Antibodies for Infectious Diseases

Antibodies in Infectious Diseases aims to inform, update, and inspire students, teachers, researchers, pharmaceutical developers, and health care professionals on the status of the development of antibody-based therapies for treating infectious diseases and the potential for these in times of growing antibiotic resistance to provide alternative treatment solutions to the currently used antibiotics and new treatments for infectious diseases where no proper treatments are available.

In the setting of infectious diseases, antibody function refers to the biological effect that an antibody has on a pathogen or its toxin. Thus, assays that measure antibody function are differentiated from those that strictly measure the ability of an antibody to bind to its cognate antigen. Examples of antibody functions include neutralization of infectivity, phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), and complement-mediated lysis of pathogens or of infected cells.

Currently, well over 1,000 antibody Fab or Fab variable (Fv) structures have been determined and deposited in the Protein Data Bank (www.rcsb.org). Many of these antibodies target proteins found on or secreted by infectious agents, such as viruses or bacteria. At first glance, one antibody Fab fragment may look just like another, but closer inspection shows that these workhorses of the adaptive immune system can capitalize on novel structural features to tailor their binding sites to accommodate targets of diverse shapes, sizes, and properties. Recent crystallographic and electron microscopy studies of human antibodies against human immunodeficiency virus type 1 (HIV-1) and influenza viruses have revealed some unusual structural features and modes of antigen recognition that enable these antibodies to effectively and broadly neutralize their rapidly evolving targets. We will focus here on immunoglobulin G (IgG) antibodies, as these are the best characterized from a structural perspective.

The complement system is an integral and evolutionarily ancient component of the innate immune system, serving as the first line of defense against common pathogens ( Fig. 1 ) ( 1 ). The prime functions of complement in innate host defense are accomplished through three effector pathways. These include lysis, inflammation, and opsonization ( Fig. 2 ). The latter is central to microbial recognition and clearance by phagocytic cells. Complement further cooperates with Toll-like receptors in response to microbial structure and infection, in which immune responses are determined through both synergistic and antagonistic manners ( 1 ). Complement is also a functional bridge between innate and adaptive immunity, orchestrating an integrated host defense response to pathogenic challenges. For instance, complement can modulate adaptive immunity by providing signals that reinforce humoral responses to antigens by priming and regulating T cells and lowering the B-cell activation threshold ( 2 , 3 ). It is also an important integral point for cross talk with other biological cascades to ensure homeostasis is maintained. One example is the interplay between the complement and coagulation cascades, whereby the complement system can amplify coagulation by enhancing local clotting and, as a result, preventing microbial spread through the systemic circulation. Likewise, the activated clotting factor XII can activate the classical complement pathway and thrombin can directly cleave the third and fifth complement components (C3 and C5, respectively) ( 4 , 5 ).

Allergic inflammation, caused by development of an allergen-induced immune response, is largely driven via immunoglobulin E (IgE)-dependent mechanisms. It manifests clinically as asthma, rhinoconjunctivitis (more commonly known as hay fever), allergic skin inflammation (the main example of which is atopic dermatitis), food allergy, urticaria, and/or anaphylaxis, with several known disease variants caused by different underlying cellular and molecular mechanisms ( 1 ). Increased levels of circulating IgE, allergen-specific IgE reactivity profiles measured with radioallergosorbent tests and positive skin prick tests for specific allergens, together with auxiliary ex vivo and in vitro mast cell and basophil activation functional readouts, support the importance of IgE antibodies in the clinical manifestation of allergies ( 2 , 3 ). Allergic inflammation can be local (that is, within the target organ), as is the case for allergic rhinoconjunctivitis and allergic asthma, or systemic, as is the case for anaphylaxis. The etiology of allergic immune responses has been shown to be influenced by several factors, including genetic susceptibility ( 4 ), route of exposure, dose of the allergen, and in some cases, structural characteristics of the allergen ( 5 ).

