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Memory is the hallmark of the adaptive immune system, and the observation that infectious diseases often lead to lifelong immunity in individuals who survive a first infection became the genesis for the development of vaccines. Immunization, which is the iatrogenic engineering of a protective memory immune response to a pathogen, became a standard part of medical care in the twentieth century, and has had an almost incalculable positive effect on human health and wellness. Vaccines to many, but by no means all, infectious diseases have been developed and are in common use. Smallpox vaccine, arguably the most effective vaccine in human history, was (and still is) delivered through disrupted epidermis in a process called scarification. Virtually all vaccines today are delivered by means of a hypodermic needle and syringe into muscle, in a process that bypasses the epidermis and dermis and their attendant innate and adaptive immune attributes. This article discusses vaccines in the context of the newly appreciated paradigm of tissue-resident memory T cells, and specifically discusses the role of these cells in skin and other epithelial interfaces with the environment in the maintenance of protective immunity.
Innovation is defined as “the introduction of something new”. Investigative dermatology, similar to all other biomedical scientific disciplines, depends existentially on a steady influx of new techniques, novel approaches, new theories, and paradigm-shifting ideas to move forward as a discipline. Without question, our understanding of skin biology at present is significantly different from what it was in 1937, the year the Journal of Investigative Dermatology was first published. This is perhaps most strikingly evident in our current appreciation of the skin as a complex immunological organ. This article will highlight some of the translational innovations that have occurred during those years, particularly those relevant to vaccine biology. Because a comprehensive review cannot be accommodated in a short article, this manuscript reflects the authors’ personalized views.
As America was beginning to emerge from a long economic depression in the late 1930s, and was increasingly threatened by international pressures that would soon lead to her entry into World War II, translational biomedical research was moving forward at a remarkable clip. The level of innovation in biomedical science relevant to human disease that would transpire in the twentieth century was nothing short of astonishing. Hardly a family in America was not touched by deadly childhood infectious diseases in the first few decades of the twentieth century. Nevertheless, by mid-century, antibiotics and vaccines had reduced morbidity and mortality of these diseases in children by, quite literally, orders of magnitude (Bud, 2007; Ellis, 2009). Infectious diseases, which through millennia had profoundly shaped the course of human history, were beginning to yield to innovations emerging from translational discoveries in the early twentieth century. Increasingly, bacterial diseases were beginning to respond to recently discovered antibiotics. Pneumococcal bacterial pneumonia, streptococcal cellulitis and pharyngitis, and bacterial otitis media had become readily treatable diseases (Clardy et al., 2009). By the 1950s, syphilis, an international scourge that had helped to define the specialty of dermatology (academic departments were often known as departments of Dermatology and Syphilology), could for the first time be definitively treated with penicillin (Douglas, 2009). Even tuberculosis was beginning to yield to combination chemotherapeutic approaches, although this ancient enemy remains a global challenge (Mitchison, 2005). Although certain bacteria responsible for diseases would evolve over the next several decades to develop antibiotic resistance, the spectrum and impact of infectious diseases on human health was forever altered by these new developments. However, many diseases caused by viruses, once acquired, remained impossible to treat definitively, and extensive research focused squarely on prevention of these diseases rather than their treatment, principally through innovations in vaccination.
In 1936, 1 year before the Society for Investigative Dermatology and its journal were founded, Max Thieler developed a vaccine for yellow fever, an accomplishment for which he won the Nobel prize in 1951 (Norrby, 2007). Although primarily a disease of the tropics, deadly yellow fever epidemics had hit Philadelphia, Baltimore, and New Orleans in previous decades, and the prospect of its eradication in America was enthusiastically welcomed. The overarching rationale behind vaccination, however, had developed centuries earlier from ancient clinical observations, and since has been confirmed experimentally and popularized by giants of translational medicine such as Edward Jenner and Louis Pasteur. The principle behind vaccination stemmed from observations that complete and durable immunity to certain infectious diseases was conferred to individuals by prior infection and clinical disease. Thus, the goal of vaccination was originally to iatrogenically create a subclinical infection, ideally with an attenuated pathogen that did not cause disease, thus provoking a similar protective and durable immune response without causing a life- or health-threatening disease (Hilleman, 2000).
