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In This Issue
The Convergence of Engineering and the Life Sciences
Nanotechnology An Enduring Bridge Between Engineering and Medicine
Understanding and Harnessing the Immune System for the Rational Design of Therapies and Vaccines
Systems Biology and Systems Pharmacology
Regenerative Engineering Materials, Mimicry, and Manipulations to Promote Cell and Tissue Growth
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Fall Issue of The Bridge on the Convergence of Engineering and the Life Sciences
October 1, 2013 Volume 43 Issue 3

Understanding and Harnessing the Immune System for the Rational Design of Therapies and Vaccines

Tuesday, October 1, 2013

Author: Arup K. Chakraborty and Mark M. Davis

The immune system provides protection from infectious pathogens, and its aberrant regulation is implicated in several diseases. Vaccination, one of the greatest triumphs of modern medicine, is evidence that the immune system can be harnessed and manipulated to improve the human condition. But doing this in a systematic way to address major health challenges requires a comprehensive understanding of human immune responses of both healthy and ill persons to evolving pathogens and diseases. We assert that achieving this goal will require creative approaches at the convergence of the life and physical sciences, engineering, and clinical practice and research.

The Immune System as Sentinel of Human Health

Humans are constantly exposed to infectious pathogens and cancer cells routinely emerge. Yet most people are rarely sick, thanks to the protection provided by the immune system.

But the human immune system is not infallible. Some pathogens can defeat it, resulting in pandemics. For example, the human immunodeficiency virus (HIV), the causative agent of AIDS, has not been cleared by any known person’s immune system and over 30 million people have succumbed to it. The immune system can also mistakenly attack the host body’s own tissues or organs, leading to autoimmune diseases such as lupus and multiple sclerosis, which afflict increasing numbers of people in developed nations.

Figure 1

The desire to combat infectious and autoimmune diseases and the corresponding drive to decipher the complex basic science puzzles presented by the immune system have led to a great deal of experimental research aimed at understanding how the immune system functions. Some spectacular discoveries have been made, resulting in a basic understanding of the key parts of the immune system (sketched in simplified form in Figure 1).

The Innate and Adaptive Immune Systems

The human immune system can roughly be partitioned into two inextricably linked parts called the innate and adaptive immune systems. The innate immune system is the first line of defense; its components eliminate pathogens in a crudely specific manner by recognizing common molecular signatures on the surface of many pathogens, which are distinct from host molecules. Although this system is efficient, many bacteria and viruses have evolved strategies to evade the innate immune response, enabling them to establish infection.

Now the adaptive immune system gets in the game. Consider what happens upon infection with a virus: the virus hijacks the transcriptional machinery of the infected host cell, enabling synthesis of its own proteins and assembly of new virus particles.

B cells play an important role in adaptive immune responses. Each B cell has its own distinct surface receptor, the B cell receptor (BCR). If a BCR can bind sufficiently strongly to spikes on the surface of a virus, a complex set of intracellular biochemical reactions may trigger genes that “activate” the B cell. Activated B cells proliferate and form intricate structures called germinal centers (GCs), where evolution occurs in short order. Mutations are introduced into the BCR genes of the proliferating B cells. The mutated cells compete to bind to virus particles (on the surface of cells in the GC) and higher-affinity variants (i.e., those that bind more strongly) are selected. Many rounds of this process, called affinity maturation, ensue. Thus, B cells that bind strongly to this virus develop. Soluble forms of their BCR, called antibodies (Abs), are then secreted. Abs bind to the spikes on the virus and eliminate it using various components of the innate immune system.

Today, protein engineers mimic affinity maturation in vitro to engineer Abs and enzymes, which then perform functions like catalysis and therapy in an optimal fashion. Antibodies are commonly used as therapies for diverse diseases like cancers, with sales of over $44 billion in 2011.

