Poliovirus escapes antibodies

Antibodies bound to poliovirus.

Antibodies (purple) bound to poliovirus. Image credit: Jason Roberts

Antigenic variation is a hallmark of influenza virus that allows the virus to evade host defenses. Consequently influenza vaccines need to be reformulated frequently to keep up with changing viruses. In contrast, antigenic variation is not a hallmark of poliovirus – the same poliovirus vaccines have been used for nearly 60 years to control infections by this virus. An exception is a poliovirus type 1 that caused a 2010 outbreak in the Republic of Congo.

The 2010 outbreak (445 paralytic cases) was unusual because the case fatality ratio of 47% was higher than typically observed (usually less than 10% of patients with confirmed disease die). The first clue that something was different in this outbreak was the finding that sera from some of the fatal cases failed to effectively block (neutralize) infection of cells by the strain of poliovirus isolated during this outbreak (the strain is called PV-RC2010). The same sera effectively neutralized the three Sabin vaccine viruses as well as wild type 1 polioviruses isolated from previous outbreaks. Therefore gaps in vaccination coverage were solely not responsible for this outbreak.

Examination of the nucleotide sequence of the genome of type I polioviruses isolated from 12 fatal cases revealed two amino acid changes within a site on surface of the viral capsid that is bound by neutralizing antibodies (illustration). The sequence of this site, called 2a, was changed from ser-ala-ala-leu to pro-ala-asp-leu. This particular combination of amino acid substitutions has never been seen before in poliovirus. Virus PV-RC2010, which also contains these two amino acid mutations, is completely resistant to neutralization with monoclonal antibodies that recognize antigenic site 2 (monoclonal antibodies recognize a single epitope, as opposed polyclonal antibodies which is a mixture of antibodies that recognize many epitopes. The antibodies in serum are typically polyclonal).

Poliovirus neutralization titers were determined using sera from Gabonese and German individuals who had been immunized with Sabin vaccine. These sera effectively neutralized the type I strain of Sabin poliovirus, as well as type 1 polioviruses isolated from recent outbreaks. However the sera had substantially lower neutralization activity against PV-RC2010. From 15-29% of these individuals would be considered not to be protected from infection with this strain.

Nucleotide sequence analysis of PV-RC2010 reveals that it is related to a poliovirus strain isolated in Angola in 2009, the year before the Republic of Congo outbreak. The Angolan virus had just one of the two amino acid changes in antigenic site 2a found in PV-RC2010.

It is possible that the relative resistance of the polioviruses to antibody neutralization might have been an important contributor to the high virulence observed during the Republic of Congo outbreak. The reduced ability of serum antibodies to neutralize virus would have lead to higher virus in the blood and a greater chance of entering the central nervous system. Another factor could also be that many of the cases of poliomyelitis were in adults, in which the disease is known to be more severe.

An important question is whether poliovirus strains such as PV-RC2010 pose a global threat. Typically the fitness of antigenically variant viruses is not the same as wild type, and therefore such viruses are not likely to spread in well immunized populations. Today some parts of the world have incomplete poliovirus immunization coverage, which together with the reduced circulation of wild type polioviruses leads to reduced population immunity. Such a situation could lead to the evolution of antigenic variants. This situation occurred in Finland in 1984, when an outbreak caused by type 3 poliovirus took place. The responsible strains were antigenic variants that evolved due to use of a sub-optimal poliovirus vaccine in that country.

The poliovirus outbreaks in the Republic of Congo and Finland were stopped by immunization with poliovirus vaccines, which boosted the population immunity. These experiences show that poliovirus antigenic variants such as PV-RC2010 will not cause outbreaks as long as we continue extensive immunization with poliovirus vaccines, coupled with environmental and clinical testing for the presence of such viruses.

TWiV 257: Caveat mTOR

On episode #257 of the science show This Week in Virology, the TWiV team consider how the kinase mTOR modulates the antibody response to provide broad protection against influenza virus, and explore the problems with scientific research.

You can find TWiV #257 at www.microbe.tv/twiv.

TWiV 218: Monkeys turning valves and pushing buttons

On episode #218 of the science show This Week in Virology, Vincent, Alan, and Welkin discuss how endogenous retroviruses in mice are held in check by the immune response.

