Rosenthal, Ken S. PhD
Vaccination is probably the most beneficial therapy physicians can provide to their patients. Immunization programs have led to the elimination and/or control of infectious agents including smallpox, polio, measles, mumps, rubella, influenza, Hemophilus influenzae B, pertussis, tetanus, and diphtheria (Table 1).1,2 Vaccines are being developed to prevent or protect against many other infectious agents and as therapies against infections, autoimmunity, cancer, hypertension, and other diseases. Vaccines can even be used to establish means for contraception and promote the cessation of bad habits, such as smoking. With all of these wonderful accomplishments, people ask why successful vaccines have not been developed against HIV, CMV, and SARS-coronavirus, bacteria such as Pseudomonas aeruginosa, Neisseria gonorrhea, or Mycobacterium tuberculosis and parasitic diseases, such as malaria. Why can't we develop a "flu vaccine" which will protect us from all influenza virus infections? Part of the answer is that vaccine development has become very expensive, requiring over 500 million dollars per vaccine, takes a long time to get through phase 3 trials, and the profit margin for such an investment is 1/10 that of a successful drug, such as a cholesterol-lowering statin derivative. The other issue is that the answers to the following key biologic questions are sometimes difficult to obtain.
1. Which type of immune response is necessary to prevent the microbe from causing disease? (Note that immunization does not usually prevent infection, but prepares the host to defend itself upon challenge.)
2. What molecule will elicit a protective immune response toward the microbe?
3. How can a vaccine be prepared to deliver the immunogen and elicit the proper response in a safe, relatively inexpensive, well-tolerated manner?
The purpose of this review is to answer these 3 questions by describing the biology relevant to vaccination and some of the newer approaches to vaccination.
1. Which Type of Immune Response?The immune response evolved to prevent the spread of an infection beyond the control of innate protections (eg, barriers, complement, neutrophils). The type of immune response required to control an infection is determined by the microbe (Fig. 1) Some infections can be resolved by antibody, whereas others require both antibody and cell-mediated immunity. Antibody can limit the spread of a microbe and promote the resolution of extracellular microbes, especially those in the blood stream or at mucosal surfaces but is not effective in controlling intracellular microbes, such as mycobacteria and viruses, or fungi for which cell-mediated immune responses are also necessary. This is illustrated in Figure 2 for a virus such as polio, which kills its host cell to be released and for measles or even human immunodeficiency virus, which are released without killing the host cell. Antibody is sufficient to resolve the polio infection because the virus kills the cellular factory and the antibody controls the extracellular virus. For measles or human immunodeficiency viruses, antibody can neutralize extracellular virus but unless cell-mediated immunity kills the cellular factory, virus will continue to be synthesized, released, and the infection will persist.As will be discussed below, the type of response that is generated to an infection or a vaccine is determined by the cytokine response to the immunogen. A response which includes both antibody and cellular immunity is termed a TH1 type of response (1 = first = early = local = antibody and cell-mediated responses), whereas a predominantly antibody response is termed a TH2 type of response (2 = later = systemic = antibody). A person who is more prone to TH2-type responses is likely to endure more severe disease presentations with certain intracellular bacteria, viruses, fungi, and parasites, such as mycobacteria leprae, herpes viruses, or leishmania.
2. What Molecule?The target for eliciting protective immunity differs for an antibody response and a cell-mediated response. Antibody is very effective in limiting the spread of a microbe in the blood (bacteremia or viremia) or other body fluid and in preventing the adhesion or entry of the microbe into cells or across membranes. The most effective protective antibody is directed toward molecules which promote the attachment of the microbe to cells, such as a bacterial adhesion protein or the viral protein which binds to the cellular receptor (eg, hemagglutinin of influenza). Unfortunately, mutants with small changes in the structure or sequence that is recognized by the antibody can become resistant to this type of protection. This is especially true for influenza and HIV.The targets for cell-mediated immune responses are less obvious. Cell-mediated immune responses are generated against microbial debris that is processed by phagocytes (described below) or within infected cells and presented to the T cell by major histocompatibility (MHC) molecules. In essence, T cells respond to microbial trash rather than the microbe itself. The structures that are recognized by T cells are less likely to differ between microbial strains. As a result, immunization of T cells can produce a more generic protective response than antibody.
3. How to Activate Protective Responses by Immunization.Since the immune response evolved to provide protection against infectious diseases, the optimal development of a protective immune response by a vaccine should mimic the steps and processes elicited during the establishment of natural immunity. Development of a natural immune response is initiated by microbial infection which triggers innate responses to the infection and progresses through a series of stages, with defined cellular characters and a molecular text, consistent with the development of the action in a drama.