Bacteriophage (phage) are viruses that infect and replicate within bacterial cells. Filamentous phage particles inject single-stranded DNA into target bacterial cells for subsequent replication and assembly of new virions within the host cytoplasm. The filamentous phage species capable of infecting Escherichia coli manifest as long, thin filaments that are secreted from host bacteria without cell lysis. Due to their ease of manipulation and stability in a range of temperatures and pH, F pilus-specific filamentous phage species, including f1, fd, and M13, serve as reliable vehicles for combinatorial technologies, such as phage display ( 1 – 4 ).

Today, several methods are available to isolate human monoclonal antibodies. The first efficient method described was the panning of phage display libraries constructed from the Ig variable genes of immunized or infected individuals ( 1 ) or from random synthetic libraries ( 2 ). While this method has led to the isolation of several neutralizing antibodies against multiple pathogens, the resulting antibodies do not represent necessarily the natural antibody repertoire, since the antibody fragments are generated from the random pairing of immunoglobulin VH and VL variable regions. Thus, in the case of phage libraries, it is unlikely that a given VH/VL pair went through a selection process, including the negative selection for self-reactivity. Another significant drawback is that target antigens must be known a priori, since the selection is based on binding to a purified antigen, rather than for instance neutralization. Consequently, this system is not suitable to identify new neutralizing targets within complex pathogens. In addition, selection for high-affinity binding does not necessarily translate into higher protection if the epitope recognized is not readily available on the viral spikes. An additional problem of this approach that was frequently encountered is that the antibodies isolated in Escherichia coli or yeasts may be expressed suboptimally in mammalian cells.

Monoclonal antibodies (mAbs) have revolutionized the conduct of science since their first description in 1975 ( 1 ). The use of these specific reagents also has made possible improved clinical diagnostics in the medical arena, and many antibodies have found their way to clinical use as prophylactic or therapeutic agents. Nevertheless, the potential of mAbs derived specifically from technology based on human hybridomas remains largely unfulfilled. The principal reason for the lack of a large number of hybridoma-derived mAb therapeutics has simply been the technical difficulty in generating stable hybridomas that secrete human mAbs of high affinity and functional activity. This chapter reviews recent efforts to develop and employ novel methods for the efficient generation of human hybridomas secreting human mAbs for clinical use.

The potential uses of human pathogen-specific antibodies are enormous in terms of both diagnostics and therapeutics. Early applications used polyclonal sera for prophylaxis and therapies, but problems such as allergic reactions, cost, and difficulty in their generation have led to the use of mouse-derived monoclonal antibodies that were humanized by various methods ( 1 ). These methods involved substituting part or all of the murine antibody backbone with its human equivalent to derive chimeric or fully humanized antibodies. Less labor-intensive methods used transgenic mice harboring human immunoglobulin genes for immunization to derive human antibodies ( 2 ). While this has hastened human antibody generation, some limitations exist, such as differences in the maturation processes between the mouse B cells expressing human antibodies and human B cells secreting human antibodies. Therefore, an ideal way to produce authentic affinity-matured human antibodies is to identify and harness the specific antibody-producing human B-cell clones themselves. Conventional methods involved immortalizing antigen-specific B cells from individuals who either recovered from a disease or were vaccinated with a desired antigen to derive stable antibody-producing cells. Alternatively, more recent high-throughput methods involved rescuing the specific antibody genes from either specific plasma cells or memory B cells ( 3 ). While these methods have now become routine, they both require collecting B cells from suitable human subjects. In addition to the paucity of specific pathogen-exposed human subjects when needed and the existence of low numbers of antigen-specific cells, there are other practical and ethical considerations. One such consideration is the derivation of antibodies against dangerous pathogens such as Ebola virus. These limitations pointed out the need for a more practical experimental system that permits isolation of large quantities of antigen-specific B cells against any pathogen or antigen of interest. In this regard, newer-generation humanized mice harboring a transplanted human immune system with a capacity to yield antigen-specific B and plasma cells are expected to fill this need ( 4 , 5 , 6 ) and are discussed here.

