Secondary response (second inoculation of antigen)
Fig. 7.7 Immune memory. The first exposure to an antigen results in the primary response, which occurs after a week or so. During this time, maturation of immune-reactive cells is taking place. Once the primary response occurs, antibody and reactive T and B cells decline to a low level. Upon restimulation with the same antigen, the memory lymphocytes are rapidly mobilized and a more intense and more rapid immune response follows.
function too well, inadequate immunity may result. Several autoimmune diseases are caused by a lack of regulatory T cells, which normally comprise 1—3% of the total population of CD4+ T cells.
Other types of immune pathologies include autoimmune diseases where the immune system destroys seemingly healthy tissue in the body. This can be due to the immune system attempting to destroy cells that express viral antigens but that are otherwise healthy.
An example of an autoimmune pathology due to viral infection and persistent presentation of antigen is subacute sclerosing panencephalitis (SSPE), which is a pathological response to persistence of measles virus antigen in neural tissue. This was briefly described in Chapter 4. Some other autoimmune diseases, such as multiple sclerosis, are thought to be caused by a previous virus infection and apparent recovery. It has been suggested that a previous infection with a virus (perhaps years before) can lead to immune pathology — in this case demylenation of neurons. The exact mechanism of such pathology is not known, but a process termed "molecular mimicry" where a specific epitope of the pathogen bears similarity to one in the host tissue is suspected. Here, during the course of a normal immune response against the invading pathogen, normal tissue is also now recognized as foreign. This is known to be the mechanism for the role of group A Streptococcus in rheumatic fever where the robust immune response to the bacterial epitope leads to problems because of similarity to an epitope found in a protein in heart tissue. This mechanism has not been proved for multiple sclerosis, and indeed, such cases require very careful statistical evaluation of long-term medical records to demonstrate correlations.
The immune response is an effective one, and plays a constant role in selection against viruses that do not mount an efficient infection. Despite the effectiveness of the immune response, it is clear that many virus infections survive and thrive in the setting of the host's immune capacity. Indeed, the great majority of nuclear-replicating DNA viruses establish longlasting associations with their hosts. Clearly they are able to deal with host attempts to clear the infection. A major factor in virus survival is the fact that viruses mount many effective counter responses to the immune response. Some of these are essentially passive while others involve virus-mediated blockage of specific portions of the immune response.
Passive evasion of immunity — antigenic drift
All animal viruses occur in antigenically distinct forms or serotypes. The number of forms varies with the type of virus. For example, there is only one strain of measles virus, three major serotypes of poliovirus, more than 40 for adenovirus, and as many as 100 for papillomaviruses. A serotype is stable and may be confined to a specific geographic location, and prior infection with one serotype of a specific virus will lead to no or only partial protection from reinfection with another.
Because RNA-directed RNA replication has no built-in enzymatic error-correction mechanism, in contrast to DNA replication, RNA viruses are generally more susceptible to the generation of mutations leading to serotype formation than are DNA viruses. This process is often termed antigenic drift, and such drift is probably responsible for the large number of serotypes of rhinoviruses (more than 100), and is clearly responsible for the drift in influenza virus serotypes.
This mechanism for drift is countered by other factors that tend to favor antigenic "conservatism." For example, many RNA viruses (e.g., measles and poliovirus) do not exhibit large numbers of serotypes, and even where there is extensive drift, as with influenza, the internal proteins are antigenically relatively stable.
One factor in stabilizing protein sequences even when they are encoded by highly mutable RNA sequences is that important functional constraints on the amino acid sequence of viral proteins are imposed by enzymatic or precise structural functions. Such constraints do not operate with the same lack of tolerance for variation in the external glycoproteins of enveloped viruses.
Passive evasion of immunity — internal sanctuaries for infectious virus
Some viruses can evade the immune response of the host by establishing persistent or latent infections in tissue that is not subject to extensive immune surveillance. A classic example is the ability of HSV to establish latent infection in nondividing sensory neurons. Another example is the ability of respiratory syncytial virus to replicate at low levels in the mucous membranes of the nasopharynx where secretory antibodies provide protection against invasion by the virus, but cannot clear it. The highly localized replication of papillomaviruses, such as those causing skin warts, is another example of virus infection in a localized area that is removed from intense immune surveillance.
