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Immunological memory to viral infection
Immunological memory to viral infection Commentary Mark K Slifka Immunological memory is defined by the ability of a host to remember a past encounter with a specific pathogen and to respond to it in an effective manner upon re-exposure. How long immunological memory can be maintained in the absence of re-infection continues to be a subject of great controversy. Recent studies on immunity following smallpox vaccination demonstrate that T-cell memory declines steadily with a half-life of 8–15 years, whereas antiviral antibody responses are maintained for up to 75 years without appreciable decline. By combining recent advances in quantitative immunology with historical accounts of protection against smallpox dating back to the time of Edward Jenner, we are gaining a better understanding of the duration and magnitude of immunological memory and how it relates to protective immunity.
immunological memory. Bearing these two factors in mind, smallpox vaccination (using the vaccinia virus) represents an excellent model for study because the virus is typically cleared from the site of infection within a month [1,2] and is not known to spread systemically or persist in most normal healthy individuals [3]. Smallpox vaccination of the general public in the US ceased in 1972, thus reducing the remote possibility of re-exposure to vaccinia [4,5]. In addition, the last case of smallpox in the United States occurred in 1949, more than 50 years ago [6]. Together, this provides an important opportunity to measure antiviral immunity over the course of many decades in the absence of re-exposure to viruses that could potentially reactivate the immune response.
Addresses Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006, USA e-mail: [email protected]
The risks of smallpox vaccination
Current Opinion in Immunology 2004, 16:443–450 This review comes from a themed issue on Host–pathogen interactions Edited by Tom Ottenhoff and Michael Bevan Available online 15th June 2004 0952-7915/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2004.05.013
Abbreviations AIDS acquired immunodeficiency syndrome CMV choriomeningitis virus ELISPOT enzyme-linked immunosorbent spot HIV human immunodeficiency virus IFN interferon LCMV lymphocytic CMV RSV respiratory syncytial virus VN vaccinia necrosum
Introduction To study the true duration of immunological memory at least two principal criteria must be met. First, the virus that is being studied must not persist in the host, as persistent viruses, such as cytomegalovirus (CMV) or Epstein-Barr virus (EBV), can re-activate and viral replication could then result in periodically boosting the host immune response over time. Second, the virus in question should not be endemic to the population under study, otherwise intermittent re-infection from the community could result in skewing of the calculated duration of www.sciencedirect.com
Smallpox vaccination consists of a live infection by the vaccinia virus and it induces cross-reactive immunity to many orthopoxviruses including smallpox, monkeypox, camelpox and cowpox, among others. Vaccinia has been described as the most dangerous vaccine in use today, killing approximately one person out of every one million people vaccinated. Surprisingly, a recent survey of the general public found that 19% of people felt that it was ‘somewhat likely’ that they would die if vaccinated/revaccinated and 6% felt that it was ‘very likely’ that they would die of complications due to smallpox vaccination [7]. In an attempt to curb these exaggerated fears, we asked the following question: how dangerous is vaccinia compared to other viruses, such as respiratory syncytial virus (RSV) or influenza – viruses that we often consider relatively benign? In an extensive epidemiological study [8] it was determined that RSV and influenza cause substantial levels of mortality and our comparison (Figure 1) shows that in people <65 years of age, RSV is more than 100-fold more deadly than vaccinia, whereas influenza is approximately 90 times more lethal. The mortality rates for RSV and influenza are based on person-years (not per infection) and, as only a fraction of people are infected with either of these viruses in any one particular year, the actual mortality rate per infection is likely to be substantially higher than the numbers shown here. Similar to vaccinia, the high mortality rates of RSV and influenza are due, at least in part, to the risks that these viruses pose for young infants as well as the elderly over the age of 65. If we narrow our comparisons to individuals between the ages of 1 and 49, then the mortality rates change to approximately five deaths associated with RSV infections and nine deaths associated with influenza infections per million person-years [8]. However, by Current Opinion in Immunology 2004, 16:443–450
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A comparison of virus-associated mortality rates following infection with RSV, influenza, or vaccinia. The hazards of smallpox vaccination using live vaccinia virus are well-documented [9–11] and of considerable concern to health care professionals [5,55,80] as well as the general public [7]. In this analysis, we compared the mortality rates associated with vaccinia infection (i.e. smallpox vaccination) to two other common viral pathogens, respiratory syncytial virus (RSV) and influenza. Although 90% of influenza-associated deaths and 78% of RSV-associated deaths occur in the elderly, 65 years of age [8], this age group was not routinely vaccinated against smallpox and therefore this figure shows only the lethal events occurring in individuals <65 years old. The mortality rate for vaccinia is estimated per million immunizations [9–11] and the mortality rates for RSV and influenza are calculated from underlying respiratory and circulatory deaths associated with RSV or influenza per million person-years for the 1990–1991 through 1998–1999 seasons [8]. As only a fraction of the population is infected with RSV or influenza in any given year, these mortality estimates are considered to be highly conservative.
removing children <1 year of age from the equation, this also reduces vaccinia-associated deaths due to smallpox vaccination by approximately one-half, resulting in a mortality rate of 0.5 per million [9], which is still 10–20-fold lower than the mortality rates that are observed following RSV or influenza infections. In adults over age 20, the mortality rate associated with smallpox vaccination is 0 per 605 000 primary vaccinations [10,11] and 1 per 7 250 000 re-vaccinations [10,11]. Recently, it was noted that smallpox vaccination may cause myopericarditis [12] and there was concern that vaccination may have caused three fatal heart attacks in recent vaccinees, but this trepidation appears to be scientifically unfounded [13]. During a nine-year span of inquiry [9], 88% (60/68) of recorded deaths due to smallpox vaccination occurred in individuals <20 years of age Current Opinion in Immunology 2004, 16:443–450
and 73% (43/68) occurred in children <4 years of age. Of the eight adults who died of vaccinia-related complications, five were over the age of 60 and four had been diagnosed with cancer at the time that they were vaccinated [9]. This indicates that out of a large population, there will be susceptible individuals at risk of microbial infections — and vaccinia, although dangerous in the sense that it is a live viral infection, is actually less virulent than RSV or influenza in terms of the risk for developing a lethal infection.
Early critics of smallpox vaccination In his first report on the theory that cowpox inoculation could protect against smallpox [14], Edward Jenner provided several case studies in which he demonstrated that people were fully protected against direct smallpox inoculation even though it had been 25–38 years since they had been infected with cowpox. He realized some critics might assume that endemic smallpox would skew his results, and to this issue he stated, ‘Had these experiments been conducted in a large city, or in a populous neighbourhood, some doubts might have been entertained; but here, where population is thin, and where such an event as a person’s having had the smallpox is always faithfully recorded, no risk of inaccuracy in this particular can arise’ [14]. The duration of protective immunity, however, continued to be a point of contention and in Jenner’s third study, published in 1800 [15], he again addressed this issue by stating, ‘Some there are who suppose the security from the smallpox obtained through the cow-pox will be of a temporary nature only. This supposition is refuted not only by analogy with respect to the habits of diseases of a similar nature, but by incontrovertible facts, which appear in great numbers against it’ [15]. To further his point, he then presented new evidence, ‘But among the cases I refer to, one will be found of a person who had the cow-pox fifty-three years before the effect of the smallpox was tried upon him. As he completely resisted it, the intervening period I conceive must necessarily satisfy any reasonable mind.’ Despite the elegant experiments performed by this remarkable individual, the issue of how long protective immunity against poxviruses can persist continues to be heatedly debated even today — over 200 years after Edward Jenner first demonstrated protective, long-term memory against smallpox.