You can find TWiV #218 at www.microbe.tv/twiv.

TWiV 168: Super CalTech prophylaxis and ferret runny noses

adeno-associated virusHosts: Vincent Racaniello, Dickson DespommierRich ConditAlan Dove, and Welkin Johnson

Welkin joins the TWiV team for a discussion of HIV prophlaxis using vectored antibodies, and the influenza H5N1 virus studies in ferrets that were not redacted.

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Welkin – Virtual PI (Nature)
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TWiV 161: Concerto in B

antibodyHosts: Vincent Racaniello, Rich ConditAlan Doveand Gabriel Victora

Vincent, Rich, Alan and Gabriel review the production of antibodies by B cells, and how high affinity antibodies are selected in the germinal centers of lymph nodes.

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Gabriel – Antibody-based protection against HIV (Nature)
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Gut microbes influence defense against influenza

modelThe bacteria in our intestines outnumber by tenfold the 100 trillion cells that comprise the human body. This gut microbiota has many beneficial functions, including the production of vitamins and hormones, fermentation, regulation of gut development, and shaping intestinal immune responses. They also play a role in pathological conditions such as diabetes and obesity, and influence the immune functions of distal mucosal surfaces such as the lung. Examples include the amelioration of allergen-induced asthma by colonization of the stomach with Helicobacter pylori, and involvement of the gut microbiota in development of immune defenses against influenza virus infection.

When the gut microbiome of mice is altered by treatment with antibiotics, subsequent intranasal infection with influenza A virus leads to reduced antiviral antibody and T-cell responses. The antibiotic treatment does not cause a general immunodeficiency – the mice can respond normally to protein antigens.

The defective immune response to influenza virus in antibiotic treated mice can be rescued by treating the mice with compounds that stimulate the innate immune response – such as lipopolysaccharide, a bacterial product. These compounds rescue the immune defect when administered either intransally or rectally at the time of influenza virus infection. Apparently stimulating the innate immmune response in the gut is sufficient to correct an immune defect in the lung.

How might gut bacteria be important for immune responses to a lung infection? When influenza virus infects the lung, development of immune defenses depend upon a complex of several proteins called the inflammasome. This structure is needed for the production of cytokines that promote adaptive immune defenses: antibodies and T cells. These cytokines are also needed for the activity of dendritic cells, sentinels that sense a virus infection, and travel to the nearby lymph nodes to inform T cells that there is a problem.

Antibiotic treatment of mice impairs the influenza virus-induced production of inflammasome-dependent cytokines. These results are consistent with the finding that antibiotic-treated mice respond normally to infection with herpes simplex virus type 2 and Legionella pneumophila, two pathogens for which the inflammasome is not required for adaptive immune responses. Furthermore, microbe-mediated inflammasome activation is needed for migration of lung dendritic cells to lymph nodes. In antibiotic treated mice, lung dendritic cells fail to migrate to local lymph nodes. Hence T cells are not informed of the infection, leading to poor antibody and cellular responses.

These findings reveal a link between the gut microbial community and inflammasome-dependent activation of cytokines. How gut bacteria effect this process is not understood. One idea is that bacterial products stimulate white blood cells in the intestine to produce compounds that migrate to the lung and activate the inflammasome.

If you are wondering about the practical consequences of these findings, read the last paragraph of the paper:

Because antibiotic use is prevalent in the treatment of respiratory infections, our results imply a possible deleterious effect of such treatment in initiating proper immune responses to influenza virus. Conversely, it will be important to determine whether probiotic therapy can be explored for immune-stimulating effects during the flu season.

Ichinohe T, Pang IK, Kumamoto Y, Peaper DR, Ho JH, Murray TS, & Iwasaki A (2011). Microbiota regulates immune defense against respiratory tract influenza A virus infection. Proceedings of the National Academy of Sciences of the United States of America, 108 (13), 5354-9 PMID: 21402903

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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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

Detecting viral proteins in infected cells or tissues by immunostaining

Many virological techniques are based on the specificity of the antibody-antigen reaction. Examples in our virology toolbox include western blot analysis and ELISA. While very useful, these methods cannot be used to visualize viral proteins in infected cells or tissues. To do that we must turn to immunostaining.

