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Arvin A, Campadelli-Fiume G, Mocarski E, et al., editors. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press; 2007.

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Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis.

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Chapter 71Human cytomegalovirus vaccines

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Division of Infectious Diseases and Immunology Saint Louis University School of Medicine, Missouri, USA

Efforts to develop a human cytomegalovirus (HCMV) vaccine began more than 30 years ago in response to then recent reports that HCMV was capable of causing severe congenital disease. During the intervening years, our understanding of HCMV biology and immunology has increased dramatically. That knowledge, coupled with the introduction of several new vaccine methodologies, opened the door to an impressive expansion of HCMV vaccine research, particularly during the past decade. This chapter focuses on the principles underlying HCMV vaccine development and on the vaccine approaches that are currently under investigation.

Cytomegalovirus and human disease

The manifestations of HCMV infection vary with the age and immunocompetence of the host. In both adults and children, HCMV infection is usually asymptomatic. On rare occasions, otherwise healthy adults with primary HCMV infection will experience an infectious mononucleosis-like syndrome, with prolonged fever and mild hepatitis (Cohen and Corey, 1985). However, HCMV can cause serious morbidity and mortality when the host is unable to mount an adequate immune response or when infection is acquired in utero.

Congenital HCMV infection occurs in about 1% of children born in the USA, resulting in approximately 40 000 new infections each year (Pass and Burke, 2002; Plotkin, 1999). More than 90% of infected infants are asymptomatic at birth, and most will escape serious consequences of HCMV infection. However, even among initially asymptomatic children, 5%–15% will eventually develop sequelae of infection including hearing loss, mental retardation, chorioretinitis or cerebral palsy (Fowler et al., 1992; Revello and Gerna, 2002; Pass and Burke, 2002; Stagno et al., 1982, 1986). Children born with symptomatic HCMV disease have a substantially worse prognosis. Approximately 10% will die, and most of the survivors will display profound deficits as the result of central nervous system damage (Istas et al., 1995; Pass and Burke, 2002). All together, 4000 to 8000 children in the USA develop HCMV-related neurological disease each year, making HCMV the leading infectious cause of congenital mental retardation and deafness (Fowler et al., 1992; Plotkin, 1999).

Despite the availability of antiviral therapy, HCMV disease also remains a feared complication in persons undergoing immunosuppressive therapy for malignancies or organ transplantation, and in persons with AIDS. In these individuals, who have severely impaired cellular immunity, HCMV can affect almost any organ system, and it commonly causes pneumonia, retinitis, hepatitis and ulcerative lesions of the gastrointestinal tract (Pass, 2001). For example, HCMV causes symptomatic illness in 35%, and death in 2% of all renal transplant recipients (Adler, 1996). In hematopoietic stem cell transplant (HSCT) recipients, the most common disease manifestation of HCMV infection is interstitial pneumonia (Leather and Wingard, 2001). Historically, this disease typically occurs during the first 100 days following transplantation. However, late-onset HCMV pneumonia is becoming more common due to the use of HCMV antiviral prophylaxis or pre-emptive therapy during the first 3 months following HSCT (Boeckh et al., 1996; Leather and Wingard, 2001).

The case for a cytomegalovirus vaccine

Recognition of HCMV disease as a major public health problem has grown in the medical and scientific communities, if not among the general public. HCMV infection causes more CNS disease than did either Hemophilus influenzae b or congenital rubella prior to their near eradication in the USA through vaccination (Pass and Burke, 2002). Moreover, it has been observed that HCMV infection now causes as many cases of mental retardation as the common genetic syndromes, trisomy 21 and fragile X chromosome (Plotkin, 1999). The disease burden associated with cytomegalovirus infection is estimated to cost the US healthcare system at least 4 billion dollars annually, with the majority of the cost attributable to long-term sequelae experienced by individuals who acquire congenital HCMV disease. These data placed HCMV in the highest priority grouping of vaccine targets in a recent Institute of Medicine report (Stratton et al., 2000).

One alternative to vaccination, prevention of HCMV infection through public health measures, is complicated by the high prevalence of HCMV, its persistence following primary infection, its many avenues of transmission (including blood, urine, saliva, semen, breast milk, donated organs) and its propensity to be shed for long periods of time following primary infection, especially by children (Pass, 2001). Of particular relevance, non-pregnant women evaluated in a randomized, controlled trial who were given explicit instructions on methods to avoid HCMV infection nonetheless acquired HCMV infection at rates equivalent to women who received no counseling (Adler et al., 1996).

The successful prevention and treatment of HCMV disease in immunocompromised patients has greatly improved in recent years due to continuing refinements in prophylactic and preemptive therapy for high risk individuals as well as an expanding arsenal of antiviral agents. Despite this, as many as 50% of solid organ transplant recipients develop symptomatic HCMV disease resulting in significant morbidity (Fishman and Rubin, 1998; Patel and Paya, 1997; Sia and Patel, 2000; Simon and Levin, 2003), and even with optimal therapy, mortality from HCMV pneumonia in hematopoietic stem cell transplant recipients may exceed 40% (Leather and Wingard, 2001). Recent data also suggest that antiviral therapy offers some benefit to newborns with HCMV disease (Michaels et al., 2003; Kimberlin et al., 2003). However, treatment alone will undoubtedly benefit only a minority of infants with congenital HCMV disease. Indeed, most infants who ultimately develop sequelae of HCMV infection are asymptomatic at birth and, thus, would not be considered for treatment (Griffiths, 2002). Also, treatment requires long-term antiviral therapy, which carries a substantial risk of complications, and treatment after birth is unlikely to repair organ damage, especially to the central nervous system, that occurred in utero (Kimberlin et al., 2003; Michaels et al., 2003). For all these reasons, then, a vaccine that prevents infection with HCMV, or at least mitigates its effects in vulnerable persons, is essential for eradicating the often devastating disease caused by HCMV.