Antibodies can only attack viruses that are in the blood or extracellular spaces, not infected cells and the pathogens they harbor. Some viral proteins are processed by an intricate mechanism inside infected cells and cut up into short peptide fragments, which are displayed on the surface of infected cells, bound to large cell surface proteins called major histocompatibility complex (MHC) molecules. Cells of the innate immune system (e.g., dendritic cells) engulf pathogens and display peptide-MHC (pMHC) complexes, molecular signatures of infection that are recognized by T cells, which are also key players in adaptive immunity. It is difficult for pathogens to hide important parts of their anatomy from T cells, since their molecules are chopped up into such small pieces.

As with the B cells, humans have many clones of T cells, each with a specific receptor on its surface called a T cell receptor (TCR). If a TCR binds sufficiently strongly to pMHC presented by a particular virus, biochemical reactions similar to those in B cells occur, resulting in T cell activation. The activated T cells proliferate but, unlike the B cells, do not mutate their TCR.

There are two major types of T cells. One is the cytotoxic T lymphocyte (CTL); if an activated CTL encounters an infected cell displaying the same peptide that activated its TCR, it secretes products that kill the infected cell. The other kind of T cell, the T helper cell, plays a key role in the affinity maturation of B cells and the activation of CTLs.

When an infection is cleared by the immune system, most of the B and T cells that proliferated in response to the particular virus die, but a few remain as long-lived “memory” cells that can mount robust and rapid responses upon reinfection with the same pathogen. This memory is the basis of vaccination: a vaccine induces pathogen-specific immune responses, which then lie “ready and waiting” to prevent or abort infection.

While the network of interacting cells shown in Figure 1 is complex, the immune system is actually considerably more complicated, with many more cell types, diverse secreted products of immune cells influencing the function of others, and intricate processes that occur during the development of B and T cells, which train the immune repertoire to not be rampantly autoreactive.

Progress, Goals, and Challenges in Understanding the Immune System

Despite major advances over the past 50 years in understanding of adaptive immunity (summarized in Figure 1), the principles that determine the emergence of a systemic immune or autoimmune response remain elusive.

A major barrier to the quest for mechanistic principles is that the processes underlying a systemic immune response involve cooperative dynamic events, with many participating components that must act in concert. Moreover, these collective processes span a spectrum of scales—a myriad of proteins inside a single cell must act in precise collective ways to initiate gene transcription events that result in activation, many types of cells must act cooperatively in tissues, and of course there is the scale of the entire organism. This hierarchy of cooperative processes, with feedback between the scales, often makes it difficult to intuit underlying mechanisms from a few experimental observations. Further confounding intuition is that many of these processes are inherently stochastic in character.

New technologies and approaches, with immunologists working closely with engineers, physicists, and computational scientists, are beginning to describe this complex system using cell lines and mice as model animals. Due to space constraints, we mention only a few examples where such efforts have recently been fruitful in basic immunology.

Video microscopy, two-photon microscopy, 3D electron microscopy (EM) tomography, photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) have yielded vivid images of immunological processes and new mechanistic understanding (Grakoui et al. 1999; Halin et al. 2005; Lillemeier et al. 2010). Methods developed by engineers and physicists are beginning to provide detailed information about how cell-cell interactions occur across 2-dimensional interfaces to initiate key biochemical reactions (Huang et al. 2010; Huppa et al. 2010). Methods to assay single cells are revealing the heterogeneity of responses generated with the same stimulus (Han et al. 2012). Computational methods have been coupled with experiments to shed light on the role of mechanistic features in biochemical signaling reactions that mediate important phenomena in immune cells (Altan-Bonnet and Germain 2005; Das et al. 2009), the development of immune repertoires (Košmrlj et al. 2010), and host-pathogen dynamics (Ho et al. 1995). These advances are very important, but it is not yet possible to achieve clinically important goals such as

  1. the rational design of vaccines and therapies for autoimmune diseases and cancer, and
  2. the ability to predict disease states and guide clinical decisions based on monitoring the immune response.