Passive evasion of immunity — immune tolerance
The immune system of fetuses and neonates is immature. This is an important strategy in the survival of the fetus as it develops in an antigenically distinct individual: its mother. Fetal and neonatal infections with viruses that normally cause generally mild infections in an immune-competent individual can be devastating in neonates. Rubella causes severe developmental abnormalities of the nervous system when it infects a developing fetus, and the fact that the virus does not evoke lasting immunity in adults means that it is a threat even to a mother who has been infected previously. A primary or reactivating HSV infection of the mother at the time of birth can lead to neonatal encephalitis with grave prognosis, and neonatal and uterine infections with cytomegalovirus are strongly linked to neurologically based developmental disorders. Active HIV replication at the time of delivery is the major mechanism of mother to child transmission of this virus, also.
At least one group of viruses, the arenaviruses, utilizes the ability to selectively accommodate themselves to the developing immunity of the neonate. These viruses, of which lymphocytic choriomeningitis virus (LCMV) is the best-characterized laboratory model, persist in populations of rodents and are transmitted to newborns from the infected mother. The mouse develops relatively normally with persistent viremia and shows an impaired immune response to LCMV. The tolerant mouse has circulating antibody that is reactive with the virus but cannot neutralize it. Further, there is a lack of T-cell responsiveness to the virus. If an immune-competent adult mouse is infected with LCMV, a robust immune response is mounted, but the infection is usually fatal! (see Chapter 23, Part V).
The mechanism for establishing immune tolerance is complex; it involves selection of specific viral genotypes with the ability to infect macrophages and some other cells of the immune system during the early stages of infection of the infant. This infection results in suppression of specific immunity against the virus.
Interestingly, the virus that is spread between individuals has tropism for neural tissue. These neurotropic and lymphotropic viruses differ only in a single amino acid in both the viral glycoprotein and the viral polymerase. The two variants are generated by random periodic mutations during replication of the resident virus in the animal, and while the neurotropic variant has little effect in the immune-tolerant animal, it causes severe disease in an uninfected adult. Similar patterns of infection are seen with other arenaviruses, several of which — including Lassa fever virus — are pathogenic for humans.
Active evasion of immunity — immunosuppression
Infections with a number of viruses lead to a transitory or permanent suppression of one or several branches of host immunity. Infectious mononucleosis caused by primary infection with EBV is a self-limiting generalized infection characterized by a relatively large induction of regulatory T lymphocytes. This not only results in the virus being able to maintain its infection effectively, but also results in the individual who has the infection being more susceptible to other infections. Some retroviruses, especially HIV, are able to specifically inhibit T-cell proliferation by the expression of suppressor proteins. Further, the continued destruction of T lymphocytes by HIV replication eventually leads to profound loss of immune competence: AIDS.
The polydnaviruses of certain wasps illustrate an evolutionary adaptation between virus and host based on the virus's ability to actively suppress immunity. This virus (mentioned in Chapter 1) is maintained as a persistent genetic passenger in the ovaries and egg cells of parasitic wasps. These wasps lay eggs in caterpillars of another insect species, and the developing larvae feed on the caterpillar as they develop. The polydnavirus inserted into the caterpillar along with the wasp egg induces a systemic, immunosuppressive infection so that the caterpillar cannot eliminate the embryonic tissue at an early stage of development. If wasps without such viruses inject eggs into the caterpillar host, there is a significant reduction in larval survival.
Active evasion of immunity — blockage of MHC antigen presentation
Adenovirus, HIV, and HSV specifically inhibit MHC-I antigen presentation. In each case, a specific virus protein that mediates this blockage is expressed. While it is apparent that the slowly replicating adenovirus will greatly benefit from its ability to interfere with host immunity, it requires a moment of reflection to see the importance of the blockage of MHC-I antigen presentation by HSV, which replicates very rapidly and efficiently in the cells it infects. Here, it is likely that the value is found in the earliest stages of reactivation from latent infection where small amounts of virus must be able to initiate infection in a host that has a powerful immune memory biased against HSV replication. Similarly, one of the earliest genes expressed by HIV encodes a protein (Nef), which downregulates MHC-I expression, thus evading cytotoxic T cell responses.