IMMUNIZATION: A PLAY IN 3 ACTS
For this review, the different stages in the establishment of an immune response to a microbe or a vaccine will be described within a three-act play (Table 2). The outcome of this play is determined by the early interactions of the microbial protagonist and the cellular actors which respond to the challenge. The interactions between these actors will determine whether a predominantly humoral response (antibody) (TH2) or an immune response with both antibody and cell-mediated responses (TH1) will occur. The vaccine developer, like a playwright, devises a mock protagonist which will elicit an immunologic development similar to the microbe. The cast for this drama is made up of white blood cells from individuals differing in age, genetic makeup (eg, MHC antigens), and infection experience.
The prologue of the play takes place at the site of infection or immunization and portrays the initial responses to the immunogen, the microbe or vaccine. In Act 1, the immunogenic presence is processed for presentation to the actors of the immune response. Act 2, Scene 1 begins with the development of immune responses that reinforce the protections at the local site promoting both cell-mediated and antibody responses, a TH1 response. In Scene 2, the plot expands to systemic antibody protections. The final act, Act 3, includes the resolution of the drama and development of immune memories to the production. As with any drama, each act builds upon the previous one, especially the first act, and the outcomes depend upon a successful development and progression of the previous acts. A successful immunization must elicit appropriate character and plot development that can be retold whenever the protagonist initiates another performance of the immunologic drama (immunologic memory).
The cast of characters for the immune system consists of the cells that deliver the response (Table 3). As with the characters of a play, the different immune cells can be distinguished by their costumes, which consist of their cell surface antigens. In addition to allowing the laboratory audience to distinguish the different cells, these cell surface molecules also determine the role (function) that the cell plays and the types of interactions that the cells make with each other in the immune drama. For example, the T cell is identified by the molecule that recognizes immunogens, its T-cell receptor (TCR), and is distinguished into functional subsets by expression of either CD4 or CD8, which interact with MHC II or MHC I proteins, respectively. Although the MHC I (major histocompatibility complex) protein is present on all nucleated cells, the MHC II protein is only present on cells that can present antigen to the TCR (eg, dendritic cells [DCs], macrophages, and B cells). The B cell, a potential antibody-producing factory, can be identified by the surface expression of the immunoglobulin that it produces.
As indicated above, the cast of characters are introduced within the action of the different scenes of the play (Table 4). Recent studies have shown that the principal actor in the immunization process is the DC and much of this review will discuss its role and interactions with T cells to initiate the development of the immune response. During the prologue, the neutrophils, natural killer (NK) cells, NK T cells, monocytes, macrophages, and DCs are introduced. The main characters of Acts 1 and 2 are the DCs and CD4 T cells. New roles for CD8 T cells and B cells are introduced toward the end of Act 1. In the final act, our lymphocytes mature. Some of these develop into plasma and memory cells.
Dendritic cells are very important members of the cast but have remained somewhat reclusive and difficult to work with until recently. DCs include the myeloid, plasmacytoid, and follicular DCs. This immune drama features primarily the myeloid DCs. These cells are derived from monocytes and monocyte-like predendritic cells, which differentiate into immature DCs, which can reside in tissues and organs. The immature DCs are actively phagocytic and can be identified by expression of myeloid markers, including an LPS receptor (CD14). Some DCs express CD4, others express CD8, and some express neither molecule. Mature DCs become potent antigen-presenting cells. They extend their membrane processes to increase the cell's surface area and express proteins to facilitate interactions with T cells to promote presentation of antigen.
The immunization drama is written in the molecular language of small proteins and the dialogue of each act is written with its own predominant chemokine and cytokine language. Chemokines are small proteins that activate and guide cells to certain body locations and cytokines are the hormones of the immune response. Production of a cytokine, or a defined group of cytokines, can direct the action of another cell type, or an entire cast of cells. As the star of the immune drama, the cytokine language of the DC determines the plot direction of the play. The T-cell costars take their cues from the DC and its cytokines. For example, IL-6 is essential for awakening the naive T cell to develop its function and without IL-12, the drama will skip the type of cell-mediated, local-acting immune response (TH1) that is described in Act 2, Scene 1 and will proceed by default to a more systemic, antibody type of response (TH2), as described in Act 2, Scene 2. By the time Act 3 comes, most of the language has been introduced. A list of the most relevant cytokines spoken during each of the acts, and a description of their source and function, is presented in Table 4.
The prologue for this play takes place in a quiet section of the skin. Macrophages, monocytes, and immature DCs (myeloid cells) are strolling through the region on patrol. Macrophages and DCs are constantly gobbling up the occasional suicidal cell (apoptosis) or protein trash expelled from cells into the lymph. They break down the proteins into peptides and amino acids to clean up the neighborhood of its normal protein trash. NK cells and NK T cells are bumping up against the cells on their regular patrol, like policemen, checking with their inhibitory receptors (KIR) to make sure that all cells have the appropriate MHC markers indicative of a normal cell. If they come across an occasional cell which lacks sufficient MHC I proteins (eg, tumor cells or viral infected cells), they become activated and kill the cell. Complement proteins constantly wash up on the cells and an occasional C3 will break into pieces but our cells are protected by complement regulatory proteins. The scene is quiet and controlled by the many regulatory processes that prevent abnormal responses. The characters of the local immune cells, and especially the DC, remain uncomplicated and naive unless forced to mature by interaction with the protagonist, microbes, or their components.