The study of small animal models and cell lines has revealed some of the mechanisms and parts involved in the function of key modules of the immune system (and these often carry over to humans). However, achieving the goals above will require determining how the systemic immune response develops and how it interacts with new and evolving pathogens and disease states. This, in turn, necessitates an understanding both of the complex processes that underlie a systemic immune response in humans and of the ways pathogens and cancers evolve to circumvent natural or vaccine-/therapy-induced responses.

Importantly, humans exhibit considerable genetic diversity (their “genotype”) in their immune system as well as different histories of infections and resulting states of immunological memory. In contrast, inbred mice—the major experimental model for immunology—are genetically homogeneous and kept in ultraclean housing. How genetic factors and history of infections influence systemic immune responses to new and evolving pathogens and disease states is poorly understood and presents unique challenges in experimental design, technology development, and computational studies.

The Triumph and Challenges of Vaccines

Following the seminal work by Jenner, Pasteur, and others, vaccination eradicated smallpox in 1977, thus ending a scourge that had afflicted humans since ancient times. It is close to doing the same for polio, and is substantially reducing mortality and severe illness, especially in young children and the elderly. A vaccine-induced immune response protects the host against infection by a specific pathogen (e.g., influenza, yellow fever, measles, smallpox) so effectively that often a person does not even notice reinfection.

But vaccine development today uses roughly the same paradigm and methodology developed by Pasteur in the 1870s, which is to grow a particular pathogen in culture such that it loses its virulence and inject humans with this product to induce memory immune responses against the pathogen. This was developed purely empirically and without any knowledge of the immune system. In the early 1900s, a second innovation, an adjuvant, which generally stimulates components of the innate immune system, was added to some vaccines. How adjuvants work and how they should be chosen remains poorly understood, resulting in many failures.

Thus, although vaccination has been a great success, important challenges remain. Broadly effective vaccines do not exist for serious diseases such as HIV, hepatitis C virus (HCV), malaria, and tuberculosis (TB). Even for influenza, vaccines that can target all strains are not available, which means that every year a new vaccine is formulated and manufactured based on educated guesses about what strains will be prevalent. The guesses are not always correct.

Blueprint for Rational Design of a New Generation of Vaccines

Systematic design—like the design of a bridge or an aircraft—of successful vaccines and immunotherapies against diverse pathogens and diseases is not possible in the absence of adequate information about how systemic human immune (or autoimmune) responses develop and interact with evolving pathogens. A comprehensive blueprint showing how the immune system functions would enable the determination of design principles that could be used to engineer such therapies and vaccines. Creation of this knowledge, and the optimal deployment of resulting strategies, will require intensive research that brings together approaches from science, engineering, and clinical medicine.

We imagine a future where vaccine development will become a systematic scientific and engineering discipline that harnesses deep knowledge of the immune system. Achieving this goal will require major advances in a spectrum of activities, which we call the three Ds of vaccine development: discovery, design, and delivery.

Discovery

Why does standard vaccination not work for some pathogens? HIV, the causative agent of AIDS, vividly illustrates the answer. HIV is a rapidly mutating virus. The diversity of circulating strains of influenza in the entire world in 1996 is comparable to the diversity of HIV strains in a single chronically infected patient, and is completely dwarfed by the diversity of strains in a single region of Africa. This extremely high mutability presents a severe challenge for vaccination because it enables HIV to evade vaccine-induced immune responses specific for a particular strain. Moreover, HIV targets and eventually kills helper T cells, important orchestrators of the immune response.

Other diseases also resist vaccination. HCV has a very high mutation rate and some less defined ability to disable critical immune functions. Malaria and sleeping sickness exhibit a variant of the mutation strategy, expressing interchangeable surface proteins. TB also mutates fairly rapidly, and encapsulates itself in cysts that are resistant to immune attack. So vaccination efforts are challenged by a category of infectious diseases that are unusually mutable and have developed ways to disable or evade the immune system.

The rational design of vaccines against such pathogens requires the development of systematic ways to define the mutational vulnerabilities of pathogens and the diversity of human immune responses (which depend on people’s genotype). Vaccine design can be guided by learning how effective immune responses develop upon vaccination and then focusing the responses to target the pathogen’s mutational vulnerabilities in diverse people. The knowledge required to achieve this goal is currently missing, but recent studies suggest that it can be obtained through multidisciplinary research approaches, as described below.