However, different strategies were required for different types of infection, based on the mechanism. When the morbidity and mortality of an infectious disease is caused by toxins, as is the case with tetanus, diphtheria, and pertussis (whooping cough), generating a neutralizing antibody response to the toxin is sufficient, although multiple booster injections are often needed. When the disease is caused by a viral infection of a peripheral tissue, however, the most effective vaccines have typically involved an intact virus, either attenuated (live but less virulent) and thus capable of infecting host cells or rendered noninfectious by thermal or chemical treatment (Hilleman, 2000). Measles, mumps, and rubella vaccines are attenuated viruses, as are rotavirus, oral polio, yellow fever, some influenza vaccines, and varicella zoster vaccines. Relatively fewer vaccines, in contrast, involve an inactivated virus that cannot infect cells; these include vaccines for hepatitis A and B, seasonal influenza, and human papilloma virus vaccines (e.g., Gardisil; Liu, 1998).
In fact, vaccines date back to the tenth century, and thus it cannot be claimed that vaccination is a twenty-first (or even twentieth) century innovation. Certainly, vaccines designed to lower the risk of cervical cancer from human papilloma virus infection (i.e., papilloma virus 6, 11, 16, 18; Gardasil) are novel, and intranasal influenza virus vaccine for influenza (i.e., Flumist) are relatively recent developments. However, there are still a number of diseases for which an effective vaccine is elusive. HIV-1 is the best example, a virus whose strategies to elude the immune system have thwarted some of the best minds in vaccine biology (Bojak et al., 2002). In addition, Dengue fever, Ebola, and other hemorrhagic fevers still elude reliable vaccines. Nonviral diseases such as malaria, tuberculosis, and third-world scourges such as tyrpanosomiasis and leishmaniasis continue to take enormous human tolls without a candidate vaccine in sight. Therefore, why and how can the immune system be apparently so readily manipulated to protect humans against some infectious diseases, but not others? To begin to answer these questions, it is useful to turn to a new set of innovations, not in vaccines, but in the science of translational immunology.
Virtually all vaccines developed in the past century were tested by their capacity to generate an antibody response (Kayhty, 1998). For pathogens that have a requisite blood-borne phase, this is critically important. For example, tetanus toxin must enter extracellular space and blood from infected skin to mediate its neurological effects, and thus circulating antibodies generated by prior vaccination can be completely protective. Similarly, such circulating preformed antibodies can protect against viremia or bacteremia, and thus an antibody response is clearly important for many diseases. However, for viral diseases such as HIV, although antibodies may limit viremia, they cannot prevent infection of dendritic cells and T cells at the mucosal interface (Keating and Noble, 2003). Similarly, pathogens that have an intercellular mode of survival, such as mycobacterium tuberculosis, and the parasites that cause malaria and leishmaniasis, cannot be addressed or controlled conclusively by preformed antibodies. Clearly, although antibodies have an important place in vaccine efficacy, the focused attention on humoral response to the exclusion of cell-mediated tissue-specific responses may be the “Achilles heel” of vaccine science.
Investigative dermatology has had a central role in clarifying the complexity and diversity of the immune system in peripheral epithelial tissues. For many years, the study of immunity in skin and gut was thought to be specialized and esoteric, and this science was often relegated to “Regional Immunity” sessions at national immunology meetings. The prevailing principle was that fundamental immunology is really the cell biology of lymphocytes, dendritic cells, and other cellular elements of the immune system, which could be studied in tissue culture and were not fundamentally dependent on any non-lymphoid anatomic context. In retrospect, this view did not accommodate the reality that the immune system evolved to protect the host against microbial infection, and that challenge by such pathogens invariably occurs at interfaces of the organism and the environment. These epithelial barriers to the environment—skin, gut, lung, reproductive mucosa—were precisely the sites where the host would first encounter microbial challenges (Kupper and Fuhlbrigge, 2004). Infectious challenges by pathogens, which cause morbidity, had to be distinguished by the immune system from colonization by commensal organisms, which do not. This has been most recently highlighted by seminal work on the diversity of the human gut and skin microbiomes (Grice et al., 2009; Tschop et al., 2009), indicating a level of complexity in commensal organisms that live harmoniously upon us or within us, which had not been previously appreciated.