Upon bacterial attack, complement becomes activated via the alternate or lectin routes by touching the surface of the bacteria. Chemotactic and anaphylactic factors (C3a, C5a) are produced, complement fragments stick to the microbial surface to make them easier to be phagocytosed, and the complement builds a molecular structure to drill holes in the bacteria. In addition, a B-lymphocyte activation protein (C3d) is produced to facilitate antibody production. Neutrophils become alarmed and jump into the fray, gobbling bacteria and blowing them up within their vesicles. These processes fill the stage with microbial fragments and carcasses which are large enough to be phagocytosed by DCs and macrophages. Unlike the normal detritus of their environs, the microbial material is recognized by receptors on dendritic and other cells which include Toll-like receptors (TLRs), lectins, and scavenger receptors, and triggers a rapid response. These pathogen-associated molecular patterns are present in bacterial or viral components such as lipopolysaccharide (LPS), peptidoglycan, undermethylated guanosine-cytosine DNA sequences (CpG), double-stranded RNA, and other molecules. LPS is probably the most potent TLR activator. Different TLRs are present on the cell surface or within the phagocytic vesicles of DC, macrophages, and other cells to interact with the bacterial and viral components.5
The language of the prologue is triggered by the TLR activation of myeloid and endothelial cells. The text is written as a rapid banter of the proteins of the complement systems, the acute phase proteins, chemokines such as IL-8, and the cytokines, IL-1, IL-6, TNF-α, IL-12, and IL-18. These cytokines determine the subsequent plot direction.
Proper TLR stimulation is an essential step in the activation and proper development of the DC's character. Binding to the TLR activates molecular pathways that initiate the maturation of the DC, production and release of cytokines, and the movement of DCs to the lymph node.6-8 Without the challenge from the microbial protagonist to trigger the TLR activation pathways, the DC character remains complacent, does not mature properly, and cannot produce IL-12 necessary to stimulate the TH1 response in Act 2, Scene 1. As a result, the immunization drama skips directly to Scene 2 and the play is less memorable (poor memory immune response).
Immunization with most inactivated vaccines consists of in-jection of a bolus of protein and does not include a mechanism for TLR stimulation. These vaccines can only generate TH2 responses, production of antibody, and limited memory response.
ACT 1: ANTIGEN PROCESSING
Act 1, Scene 1: DC Presentation of Antigen to T Cells
The prologue ends with the activation and maturation of the DC. The main characters of Act 1 are the DC and CD4 and CD8 T cells.
Having been activated by TLR ligands, the DC takes a final phagocytic survey of the proteins at the infection site and shuttles these proteins into a slow proteolytic processing cascade to produce peptides of approximately 12 amino acids in length. The DC matures and moves to the lymph node where it will present the microbial peptides to CD4 and CD8 T cells as evidence of the action occurring at the site of infection. The DC processes the microbial proteins slowly and carefully to ensure that appropriate peptides are available for presentation in the lymph node. The more mature DC also has an enhanced costume, with increased cell surface expression of the MHC II, CD80, CD83, and CD86 molecules consistent with its maturation and the new role it will play presenting the peptides to T cells and to initiate the immune response in Act 2.
Presentation of immunogenic (microbial) proteins to T cells requires that they be simplified to individual immunologic recognition structures (epitopes) so that they can be recognized by T cells in the lymph node. Like a bureaucratic military officer accepting a password, the T cell will only recognize or become activated by a peptide properly presented to its TCR from within the cleft of an MHC molecule. The CD4 T cell recognizes peptides in an MHC II molecule (CD4 interacts with MHC II) and the CD8 T cell recognizes peptides in an MHC I molecule (CD8 interacts with MHC I). These peptide passwords are always contiguous peptide sequences from a protein.
The MHC II molecule clamps an 11-13 amino acid peptide in a hotdog bun-like cleft at the top of the molecule presenting a side of the peptide to the TCR on CD4 T cells. The peptides that are presented by MHC II molecules to CD4 T cells are obtained by phagocytosis of extracellular debris. The MHC I molecule holds an 8-9 amino acid peptide in a pita bread-like pocket on the top of the molecule for presentation to CD8 T cells. The peptides that are presented by MHC I molecules on DC to CD8 T cells arise from 2 sources, intracellular proteins and extracellular proteins that enter the cross-presentation pathway. Only DCs have the cross-presentation pathway.
