Bone marrow produces immature lymphocyte precursors of B cells, whereas T cells are produced in the thymus (McHeyzer-Williams and McHeyzer-Williams 2005). Prior to immunization, the host immune system contains millions of naïve B cells circulating through the spleen and lymph nodes via the bloodstream. Each naïve B cell expresses a transmembrane form of antibody (immunoglobulin or Ig) called the B cell receptor (BCR) that is unique to each cell (Pape et al. 2011; Taylor et al. 2012a). The presence of a functional BCR defines a B cell identity. To enable adaptive immune responses against infections, vaccination, or the variety of antigens that individuals may encounter during a lifetime, the naïve (i.e., pre-immunization or in unimmunized subjects) B cell pool requires a large BCR repertoire. In mammals, BCR diversity is established through rearrangement of immunoglobulin heavy and light chain V, D, and J gene segments, a process known as V(D)J recombination (Tonegawa 1983). In addition to V(D)J recombination, other mechanisms contribute to the highly diverse BCR repertoire, such as addition or subtraction of nucleotides at gene segment junctions. Both B and T cells are main players in humoral adaptive immune responses to infections and vaccines. Yet B cells are the only immune cell type capable of generating antibodies against antigens, including drugs of abuse.

In mammals, B cells are derived from two lineages, B-1 and B-2, and their commitment to either is based on complex processes (Scholz et al. 2014). It is not clear the extent to which B cells derive from a common precursor cell type and evolve into separate lineages driven by antigen selection, or alternatively originate from distinct precursors. The B-1 cell lineage has been well characterized in mice, whereas its relevance to the human immune system is less understood. In contrast, the B-2 lineage has been studied in depth in both mice and humans. In mice, prenatal production of the B-1 lymphocytes occurs in the liver, while the B-2 lineage emerges from the bone marrow in adults, and its is responsible for the generation of B cell pools found in the lymph nodes and spleen (Scholz et al. 2014). The characteristics of B-2 cell development and rearrangement of the immunoglobulin variable regions in mice provide a close preclinical model of human B cell and BCR processes.

After immunization with the antigen of interest, T cell-dependent B cell activation largely occurs within secondary lymphoid organs in the germinal center (GC) (McHeyzer-Williams and McHeyzer-Williams 2005). The GC-dependent T cell-dependent B cell activation is a critical aspect of the adaptive immune response to generate mature switched immunoglobulin memory B cells and antibody-secreting plasma B cells, which are B cell types essential for the generation of long-term high-affinity antibody responses against antigens and responding to booster vaccine injections (McHeyzer-Williams and McHeyzer-Williams 2005).

Shortly after immunization (i.e., 3–7 days), within secondary lymphoid organs, proliferating antigen-specific B cells move toward the B/T cell border, a specific anatomical area of B and T cell interaction, and migrate deeper into follicles to form GC (McHeyzer-Williams and McHeyzer-Williams 2005). Germinal centers are dynamic hot spots where T cell-dependent B cell activation, somatic hypermutation, and affinity maturation processes occur (Victora and Nussenzweig 2012). The formation of GC involves coordinated interaction between activated antigen-specific B and T cells in the presence of a professional antigen-presenting cell (APC) displaying the major histocompatibility complex II (MHCII) receptor. In the GC, antigen-specific B cells interact with a subset of CD4⁺ T cells, known as T follicular helper cells (T_fH), which specialize in sustaining B cell maturation (McHeyzer-Williams et al. 2012; Crotty 2011). CD4⁺ T cells promote GC B cell activation by a variety of signaling mechanisms that include either direct interaction through membrane receptors (e.g., CD40) or secretion of interleukins (e.g., IL-4) and other soluble molecules (Victora and Nussenzweig 2012).