Duration of antiviral T-cell memory Analysis of T-cell memory requires quantitation of virusspecific T cells, preferably examined directly ex vivo in order to avoid artifacts of prolonged in vitro stimulation. This was not feasible before the advent of vacciniaspecific enzyme-linked immunosorbent spot (ELISPOT) assays [16–18,19,20], intracellular cytokine staining analysis (ICCS; [21–23,24], or peptide–MHC tetramer staining [19,22]. Early studies instead relied on limiting dilution analysis and the development of cytotoxic T-cell clones as a means of demonstrating long-term immunity. www.sciencedirect.com
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Although one study identified cytotoxic CD4þ T-cell memory up to 50 years post-vaccination [25], another study only identified T-cell memory for up to three years and failed to elicit memory CD8þ T-cell responses in subjects examined more than 20 years after smallpox vaccination, thus suggesting that T-cell memory was short lived [26]. The discrepancy between these earlier studies may reflect differences in in vitro cultivation of T-cell clones, the low frequency of virus-specific T cells maintained in vivo, and/or the small number of volunteers that were examined. To date, the most commonly used technique used to study vaccinia-specific T-cell responses in humans is the IFN-g ELISPOT assay. This provides a very sensitive calculation of the number of virus-specific IFN-g-producing cells [16–18,19,20]; however, one drawback is that it is problematic to phenotype the IFN-gþ cells unless they are purified before the assay, otherwise one cannot distinguish between virus-specific CD4þ and CD8þ subsets. An alternative to the ELISPOT assay is to use intracellular cytokine staining. This technique quantifies virus-specific T cells and distinguishes between CD4þ and CD8þ T-cell subsets, but care should be taken to use anti-CD8b detection antibodies rather than anti-CD8a when working with human peripheral blood monocyte (PBMC) samples, as IFN-gþCD8alow cells that respond to vaccinia directly ex vivo might actually be CD8aþCD56þ natural killer (NK) cells [27]. Recent studies have identified potential human CD8þ T-cell epitopes in HLA-
A*0201 transgenic mice [22] and in recently vaccinated human volunteers [19]. The advantage of using peptide– MHC tetramers is that they quantify T cells directly ex vivo without requiring re-stimulation, and they measure T-cell numbers regardless of their functional capabilities. Although vaccinia expresses nearly 200 proteins, the combined use of just two recently identified immunodominant peptide epitopes provided quantitation of Tcell numbers equivalent to 35%, 14% and 6% of the total virus-specific ELISPOT response observed in three HLA-A*0201þ individuals after live virus stimulation in vitro [19]. Although the HLA-A*0201-specific peptide epitope identified in transgenic mice [22] was apparently not observed in the three humans tested [19], these results show that peptide–MHC tetramer staining is an effective technique for quantifying vaccinia-specific CD8þ T cells. One of the most intriguing questions regarding T-cell memory following smallpox vaccination is how long it persists and how it correlates with protective immunity (Figure 2). A recent study followed vaccinia-specific T-cell responses in groups of volunteers who received 1, 2, 3–5, or up to 6–14 smallpox vaccinations [24]. T-cell memory was not permanent, but instead declined slowly with a half-life of 8–15 years, regardless of how many vaccinations were given. Remarkably, virusspecific T cells could still be identified in some individuals for up to 75 years after a single vaccination. Similar results were obtained in another study which followed
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Relationship between immunological memory and protective immunity following smallpox vaccination. Two independent studies [20,24] have quantitated the duration of T-cell- and B-cell/antibody-mediated immunity over the course of several decades and came to remarkably similar conclusions: T-cell memory declines slowly over time, with a half-life of 8–15 years (representative thin line), whereas serum antibody responses (and B-cell memory; [20]) are maintained essentially for life with little or no observable decline (representative bold line). Immunological memory quantitated directly ex vivo does not necessarily demonstrate protective immunity; this can only be accomplished by natural exposure or experimental challenge experiments with the virulent pathogen of interest. In this regard, the protection afforded by smallpox vaccination was determined at the indicated intervals (bar graph inset) following immunization and shows that >90% of vaccinees are protected against lethal smallpox (normally 30% mortality in unvaccinated individuals) for at least 60 years post-vaccination [47,48]. Similar results showing long-term immunity were observed during imported smallpox outbreaks throughout Europe between 1950 and 1971 [49,50], decades after endemic smallpox had been eradicated [49]. www.sciencedirect.com
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vaccinia-specific T-cell responses by ELISPOT analysis for over 50 years; T-cell memory declined with a halflife of 14 years overall [20]. Interestingly, when Tcell subsets were compared within the same individual by ICCS, CD4þ T-cell memory appeared to be preferentially maintained over CD8þ T-cell memory [24]. This may explain why an earlier study showing longterm T-cell memory of up to 50 years [25] identified only CD4þ cytotoxic T-cell clones and why others found it difficult to culture CD8þ T-cell clones after 20 years post-vaccination [26]. The loss of CD8þ T-cell memory over CD4þ T-cell memory was unexpected; an elegant study in mice infected with lymphocytic choriomeningitis virus (LCMV) found that CD4þ T-cell memory was less stable than CD8þ T-cell memory [28]. This may be due to differences between mice and humans or to comparing different viruses (LCMV versus vaccinia) but, in either case, it will be interesting to see if these differences are directly related to the virus in question or to the host in which it is analyzed.
Duration of antiviral antibody responses In contrast to slowly declining T-cell memory, antiviral antibody responses [2,20,2429–31] and B-cell memory [20] following smallpox vaccination are maintained for decades and vaccinia-specific antibody responses are easily identified even 75 years after a single vaccination (Figure 2; [24]). Although booster vaccinations resulted in rapid and large anamnestic antibody responses, the level of antibody maintained during the plateau phase of memory was only marginally improved by a second vaccination, and no significant differences were observed after further booster vaccinations — remarkably, even 6–14 smallpox vaccinations failed to achieve appreciably higher long-term antibody responses than those obtained by just one or two vaccinations. This suggests that booster vaccinations may improve the antibody responses elicited by a suboptimal primary vaccination, but there may be a physiological limit to the maximum level of antibodies that an individual will maintain against any one particular pathogen. The mechanism underlying such long-term persistence of antibody production is unknown but could be due to continuous stimulation/differentiation of memory B cells [32,33], survival of long-lived plasma cells [34–37], or some combination of these potential mechanisms.
Duration and mechanisms of protective immunity The duration of protective immunity following smallpox vaccination was thought to wane rapidly, with substantial loss of protection within 3–5 years [38–43]. This viewpoint was supported by a study showing that 75.5% of re-vaccinated volunteers lacked immunological memory after 30 years because they experienced a ‘take’ (i.e. vesicle formation at the scarification site) after re-vaccination [44]. Much older data, however, show that this is Current Opinion in Immunology 2004, 16:443–450
not an accurate measure of true immunological memory, as 85% of healthy adults still experience a ‘take’ when re-vaccinated just one year after a successful primary vaccination [45] or just a few months after vaccination/ re-vaccination [46]. These studies show that skin immunity has no correlation with protective systemic immunity, as it is well established that strong protection against smallpox exists at one year or less after vaccination. The concept that immunity after smallpox vaccination is short lived was not necessarily based on immunological data, as neutralizing serum antibody has long been known to persist for decades [2,20,24,29–31]. Likewise, this notion was not based on quantitation of T-cell memory, as analysis of long-term T-cell memory in large clinical groups has just recently been published [20,24]. In smallpox hospitals, vaccinations were often administered annually to assure full protection, whereas booster vaccination was required every three years for international travel, and re-vaccination every 10 years was recommended for poxvirus researchers. So what really is the duration of protective immunity following smallpox vaccination? One of the most extensive studies on protective immunity was performed during the smallpox outbreaks that occurred in Liverpool, England in the early 1900s (Figure 2; [47,48]). Although smallpox infection typically caused 30% mortality, there were no reported deaths due to smallpox during the first 20 years after a single vaccination (206 cases, 100% survival). Of those vaccinated 20–30 years previously, 99% (330/333) survived the disease, as did 94% (361/384) of people vaccinated 30–60 years previously. One must keep in mind that these studies were conducted at a time and place where smallpox was endemic and one could argue that long-term immunity may have been afforded by repeated exposure to smallpox over a lifetime. However, analysis of imported smallpox outbreaks in non-endemic European countries provided surprisingly similar results [49,50]. For example, during outbreaks that resulted in a staggering 55% overall mortality rate in unvaccinated individuals, the mortality rates in previously vaccinated hospital staff and the general public were markedly reduced; of individuals vaccinated 0–10, 11–20 or >20 years previously, the survival rates were 98%, 94% and 93%, respectively (excluding hospital patients of unknown pre-existing health status; [50]). Moreover, these studies only take into account the previously vaccinated individuals with the lowest levels of immunity and are therefore susceptible to infection; a meta-analysis of 10 epidemiological studies (see page 200 in [51]) showed that only 4% of previously vaccinated household contacts actually develop clinical symptoms of smallpox. Even if one assumed an attack rate of 10% (instead of 4%) and a mortality rate of 10% (instead of 7%) in individuals presenting with clinical symptoms, this would still be indicative of a 99% overall survival rate. Data from the www.sciencedirect.com