The central role antibodies play in our immune system makes them important targets for computation-based structural modeling. Antibodies consist of a “constant” and a “variable” region ( Fig. 1 ). The constant region is virtually identical in all antibodies of the same isotype, while the variable region differs from one B-cell-derived antibody to the next. The variable region of an antibody is the “business end,” the region that recognizes its antigen via so-called complementarity-determining regions (CDRs). Their large size (∼150 kDa) and inherent variability, in particular in the CDRs, make antibodies a formidable challenge for molecular modeling. Before we begin to model antibodies, it is useful to briefly review their overall structure.

HIV continues to be a major global public health issue, with an estimated 35 million people living with the virus and more than 2 million new infections occurring yearly ( 1 ). As part of the natural immune response, antibodies (Abs) exert immune pressure on HIV and play a key role both in controlling the virus and in driving escape mutations in the viral envelope glycoproteins. Therefore, and because the elicitation of Abs is believed to be crucial for an effective vaccine against HIV, Abs targeting HIV have been the focus of intense research in the past years.

Influenza hemagglutinin (HA) is the major glycoprotein on the surface of influenza virions. It mediates receptor binding and fusion. The surface glycoprotein neuraminidase (NA) is a receptor-destroying enzyme. Even though humoral immunity to NA and other proteins and cellular immunity to several viral proteins contribute to protection against influenza infection, neutralizing antibodies directed against influenza HA are sufficient to protect against disease. The H3 HA crystal structure was solved in 1981 at 3-Å resolution ( 1 ). Since then, the crystal structures of HA molecules from H2, H5, H7, and several different H1 strains including the pandemic 1918 H1 and the pandemic 2009 H1 ( 2 ) have been determined. In brief, HA is a trimeric type I membrane glycoprotein made of three identical subunits ( Fig. 1 ). Each subunit is synthesized as an HA0 precursor and cleaved proteolytically into an HA1 subunit that composes the membrane-distal globular head and part of the membrane-proximal stem region, and an HA2 subunit that only contributes to the stem region ( Fig. 1 ).

Respiratory syncytial virus (RSV) poses a serious and significant health problem. RSV was discovered in 1956 and quickly became recognized as the leading cause of lower respiratory tract disease in infants and young children ( 1 , 2 ). Preterm infants and young children with bronchopulmonary dysplasia (BPD) or congenital heart disease (CHD) are at high risk of serious RSV infection and may require hospitalizations and stays in the pediatric intensive care unit ( 3 , 4 ). Although these high-risk groups experience an increased incidence of RSV disease, it is important to note that the majority of infants hospitalized for RSV are previously healthy, nonpremature children ( 5 ). In children less than 5 years of age, RSV infections account for 50 to 80% of winter bronchiolitis hospitalizations and 30 to 60% of pneumonia hospitalizations ( 6 , 7 ). RSV bronchiolitis is reported as the leading cause of hospitalization for infants less than 12 months of age ( 8 , 9 , 10 , 11 ). Hospitalization for RSV can reach rates of 1 to 20 per 1,000 infants less than 1 year of age in developed countries ( 12 ). Although not common, RSV can be fatal, as 140 to 500 infant deaths are attributed to RSV each year in the United States ( 13 , 14 ). In addition to infants and young children, another risk group for RSV disease is the elderly. In fact, while mortality in children due to RSV disease has decreased over the years, mortality due to RSV disease among the elderly is still a significant problem ( 15 , 16 , 17 , 18 , 19 , 20 ). Also, immunosuppressed leukemia patients or patients receiving stem cell transplant therapy experience as much as 80 to 100% mortality upon RSV infection and are therefore a high-risk group for RSV disease ( 21 ).

