Antigen is a molecule recognized by the receptors of B and T lymphocytes.

Activation of the adaptive immune defenses is triggered by pathogen-specific molecular structures called antigens. Antigens are similar to the pathogen-associated molecular patterns (PAMPs); however, whereas PAMPs are molecular structures found on numerous pathogens, antigens are unique to a specific pathogen. The antigens that stimulate adaptive immunity to chickenpox, for example, are unique to the varicella-zoster virus but significantly different from the antigens associated with other viral pathogens.

The term antigen was initially used to describe molecules that stimulate the production of antibodies; in fact, the term comes from a combination of the words antibody and generator, and a molecule that stimulates antibody production is said to be antigenic. However, the role of antigens is not limited to humoral immunity and the production of antibodies; antigens also play an essential role in stimulating cellular immunity, and for this reason antigens are sometimes more accurately referred to as immunogens. In this text, however, we will typically refer to them as antigens.

Pathogens possess a variety of structures that may contain antigens. For example, antigens from bacterial cells may be associated with their capsules, cell walls, fimbriae, flagella, or pili. Bacterial antigens may also be associated with extracellular toxins and enzymes that they secrete. Viruses possess a variety of antigens associated with their capsids, envelopes, and the spike structures they use for attachment to cells.

Antigens may belong to any number of molecular classes, including carbohydrates, lipids, nucleic acids, proteins, and combinations of these molecules. Antigens of different classes vary in their ability to stimulate adaptive immune defenses as well as in the type of response they stimulate (humoral or cellular). The structural complexity of an antigenic molecule is an important factor in its antigenic potential. In general, more complex molecules are more effective as antigens. For example, the three-dimensional complex structure of proteins make them the most effective and potent antigens, capable of stimulating both humoral and cellular immunity. In comparison, carbohydrates are less complex in structure and therefore less effective as antigens; they can only stimulate humoral immune defenses. Lipids and nucleic acids are the least antigenic molecules, and in some cases may only become antigenic when combined with proteins or carbohydrates to form glycolipids, lipoproteins, or nucleoproteins.

One reason the three-dimensional complexity of antigens is so important is that antibodies and T cells do not recognize and interact with an entire antigen but with smaller exposed regions on the surface of antigens called epitopes. A single antigen may possess several different epitopes (Figure), and different antibodies may bind to different epitopes on the same antigen (Figure). For example, the bacterial flagellum is a large, complex protein structure that can possess hundreds or even thousands of epitopes with unique three-dimensional structures. Moreover, flagella from different bacterial species (or even strains of the same species) contain unique epitopes that can only be bound by specific antibodies.

An antigen’s size is another important factor in its antigenic potential. Whereas large antigenic structures like flagella possess multiple epitopes, some molecules are too small to be antigenic by themselves. Such molecules, called haptens, are essentially free epitopes that are not part of the complex three-dimensional structure of a larger antigen. For a hapten to become antigenic, it must first attach to a larger carrier molecule (usually a protein) to produce a conjugate antigen. The hapten-specific antibodies produced in response to the conjugate antigen are then able to interact with unconjugated free hapten molecules. Haptens are not known to be associated with any specific pathogens, but they are responsible for some allergic responses. For example, the hapten urushiol, a molecule found in the oil of plants that cause poison ivy, causes an immune response that can result in a severe rash (called contact dermatitis). Similarly, the hapten penicillin can cause allergic reactions to drugs in the penicillin class.

An antibody is a protein component of the immune system that circulates in the blood, recognizes foreign substances like bacteria and viruses, and neutralizes them. After exposure to a foreign substance, called an antigen, antibodies continue to circulate in the blood, providing protection against future exposures to that antigen.

Antibody is a part of the host cell's defense. It's made by a certain type of white blood cell that's called a B cell. The structure of the antibody consists of two light chains and two heavy chains, and at the very tip of the antibody is a hypervariable region, and this hypervariable region allows the antibody to make different types of antibodies that will respond to all of the antigens that will assault the body. An antigen is anything that is foreign to the human body. It can be a virus, it can be a bacteria, and in some cases your own body will appear as foreign. And so you can have in certain instances where your own body will make antibodies against parts that are part of you.