Natural immunity confers protection

When considering the feasibility and design of an HCMV vaccine, it is important to first establish that natural immunity prevents disease. This issue is complicated for HCMV because virus capable of causing disease may arrive through three distinct avenues: primary infection, reactivation of virus already residing within the host, or reinfection of a previously infected individual with a different strain of HCMV. Data directly addressing the protective efficacy of pre-existing immunity in healthy adults is sparse. However, compelling evidence that previous infection prevents reinfection comes from a study of mothers of children shedding HCMV (Adler et al., 1995). During the course of this study, 9 of 19 (47%) seronegative women developed primary infection, whereas only 3 of 42 (7%) seropositive women showed evidence of new HCMV infection, indicating 85% protection attributable to prior immunity (Plotkin, 2002). In addition, healthy adults with pre-existing immunity to HCMV were significantly protected from HCMV disease compared to seronegative individuals when challenged with the non-attenuated Toledo strain of HCMV (Plotkin et al., 1989; Quinnan et al., 1984). In this case, seropositive persons were also protected from HCMV infection, albeit to a lesser degree.

Since the primary goal of HCMV vaccination is to prevent congenital HCMV disease, the protection offered by preconceptual maternal immunity should predict the potential value of vaccination in this setting. Preconceptual immunity could protect newborns by preventing transmission of the virus to the fetus or by mitigating the effects of infection. Data indicate that maternal immunity to HCMV prior to conception provides both of these elements of protection. Primary infection during pregnancy results in transmission of HCMV to the fetus 15%–40% of the time, whereas women with preexisting immunity transmit HCMV only about 1%–2% of the time (Stagno et al., 1982; Plotkin, 2002). In accordance with these data, a recent study showed that women who have naturally acquired immunity to HCMV prior to conception are 69% less likely to give birth to an infant with congenital HCMV infection than women who are initially seronegative (Fowler et al., 2003). Moreover, congenital HCMV infections in infants born to women with HCMV immunity at the time of conception are considerably less likely to cause symptomatic disease at birth (Fowler et al., 1992; Stagno et al., 1982, 1997). In general, these children also have both fewer and less severe sequelae of HCMV infection, even when considering that adverse outcomes, such as hearing loss and mental retardation, may become apparent only months or years later (Fowler et al., 1992). Maternal immunity prior to conception, however, does not confer complete protection against HCMV transmission to the fetus. Of note, a recent report concluded that while the severity of hearing loss in HCMV congenitally infected children was less if their mothers had preexisting immunity, the incidence of hearing loss was unaffected by maternal serostatus (Ross et al., 2006). Indeed, in populations with high rates of HCMV seropositivity the majority of HCMV infections may occur in infants born to seropositive mothers even given the relatively low risk of transmission (Demmler, 1991; Pass and Burke, 2002; Stagno et al., 1977; Schopfer et al., 1978; Plotkin, 2002; Boppana et al., 2001). Many of these infections presumably occur through the transmission of reactivated maternal virus, although a significant proportion undoubtedly arise from reinfection of the mother with a different strain of HCMV followed by transmission of the new virus to the fetus (Marshall and Plotkin, 1993; Boeckh et al., 1996; Stagno et al., 1982; Boppana et al., 2001). In addition, symptomatic HCMV disease has been well documented in children born to mothers with preconceptual immunity to HCMV (Boppana et al., 1999, 2001; Schopfer et al., 1978; Ahlfors et al., 1999; Morris et al., 1994).

In conclusion, prior immunity to HCMV provides substantial protection against HCMV infection and disease, with the degree of protection estimated to be between 70% and 90% (Adler et al., 1995; Fowler et al., 2003; Plotkin, 2002). This conclusion engenders optimism that vaccination of seronegative girls prior to pregnancy may prevent a substantial proportion of cases of congenital HCMV disease and the attendant early and late sequelae. Moreover, vaccination may ultimately reduce asymptomatic infection and shedding by young children, which would lessen the reservoir of virus available to infect the fetuses of other mothers (Adler, 1988; Pass et al., 1986). Indeed, it has been calculated that a vaccine that is only 60% effective against primary infection would be sufficient to eradicate HCMV from a given community within a developed country (Griffiths et al., 2001).

Immunology of HCMV protection

Both neutralizing antibodies and cell-mediated immunity contribute to protection against HCMV disease (Table 71.1; for review see Gonczol and Plotkin, 2001; Plotkin, 2002; Pass and Burke, 2002). The importance of antibodies in preventing HCMV disease was first suggested by the observation that serious HCMV disease in newborn blood transfusion recipients was less frequent in infants born to seropositive mothers (Yeager et al., 1981). This protection was presumably due to antibodies that had been transferred from the mother to the infant prior to birth. The protective benefit of HCMV antibodies in neonates was reinforced by subsequent data showing that passively administered antibodies, in the form of HCMV immune globulin, protected premature infants from HCMV disease (Snydman et al., 1995). In addition, a nonrandomized study suggested that administration of HCMV-specific hyperimmune globulin to pregnant women may be effective in the treatment and prevention of congenital HCMV infection (Nigro et al., 2005). HCMV immune globulin also appears to offer renal, liver, heart and bone marrow transplant recipients some protection from the most severe effects of HCMV disease (Snydman, 1993; Falagas et al., 1997; Valantine, 1995; Bowden et al., 1986; Glowacki and Smaill, 1994; Messori et al., 1994). Protective levels of antibodies have not been established, but some evidence suggests that higher levels of neutralizing antibodies correlate with a lower risk for reinfection (Adler et al., 1995). Regardless of the clinical setting, however, antibodies alone offer only partial protection and appear to be more effective in mitigating serious HCMV disease than in preventing infection.

Table 71.1. Known targets of human immune responses to HCMV.

Table 71.1

Known targets of human immune responses to HCMV.

Analysis of the HCMV genomic sequence suggests that is capable of encoding in excess of 60 glycoproteins, although how many are actually expressed is unknown (Chee et al., 1990; Cha et al., 1996; Davison et al., 2003). Most HCMV neutralizing antibodies, however, appear to recognize a tiny subset of these proteins, namely, glycoprotein B (gB), glycoprotein H (gH) and the glycoprotein M-N (gM-gN) complex (Britt et al., 1990; Kari and Gehrz, 1990; Mach et al., 2000; Marshall et al., 1992, 1994; Rasmussen et al., 1991; Urban et al., 1996).