Human metapneumovirus (HMPV), a paramyxovirus first discovered in 2001, is a significant cause of respiratory tract disease in children and adults ( 1 ). Humoral immunity plays an important role in HMPV infection, and the study of HMPV antibodies provides important clinical information including the seroprevalence of HMPV, age of primary infection, serological cross-protection between HMPV subgroups, evaluation of vaccine immunogenicity, and strategies for prophylaxis and therapy using monoclonal antibodies (mAbs).

Antibody-dependent enhancement (ADE) is a phenomenon involving infectious IgG antibody immune complexes that mediate the worsening of diseases involving a wide spectrum of microbes and vertebrates. ADE is a new type of Gell-Coombs immunopathology: type I, IgE-mediated immediate hypersensitivity; type II, antibody-mediated acute immune complex disease; type III, IgG-mediated complement-dependent foreign antigen immune complex disease; type IV, cell-mediated immune and autoimmune diseases; and type V, IgG immune complex enhancement of microbial infection in Fc-receptor (FcR)-bearing cells. Three of these immunopathologies are mediated by IgG antibodies. Type V immunopathology differs in function from type II and III immunopathologies in that immune complexes are not directly cytotoxic but serve to increase disease severity by regulating the productivity of intracellular microbial infection. In type II immunopathologies, IgG antibodies are often directed at autoantigens and include acute rheumatic fever where microbial antigens mimic antigens in various human tissues, generating an immune response that breaks down immune tolerance. In type III immunopathologies foreign antigen-antibody complexes are often trapped in the basement membranes of endothelial linings. Examples include acute serum sickness, glomerular nephritis, and postimmunization diseases such as breakthrough measles and respiratory syncytial virus infections in vaccine recipients that result in destructive complement-fixing virus-IgG immune complexes predominantly in the lung ( 1 ).

First visualized in 1904 as large inclusions in tissue sections from luetic infants and isolated in 1957 ( 1 ), the human cytomegalovirus (CMV) is a remarkably successful pathogen. Worldwide, there is a 50 to 90% probability of infection by age 50 without any clear markers of genetic susceptibility. Primary infection results in life-long latency, requiring continuous vigilance by the host immune system and characterized by serum antibody titers and a strong cytotoxic T-cell response. While most individuals will be infected with at least one strain of CMV, infection rarely leads to disease in immunocompetent individuals. However, CMV is a primary cause of congenital neurological defects and causes disease in those with compromised immune systems, such as transplant patients, with only limited therapies available.

Rotaviruses (RV) are ubiquitous highly infectious double-stranded RNA viruses of importance in public health because of the severe acute gastroenteritis (GE) they cause in young children and many other animal species. They are very well adapted to their host, causing frequent symptomatic and asymptomatic reinfections. Antibodies are the major component of the immune system that protects infants against RV reinfection. The relationship between the virus and the B cells (Bc) that produce these antibodies is complex and incompletely understood ( 1 ). In this review, the following basic aspects of RV-specific Bc (RV-Bc) will be addressed: (i) ontogeny; (ii) use of immunoglobulin (Ig) genes; (iii) differential distribution (compartmentalization) in the intestinal and systemic immune systems; (iv) specificity of RV-Ig produced and the mechanisms by which it mediates protection; and finally, (v) practical applications for the use of RV-Ig, including RV-Ig as a prophylactic or therapeutic agent and as a correlate of protection. The immune response generated against RV vaccines has been recently reviewed ( 2 , 3 ) and will only be briefly discussed. The focus of this review is antibodies induced by natural RV infection in humans, but reference to studies of the murine and porcine animal models of RV infection will be made when necessary.