Dendritic cells, macrophages, and B lymphocytes have the machinery to acquire microbial detritus (trash), process antigenic proteins into peptides, and present these peptides to T cells on MHC II molecules. The microbial trash is internalized by phagocytosis or pinocytosis, transferred to lysosomal vesicles where it is digested. At the same time, MHC II molecules are assembled in the endoplasmic reticulum with a packaging component, termed the invariant chain (Ii), that occupies the hotdog bun-like peptide-binding cleft. Vesicles containing the MHC II molecules leave the endoplasmic reticulum and fuse with the antigen containing lysosomal vesicles. Microbial peptides displace the invariant chain and the MHC II molecule is delivered to the cell surface to present its peptide to CD4 T cells.
The peptides presented by MHC I molecules are derived from intracellular proteins, misfolded proteins, signal sequences, and other proteins. These proteins are proteolytically processed by all nucleated cells into epitope passwords of 8-9 amino acids which distinguish friend (self) from foe (virus, tumor cell, or foreign tissue graft). These peptides enter the endoplasmic reticulum through the TAP (transporter associated with processing) and some of them are able to bind within the pocket on the top of MHC I molecules. Once the cleft is filled, the MHC I molecule picks up an additional subunit, the beta-2-microglobulin, and is transported through the Golgi apparatus to the cell surface to express the "self password" to CD8 T cells. During a viral infection, the cell is filled with an abundance of a limited number of foreign proteins which are also targeted to this pathway. As a result, the viral peptides will fill most of the MHC I molecules and provide a "nonself" signal to activated CD8 T cells to trigger the killing of the infected cell.
Unlike any other cell, the DC can process viral and tumor cell proteins in a manner to activate a CD8 T-cell response. In a process called cross presentation, the DC shuttles microbial peptides that were phagocytosed at the site of infection into the endoplasmic reticulum where the peptides can fill the pita bread-like pocket of MHC I molecules. The DC can also phagocytose cells that have died by apoptosis, including tumor cells, and present antigenic tumor proteins (eg, HER-2 Neu from breast cancers) in a similar manner.
It is at this point in the immune response drama that each person defines the plot of the play. Everyone has different MHC I molecules, a different pair of HLA A and B (MHC I) molecules and MHC II molecules, HLA DP, DQ and DR, one obtained from each of their parents. Different MHC molecules bind different repertoires of epitope peptides. This potentially allows some individuals to initiate a more effective protective response than others (eg, against an influenza infection). This consideration also has important ramifications for vaccine design. The vaccine must contain peptides that can be presented by the predominant MHC I and/or MHC II molecules in the population to be effective.
The viral or other microbial peptides that fill the MHC I and MHC II molecules during an infection include epitopes which can be incorporated into vaccines to activate protective T-cell responses. Manipulation of the mechanisms for delivering the epitope into the cleft on the MHC I or MHC II molecule is the basis for several new approaches to vaccine construction (as discussed later).
Act 1, Scene 2: IgM Antibody Production
At the same time as the peripheral DC is maturing and moving to the lymph node, the microbial debris in lymph fluid is filtered by the lymph nodes and may come into contact with the B lymphocyte. Each B cell speaks in a monotone, producing only one specificity of antibody and capable of responding to only 1 epitope structure. A chorus of B cells produce responses to the many different epitopes on an immunogen. In the absence of T cells and appropriate antigenic stimulation, the costume worn by the B-cell character is limited to surface IgD and IgM and these antibodies advertise the antigen specificity of the cell. The surface immunoglobulin can sense the presence of antigenic cues but will only heed a signal and activate the cell if several surface immunoglobulin molecules are bound together (crosslinking them) as for the repetitive structures of peptidoglycan, the O antigen of LPS, or capsule polysaccharide. These responses are enhanced several thousand fold by the presence of the C3d molecule produced during complement activation. The C3d molecule binds to a receptor found almost exclusively on B cells. Without the help provided by T-cell responses, the B cells are restricted to producing IgM. IgM is a large, evolutionarily ancient immunoglobulin which is too big to distribute efficiently throughout the body. The mature, more diverse IgG, IgE, and IgA antibody responses are generated in Act 2 with the help of T-cell-derived cytokines.
IgM is the only type of antibody that is generated by capsular polysaccharides and glycolipids as these nonprotein molecules cannot be presented by DC to activate T cells. As a result, only IgM will be produced in response to the capsular vaccines (eg, pneumococcus, meningococcus, and Hemophilus influenzae). As described below, conjugation of the polysaccharide to a protein provides the bridge necessary to allow the T cell to provide the signals to enhance the immunoglobulin response to the polysaccharide.
Act 2, Scene 1: Activation of CD4 TH1 Responses in the Lymph Node
The setting for Act 2 is in the lymph node, a short distance from the site of infection. B cells sit quietly in follicles of the lymph node and T cells, mostly CD4 T cells but some CD8 T cells, are resting between the follicles in the cortex surrounded by DCs. Microbial components start to wash into the lymph node and DCs make the trip from the periphery to activate antigen-specific T cells. These stimuli initiate the movement and interaction of the B and T lymphocytes and the DCs that are resident in the lymph node.
