Chapter 10: Host Defenses against Viral Infection and Viral Counterdefenses - Introduction - Adaptive Immune System

Host Defenses against Viral Infection

and Viral Counter defenses

INTRODUCTION

Major Histocompatibility Complex

Both cellular immunity and humoral immunity require

On infection of a mammal by a microorganism, a complex

the activation of a class of lymphocytes called T cells

response is generated that attempts to eliminate the infec-

(T from thymus, where these cells mature). T cells recognize

tious agent. These responses can be conveniently grouped

peptide antigens 820 amino acids in length that are pre-

into two categories, innate immune responses and adap-

sented to them by cell surface proteins encoded in the major

tive immune responses. The adaptive or acquired immune

histocompatibility complex (MHC) locus (in humans, the

response requires time to develop, is specific to the invading

MHC is called HLA, from human lymphocyte antigen, and

pathogen, and is followed by immunologic memory that usu-

is encoded in chromosome 6). The two types of MHC mol-

ally renders the host immune, or at least less susceptible, to

ecules that present antigenic peptides are called class I and

subsequent infections by the same organism. The two most

class II. Both class I and class II MHC molecules are integral

important components of this response are the production

membrane proteins that are composed of two polypeptide

of cytotoxic T lymphocytes (CTLs) and the production of

chains.

humoral antibodies. Innate responses, in contrast, generate

early responses to infection, are not specific to the patho-

gen, and do not render the organism resistant to subsequent

Class I and Class II MHC

infection by the same pathogen. Cytokines such as inter-

The MHC class I molecule is a heterodimer composed of

feron are among the most effective innate responses. Innate

a heavy chain of about 350 amino acids, which is encoded

and adaptive immune responses do not function indepen-

within the MHC locus, and a light chain of about 100 amino

dently of one another: The proper functioning of the immune

acids, β2 microglobulin, which is encoded elsewhere. The

system requires the activities of both, and, in fact, the activa-

structure of an MHC class I molecule is shown schematically

tion of the adaptive response requires the prior activation of

in Fig. 10.2A, and as determined by X-ray crystallography in

the innate system. The various activities of the two systems

Fig. 10.2B. The MHC class I heavy chain consists of three

constitute a multifaceted, interactive, and complex series of

extracellular domains called α1, α2, and α3, a transmem-

responses to infection by a pathogen. Figure 10.1 illustrates

brane domain, and a cytoplasmic domain. β2 microglobulin

some of the cells and other participants involved in innate

forms a fourth extracellular domain and is held in the com-

and acquired immunity.

plex by noncovalent interactions. The α1 and α2 domains,

which are structurally related to one another, form a platform

ADAPTIVE IMMUNE SYSTEM

with helical walls. The walls form a groove in which the

antigenic peptide, consisting usually of 810 amino acids, is

anchored. The α3 domain and β2 microglobulin are homolo-

The adaptive immune system contains two major arms.

The cellular arm leads to the production of CTLs, also called

gous (derived from a common ancestral polypeptide by gene

killer T cells. The humoral arm leads to the production of

duplication) and are members of the immunoglobulin (Ig)

antibodies that are secreted by B cells. T-helper cells are

superfamily. They share some sequence identity and have

important players in orchestrating both of these arms.

common structural features.

Innate Immunity

Complement

Immune System

Macrophages

Natural Killer

Monocytes

Cascade

Players

Cells

Eosinophils

Neutrophils

Cytokines

PRRs

Acquired Immunity

Pathogen/Antigen

Intracellular Antigens

Extracellular

Cells infected with

Antigens

Viruses, Rickettsia, or Mycoplasma

Bacteria, Viruses

Immune System

TH1

TH2

B Cell

CTL

Players

Immediate Effect

Cell killing by CTLs

Soluble Antigen, Activated B Cell

Result

Humoral Immunity

Cell-Mediated Immunity

Antigen Presentation

Class II MHC

Class I MHC

FIGURE 10.1 The mechanisms of innate and acquired immunity are integrated to provide the basis for humoral and

cellular immunity. PRRs are pattern recognition receptors. Adapted from Mims et al. (1993), p. 5.8.

The structure of MHC class II molecules is similar to

exceedingly polymorphic and there are hundreds of different

that of class I molecules, as illustrated in Fig. 10.2A. Class

alleles within the human population. The haploid number

II molecules are composed of a heterodimer of two proteins

of genes in humans that encode heavy chains used in class

encoded within the MHC locus. These two proteins, des-

I MHC molecules is three (called HLA-A, -B, -C), and a dip-

ignated α and β, each contain two extracellular domains

loid individual can make up to six different class I MHC

(and thus the assembled molecule contains four extracel-

molecules with differing requirements for anchor residues.

In the case of class II MHC, six α and seven β genes have

lular domains as does class I MHC). Both proteins are

anchored in the plasma membrane by membrane-spanning

been identified in humans (of which the most important

anchors and have cytoplasmic domains. The distal α1 and

loci are called DR, DQ, and DP). Thus, any individual can

β1 domains form a platform with a groove that binds an

present very many different peptides to T cells. Note that

antigenic peptide for presentation to T cells, but in this case

since the MHC is polymorphic, different individuals present

the peptide is longer, usually 1418 amino acids in length.

different peptides to T cells.

The proximal domains, α2 and β2, are members of the Ig

superfamily.

T-Cell Recognition of Peptide Antigens

The number of peptides that can be presented by an

individual class I or class II molecule is large. Only cer-

T cells express a T-cell receptor on their surface, which is

tain residues in the peptide, called anchor residues, inter-

able to recognize a specific peptide presented in the context

act specifically with the MHC molecule. The remainder of

of class I or class II MHC molecules. The T-cell receptor

is a heterodimer formed by one α and one β chain, or by

the peptide can vary in sequence. Furthermore, the MHC is

Class II MHC

Class I MHC

2 S

b 2m

Plasma

membrane

Cell cytoplasm

Peptide-binding

cleft

a 2 domain

a 1 domain

b 2 microglobulin

a 3 domain

FIGURE 10.2

Upper panels: schematic representations of MHC class I and MHC class II molecules.

The orientation of these molecules at the cell surface is indicated, as are the domain structures of the extracellular

portions of the proteins, the membrane-spanning domains and the cytoplasmic domains, and how they function in

the cell. The yellow and green spheres represent bound peptide antigens. Lower panel: three-dimensional ribbon

diagram of the structure of MHC-1 (HLA-A2), as determined by X-ray crystallography. From Bjorkman et al.

(1987) as reprinted in Kuby (1997).

one γ and one δ chain, as illustrated in Fig. 10.3. The great

domains. V and J, or V, D, and J, are first joined at the DNA

majority of circulating T cells possess receptors formed by

level by a process that deletes the intervening DNA. The

αβ dimers.

combined VJ or VDJ is then joined to C by splicing of the

During maturation of T cells, three or four separate regions

pre-mRNA transcript. Multiple copies of each of the four

of the genes for α or β, respectively, are brought together by

segments exist in the germ line and combinatorial joining

of these results in a very large number of possible α or β

deletion of the intervening sequences, with the rearrange-

ments to form the β chain occurring first. This process is

subunits. Furthermore, the joining of VJ and VDJ is impre-

illustrated in Fig. 10.4. The regions are the V (variable), D

cise, and additional diversification results from repair of

(diversity), J (joining), and C (constant) regions for β, or

the joining regions. Combinatorial joining of α and β subu-

the V, J, and C regions for α. The V and C regions belong to

nits results in the production of an even larger repertoire of

the Ig superfamily, whereas D and J are shorter, unrelated

T-cell receptors. An estimate of the possible diversity of

a or g chain

b or d chain

T-cell receptors, sometimes referred to as the theoretical

repertoire, is shown in Table 10.1. The theoretical repertoire,

NH2 NH2

how many T-cell receptors could possibly be formed, is

Variable

perhaps 1018, which is an exceedingly large number.

Regions

Once any individual T cell develops and expresses a

T-cell receptor, no further recombination occurs and the

receptor does not change thereafter. The function of T cells,

Constant

simply stated, is to discriminate self from nonself, and

Regions

T cells that express an appropriate receptor that serves

this function are selected in the thymus (see later) and

Plasma

released into the circulation, where they are long lived.

membrane

Any individual has many, many T cells in the body and

these continue to develop throughout life. The repertoire

Cell cytoplasm

COOH COOH

present in any human at any one time certainly exceeds

108 different T-cell receptors, expressed on different T

cells.

Each different T-cell receptor is potentially able to

recognize a different peptide epitope (the epitope is the

FIGURE 10.3

Structure of the T-cell receptor (TCR). Receptors are

surface of the peptide that interacts with the T-cell recep-

heterodimers of α/β or γ/δ TCR chains. Each chain has a transmembrane

tor). The T-cell receptor recognizes the peptide epitope in

anchor (TM). The C-terminal domains and the TM are relatively constant in

the context of class I or class II MHC; that is, the T-cell

sequence, but the N-terminal domains are variable. Adapted from Chen and

Alt (1997) Figures 7.6 and 7.7 on pp. 344 and 345.