Glycoprotein B is the most abundant membrane protein in the HCMV envelope (Britt and Mach, 1996). It is highly conserved among all mammalian herpesviruses, and participates in several facets of the virus life cycle including entry and cell–cell spread (Bolds et al., 1996; Compton et al., 1993; Navarro et al., 1993). Recently, it was shown that HCMV entry into cells is mediated by gB binding to the cellular epidermal growth factor receptor (Wang et al., 2003). HCMV gB, like the gB homologues in other herpesviruses, is a large type Ⅰ membrane protein (for review see Britt and Mach, 1996; Spaete, 1994). It is cleaved by a host cell protease into two peptides, which remain disulfide-linked. Glycoprotein B is modified by N- and O-glycosylation and forms homodimers in both virions and infected cells (Mocarski and Courcelle, 2001). Glycoprotein B appears to be the most immunogenic HCMV protein. Almost all persons develop antibodies to gB following HCMV infection, and gB-specific antibodies account for 40%–70% of the total HCMV neutralizing activity in HCMV seropositive individuals (Britt et al., 1990; Cremer et al., 1985; Kniess et al., 1991; Marshall et al., 1992). Glycoprotein B contains two well-characterized major antigenic domains, AD-1 and AD-2, that are capable of inducing neutralizing antibodies during infection (Britt et al., 1988; Kniess et al., 1991; Marshall et al., 2000; Meyer et al., 1992; Wagner et al., 1992). The antibody response after natural infection is directed most frequently to AD-1, which is highly conserved in clinical isolates (Schoppel et al., 1996; Wada et al., 1997; Chou and Dennison, 1991). However, 11% of HCMV seropositive persons lacked antibodies to linear epitopes on either AD-1 or AD-2, but had neutralizing activity suggesting that for some individuals, different epitopes in gB or epitopes in other HCMV proteins may be more important in the generation of virus neutralizing responses (Marshall et al., 2000).

Glycoprotein H is a relatively abundant component of the virion envelope and is also conserved among the mammalian herpesviruses, although it is much more divergent than gB (Mocarski and Courcelle, 2001). HCMV gH has been shown to participate in membrane fusion, and it may play role in virus entry at a step following attachment (Britt and Mach, 1996; Mocarski and Courcelle, 2001; Keay and Baldwin, 1991; Rasmussen et al., 1984). Glycoprotein H is also an important target of host immune responses, and almost all persons infected with HCMV develop gH-specific antibodies (Urban et al., 1996). In some cases, gH may be the dominant target of neutralizing antibodies as it has been shown to account for 0–58% of the total virus neutralizing activity in persons with a past history of HCMV infection (Urban et al., 1996). Like gB, antigenic domains have been identified in gH (Simpson et al., 1993). Interestingly, HIV-infected persons with CD4 counts less than 100 cells/mm3 who had histories of past HCMV infections rarely had detectable gH antibody titers compared to persons with higher CD4 counts, while gB titers were unaffected by the CD4 count (Rasmussen et al., 1994). Given that HIV-infected persons with CD4 counts less than 100 cells/mm3 are at high risk for retinitis due to reactivated HCMV, this finding raises the possibility that gH antibodies may be necessary for containing reactivated virus in some settings.

Recently, the gM-gN complex was recognized as an important target of antibody responses in seropositive adults (Kari and Gehrz, 1990; Mach et al., 2000). Sera from HCMV-infected adults failed to recognize gM or gN when they were expressed alone; however, sera from 62% of previously infected individuals reacted with the gM–gN complex. The importance of gM–gN antibodies in preventing HCMV disease is unknown. A possible link between gM–gN antibodies and human disease, however, was suggested by the observation that the 14 of 16 congenitally infected infants lacked detectable antibodies against this complex, whereas most adults in the same study had gM–gN antibodies (Mach et al., 2000).

In addition to gB, gH and gM–gN, antibodies to several non-envelope HCMV proteins, including pp65, IE1, pp150, pp28, pp71 and pp52, are commonly detected in seropositive people (Pass and Burke, 2002). It remains to be determined whether these antibodies contribute to protection against HCMV infection and disease.

The pivotal role for cell-mediated immunity in the control of HCMV infection is underscored by the fact that virtually all cases of severe HCMV disease not associated with congenital infection occur in persons with profoundly impaired cellular immunity, and the severity of HCMV disease typically correlates with the degree of immunosuppression. Specifically, HLA-restricted CD8+ cytotoxic T lymphocyte (CTL) responses are crucial for the control of HCMV disease in immunocompromised persons (Quinnan et al., 1982; Li et al., 1994; Reusser et al., 1991). Allogeneic marrow transplant recipients are at high risk for HCMV disease until their CD8+ CTLs return. The fundamental importance of CTL responses in controlling HCMV disease was directly assessed by an adoptive transfer study in which marrow transplant recipients received serial transfusions of HCMV-specific CTLs (Walter et al., 1995). None of the 14 very high-risk patients in this study developed HCMV viremia or disease. Therefore, considerable effort has been devoted in recent years to identifying the HCMV targets of CTL responses since the induction of such responses may be imperative for the success of an HCMV vaccine. This work led to the discovery that the tegument protein pp65 is the dominant target of virus-specific CTLs. In persons with past HCMV infection, approximately 70–90% of all CTLs that recognize HCMV-infected cells are specific for this protein (Boppana and Britt, 1996; Kern et al., 2002; McLaughlin-Taylor et al., 1994; Wills et al., 1996). Recently, specific peptides derived from the pp65 sequence have been identified that are able to induce HCMV-specific CTL in an HLA-A24-restricted manner (Akiyama et al., 2002; Masuoka et al., 2001).

While it is striking that a single protein induces so much of the CTL response directed against such a complex virus, other HCMV proteins have also been shown to contain CTL epitopes. Most notable among these are the HCMV immediate-early protein, IE1, and the tegument protein, pp150, which in some individuals appears to induce CTL responses with similar precursor frequencies to pp65 (Kern et al., 2000; Gyulai et al., 2000; La Rosa et al., 2005). Like pp65, IE1- and pp150 derived peptides that induce CTL in an HLA-restricted manner have been identified (Frankenberg et al., 2002; La Rosa et al., 2005). In addition, other HCMV antigens that are capable of inducing CTL responses including gB, gH, pp150, pp28, pp50, US2, US3, US6, and US18 (Boppana and Britt, 1996; Gyulai et al., 2000; Elkington et al., 2003).

The effectiveness of an HCMV vaccine is likely to be enhanced by, and may absolutely require, the induction of HCMV-specific CTL responses. Such responses would be expected following inoculation with live attenuated vaccines. However, other vaccine approaches, such as vectored vaccines and DNA vaccines, as discussed below, may require the inclusion of specific CTL epitopes to achieve similar results. Moreover, multiple CTL epitopes may need to be included to ensure maximal coverage in the community at large.