Antigens

Antigens on pathogens are usually large and complex, and consist of many antigenic determinants. An antigenic determinant (epitope) is one of the small regions within an antigen to which a receptor can bind, and antigenic determinants are limited by the size of the receptor itself. They usually consist of six or fewer amino acid residues in a protein, or one or two sugar moieties in a carbohydrate antigen. Antigenic determinants on a carbohydrate antigen are usually less diverse than on a protein antigen. Carbohydrate antigens are found on bacterial cell walls and on red blood cells (the ABO blood group antigens). Protein antigens are complex because of the variety of three-dimensional shapes that proteins can assume, and are especially important for the immune responses to viruses and worm parasites. It is the interaction of the shape of the antigen and the complementary shape of the amino acids of the antigen-binding site that accounts for the chemical basis of specificity (image).

A typical protein antigen has multiple antigenic determinants, shown by the ability of T cells with three different specificities to bind to different parts of the same antigen.

Antigen Processing and Presentation

Although image shows T cell receptors interacting with antigenic determinants directly, the mechanism that T cells use to recognize antigens is, in reality, much more complex. T cells do not recognize free-floating or cell-bound antigens as they appear on the surface of the pathogen. They only recognize antigen on the surface of specialized cells called antigen-presenting cells. Antigens are internalized by these cells. Antigen processing is a mechanism that enzymatically cleaves the antigen into smaller pieces. The antigen fragments are then brought to the cell’s surface and associated with a specialized type of antigen-presenting protein known as a major histocompatibility complex (MHC) molecule. The MHC is the cluster of genes that encode these antigen-presenting molecules. The association of the antigen fragments with an MHC molecule on the surface of a cell is known as antigen presentation and results in the recognition of antigen by a T cell. This association of antigen and MHC occurs inside the cell, and it is the complex of the two that is brought to the surface. The peptide-binding cleft is a small indentation at the end of the MHC molecule that is furthest away from the cell membrane; it is here that the processed fragment of antigen sits. MHC molecules are capable of presenting a variety of antigens, depending on the amino acid sequence, in their peptide-binding clefts. It is the combination of the MHC molecule and the fragment of the original peptide or carbohydrate that is actually physically recognized by the T cell receptor (image).

Two distinct types of MHC molecules, MHC class I and MHC class II, play roles in antigen presentation. Although produced from different genes, they both have similar functions. They bring processed antigen to the surface of the cell via a transport vesicle and present the antigen to the T cell and its receptor. Antigens from different classes of pathogens, however, use different MHC classes and take different routes through the cell to get to the surface for presentation. The basic mechanism, though, is the same. Antigens are processed by digestion, are brought into the endomembrane system of the cell, and then are expressed on the surface of the antigen-presenting cell for antigen recognition by a T cell. Intracellular antigens are typical of viruses, which replicate inside the cell, and certain other intracellular parasites and bacteria. These antigens are processed in the cytosol by an enzyme complex known as the proteasome and are then brought into the endoplasmic reticulum by the transporter associated with antigen processing (TAP) system, where they interact with class I MHC molecules and are eventually transported to the cell surface by a transport vesicle.

Extracellular antigens, characteristic of many bacteria, parasites, and fungi that do not replicate inside the cell’s cytoplasm, are brought into the endomembrane system of the cell by receptor-mediated endocytosis. The resulting vesicle fuses with vesicles from the Golgi complex, which contain pre-formed MHC class II molecules. After fusion of these two vesicles and the association of antigen and MHC, the new vesicle makes its way to the cell surface.

Professional Antigen-presenting Cells

Many cell types express class I molecules for the presentation of intracellular antigens. These MHC molecules may then stimulate a cytotoxic T cell immune response, eventually destroying the cell and the pathogen within. This is especially important when it comes to the most common class of intracellular pathogens, the virus. Viruses infect nearly every tissue of the body, so all these tissues must necessarily be able to express class I MHC or no T cell response can be made.