Vd n Dd1Dd 2 Jd1 JdD Cd

Ja1 Ja 2

Va n Vd 1

Ja n

Germ-line a-chain DNA

V-J Joining

Va1

Va 2

Va Ja Ja n

Rearranged a-chain DNA

Transcription

mRNA splicing

a chain

Translation

Ja Ca

Protein product heterodimer

b chain

Transcription

mRNA splicing

Translation

Cb 2

Cb1

Db 2

Vb 14 3

Vb1

Rearranged β-chain DNA

V-DJ joining

-J joining

Vb14 3

Cb1

Cb2

Vb1

Vb n

Germ-line

b -chain DNA

FIGURE 10.4 Gene rearrangements to generate T-cell receptor diversity. The example shown generates an αβ T-cell

receptor. The α chain undergoes a VαJα joining, whereas the β chain undergoes two joins: Dβ to Jβ followed by Vβ to

DβJβ. Primary RNA transcripts are spliced to give mRNAs in which RNA sequences encoding VJ or VDJ are joined to

RNA sequences encoding constant domains, Cα or Cβ. These spliced mRNAs are then translated into the α and β chains

of the TCR. Adapted from Kuby (1997) Figure 11.6 on p. 269.

TABLE 10.1

Comparison of Diversity in Human Immunoglobulin and T-Cell Receptor Genes

Immunogobulins

αβ T-cell receptors

γ δ T-cell receptors

Light chains

κ chain

λ chain

α chain

β chain

γ chain

δ chain

Mechanism of diversity

Heavy chains

Multiple germ-line gene segments

~70

3,2

Combinatorial joining

Combinatorial VJD

65 × 27 × 6 =

40 × 5 =

30 × 4 =

70 × 61 =

52 × 2 × 13 =

12 × 3 = 36

4×3×3=

1.0 × 104

1.3 × 103

combinations

200

120

~430

D segments read in

Rarely

Often

3 frames

Joints with N and P

(1)

nucleotides

1.0 × 104 × 320 = 3.2 × 106

430 × 1.2 × 103 = 5.8 × 106

36 × 36 = 1.3 × 103

V gene pairs

~3 × 107

~2 × 1011

Junctional diversity

???

~1014

~1018

Total diversity

???

Source: Data from Janeway et al. (1999) pp. 62, 93, 158.

receptor interacts with both the peptide and the MHC mol-

Cytotoxic T Cells

ecule (Fig. 10.5). The T cell cannot recognize the peptide

The majority of CD8+ T cells are or become CTLs,

alone or even recognize the peptide presented by the wrong

although a minority of CD4+ T cells are also CTLs. CD8+

MHC molecule. Similarly, the T cell cannot recognize a

T cells are class I MHC restricted, as described. Because

class I or class II MHC molecule with the wrong peptide

class I MHC molecules are expressed on most mammalian

in its cleft. The discovery of this requirement for dual rec-

cells, the major exception being neurons and, in humans, red

ognition, which was made using a viral system, resulted

blood cells, which express little or no class I MHC, most

in a Nobel Prize for Doherty and Zinkernagel (Table 1.1).

cells in an individual are capable of presenting peptides to

Such a requirement for multiple interactions is a recur-

T cells in a class I MHC context.

ring theme in the immune system, and it has evolved

The peptides presented by class I MHC are derived from

because this potent and potentially harmful system must

intracellular proteins and represent a sampling of all proteins

be carefully regulated.

being synthesized within the cell. The pathway involved is

The T-cell receptor is part of a complex containing

illustrated in Fig. 10.6. The peptides are generated by prote-

accessory molecules that are required for the function of the

olysis of intracellular proteins by an enzyme system referred

receptor. Two such molecules are CD4 and CD8, and mature

to as the proteasome. The proteasome is a large complex,

T cells possess either CD4 or CD8 (but not both). CD8 con-

possessing many subunits, that is present in the cytoplasm. It

tains one Ig domain attached to a stalk region, whereas CD4

possesses ATP-dependent proteolytic activity and is the major

contains four Ig domains (Fig. 10.5). CD8+ T cells recog-

cellular proteolytic site other than the lysosome. In addition

nize peptides presented by class I MHC (Fig. 10.5A). CD4+

to its function in the immune response, the proteasome is

T cells, in contrast, recognize peptide epitopes presented

important for turnover of many proteins within the cell and

in the context of class II MHC (Fig. 10.5B). CD8 or CD4

for degradation of misfolded proteins. Peptides resulting from

interacts with constant regions of class I or class II MHC

degradation of intracellular proteins are actively secreted, in

molecules, respectively, and increases the binding affinity

a process that requires hydrolysis of ATP, into the lumen of

of the T cell for its cognate MHCpeptide complex by about

the endoplasmic reticulum (ER) by a transporter called TAP

100-fold. Class I and class II MHC molecules acquire the

(transporter associated with antigen presentation). TAP is

peptides that they present in fundamentally different ways

encoded in the MHC and consists of a heterodimer anchored

and are components of two different responses to infection

in membranes of the ER.

by microorganisms

Antigen-presenting cell

Almost any host cell

CD8

MHC Class I

MHC Class II

dimer

Antigenic

peptide

CD4

TCR

chain

chain TCR

chain

COOH

Cytotoxic CD8 T cell

CD4 Helper T cell

FIGURE 10.5

(A) Interaction of the TCR on a cytotoxic CD8+ T cell with an MHC class I molecule complexed with

an antigenic peptide on almost any cell. The TCR interacts with both the peptide and with the MHC molecules. The CD8

homodimer interacts with a conserved region of the MHC α3 domain. (B) Interaction of the TCR on a CD4+ helper T cell

with an MHC class II molecule complexed with an antigenic peptide on the antigen-presenting cell. The TCR interacts

with both the peptide and with the MHC molecule. The membrane distal domain of CD4 recognizes a conserved region

of the MHC β2 domain. Adapted from Chen and Alt (1997) pp. 344 and 345; and data from Kuby (1997) pp. 275277.

Proteins secreted into the lumen of the ER during syn-

proteins including the two subunits of TAP (TAP1 and

thesis are also sampled. There is a pathway that recycles

TAP2), a transmembrane glycoprotein called tapasin, the

lumenal proteins back to the cytoplasm. This pathway may

soluble chaperone calreticulin, and thiol oxidoreductase

serve to rid the ER of misfolded proteins as well as enabling

Erp57. Binding of peptide stabilizes the class I molecule and

the sampling of proteins destined for the plasma membrane

facilitates its release and transport to the cell surface. Class

or other intracellular organelles. On reentry into the cyto-

I MHC that is transported without a peptide is unstable at

plasm, a cellular glyconase removes carbohydrates from

37°C and is degraded.

glycoproteins, and the protein backbone is degraded by the

The end result is that class I MHC presents a random

proteasome. Thus, viral proteins that are inserted into the

sampling of peptides derived from proteins being synthe-

lumen of the ER during synthesis, such as glycoproteins used

sized within the cell for inspection at the cell surface by any

to assemble progeny virions, are also sampled by the

T cell that may be in the vicinity. The peptides bound to a

proteasomeTAP pathway.

single isoform of class I MHC molecules present on the sur-

A peptide delivered to the lumen of the ER by TAP can be

face of cells in culture have been analyzed by very sensitive

bound by a class I MHC molecule if it has the right anchor

techniques. More than 10,000 different peptides, present at

residues. Delivery of the peptide to the MHC is a complex

2 to 4000 copies per cell, were identified. This great diver-

process that ensures that the MHC receives a high-affinity

sity of peptides consists mostly of self-peptides, but peptides

peptide. The peptide can be shortened to the proper length

derived from intracellular viruses or other intracellular path-

(810 residues) by a protease called ERAAP if it is too long.

ogens will be represented if the cell is infected. If a patrolling

The MHC heavy chain is bound by the membrane-associated

T cell has a receptor that binds specifically to a peptide being

chaperone calnexin upon synthesis and folds into its proper

presented by the class I MHC on another cell, the T cell may

conformation. Calnexin then dissociates, β2 microglobulin

become activated and may proliferate. Once activated, CTLs

associates with the heavy chain, and the MHC is delivered

kill cells that present the epitope they recognize. In different

to the peptide-loading complex, which consists of several

assays, the number of MHCpeptide complexes required for

External antigen or

Infected cell

pathogen

Viral protein

synthesized in

the cell

NUCLEUS

Proteasome

Acidic vesicle

TAP

(Invariant

chain

peptide)