Vaccine development

HCMV has been the target of active vaccine development efforts since the 1970s. Early work focused on the development of live-attenuated vaccines, which have now been tested in numerous human trials and, as a family, continue to show considerable promise. With advances in molecular techniques, and rapidly expanding knowledge of HCMV biology and immunology, several other approaches are currently being applied to the development of safe and effective HCMV vaccines (Table 71.2).

Table 71.2. Status of HCMV vaccines currently being tested.

Table 71.2

Status of HCMV vaccines currently being tested.

Replicating vaccines

The first HCMV vaccine tested in humans was AD169, a laboratory-adapted strain of HCMV made by passaging virus isolated from human adenoidal tissue a total of 54 times in four different cultured human fibroblast cell lines (Elek and Stern, 1974). A lysate containing infectious virus derived from sonicated AD169-infected cells was administered to HCMV seronegative adults. Twenty-five of 26 volunteers (96%) who received 10 000 plaque forming units (pfu) of virus subcutaneously seroconverted. The vaccine was safe and well tolerated with 12 of 26 recipients exhibiting minor local reactions and one person developing lymphadenopathy and lymphocytosis. None of the vaccinees excreted virus based on cultures of throat washings and urine. Two vaccinees tested a year later showed no reduction in antibody titers. However, evaluation of some of these subjects 8 years later revealed that only half had detectable HCMV antibody or lymphocyte transformation responses (Stern, 1984).

A second clinical trial of AD169 was conducted a few years after the first (Neff et al., 1979). The virus used in this trial was passaged an additional five times and prepared as a filtered sonicate of infected cells. Twenty-four adult men were vaccinated subcutaneously, and all 20 of the initially seronegative individuals developed antibodies to HCMV by one month following vaccination. One year later, immune adherence antibodies had declined slightly while complement-fixing antibodies had declined significantly. Participants with pre-existing immunity to HCMV exhibited no antibody response to vaccination. The vaccine virus could not be detected in leukocytes, urine or throat specimens from the vaccinated seronegative persons and was not transmitted to any of the 10 seronegative contacts of the vaccine recipients, which was taken as evidence for lack of contagiousness of the vaccine virus. AD169 was not pursued further as a vaccine candidate. Instead, attention turned to the HCMV Towne strain as a potential live attenuated vaccine, and Towne remains today the best-studied HCMV vaccine candidate.

The Towne strain of HCMV was isolated in 1970 from the urine of a 2-month old infant with congenital disease, then passaged 125 times exclusively in WI-38 human diploid fibroblasts, including three clonings (Plotkin et al., 1975). It was characterized in 1975 prior to its use in clinical trials and was shown to possess several characteristics that distinguished it from native HCMV indicating that it had been altered by cell culture passage. These included increased production of cell-free virus, thermostability and trypsin resistance. In addition, safety tests in various animals and cell lines showed the virus stocks to be free of adventitious agents.

In the first published human trial describing Towne vaccination, all 10 seronegative adults inoculated intramuscularly seroconverted within four weeks while 11 seronegative adults inoculated both intranasally and orally failed to seroconvert (Just et al., 1975). Five persons with pre-existing immunity to HCMV were vaccinated with Towne and showed no increase in antibody titers. None of the participants had systemic symptoms or atypical lymphocytosis; however, 7 of the original 10 seronegative vaccinees developed mild local reactions beginning 14–16 days following vaccination and lasting about a week. As with the AD169, Towne could not be isolated from the leukocytes or urine of any vaccinee, suggesting that Towne is attenuated and unable to persist in vaccinated persons.

A second early trial with Towne largely reinforced the findings from the first trial (Plotkin et al., 1976). Once again, persons inoculated intranasally failed to seroconvert whereas all seronegative volunteers who were vaccinated subcutaneously acquired HCMV-specific antibodies, and no excretion of vaccine virus from these individuals could be documented. In contrast to the earlier study, however, all 4 seronegative participants in this trial acquired complement-fixing antibodies compared to 1 of 10 in the first trial. Also, both initially seropositive vaccinees developed IgM antibodies, raising the possibility that Towne may reinfect persons who had previously been infected with native HCMV.

Subsequent human trials with Towne confirmed its ability to elicit both binding and neutralizing antibodies, and demonstrated that Towne-induced antibodies have similar specificities to the antibodies arising from natural HCMV infection (Gonczol et al., 1989). Early studies showed that both binding and neutralizing antibody titers in response to Towne vaccination were substantially lower than those following natural infection (Gonczol et al., 1989; Adler et al., 1995). However, a more recent study showed that a new lot of Towne vaccine induced titers of neutralizing antibodies comparable to those induced by natural infection and that the response was dose dependent (Adler et al., 1998). In this study, as in earlier trials, the level of Towne-induced antibodies waned over the course of a year, while those in naturally seropositive women remained stable.

The ability of Towne to induce cellular immune responses is well documented. Towne vaccination of healthy seronegative adults uniformly results in HCMV-specific lymphocyte responses, a surrogate for CD4+ cell activation, which persist for at least 10 months (Gehrz et al., 1980; Starr et al., 1981; Fleisher et al., 1982; Plotkin et al., 1989; Adler et al., 1995, 1998). In addition, Towne consistently elicits HCMV-specific CD8+ CTL responses in immunocompetent individuals (Quinnan et al., 1984; Adler et al., 1998).

Towne has also been tested in a series of studies in prospective kidney transplant recipients, a population that is at high risk for HCMV disease following transplantation (Glazer et al., 1979; Marker et al., 1981; Starr et al., 1981; Plotkin et al., 1984, 1991, 1994; Brayman et al., 1988; Balfour, 1991). Most renal transplant candidates developed humoral and cellular immune responses following Towne vaccination. The responses, however, tended to be delayed or diminished when compared to those of healthy vaccinees (Starr et al., 1981; Plotkin et al., 1984, 1991). Three controlled trials have been conducted in which renal transplant candidates received either Towne vaccine or a placebo (Balfour, 1991; Plotkin et al., 1991, 1994). Each of these trials yielded similar results (Table 71.3). Vaccination with Towne failed to prevent HCMV infection following transplantation, and while the incidence of total HCMV disease was decreased, this effect did not achieve statistical significance. However, in the highest risk population, namely HCMV seronegative persons who received a kidney from a seropositive donor, the incidence of severe HCMV disease was reduced by 72%–100%, a degree of protection comparable to that stimulated by natural infection (Plotkin et al., 1991).