Staphylococcus aureus is a nonmotile, ubiquitous, gram-positive coccus which is a major human pathogen responsible for a wide range of infections, including skin and soft tissue infections, bacteremia, pneumonia, and several toxin-mediated diseases. Among many extracellular proteins, S. aureus strains also secrete a variety of potent toxins which include alpha hemolysin, enterotoxins, leukocidins, and exfoliative toxins, all of which are directly associated with particular disease manifestations. To date, more than 33 enterotoxin sequences have been described in various S. aureus genomes. Enterotoxins are heat stable and exert their effect on the epithelium of the intestinal tract when ingested, and thus, they are a common cause of food poisoning. Several enterotoxins are potent superantigens (SAgs) that, in a non-antigen (Ag)-dependent way, predominantly activate CD4+ T cells ( 1 ) but also activate other immune cells. The SAgs of S. aureus include toxic shock syndrome toxin 1 (TSST-1), enterotoxin serotypes A to E and I (sea, seb, sec, sed, see, and sei), and enterotoxin-like serotypes G (selG), H (selH), and J to U (selJ to selU). Of these SAgs, sea to see have the ability to induce emesis in monkeys and are thus referred to as classic enterotoxins. The remaining SAgs either have not been tested for emetic activity or lack emetic activity and are therefore referred to as enterotoxin-like proteins (selG, selH, and selJ to selU). For the most part, staphylococcal SAgs are encoded by mobile genetic elements, which include extrachromosomal plasmids as well as chromosomal prophages, transposons, and pathogenicity islands. It is noteworthy that a chromosomally carried enterotoxin-like gene (selX) was recently identified ( 2 ). The seb gene is carried on the pathogenicity island SaPI3. The genes of SAgs selG, selI, selM, selO, and selU are located in the enterotoxin gene cluster (egc) and are among the most prevalent SAgs in clinical S. aureus isolates. They are expressed by S. aureus during logarithmic growth and shut off expression once a certain bacterial density is reached. Consequently, they do not induce a humoral response in the human host. In contrast, non-egc-associated SAgs (e.g., sea, seb, sec, and tsst-1) are expressed in late-logarithmic and stationary growth, induce a specific antibody (Ab) response in the human host, and are a prominent cause of cause toxic shock ( 3 ).

Monoclonal antibodies are enjoying outstanding commercial success, with 4 of the top 10 best-selling drugs. Although most of the preclinical and clinical experiences with therapeutic antibodies have been gained from the treatment of cancer and inflammatory diseases, effective therapy is not limited to these indications. For example, palivizumab (Synagis) has been approved for the prophylactic treatment for respiratory syncytial virus (RSV) infections and remains the only therapeutic antibody for the treatment of infectious diseases to date.

New high-throughput DNA sequencing (HTS) technologies developed in the past decade have rapidly increased the scale of data collection for all aspects of human genetics ( 1 , 2 ). The complex somatic gene rearrangements of immunoglobulin (Ig) and T-cell antigen receptors (TCRs) in the adaptive immune system are particularly appropriate targets for investigation using these new technologies. The antigen specificity of adaptive human immune responses and the storage of specific immunological memory depend on the sequences of the Ig and TCR gene rearrangements expressed by B cells and T cells. Until recently, the difficulty and cost of obtaining sequence data limited the kinds of immunological research questions that could be studied. Pioneering work examining dozens to hundreds of Ig rearrangements with Sanger sequencing has revealed some overall features of the repertoires of these receptors, while physical selection and sorting of B-cell populations of interest has led to the identification of antibodies specific for a variety of infectious agents and vaccine components. However, given that a single human body contains an estimated 1011 B cells representing, at a minimum, millions of distinct clonal populations, experiments using Sanger sequencing were underpowered to evaluate the full scale of antibody repertoires. This chapter first reviews genetic features of Ig loci and the HTS technologies that have been applied to human repertoire studies, then discusses experimental design, data analysis choices in these experiments, and insights gained in immunological and infectious disease studies using these approaches.