Discovery of Mutational Vulnerabilities of Pathogens

For highly mutable pathogens (e.g., viruses), the goal is for immune responses to target residues (or regions) of their proteins, or proteome, so that mutations result in an unviable pathogen (the pathogen is then cornered between being killed by the immune response or making mutations that cripple it). It is difficult to determine mutational vulnerabilities by simply searching for residues in viral proteins that exhibit a low frequency of mutation, because the fitness costs incurred by making a primary mutation that evades the immune response can be compensated by other mutations due to synergistic effects.

What is required is knowledge of the replicative fitness of mutant strains bearing multiple mutations. This information can be represented as a fitness “landscape” (Figure 2A), which defines mutational vulnerabilities of pathogens and identifies potent immune responses against them. Such responses should target multiple residues in a virus’s proteins, where strains with mutations at these residues correspond to “valleys” in the fitness landscape due to the deleterious effects of simultaneous mutations on viability. Thus, the immune response would push the virus from one of the fitness “hills” into an adjacent fitness valley. Knowledge of the fitness landscape also enables the design of immune responses that can block the “mountain passes” that pathogens must traverse by mutation in going from one hill (when it is subject to immune attack) to an adjacent one.

One way to determine the viral fitness landscape is to systematically create mutant strains using modern molecular biology techniques and measure the viability of each strain in vitro. Regression algorithms can then be used to fit these experimental data. But it is difficult to imagine how the sequence space of viruses could be comprehensively sampled in this way. For example, consider a single viral protein made up of 500 residues; even if each residue could mutate to just one other amino acid, the number of possible mutant strains would equal 2500 (~10150), an astronomical number!

Information about how mutations affect the prevalence of circulating viruses is contained in the sequences of viral proteins derived from viruses extracted from diverse patients. But the information is influenced by statistical noise, the patients’ immune responses, and virus phylogeny. Recently, it has been suggested that for several HIV proteins these effects can be deconvoluted to translate sequence data into fitness landscapes by combining computational methods rooted in statistical mechanics (e.g., spin glass models and random matrix theory) with sequence data, in vitro experiments, and data on infected patients (Dahirel et al. 2011; Ferguson et al. 2013). The results are very encouraging (e.g., Figure 2B). The cost of sequencing and computation are going down dramatically, and thus this approach offers the possibility of defining fitness landscapes of highly mutable pathogens (and cancers) in order to facilitate rational design of vaccines and therapies.

Figure 2

But many challenges remain. Chief among them are the needs for

  • high-throughput sequencing of pathogen samples extracted from patients of diverse genotypes;
  • improved computational methods that combine machine learning algorithms and statistical mechanics to translate sequence data to fitness landscapes; and
  • development and maintenance of patient cohorts at various stages of disease so pathogen evolution and host-pathogen dynamics can be followed.

Discovery of Effective Immune Responses in Healthy, Vaccinated, and Diseased Humans

The mutational vulnerabilities of pathogens can be exploited only if immune responses in humans can be directed against them by vaccination. This will require major cross-disciplinary efforts aimed at defining how systemic immune responses develop and determining which regions of a pathogen’s proteome can be specifically targeted by human Abs and T cells (i.e., their specificities).

One technique involves monitoring the human immune response upon vaccination to discover how potent systemic immune responses emerge. This is a “systems biology” approach, as it measures a wide range of parameters in vaccinated human subjects to gauge how the different components of the system react and which correlate with each other positively or negatively (Davis 2008).

Pulendran, Sékaly, and colleagues pioneered this area in their separate analyses of the yellow fever vaccine, one of the most successful vaccines ever developed. These studies identified a number of early events in innate immune pathways and T cell responses that correlated with better outcomes (Gaucher et al. 2008; Querec et al. 2009). A similar study for influenza also found that strong innate immune activation correlates with a robust Ab response (Nakaya et al. 2011).