Naive T cells, never having been involved in an immune response, are very susceptible to control by regulatory T cells, exemplified by the CD4-CD25 TR cells. The TR cells tightly control the initiation of a new immune response and maintain complacency by putting a cytokine inhibitory spell (IL-10 and TGF-β) on the naive T cells. The TR are part of an extensive system of checks and balances that are used to ensure that inappropriate (eg, autoimmune) responses are not activated. This block can only be lifted by the combination of IL-6 and antigen presentation by the DC.9 Hence, only a DC can initiate a primary immune response. This is another reason why a vaccine or its adjuvants should activate DCs to ensure proper immunization.
The T cell also has its own failsafe mechanism to limit spurious responses. The T cell requires at least 2 interactions, much like the 2 keys required for launching a missile, to initiate the activation of the cell. The first key for activation of the T cell is the unique interaction of the TCR with the antigenic peptide nestled in the cleft of the MHC molecule. The second signal comes from the interaction of CD28 on the T cell with the B7-1 (CD80) or B7-2 (CD86) from the DC. Other interactions that reinforce the activation dialogue between the T cell and the DC include the interaction of the CD4 or CD8 co-receptor molecule on the T cell with the MHC II or MHC I molecule on the DC, and interactions of adhesion molecules on both the T cell and the DC.
Each T cell has a different TCR and, like an antibody molecule, they determine the antigenic response of the T cell. The TCR on CD4 T cells recognizes a linear peptide sequence from within a protein presented by MHC II molecules. Binding of the peptide-MHC II complex on the DC to the TCR activates the adjacent CD3 complex to initiate a cascade of molecular events, which provides the primary activation stimulus for the cell. Binding of the B7-1 (CD80) or B7-2 (CD86) molecule on the DC with the CD28 molecule on the T cell provides the second key for activation. The response is reinforced by other cell-cell interactions and cytokine signals (eg, IL-6) provided by the DC.
A key plot determinant in the immune drama occurs at this point. If the cytokine signals from the DC includes IL-12, then the T cell will continue with Act 1 and the development of a TH1 type of response. If not, then the T cell skips the action of Scene 1 and proceeds to Act 2 and generates the cytokines characteristic of a TH2 response.
Upon stimulation in the presence of IL-12, the CD4 TH1 cell makes IL-2 and IFN-γ to help initiate the immune response. IL-2 stimulates the cell division and expansion of the lymphocyte cast, including T, B, and NK cells, whereas IFN-γ directs several other actions. IFN-γ, also known as macrophage activation factor, enhances the antimicrobial activity of macrophages, so that they can deliver proper antimycobacterial, antifungal, and antiviral action at the site of infection. Many of these macrophage functions are also associated with the Type IV hypersensitivity reactions, also known as delayed-type hypersensitivity reactions. The IFN-γ also stimulates the macrophage and DC to make more IL-12, reinforcing the TH1 setting.
Scene 1 also includes the activation of CD8 T cells in the lymph node. Antigenic peptides prepared by the cross-presentation pathway are presented by DC on MHC I molecules to the CD8 T cells. Once activated, they can respond to the IL-2 produced by the CD4 T cells and their numbers will multiply. The number of CD8 T cells may increase as much as 100,000 times in response to a single microbial antigen. CD8 T cells can produce cytokines similar to CD4 T cells or can move to the site of infection, interact with, and kill virally infected cells.
While the T-cell action is occurring, a B-cell subplot is initiated. This subplot starts with a ballet of sorts between the T cells and the B cells. The activated, antigen-specific T cells move toward the B-cell zones of the lymph node and wander around until they meet and interact with B cells that recognize parts of the same antigen. The antigens will bind to the surface immunoglobulin on the B cells, will be internalized, and then processed for presentation on MHC II molecules to CD4 T cells. Although similar to the processing performed by DCs, the repertoire of antigenic peptide epitopes produced by the B cell will be limited to a single protein. The B cell presents these peptides on MHC II molecules to CD4 T cells while its costimulator B7-1 or B7-2 proteins bind to their receptors on the T cell. The T cell accepts these interactions and links its CD40 ligand to the CD40 on the B cell. This CD40-CD40 ligand interaction provides permission to the B cell to make antibody. At the same time, cytokines from the T cell, such as IL-2 produced as part of a TH1 response or IL-4 produced as part of a TH2 response, stimulate the growth of the B cell which increases the number of cells producing this particular antibody molecule. Other cytokines, such as IFN-γ for TH1 responses or IL-4, and IL-5, for TH2 responses, promote an irreversible transformation of the B cell's character, a class switch. The class switch occurs by a deletion of portions of the immunoglobulin gene and results in an irreversible change in the antibody molecule. The portion of the immunoglobulin responsible for recognizing the epitope is now attached to a different heavy chain gene. IFN-γ promotes the switch from IgM and IgD production to production of IgG 2a in the mouse, an antibody isotype which interacts with complement and reinforces the local antimicrobial response. This process is termed T-cell-dependent, immunoglobulin class switching. In Scene 2, other T-cell-derived cytokines will promote the deletion of other intervening sequences and the production of other immunoglobulins.