Table 71.3. Protective efficacy of Towne vaccination in seronegative renal transplant recipients who received kidneys from seropositive donors.

Table 71.3

Protective efficacy of Towne vaccination in seronegative renal transplant recipients who received kidneys from seropositive donors.

The efficacy of Towne vaccination was also assessed in a controlled challenge study. In that study, seronegative adults were vaccinated with Towne and challenged with the non-attenuated, low-passage Toledo strain of HCMV (Plotkin et al., 1989). The ability of Toledo to cause disease was confirmed when all 6 seronegative, unvaccinated persons challenged subcutaneously with 10 or 100 pfu of Toledo developed clinical symptoms of HCMV disease as well as evidence of infection based positive viral cultures from their blood, urine and/or saliva. Twelve adults who had been vaccinated with Towne 1 year earlier were challenged with Toledo. All 5 who were challenged with 10 pfu of Toledo were protected from infection and disease. However, of the 7 Towne-vaccinated persons who were challenged with 100 pfu of Toledo, most (4/7) showed evidence of infection based on virus culture, one had clinical illness and 3 had laboratory abnormalities suggestive of HCMV infection. In contrast, naturally seropositive persons were protected from challenge with 100 pfu of Toledo; however, 5/5 seropositive individuals challenged with 1000 pfu of Toledo exhibited clinical disease, laboratory abnormalities and/or positive HCMV cultures. Therefore, in this small study, Towne appeared to afford some protection against HCMV infection and disease, but less than natural infection. This study and an earlier challenge study in seropositive, unvaccinated individuals (Quinnan et al., 1984) provided valuable information not only on the protective efficacy of Towne relative to natural infection, but also on the dose dependence and natural history of HCMV disease in immunocompetent adults. However, challenge studies with HCMV may no longer be possible given contemporary regulatory standards designed to ensure volunteer safety.

To assess the protective efficacy of Towne in a more natural setting, a placebo-controlled study was performed in seronegative women with children in daycare. This was, in effect, a challenge study as the parents of children in daycare are at high risk for HCMV infection (Pass et al., 1986). In this population, Towne vaccination failed to protect women from HCMV infection, while natural infection was highly protective against re-infection with HCMV (Adler et al., 1995).

Towne remains the only HCMV vaccine candidate that has completed efficacy testing in any human population. In the final estimation, Towne vaccination induces both humoral and cellular immunity, and it is capable of providing some protection against HCMV disease in certain settings. However, it is clearly less protective than natural infection, particularly in its ability to prevent infection with native HCMV, which may be critical for preventing congenital disease. In an effort to enhance the immunogenicity of Towne, the co-administration of recombinant human IL-12 with Towne was evaluated in a recent clinical trial (Jacobson et al., 2006). This combination proved to be well tolerated and resulted in enhanced HCMV-specific antibody and T cell responses.

The nature of the deficit in Towne’s ability to stimulate protective immunity is unknown, but it has been suggested that the lower neutralizing antibody titers induced by Towne compared to natural infection may be at fault (Wang et al., 1996). Regardless, the experience with Towne suggested that it is overly attenuated and prompted researchers to pursue less attenuated live vaccines that will, ideally, retain the excellent safety and tolerability profiles of Towne.

The high level of attenuation exhibited by Towne is presumably due to genetic mutations introduced during its extensive passage in cultured cells (Huang et al., 1980; Prichard et al., 2001). Furthermore, recent data indicate that HCMV Towne contains numerous mutations throughout in its genome when compared to the non-attenuated Toledo strain of HCMV (G. Kemble, personal communication, 2003); however, the specific mutations causing attenuation are not known. In an effort to produce a vaccine that is intermediate in attenuation between HCMV Towne and wild-type virus, genetic recombinants were constructed in which regions from the HCMV Toledo genome were substituted for the corresponding regions of the Towne genome using cosmid-based mutagenesis (Kemble et al., 1996). Four independent chimeric viruses (referred to as Chimeras I–IV) were produced in which every region of the Towne genome was replaced sequentially by Toledo sequences (Fig. 71.1). Each of the four chimeric vaccine candidates made using this approach contains the UL/b region of the HCMV genome. This region, which is predicted to encode 19 proteins, is universally found in the genomes of circulating HCMV isolates but is not present in it entirety in many HCMV strains that have undergone extensive passage in cell culture, such as AD169 (Cha et al., 1996). It was hypothesized that by using this approach, one or more of the Towne/Toledo recombinants would contain some, but not all, of the mutations that confer attenuation on Towne. This, in turn, should result in a chimeric virus that is attenuated relative to Toledo, but less attenuated than Towne.

Fig. 71.1. Genomic structures of the two parental strains and four chimeras.

Fig. 71.1

Genomic structures of the two parental strains and four chimeras. The specific regions of each chimera’s genome derived from Towne( Image 9780521827140c71_figu001.jpg) and Toledo( Image 9780521827140c71_figu002.jpg) are shown. The UL/b’ region( Image 9780521827140c71_figu002.jpg) of Toledo is marked(courtesy of George Kemble)

The four Towne/Toledo recombinant viruses were evaluated in a recently completed double-blinded, placebo controlled clinical trial (Heineman et al., 2003). The study was designed to determine whether the vaccine candidates are safe and well tolerated, whether they are attenuated relative to Toledo and whether they are shed in the blood, urine or saliva of vaccinees. Healthy HCMV seropositive adults each received a single dose of 1000 pfu of one of the four investigational vaccines or an inactive placebo. Participants were evaluated weekly for 8 weeks, then less frequently for the remainder of a year, for clinical or laboratory evidence of HCMV infection and disease. All four vaccine candidates were safe and well-tolerated although as a group they produced more local reactogenicity than the placebo. As predicted, each of the Towne/Toledo chimeric vaccine candidates was attenuated relative to Toledo based on comparison to the previous Toledo challenge data discussed above (Quinnan et al., 1984; Plotkin et al., 1989). This attenuation was evident from the paucity of laboratory abnormalities suggestive of HCMV infection in the recipients of the chimeric vaccine candidates in contrast to those noted in HCMV seropositive persons who had received the same dose of Toledo (Table 71.4). However, the degree of attenuation of the four Towne/Toledo chimeras relative to each other could not be discerned from this initial trial. Like Towne, none of the vaccine candidates was cultured from the blood, urine or saliva of any vaccinee or any of their close contacts suggesting that systemic infection did not occur in this population. Immunogenicity data from this trial are pending. Future studies are planned to address the safety and immunogenicity of these vaccine candidates in seronegative persons, who comprise the target population for vaccination.