Through the highly innovative work of investigative dermatologists, skin was characterized as an organ that contained specialized sets of dendritic cells (reviewed in Larregina and Falo, 2005; Zaba et al., 2009; Kaplan, 2010), specialized cells capable of activating T cells, the diversity of which continues to be revealed. More recently, it became clear that “memory” as a descriptor for T cells could refer not only to antigenic memory but also to anatomic memory—antigens that were first encountered through skin, and therefore found their way to skin draining lymph nodes, provoked an expansion of memory T cells in the blood that expressed unique cell surface markers that not only identified them as skin-homing T cells but also actually helped them extravasate into skin through interactions with skin microvasculature (Picker et al., 1993; Rossiter et al., 1994; Butcher et al., 1999; Kupper and Fuhlbrigge, 2004). Analogous populations of circulating T cells that homed to the gastrointestinal tract were also characterized (Butcher et al., 1999), and populations that homed to the lung were identified as well (Purwar et al., 2011).
Yet another level of innovation would follow. Most recently, investigative dermatologists again made an important and unexpected discovery: many if not most skin-homing T cells were, in effect, “skin-resident” T cells, and that skin contained far more skin-homing memory T cells at steady state than could be found in blood (Clark et al., 2006a, b). Similar observations were being made for gut and lung, and resident populations of T cells were even observed in the central nervous system after infection (Masopust et al., 2010; Wakim et al., 2010; Purwar et al., 2011). Many of these studies have been cross-validated in mouse models and human tissue (Zhu et al., 2007; Gebhardt et al., 2009; Liu et al., 2010; Masopust et al., 2010). These “tissue-resident” T cells, then, were enriched in specificity for pathogens previously encountered through their respective epithelial tissues (Clark, 2010; Bevan, 2011).
At steady state, both these T cells and the tissue-resident dendritic cells capable of presenting antigen to them are in a resting state. It is here that innate immunity has an important role: cytokines produced by keratinocytes (Gutowska-Owsiak and Ogg, 2012) and other non-bone marrow–derived resident cells have a vital role in the generation of immune responses. In the face of pathogenic challenge, dendritic cells activated by the innate immune system (e.g., Toll-like receptors, epithelial cytokines, other endogenous adjuvants; Modlin, 2011) can present pathogen/antigen to tissue-resident T cells, thus producing a rapid T-cell effector response that does not require memory T-cell extravasation from peripheral blood (Sheridan and Lefrancois, 2011). These observations have added a completely new level of importance to “regional immunology”. Tip O’Neill was famous for quipping that “all politics is local”. In the same sense, it appears that with regard to host defense against infectious diseases, all immunology is “regional”. Anatomical context of immune responses is now considered central to understanding the pathophysiology of infectious and autoimmune disease.