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two epidemiological studies presented here [47,50] has recently been incorporated into a sophisticated mathematical model of long-term immunity [52]. Similar to the immunological and epidemiological data presented in Figure 2, these results indicate that, although immunity does wane gradually over time, protective immunity against lethal infection can clearly exist for up to a lifetime — as first demonstrated by Edward Jenner himself [15]. The mechanism of protective immunity following acute viral infection is perhaps even more controversial than the duration of protective immunity. It is generally agreed that T-cell immunity is critical for recovery from primary viral infections [53], but the relative role of pre-existing T-cell- and B-cell-mediated immunity during re-exposure to a specific pathogen remains an area of intense deliberation — although many are coming to realize that pre-existing antibody may play a more important role than previously realized. In terms of primary vaccinia infections, T-cell-mediated immunity is very important; when an HIVþ military recruit almost died of disseminated vaccinia following smallpox vaccination in 1984, it was probably due to low T-cell immunity [54]. Retrospective analysis indicates that HIV infection itself is not an absolute death sentence [55] because it is estimated that 300 HIVþ recruits were vaccinated prior to 1990 without any recorded incident [56]. In contrast, when recombinant vaccinia was administered to AIDS patients, 3/8 patients died of complications; all 3 had CD4þ T-cell counts of <50 cells/mm3 [57]. These findings resemble older observations of vaccinia necrosum (VN), a typically fatal disease caused by smallpox vaccination of individuals lacking cellular immunity. As the affected individuals lacked T-cell responses, they also universally lacked effective antibody responses (18/18 VN patients failed to mount neutralizing antibody responses above 1:4; [58]). Remarkably, up to 80% of children with VN could be saved by administering vaccinia immune globulin (VIG), even in cases in which they were genetically incapable of mounting an antiviral T-cell response [58]. The role of pre-existing T-cell memory in protection from re-infection is not as clear. In a murine model of intranasal vaccinia infection, T cells were important during primary infection but were unnecessary for protection against re-infection — if pre-existing antibody was present [59]. In humans, >90% of immunized individuals achieve lifelong protection against lethal smallpox infection (Figure 2; [47–50]), even though CD8þ T-cell memory drops below the limits of detection in 50% of individuals within 20 ysears after vaccination [24]. This would suggest that CD4þ T-cell memory and/or preexisting antiviral antibody titers (both retained in >90% of individuals for decades) might be responsible for this extended protection afforded after CD8þ T-cell memory has declined. One might question the effectiveness of www.sciencedirect.com
CD4þ T-cell memory, as these cells are restricted to MHC class IIþ targets. However, vaccinia-specific cytotoxic MHC class II-restricted CD4þ T cells have been observed in humans [25,26,60] and chimpanzees [61], and it is thought that poxviruses disseminate from the lungs via transport in phagocytic cells, such as macrophages that are MHC class IIþ and therefore susceptible to CD4þ T-cell-mediated immunosurveillance. Moreover, human respiratory epithelial cells can express MHC class II [62–67] and may also be recognized by cytotoxic CD4þ T cells in the context of a respiratory infection. Despite the obvious virtues of CD4þ and CD8þ T-cell memory and their necessity during primary viral infection, antiviral antibody production is more long lived and appears to be more important in protection against reinfection by poxviruses. During primary vaccinia infection of rhesus macaques with or without concomitant simian immunodeficiency virus (SIV) infection, clinical outcome correlated with CD4þ T-cell counts, whereas during re-infection there was no correlation between CD4þ T cells and protection. Instead, in previously vaccinated animals there was a strong correlation between the levels of pre-existing antibody and resolution of infection [68]. In humans, two independent studies demonstrated that individuals with pre-existing antibody titers of 1:32 [69] or 1:20 [70] were protected from contracting smallpox. This may have been due directly to the antibody itself or the high antibody titers may have simply been indicative of higher T-cell memory as well. However, comparisons between antibody levels and T-cell memory within the same individual indicate that there is no direct correlation between these two arms of the immune response [24]. In previously vaccinated human subjects who later contracted smallpox, 2/6 individuals (33%) with low antibody titers (<70% neutralization of smallpox) within 8 days of onset of smallpox symptoms eventually died of complications, whereas only 1/18 (5.6%) with high antibody titers (>70% neutralization) within 8 days of onset of symptoms later succumbed to the disease [71]. Adoptive transfer of neutralizing poxvirus-specific antibody is highly protective in animal models [59,72–74] and in humans who are subsequently exposed to smallpox [75–79]. Perhaps the most impressive proof of the effectiveness of passive immunotherapy against smallpox was demonstrated by Couzi and Kircher in 1941 [75]. During a smallpox outbreak, 3/10 patients died when given standard care. In an attempt to reduce mortality, standard care was then changed to include the use of convalescent serum or blood from recent smallpox survivors (obtained 10 days after lesions had scabbed over) to treat the rest of the incoming patients. The result: there were no further deaths recorded — 250/250 patients survived. This outcome required relatively large quantities of serum, which would have probably been impractical under most field conditions and may explain why this treatment was not adopted on a wider scale. Most Current Opinion in Immunology 2004, 16:443–450
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importantly, this study [75] clearly demonstrates how therapeutic antibody-mediated intervention can reduce smallpox mortality from 30% to less than 0.4% when performed under the proper conditions; at an early stage of disease, and using convalescent serum of high potency [53].
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Conclusions
12. Halsell JS, Riddle JR, Atwood JE, Gardner P, Shope R, Poland GA, Gray GC, Ostroff S, Eckart RE, Hospenthal DR et al.: Myopericarditis following smallpox vaccination among vaccinia-naive US military personnel. Jama 2003, 289:3283-3289.
Quantitative analysis of long-term immunity following smallpox vaccination clearly demonstrates that antiviral T cell, B cell, and antibody defense mechanisms remain in place for decades, and in some instances, even for life. These results are further substantiated by historical records dating back to the time of Edward Jenner, each demonstrating protective immunity against lethal smallpox infection that lasts for many years, often in the absence of re-vaccination or natural exposure to orthopoxviruses. These results prove that lifelong immunity is not only attainable (at least for certain microbes such as poxviruses) but it also sets the bar for future vaccine development against other microbial pathogens.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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2.
Frey SE, Newman FK, Yan L, Belshe RB: Response to smallpox vaccine in persons immunized in the distant past. Jama 2003, 289:3295-3299. This study demonstrated long-term immunity in individuals immunized over 30 years ago, as indicated by high levels of pre-existing serum antibody, a significantly lower incidence of fever following re-vaccination, significantly smaller skin lesions, and a shorter duration of viral shedding with lower virus titers compared to naı¨ve, previously unvaccinated controls. 3.
Cummings JF, Polhemus ME, Hawkes C, Klote M, Ludwig GV, Wortmann G: Lack of vaccinia viremia after smallpox vaccination. Clin Infect Dis 2004, 38:456-458.
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Blendon RJ, DesRoches CM, Benson JM, Herrmann MJ, Taylor-Clark K, Weldon KJ: The public and the smallpox threat. N Engl J Med 2003, 348:426-432. This survey provides substantial insight into how the general public perceives the dangers of smallpox and the risks associated with smallpox vaccination. The lay public has many false perceptions about this subject and the author advocate that further education of the public is necessary. 8.
Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, Fukuda K: Mortality associated with influenza and respiratory syncytial virus in the United States. Jama 2003, 289:179-186.
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10. Neff JM, Lane JM, Pert JH, Moore R, Millar JD, Henderson DA: Complications of smallpox vaccination. I. National survey in the United States, 1963. N Engl J Med 1967, 276:125-132. 11. Lane JM, Ruben FL, Neff JM, Millar JD: Complications of smallpox vaccination, 1968. N Engl J Med 1969, 281:1201-1208.
