti-adenovirus antibodies, and requires the antibody Fc domain. These observations show that adenovirus neutralization by antibodies occurs in the cell cytoplasm, and is dependent upon the binding of antibodies to TRIM21 protein.

How does the interaction of TRIM21 with antibodies bound to adenovirus neutralize viral infectivity? TRIM21 is known to target proteins for degradation by linking them to a small protein called ubiquitin, which labels them for elimination. Cells have two different pathways for degrading ubiquitinated proteins: autophagy and the proteasome. Neutralization of antibody-coated adenovirus was not affected by an inhibitor of autophagy, but was blocked by a proteasome inhibitor. Consistent with this observation, antibody-coated adenovirions in the cell cytoplasm contained both TRIM21 and ubiquitin. Such virions are rapidly degraded, destroying their infectivity.

When antibodies were introduced in uninfected cells, they still associated with TRIM21 protein. This observation means that a virus particle is not needed for the interaction of TRIM21 with antibody. The importance of this finding is that it is possible that other viruses are neutralized by a TRIM21-dependent mechanism. Answering this question could have practical value, because stimulation of TRIM21 immunity might be an important property of effective vaccines.

TRIM21 is an example of a protein that bridges the innate and adaptive immune responses. It is induced by interferons, which are produced early in infection as foreign molecules are detected by the innate immune system. Furthermore, TRIM21 assists in viral neutralization by binding to antibodies, which are products of the adaptive immune response. As would be expected, antibody neutralization of adenovirus is more efficient when cells are treated with interferon.

The participation of cytoplasmic TRIM21 in antibody-mediated virus neutralization might explain a variety of previously unexplained observations. These include:

  • There is a linear-log relationship between antibody dilution and neutralization of adenovirus, and longer incubation does not result in more neutralization
  • Antibody neutralization of poliovirus is observed even when antibodies are added after attachment of virions to cells
  • A single IgG antibody molecule is enough to neutralize poliovirus and adenovirus infectivity
  • 5-6 IgG molecules are enough to neutralize rhinovirus
  • Intact antibody molecules are more effective at neutralizing viruses than those which have been cleaved to produce Fab and Fc fragments

It has always been difficult to understand how just a few antibody molecules can neutralize viral infectivity. The TRIM21 dependent mechanism provides the first plausible mechanism.

An important issue that is not addressed by these studies is the relationship between viral entry and TRIM21 mediated neutralization. Adenovirus is taken into the cell by endocytosis, and then released into the cytoplasm as a partially disassembled particle which docks onto the nuclear pore complex, leading to entry of DNA into the nucleus. Does TRIM21 accompany the virion throughout the endocytic process, targeting the capsid to the proteasome after it is released from the endosome? If TRIM21 were shown to be involved in neutralization of poliovirus, it would not be consistent with the observation that poliovirions do not exit endosomes – the viral RNA is simply translocated across the endosome membrane.

TRIM21-dependent antibody neutralization of viruses is a fascinating new mechanism that could apply to a wide range of viruses. But a number of questions must be answered before it enters the virology textbooks.

Mallery DL, McEwan WA, Bidgood SR, Towers GJ, Johnson CM, & James LC (2010). Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21). Proceedings of the National Academy of Sciences of the United States of America PMID: 21045130

Virus neutralization by antibodies

The antibody response is crucial for preventing many viral infections and may also contribute to resolution of infection. When a vertebrate is infected with a virus, antibodies are produced against many epitopes on multiple virus proteins. A subset of these antibodies can block virus infection by a process that is called neutralization.

Antibodies can neutralize viral infectivity in a number of ways, as summarized in the illustration. They may interfere with virion binding to receptors, block uptake into cells, prevent uncoating of the genomes in endosomes, or cause aggregation of virus particles. Many enveloped viruses are lysed when antiviral antibodies and serum complement disrupt membranes.


Non-neutralizing antibodies are also produced after viral infection. Such antibodies bind specifically to virus particles, but do not neutralize infectivity. They may enhance infectivity because antibodies can interact with receptors on macrophages. The entire virus-antibody complex is brought into the cell by endocytosis. Viral replication can then proceed because the antibody does not block infectivity. This pathway may allow entry into cells which normally do not bear specific virus receptors.

Where in the body does antibody neutralization of viruses take place? Virions that infect mucosal surfaces encounter secretory IgA antibodies present at the apical surfaces of epithelial cells. Viruses that spread in the blood will be exposed to IgG and IgM antibodies. In fact, the type of antibody that is produced can influence the outcome of viral infection. Infection with poliovirus causes IgM and IgG responses in the blood, but mucosal IgA is vital for blocking infection. This antibody can neutralize poliovirus in the intestine, the site of primary infection. The live attenuated Sabin poliovirus vaccine is effective because it elicits a strong mucosal IgA response and provide intestinal immunity.  In contrast, the injected (Salk) polio vaccine does not produce intestinal immunity, and therefore is less effective at preventing spread of poliovirus in a population.

The vast majority of influenza vaccines are administered by injection and stimulate the production of IgG antibodies; they are poor inducers of mucosal IgA antibodies. The efficacy of influenza vaccines would likely be markedly improved if they could be designed to stimulate mucosal immunity. Flumist is a licensed, intranasally-administered infectious virus vaccine that has been shown to stimulate both mucosal and systemic immunity. Nine clinical trials have been conducted in children comparing the efficacy of Flumist with inactivated vaccine or placebo. An analysis of the results suggests that the intranasally administered vaccine is more effective in preventing influenza. These results underscore the role of local immunity in resistance to respiratory pathogens.

Rhorer, J., Ambrose, C., Dickinson, S., Hamilton, H., Oleka, N., Malinoski, F., & Wittes, J. (2009). Efficacy of live attenuated influenza vaccine in children: A meta-analysis of nine randomized clinical trials Vaccine, 27 (7), 1101-1110 DOI: 10.1016/j.vaccine.2008.11.093

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:


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.


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