Consequences of immune suppression to virus infections
While some viruses are able to either mildly or profoundly suppress immunity during the course of infection and pathogenesis, immune suppression is also an important tool in certain medical conditions. Examples include the need to suppress host cell-mediated immunity prior to organ or tissue transplantation. Immune suppression also results from some types of intravenous drug abuse.
Major complications from immune suppression are reactivating herpesvirus infections such as varicella zoster (chicken pox) and cytomegalovirus infection. Of course, the same problems can occur when the immune system is disrupted by viral infections such as with HIV. A potentially more critical complication of significant populations of individuals evidencing immune suppression results from their serving as potential selective reservoirs for the development of antigenic and drug-resistant strains of pathogens. For example, the current increase in appearance of antibiotic-resistant tuberculosis is linked definitively to a combination of incomplete drug therapy, HIV infection, and drug-induced immunosuppression in critical urban, prison, and Third World populations.
MEASUREMENT OF THE IMMUNE REACTION Measurement of cell-mediated (T-cell) immunity
Cell-mediated immunity requires incubation of immune lymphocytes with a target cell and then measurement of a specific T-cell response. This can be difficult and tricky, but for measurement of T-cell-mediated cell lysis, the release of radioactive chromium from target cells is a convenient method. Target cells are incubated under conditions such that they incorporate the radioactive metal. The cells are rinsed so that the only radioactivity is inside the cells. Thus, the radioactivity will sediment to the bottom of a centrifuge tube under low gravity force (low speeds). In the presence of reactive killer T cells, the target cells are lysed and the "hot" chromium enters the solution and cannot be sedimented under low speeds.
A numerical assessment of the number of reactive lymphocytes can also be carried out by measuring cell replication as a response to a specific antigen. White blood cells are incubated with antigen and a radioactive nucleoside precursor to DNA. As T lymphocytes proliferate in response to antigen, they will incorporate this radioactive precursor. A measure of the incorporation of radioactivity in comparison to a control culture can be made and expressed as a lymphocyte stimulation index.
Another method for measuring T-cell immunity is to incubate antigen-bearing cells with lymphocytes. Reactive T lymphocytes will form rosettes around the antigen-bearing cell, and these can be observed and counted in the microscope.
Antibody molecules are secreted glycoproteins that have the capacity to recognize and combine with specific portions of viral or other proteins foreign to the host. As described in Chapter
12, Part III, antibody molecules have a very specific structure in which the antigen-combining sites, which comprise variable amino acid sequences, are at one location on the antibody molecules while a region of fixed amino acid sequence is found at another location. This constant region (Fc region) has a major function in mediating secretion of the antibody molecule from the B lymphocyte expressing it. Another major function of the Fc region is to serve as a signal to cells and other specific cellular proteins that the molecule bound to the antigen is, indeed, an antibody.
Enzyme-linked immunosorbent assays (ELISAs)
A number of methods to measure antibody reactions involve use of the antibody molecule's Fc region as a "handle." Extremely sensitive methods known collectively as enzyme-linked immunosorbent assays (ELISAs) use enzymes that can process a colorless substrate into a colored product bound to the Fc region of an antibody molecule. When the antibody is bound to an antigen, the enzyme affixed to the Fc region will also be bound. If the antigen—antibody complex is then incubated with appropriate substrates for the bound enzyme, the generation of color can be used as a measure of the antibody present. Examples of the method are outlined in Fig. 7.9.
ELISAs are of tremendous value for rapid diagnosis, and have great commercial significance. For example, if an antigenic peptide is bound to an insoluble matrix such as a flexible plastic strip onto which dry reagents are included and this strip is dipped into a plasma preparation
Fig. 7.9 An enzyme-linked immunosorbent assay (ELISA): the method of using a color reaction mediated by an enzyme bound to the Fc region of the antibody molecule. P = colored product; S = substrate.