Table 71.4. Towne/Toledo chimeric vaccine candidates are attenuated relative to Toledo in natural seropositives inoculated with 1000 pfu.

Table 71.4

Towne/Toledo chimeric vaccine candidates are attenuated relative to Toledo in natural seropositives inoculated with 1000 pfu.

Subunit vaccines

Subunit vaccines, in which a single or a few specific proteins are used in combination with an adjuvant to stimulate protective immunity, are attractive for several reasons. Most importantly, they are not infectious and contain no genetic material thus eliminating some safety concerns. Also, using modern molecular genetic methods, large amounts of the vaccine antigens can be produced easily and cheaply. Finally, subunit vaccines for numerous infectious diseases have been studied extensively in animals and, in some cases, have progressed to human trials. Of particular relevance, recent data suggest that a subunit vaccine derived from a single herpes simplex virus (HSV) type 2 protein may protect seronegative women from disease caused by primary HSV infection (Stanberry et al., 2002).

As discussed above, HCMV gB is both highly immunogenic and highly conserved between HCMV isolates. For these reasons, and because it is the best studied HCMV glycoprotein, gB has been the primary antigen used in most HCMV subunit vaccine studies. Glycoprotein B used in vaccine studies has been modified to facilitate its purification. To that end, its hydrophobic transmembrane domain has been removed, and it has been mutated to eliminate its internal proteolytic cleavage site. Thus, the form of gB used in human trials is expressed in Chinese hamster ovary cells and is purified as an excreted protein consisting of a single 807 amino acid peptide (Pass et al., 1999).

The choice of vaccine adjuvants has considerable impact on the immunogenicity of subunit vaccines. In the human trials reported to date, HCMV gB has been combined with either aluminum hydroxide (alum), the adjuvant used in the licensed hepatitis B vaccines, or MF59, a proprietary oil-in-water emulsion of squalene (Chiron Vaccines, Emeryville California) (Ott et al., 1995). MF59 was previously shown to induce higher antibody titers than alum when combined with a variety of viral antigens (McElrath, 1995). Accordingly, virtually all healthy seronegative adults inoculated at 0, 1 and 6 months with HCMV gB combined with MF59 developed levels of binding and neutralizing antibodies comparable to those induced by natural infection, whereas persons vaccinated similarly with gB/alum produced significantly lower titers of gB-specific antibodies (Pass et al., 1999). Also, IgG and IgA antibodies to gB were present in the saliva or nasal washes of most gB/MF59 recipients (Wang et al., 1996). Dose comparison studies demonstrated that low doses of gB (5 μg) combined with MF59 elicited antibody responses similar to those observed with higher doses (30 or 100 μg) (Pass et al., 1999; Frey et al., 1999). Toddlers who received three 20 μg doses of gB/MF59 developed mean gB binding and neutralizing antibody titers six-fold higher than were observed in earlier adult studies (Mitchell et al., 2002). Following vaccination with gB/MF59, neutralizing antibody titers waned rapidly, and it was suggested that insufficient CD4+ responses might have contributed to this decline. Nonetheless, neutralizing antibody titers rebounded dramatically after an additional dose of vaccine (Plotkin, 2001; Pass and Burke, 2002). Vaccination with gB/MF59 also induced strong lymphocyte proliferative responses to both gB and HCMV, and these declined little during the year following vaccination (Pass and Burke, 2002). The HCMV subunit vaccines caused more injection site pain than placebo; however, both were generally well tolerated.

The studies described above laid the groundwork for efficacy trials of the gB/MF59 subunit vaccine. Currently, the ability of gB/MF59 to prevent congenital HCMV infection in the children of healthy seronegative women is being assessed in a double-blinded, placebo controlled trial (R. Pass, personal communication, 2006). In addition, a study is being planned to determine whether gB/MF59 vaccination protects adolescents from HCMV infection, a population that may ultimately be targeted for vaccination (D. Bernstein, personal communication, 2003). While HCMV subunit vaccines have thus far focused on the use of gB as the immunogen, future subunit vaccines may include gH, gM-gN, and perhaps other HCMV antigens.

Vectored vaccines

A number of viruses have been utilized as vectors to express potential vaccine antigens. Of these, the attenuated ALVAC strain of canarypox has been most extensively employed as a vector for the delivery of HCMV antigens (Baxby and Paoletti, 1992; Tartaglia et al., 1992; Plotkin et al., 1995). The ALVAC genome will accommodate the insertion of large exogenous DNA fragments providing great flexibility in the choice of antigen genes or combinations of genes. While it can infect human cells and express foreign antigens, its own genome is not replicated and progeny virions are not produced, thus reducing the risk of vaccine-associated complications. Most importantly from the perspective of vaccine efficacy, foreign antigens expressed by ALVAC are transported and processed authentically within cells allowing their presentation in the context of MHC class Ⅰ molecules. This, in turn, may facilitate the stimulation of CTL responses that mimic those of natural infection (Pialoux et al., 1995; Clements-Mann et al., 1998; Taylor et al., 1995; Plotkin et al., 1995).

Because of its preeminence as target for neutralizing immunity, gB was the first HCMV antigen chosen for expression by ALVAC. Studies in mice and guinea pigs showed that two doses of ALVAC-gB induced neutralizing antibodies and also high levels of HCMV-specific CD8+ CTL responses (Gonczol et al., 1995). UV-inactivated ALVAC-gB failed to induce CTLs indicating that de novo synthesis of gB was required. In addition, prior vaccinia virus exposure did not inhibit the gB-specific immunity induced by ALVAC-gB in mice, addressing an issue that may become more important if vaccinia vaccination rates increase in response to bioterrorism concerns. The promise of the initial animal studies, however, was not fully realized in early human trials. Even after three doses of ALVAC-gB, seronegative adults failed to develop significant HCMV neutralizing antibody titers perhaps reflecting low levels of antigen production (Adler et al., 1999).