13. Centers for Disease Control and Prevention (CDC): Cardiac deaths after a mass smallpox vaccination campaign–New York City, 1947. MMWR Morb Mortal Wkly Rep 2003, 52:933-936. This study examined the number of cardiac deaths that occurred immediately following the vaccination of more than six million people within a four week period in 1947 and provides compelling evidence that the three heart attacks observed in recently vaccinated individuals in 2003 were probably only coincidence and not directly related to receiving smallpox vaccination. 14. Jenner E: An inquiry into the causes and effects of the variole vaccine, or cow-pox. London; 1798. 15. Jenner E: A continuation of facts and observations relative to the variole vaccine, or cow-pox. London, 1800. 16. Ennis FA, Cruz J, Demkowicz WE Jr, Rothman AL, McClain DJ: Primary Induction of Human CD8R Cytotoxic T Lymphocytes and Interferon- gamma-Producing T Cells after Smallpox Vaccination. J Infect Dis 2002, 185:1657-1659. 17. Frey SE, Newman FK, Cruz J, Shelton WB, Tennant JM, Polach T, Rothman AL, Kennedy JS, Wolff M, Belshe RB et al.: Dose-related effects of smallpox vaccine. N Engl J Med 2002, 346:1275-1280. 18. Weltzin R, Liu J, Pugachev KV, Myers GA, Coughlin B, Blum PS, Nichols R, Johnson C, Cruz J, Kennedy JS et al.: Clonal vaccinia virus grown in cell culture as a new smallpox vaccine. Nat Med 2003, 9:1125-1130. 19. Terajima M, Cruz J, Raines G, Kilpatrick ED, Kennedy JS, Rothman AL, Ennis FA: Quantitation of CD8R T Cell Responses to Newly Identified HLA-A*0201-restricted T Cell Epitopes Conserved Among Vaccinia and Variola (Smallpox) Viruses. J Exp Med 2003, 197:927-932. This study was the first to identify vaccinia-specific CD8þ T-cell epitopes in humans and shows the utility of peptide tetramer staining analysis in quantitation of virus-specific CD8þ T-cell populations. 20. Crotty S, Felgner P, Davies H, Glidewell J, Villarreal L, Ahmed R: Cutting Edge: Long-Term B Cell Memory in Humans after Smallpox Vaccination. J Immunol 2003, 171:4969-4973. This study demonstrated long-term T-cell- and B-cell-mediated immunity following smallpox vaccination with two main conclusions: antibody production and B-cell memory are maintained at a very stable level, but T-cell memory declines slowly over time with a half-life of about 14 years. Similar results were observed in [24]. 21. Harrington LE, van der Most R, Whitton JL, Ahmed R: Recombinant vaccinia virus-induced T-cell immunity: quantitation of the response to the virus vector and the foreign epitope. J Virol 2002, 76:3329-3337. 22. Drexler I, Staib C, Kastenmuller W, Stevanovic S, Schmidt B, Lemonnier FA, Rammensee HG, Busch DH, Bernhard H, Erfle V et al.: Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines. Proc Natl Acad Sci USA 2003, 100:217-222. 23. Poccia F, Gioia C, Montesano C, Martini F, Horejsh D, Castilletti C, Pucillo L, Capobianchi MR, Ippolito G: Flow cytometry and T-cell response monitoring after smallpox vaccination. Emerg Infect Dis 2003, 9:1468-1470. 24. Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, Sexton GJ, Hanifin JM, Slifka MK: Duration of antiviral immunity after smallpox vaccination. Nature Medicine 2003, 9:1131-1137. This study of over 300 individuals demonstrated that antiviral immunity could be maintained for up to 75 years after a single smallpox vaccination. www.sciencedirect.com
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Moreover, analysis of multiple vaccinations revealed that, although immunity is boosted by re-vaccination, the half-life of T-cell memory remains essentially the same (T1/2 ¼ 8–15 years) and antibody levels appear to return to a certain plateau, with little or no change in the total magnitude of long-term antibody production following multiple rounds of vaccination. Similar results were observed in [20].
46. Kaplan C, Benson PF, Butler NR: Immunogenicity of Ultraviolet-Irradiated, Non-Infectious, Vaccinia-Virus Vaccine in Infants and Young Children. Lancet 1965, 191:573-574.
25. Demkowicz WEJ, Littaua RA, Wang J, Ennis FA: Human cytotoxic T-cell memory: long-lived responses to vaccinia virus. J Virol 1996, 70:2627-2631.
48. Hanna W, Baxby D: Studies in smallpox and vaccination. 1913. Rev Med Virol 2002, 12:201-209.
26. Erickson AL, Walker CM: Class I major histocompatibility complex-restricted cytotoxic T cell responses to vaccinia virus in humans. J Gen Virol 1993, 74:751-754. 27. Speller SA, Warren AP: Ex vivo detection and enumeration of human antigen-specific CD8R T lymphocytes using antigen delivery by a recombinant vaccinia expression vector and intracellular cytokine staining. J Immunol Methods 2002, 262:167-180. 28. Homann D, Teyton L, Oldstone MB: Differential regulation of antiviral T-cell immunity results in stable CD8R but declining CD4R T-cell memory. Nat Med 2001, 7:913-919. 29. McCarthy K, Downie AW: The antibody response in man following infection with viruses of the pox group. II. Antibody response following vaccination. J Hyg 1958, 56:466-478. 30. el-Ad B, Roth Y, Winder A, Tochner Z, Lublin-Tennenbaum T, Katz E, Schwartz T: The persistence of neutralizing antibodies after re-vaccination against smallpox. J Infect Dis 1990, 161:446-448. 31. Stienlauf S, Shoresh M, Solomon A, Lublin-Tennenbaum T, Atsmon Y, Meirovich Y, Katz E: Kinetics of formation of neutralizing antibodies against vaccinia virus following re-vaccination. Vaccine 1999, 17:201-204. 32. Gray D, Skarvall H: B-cell memory is short-lived in the absence of antigen. Nature 1988, 336:70-73. 33. Bernasconi NL, Traggiai E, Lanzavecchia A: Maintenance of serological memory by polyclonal activation of human memory B cells. Science 2002, 298:2199-2202. 34. Slifka MK, Ahmed R: Long-term humoral immunity against viruses: revisiting the issue of plasma cell longevity. Trends Microbiol 1996, 4:394-400. 35. Manz RA, Thiel A, Radbruch A: Lifetime of plasma cells in the bone marrow. Nature 1997, 388:133-134. 36. Slifka MK, Antia R, Whitmire JK, Ahmed R: Humoral immunity due to long-lived plasma cells. Immunity 1998, 8:363-372. 37. Manz RA, Arce S, Cassese G, Hauser AE, Hiepe F, Radbruch A: Humoral immunity and long-lived plasma cells. Curr Opin Immunol 2002, 14:517-521. 38. Henderson DA: The looming threat of bioterrorism. Science 1999, 283:1279-1282. 39. Meltzer MI, Damon I, LeDuc JW, Millar JD: Modeling potential responses to smallpox as a bioterrorist weapon. Emerg Infect Dis 2001, 7:959-969. 40. Gani R, Leach S: Transmission potential of smallpox in contemporary populations. Nature 2001, 414:748-751. 41. Kaplan EH, Craft DL, Wein LM: Emergency response to a smallpox attack: the case for mass vaccination. Proc Natl Acad Sci USA 2002, 99:10935-10940. 42. O’Toole T, Mair M, Inglesby TV: Shining light on ‘‘Dark Winter’’. Clin Infect Dis 2002, 34:972-983. 43. Smith GL, McFadden G: Smallpox: anything to declare? Nat Rev Immunol 2002, 2:521-527. 44. Sauri M, Sibley C, Monk B, Nichols M, Lai S: Durability of vaccinia immunization based on reaction at the rechallenge site. Md Med 2002, 3:44-51. 45. Cross RM, Kaplan C, McClean D: Studies with dried and glycerinated smallpox vaccines of full and diminished potencies. Bull World Health Organ 1958, 19:123-128. www.sciencedirect.com