Within the GC, antigen-specific B cells go through affinity maturation to become either switched immunoglobulin (swIg) memory B cells carrying specialized immunoglobulins (e.g., IgG, IgA, or IgE) or long-lived antibody-secreting plasma B cells (Taylor et al. 2012a). Both GC-dependent B cell maturation and T_fH activation are dependent upon a complex transcriptional program involving balanced activity of a variety of downstream signals (McHeyzer-Williams et al. 2012; Crotty 2011). These pathways include the signal transducers and activators of transcription (STAT), the transcription factors B cell 6 lymphoma protein (bcl-6) and B lymphocyte-induced maturation protein 1 (blimp-1) (McHeyzer-Williams et al. 2012; Crotty 2011). Affinity maturation is a complex process that involves somatic hypermutation, class switch recombination, and clonal selection (Victora and Nussenzweig 2012). Somatic hypermutation, which is characterized by high mutations rates in B cells, involves the key effector activation-induced deaminase (AID) which introduces mutations in the variable regions of the V(D)J gene segments, to modify the binding affinity of the BCR (Maul and Gearhart 2010). Somatic hypermutation is critical to increase the diversity of the BCR within the B cell repertoire and generate immunoglobulins with high affinity for the target antigen. Similarly, class switch recombination is instrumental in generating B cells that switched their immunoglobulin from the low-affinity IgM, or IgD, to the more specialized IgG, IgA, and IgE. After the occurrence of somatic hypermutation and class switch recombination events, B cells will compete for the antigen and other resources through clonal selection. Only the antigen-specific B cell clones that have the highest affinity for the antigen will survive (McHeyzer-Williams and McHeyzer-Williams 2005; Victora and Nussenzweig 2012; McHeyzer-Williams et al. 2012).

Addiction vaccines consist of drug-derived haptens chemically bound to larger foreign immunogenic structures that stimulate T cell-dependent B cell activation to generate antibodies against drugs of abuse (Fig. 19.1). The physical and chemical structure of the conjugate immunogen (i.e., the vaccine) is crucial for the coordinated activation of B and T cells, in the presence of APC. As shown in Fig. 19.1, a conjugate vaccine is composed of (1) a hapten derived from the structure of the targeted drug of abuse, which represents the B cell epitope that binds hapten-specific B cells that, in turn, will proliferate and mature into memory and antibody-secreting plasma B cells; (2) a larger immunogenic structure, called a carrier, which activates carrier-specific CD4⁺ helper T cells by providing T cell epitopes in the form of smaller peptide sequences presented by MHCII receptors on APC; and (3) an adjuvant that enhances immunogenicity of the conjugate through various mechanisms.

After immunization, haptens can directly bind to the BCR and activate hapten-specific B cells. However, the generation of antibodies against the drug-based hapten and free drug requires T cell-dependent B cell activation involving professional APC that display surface MHCII receptors, including macrophages, dendritic cells, and B cells. After immunization, macrophages and dendritic cells uptake the conjugate in a nonselective manner or carry the whole immunogen to the draining lymph node for processing. Alternatively, the antigen passively drains to the lymph nodes through the lymphatic system. After internalization, APC digest the carrier protein to shorter peptides, which are displayed on their MHCII receptors. Peptide presentation on MHCII receptors is necessary for engagement of CD4⁺ T helper cells. MHCII presentation by either B cells or other APC will engage T cell help through recognition by T cell receptors (TCR) specific for the peptide sequence derived from the carrier protein. MHCII-dependent activation of naïve T cells specific for peptides derived from carrier proteins promotes their differentiation into mature effector T cells including antigen-specific CD4⁺ T helper (T_H) cells. CD4⁺ T_H cells can be further subdivided in T_H1 or T_H2 cells, which activate either the cellular (e.g., antigen-specific CD8⁺ T cells or NK cells) or the humoral (i.e., B cells and antibodies) branch of the adaptive immune response, respectively. This is of particular importance for understanding the mechanisms underlying vaccine efficacy since most adjuvants are characterized by their ability to activate the T_H1, T_H2, or T_H1/T_H2 (for details on adjuvants, see Chap. 16). For example, alum is a known T_H2 adjuvant while complete Freund’s adjuvant (CFA) activates a mixed T_H1/T_H2 response. Activated antigen-specific CD4⁺ T_H cells support hapten-specific B cell clonal expansion and maturation by direct interaction through membrane receptors, such as CD40:CD40L, or by soluble molecules such as interleukins (e.g., IL-4, IL-6, and IL-21) and other inducible costimulatory signals (e.g., ICOS:ICOSL). Supported by T_fH cells, activated B cells will then go through expansion, maturation, and differentiation in various B cell phenotypes including swIg memory B cells displaying high-affinity BCR or antibody-secreting B cells capable of secreting high-affinity switched immunoglobulins, such as IgG, specific for drugs of abuse. The next paragraphs will cover in detail the initiation and progression of hapten-specific B cell responses to conjugate vaccines for drug addiction.