If tissue-resident memory T cells indeed have an important role in host defense in epithelial tissues, then generating these cells must be a goal of vaccination. Given what we know about the anatomic imprinting of T cells, it stands to reason that how and where a vaccine is delivered makes a difference. Although injection into muscle or subcutaneous fat may be an acceptable way to make a robust circulating antibody response, it is not a logical way to generate populations of relevant tissue-resident T cells. Certainly, neither subcutaneous fat nor skeletal muscle has had to evolve the highly sophisticated immunologic machinery to protect against infection that peripheral epithelial tissues that interface with the environment have had to. The observation that the tissue, as well as the lymph node that drains the tissue, has a role in fashioning the long-lived T-cell memory response to infection, makes delivery into non-epithelial tissues significantly less attractive. If the goal of vaccination is to generate not just antibodies but also the robust and tissue-relevant long-lasting immunity that the infectious disease normally generates (without the morbidity and potential mortality), how and where (anatomically) the vaccine is delivered makes a difference. Moreover, not harnessing the elegant and highly evolved endogenous machinery of the skin to generate durable immunologic memory is a missed opportunity. Indeed, a number of groups are beginning to appreciate the fact that skin immunization has demonstrable advantages over other routes of administration (Amorij et al., 2010; Liu et al., 2010).
Sometimes, it is important to get the perspectives of the oldest person in the room. In the rush to innovate, sometimes critical elements of existing technologies are jettisoned because their importance is underestimated or unappreciated. The smallpox vaccine is an illustrative case in point. Smallpox, a disease caused by the poxvirus Variola major, was quite literally one of the great scourges of humanity (Stewart and Devlin, 2006). Civilizations rose and fell under the pressure and sway of smallpox epidemics, and entire wars were sometimes decided based on regional smallpox outbreaks (Radetsky, 1999). For centuries, this highly contagious disease decimated populations across the globe, principally in the Eastern hemisphere, and was particularly virulent in populations that had never been exposed previously (e.g., native Americans, the Western hemisphere; Eyler, 2003). It has been proposed that the conquest of Latin and South America by Spain could not have occurred without the transmission, albeit unintentional, of smallpox from infected sailors to indigenous populations. The worldwide case fatality rate of smallpox averaged roughly 33%, and survivors were often disfigured and/or blinded. Among infants, case fatality ratios of 90% were common. In addition, among naive populations in the Western hemisphere, case fatality ratios in healthy adults of >80% were not uncommon, and native American populations were routinely decimated by the disease. In the first half of the twentieth century alone, more lives were lost to smallpox globally than were lost in both world wars and the 1918 influenza pandemic combined. It is therefore not an understatement that one of the greatest achievements of modern medicine was the global eradication of smallpox, which was finally achieved in the 1970s. The last case in the United States of America was in 1949, more than 10 years after the JID was first published. A highly effective vaccine, fashioned from a related poxvirus vaccinia virus (VACV), was responsible for this incredible achievement.
On the basis of the observation that survivors of infection with Variola major had subsequent lifelong immunity to smallpox, application of fluid from a smallpox lesion to abraded skin of a naive recipient was first recorded in the tenth century in China. This process, know as “variolation”, predictably led to full-blown protective immunity, but more than occasionally led to clinical infection and sometimes death from smallpox. Variolation, sometimes just known as inoculation, fell in and out of favor over the next several centuries after its use spread to other continents, and was practiced sporadically in many countries. Its use in Britain and other European countries was championed by Lady Montague, who learned of the practice while living in Turkey in the eighteenth century. However, despite a fatality rate of 2–3% from variolation, its success in protecting against smallpox (with a case fatality rate of 20–30%) could not be ignored (Cantey, 2011). In 1797, Edward Jenner VACV used the contents of a cowpox blister, derived from the infected hand of a milkmaid, and applied it to the disrupted epidermis of a young boy (i.e., vaccination). The boy experienced mild illness, but recovered completely. Further, in an experiment that would not pass institutional review board muster today, Jenner challenged the young boy by inoculation with Variola major (Bloch, 1993). The boy did not contract smallpox, either locally or systemically, and Jenner concluded that protection was complete. In a striking example of innovation not being appreciated by established science, Jenner’s manuscript describing the experiment to the Royal Society of Medicine in London was summarily rejected without review. He privately published a booklet describing the experiment and the theory behind it (Gross and Sepkowitz, 1998), but it took a number of years for vaccination to be accepted as a novel and highly effective protective therapy for this deadly disease. It should be noted that neither Lady Montague nor Edward Jenner, nor other proponents of inoculation and vaccination, had any knowledge of viruses, least of all the significant genetic homology between the different poxvirus species.