The efficiency of the adaptive immune response and its capability of recognizing a large number of different antigens depend on the huge diversity of the antigen receptors, immunoglobulins (IG), or antibodies of the B lymphocytes and T cell receptors (TR) of the T lymphocytes. The genes that code the IG and TR are highly polymorphic and are organized in clusters in several loci (three loci for IG and four for TR in humans) located on different chromosomes (four in humans) in the genome ( 1 , 2 ). The molecular synthesis of the IG and TR chains is particularly complex and unique. It includes several mechanisms that occur at the DNA level: combinatorial rearrangements of the variable (V), diversity (D), and joining (J) genes that code the IG and TR variable domain, exonuclease trimming at the ends of the V, D, and J genes, and the random addition of nucleotides by the terminal deoxynucleotidyltransferase (TdT) that create the junction N diversity and, for IG, somatic hypermutations ( 1 , 2 ). The IG and TR repertoires show an extraordinary diversity with a potential of 1012 IG and 1012 TR per individual, and the only limiting factor is the number of genetically programmed B and T cells for an organism. Therefore, the analysis of the IG and TR genes and of their expression represents a crucial challenge for the understanding of the immune response in normal and pathological situations.

Antibodies bind antigens via noncovalent bonds, such as hydrogen bonds, ionic, hydrophobic, and Van der Waals forces, and their interactions depend strongly on the distance between two interacting molecules. While each individual bond is weak, the collective noncovalent bonds between the antibody and antigen can be strong when all the interacting molecules work together synergistically. Because there are a very large number of these interactions and the antigen and antibody are large, flexible, dynamic molecules, binding between an antibody and antigen is a very complex process. The binding interactions may also be time dependent, because formation of an antibody-antigen complex may involve a sequential series of interactions which induce conformational changes that generate some bonds while breaking others. Because of the dynamic and transient state of antibody-antigen interactions, measurements of the antibody-antigen interactions can be quite complicated and inconsistent, and the results may vary depending on the sample treatment conditions and technique utilized ( 1 ).

There is a growing need for alternatives to conventional antibiotics for the treatment of infectious diseases. The number of bacterial pathogens that are resistant even to the most powerful antibiotics is growing each year. HIV remains an incurable disease more than 30 years since its identification. Since 1979 there has been a >200% increase in the annual number of cases of invasive fungal infections in the United States. To exacerbate these problems, the number of patients who cannot fight infections because of impaired immunity is growing and includes HIV patients, patients who have been through cancer chemotherapy, and organ transplant recipients.

Production and evaluation of monoclonal antibodies (MAbs) produced in plants to combat infectious diseases (IDs) has been ongoing for almost 20 years ( 1 ). With the recent FDA approval of the first plant-derived biologic ( 2 ), development of rapid manufacturing technology ( 3 ), and the capability of producing MAbs with homogenous mammalian glycosylation ( 4 ), a wave of infectious disease (and other) MAbs that are plant-derived are expected to enter clinical trials in the next several years. This review is intended to summarize the results of research on plant-derived MAbs to infectious pathogens that have completed animal or clinical studies.

The holy grail of human immunodeficiency virus (HIV) vaccine development is an immunogen that elicits antibodies that neutralize field strains of the virus. In recent years, we have gained tremendous insights into the structure and function of the HIV envelope glycoprotein, but limited progress has been made in designing such immunogens. These sobering observations underscore the tremendous hurdles that must be overcome to develop an effective HIV vaccine ( 1 , 2 , 3 , 4 , 5 ). Foremost among these hurdles is the inability to induce antibodies that neutralize a wide array of HIV field isolates. Such antibodies are rare, and, until recently, only a handful of these antibodies had been isolated ( 6 , 7 , 8 , 9 ). Over the past few years, a much larger number of HIV antibodies have been identified that have a much broader range of neutralization and are orders of magnitude more potent than the previously identified group ( 10 , 11 , 12 , 13 ). These antibodies were isolated from the high-throughput screening of sera from HIV-1-infected individuals and categorized as “elite neutralizers” based on their neutralization breadth and potency ( 14 ). Extensive sequence analysis of these potent, broadly neutralizing antibodies revealed that high levels of somatic mutations were involved to generate the mature antibody ( 11 ). Furthermore, the maturation may have involved repeated rounds of antibody selection through HIV antigen interaction, a process that may not be possible to duplicate from a traditional HIV protein subunit or viral vector vaccine.