Because T-cell help requires a peptide linkage between the T cell and the B cell, polysaccharide capsules cannot elicit IgG antibodies. Protein-conjugated polysaccharide vaccines, such as the HiB vaccine, can override this problem. The HiB vaccine consists of the Hemophilus influenzae capsular polysaccharide attached to the inactivated diphtheria toxin protein. The B-cell-making antibody against the capsular polysaccharide will bind and internalize the vaccine conjugate. The diphtheria toxoid protein will then be processed and presented on MHC II molecules to a T-cell partner. This will initiate a handshake between the cells which will allow the T cell to provide the help necessary to stimulate the class switch from anticapsular polysaccharide IgM antibody to IgG production and the potential for memory cell development. Pneumococcal and meningococcal conjugated vaccines have also been developed.
Act 2, Scene 2: TH2 Responses
Scene 2 is devoted primarily to expanding the role played by the B cell and its antibodies. CD4 T cells producing IL-4, IL-5, IL-10, and IL-13 promote the further differentiation of the B cell to produce other IgG subtypes or IgE antibodies. The relative concentrations of IL-4 and IL-5 determine which IgG subtype or IgE is produced by the B cell. IgA production requires additional stimuli [eg, TGF-β (transforming growth factor beta)].
For the usual immune drama, the TH1 response that developed in Scene 1 segues into a TH2 type of response in Scene 2. These responses occur in the absence of IL-12. Although the definitive screen play for the transition from Scene 1 to Scene 2 has not been clearly written, the change in action may occur due to the distribution of the immunogenic components in the lymph to distant lymph nodes or the spleen without the IL-12-producing DCs introduced in the prologue and Act 1. This can also occur for an inactivated vaccine administered as a bolus of protein without a corresponding TLR activation of DCs.
ACT 3 EXPANSION OF IMMUNITY AND MEMORY
Act 3, Scene 1: Immunologic Outcomes
Scene 1 takes place soon after the establishment of the immune response. At this point, macrophages and B cells have joined the DCs in presenting antigen to T cells. T cells and B cells expressing antigen-specific TCRs or immunoglobulins receive signals to grow and multiply promoting expansion of the response (clonal expansion). As the B cells divide, their immunoglobulin monologues are edited by small changes caused by somatic mutations in the immunoglobulin gene. This improves their specificity for antigen. Many of the B cells terminally differentiate into plasma cells to increase their antibody production. Activated CD4 and CD8 T cells wander to the site of infection to turn on macrophages with cytokines or to kill infected target cells. Some of the T and B cells stop growing and become memory cells, others continue the response and ultimately burn themselves out and commit apoptosis (hari kari).
Resolution of the infection occurs with antibody neutralization of extracellular organisms, especially in the blood. Antibody also opsonizes the bacteria, enhancing their uptake by macrophages and neutrophils. IFN-γ activates macrophages to enhance their antimicrobial activity against intracellular infections and fungi. CD4 and CD8 T cells, with help from NK cells and activated macrophages, will kill virally infected cells to eliminate cellular factories for the pathogen. But we pay a price for all of these responses!! As described in my review article in the last edition of the journal, "Microbial Diseases, Are They Self Inflicted?," the immune response also makes us tired and causes immunopathology.10
Act 3, Scene 2: Future Immunologic Outcomes
Scene 2 occurs several years later and is triggered by the return of the microbial protagonist. Having already developed the T cell and B cell characters and generated memory cells in Acts 2 and 3, less time is required to introduce and mature these cells and their response. Unlike the naive T cells, memory T cells are not under the spell of TR cells and they can rapidly respond to interactions with antigen-presenting macrophages and B cells in addition to DCs. This is one reason why a much stronger memory response can be initiated much faster than a primary response. Ultimately, the role of a vaccine is to generate memory T and B cells so that a quicker, stronger immune response can be generated later in life to prevent the spread of the disease produced by a microbial challenge.
VACCINES AND THE IMMUNE DRAMA
Live vaccines initiate the immunization drama as weakened protagonists. Almost all live vaccines are attenuated viruses which are capable of infection but unable to cause significant disease because they do not replicate efficiently at body temperature, in human cells. Alternatively, these microbes cannot spread, infect, or damage the tissue associated with significant disease (eg, polio vaccine and the brain). The infection will initiate the natural immune drama and their immunogenic components will be internalized by DC and activate the DC to allow the immune drama to progress to a successful, natural conclusion.