47. Hanna W: Studies in smallpox and vaccination. New York: William Wood and Company; 1913.
49. CDC: Smallpox - Stockholm, Sweden, 1963. MMWR 1963, 12:172, 174-176, 183, 188, 191, 220, 236. 50. Mack TM: Smallpox in Europe, 1950-1971. J Infect Dis 1972, 125:161-169. 51. Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID: Smallpox and its eradication. In The pathogenesis, immunology, and pathology of smallpox and vaccinia. Geneva: World Health Organization; 1988. 52. Eichner M: Analysis of historical data suggests long-lasting protective effects of smallpox vaccination. Am J Epidemiol 2003, 158:717-723. 53. Whitton JL, Slifka MK, Liu F, Nussbaum AK, Whitmire JK: The regulation and maturation of antiviral immune responses. Adv Vir Res 2004, in press. This review of antiviral immunity provides a comprehensive overview of how antibody and T-cell-mediated immunity evolve, mature and work together to overcome many viral pathogens. 54. Redfield RR, Wright DC, James WD, Jones TS, Brown C, Burke DS: Disseminated vaccinia in a military recruit with human immunodeficiency virus (HIV) disease. N Engl J Med 1987, 316:673-676. 55. Amorosa VK, Isaacs SN: Separate worlds set to collide: smallpox, vaccinia virus vaccination, and human immunodeficiency virus and acquired immunodeficiency syndrome. Clin Infect Dis 2003, 37:426-432. 56. Bartlett JG: Smallpox vaccination and patients with human immunodeficiency virus infection or acquired immunodeficiency syndrome. Clin Infect Dis 2003, 36:468-471. 57. Zagury D: Anti-HIV cellular immunotherapy in AIDS. Lancet 1991, 338:694-695. 58. Kempe CH: Studies on smallpox and complications of smallpox vaccination. Pediatrics 1960, 25:176-189. 59. Belyakov IM, Earl P, Dzutsev A, Kuznetsov VA, Lemon M, Wyatt LS, Snyder JT, Ahlers JD, Franchini G, Moss B et al.: Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses. Proc Natl Acad Sci USA 2003, 100:9458-9463. This study shows that, when antiviral antibody is present, CD4þ or CD8þ T cells are not necessary for controlling vaccinia infection. However, in the absence of antibody or in unvaccinated animals lacking pre-existing antibody, T-cell-mediated immunity is important for controlling a sublethal infection. 60. Littaua RA, Takeda A, Cruz J, Ennis FA: Vaccinia virus-specific human CD4R cytotoxic T-lymphocyte clones. J Virol 1992, 66:2274-2280. 61. Zarling JM, Eichberg JW, Moran PA, McClure J, Sridhar P, Hu SL: Proliferative and cytotoxic T cells to AIDS virus glycoproteins in chimpanzees immunized with a recombinant vaccinia virus expressing AIDS virus envelope glycoproteins. J Immunol 1987, 139:988-990. 62. Rossi GA, Sacco O, Balbi B, Oddera S, Mattioni T, Corte G, Ravazzoni C, Allegra L: Human ciliated bronchial epithelial cells: expression of the HLA-DR antigens and of the HLA-DR alpha gene, modulation of the HLA-DR antigens by gammainterferon and antigen-presenting function in the mixed leukocyte reaction. Am J Respir Cell Mol Biol 1990, 3:431-439. 63. Wang D, Levasseur-Acker GM, Jankowski R, Kanny G, Moneret-Vautrin DA, Charron D, Lockhart A, Swierczewski E: HLA class II antigens and T lymphocytes in human nasal epithelial cells. Modulation of the HLA class II gene transcripts by gamma interferon. Clin Exp Allergy 1997, 27:306-314. 64. Striz I, Mio T, Adachi Y, Carnevali S, Romberger DJ, Rennard SI: Effects of interferons alpha and gamma on cytokine production Current Opinion in Immunology 2004, 16:443–450
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and phenotypic pattern of human bronchial epithelial cells. Int J Immunopharmacol 2000, 22:573-585. 65. Papon JF, Coste A, Gendron MC, Cordonnier C, Wingerstmann L, Peynegre R, Escudier E: HLA-DR and ICAM-1 expression and modulation in epithelial cells from nasal polyps. Laryngoscope 2002, 112:2067-2075. 66. Hegde NR, Johnson DC: Human cytomegalovirus US2 causes similar effects on both major histocompatibility complex class I and II proteins in epithelial and glial cells. J Virol 2003, 77:9287-9294. 67. Rees LE, Ayoub O, Haverson K, Birchall MA, Bailey M: Differential major histocompatibility complex class II locus expression on human laryngeal epithelium. Clin Exp Immunol 2003, 134:497-502. 68. Edghill-Smith Y, Venzon D, Karpova T, McNally J, Nacsa J, Tsai WP, Tryniszewska E, Moniuszko M, Manischewitz J, King LR et al.: Modeling a safer smallpox vaccination regimen, for human immunodeficiency virus type 1-infected patients, in immunocompromised macaques. J Infect Dis 2003, 188:1181-1191.
72. Czerny CP, Mahnel H: Structural and functional analysis of orthopoxvirus epitopes with neutralizing monoclonal antibodies. J Gen Virol 1990, 71(Pt 10):2341-2352. 73. Galmiche MC, Goenaga J, Wittek R, Rindisbacher L: Neutralizing and protective antibodies directed against vaccinia virus envelope antigens. Virology 1999, 254:71-80. 74. Ramirez JC, Tapia E, Esteban M: Administration to mice of a monoclonal antibody that neutralizes the intracellular mature virus form of vaccinia virus limits virus replication efficiently under prophylactic and therapeutic conditions. J Gen Virol 2002, 83:1059-1067. 75. Couzi G, Kircher JP: Immunotherapie de la Variole. Bulletin de l’Institut d’hygiene du Maroc 1941, 1:59-68. 76. Kempe CH, Berge TO, England B: Hyperimmune vaccinial gamma globulin. Pediatrics 1956, 18:177-188. 77. Peirce ER, Melville FS, Downie AW, Duckworth MJ: Antivaccinial gamma-globulin in smallpox prophylaxis. Lancet 1958, 2:635-638.
69. Mack TM, Noble J Jr, Thomas DB: A prospective study of serum antibody and protection against smallpox. Am J Trop Med Hyg 1972, 21:214-218.
78. Kempe CH, Bowles C, Meiklejohn G, Berge TO, St. Vincent L, Babu BVS, Govindarajan S, Ratnakannan NR, Downie AW, Murthy VR: The use of vaccinia hyperimmune gammaglobulin in the prophylaxis of smallpox. Bull WHO 1961, 25:41-48.
70. Sarkar JK, Mitra AC, Mukherjee MK: The minimum protective level of antibodies in smallpox. Bull World Health Organ 1975, 52:307-311.
79. Marennikova SS: The use of hyperimmune antivaccinia gamma-globulin for the prevention and treatment of smallpox. Bull World Health Organ 1962, 27:325-330.
71. Downie AW, McCarthy K: The antibody response in man following infection with viruses of the pox group. III. Antibody response in smallpox. J Hyg 1958, 56:479-487.
80. Kwon N, Raven MC, Chiang WK, Moran GJ, Jui J, Carter RA, Goldfrank L: Emergency physicians’ perspectives on smallpox vaccination. Acad Emerg Med 2003, 10:599-605.
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immunological memory. Bearing these two factors in mind, smallpox vaccination (using the vaccinia virus) represents an excellent model for study because the virus is typically cleared from the site of infection within a month [1,2] and is not known to spread systemically or persist in most normal healthy individuals [3]. Smallpox vaccination of the general public in the US ceased in 1972, thus reducing the remote possibility of re-exposure to vaccinia [4,5]. In addition, the last case of smallpox in the United States occurred in 1949, more than 50 years ago [6]. Together, this provides an important opportunity to measure antiviral immunity over the course of many decades in the absence of re-exposure to viruses that could potentially reactivate the immune response.