MHC Class I

MHC Class II

FIGURE 10.6 Antigen processing by the MHC. Both class I and class II pathways are shown. On the left, a viral

protein is synthesized in the cell (a) degraded (b) and viral peptides are bound by TAP in the ER and transported into the ER

lumen, where they are bound by MHC class I molecules (c), MHC containing bound peptide is transported by cellular

vesicles through the Golgi apparatus (d) to the cell surface (e) where it is expressed for inspection by T cells. On the

right, an external antigen or pathogen is endocytosed into the cell (A) and is degraded in an acidic vesicle (B). Class II

molecules are synthesized and imported into the ER (C) with a trimer of an invariant chain peptide in the binding site, and

transported to the trans-Golgi (D) where the invariant chain is lost. The Golgi vesicle fuses with the endosome containing

the antigenic peptide (E), and the peptideMHC class II complex is transported to the cell surface. Adapted from Fields

et al. (1996) p. 351.

recognition by a CTL has been estimated to be between one

Inside the APC, the viral antigen may enter the cytoplasm

and several hundred, and may depend on the affinity of bind-

and be processed by the methods described earlier, or may

ing of the MHCpeptide target by the T-cell receptor as well

be processed and bound by MHC class I within the endo-

as the state of activation of the T cell.

somal compartment. Activation of a T cell by an APC

The activation of a T cell is a multistage process. Naïve

requires not only the recognition of the cognate antigen in

T cells are activated to become effector cells by interaction

a class I context, but also that additional immunostimula-

with professional antigen presenting cells (APCs), usually

tory signals be present. For example, if Toll-like receptors,

macrophages or dendritic cells, that present antigen in the

described later, have been activated in the APC, it expresses

context of class I or class II MHC. APCs can present anti-

additional proteins that enable it to activate the T cell. In the

gens in a class I context that are synthesized within the cell

absence of such prior activation of the APC, it will tolerize

and processed as described before, but they can also present

a T cell rather than activating it, illustrating another layer of

antigens in a class I context that they acquire from the exter-

control to avoid possible autoimmune responses.

nal environment (called cross-presentation). Cross-presenta-

Proliferation of activated CTLs requires further stimula-

tion allows APCs to respond to a viral antigen, for example,

tion by cytokines such as interleukin-2 (IL-2). The source of

even if the virus does not replicate within the APC. These

IL-2 is usually a class of T-helper (TH) cells (most TH cells are

CD4+, as described later). Activated TH-1 cells secrete IL-2 as

antigens may arise from the dissolution of an infected cell

well as tumor necrosis factor β (TNF-β), interferon γ (IFN-γ),

that dies, and enter the APC by phagocytosis or pinocytosis.

and other cytokines. Proliferation of T cells that recognize a

are released into the circulation to patrol for cells that are

specific peptide derived from a viral protein means that a vig-

infected by viruses or other intracellular pathogens.

orous CTL response against an invading pathogen ensues.

A second level of control that reduces the incidence of

CTLs kill target cells by inducing apoptosis, a cell sui-

reaction against self lies at the level of cytokine induction.

cide pathway described in a later section of this chapter.

Virus infection or infection by other parasites activate innate

One of three different mechanisms is used to induce apop-

response elements, of which Toll-like receptors are the

tosis. These mechanisms are listed in outline form here but

best known, that result in the production of inflammatory

are described in more detail when the apoptotic pathway

cytokines. Cytokines such as IL-2 or IFN are required as a

is considered. In one mechanism, the T cell releases the

second signal for CTL activation, and IFNs also upregulate

contents of granules, which contain perforins and proteases

the presentation of antigens by MHC molecules as well as

among other components, into the target cell. Perforins

other aspects of the immune response. Thus, the inflamma-

form pore structures in the plasma membrane of the target

tory response makes it more likely that T cells will respond

cell that allow ions to leach out of the cell, and the pro-

to antigens that they recognize. As described before, APCs

teases participate in the activation of cell pathways leading

may tolerize a T cell to an antigen rather than activating

to apoptosis. In a second mechanism, apoptosis is induced

it if the appropriate immunostimulatory factors are not

by triggering the Fas death receptor on the surface of the

present, another layer of control to prevent autoimmunity.

target cell. These two mechanisms lead to cell death within

Furthermore, once activated, CTLs undergo apoptosis if the

/ 6 hours. A third mechanism utilizes the TNF-α death

cytokine signals are no longer present, thus damping out any

pathway and is a slower process, leading to cell death in

autoimmune responses that might occur.

1824 hours. Killing of target cells is a drastic response

Killing of virus-infected cells by CTLs in an effort to

and the CTL pathway is directed toward eliminating inter-

eradicate viral infection relies on the ability of the cells of

nal pathogens, usually viruses. Because early proteins

most organs to regenerate from progenitor cells. In this con-

encoded by the virus can be sampled by the MHCT-cell

text it makes sense that neuronal cells express only low lev-

receptor pathway as well as late proteins, it is possible for

els of class I MHC. These cells are terminally differentiated

a T cell to kill a virus-infected cell before it has time to

and nondividing, and if killed by a CTL they are unable to

synthesize much progeny virus. Many viruses counter this

regenerate.

pathway by interfering with the ability of an infected cell

Although the ability of CTLs to kill virus-infected cells

to express class I MHC at its surface, as described later in

is well established, recent findings indicate that CTLs, as

this chapter.

well as other activated cells of the immune system, may

Although three killing mechanisms are used by differ-

also use noncytolytic means to control and clear many

ent CTLs, they are not redundant. Mice that lack the per-

virus infections. This control is thought to be achieved

by the secretion of cytokines such as IFN-γ and TNF-α.

forin gene are unable to control infection by lymphocytic

choriomeningitis virus and half die within a month of infec-

Dengue virus infection in the brains of mice is one exam-

tion. This virus is not pathogenic in normal mice or even

ple in which noncytolytic clearance appears to be impor-

in immunocompromised mice. Thus death must result from

tant. During dengue virus infection of neurons, CTLs are

immunopathology caused by an unbalanced or incomplete

actively recruited into the brain and are essential for the

T-cell response.

clearance of virus in immunized mice, at least under some

Since the class I pathway presents peptides derived from

conditions. Neurons are immunologically privileged, as

self as well as from viruses that may have infected the cell,

noted before, and noncytolytic mechanisms of control are

how then do the CTLs know not to kill cells expressing self-

important. Hepatitis B virus infection of hepatocytes is

antigens? The answer lies in part in the selection of an appro-

another system in which there is evidence that noncytolytic

priate repertoire of T cells. T cells bearing T-cell receptors

control is important, even though hepatocytes do regener-

recognizing many different possible peptide antigens arise

ate and killing of infected hepatocytes does occur, causing

by random combinatorial joining and diversity-inducing

the symptoms of hepatitis. Other examples are also known.

processes. T cells undergo their early differentiation in the

Noncytolytic clearance does not appear to be universal,

thymus, where selection occurs. Only T cells that express

however. It appears to be possible only in some tissues and

a T-cell receptor capable of recognizing class I MHC (or

for some viruses.

class II MHC) bearing a peptide are selected (called posi-

tive selection); T cells that do not express an appropriate

T-Helper Cells

receptor die. However, if such a T-cell receptor has a high

Most CD4+ T cells are helper cells. Some CD8+ T cells

affinity for self-peptides present in the thymus, then that T

are also helper cells, but most helper cells are CD4+. CD4+ T

cell also dies (called negative selection). In this process of

selection only about 2% of T cells survive and most of these

cells recognize peptides presented in the context of class II

encode receptors that recognize nonself antigens. These T cells

MHC molecules--they are referred to as class IIrestricted

cells (see Fig. 10.5). Class II MHC molecules, unlike class I,

make antibody (see later). It is the disappearance of TH cells

are present on only a restricted set of cells within the organism

during HIV infection that leads to the symptoms of AIDS.

and are most abundant on B cells, macrophages, dendritic

TH cells are not homogeneous. Different TH cells secret

cells, and, in humans, activated T cells, that is, cells of the

different panels of cytokines and have different functions in

immune system itself.

the immune response. Two main types have been recognized,

The peptides presented by class II MHC molecules are

which perhaps represent extremes in function. TH-1 cells are

derived from extracellular proteins, and thus these MHC

highly effective for CTL activation and function in the cel-

molecules sample the extracellular environment. Proteins,

lular immune pathway. TH-2 cells are optimal for the activa-

whole viruses, or other microorganisms are taken up by

tion of B cells and function in the humoral immune pathway.

antigen-presenting cells and degraded within intracellu-

They secrete IL-4, IL-5, IL-6, and IL-10. TH-1 and TH-2 cells

lar organelles. Peptides derived from these sources can be

can be mutually antagonistic. The cytokines secreted by one

bound by class II MHC molecules being transported to the

suppress the other, and a balanced immune response often

cell surface. The process of producing a peptide and trans-

requires a balanced activation of these two classes of help-

ferring it to a class II MHC molecule is complicated, as is

ers. TH-2 cells usually deliver their cytokine signals directly

the case for class I MHC, but will not be described here since

to the B cells that they help, following cellcell contact. TH-1

viruses are not known to interfere with the class II pathway,

cells, in contrast, do not deliver their signals directly to the

whereas they do interfere with the class I pathway. The pep-

T cells that they help.