On the other hand, class II MHC molecules are expressed only on the cells of the immune system, specifically cells that affect other arms of the immune response. Thus, these cells are called “professional” antigen-presenting cells to distinguish them from those that bear class I MHC. The three types of professional antigen presenters are macrophages, dendritic cells, and B cells (image).

Macrophages stimulate T cells to release cytokines that enhance phagocytosis. Dendritic cells also kill pathogens by phagocytosis (see image), but their major function is to bring antigens to regional draining lymph nodes. The lymph nodes are the locations in which most T cell responses against pathogens of the interstitial tissues are mounted. Macrophages are found in the skin and in the lining of mucosal surfaces, such as the nasopharynx, stomach, lungs, and intestines. B cells may also present antigens to T cells, which are necessary for certain types of antibody responses, to be covered later in this chapter.

• Major histocompatibility complex (MHC) is a collection of genes coding for glycoprotein molecules expressed on the surface of all nucleated cells.
• MHC I molecules are expressed on all nucleated cells and are essential for presentation of normal “self” antigens. Cells that become infected by intracellular pathogens can present foreign antigens on MHC I as well, marking the infected cell for destruction.
• MHC II molecules are expressed only on the surface of antigen-presenting cells (macrophages, dendritic cells, and B cells). Antigen presentation with MHC II is essential for the activation of T cells.
• Antigen-presenting cells (APCs) primarily ingest pathogens by phagocytosis, destroy them in the phagolysosomes, process the protein antigens, and select the most antigenic/immunodominant epitopes with MHC II for presentation to T cells.
• Cross-presentation is a mechanism of antigen presentation and T-cell activation used by dendritic cells not directly infected by the pathogen; it involves phagocytosis of the pathogen but presentation on MHC I rather than MHC II.

As discussed in Cellular Defenses, major histocompatibility complex (MHC) molecules are expressed on the surface of healthy cells, identifying them as normal and “self” to natural killer (NK) cells. MHC molecules also play an important role in the presentation of foreign antigens, which is a critical step in the activation of T cells and thus an important mechanism of the adaptive immune system.

Major Histocompatibility Complex Molecules

The major histocompatibility complex (MHC) is a collection of genes coding for MHC molecules found on the surface of all nucleated cells of the body. In humans, the MHC genes are also referred to as human leukocyte antigen (HLA) genes. Mature red blood cells, which lack a nucleus, are the only cells that do not express MHC molecules on their surface.

There are two classes of MHC molecules involved in adaptive immunity, MHC I and MHC II (Figure 18.11). MHC I molecules are found on all nucleated cells; they present normal self-antigens as well as abnormal or nonself pathogens to the effector T cells involved in cellular immunity. In contrast, MHC II molecules are only found on macrophages, dendritic cells, and B cells; they present abnormal or nonself pathogen antigens for the initial activation of T cells.

Both types of MHC molecules are transmembrane glycoproteins that assemble as dimers in the cytoplasmic membrane of cells, but their structures are quite different. MHC I molecules are composed of a longer α protein chain coupled with a smaller β₂ microglobulin protein, and only the α chain spans the cytoplasmic membrane. The α chain of the MHC I molecule folds into three separate domains: α₁, α₂ and α₃. MHC II molecules are composed of two protein chains (an α and a β chain) that are approximately similar in length. Both chains of the MHC II molecule possess portions that span the plasma membrane, and each chain folds into two separate domains: α₁ and α₂, and β₁, and β₂. In order to present abnormal or non-self-antigens to T cells, MHC molecules have a cleft that serves as the antigen-binding site near the “top” (or outermost) portion of the MHC-I or MHC-II dimer. For MHC I, the antigen-binding cleft is formed by the α₁ and α₂ domains, whereas for MHC II, the cleft is formed by the α₁ and β₁ domains (Figure 18.11).

Figure 18.11 MHC I are found on all nucleated body cells, and MHC II are found on macrophages, dendritic cells, and B cells (along with MHC I). The antigen-binding cleft of MHC I is formed by domains α₁ and α₂. The antigen-binding cleft of MHC II is formed by domains α₁ and β₁.