Addresses Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006, USA e-mail: [email protected]
The risks of smallpox vaccination
Current Opinion in Immunology 2004, 16:443–450 This review comes from a themed issue on Host–pathogen interactions Edited by Tom Ottenhoff and Michael Bevan Available online 15th June 2004 0952-7915/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2004.05.013
Abbreviations AIDS acquired immunodeficiency syndrome CMV choriomeningitis virus ELISPOT enzyme-linked immunosorbent spot HIV human immunodeficiency virus IFN interferon LCMV lymphocytic CMV RSV respiratory syncytial virus VN vaccinia necrosum
Introduction To study the true duration of immunological memory at least two principal criteria must be met. First, the virus that is being studied must not persist in the host, as persistent viruses, such as cytomegalovirus (CMV) or Epstein-Barr virus (EBV), can re-activate and viral replication could then result in periodically boosting the host immune response over time. Second, the virus in question should not be endemic to the population under study, otherwise intermittent re-infection from the community could result in skewing of the calculated duration of www.sciencedirect.com
Smallpox vaccination consists of a live infection by the vaccinia virus and it induces cross-reactive immunity to many orthopoxviruses including smallpox, monkeypox, camelpox and cowpox, among others. Vaccinia has been described as the most dangerous vaccine in use today, killing approximately one person out of every one million people vaccinated. Surprisingly, a recent survey of the general public found that 19% of people felt that it was ‘somewhat likely’ that they would die if vaccinated/revaccinated and 6% felt that it was ‘very likely’ that they would die of complications due to smallpox vaccination [7]. In an attempt to curb these exaggerated fears, we asked the following question: how dangerous is vaccinia compared to other viruses, such as respiratory syncytial virus (RSV) or influenza – viruses that we often consider relatively benign? In an extensive epidemiological study [8] it was determined that RSV and influenza cause substantial levels of mortality and our comparison (Figure 1) shows that in people <65 years of age, RSV is more than 100-fold more deadly than vaccinia, whereas influenza is approximately 90 times more lethal. The mortality rates for RSV and influenza are based on person-years (not per infection) and, as only a fraction of people are infected with either of these viruses in any one particular year, the actual mortality rate per infection is likely to be substantially higher than the numbers shown here. Similar to vaccinia, the high mortality rates of RSV and influenza are due, at least in part, to the risks that these viruses pose for young infants as well as the elderly over the age of 65. If we narrow our comparisons to individuals between the ages of 1 and 49, then the mortality rates change to approximately five deaths associated with RSV infections and nine deaths associated with influenza infections per million person-years [8]. However, by Current Opinion in Immunology 2004, 16:443–450
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A comparison of virus-associated mortality rates following infection with RSV, influenza, or vaccinia. The hazards of smallpox vaccination using live vaccinia virus are well-documented [9–11] and of considerable concern to health care professionals [5,55,80] as well as the general public [7]. In this analysis, we compared the mortality rates associated with vaccinia infection (i.e. smallpox vaccination) to two other common viral pathogens, respiratory syncytial virus (RSV) and influenza. Although 90% of influenza-associated deaths and 78% of RSV-associated deaths occur in the elderly, 65 years of age [8], this age group was not routinely vaccinated against smallpox and therefore this figure shows only the lethal events occurring in individuals <65 years old. The mortality rate for vaccinia is estimated per million immunizations [9–11] and the mortality rates for RSV and influenza are calculated from underlying respiratory and circulatory deaths associated with RSV or influenza per million person-years for the 1990–1991 through 1998–1999 seasons [8]. As only a fraction of the population is infected with RSV or influenza in any given year, these mortality estimates are considered to be highly conservative.
removing children <1 year of age from the equation, this also reduces vaccinia-associated deaths due to smallpox vaccination by approximately one-half, resulting in a mortality rate of 0.5 per million [9], which is still 10–20-fold lower than the mortality rates that are observed following RSV or influenza infections. In adults over age 20, the mortality rate associated with smallpox vaccination is 0 per 605 000 primary vaccinations [10,11] and 1 per 7 250 000 re-vaccinations [10,11]. Recently, it was noted that smallpox vaccination may cause myopericarditis [12] and there was concern that vaccination may have caused three fatal heart attacks in recent vaccinees, but this trepidation appears to be scientifically unfounded [13]. During a nine-year span of inquiry [9], 88% (60/68) of recorded deaths due to smallpox vaccination occurred in individuals <20 years of age Current Opinion in Immunology 2004, 16:443–450
and 73% (43/68) occurred in children <4 years of age. Of the eight adults who died of vaccinia-related complications, five were over the age of 60 and four had been diagnosed with cancer at the time that they were vaccinated [9]. This indicates that out of a large population, there will be susceptible individuals at risk of microbial infections — and vaccinia, although dangerous in the sense that it is a live viral infection, is actually less virulent than RSV or influenza in terms of the risk for developing a lethal infection.
Early critics of smallpox vaccination In his first report on the theory that cowpox inoculation could protect against smallpox [14], Edward Jenner provided several case studies in which he demonstrated that people were fully protected against direct smallpox inoculation even though it had been 25–38 years since they had been infected with cowpox. He realized some critics might assume that endemic smallpox would skew his results, and to this issue he stated, ‘Had these experiments been conducted in a large city, or in a populous neighbourhood, some doubts might have been entertained; but here, where population is thin, and where such an event as a person’s having had the smallpox is always faithfully recorded, no risk of inaccuracy in this particular can arise’ [14]. The duration of protective immunity, however, continued to be a point of contention and in Jenner’s third study, published in 1800 [15], he again addressed this issue by stating, ‘Some there are who suppose the security from the smallpox obtained through the cow-pox will be of a temporary nature only. This supposition is refuted not only by analogy with respect to the habits of diseases of a similar nature, but by incontrovertible facts, which appear in great numbers against it’ [15]. To further his point, he then presented new evidence, ‘But among the cases I refer to, one will be found of a person who had the cow-pox fifty-three years before the effect of the smallpox was tried upon him. As he completely resisted it, the intervening period I conceive must necessarily satisfy any reasonable mind.’ Despite the elegant experiments performed by this remarkable individual, the issue of how long protective immunity against poxviruses can persist continues to be heatedly debated even today — over 200 years after Edward Jenner first demonstrated protective, long-term memory against smallpox.
Duration of antiviral T-cell memory Analysis of T-cell memory requires quantitation of virusspecific T cells, preferably examined directly ex vivo in order to avoid artifacts of prolonged in vitro stimulation. This was not feasible before the advent of vacciniaspecific enzyme-linked immunosorbent spot (ELISPOT) assays [16–18,19,20], intracellular cytokine staining analysis (ICCS; [21–23,24], or peptide–MHC tetramer staining [19,22]. Early studies instead relied on limiting dilution analysis and the development of cytotoxic T-cell clones as a means of demonstrating long-term immunity. www.sciencedirect.com
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Although one study identified cytotoxic CD4þ T-cell memory up to 50 years post-vaccination [25], another study only identified T-cell memory for up to three years and failed to elicit memory CD8þ T-cell responses in subjects examined more than 20 years after smallpox vaccination, thus suggesting that T-cell memory was short lived [26]. The discrepancy between these earlier studies may reflect differences in in vitro cultivation of T-cell clones, the low frequency of virus-specific T cells maintained in vivo, and/or the small number of volunteers that were examined. To date, the most commonly used technique used to study vaccinia-specific T-cell responses in humans is the IFN-g ELISPOT assay. This provides a very sensitive calculation of the number of virus-specific IFN-g-producing cells [16–18,19,20]; however, one drawback is that it is problematic to phenotype the IFN-gþ cells unless they are purified before the assay, otherwise one cannot distinguish between virus-specific CD4þ and CD8þ subsets. An alternative to the ELISPOT assay is to use intracellular cytokine staining. This technique quantifies virus-specific T cells and distinguishes between CD4þ and CD8þ T-cell subsets, but care should be taken to use anti-CD8b detection antibodies rather than anti-CD8a when working with human peripheral blood monocyte (PBMC) samples, as IFN-gþCD8alow cells that respond to vaccinia directly ex vivo might actually be CD8aþCD56þ natural killer (NK) cells [27]. Recent studies have identified potential human CD8þ T-cell epitopes in HLA-
A*0201 transgenic mice [22] and in recently vaccinated human volunteers [19]. The advantage of using peptide– MHC tetramers is that they quantify T cells directly ex vivo without requiring re-stimulation, and they measure T-cell numbers regardless of their functional capabilities. Although vaccinia expresses nearly 200 proteins, the combined use of just two recently identified immunodominant peptide epitopes provided quantitation of Tcell numbers equivalent to 35%, 14% and 6% of the total virus-specific ELISPOT response observed in three HLA-A*0201þ individuals after live virus stimulation in vitro [19]. Although the HLA-A*0201-specific peptide epitope identified in transgenic mice [22] was apparently not observed in the three humans tested [19], these results show that peptide–MHC tetramer staining is an effective technique for quantifying vaccinia-specific CD8þ T cells. One of the most intriguing questions regarding T-cell memory following smallpox vaccination is how long it persists and how it correlates with protective immunity (Figure 2). A recent study followed vaccinia-specific T-cell responses in groups of volunteers who received 1, 2, 3–5, or up to 6–14 smallpox vaccinations [24]. T-cell memory was not permanent, but instead declined slowly with a half-life of 8–15 years, regardless of how many vaccinations were given. Remarkably, virusspecific T cells could still be identified in some individuals for up to 75 years after a single vaccination. Similar results were obtained in another study which followed
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Relationship between immunological memory and protective immunity following smallpox vaccination. Two independent studies [20,24] have quantitated the duration of T-cell- and B-cell/antibody-mediated immunity over the course of several decades and came to remarkably similar conclusions: T-cell memory declines slowly over time, with a half-life of 8–15 years (representative thin line), whereas serum antibody responses (and B-cell memory; [20]) are maintained essentially for life with little or no observable decline (representative bold line). Immunological memory quantitated directly ex vivo does not necessarily demonstrate protective immunity; this can only be accomplished by natural exposure or experimental challenge experiments with the virulent pathogen of interest. In this regard, the protection afforded by smallpox vaccination was determined at the indicated intervals (bar graph inset) following immunization and shows that >90% of vaccinees are protected against lethal smallpox (normally 30% mortality in unvaccinated individuals) for at least 60 years post-vaccination [47,48]. Similar results showing long-term immunity were observed during imported smallpox outbreaks throughout Europe between 1950 and 1971 [49,50], decades after endemic smallpox had been eradicated [49]. www.sciencedirect.com
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vaccinia-specific T-cell responses by ELISPOT analysis for over 50 years; T-cell memory declined with a halflife of 14 years overall [20]. Interestingly, when Tcell subsets were compared within the same individual by ICCS, CD4þ T-cell memory appeared to be preferentially maintained over CD8þ T-cell memory [24]. This may explain why an earlier study showing longterm T-cell memory of up to 50 years [25] identified only CD4þ cytotoxic T-cell clones and why others found it difficult to culture CD8þ T-cell clones after 20 years post-vaccination [26]. The loss of CD8þ T-cell memory over CD4þ T-cell memory was unexpected; an elegant study in mice infected with lymphocytic choriomeningitis virus (LCMV) found that CD4þ T-cell memory was less stable than CD8þ T-cell memory [28]. This may be due to differences between mice and humans or to comparing different viruses (LCMV versus vaccinia) but, in either case, it will be interesting to see if these differences are directly related to the virus in question or to the host in which it is analyzed.