tides derived from this pathway are kept separate from pep-

tides generated through the proteasomeTAP pathway, and

B Cells and Secretion of Antibodies

the end result is that the class II pathway presents peptides

derived from the external environment, whereas the class I

B cells, so named because they are derived from the bone

pathway normally presents peptides derived from the intrac-

marrow in mammals, secrete antibodies, which are members

ellular environment (Fig. 10.6) (but see the previous discus-

of the Ig superfamily. The essential subunit of an antibody is

sion of cross-presentation).

a heterodimer of a heavy (H) chain (which has four or five Ig

Professional APCs, which may be macrophages, B cells,

domains) and a light (L) chain (which has two Ig domains).

or dendritic cells, present peptides to T cells. They also

This subunit is always present as a dimer in which two H-L

express accessory proteins that deliver a second signal to the

heterodimers are linked through the H chains. The structure

T cell that is required for its activation. When a TH cell is acti-

of a light chain, as determined by X-ray crystallography, is

vated by the interaction of its receptor with its target peptide

shown in Fig. 10.7. This structure illustrates the Ig fold that

presented by class II MHC, and the appropriate second sig-

is common to all Ig domains.

nals are present, as described before, the cell proliferates and

The five classes of antibodies are illustrated in Fig. 10.8.

secretes cytokines that are important for eliciting an immune

An IgG molecule consists of a dimer of H-L heterodimers in

which the H chain is of the γ class. IgD and IgE antibodies

response. Activated TH cells are essential for any immune

response, whether CTLs to kill infected cells or B cells to

are also dimers of H-L heterodimers, but in this case the H

CL domain

VL domain

Loops

β strands

C-terminus

N-terminus

CDRs

Disulfide bond

FIGURE 10.7

Ribbon diagram of an immunoglobulin light chain depicting the immunoglobulin-fold structure of its

variable and constant domains. Two β-pleated sheets in each domain (colored in red and pink in the constant domain and

yellow and brown in the variable domain) are held together by hydrophobic interactions and a single disulfide bond (dark

bar). The hypervariable regions also known as complementarity determining regions (CDRs), shown in blue, form part of

the antigen-binding site. Adapted from Kuby (1997) p. 113.

IgG

IgD

IgE

The two Ig domains of the L chain are called V (for variable)

and C (for constant) and between these two domains is a J

VH CL

(for joining) domain. All three domains are encoded sepa-

Cg 1

Ce 1

Cd 1

rately in the genome. There are multiple copies of V, J, and

Ce 2

C in the genome (Table 10.1), and these gene segments are

Cg 2

Cd 2

Ce 3

polymorphic in the population. The light chain genes fall into

Cg 3

Cd 3

two different families, called κ and λ (Table 10.1), which are

Ce 4

encoded on two different chromosomes in humans. During

maturation of a B cell, a V-gene segment, a J-gene segment,

IgA Dimer

IgM Pentamer

and a C region of the light chain are brought into juxta-

position to one another, as illustrated in the top panel of

VH CL

Fig. 10.10. In this process, a V-gene segment is fused to a

Cm 1

Ca 1

J-gene segment by deleting the intervening DNA. The C

Cm 2

region is brought into play by RNA splicing: transcription of

Ca 2

the VJ region continues through the C region, and splicing of

Cm 3

Ca 3

the pre-mRNA joins the J region to the C region.

Cm 4

The heavy chain, which is the first to rearrange, is formed

S S

by a similar sequence of events, but in this case there is an

additional gene segment D (for diversity) that introduces

additional diversity in the recombination process. As for the

light chain, there are multiple copies of V, D, and J in the

genome (Table 10.1), and the population is polymorphic for

these gene segments. During B-cell maturation, a D-gene

segment is first joined to a J segment, and the DJ segment is

then joined to a V segment (bottom panel of Fig. 10.10).

Recombinational rearrangements to form the antigen-

binding site occur only during the maturation of the B cell.

Once a B cell is mature, no further rearrangements occur in

FIGURE 10.8

Structure of the different classes of secreted immuno-

this region, although during an immune response the anti-

globulin molecules. Note that IgM and IgA are secreted as pentamers and

dimers, respectively, linked by a J-chain. Adapted from J. Gally (1973).

gen-binding site is subjected to hypermutation in germinal

IgG, IgA, and IgD heavy chains have four domains and a hinge region; IgE

centers, as described later.

and IgM lack the hinge. Not shown are intrachain disulfide bonds and bonds

The many combinatorial possibilities of V and J light-

linking the light and heavy chains.

chain gene segments, and of V, D, and J heavy-chain gene

segments, lead to the possible production of a very large

number of light chains and heavy chains (Table 10.1). In

chains are of the δ or ε class, respectively. IgA antibodies

addition, joining V with J, or joining V, D, and J, is impre-

contain four H-L heterodimers (it consists of a dimer of the

cise, leading to additional diversity. Finally, joining an L

dimeric unit, as shown). In this case, the H chain is of the α

chain with an H chain to form the heterodimer generates still

class. IgM contains 10 H-L heterodimers (it is a pentamer of

more possible antigen recognition sites, since the antibody

dimeric units) formed with µ H chains.

recognition site is formed by the V regions of both the

A more detailed representation of an IgG molecule is

H and the L chains. The total number of possible combinations

shown in Fig. 10.9. The terminal domains of both H and

is very large (Table 10.1). It is important to note that any

L chains are variable. Within the variable domains, there

individual B cell usually produces only one H chain and one

are regions that are more variable than other regions, called

L chain, and thus each B cell produces only one antibody

hypervariable regions, also known as complementarity-

recognition site.

determining regions (CDRs) (see Fig. 10.7). The combining

site of the antibody, the region that specifically binds to an

Activation of B Cells

antigen recognized by the antibody, is formed by the vari-

able regions of both the H and L chains.

During maturation of B cells, a large population of cells

results, each of which has one antibody-combining site. The

theoretical repertoire, how many different types of B cells

Formation of Light and Heavy Chains

could conceivably be produced using the known mecha-

nisms that are active during B-cell development, is thought

The L and H chains of antibody molecules are formed in

to be much larger than the minimal estimate of 1014 shown

a process that is similar to that used to form T-cell receptors.

Variable regions

Antigen-binding

domain

CH1

Light chain

Heavy chain

variable

Heavy Chains

region

Constant

CDR1

Variable

CDR2

Hypervariable (CDR's)

CH2

Hinge

Effector

function

Light Chains

domain

Constant

CH3

Variable

Hypervariable (CDR's)

FIGURE 10.9 Diagrammatic representation of an IgG molecule. Each antibody molecule is composed of two heavy

chains (which contain four domains, each consisting of an Ig fold like that shown in Fig. 10.7) and two light chains, which

each have two Ig domains. The distal domains of both the light and heavy chains are the variable regions, composed of

interspersed framework regions and hypervariable regions (diagonal shading) known as complementarity-determining

regions or CDRs. The right part of the figure illustrates the role of the V, D, and J gene segments in encoding CDR1, 2,

and 3 regions of Ig variable region genes. Adapted from Chen and Alt (1997) pp. 340 and 345.

in Table 10.1. In humans there may be 1010 B cells with

that recognize this peptide will secrete cytokines that stimu-

differing specificities circulating at any time, and it is esti-

late the B cell to proliferate and secrete antibodies. Note that

mated that about 1 in 105 cells produces an antibody that will

the peptide displayed by the class II molecule does not have

bind, with differing affinities, to any particular antigen that

to be related to the epitope recognized by the antibody dis-

is being examined.

played on the B-cell surface. It may, in fact, be derived from

The antibody molecule is first produced as an integral

an entirely different protein. Thus, while T cells respond to

membrane protein that is displayed on the surface of the

peptide epitopes, the antibody molecules can recognize much

B cell, anchored through a membrane-spanning region and

more complex antigens, such as whole proteins or viruses

containing an intracellular cytoplasmic domain. If the anti-

or even nonprotein antigens like carbohydrates. The pro-

body displayed on the surface of the cell binds antigen, the

tein epitopes recognized by antibodies are most often what

B cell is activated. If the cell receives a second signal from

are called conformational or nonlinear epitopes, which are

a TH-2 cell, it proliferates to form cells that secrete antibody.

formed by residues physically located at different places in

Memory cells also arise that serve to protect the organism

the linear sequence of a protein but which form a contiguous

from future infection by the same pathogen.

surface in the protein after it folds into its three-dimensional

The TH-2 cell signal may be delivered to the B cell either

conformation. Such discontinuous epitopes are destroyed if

through a specific pathway or through a nonspecific path-

the protein is denatured and the different components of the

way. Antigen, which could be in the form of a whole virus or

epitope separated from one another. A certain fraction of

in the form of a protein, that is bound to antibody present on

antibodies, however, recognize continuous epitopes, which

the surface of the B cell can be internalized by the B cell and

are formed by a linear sequence of amino acids present in

degraded by the MHC class II antigen-processing pathway.

the protein.