Antigen-Presenting Cells (APCs)

All nucleated cells in the body have mechanisms for processing and presenting antigens in association with MHC molecules. This signals the immune system, indicating whether the cell is normal and healthy or infected with an intracellular pathogen. However, only macrophages, dendritic cells, and B cells have the ability to present antigens specifically for the purpose of activating T cells; for this reason, these types of cells are sometimes referred to as antigen-presenting cells (APCs).

While all APCs play a similar role in adaptive immunity, there are some important differences to consider. Macrophages and dendritic cells are phagocytes that ingest and kill pathogens that penetrate the first-line barriers (i.e., skin and mucous membranes). B cells, on the other hand, do not function as phagocytes but play a primary role in the production and secretion of antibodies. In addition, whereas macrophages and dendritic cells recognize pathogens through nonspecific receptor interactions (e.g., PAMPs, toll-like receptors, and receptors for opsonizing complement or antibody), B cells interact with foreign pathogens or their free antigens using antigen-specific immunoglobulin as receptors (monomeric IgD and IgM). When the immunoglobulin receptors bind to an antigen, the B cell internalizes the antigen by endocytosis before processing and presentting the antigen to T cells.

Antigen Presentation with MHC II Molecules

MHC II molecules are only found on the surface of APCs. Macrophages and dendritic cells use similar mechanisms for processing and presentation of antigens and their epitopes in association with MHC II; B cells use somewhat different mechanisms that will be described further in B Lymphocytes and Humoral Immunity. For now, we will focus on the steps of the process as they pertain to dendritic cells.

After a dendritic cell recognizes and attaches to a pathogen cell, the pathogen is internalized by phagocytosis and is initially contained within a phagosome. Lysosomes containing antimicrobial enzymes and chemicals fuse with the phagosome to create a phagolysosome, where degradation of the pathogen for antigen processing begins. Proteases (protein-degrading) are especially important in antigen processing because only protein antigen epitopes are presented to T cells by MHC II (Figure 18.12).

APCs do not present all possible epitopes to T cells; only a selection of the most antigenic or immunodominant epitopes are presented. The mechanism by which epitopes are selected for processing and presentation by an APC is complicated and not well understood; however, once the most antigenic, immunodominant epitopes have been processed, they associate within the antigen-binding cleft of MHC II molecules and are translocated to the cell surface of the dendritic cell for presentation to T cells.

Figure 18.12 A dendritic cell phagocytoses a bacterial cell and brings it into a phagosome. Lysosomes fuse with the phagosome to create a phagolysosome, where antimicrobial chemicals and enzymes degrade the bacterial cell. Proteases process bacterial antigens, and the most antigenic epitopes are selected and presented on the cell’s surface in conjunction with MHC II molecules. T cells recognize the presented antigens and are thus activated.

Antigen Presentation with MHC I Molecules

MHC I molecules, found on all normal, healthy, nucleated cells, signal to the immune system that the cell is a normal “self” cell. In a healthy cell, proteins normally found in the cytoplasm are degraded by proteasomes (enzyme complexes responsible for degradation and processing of proteins) and processed into self-antigen epitopes; these self-antigen epitopes bind within the MHC I antigen-binding cleft and are then presented on the cell surface. Immune cells, such as NK cells, recognize these self-antigens and do not target the cell for destruction. However, if a cell becomes infected with an intracellular pathogen (e.g., a virus), protein antigens specific to the pathogen are processed in the proteasomes and bind with MHC I molecules for presentation on the cell surface. This presentation of pathogen-specific antigens with MHC I signals that the infected cell must be targeted for destruction along with the pathogen.

Before elimination of infected cells can begin, APCs must first activate the T cells involved in cellular immunity. If an intracellular pathogen directly infects the cytoplasm of an APC, then the processing and presentation of antigens can occur as described (in proteasomes and on the cell surface with MHC I). However, if the intracellular pathogen does not directly infect APCs, an alternative strategy called cross-presentation is utilized. In cross-presentation, antigens are brought into the APC by mechanisms normally leading to presentation with MHC II (i.e., through phagocytosis), but the antigen is presented on an MHC I molecule for CD8 T cells. The exact mechanisms by which cross-presentation occur are not yet well understood, but it appears that cross-presentation is primarily a function of dendritic cells and not macrophages or B cells.