In another “first of its kind” study, many immune parameters were measured in many humans prior to vaccination, and statistical methods and gene modules were used to predict which persons would mount effective or ineffective Ab responses—with slightly better than 80 percent accuracy (Furman et al. 2013). These studies have involved close collaborations among immunologists, clinicians, technologists, biostatisticians, and bioinformaticians, and appear highly promising in characterizing how vaccination may induce effective responses.

Such research must be coupled with efforts to define possible Ab and T cell repertoires in humans with different genotypes because this is the menu of specificities available for attacking mutational vulnerabilities of pathogens. The variety of circulating Abs in a person is determined by many factors: the kinds of BCRs originally generated, the history of infections that induced affinity maturation, and the types of mutated BCR proteins generated by affinity maturation that can fold into functional receptors. Knowledge of how these processes shape the Ab repertoire, and how they can be manipulated by vaccination protocols to induce new potent Abs, is grossly incomplete. High-throughput sequencing of Ab repertoires of healthy, diseased, vaccinated, and aging humans of diverse genotypes is necessary.

Quake and coworkers have developed high-throughput sequencers that enabled them to sequence every antibody in a number of zebrafish, and the data were analyzed using sophisticated theoretical methods (Jiang et al. 2011; Mora et al. 2010; Weinstein et al. 2009; also see Quake 2013, in this issue). Their studies suggest that correlations between amino acid substitutions at different residue positions greatly limit sequence diversity, and that the repertoire can be clustered into groups of related Abs that are often shared between fishes. Efforts are under way to obtain this type of information for humans in various states of health and disease (Jiang et al. 2013).

Similar studies for T cells are also critically important, especially because T cell repertoires are much more likely to be dependent on genotypes, as human MHC genes are highly polymorphic. A recent methodological breakthrough may offer a solution to high-throughput sequencing of MHC genes (Wang et al. 2012). A recent study shows that humans with certain MHC genotypes are more likely to have T cell repertoires that help protect them from mutable pathogens but also make them more prone to autoimmunity (Košmrlj et al. 2010). Another study in humans indicates that autoreactive T cells and memory cells specific for pathogens that the individual has never encountered are more frequent than previously thought (Su et al. 2013).

These studies are just the tip of the iceberg. Many cross-disciplinary efforts are needed, particularly in the following areas:

  • New instrumentation. Many of the technologies used to monitor human clinical samples are decades old; for example, the cost of sequencing has dropped precipitously, but 1960s technology is still widely used to isolate white blood cells.
  • Novel software. The immune system involves many cells, molecules, and mechanisms, with hundreds or thousands of moving parts. Software (using machine learning algorithms) is needed to learn the phenomenological behavior revealed by the data.
  • New research approaches. Determination of the correlates of T cell protection faces many difficult technical challenges with currently known approaches.
  • High-throughput, single-cell sequencing. This will define the range of Ab and T cell repertoires in humans.

Design

Knowledge of the range of possible immune responses and pathogenic fitness landscapes, combined with monitoring of systemic immune responses in humans, will begin to provide a detailed picture of host-pathogen riposte and how one might manipulate it. Such manipulation will entail matching up vulnerable regions of the pathogen with the range of immune responses possible in humans of diverse genotypes. The active component of the vaccine, the immunogen, can then be designed to induce responses that target the vulnerable regions. But important challenges need to be addressed to make this a reality:

  • The immunogen must be designed such that affinity maturation produces Abs that target different strains of the same mutable pathogen. Such a design will likely depend on collaboration among protein engineers, theorists, immunologists, and clinicians.
  • If whole proteins of the pathogen are used as the immunogen, in people with many genotypes (MHCs) it may be easier to display peptides from regions of the proteome where mutations do not incur a large fitness cost. Thus, the pathogen will evade the vaccine-induced T cell response. So only short regions of the virus containing mutationally vulnerable regions that can also be exhibited by diverse individuals in a population need to be used as an immunogen. Knowledge of how the genotype affects the display of peptides is incomplete.