Peptides derived from the degraded virus or protein can then

In addition to specific activation of B cells by T-helper

be presented on the surface of the B cell in the context of

cells, B cells can also be activated through area stimu-

class II MHC molecules. Class IIrestricted T-helper cells

lation. If a B cell is in the vicinity of T-helper cells that

Light Chain

Cκ

LVk1

LVkn

Germline k-Chain DNA

V-J Joining

VkJk

LVk1

Rearranged k-Chain DNA

Transcription, RNA Splicing, Polyadenylation

L VJ

Light Chain (k) mRNA

Cap

Translation

Light Chain (k) Protein

VH Cm1 Cm2 Cm3 Cm4

Heavy Chain (m) Protein

IgM

Translation

LV DJ Cm

Heavy Chain (m) mRNA

Cap

Transcription, RNA Splicing, Polyadenylation

LVH179 V DJ J

L VH1

Rearranged Η-Chain DNA

V-DJ Joining

DH1 DH6 DJ JH

VH180 VHn

LVH1

D-J Joining

Germline Η-Chain DNA

Cg3 Cg1 Cg2b Cg2a Ce

VHn DH1 DH7 DH13

LVH1

Heavy Chain

FIGURE 10.10 Formation of the IgM heavy and light chains. (Top) Illustration of the germ-line genes encoding κ

immunoglobulin light chains encoded on human chromosome 2. The first line shows the germ-line genes, and the next

line illustrates the B-cell genes after VJ recombination. The next line down shows the RNA transcript after splicing to

join the VJ region to the C region. This mRNA is translated by cytoplasmic ribosomes into the light chain, which then

combines with a heavy chain. The leader, L, is translated into a signal sequence that is removed posttranslationally.

(A similar series of rearrangements occurs among the gene segments for the λ light chains encoded on human chromosome

22.) (Bottom) A comparable illustration of the heavy chain genes in the germ line and in the B cell, after two rounds of

rearrangement known as "DJ joining," and "VDJ joining." The final IgM molecule is a pentamer held together with

disulfide bonds and a "J chain" which links the Fc regions. Adapted from Kuby (1997) Figures 7.4 and 7.5 on pp. 172

and 173.

are releasing cytokines to activate B cells, it may also be

antibodies. The first antibodies secreted are IgM, whose

stimulated. The importance of area stimulation, and the fre-

structure is illustrated in Fig. 10.8. IgM antibodies, which

quency with which it occurs, in the context of fighting off a

circulate in the blood, can be detected as early as a few days

viral infection is not clear. In many cases of viral infection,

after virus infection. Their production quickly wanes and

a generalized and active inflammatory response occurs that

over a period of weeks or months the concentration of IgM

involves the release of many cytokines, and in which many

in the blood decreases to very low or undetectable levels, as

different antigens are being presented. Area stimulation

illustrated schematically in Fig. 10.11. Thus, the presence of

could be important in developing a rapid response during

IgM antibodies specific for a virus is usually a sign of acute,

such events. However, in such a process antibodies against

or at least very recent, infection.

self might also be produced, and such processes must be

During further maturation of the B cell, class switching

controlled.

occurs, as illustrated in Fig. 10.12. Recombinational events

in the heavy-chain region lead to the substitution of the

IgG, IgE, or IgA heavy chain for that of IgM. Homologous

Secretion of Antibodies

recombination within the intron just downstream of the J

A B cell stimulated by exposure to its cognate antigen and

gene results in deletion of the intervening DNA such that the

by help from a TH-2 cell proliferates and begins to secrete

active VDJ gene is brought into contact with the C region

IgA) or directly (e.g., IgM to IgE without passing through

100

Total

an IgG phase). The situation in the human chromosome is

more complicated (Fig. 10.12). There are two α genes, the

first of which lies between two of the four γ genes. Thus,

Secondary

Primary

there are more ways to switch from one class of antibody

Response

Response

to another.

Production of IgG (or of IgE or IgA) thus occurs later

Total

after infection (illustrated schematically in Fig. 10.11). At

IgG

least 2 weeks are required before there is production of

0.1

IgM

IgG

large amounts of IgG. Once a B cell begins to make IgG,

IgM

it is no longer able to make IgM because the gene encod-

ing the M heavy chain (Cµ in Fig. 10.12) has been deleted.

Note that the antigen-combining site of an antibody is not

1 Antigen

2 Antigen

changed by class switching because the V region of the H

Time after immunization

chain is not involved. However, hypermutation in the com-

FIGURE 10.11 Time course of development of circulating antibodies

bining site to increase the affinity of the antibody for the

after primary and secondary immunizations. No timescale has been shown,

antigen, which occurs in germinal bodies, is associated with

since the actual results vary with antigen, adjuvant, site of injection, and

class switching because a deaminase that is induced upon

animal species. From Kuby (1997) Figure 16.19 on p. 398.

activation of the B cell is involved in both class switching

and hypermutation.

IgG circulates in the blood and therefore very many cells

of a different class of heavy chain. The order of heavy-chain

exposed to blood and blood products are exposed to IgG.

genes in the mouse chromosome is µδγεα, and class switch-

IgG lasts only 2030 days in the blood, but B cells continue

ing can happen either sequentially (IgM to IgG to IgE to

ΨCε

Cµ

Cγ 1

Cγ 3

Cγ 2

Cα 2

Cα 1

Cδ

Cγ 4

Cε

VDJ

H-Chain DNA

Sµ

Sα 1

Sγ 3

Sγ 1

Sγ 2

Sγ 4

Sε

Sα 2

Recombination between Sµ and Sγ1

Cγ 1 Ψ C ε

Cγ 2

Cα 2

Cα 1

Cγ 4

Cε

VDJ

Class-switched

Sα 1

H-Chain DNA

Sγ 4

Sε

Sα 2

SS 2

Sγ 1

Recombination between Sγ 1 and Sε

Transcription,

Splicing,

Polyadenylation

VDJJH

Cα 2

Cε

γ1

Sα 2

Cγ 1

VDJ

Transcription,

IgG1 mRNA

Cap

Splicing,

Polyadenylation

Cε

VDJ

ranslation

gE mRNA

Cap

Translation

IgG Heavy Chains

IgE Heavy Chains

FIGURE 10.12

Immunoglobulin class switching to produce heavy chains for IgG and IgE, following initial

rearrangements to produce IgM. Switch sites, designated "S", are located upstream of each CH gene except Cδ. In

humans there is only one allele for Cµ, Cδ, and Cε, but there are two alleles for Cα, designated α1 and α2 and four alleles

for Cγ, designated γ1, γ2, γ3, and γ4, which differ somewhat in sequence. In addition there is a pseudogene related to Cε.

Because there is no switch site for Cδ, IgD is produced only in conjunction with IgM, by alternative mRNA splicing.

Adapted from Kuby (1997) Figure 7.17 on p. 185; and Janeway et al. (2004) Figure 4.18.

to produce antibody for long periods of time, and although

Antibodies may bind to a virus and neutralize its infec-

production wanes with time, IgG remains circulating in the

tivity, and such neutralizing antibodies are thought to be of

blood for years or decades. Because of this, and the ease

critical importance in controlling virus infection. However,

of obtaining blood samples, IgG has been extensively used

other antibodies may bind to a virus without inactivating it

to monitor past exposure to different pathogens and the

and such nonneutralizing antibodies can also be protective.

immune status of individuals for any particular pathogen.

There are several mechanisms by which nonneutralizing

As illustrated in Fig. 10.11, on a second exposure to an anti-

antibodies may protect. Aggregation of virions by antibodies

gen, IgG concentrations rise dramatically and remain in the

leads to a reduction in the total number of infectious viruses.

circulation at much higher levels. IgM concentrations do not

The maturation of enveloped viruses by budding through the

show this anamnestic effect.

cell plasma membrane can be inhibited by the binding of

Because of its widespread circulation within the body, its

antibodies to viral proteins present on the surface of the cell.

high concentrations, and the anamnestic effect that occurs

Antibody-dependent lysis of an infected cell can occur by

on secondary exposure, IgG is important in the control of

a complement-mediated pathway or by a natural killer cell

viral diseases. It is also important in protecting the fetus and

pathway called antibody-dependent cell-mediated cytotox-

the very young. IgG crosses the placenta during pregnancy

icity. These pathways are triggered by the binding of anti-

and is present in fetal blood. This transfers maternal immu-

body to viral protein expressed on the cell surface. Binding

nity to the fetus, and this immunity lasts for the first few

of antibody or of certain components of complement to a

months of postnatal life.

virus can also increase the rate of phagocytosis by macro-

IgA is also present in the blood, but its importance lies in

phages, which results in the destruction of the antigen, a

the fact that it is secreted. It is present on mucosal surfaces

process called opsonization. Complement and natural killer

where it helps prevent viral diseases such as those caused

cells, which are components of innate immunity as well as

by rhinoviruses or influenza viruses in the respiratory tract

of acquired immunity, are described later. Other mechanisms

or by rotaviruses or enteroviruses in the intestinal tract. It is

that result in protection by antibodies also exist.

present in secretions such as tears, saliva, and genital tract

secretions, where it plays an antiparasite role. Because it is

Immunologic Memory

present in milk, it also serves to transfer maternal immunity

to the gut of the infant.