Enzyme^^^ Fc region ^k ^ntigen
^ A gV\ A J
Wash away un
^ A A A J
_ , , , , Add colorle Colored product
y A A A J
Enzyme converts into colored product
^ No enzyme, no color ^
that contains antibody against the peptide, a color will develop. Even if the amount of antibody is very low, incubation for a long enough period will generate some color as long as the enzyme used is relatively stable. The method is quite adaptable to quantitative as well as qualitative analysis, and can be adapted for use with automated equipment. A number of kits are currently commercially available where a small sample of body fluid that might contain either a virus or an antibody of interest can be spotted and dried. The kit is then sent to a laboratory where it can be quantitatively analyzed.
The use of lasers and microtechnology developed in the electronics industry promises to provide even more revolutionary changes to our ability to detect extremely small amounts of viral antigens or antibodies in test material. A microchip can be synthesized with a huge number of different potential antigens bound to it, and this can be incubated with unknown antibody and then subjected to either an ELISA or another method to generate a fluorescent signal where an antigen—antibody complex is formed. This can be rapidly scanned with a laser beam and fluorescent microscope, or alternatively in a solid-state detection device. Such methods make it potentially possible to screen a given serum sample for the presence of antibodies directed against all or nearly all identified pathogenic agents in a few hours! Detection of such antibodies indicates current or previous exposure to the corresponding pathogen.
Some ways to measure the reaction between specific antibody molecules and an antigen involve the loss of specific functions by the target virus. Many antibodies will block the ability of a virus to initiate an infection in a cultured cell, and thus block the formation of a center of infection or virus plaque. Plaque assays are described in Chapter 10, Part III, and the inhibition of plaque formation is termed an infectivity neutralization or neutralization of a virus. Here, a target virus with a known titer is incubated with test antibody dilutions. The more concentrated and specific the antibody, the more the initial antibody solution can be diluted and still block viral infectivity (and thus formation of plaques). Neutralization is illustrated schematically in Fig. 7.10.
Some methods for the measurement of antibody against viruses are based on the ability of the antibody to block some property of the virus. For example, it has been known since the first part of this century that many enveloped viruses will stick to red blood cells and cause them to agglutinate. This property of hemagglutination can be used as a crude measure of viral particle concentration in solution, as described in Chapter 9, Part III.
Many antibodies against enveloped viruses will inhibit virus-mediated agglutination of red blood cells, and this hemagglutination inhibition (HI) test can be used to measure antibody levels. The basic method was worked out long before a detailed understanding of the immune response was available, but it is based on the fact that many antibody molecules bind to the surface of viruses and physically mask them. If a virus that can cause hemagglutination is pre-incubated with an antibody to it, the virus will be coated with antibody and will not be able to stick to the red blood cells. This happens because the surface of the virus particle is relatively small, and once a protein molecule is stuck to it, that protein will block access to portions of the surface. If enough antibody sticks, the whole surface is obscured.
An experiment utilizing inhibition of hemagglutination (also called an HI test) is shown in Fig. 7.11. All that is required to measure a patient's immune response is a standard virus stock and blood serum. The basic procedure is as follows: Standard samples of red blood cells
Neutralizing antibody can affect different stages in the entry process
Fig. 7.10 Antibody neutralization of virus infectivity. Specific types of antibody molecules, called neutralizing antibodies, can bind to surface proteins of the virus and block one or another aspect of the early events of virus-cell recognition or effective internalization of the virus.
(e.g., guinea pig or chicken red blood cells for influenza virus) are mixed with a known amount of virus stock and different dilutions of an unknown antibody, which could be in a patient's serum. After a suitable period of time, the solution is gently shaken and subjected to low-speed centrifugation. If the red blood cells are agglutinated, the cells make a jelly-like clump and cannot sediment. Agglutination is characterized by a diffuse red or salmon-pink solution. If the red blood cells do not agglutinate because of sequestration of virus by the antibody, the cells' pellet forms a red "button" at the bottom of the tube. The beauty of using HI is not accuracy; it is relative speed, ease, and low cost of performance, which is very important in small clinical laboratories, especially in developing countries.