Delivery

Novel immunogens, such as those composed of short sections of pathogenic proteins, often do not result in strong responses. But a number of sophisticated methods are emerging to address this challenge.

Chemistry and materials science–based approaches to immunomodulation have the potential to greatly enhance the safety and efficacy of immunotherapies and vaccines (Kwong et al. 2013; Stephan et al. 2010). One such method is nanoparticle delivery of immunomodulators, which can block systemic toxicity and coordinate the action of potent immunomodulatory drugs and biologics. Vaccines based on synthetic nanoparticles have been shown to enhance innate and adaptive immune responses, and synthetic polymer scaffolds induce coordinated steps in the recruitment, activation, and antigen loading of dendritic cells to prime unprecedented antitumor immunity in small animal models. These engineered changes in vaccine delivery have been shown to result in responses that are unachievable with existing adjuvants. However, these approaches have yet to be translated for human use.

Finally, we note that, because of the challenge of manufacturing GMP-grade biologic materials, it takes roughly two years for a vaccine candidate that has passed all preclinical tests to proceed to the start of a human trial. Engineered synthetic carriers of vaccines such as nanoparticles may shorten this time—and the associated cost—dramatically.

Leveraging Vaccine Development Efforts to Monitor Human Health and Develop Therapies

Work done to make vaccine development systematic could also be used to develop metrics of immune health, which currently do not exist. The immune system is relevant not just to infectious diseases and autoimmunity but also cardiovascular and neurological diseases, and other areas are recognized almost every day. Thus there is a critical need for a simple test of immune functionality that can predict disease states and indicate interventions before a medical condition becomes serious. Knowledge gained from monitoring immune responses to vaccination could be leveraged toward this end. One example is juvenile diabetes, whose incidence is rapidly rising; while it can be controlled by insulin injection, the disease has serious long-term health problems for many. There is already evidence that circulating autoantibodies predict disease years before insulin-producing cells are destroyed, but knowledge is limited. This is a situation where a comprehensive systems approach to sift through every aspect of the immune system would have great clinical impact (Davis 2008).

Progress through Convergence

The research efforts outlined above require a convergence of disciplines. New experimental probes and reagents are required, which engineers, physicists, and chemists can develop. Computational efforts, particularly those rooted in statistical physics, information theory, and machine learning, are needed to make sense of large datasets and unearth the mechanisms of the stochastic, cooperative, and multiscale processes that underlie these observations. New computational and experimental tools must be deployed in concert with a deep knowledge of basic immunology. Finally, and importantly, a new convergence-based paradigm (Figure 3) can become a reality only if there is a tight coupling of research efforts with a knowledge of pathogenesis and therapeutic possibilities possessed by clinicians and clinician-scientists. No single laboratory in academia or industry can master this complex array of skills at the frontier of human knowledge to carry out such a paradigm of research and development. Collaborative efforts are needed among engineers, scientists, and clinicians in order to progress toward harnessing the immune system to combat pathogens and diseases. Only then can the systematic design of immunomodulating vaccines and therapies become like the design of aircraft and microelectronics today. In addition to vaccines and therapeutics, new commercial products will make immune system monitoring devices routinely available in the clinic. The established “silos” that currently define scientific endeavor, funding models, and the reward system are hard to break down, but success depends on integrated and collaborative interdisciplinary efforts.

Figure 3

If research succeeds in achieving the goals described, human health care will be well advanced.

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About the Author:Arup K. Chakraborty (NAE) is the Robert T. Haslam Professor of Chemical Engineering in the Departments of Chemical Engineering, Physics, Chemistry, and Biological Engineering, and director of the Institute for Medical Engineering and Science, Massachusetts Institute of Technology; and leader of the computational biology program at the Ragon Institute of MGH, MIT, and Harvard in Boston. Mark M. Davis (NAS/IOM) is the Avery Family Professor of Immunology in the Department of Microbiology and Immunology at Stanford University School of Medicine, an investigator in the Howard Hughes Medical Institute, and director of the Institute of Immunity, Transplantation, and Infection at Stanford University.