In the course of the B-cell response, memory B cells

IgE is important for control of infection by multicellular

are formed. Memory B cells persist after the antigen disap-

parasites. It can bind to mast cells via specific receptors and

pears from the body, and are primed to react quickly and

cause an inflammatory response, leading to the destruction

vigorously on renewed stimulation by the cognate antigen.

of parasites. It is IgE that produces allergic symptoms that

Renewed activity of memory B cells still requires T-helper

occur when pollen granules or mites or other comparatively

cell stimulation, but the secondary response leads to imme-

large particles are recognized by IgE, producing an inflam-

diate production of antibody of high affinity, because there

matory response.

is no need for a long maturation process, and is so vigor-

IgD is present in a membrane-bound form at the surface

ous that it results in the production of much larger amounts

of immature B cells, along with IgM, where it helps in the

of antibody than are produced during a primary response

activation of the cell on exposure to antigen as described

(Fig. 10.11).

before. It is also present in very small amounts in the blood.

Memory T cells are also generated in the course of an

There is no class switching mechanism to express IgD and it

immune response. After expansion of T-cell clones following

is only produced in combination with IgM by means of dif-

stimulation by the cognate antigen, most activated T cells die

ferential splicing of mRNAs.

by apoptosis when no longer stimulated by the presence of

Although the B-cell repertoire first arises by combinato-

antigen and cytokines. Immunologic memory remains, how-

rial joining of the V and J light-chain gene segments and of V,

ever. It had been widely thought that such memory requires

D, and J heavy-chain gene segments, the antibody response

a continuous supply of antigen with which T cells interact.

is fine-tuned once a B cell has been activated. There is an

This antigen might be present because the virus establishes a

error-inducing mechanism during B-cell replication in which

chronic infection, or antigen might be sequestered in regions

the genes encoding the V segments of both heavy and light

of the body and made available over an extended period of

chains undergo hypermutation. This activity takes place in

time. It was thought even possible that T cells continued to

germinal centers of the lymph node and there is concurrent

be stimulated because of cross-reactivity with self antigens.

selection for B cells that bind more tightly to the antigen. Over

Data have now accumulated, however, that memory T cells

a period of time B cells are selected that bind to the antigen

exist in a quiescent state for long periods of time and are

with higher and higher affinity. Hypermutation in germinal

capable of rapid reactivation on renewed exposure to anti-

centers has been postulated to play a role in the development

gen. Thus, memory in T cells is now thought to be similar to

of Burkitt's lymphoma, as described in Chapter 7.

memory in B cells.

It is the existence of memory cells that renders an ani-

other than cell lysis. Some products enhance the neutrali-

mal immune to the virus or other pathogen that first evoked

zation of viruses by antibody. By binding to virus that is

the immune response. Memory cells are primed for such a

coated with antibody, they render the virus less capable

rapid and vigorous response that the invading organism is

of binding to its receptor; cause aggregation of the virus,

stopped early during the infection process, before disease

resulting in fewer infectious units; and increase the uptake of

is established. Residual antibodies circulating in the blood

viruses by phagocytic cells (opsonization). Other molecules

or present on mucosal surfaces may even prevent infection

induce an inflammatory response, in part by inducing the

altogether (sterilizing immunity).

release of agents by mast cells and basophils, or take part in

The immune status of an individual is often tested by

the activation of B cells, or help in the clearing of immune

examining the blood for the presence of antibodies. Such

complexes.

an assay is imperfect, however. The presence of antibod-

Numerous studies have shown that complement is

ies in the blood is usually a good indication that a person is

required for an effective immune response. As one example,

immune. However, although such antibodies fade with time,

mice lacking a receptor for an early product of the comple-

in many cases a person remains immune despite the absence

ment cascade, called C3d, are unable to mount an antibody

of detectable antibodies in the blood, because memory cells

response. As a second example, depletion of complement in

that are primed to react quickly are still present.

mice infected with Sindbis virus leads to a prolonged viremia

and a more severe central nervous system disease. As a third

example, people are known who are genetically deficient for

Complement System

components of the complement pathway. Many suffer from

The complement system is composed in part of more than

immune-complex diseases as a consequence, because they

20 soluble proteins that circulate in the blood. These pro-

are unable to effectively clear immune complexes. They also

teins are activated through a proteolytic cascade to produce

suffer from an increased incidence of bacterial infections.

effector molecules that aid in the control of viral infection

or infection by other pathogens. Complement forms part of

Adaptive Immunity in the Control of

both the adaptive immune system and the innate immune

Virus Infection

system. Its activities turn antibodies into effective kill-

ers of viruses or of virus-infected cells, as well as of other

It has been conjectured that the two arms of the adaptive

pathogens, and in this role it is a component of the adaptive

immune system evolved to fight off different pathogens. The

response. Complement can also be activated by interaction

CTL response seems well adapted for the control of viral

with parasites in the absence of antibody, however, and in

infections because these pathogens replicate intracellularly,

this role it is a component of the innate responses.

but less well adapted for controlling extracellular patho-

The classical pathway of complement activation (an adap-

gens such as bacteria or protozoa. Conversely, the humoral

tive response) involves interaction of a complex of comple-

response and the associated complement system seem better

ment molecules called C1 with IgG or IgM. This could be

adapted for the control of extracellular pathogens. Consistent

IgG or IgM bound to antigen present at the surface of an

with this model, children who are deficient in the production

infected cell or a bacterium, for example. The alternative

of antibodies do not in general show an increased suscep-

pathway of complement activation (innate response) does

tibility to viral diseases but do show a marked increase in

not involve interaction with antibody, but rather requires the

susceptibility to bacterial infection. Children unable to make

deposition of a molecule called C3b on the surface of a parti-

gammaglobulin, for example, recover normally from infec-

cle, such as a parasite. Once bound, C1 or C3b interacts with

tion by measles virus and are immune to reinfection, dem-

other components of the complement system. The result

onstrating the importance of T-cell immunity in this disease.

is the activation of a cascade of proteases whose cleavage

Conversely, impairment of CTL function in children often

activities result in the formation of effector molecules. One

leads to increased frequency and severity of virus infec-

group of effector molecules forms a complex that inserts into

tions. Such findings suggest that CTLs evolved primarily to

the lipid bilayers of cell membranes and results in the lysis

deal with viral infections and remain of prime importance

of the cell. Many enveloped viruses can also be killed by

in dealing with viral infections, whereas the humoral sys-

this lytic mechanism. The system must be finely regulated so

tem evolved to deal with infections by free-living organisms

that activated components of complement are produced only

such as bacteria, protozoa, and yeast.

in response to pathogens and so that cell killing is confined

Although this model may well be correct, it is clear that

to infected cells or parasites. Control of complement activa-

humoral antibodies are also important in the control of viral

tion and action is therefore suitably complicated.

disease. Many experiments have shown that passively trans-

Other effector molecules that result from activation of

ferred antibodies alone can protect against viral infection.

complement have activities that aid in the control of viral

Further, whether reinfection by a virus results in disease or

infection or infection by other parasites by mechanisms

asymptomatic infection is often correlated with the level of

antibodies against the virus in the blood. These observations

people concerned with their appearance). This technique

are particularly relevant to the protection of an unborn or

was greatly refined about 200 years ago by Jenner, who

newborn child from viral disease. Maternal antibodies are

immunized people against smallpox with a nonhuman virus

actively introduced into the fetal bloodstream during intra-

derived from cows. Cowpox virus is antigenically related

uterine development. Such antibodies are critical for the pro-

to smallpox virus and induces immunity to smallpox, but

tection of the fetus and the newborn against viral infections

does not cause such severe disease as smallpox. This immu-

early in life before its own immune system develops. It is

nization procedure was very successful and smallpox has

also clear that neutralizing antibodies are of prime impor-

now been eradicated using modern versions of Jenner's

tance in preventing reinfection by at least some viruses, such

original vaccine. The process of using a nonhuman virus to

as influenza virus. Previous infection leading to a vigorous

induce immunity in humans against a related human virus

T-cell response directed against many of the viral proteins

has been referred to as "the Jennerian approach." Jenner's

does not protect against subsequent reinfection by vari-

use of cowpox virus to immunize against smallpox gave us

ants which are altered only in their surface glycoproteins.

the name "vaccination" and "vaccine," from the Latin word

As another example of the importance of humoral antibod-

vacca meaning cow.