In direct immunostaining (illustrated), an antibody that recognizes a viral antigen is coupled directly to an indicator (a fluorescent dye or an enzyme). Indirect immunostaining is a more sensitive method because a second antibody is coupled to the indicator. The second antibody recognizes a common epitope on the virus-specific antibody. Multiple second antibodies can bind to the first antibody, leading to an increased signal from the indicator compared to direct immunostaining.

To carry out immunostaining, virus-infected cells are fixed to preserve cell morphology or tissue architecture. This step is usually accomplished with acetone, methanol, or paraformadehyde. After incubation of fixed cells with the appropriate antibody, excess antibody is removed by washing, followed by microscopy. Common indicators that are coupled to antibody molecules include fluorescein and rhodamine, which fluoresce on exposure of the cells to ultraviolet light. Filters are placed between the specimen and the eyepiece to remove blue and ultraviolet light; this ensures that the field is dark, except for cells that have bound antibody. These emit green (fluorescein) or red (rhodamine) light.

Antibodies can be coupled to indicators other than fluorescent molecules. Examples are enzymes such as alkaline phosphatase, horseradish peroxidase, and β-galactosidase. These enzymes can convert an added substrate to a colored dye. For example, the bacterial enzyme β-galactosidase converts the chromogenic substrate X-gal to a blue product, which can be visualized by microscopy.

Immunostaining is widely used in the research laboratory to determine subcellular location of proteins in cells. An example is the location of the herpes simplex viral protein VP22 in the nucleus of infected cells. To produce this image, virus-infected cells were stained with an antibody against VP22 and a mouse monoclonal antibody against α-tubulin, a cellular protein. Second antibodies bound to indicator molecules were then added: fluorsecein-conjugated anti-rabbit antibody, and Texas red-conjugated anti-mouse antibody (Texas red is another red fluorescent dye). The stained cells were then photographed with a microscope using ultraviolet light. The results show that VP22 (green) is located in the cell nucleus. Cellular α-tubulin is stained red. Photo courtesy of John Blaho.

Other uses of immunostaining include monitoring the synthesis of viral proteins, determining the effects of mutation on protein production, and investigating the sites of virus replication in animal hosts. Immunostaining of viral antigens in clinical specimens is also used to diagnose viral infections. Direct and indirect immunofluorescence assays with nasal swabs or washes are routine for diagnosis of infections with respiratory syncytial virus, influenza virus, parainfluenza virus, measles virus, and adenovirus.

When cultured cells are examined by this technique it is called immunocytochemistry; when tissues are studied, the procedure is immunohistochemistry. Flow cytometry is yet another way to use immunostaining to study the synthesis of one or more proteins in cells.

Detection of antigens or antibodies by ELISA

A more rapid method than Western blot analysis to detect a specific protein in a cell, tissue, organ, or body fluid is enzyme-linked immunosorbent assay, or ELISA. This method, which does not require fractionation of the sample by gel electrophoresisis, is based on the property of proteins to readily bind to a plastic surface.

To detect viral proteins in serum or clinical samples, a capture antibody, directed against the protein, is linked to a solid support such as a plastic 96 well microtiter plate, or a bead. The clinical specimen is added, and if viral antigens are present, they will be captured by the bound antibody. The bound viral antigen is then detected by using a second antibody linked to an enzyme. A chromogenic molecule – one that is converted by the enzyme to an easily detectible product – is then added. The enzyme amplifies the signal because a single catalytic enzyme molecule can generate many product molecules.

To detect antibodies to viruses, viral protein is linked to the plastic support, and then the clinical specimen is added. If antibodies against the virus are present in the specimen, they will bind to the immobilized antigen. The bound antibodies are then detected by using a second antibody that binds to the first antibody.

ELISA is used in both experimental and diagnostic virology. It is a highly sensitive assay that can detect proteins at the picomolar to nanomolar range (10-12 to 10-9 moles per liter). It is the mainstay for the diagnosis of infections by many different viruses, including HIV-1, HTLV-1, adenovirus, and cytomegalovirus.