Serum complement is made up of a number of soluble proteins that are able to stick to cells bearing antibody—antigen complexes. As this binding occurs, the complement proteins undergo structural changes and, finally, the last protein bound is activated to become a protease, which then lyses the cell. The ability of complement to bind to antibody—antigen complexes at the Fc region of the antibody is termed fixation because once bound, the complement is no longer free in solution. This property can be used as a relatively simple and inexpensive method to measure antibody—antigen reactions called complement fixation (CF) titration.
In a CF assay, sheep red blood cells are used to make an antibody against their surface proteins, often in a horse, goat, or other large animal. The red blood cells are then "standardized"
No virus Control
Dilution of Antiserum 1 to:
Fig. 7.11 The hemagglutination inhibition assay for measuring antibody against a virus in serum. The assay is carried out by mixing constant amounts of a known hemagglutinating virus with serial dilutions of serum; then the virus—serum mixture is added to red blood cells. Low dilutions of serum result in sequestering the virus so that it is not available for hemagglutination, and red blood cells in the wells pellet to the bottom under low centrifugal fields. Higher dilutions of the antiserum dilute the antibody concentration to a point where enough virus remains to cause a positive hemagglutinin reaction. If there were more antibody in the serum, a higher dilution would be required to accomplish this. Thus, the hemagglutination inhibition titer of the serum is a measure of how far it can be diluted and still block the hemagglutinin reaction. This is a measure of antibody concentration. In the example shown, a 1 : 3200 dilution of the original sample (asterisks) was the last one in which agglutination was inhibited. This is the endpoint of the antiserum dilution. Since a 1 : 3200 dilution was the endpoint, there were 3200 hemagglutination inhibition units in the original stock. (Based on a figure in Dimmock NJ, Primrose SB. Introduction to modern virology, 4th ed. Boston: Blackwell Science, 1994.)
so that when a specific amount of antibody is added to them and the mix is incubated with guinea pig complement, the red blood cells lyse. Lysis of the red blood cells is readily assayed because when a solution of lysed red blood cells is centrifuged at low speed, the solution will stay red because there are no cells to take the hemoglobin to the bottom of the tube to form a pellet.
After the red blood cells, anti-red blood cell serum, and complement are standardized, they can be stored for relatively long periods in the cold. When they are used to assay an antibody-antigen reaction, the following process is carried out. Serial dilutions of either a solution of antibody of unknown strength and a fixed amount of known virus, or a solution with an unknown amount of virus and a fixed amount of known antibody, are incubated together. Then they are mixed with a known amount of guinea pig complement. If an antibody-antigen complex has formed, the complement will be fixed (i.e., bound) by it. If not, the complement will stay in solution. If there is an intermediate level of complex, then some complement will be fixed and some will be free.
Following incubation of the unknown antibody-antigen mix with the known amount of complement, the whole "mess" is incubated with standard amounts of red blood cells and anti-red blood cell antibody. If all the complement is fixed, there will be no lysis of the red blood cells. If some is fixed, there will be partial lysis of the red blood cells. If none is fixed, there will be complete lysis. Measurement of the degree of lysis (by measuring the amount of red color in solution following low-speed centrifugation) can be used to measure the amount of unknown antibody—antigen reaction and provides the CF titer.
Like HI, this method is not extremely precise or sensitive, but it is cheap, fast, and requires few expensive pieces of equipment. It is an ideal method for getting quick results in small laboratories or those with limited resources. It is still used in all modern hospitals
QUESTIONS FOR CHAPTER 7
2 Why are soluble antibodies (the products of the humoral response) good antiviral agents?
4 What protein structural features are involved in the antigenic nature of epitopes?
5 What steps occur in the immune response following the primary infection of a vertebrate by a virus?
6 Assume you know that for a particular nonenveloped virus, gene A codes for a transcriptional activator, gene B for an origin binding protein, and gene C for a capsid protein. Following a normal infection in an animal, what would most likely generate a neutralizing antibody?
7 What are some of the problems that arise in considering vaccination strategies for viral diseases?
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