ALVAC-gB proved far more successful when it was used to “prime” the immune system in so-called prime-boost vaccination strategies. HCMV seronegative adults who were primed with two doses of ALVAC-gB at days 0 and 30, then boosted with a single dose of Towne at day 90 developed binding and neutralizing antibody titers at least as high as naturally seropositive individuals (Adler et al., 1999). Similarly, individuals primed with two doses of ALVAC-gB, then boosted with gB/MF59 subunit vaccine developed high antibody titers and lymphoproliferative anti-HCMV responses. These humoral and cellular responses, however, were not significantly different from those elicited by three doses of gB/MF59 alone, thus demonstrating no clear benefit to priming with ALVAC-gB (Bernstein et al., 2002).

The greatest value of canarypox-based vaccines may derive from their ability to stimulate specific CTL responses. ALVAC expressing pp65, an abundant tegument protein that is a major CTL target in naturally HCMV seropositive persons, induced CD8+ CTL responses in all seronegative persons tested (Berencsi et al., 2001). The CTL responses were detected after two doses of ALVAC-pp65 and were present at frequencies comparable to those seen in naturally seropositive individuals. Ultimately, canarypox-based vaccines that express both CTL and neutralizing antibody targets, such as pp65 and gB, may be used in prime-boost vaccination protocols. For example, two doses of an ALVAC-based vaccine may be followed by a boost with a subunit or live-attenuated vaccine, to confer high levels of both cellular and humoral immunity.

Peptide vaccines

The development of peptide vaccines for HCMV represents an effort to directly stimulate a protective CTL response. Toward this end, a 9 amino acid minimal cytotoxic epitope derived from HCMV pp65 was identified and lipidated at its amino terminus to allow its administration without an adjuvant (Diamond et al., 1997; Martinon et al., 1992). HLA A2.1 transgenic mice immunized with this peptide, in combination with the PADRE pan-HLA-DR T-helper epitope, developed HCMV-specific CTL. Subsequent studies showed that linking the HCMV pp65 epitope directly to the PADRE epitope in the same peptide elicited vigorous HCMV-specific CTL responses in HLA transgenic mice when delivered either subcutaneously or intranasally (La Rosa et al., 2002). In either case, the response was significantly enhanced by the co-administration of CpG-containing single stranded DNA, which enhances the immune response and biases it in the direction of Th1 activity (Klinman, 2003). A repertoire of HCMV epitopes specific for different HLA alleles has been defined that should provide 90% coverage to the Caucasian population, although the derivation of four or more additional CTL epitopes would be needed to attain 90% coverage for African Americans or Asians (Longmate et al., 2001). Peptide vaccination may have its primary utility in therapeutic rather than prophylactic vaccination as HLA allele-specific peptides may be limited in their ability to elicit immunity in the population at large (Gonczol and Plotkin, 2001). To that end, the HCMV-pp65-specific memory CTL response could be amplified in a hematopoietic stem cell donor prior to transplantation by administration of a peptide vaccine, thereby providing protection against HCMV disease in the recipient (La Rosa et al., 2002).

DNA vaccines

The initially surprising discovery that direct injection of purified DNA encoding specific antigens can induce protective immunity led to the development of DNA vaccination strategies for many pathogens including HCMV (Wolff et al., 1990; Ulmer et al., 1993). In this approach, as with live attenuated and vectored vaccines, HCMV antigens are synthesized and processed using authentic cellular pathways thus allowing their expression in the context of MHC class Ⅰ molecules (Tang et al., 1992; Ulmer et al., 1993; Raz et al., 1994). This approach, therefore, has the potential to induce both humoral and cellular immunity to HCMV. While DNA vaccines for HCMV have yet to be studied in humans, compelling data, some of which is discussed below, has been generated in animal models.

The first DNA vaccine for HCMV consisted of a plasmid containing the gene for pp65 (Pande et al., 1995). Most of the mice injected intramuscularly with this plasmid developed antibodies to pp65 confirming that vaccination with HCMV DNA was capable of eliciting an antigen-specific immune response. The protective efficacy of DNA vaccination was subsequently demonstrated in mice (Gonzales Armas et al., 1996). After inoculation with the murine CMV (MCMV) pp89 gene, the major target for CD8+ T-cells (Reddehase and Koszinowski 1984; Holtappels et al., 1998), mice exhibited 45% protection against lethal challenge as well as significantly lower viral titers in the spleen and salivary glands. A DNA vaccine expressing a different MCMV antigen, M84, which is homologous to HCMV pp65, also afforded some protection to mice. Coimmunization with both pp89 and M84 DNA vaccines provided the best protection suggesting that the most successful DNA vaccines may express multiple antigens (Morello et al., 2000; Ye et al., 2002).

Considerable evidence suggests that both humoral and cell-mediated immunity participate in the control of HCMV disease. Accordingly, DNA vaccines expressing gB were tested in mice and shown to elicit neutralizing antibodies (Hwang et al., 1999). Therefore, a second generation of HCMV DNA vaccines was designed to stimulate both neutralizing antibodies to HCMV and HCMV-specific CTL responses. These vaccines consisted of a cocktail of plasmids encoding pp65, to stimulate CTL responses, and gB, to induce neutralizing antibodies (Endresz et al., 1999; Schleiss et al., 2000). After three immunizations, all mice developed gB- and pp65-specific antibodies, and about 60% developed pp65-specific CTL responses. Similarly, guinea pigs also developed antibodies to both antigens.