ies in combating viral infections, CTL-induced cytolysis

Since Jenner's time, other approaches to vaccination have

is not very effective in the control of viral infection of the

been developed. Rather than using a nonhuman virus as a

brain. Neurons are terminally differentiated and cannot be

vaccine, it is more common to use an attenuated strain of

replaced, and express only low levels of MHC class I mol-

a virulent human virus. Attenuation has classically been

ecules. However, CTLs do appear to be important in control

achieved by passing the virus in animals or in cultured cells

of viral infections in the brain, perhaps because they secret

from animals. Passage selects for viruses better adapted to

IFN-γ when activated. Other mechanisms involving humoral

grow in the nonhuman host and often results in a virus that is

antibodies are also probably important. It has been shown in

attenuated in humans. One of the earliest vaccines to be devel-

a mouse model that humoral antibodies can cure persistently

oped in this way was a rabies vaccine developed by Pasteur

infected neurons of viral infection.

by passing the virus in rabbits. A more modern method for

Thus, a broad and varied immune response to viral infec-

producing a vaccine strain, which is still often used today,

tion is important for both the suppression of the original

was introduced by Theiler and Smith, who passaged virulent

virus infection and in evoking a status of immunity to sub-

yellow fever virus in chicken tissue and chicken cells in cul-

sequent reinfection by the virus. Experiences with vaccines

ture. After 100 passages, a marked change in virulence of the

used in humans support this idea. Some vaccines have been

virus occurred. Although the passaged virus retained its abil-

found to provoke an unbalanced response that renders sub-

ity to infect humans, it no longer caused disease. Passage of

sequent infection by the virulent virus more serious, such as

virus in tissue culture cells and selection of attenuated vari-

early vaccines against measles virus and respiratory syncy-

ants has been used to produce vaccines for measles, mumps,

tial virus, discussed elsewhere.

and rubella, among others. With modern technology, it is

now possible to introduce mutations into a viral genome

that might be expected to attenuate the virus and to test the

Vaccination against Viruses

effects of such mutations in model systems (see Chapter 11).

Although no currently licensed human vaccines have been

For most viruses, once a person has been infected and

produced in this way, it is expected that this approach will be

recovered, he or she is immune to subsequent reinfection

useful for future vaccines.

by the same virus. This is the concept behind immunization,

In a few cases it is possible to infect humans with a viru-

also called vaccination, in which a person is exposed to a

lent virus in a way that does not lead to disease. Oral vaccines

virus, either live attenuated virus or inactivated virus, or to

have been developed for adenoviruses 4 and 7 in which the

components of the virus, in order to establish the immune

virus is encapsulated in a protective coating that does not dis-

state.

solve until the virus reaches the intestine. The viruses repli-

cate in the intestine but do not produce disease, although they

Live Virus Vaccines

do induce immunity against adenovirus respiratory disease.

Live virus vaccines in general induce a more protective

Immunization has been practiced for centuries, having

and longer lasting immunity than do inactivated virus vac-

been introduced a millennium ago for smallpox. In a proc-

cines. They replicate and therefore produce large amounts

ess called variolation, less virulent strains of smallpox virus

of antigen over a period of days or weeks that continues to

were introduced into humans by intranasal inoculation.

stimulate the immune system. Furthermore, the viral anti-

The disease induced by this procedure had a lower fatal-

gens are presented in the context of the normal viral infec-

ity rate than that caused by the epidemic disease (although

tion and these vaccines induce the full range of immune

the fatality rate was still significant), and the extent of

responses, which includes production of CTLs as well as

pocking or scarring was less (which was of importance to

antibody. Live virus vaccines may be more effective than

taminated with SV40, for example, which was present in

inactivated vaccines in eradicating the wild-type virus from

the monkey kidney cells being used to prepare the vaccine.

a society. As described in Chapter 3, an inactivated poliovi-

Millions of people were unknowingly infected with SV40,

rus vaccine protects the individual from disease but it does

which fortunately appears to cause no disease in humans,

not prevent the wild-type virus from circulating. Finally, live

or at least to cause disease (some rare brain tumors) only

virus vaccines are cheaper to produce and administer than

very rarely. Finally, living viruses are often unstable, mak-

inactivated virus vaccines because a single dose containing

ing it difficult to transport vaccines over long distances

smaller amounts of (live) virus is usually sufficient to induce

and to store them so that they maintain their potency, a

immunity.

problem that is more acute in developing tropical coun-

Although they have many advantages, live virus vac-

tries.

cines also suffer from a number of potential problems.

Although there are potential difficulties, live virus vac-

Attenuating the virus sufficiently so that it does not cause

cines have many advantages and have been extremely suc-

disease while retaining its potency for inducing immunity

cessful in the control of viral diseases. Smallpox virus has

can be difficult to achieve. The human population is outbred

been eradicated, measles virus and poliovirus are on the brink

and individuals differ greatly in health status, immune com-

of eradication, and many other serious diseases have been

petence, and ability to fight off viral infections, yet a single

controlled by live virus vaccines. A partial list of currently

vaccine must be useful for all, or at least most, individu-

licensed virus vaccines, many of which use live viruses, is

als in the population. Furthermore, many viruses quickly

given in Table 10.2.

become overattenuated on passage, losing their ability to

induce immunity on infection of humans. Another problem

is the potential for reversion of the virus to virulence and

the possible virulence of the attenuated virus in normal or,

TABLE 10.2 Available Viral Vaccines

especially, immunocompromised people. In the case of the

Live attenuated

Sabin polio vaccine, about 10 vaccine-related cases of para-

virus

Killed virus

Subunit vaccines

lytic polio occurred every year in the United States due to

reversion to virulence of the virus (Fig. 3.4) before it was

Poliovirus (Sabin)

Polio (Salk)

Hepatitis B

replaced with an inactivated vaccine. Certain lots of yellow

West Nilea

Measles

Rabies

fever virus vaccine have also been found to contain partial

Human papillomavirusb

Mumps

Influenza

revertants that can cause encephalitis, especially in infants.

Rubella

Hepatitis A

Because of this, each lot of yellow fever vaccine must be

Yellow fever

Japanese encephalitis

carefully monitored for virulence and vaccination of infants

Vaccinia

Western equine

under 6 months of age is not recommended. Despite this

encephalitis

Varicellac

care, a few deaths from vaccine-induced yellow fever have

Rotavirusd

occurred very recently, which appear to be due to residual

virulence of the virus in persons with immune systems

Adenovirus (in

military recruits)

that are inadequate to handle the infection with this virus

Junin (Argentine

because of age or other reasons.

hemorrhagic fever)

Interference caused by activation of the innate immune

Influenzae

system can also cause problems. The effectiveness of a

live virus vaccine may be diminished because of interfer-

Recombinant vaccine on yellow fever 17D backbone in clinical trials;

ence from a preexisting infection. Interference also makes

already in use as veterinary vaccine.

it difficult (although not impossible) to immunize simul-

Recombinant vaccine (Gardasil) based on virus-like-particles composed

taneously against multiple viruses when using live virus

of proteins L1 and L2 approved for girls 926 years old by the FDA

vaccines. This becomes a particular problem with vac-

on June 8, 2006. Quadrivalent vaccine contains antigens from HPV16,

cines that contain multiple components, such as vaccines

HPV18, and types 6 and 11 of HPV 6.

Live attenuated vaccine (Zostavax) to protect adults over 60 who have

for the four serotypes of dengue virus or for four serotypes

previously had varicella from contracting shingles, approved May 25,

of rotaviruses. However, the mumpsmeaslesrubella vac-

2006.

cine contains three live, attenuated viruses and this vaccine

Second generation pentavalent vaccine (Rota Teq) approved by the FDA

has been very successful in controlling these three viruses

on Feb. 3, 2006.

with a single inoculation (albeit that a booster is recom-

New generation live vaccines composed of reassortants of previous

vaccine strains with HA and NA of current epidemic strain being

mended after 10 years or so). Problems can also arise from

developed.

the presence of adventitious infectious agents in the vac-

Source: Data for this table came from Granoff and Webster (1999)

cine, since it has not been treated with inactivating agents.

p. 1862; from Fields et al. (1996), p. 371; and from Arvin and Greenberg

Early lots of the live Sabin poliovirus vaccine were con-

(2006).