“Antigens” are molecular structures on the surface of viruses that are recognized by the immune system and are capable of triggering one kind of immune response known as antibody production. Two proteins (hemagglutinin and neuraminidase) on the surface of influenza viruses contain the major antigens targeted by antibodies (see Figure 1).

When someone is exposed to an influenza virus (either through infection or vaccination) their immune system makes specific antibodies against the antigens (surface proteins) on that particular influenza virus. The term “antigenic properties” is used to describe the antibody or immune response triggered by the antigens on a particular virus. “Antigenic characterization” refers to the analysis of a virus’ antigenic properties to help assess how related it is to another virus.

CDC antigenically characterizes about 2,000 influenza viruses during a typical flu season to monitor for changes in circulating viruses and to compare how similar these viruses are to those included in vaccines. Antigenic characterization can give an indication of the flu vaccine’s ability to produce an immune response against the influenza viruses circulating in people. This information also helps experts decide what viruses should be included in the upcoming season’s influenza vaccine.

Serology tests using human sera and genetic sequencing provide additional information about how similar circulating flu viruses are to vaccine viruses or other influenza viruses.

Figure 1. Influenza Virus Features

The above image shows the different features of an influenza virus, including the main surface proteins, hemagglutinin (HA) and neuraminidase (NA). Following influenza infection or receipt of a flu vaccine, the body’s immune system develops antibodies that recognize and bind to “antigenic sites,” which are regions found on an influenza virus’ surface proteins. By binding to the HA antigenic sites, antibodies can neutralize influenza viruses, which prevents them from causing further infection. (Antibodies that bind to the NA antigenic sites can also reduce further spread.)

The Hemagglutination Inhibition Assay (HI Test)

Scientists use a test called the hemagglutination inhibition assay (HI test) to antigenically characterize influenza viruses. HA proteins on the surface of influenza viruses can bind to red blood cells and “glue” them together, forming a lattice structure (this is known as “hemagglutination”). The picture below provides an example of hemagglutination. The rightmost section of the image shows what hemagglutination looks like in the well of a microtiter plate (this is explained in greater detail further below).

The HI test works by measuring how well antibodies bind to the HA proteins and prevent them from “gluing” red blood cells together (i.e., hemagglutination inhibition). The picture below provides an example of hemagglutination inhibition. The rightmost section of the image shows what hemagglutination inhibition looks like in the well of a microtiter plate (this is explained in greater detail further below).

Scientists use the HI test to assess the antigenic similarity between influenza viruses. This test helps to select the candidate vaccine viruses (CVVs) included in seasonal flu vaccines. HI test results can tell us whether antibodies developed after vaccination with one virus recognize, bind to, and therefore, are similar to other circulating influenza viruses. Scientists also use the HI test to compare the antigenic properties (i.e., the virus’ ability to be recognized by antibodies) of currently circulating influenza viruses with those of influenza viruses that have circulated in the past.

The HI test involves three main components: antibodies, influenza virus, and red blood cells that are mixed together in the wells (i.e., cups) of a microtiter plate. (See Image 1.)

Image 1. A Microtiter Plate

A microtiter plate is used to perform the HI test. The plate contains wells (i.e., cup-like depressions that can hold a small amount of liquid) where the solution of antibodies, influenza virus and red blood cells are inserted and allowed to interact. These wells are arranged according to rows and columns (which are identified on the microtiter plate by letters and numbers, respectively). The rows of the plate can be used to test different influenza viruses against the same set of antibodies. The columns can be used to differentiate between greater dilutions of antibodies, like a scale from low to high going from left to right (see Figures 3 and 4 for an example).