Among the polyclonal naïve B cell population, very few B cells display a BCR with high affinity for the hapten of interest. After immunization with drug hapten-protein conjugates, successful activation and maturation of B cells that recognize the vaccine will generate high-affinity antibodies against the target drug. In secondary lymphoid organs, such as the spleen and lymph nodes, hapten-specific naïve B cells expressing surface immunoglobulins, which recognize and bind the vaccine, undergo activation and proliferation with the help of CD4⁺ T cells. Based on studies of protein-based antigens, we know that these processes generate (1) short-lived antibody-secreting B cells, (2) GC-independent differentiation in low-affinity IgM⁺ memory B cells, and (3) GC B cells (Fig. 19.2) (Taylor et al. 2012a). Germinal center B cells then mature into either long-lived antibody-secreting B cells (ASC or plasma cells) that maintain serum immunoglobulin levels or long-lived memory B cells which will switch their Ig constant region from IgM to IgG, IgA, or IgE while acquiring somatic mutations in their variable region (i.e., swIg B cells) (McHeyzer-Williams and McHeyzer-Williams 2005; Maul and Gearhart 2010; Tarlinton 2008). Long-lived antibody-secreting and memory B cells will then transit and reside to the bone marrow (i.e., homing). After subsequent boost immunizations, the IgM⁺ or swIg memory B cell subsets respond to the antigen challenge and generate more antibody-secreting B cells, which boost the amount of antigen-specific serum antibodies (Taylor et al. 2012a). Depending upon the timing of the secondary antigen challenge, memory B cells may form more IgM⁺ or swIg memory B cells and also GC B cells (Taylor et al. 2012a). These models are likely to apply to hapten-specific B cells elicited by addiction vaccine; however, their validity still remains to be tested.

In subjects immunized against infectious diseases, exposure to the infectious agent (e.g., the hepatitis B virus) will activate antigen-specific memory B cells and induce an antigen-specific antibody response that will contain the infection. In contrast, in subjects immunized with vaccines for drug addiction, subsequent exposure to drugs of abuse, lacking T cell epitopes, is not sufficient to boost memory B cells and induce differentiation in antibody-secreting B cells. In fact, to maintain drug-specific antibody responses is necessary to administer boost immunizations with hapten-carrier conjugate immunogens. Current animal and human studies of addiction vaccines have employed immunization protocols that involved multiple repeated immunizations to rapidly achieve the required high antibody titers required to counteract plasma concentrations of drugs. Clinical trials of vaccines for nicotine and cocaine involved immunization regimens of 3–5 monthly doses followed by booster injections every 2 or 6 months to preserve serum anti-drug antibody levels (Cornuz et al. 2008; Hatsukami et al. 2011; Martell et al. 2009). Similar immunization regimens, involving repeated immunizations every few weeks with hapten-protein conjugates, have also been used to generate antibody responses against tumor-associated carbohydrate and peptide small antigens and peptide derived from Alzheimer’s pathological proteins, which are also known as weak immunogens. The rationale for this approach has been that to curb addiction, an aggressive immunization protocol may elicit a fast and robust anti-drug antibody response that would rapidly block the reinforcing effects of drugs from the initiation of treatment.

Future evaluation of new addiction vaccines may include screening of different vaccination schedules (e.g., longer time intervals between immunizations) with the goal of achieving long-term antibody and memory responses. Future studies may also test the challenging hypothesis of generating high-affinity swIg memory B cells that would recognize unconjugated drugs of abuse, so that exposure to drug would boost a protective drug-specific antibody response. Tracking the hapten-specific B cell population will provide data to support analysis of antibody quantity and quality. For instance, B cell analysis may be performed prior to immunization when antibodies are not yet present or shortly after immunization when antibody levels are below the detection threshold of current assays. Alternatively, B cells may be measured in subjects long after the antibody response waned to determine whether vaccine formulations or immunization protocols elicited long-lived memory B cells.