VACV would be subsequently isolated, cultured, titrated, and purified in the next two centuries, but its mode of delivery to human subjects remained largely unchanged. In the era before hypodermic syringes, the virus was applied to skin abraded by multiple strokes of a bifurcated needle, a process known as “scarification”. Indeed, the stability of virus preparations, and the relative ease by which nonmedically trained volunteers could vaccinate recipients by scarification even in remote third-world settings, facilitated the global WHO-led vaccination strategy that led to the first eradication of a human infectious disease (Parrino and Graham, 2006). Even today, when laboratory workers must work with poxviruses during experiments, the vaccine used is delivered by scarification.
Although vaccination with VACV was much safer than variolation, it was not without risk, and patients with compromised immune systems, as well as atopic dermatitis, occasionally suffered complications leading to morbidity and sometimes mortality (Wollenberg and Engler, 2004). Thus, after the eradication of smallpox in the wild, the risk–benefit ratio of smallpox vaccination was considered to have reached a tipping point, and routine vaccination of all children in the United States of America was discontinued in the 1980s. Even with the threat of bioterrorism looming after the anthrax attacks of 2001, this decision to not universally vaccinate the public was reinforced in the absence of a clear and present danger of smallpox infection (Parrino and Graham, 2006).
Demonstrably safer replication-deficient VACVs, including modified VACV Ankara (MVA), have been used to vaccinate large populations in European countries (Kennedy and Greenberg, 2009). This virus is used at a dose 100 times greater than the dose of VACV used in scarification, and is delivered intramuscularly. Although it engenders a robust antibody response, the T-cell response to MVA is less well characterized, and its ability to protect against smallpox is (thankfully) largely hypothetical. Because of the ease through which poxviruses can be manipulated through molecular cloning, and because poxvirus vectors can accommodate large “payloads” of complementary DNAs encoding for antigens, their use as vaccines for other infectious diseases and cancers is being pursued. However, in virtually all of the extant or proposed applications, the VACV vectors are proposed to be delivered by means of a hypodermic syringe. Indeed, in the era of the hypodermic syringe, the practice of scarification to deliver poxviruses has been increasingly viewed as a quaint and unnecessary relic of the history of medicine.
A careful review of the history of smallpox vaccination, however, would challenge this view. When hypodermic injection of VACV was directly compared with scarification, the latter was superior as judged both by surrogate immunological measures of vaccine efficiency and immunity to smallpox itself. In fact, the presence of a “pox” lesion, which we now understand as the result of epidermal keratinocyte infection with VACV and the attendant immune response, was strictly correlated with immunity to smallpox (Koplan and Marton, 1975). This was never achieved with hypodermic injection, except when virus-containing fluid was accidentally injected into the perforated epidermis upon needle withdrawal (Koplan and Marton, 1975). In mouse models, it was shown very recently that skin scarification was orders of magnitude more effective at inducing a protective T-cell–mediated immune response when directly compared with intradermal, subcutaneous, intramuscular, or even intraperitoneal delivery of VACV. This was true not only for wild-type VACV but also for the replication-deficient MVA (Liu et al., 2010). Although antibody responses were robust, the greatest differences were seen in protective T-cell– mediated responses, particularly in skin and lung (Liu et al., 2010).
Additional studies showed how these uniquely protective T-cell responses were generated. Shortly after skin scarification with VACV, T cells proliferated in draining lymph nodes, but then differentiated and separated into two groups. One group acquired skin-homing markers, entered the blood, and then migrated into skin, where they could remain in situ long term (Liu et al., 2006). The other group did not upregulate skin-homing markers, but rather retained markers of central memory T cells. These T cells entered blood and circulated into distant lymph nodes. The majority of these central memory T cells continued to recirculate between blood and multiple lymph nodes, but some acquired secondary tissue-specific imprinting; for example, T cells entering mesenteric nodes would acquire α4β7 and migrate to gut epithelium (Liu et al., 2006). Thus, scarification would generate different subset populations of T cells that would, respectively, enter the skin first, but also circulate between blood and lymph nodes, as well as entering distant epithelial tissues to become resident memory T cells. In fact, T cells generated by scarification that enter lung epithelium and remain in situ provide significant protection against a lethal respiratory challenge in the mouse model of VACV infection (Liu et al., 2010).