The antibodies used in the HI test are obtained by infecting an animal (usually a ferret) that is immunologically naïve (i.e., it has not been exposed to any influenza virus or vaccine previously in its lifetime). The animal’s immune system creates antibodies in response to the antigens on the surface of the specific flu virus that was used to infect that animal. To study these antibodies, a sample of blood is drawn from the animal, from which serum is obtained. The HI test measures how well these antibodies recognize and bind to other influenza viruses (such as, influenza viruses isolated from flu patients). If the ferret antibodies that resulted from exposure to the vaccine virus recognize and bind well to the influenza virus from a sick patient, this indicates that the vaccine virus is antigenically similar to the influenza virus obtained from the sick patient. This finding has implications for how well the vaccine might work in people. See Flu Vaccine Effectiveness: Questions and Answers for Health Professionals for more information.

As previously mentioned, the influenza viruses used in the HI test are obtained from sick people. CDC and other WHO collaborating centers collect specimens from people all over the world to track which influenza viruses are infecting humans and to monitor how these viruses are changing.

For the HI test, red blood cells (RBCs) are taken from animals (usually turkeys or guinea pigs). They are used in the HI test because influenza viruses bind to them. Normally, RBCs in a solution will sink to the bottom of the assay well and form a red dot at the bottom (Figure 2A). However, when an influenza virus is added to the RBC solution, the virus’ hemagglutinin (HA) surface proteins will bind to multiple RBCs. When influenza viruses bind to the RBCs, the red cells form a lattice structure (Figure 2B). This keeps the RBCs suspended in solution instead of sinking to the bottom and forming the red dot. The process of the influenza virus binding to RBCs to form the lattice structure is called “hemagglutination.”

Figure 2. Components of the HI Assay

The HI test involves the interaction of red blood cells (RBCs), antibody and influenza virus. Row A shows that in the absence of virus, RBCs in a solution will sink to the bottom of a microtiter plate well and look like a red dot. Row B shows that influenza viruses will bind to RBCs when placed in the same solution. This is called hemagglutination and is represented by the formation of the lattice structure, depicted in the far-right column under “Microtiter Results.” Row C shows that the antibody binds well to the virus and prevents the virus from gluing RBCs together and forming a lattice structure, therefore, hemagglutination does not occur (i.e., hemagglutination inhibition occurs instead).

When antibodies are pre-mixed with influenza virus followed by RBCs, the antibodies will bind to influenza virus antigens that they recognize, covering the virus so that its HA surface proteins can no longer bind to RBCs (Figure 2C). The reaction between the antibody and the virus inhibits (i.e., prevents) hemagglutination from occurring, which results in hemagglutination inhibition (as shown in Figure 2C). This is why the assay is called a “hemagglutination inhibition (HI) test.” Hemagglutination (as depicted in Figure 2B) occurs when antibodies do not recognize and bind to the influenza viruses in the solution, and as a result, the influenza viruses bind to the red blood cells, forming the lattice structure. When the antibodies do recognize and bind to the influenza virus in the solution, this shows that the vaccine virus (like the one the ferret was infected with) is similar to the influenza virus obtained from the sick patient. When this happens, the influenza virus being tested is said to be “antigenically similar” to the influenza virus that created the antibodies (from the ferret).

When a circulating influenza virus is antigenically different from a vaccine, the antibodies developed in response to the vaccine virus may not recognize and bind this virus. In the HI test, this will cause hemagglutination to occur (see Figure 2B). Circulating influenza viruses tested via the HI test are typically obtained from respiratory samples collected from sick patients.

Assessing Antigenic Similarity Using the HI Test

The HI test assesses the degree of antigenic similarity between two viruses using a scale based on antibody dilution. As previously mentioned, the HI test is performed using a microtiter plate. The microtiter plate contains rows and columns of wells (i.e., cups) where RBCs, influenza virus and antibodies (developed against a comparison virus, such as a vaccine virus) are mixed. Dilutions are marked across the top of the microtiter plate. These dilutions function as a scale for assessing antigenic similarity and immune response. By testing the ability of greater dilutions of antibody to prevent hemagglutination, scientists measure how well those antibodies recognize and bind to an influenza virus. The higher the dilution, the fewer antibodies are needed to block hemagglutination and the more antigenically similar the two viruses being compared are to each other. The highest dilution of antibody that results in hemagglutination inhibition of the test virus is considered an HI titer (Figure 3).