Duration of antiviral antibody responses In contrast to slowly declining T-cell memory, antiviral antibody responses [2,20,2429–31] and B-cell memory [20] following smallpox vaccination are maintained for decades and vaccinia-specific antibody responses are easily identified even 75 years after a single vaccination (Figure 2; [24]). Although booster vaccinations resulted in rapid and large anamnestic antibody responses, the level of antibody maintained during the plateau phase of memory was only marginally improved by a second vaccination, and no significant differences were observed after further booster vaccinations — remarkably, even 6–14 smallpox vaccinations failed to achieve appreciably higher long-term antibody responses than those obtained by just one or two vaccinations. This suggests that booster vaccinations may improve the antibody responses elicited by a suboptimal primary vaccination, but there may be a physiological limit to the maximum level of antibodies that an individual will maintain against any one particular pathogen. The mechanism underlying such long-term persistence of antibody production is unknown but could be due to continuous stimulation/differentiation of memory B cells [32,33], survival of long-lived plasma cells [34–37], or some combination of these potential mechanisms.
Duration and mechanisms of protective immunity The duration of protective immunity following smallpox vaccination was thought to wane rapidly, with substantial loss of protection within 3–5 years [38–43]. This viewpoint was supported by a study showing that 75.5% of re-vaccinated volunteers lacked immunological memory after 30 years because they experienced a ‘take’ (i.e. vesicle formation at the scarification site) after re-vaccination [44]. Much older data, however, show that this is Current Opinion in Immunology 2004, 16:443–450
not an accurate measure of true immunological memory, as 85% of healthy adults still experience a ‘take’ when re-vaccinated just one year after a successful primary vaccination [45] or just a few months after vaccination/ re-vaccination [46]. These studies show that skin immunity has no correlation with protective systemic immunity, as it is well established that strong protection against smallpox exists at one year or less after vaccination. The concept that immunity after smallpox vaccination is short lived was not necessarily based on immunological data, as neutralizing serum antibody has long been known to persist for decades [2,20,24,29–31]. Likewise, this notion was not based on quantitation of T-cell memory, as analysis of long-term T-cell memory in large clinical groups has just recently been published [20,24]. In smallpox hospitals, vaccinations were often administered annually to assure full protection, whereas booster vaccination was required every three years for international travel, and re-vaccination every 10 years was recommended for poxvirus researchers. So what really is the duration of protective immunity following smallpox vaccination? One of the most extensive studies on protective immunity was performed during the smallpox outbreaks that occurred in Liverpool, England in the early 1900s (Figure 2; [47,48]). Although smallpox infection typically caused 30% mortality, there were no reported deaths due to smallpox during the first 20 years after a single vaccination (206 cases, 100% survival). Of those vaccinated 20–30 years previously, 99% (330/333) survived the disease, as did 94% (361/384) of people vaccinated 30–60 years previously. One must keep in mind that these studies were conducted at a time and place where smallpox was endemic and one could argue that long-term immunity may have been afforded by repeated exposure to smallpox over a lifetime. However, analysis of imported smallpox outbreaks in non-endemic European countries provided surprisingly similar results [49,50]. For example, during outbreaks that resulted in a staggering 55% overall mortality rate in unvaccinated individuals, the mortality rates in previously vaccinated hospital staff and the general public were markedly reduced; of individuals vaccinated 0–10, 11–20 or >20 years previously, the survival rates were 98%, 94% and 93%, respectively (excluding hospital patients of unknown pre-existing health status; [50]). Moreover, these studies only take into account the previously vaccinated individuals with the lowest levels of immunity and are therefore susceptible to infection; a meta-analysis of 10 epidemiological studies (see page 200 in [51]) showed that only 4% of previously vaccinated household contacts actually develop clinical symptoms of smallpox. Even if one assumed an attack rate of 10% (instead of 4%) and a mortality rate of 10% (instead of 7%) in individuals presenting with clinical symptoms, this would still be indicative of a 99% overall survival rate. Data from the www.sciencedirect.com
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two epidemiological studies presented here [47,50] has recently been incorporated into a sophisticated mathematical model of long-term immunity [52]. Similar to the immunological and epidemiological data presented in Figure 2, these results indicate that, although immunity does wane gradually over time, protective immunity against lethal infection can clearly exist for up to a lifetime — as first demonstrated by Edward Jenner himself [15]. The mechanism of protective immunity following acute viral infection is perhaps even more controversial than the duration of protective immunity. It is generally agreed that T-cell immunity is critical for recovery from primary viral infections [53], but the relative role of pre-existing T-cell- and B-cell-mediated immunity during re-exposure to a specific pathogen remains an area of intense deliberation — although many are coming to realize that pre-existing antibody may play a more important role than previously realized. In terms of primary vaccinia infections, T-cell-mediated immunity is very important; when an HIVþ military recruit almost died of disseminated vaccinia following smallpox vaccination in 1984, it was probably due to low T-cell immunity [54]. Retrospective analysis indicates that HIV infection itself is not an absolute death sentence [55] because it is estimated that 300 HIVþ recruits were vaccinated prior to 1990 without any recorded incident [56]. In contrast, when recombinant vaccinia was administered to AIDS patients, 3/8 patients died of complications; all 3 had CD4þ T-cell counts of <50 cells/mm3 [57]. These findings resemble older observations of vaccinia necrosum (VN), a typically fatal disease caused by smallpox vaccination of individuals lacking cellular immunity. As the affected individuals lacked T-cell responses, they also universally lacked effective antibody responses (18/18 VN patients failed to mount neutralizing antibody responses above 1:4; [58]). Remarkably, up to 80% of children with VN could be saved by administering vaccinia immune globulin (VIG), even in cases in which they were genetically incapable of mounting an antiviral T-cell response [58]. The role of pre-existing T-cell memory in protection from re-infection is not as clear. In a murine model of intranasal vaccinia infection, T cells were important during primary infection but were unnecessary for protection against re-infection — if pre-existing antibody was present [59]. In humans, >90% of immunized individuals achieve lifelong protection against lethal smallpox infection (Figure 2; [47–50]), even though CD8þ T-cell memory drops below the limits of detection in 50% of individuals within 20 ysears after vaccination [24]. This would suggest that CD4þ T-cell memory and/or preexisting antiviral antibody titers (both retained in >90% of individuals for decades) might be responsible for this extended protection afforded after CD8þ T-cell memory has declined. One might question the effectiveness of www.sciencedirect.com