Just as good plays are adapted by different playwrights, there are several adaptations of live vaccines for pathogens that cannot be attenuated safely. One approach is to genetically engineer a hybrid virus consisting of a nonvirulent virus, such as vaccinia (the smallpox vaccine virus) and insert genes for the virus pathogen. For example, HIV genes encoding the gp120 immunogen for antibody production and other proteins for T-cell stimulation can be genetically engineered into vaccinia or a safe adenovirus. Adenovirus 5 and 11 strains were developed as attenuated vaccines for the military and are being used as gene and vaccine delivery vehicles. When the virus replicates, the HIV genes are expressed and the individual is immunized as if infected by both viruses.
Another approach is to infect with a virus that can initiate but not complete its replication cycle in human cells. The immunogenic proteins are synthesized, the viral proteins and genome trigger DC activation, but the complete virus cannot be made, cannot spread, and cannot cause disease. For example, disabled infectious single cycle (DISC) herpes simplex virus vaccines were developed that lack an essential gene for replication. The mutant is made in tissue culture cells that provide the necessary activity (eg, the polymerase that replicates the viral genome or a glycoprotein essential for infection). During immunization, the DISC virus initiates its replication, produces immunogenic viral proteins, but cannot complete the replication cycle. Another example is the canarypox-based hybrid vaccines. Canarypox is a relative of the smallpox and vaccinia viruses that can initiate, but not completely replicate in human cells. Canarypox vaccines containing the gp120 and other HIV genes are in clinical trials.
DNA vaccines consist of a gene for the immunogen carried in a plasmid containing DNA sequences that ensure the production of the protein in human cells. Naked DNA injected under the skin will be taken up by DCs or macrophages and these cells will express the proteins encoded by the DNA as if infected by a virus. The cells will process the proteins and present peptides on MHC I molecules to CD8 T cells. DNA vaccines initiate a T-cell response but do not elicit antibody. To enhance the efficacy of DNA vaccines, a prime-boost protocol is being used for an HIV vaccine in phase 2 trials. After receiving a DNA vaccine which expresses 4 HIV genes, the individual will receive a booster shot of an adenovirus hybrid containing these same genes.
Inactivated vaccines may consist of an inactivated microbe (eg, heat and chemically inactivated influenza virus), an inactive protein (eg, tetanus toxoid), or a peptide. Inactivated vaccines are poor actors in the immune drama because they do not provide the prompts for uptake by the DC nor the appropriate motivation to the DC (eg, binding and activation of TLR pathways). As a result, the DC cannot mature properly or initiate a complete immune response. The immune response generated to inactivated vaccines is often limited to a TH2 type of response consisting of antibody but not cell-mediated immune protections.
Individual proteins and especially peptides are too small to be seen by the DC and their uptake can be facilitated by increasing the size of the immunogen by crosslinking peptides or proteins into a larger multiantigen peptide complex. The classic approach to ensure that a peptide catches the attention of the DC is to chemically attach the peptide to a larger carrier protein, such as the hemagglutinin protein of the keyhole limpet (KLH). However, immunization with these conjugates produces antibodies to both the peptide and the KLH carrier as part of a TH2 type of response.
To facilitate uptake and provide some activation for DCs, inactivated vaccines are administered in a formulation with an adjuvant. Some adjuvants resemble TLR ligands to replace the DC activators produced during infection. Other adjuvants consist of the cytokines or chemokines produced in response to infection that will stimulate the DC and promote TH1 responses, such as GM-CSF. Unfortunately, there are very few adjuvants that are FDA approved for human usage.
The classic adjuvant, and until recently, the only FDA-approved adjuvant, is alum. Alum provides a particulate upon which the vaccine is precipitated. Although precipitation onto alum promotes uptake of the immunogen, alum is a poor activator of DC and does not induce the production of IL-12. As a result, alum-based vaccines initiate TH2-type antibody responses. Complete Freund's adjuvant (CFA) is a powerful adjuvant consisting of inactivated Mycobacterium bacillus Calmette Guerin (BCG), which contains a mixture of TLR ligands, in a mineral oil solution. Emulsification of the immunogen in the CFA provides a depot for slow antigen release to promote phagocytosis while the BCG is a strong activator of DCs and TH1 responses. CFA is not approved for human usage. Newer adjuvants approved for use in human vaccines include monophosphoryl lipid A (MPL), which consists of the endotoxin-like component of bacterial LPS (a TLR ligand); Montanide ISA51, which consists of a well-characterized mixture of the oil-like components of CFA without the BCG; and MF59, which consists of squalene microfluidized in an oil and water emulsion. These adjuvants have some of the activities represented in CFA, promote more of a cell-mediated (TH1) response, and are safer. A combination of alum and MF59, AS04, is being used with the experimental HPV and herpes simplex virus glycoprotein D subunit vaccines developed by GlaxoSmithKline.