Figure 3. HI Titer Assay

This virus has an HI titer of 1280, which means that the greatest dilution of antibody that still blocked hemagglutination from occurring was at 1280 dilution. At this dilution, the antibodies were still capable of recognizing and binding to the antigens on the virus.

Figure 4. Antigenic Characterization Assay

When CDC antigenically characterizes influenza viruses to inform decisions on the formulation of the seasonal flu vaccine, the HI test is used to compare currently circulating viruses (rows B and C) with vaccine viruses (row A). This allows scientists to quickly determine if a virus used in the seasonal flu vaccine is antigenically similar to circulating influenza viruses and therefore capable of producing an immune response against them.

Public health experts consider influenza viruses to be antigenically similar or “like” each other if their HI titers differ by two dilutions or less. (This is equivalent to a two-well (i.e., a four-fold dilution) or less difference). Using Figure 4 as an example, when circulating virus 1 is compared to a vaccine virus, circulating virus 1 differs by one dilution (a 2-fold difference) and therefore is “like” the previous season’s vaccine virus. However, circulating virus 2 differs by five dilutions (a 32-fold difference) and therefore is not “like” the previous season’s vaccine virus. Circulating viruses that are antigenically dissimilar (i.e., not “like”) to vaccine virus are considered “low reactors.”

Limitations

Antigenic characterization gives important information about whether a vaccine made using a specific vaccine virus will protect against circulating influenza viruses, but there are several limitations to antigenic characterization test methodology, which are described below.

Does not work with recent Flu A(H3N2) viruses

An important limitation of the HI test is that the majority of circulating influenza A(H3N2) viruses do not bind to RBCs, and therefore, cannot be tested using the HI test. As a result, CDC has developed a different virus neutralization assay called the “high-content imaging-based neutralization test” (HINT) to measure how circulating A(H3N2) flu viruses are evolving to evade human immunity. More information about HINT is available in a CDC spotlight article.

Egg Adaptations

Right now, most flu vaccines are made using viruses grown in chicken eggs. As human influenza viruses adapt to grow in eggs, genetic changes can occur in the viruses. These are called “egg-adapted” changes. Some egg-adapted changes may change the virus’ antigenic (or immunogenic) properties while others may not. Egg-adapted changes have become a particular problem for selection of candidate vaccine viruses (CVVs) for the influenza A(H3N2) virus component of flu vaccine made using egg-based production technology. Influenza A(H3N2) viruses tend to grow less well in chicken eggs than other influenza viruses (e.g. A(H1N1)pdm09), and they also are more prone to egg-adapted changes. Such changes can reduce the immune protection provided by the flu vaccine against circulating A(H3N2) viruses.

Time from Vaccine Virus Selection to Delivery of Vaccine

Using current egg-based vaccine production technology, it takes about six months from the time that a vaccine virus is chosen (i.e., February for the Northern Hemisphere flu vaccine) to the time when influenza vaccines become widely available. Because influenza viruses are constantly changing, circulating influenza viruses may change during this six month period. If these genetic changes cause antigenic changes, it could mean that antibodies created through vaccination may not recognize and inactivate circulating influenza viruses. New technologies that shorten the manufacturing time could reduce the chances of significant antigenic changes occurring in circulating viruses before influenza vaccines become available each year: for example, cell-based and recombinant flu vaccines.

Use of Animals

Use of the HI test to assess the similarity of circulating influenza viruses to vaccine viruses involves obtaining antibodies from animals (particularly ferrets). Ferrets are often used as an animal model for influenza infection because they can be easily infected with human influenza viruses and experience similar signs and symptoms (e.g., fever, sneezing, loss of appetite, etc.). Prior to infection, ferret serum is pre-screened to determine that it does not contain antibodies to circulating influenza viruses. When ferrets are infected with a live influenza virus, they typically produce strong antibody responses against that particular virus. Therefore, ferret antisera are commonly used to detect antigenic differences between influenza viruses. However, there are differences between the immune responses of ferrets and humans which need to be considered when assessing the antigenic properties of influenza viruses.