Inactivated Virus and Subunit Vaccines

Despite these problems, many successful vaccines

have been introduced that use inactivated viruses or sub-

The second general approach to vaccination is to use

units of viruses, and some of these are listed in Table 10.2.

inactivated virus or subunits of the virus, such as the surface

Characteristics of live virus vaccines versus nonliving

proteins of the virus. The original Salk poliovirus vaccine

vaccines are compared in Table 10.3

and current vaccines against influenza and rabies viruses

Modern biotechnology makes possible another approach

use inactivated virus. For these vaccines, virus is prepared

to subunit vaccines, the use of a nonpathogenic virus as

and purified, and virus infectivity is destroyed by treatment

a vector to express proteins from a virulent virus. This

with formalin or other inactivating agents. These inactivated

approach potentially overcomes many of the disadvantages

viruses are injected, often intramuscularly, and induce an

of inactivated virus vaccines, or of subunit vaccines based on

immune response. Subunit vaccines, on the other hand, are

purified components, because the antigens are presented in

usually produced by expressing the surface proteins of a

the context of a viral infection and in a native conformation.

virus in a cell culture system. The proteins are purified and

To date, none of the licensed vaccines use this procedure, but

injected. Licensed subunit vaccines include the modern vac-

clinical trials are in progress or are planned to begin soon for

cine against hepatitis B virus.

a number of such vaccines. Two examples will be cited. In

Inactivated virus vaccines or subunit vaccines suffer from

one series of trials, vaccinia virus is being used to express the

a different set of problems from live virus vaccines, but in

HIV surface glycoproteins. Antibodies to the HIV glycopro-

turn have a number of advantages. Their advantages include

teins are induced, but whether they are protective remains to

the fact that they are usually stable; that interference by

be determined. In another series of trials, the vaccine strain

infection with other viruses does not occur; and, with proper

of yellow fever virus has been engineered to express the sur-

monitoring, viral virulence is not a problem. Their stability

face glycoproteins of Japanese encephalitis virus, of dengue

makes transport and storage of the vaccines easier and more

virus, or of West Nile virus. Based on the successful use of

reliable. Their insensitivity to interference makes it possi-

yellow fever vaccine for more than 50 years, the prognosis is

ble to immunize against many viruses simultaneously. The

good that these new vaccines will be successful. The use of

fact that the viruses are inactivated, or that no live virus was

viruses as vectors is covered in more detail in Chapter 11.

ever present in a subunit vaccine, means that no virus infec-

tion occurs with its potential for disease. Early lots of the

Salk inactivated poliovirus vaccine were contaminated with

DNA Vaccines

live poliovirus, which resulted in a number of cases of polio

A new and potentially exciting approach to vaccination is

caused by the vaccine, but better methods of inactivation and

to inject DNA that encodes genes whose expression leads to

of monitoring residual infectivity have solved this problem.

immunity to a virus. This approach has been tested in model

Difficulties with these vaccines include the expense of pre-

systems with promising results and clinical trials in humans

paring the large amounts of material required, the necessity for

have recently begun. Plasmid DNA containing the gene for,

multiple inoculations in the case of most such vaccines, and the

failure to induce a full range of immune responses. Because

no virus replication occurs, large amounts of material must be

TABLE 10.3 Characteristics of Live and

injected to induce an adquate immune response. Further, lack

Killed Vaccines

of virus replication means that an inflammatory response that

is required for an efficient immune response must be obtained

Live attenuated

Killed virus

by using adjuvents that are incorporated into the vaccine. In

Activity

virus vaccine

and subunit

practice, inactivated virus or subunit vaccines are designed to

Antibody induction

+++

provoke only a very limited inflammatory response, and mul-

MHC class I (CD8 cells)

+++

tiple immunizations are usually required in order to achieve

Cytotoxic T-cells

+++

effective immunity. Even so, a full range of immunity is not

MHC class II (CD4 cells)

+++

achieved. Worse, in two cases immunization with inactivated

virus resulted in more serious illness on subsequent infection

Humoral antibody

+++

with epidemic virus. Inactivated virus vaccines against mea-

All viral antigens

Usually

Seldom

sles virus and respiratory syncytial virus did not protect against

Longevity of immunity

Months/years

Months

infection by the respective viruses, and led to the development

Cross-reactivity among viral

+++

of atypical disease that was more severe than that caused by

strains

the viruses in nonimmune people. The measles vaccine was

Risk of viral disease

subsequently replaced with a live virus vaccine, which has

been very successful, but no successful vaccine has yet been

Source: Data for this table came from Granoff and Webster (1999),

developed for respiratory syncytial virus.

p. 1862 and from Fields et al. (1996), p. 371.

say, a surface glycoprotein of influenza virus under the con-

transmissible gastroenteritis virus (TGEV) were better pro-

trol of a general mammalian promoter is injected intramus-

tected from TGEV infection than were animals vaccinated

cularly. Muscle cells take up DNA and import it into the

with a commercial live TGEV vaccine. Mice immunized

nucleus, a surprising finding. Expression of the influenza

with a single dose of DNA expressing the measles H protein

gene may elicit an immune reaction against the encoded

and then multiply boosted by being fed transgenic tobacco

influenza protein. Immunization with DNA might produce

plants expressing measles H protein developed significantly

protective immunity in the best of cases, or DNA immuni-

higher neutralizing antibody titers to measles virus than did

zation might serve to prime an individual for later inocula-

mice immunized with either the DNA or the plant food alone.

tion with a conventional vaccine so as to obtain a stronger

Thus, in principle plant-based vaccines are capable of induc-

response.

ing immunity. Whether practical vaccines can be developed

Immunization with DNA has many potential advantages.

using this approach remains a question for the future.

The encoded protein is made intracellularly and therefore a

CTL response is generated, because peptides are presented

Vaccine Development: The Example of

by class I MHC molecules. In addition, the proteins are made

Measles Vaccines

and are present in a native conformation, which may make

possible the production of antibodies against a wider range

Vaccines against viruses have been enormously success-

of conformational epitopes. Other advantages include the

ful in controlling viral diseases that have plagued mankind

fact that multiple proteins can be encoded in plasmid DNA,

for millennia. Smallpox virus has been eradicated world-

which allows immunization against multiple products of one

wide, poliovirus has been eradicated from the Americas and

virus or against several different viruses, and that cytokine

may be eradicated worldwide within the next few years, and

genes can also be present in the plasmid, which can serve

many epidemic diseases have been controlled, at least in

as adjuvents to obtain not only a stronger immune response

regions that have access to the vaccines. As an example of

but to direct the immune response toward desired pathways.

potential difficulties in vaccine development, however, the

Another important feature is that the expression of the for-

measles virus vaccines will be discussed. Measles virus is

eign protein continues for some time, potentially leading to

ubiquitous. Not so long ago, virtually the entire population

a better immune response. Finally, plasmid DNA is easy to

of the world was infected by the virus as children. Very few

modify to express different genes, cheap to make in quan-

people escaped infection by the virus. Measles can be a seri-

tity, and easy to purify. Once safety trials have been con-

ous illness, with significant mortality and debilitating seque-

ducted for a DNA vaccine, it should be possible to modify it

lae (Chapter 4). Concerted efforts to produce a vaccine began

to express antigens from other viruses with minimal safety

many years ago. One early measles virus vaccine, used from

concerns.

1963 to 1967, was made with inactivated virus. Vaccination

Human trials with DNA vaccines are just beginning.

with this vaccine potentiated a more serious illness when a

A Phase 1 safety trial of an HIV vaccine has just been com-

person was infected with the epidemic virus, however, char-

pleted using 86 volunteers, and the results were sufficiently

acterized by a more severe rash and called "atypical mea-

encouraging to begin Phase 2 efficacy trials. The DNA vac-

sles." The atypical disease is thought to be produced by an

cine uses multiple DNAs expressing HIV-1 genes from

atypical or unbalanced T-cell response to the virus following

three subtypes of the virus. Three intramuscular injections to

immunization. This response controls virus infection only

prime the individuals were given using a needle-free device.

poorly and leads to collateral damage from T-cell attacks on

This was followed by a boost using adenovirus type 5 that

virus-infected cells. An attenuated virus vaccine was more

expressed HIV proteins. At the highest doses used, almost

successful. The first live virus vaccine used was protective

all individuals produced antibodies against HIV as well as

but insufficiently attenuated and thus more reactogenic than

both CD4+ and CD8+ T cells that were reactive against HIV

would be ideal. It was subsequently replaced with a more

antigens.

highly attenuated vaccine.

The attenuated virus vaccine currently in use has been

highly successful in controlling measles in many develop-

Plant-Based Vaccines

ing countries as well as in the developed countries. The dis-

Another possible approach to the production of vaccines

tribution of the virus as of 1998 is shown in Fig. 10.13A,

is to use modern biotechnology to introduce genes for viral

illustrating that by this time the virus had been eradicated in

proteins into a food plant. If eating this plant induces immu-

many developed countries and had substantially reduced the

nity, it would provide a cheap and easy alternative to vaccines

incidence of measles over most of the world. By 2004, the

that require extensive purification followed by injection. No

virus had been essentially eradicated from the Americas and

human vaccines have been developed to date using this tech-

most other areas, but pockets of endemicity remain in Africa

nology, but the concept has been tested in animals. Piglets

(Fig. 10.13B) where the virus continues to be an impor-

fed transgenic corn that expresses the spike protein of swine

tant cause of infant mortality and morbidity. The failure

1998

Equator

Cases of Measles

per 100,000 Population

>100

10-100

1-10

None

No data

2004

Moroc-

Niger

Cases of Measles

per 100,000 Population

1.6-5.0

0.5-1.5

0.1-0.5

<0.1

No data

FIGURE 10.13 Incidence of measles. (A) Reported incidence rate of measles per country as of 14 August 1998. Rates

are shown as cases per 100,000 population. From Web site of World Health Organization. (B) The incidence of measles

in Africa in 2004. Note the drastically different scales. In 2004 the Americas were virtually free of measles, although

Southeast Asia (including India) still records upwards of 200,000 deaths from measles annually.

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