Recently, various innovative methods to enhance immune responses have been applied to HCMV DNA vaccines. Aluminum salts, which are licensed for use as adjuvants in humans, and CpG oligodeoxynucleotides have both been shown to enhance antibody responses to DNA vaccines (Ulmer et al., 1999; Klinman et al., 2000). A DNA vaccine containing the HCMV gB gene and administered with aluminum phosphate gel elicited significantly higher antibody responses in mice than the gB gene without aluminum phosphate gel, although no difference was seen in neutralizing antibody titers (Temperton, 2002; Temperton et al., 2003). However, the addition of CpG oligodeoxynucleotides to aluminum phosphate gel enhanced the ability of the gB gene to stimulate neutralizing antibodies. Using a related approach, several laboratories have shown that coinoculation of animals with genes encoding immunostimulatory molecules in addition to viral antigens can provide enhanced protection against disease (Tsuji et al., 1997; Xiang and Ertl, 1995; Cull et al., 2002). Applying this approach, mice coimmunized with the MCMV gB and type Ⅰ interferon genes exhibited enhanced protection against MCMV challenge when compared to mice that received the MCMV gB gene alone (Cull et al., 2002). It is interesting to note that DNA vaccination with type Ⅰ interferons alone also reduce the level of MCMV infection (Yeow et al., 1998; Cull et al., 2002; Bartlett et al., 2002). Recently, the use of full-length murine and guinea pig CMVs cloned as bacterial artificial chromosomes (BACs) have been used as DNA vaccines. In principle, BAC vaccines would deliver the full complement of viral genes thereby inducing a wide range of antiviral immune responses (Cicin-Sain et al., 2003; Schleiss et al., 2006). Preconceptual maternal immunization of guinea pigs with a replication-disable guinea pig CMV BAC significantly protected their offspring against congenital CMV disease (Schleiss et al., 2006).

DNA vaccines for HCMV are still in their infancy, and safety issues stemming from the inoculation of exogenous DNA still need to be resolved. Nonetheless, this approach is remarkably versatile and may ultimately have an important place in HCMV vaccine development.

Subviral particles

Upon infection with HCMV, cultured fibroblasts release not only infectious virions but also non-infectious particles (Craighead et al., 1972; Fiala et al., 1976; Sarov and Abady, 1975). These non-infectious particles may be either dense bodies, which are enveloped structures consisting of viral tegument proteins and glycoproteins but lacking a capsid, or non-infectious enveloped particles (NIEPs), which are similar to normal virions except for the absence of DNA and the presence of an additional polypeptide. Glycoprotein B and the tegument protein pp65 are major constituents of dense bodies, and sera from HCMV seropositive individuals react with these particles (Baldick and Shenk, 1996; Forghani and Schmidt, 1980; Gibson and Irmiere, 1984; Irmiere and Gibson, 1983; Roby and Gibson, 1986). For this reason, dense bodies have long been considered as possible HCMV vaccines (Bia et al., 1980; Fiala et al., 1976; Gibson and Irmiere, 1984; Sarov et al., 1975; Stinski, 1976). More recent studies showed that dense bodies enter cells efficiently, presumably through the interaction of envelope glycoproteins with cellular receptors, thus mimicking normal infection (Pepperl et al., 2000; Schmolke et al., 1995; Topilko and Michelson, 1994). Mice transgenic for human HLA-A2 were immunized with dense bodies without an adjuvant. After only a single inoculation, the mice developed high virus neutralization titers, and those that received three doses retained substantial virus neutralizing activity for at least a year (Pepperl et al., 2000). The immunized mice developed antibodies to gB, gH, pp65, pp150 and several unidentified proteins. Given the importance of cellular immunity in controlling HCMV disease, it is significant to note that the immunized mice also developed high levels of HCMV-specific CTLs despite the absence of HCMV protein synthesis. Recently, it was shown that genetically modified dense bodies containing foreign proteins could be generated (Pepperl-Klindworth et al., 2002). In light of this, it may be possible to produce dense body-based vaccines that are modified to improve their HCMV immunogenicity through the inclusion or enhanced expression of specific antigens. Dense body technology therefore, represents another innovative approach to HCMV vaccine development that deserves continued exploration.

Challenges for HCMV vaccine development

Species-specificity of HCMV

HCMV has a very limited host range, and no practical animal model for HCMV infection has been identified (Mocarski and Courcelle, 2001). While the CMVs of certain other animal species are well studied and very useful for addressing many aspects of vaccine development, they are genetically distinct from HCMV and may not behave identically during natural infection. Thus, for any new HCMV vaccine candidate initial safety and efficacy data can be derived from animal studies using analogous vaccines made from animal CMVs. However, this data will need to be interpreted cautiously, and valid safety and efficacy data will require human trials. Moreover, the species specificity of HCMV is an impediment for testing live, presumably attenuated vaccine candidates. Without the ability to assess HCMV virulence in an animal model, attenuation can only be confirmed through human trials. While this was accomplished for the Towne/Toledo chimeric vaccine candidates described above, it undoubtedly complicates vaccine development efforts.

Efficacy testing

The primary goal of HCMV vaccination is the prevention of congenital disease. However, given that the incidence of HCMV disease is about 0.1% of all births, a prospective trial to assess vaccine efficacy against HCMV disease would require tens of thousands of participants and a lengthy follow-up period to ensure that late sequelae of CMV infection, such as hearing and intelligence deficits, are not missed. However, an efficacy trial to determine the ability of a vaccine to protect newborns from HCMV infection could be done with a manageable number of subjects. Assuming a fetal infection rate of 1%, vaccine efficacy of 80% and a 2-year follow-up period, such a trial would require fewer than 5000 participants (Plotkin, 1999). Moreover, surrogate endpoints for vaccine efficacy could be tested initially to exclude less promising vaccine candidates from future large trials. For example, the ability of a vaccine to prevent infection in seronegative mothers with children in daycare would require less than 200 participants, given the high incidence of HCMV infection in that population (Plotkin, 1999).

When to vaccinate

To prevent congenital disease, HCMV vaccination should be administered to women prior to their becoming pregnant, and it has been proposed that vaccination of girls between the ages of 11 and 13 would be appropriate (Plotkin, 1999). However, the persistence of the immune response would need to be determined before a final schedule could be recommended. If a vaccine induced long-lived immunity, early childhood vaccination should be considered. If, however, immunity is less durable, later vaccination or regular boosters may be appropriate.

Summary

At present, vaccination remains the best hope for preventing most cases of congenital HCMV disease. Several new HCMV vaccination strategies have emerged in the past decade that take advantage of our current understanding of the host immune response to HCMV infection. Moreover, some older approaches, such as live attenuated vaccines, are being readdressed using modern molecular genetic approaches to enhance protective efficacy. More than ever, the development of a safe and effective HCMV vaccine seems to be an achievable goal. The most important scientific challenge that remains is to refine our understanding of the immunology of HCMV disease protection in order to rationally design vaccines that are both safe and effective. In addition, agreement must be reached within the medical and scientific communities on the best approaches for efficacy testing of new HCMV vaccines before the large trials necessary for licensure can be conducted.

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