CD4þ T-cell memory, as these cells are restricted to MHC class IIþ targets. However, vaccinia-specific cytotoxic MHC class II-restricted CD4þ T cells have been observed in humans [25,26,60] and chimpanzees [61], and it is thought that poxviruses disseminate from the lungs via transport in phagocytic cells, such as macrophages that are MHC class IIþ and therefore susceptible to CD4þ T-cell-mediated immunosurveillance. Moreover, human respiratory epithelial cells can express MHC class II [62–67] and may also be recognized by cytotoxic CD4þ T cells in the context of a respiratory infection. Despite the obvious virtues of CD4þ and CD8þ T-cell memory and their necessity during primary viral infection, antiviral antibody production is more long lived and appears to be more important in protection against reinfection by poxviruses. During primary vaccinia infection of rhesus macaques with or without concomitant simian immunodeficiency virus (SIV) infection, clinical outcome correlated with CD4þ T-cell counts, whereas during re-infection there was no correlation between CD4þ T cells and protection. Instead, in previously vaccinated animals there was a strong correlation between the levels of pre-existing antibody and resolution of infection [68]. In humans, two independent studies demonstrated that individuals with pre-existing antibody titers of 1:32 [69] or 1:20 [70] were protected from contracting smallpox. This may have been due directly to the antibody itself or the high antibody titers may have simply been indicative of higher T-cell memory as well. However, comparisons between antibody levels and T-cell memory within the same individual indicate that there is no direct correlation between these two arms of the immune response [24]. In previously vaccinated human subjects who later contracted smallpox, 2/6 individuals (33%) with low antibody titers (<70% neutralization of smallpox) within 8 days of onset of smallpox symptoms eventually died of complications, whereas only 1/18 (5.6%) with high antibody titers (>70% neutralization) within 8 days of onset of symptoms later succumbed to the disease [71]. Adoptive transfer of neutralizing poxvirus-specific antibody is highly protective in animal models [59,72–74] and in humans who are subsequently exposed to smallpox [75–79]. Perhaps the most impressive proof of the effectiveness of passive immunotherapy against smallpox was demonstrated by Couzi and Kircher in 1941 [75]. During a smallpox outbreak, 3/10 patients died when given standard care. In an attempt to reduce mortality, standard care was then changed to include the use of convalescent serum or blood from recent smallpox survivors (obtained 10 days after lesions had scabbed over) to treat the rest of the incoming patients. The result: there were no further deaths recorded — 250/250 patients survived. This outcome required relatively large quantities of serum, which would have probably been impractical under most field conditions and may explain why this treatment was not adopted on a wider scale. Most Current Opinion in Immunology 2004, 16:443–450
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importantly, this study [75] clearly demonstrates how therapeutic antibody-mediated intervention can reduce smallpox mortality from 30% to less than 0.4% when performed under the proper conditions; at an early stage of disease, and using convalescent serum of high potency [53].
9.
Conclusions
12. Halsell JS, Riddle JR, Atwood JE, Gardner P, Shope R, Poland GA, Gray GC, Ostroff S, Eckart RE, Hospenthal DR et al.: Myopericarditis following smallpox vaccination among vaccinia-naive US military personnel. Jama 2003, 289:3283-3289.
Quantitative analysis of long-term immunity following smallpox vaccination clearly demonstrates that antiviral T cell, B cell, and antibody defense mechanisms remain in place for decades, and in some instances, even for life. These results are further substantiated by historical records dating back to the time of Edward Jenner, each demonstrating protective immunity against lethal smallpox infection that lasts for many years, often in the absence of re-vaccination or natural exposure to orthopoxviruses. These results prove that lifelong immunity is not only attainable (at least for certain microbes such as poxviruses) but it also sets the bar for future vaccine development against other microbial pathogens.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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13. Centers for Disease Control and Prevention (CDC): Cardiac deaths after a mass smallpox vaccination campaign–New York City, 1947. MMWR Morb Mortal Wkly Rep 2003, 52:933-936. This study examined the number of cardiac deaths that occurred immediately following the vaccination of more than six million people within a four week period in 1947 and provides compelling evidence that the three heart attacks observed in recently vaccinated individuals in 2003 were probably only coincidence and not directly related to receiving smallpox vaccination. 14. Jenner E: An inquiry into the causes and effects of the variole vaccine, or cow-pox. London; 1798. 15. Jenner E: A continuation of facts and observations relative to the variole vaccine, or cow-pox. London, 1800. 16. Ennis FA, Cruz J, Demkowicz WE Jr, Rothman AL, McClain DJ: Primary Induction of Human CD8R Cytotoxic T Lymphocytes and Interferon- gamma-Producing T Cells after Smallpox Vaccination. J Infect Dis 2002, 185:1657-1659. 17. Frey SE, Newman FK, Cruz J, Shelton WB, Tennant JM, Polach T, Rothman AL, Kennedy JS, Wolff M, Belshe RB et al.: Dose-related effects of smallpox vaccine. N Engl J Med 2002, 346:1275-1280. 18. Weltzin R, Liu J, Pugachev KV, Myers GA, Coughlin B, Blum PS, Nichols R, Johnson C, Cruz J, Kennedy JS et al.: Clonal vaccinia virus grown in cell culture as a new smallpox vaccine. Nat Med 2003, 9:1125-1130. 19. Terajima M, Cruz J, Raines G, Kilpatrick ED, Kennedy JS, Rothman AL, Ennis FA: Quantitation of CD8R T Cell Responses to Newly Identified HLA-A*0201-restricted T Cell Epitopes Conserved Among Vaccinia and Variola (Smallpox) Viruses. J Exp Med 2003, 197:927-932. This study was the first to identify vaccinia-specific CD8þ T-cell epitopes in humans and shows the utility of peptide tetramer staining analysis in quantitation of virus-specific CD8þ T-cell populations. 20. Crotty S, Felgner P, Davies H, Glidewell J, Villarreal L, Ahmed R: Cutting Edge: Long-Term B Cell Memory in Humans after Smallpox Vaccination. J Immunol 2003, 171:4969-4973. This study demonstrated long-term T-cell- and B-cell-mediated immunity following smallpox vaccination with two main conclusions: antibody production and B-cell memory are maintained at a very stable level, but T-cell memory declines slowly over time with a half-life of about 14 years. Similar results were observed in [24]. 21. Harrington LE, van der Most R, Whitton JL, Ahmed R: Recombinant vaccinia virus-induced T-cell immunity: quantitation of the response to the virus vector and the foreign epitope. J Virol 2002, 76:3329-3337. 22. Drexler I, Staib C, Kastenmuller W, Stevanovic S, Schmidt B, Lemonnier FA, Rammensee HG, Busch DH, Bernhard H, Erfle V et al.: Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines. Proc Natl Acad Sci USA 2003, 100:217-222. 23. Poccia F, Gioia C, Montesano C, Martini F, Horejsh D, Castilletti C, Pucillo L, Capobianchi MR, Ippolito G: Flow cytometry and T-cell response monitoring after smallpox vaccination. Emerg Infect Dis 2003, 9:1468-1470. 24. Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, Sexton GJ, Hanifin JM, Slifka MK: Duration of antiviral immunity after smallpox vaccination. Nature Medicine 2003, 9:1131-1137. This study of over 300 individuals demonstrated that antiviral immunity could be maintained for up to 75 years after a single smallpox vaccination. www.sciencedirect.com
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Moreover, analysis of multiple vaccinations revealed that, although immunity is boosted by re-vaccination, the half-life of T-cell memory remains essentially the same (T1/2 ¼ 8–15 years) and antibody levels appear to return to a certain plateau, with little or no change in the total magnitude of long-term antibody production following multiple rounds of vaccination. Similar results were observed in [20].
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