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.

Reinfection with 2009 influenza H1N1

immune-memoryIn healthy individuals, the first encounter with a virus leads to a primary antibody response. When an infection occurs with the same or a similar virus, a rapid antibody response occurs that is called the secondary antibody response. Antibodies are critical for preventing many viral infections, including influenza. But reinfection may occur if we encounter the same virus before the primary response is complete.

Recently three cases of confirmed infection with 2009 influenza H1N1 were reported in Chile. The first patient had laboratory confirmed infection; treatment with oseltamivir resolved symptoms after 48 hours. Twenty days later the patient developed a second bout of laboratory confirmed influenza which was treated with amantadine. The second patient acquired laboratory confirmed influenza in hospital, was treated with oseltamivir and recovered. Two weeks later, while still in hospital, the patient had a new episode of laboratory confirmed influenza infection. Treatment with oseltamivir again resolved the infection. The third patient also acquired laboratory infection in hospital, was successfully treated with oseltamivir, and was discharged. He was readmitted 18 days later with confirmed pandemic H1N1 2009, and again successfully treated with oseltamivir.

These individuals were likely resusceptible to reinfection with the same strain of influenza virus due to a confluence of unusual events. First, all three were reinfected within three weeks, before their primary adaptive response had sufficiently matured. Another contributing factor was the high level of circulation of the pandemic strain. This issue was compounded for patients two and three who probably acquired both infections while in the hospital (called nosocomial transmission).

Could reinfection also occur after immunization with influenza vaccine? Yes, if the immunized individual encounters the virus before the primary antibody response matures, which occurs in 3-4 weeks. This is more likely to occur during pandemic influenza when circulation of the virus is more extensive than in non-pandemic years.

Perez CM, Ferres M, & Labarca JA (2010). Pandemic (H1N1) 2009 Reinfection, Chile. Emerging infectious diseases, 16 (1), 156-7 PMID: 20031070

Adaptive immune defenses: Antibodies

With the looming prospect of mass immunization against influenza, it’s important to understand how vaccines work. To do this we must have a good understanding of adaptive immune defenses. Today we’ll begin a discussion of the humoral arm of the adaptive immune response – antibodies.

Antibodies are large proteins produced by vertebrates that play important roles in identifying and eliminating foreign objects. The basic structural unit is composed of two heavy chains and two light chains, as shown in this diagram.

antibody

Antibodies bind other molecules known as antigens. Binding occurs in a small region near the ends of the heavy and light chain called the hypervariable region (labeled only on one arm in the figure). As the name implies, this region is extremely variable, which is why vertebrates can produce millions of antibodies that can bind many different antigens. The part of the antigen that is recognized by the antibody is known as an epitope.

There are five classes of immunoglobulin—IgA, IgD, IgE, IgG, and IgM—defined by the amino acid sequence of the heavy chain. They have different roles in immune responses; IgG, IgA, and IgM are commonly produced after viral infection.

During the first encounter with a virus, a primary antibody response occurs. IgM antibody appears first, followed by IgA on mucosal surfaces or IgG in the serum. The IgG antibody is the major antibody of the response and is very stable, with a half-life of 7 to 21 days. When an infection occurs with the same or a similar virus, a rapid antibody response occurs that is called the secondary antibody response. The specificity and memory of the antibody response are illustrated in the following graph.

immune-memory

A typical adaptive antibody response is shown as the relative concentration of serum antibodies weeks after injection of an animal with antigen A or a mixture of antigens A and B. Maximal primary response to antigen A occurs in 3 to 4 weeks. When the animal is injected with a mixture of both antigens A and B at 7 weeks, the secondary response to antigen A is more rapid and stronger than the primary response, demonstrating immunological memory. As expected, the primary response to antigen B requires 3 – 4 weeks. Antibody levels (also called antibody titers) decline with time after each immunization, a property known as self-limitation or resolution.

Antibodies are critical for preventing many viral infections, and may also contribute to the resolution of infection. We’ll next explore how antibodies accomplish these diverse activities.

Innate immune defenses

viral countermeasuresIn response to viral infection, many organisms mount a remarkable defense known as the immune response. This response to viral infection consists of an innate, or nonspecific component, and an adaptive, or specific defense. The innate response is considered the first line of immune defense because it is active even before infection begins. In fact, many viral infections are halted by the innate immune system, which responds very quickly – within minutes to hours after infection.

A key property of the innate immune system is the ability to recognize viruses as ‘foreign’. Viral proteins and nucleic acids are distinguished from cellular counterparts by cellular proteins called pattern recognition receptors (illustrated). These are proteins present either in the cell cytoplasm or on cellular membranes, where they detect viral components. For example, the cytoplasmic protein RIG-I detects double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) with a 5′-triphosphate. These types of RNAs are usually not found in the cytoplasm of unifected cells; rather they are typically products of viral replication. When RIG-I binds these viral RNAs, a series of reactions occur which lead to the synthesis of cytokines, the primary output of the innate defense system. Other detectors of viruses are the membrane-bound toll-like receptors (TLRs), which sense viral glycoproteins, dsRNA, ssRNA, and the sequence CpG in viral DNA. Engagement of TLRs by these virus-specific ligands also leads to the synthesis of cytokines, albeit by different pathways.

The presence of cytokines in the blood is typically one of the earliest indications that the host has been infected with a virus. Over 80 known cytokines are secreted by infected cells. The first that are produced after viral infection include interferon-α and -β (IFN-α, IFN-β), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-12, and IFN-γ.

Cytokines function locally by binding receptors on other cells. For example, IFN produced by infected cells engages receptors on neighboring cells. Those cells then produce hundreds of cellular proteins which have antiviral activities. When cytokines enter the circulation, they elicit symptoms typical of many viral infections, including fever, sleepiness, lethargy, muscle pain, loss of appetite, and nausea.

Another key component of the innate response are the so-called sentinel cells: dendritic cells and macrophages present in peripheral compartments such as skin and mucosal surfaces. Sentinel cells patrol the body, seeking signs of infection. Dendritic cells bind cytokines produced by virus-infected cells, and also take up viral proteins released from dying virus-infected cells. They respond by producing more cytokines to amplify the original response.

In many viral infections, the early action of cytokines produced by infected cells and dendritic cells is sufficient to eliminate the pathogen. If innate defenses are overwhelmed and virus replication continues unabated, then the second-line defenses are mobilized to ensure host survival. These comprise the adaptive immune response – antibodies and immune cells. Days to weeks are required to mount an adaptive immune response that is specifically tailored to the infecting virus. The innate response therefore serves as a crucial rapid response that provides sufficient time for the activation of the adaptive immune system.

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