The visibility of an immunogen to the DC can be increased by incorporation into a larger particle, a protein aggregate, a liposome, or a virus-like particle. For example, the immunogen can be attached to the surface or within a poly(lactide-co-glycolide) (PLG) microparticle or a liposome. Vesivax developed a technique that promotes the immunogenicity of proteins by adding a hydrophobic membrane spanning portion to the protein using genetic engineering and then inserting the purified protein into liposomal particles of defined lipid composition. ID Biomedical has developed an approach to making an aerosol-administered vaccine for the annual influenza immunizations. Detergent extracts containing the influenza HA and NA glycoproteins are mixed with detergent extracts of meningococcus outer membranes, and upon removal of the detergent, these membrane proteins aggregate to form an immunogenic particle.
Both Merck and GlaxoSmithKline have produced vaccines for Hepatitis B and human papilloma viruses. The surface antigen protein (HBsAg) and the L1 major capsid protein of the human papilloma virus are produced by genetic engineering and will self-assemble into virus-like particles. The GlaxoSmithKline vaccine consists of the L1 proteins from HPV types 16 and 18, whereas the Merck vaccine adds L1 proteins form HPV types 6 and 11. Worldwide use of the established HBV and the new HPV vaccines will prevent hepatitis and genital warts but also the cancers which they induce, primary hepatocellular and cervical carcinomas, respectively.
The adjuvant and immunogen delivery approaches described above will elicit systemic IgM and IgG types of antibodies and potentially cell-mediated immune response, but special approaches must be used to elicit mucosal immunity and IgA-type antibodies. IgA antibody can be secreted from respiratory, gastrointestinal, and other mucosal membranes to protect the surface from infection. The B subunits of cholera toxin or Escherichia coli heat-labile toxin can act as adjuvants when administered with an immunogen to promote IgA production. Alternatively, attenuated Salmonella typhimurium, which has been genetically engineered to express an immunogen, can be administered orally to promote a mucosal immune response to the immunogen.
Enhanced Immunogenicity by Manipulation of Antigen Presentation
In addition to the adjuvant or particle approaches to activating the DC for antigen presentation, some new vaccines promote the immunogenicity of peptide vaccines by manipulating the mechanisms of antigen processing and presentation (see Act 1). These approaches can convert peptides, which are often too small to be processed and presented by a DC, into immunogens. Peptide vaccines have the advantage that they can be prepared synthetically in a defined, drug-like manner to contain the epitope (recognition structure) that promotes protective, not suppressive, immunity. Several of these newer approaches promote the direct association of the peptide with MHC molecules, some of which are described below.11
The approach developed by Antigen Express (Generex Biotechnology) links the peptide immunogen to a 4-amino acid peptide called the IiKey through a 5-amino acid spacer. IiKey binds to the outside of the MHC II protein on the DC surface and opens up its peptide-binding cleft (like a key) to allow the attached immunogenic peptide to fall into and displace the contents of the cleft. In this manner, antigen-presenting cells will become loaded with the vaccine peptide to initiate the response to a defined immunogen. Epimmune attaches peptides with high affinity toward MHC II molecules (PanDR sequences) to the immunogen as a way to promote its interaction with complexes of MHC II molecules. CEL-SCI has developed the Ligand Epitope Antigen Presentation System (LEAPS) approach to promote peptide vaccine immunogenicity.12,13 Attachment of a small portion of the MHC I or MHC II molecule, termed an immune cell-binding ligand, to the peptide epitope promotes its interaction with MHC I or II molecules and this promotes presentation to CD8 or CD4 T cells to activate T-cell responses. Most interestingly, appropriate choice of the immune cell-binding ligand will determine whether a TH1 or a TH2 type of response will be initiated against the immunogen. Experimental herpes simplex virus vaccines have been developed which use the CD8 directing immune cell-binding ligand and viral peptides as small as 8 amino acids. These vaccines elicited protection from lethal challenge in mouse studies.14,15
A very specialized approach to enhancing DC interaction with antigenic proteins or peptides is being developed for cancer vaccine therapies.16 DCs purified from a patient with breast cancer, melanoma, or other tumor are mixed with the relevant tumor antigen peptide (eg, HER-2/Neu, MART-1, MEL 624) to allow binding to MHC I, and possibly, MHC II molecules. The loaded DCs are then injected back into the patient to activate antitumor immune responses. Just as for a microbial vaccine, success seems to depend upon activating the DC toward a TH1 type of response with cytokines or appropriate TLR ligands. This individualized approach to immunization has seen success in different phase 1 trials.
There are many new and powerful approaches to immunization under development and approaching FDA approval. It is hoped that this review introduced some of these new developments and the biology that must be manipulated to elicit the immunization response. Unfortunately, there remain many microbial protagonists for which vaccines are not available and these villains continue to override the immune dramas of their hosts and cause serious morbidity and mortality. New approaches to vaccination are still necessary to control or treat diseases such as malaria, HIV, M. tuberculosis, and even influenza. Continued development of new vaccines by classic and innovative approaches, improvement of old vaccines, and increased distribution to the appropriate populations can lead to the elimination or control of many of these infectious diseases.