It is increasingly being appreciated that vaccination through skin has advantages over intramuscular or subcutaneous routes (Amorij et al., 2010; Liu et al., 2010). For example, experimental flu vaccines are being delivered through skin with good success, as judged by antibody responses (interestingly, T-cell responses are rarely monitored; Amorij et al., 2010). Delivery without hypodermic needle injection requires breaching the formidable stratum corneum, and whether this feat is achieved by ultrasound approaches (Lavon and Kost, 2004), microneedle patches (Sullivan et al., 2010), or nanoneedles, considerable attention is being paid to delivery of vaccine across the top layer of skin, the epidermis, into the dendritic cell–rich dermis below. Most modern vaccines in development, however, focus on protein antigens or virus-like particles delivered with an adjuvant to activate the innate immune system. Whether any of these approaches can ever approximate the success of live attenuated virus is unknown. It may be that when a virus infects a target cell, even if it cannot replicate, a series of events occurs that cannot be reproduced or even approximated by introduction of antigen with adjuvants, even when the latter are directed at Toll-like receptors or other components of the innate immune system. Further optimization of nonviral vector vaccination will answer this question more definitively.
Vaccines to HIV have met with near-universal failure, despite a huge investment of resources, time, and intellectual energy (Girard et al., 2011). Most efforts against HIV, as against most pathogens, have focused on the antibody response, and particularly on antigens that are rarely, if ever, mutated as this wily virus adapts to the host (Girard et al., 2011). The bias of an adherent to the concept of resident memory T-cell immunity (your author) is that although circulating antibodies are important, having a population of tissue-resident effector memory T cells present in the tissue that is being infected, whether reproductive or gastrointestinal mucosa, is essential for durable and effective immunity. Very recently, a report was published indicating that a vaccine focused specifically on generating effector memory T cells was highly effective in protecting monkeys against the closely related SIV virus (Hansen et al., 2011). The senior author of this paper, Louis Picker, is famous within Investigative Dermatology for discovering that the HECA-452 antibody bound preferentially to T cells in skin; he coined the term “cutaneous lymphocyte antigen” (Picker et al., 1990). It is encouraging to see the T-cell perspective making innovative forays into the world of vaccine biology.
New hypotheses are being proposed about the true role of tissue-resident memory T cells. These hypotheses have in common the view that immune memory must contain central and peripheral elements to be successful (Clark, 2010; Bevan, 2011). The peripheral elements are dendritic cells and other antigen-presenting cells coexisting with populations of T cells that are, for all intents and purposes, resident to epithelial tissues that interface with the environment, including the skin. These resident T-cell populations are poised to rapidly respond to pathogens that attempt to breach the epithelium of skin, lung, gut, or reproductive mucosa. The central elements of immune memory include circulating central memory T cells, which are enriched for the same antigenic specificities as tissue-resident memory T cells. These cells can provide a second wave of immune response to support the first wave provided by tissue-resident T cells. They can also provide help to antibody-producing memory B cells. Finally, B cells themselves are important mediators of the immune response, as their ability to produce antibodies is an important defense against systemic spread of the localized epithelial infection. B cells can also very efficiently present antigen to T cells, and thus their potential interplay cannot be ignored. To ignore any of these elements of the immune system while developing a vaccine strategy is unlikely to meet with full success. The intersection of vaccine biology and modern immunology is an exciting and fertile substrate for further translational innovation.
CONFLICT OF INTEREST
The author states no conflict of interest.