HERPESVIRUS VACCINES

NEW VACCINES AND NEW VACCINE TECHNOLOGY

0891-5520/99 $8.00

+ .OO

HERPESVIRUS VACCINES Development, Controversies, and Applications Philip R. Krause, MD, and Stephen E. Straus, MD

Eight herpesviruses have evolved to infect humans; to persist within them; and to spread efficiently to others, generally without causing disease. Their capacity to do so reflects the genetic investment these complex DNA viruses have made in deterring host immune defenses that arise against them. The complex interplay between host and virus has made it difficult to mount useful vaccine strategies to protect against the diseases that they do occasionally inflict upon us. This article summarizes the human herpesviruses, the syndromes attributed to them, and the current state of vaccine development and prospects. The neurotropic alpha-herpesviruses include herpes simplex viruses (HSV) types 1 and 2, the causes of mucocutaneous herpes infections, and varicella-zoster virus (VZV),which causes chickenpox and zoster. Cytomegalovirus (CMV), which infects lymphocytes, monocytes, and neutrophils, and the lymphotropic human herpesviruses 6 and 7 (HHV6 and HHV-7), are classified based on their genetic content as betaherpesviruses. The two human gamma-herpesviruses, Epstein-Barr virus (EBV) and human herpesvirus 8 (also known as Kaposi‘s sarcomaassociated herpesvirus), both cause human cancers. Once a herpesvirus infects humans, it establishes a tenacious foothold, one that permits it to persist within us for life. The viruses can do so because they carry effective genetic tools to thwart the very immune The opinions expressed by Dr.Krause in this article are his own. No official endorsement by the Food and Drug Administration is implied or should be inferred.

From the Food and Drug Administration, Center for Biologics Evaluation and Research, Office of Vaccines Research and Review (PRK); and the National Institutes of Health, National Institute of Allergy and Infectious Diseases (SES), Bethesda, Maryland INFECTIOUS DISEASE CLINICS OF NORTH AMERICA VOLUME 13 * NUMBER 1 MARCH 1999

61

62

KRAUSE & STRAUS

mechanisms that successfully terminate other classical virus infections, such as polio, smallpox, and influenza. All herpesviruses establish latency, during which they express few if any protein antigens that could betray their presence to immune effector elements. In addition, they all possess means of down-regulating their presentation in the context of class I major histocompatibility complex (MHC) proteins.21,61 Some examples of these two mechanisms of immune deterrence help illustrate the rich complexity of the challenge that herpesviruses manifest as vaccine targets. HSVs rapidly spread from the mucocutaneous sites at which they are initially deposited onto human tissues to nearby sensory nerve endings. The virus core particles ascend the nerves and establish residence for life in neuronal nuclei. There, they express only one family of RNAs, ones that probably do not encode any proteins, but which enhance the ability to reactivate periodically. During latency, HSVs are truly invisible to immune surveillance. CMVs illustrate a different and highly successful series of strategic approaches to immune avoidance. It encodes at least four proteins that block viral antigen presentation in the content of MHC.5s It is so efficient at doing so that CMV-infected cells display little or no MHC proteins, stripping them, as it were, of the very labels that identify them as parts of ourselves. As such, they become susceptible to killing by natural killer cells, which are programmed to attack anything that lacks MHC proteins. CMV solves this dilemma by also encoding a protein that resembles an MHC protein, but which is not functional in antigen presentation. What capacity can a vaccine have to circumvent these formidable and highly evolved herpesvirus strategies for avoiding immune surveillance? The answer to this depends on what one hapes the vaccine to achieve. It is probably futile to presume that it could totally prevent infection, or the establishment of latency, once the infection has been initiated. But a vaccine that greatly limits the total quantity of virus that replicates at the time of initial infection may prevent significant acute illness, and there may be a smaller reservoir of latent virus from which reactivated infections might arise. Live-attenuated vaccine strains represent some of the earliest approaches to developing herpesvirus vaccines. These vaccines have the theoretical advantage of presenting all viral antigens in a natural context. The challenge is to identify a vaccine strain that is adequately immunogenic while being safe. Because of the role cell-mediated immunity (CMI) plays in containing herpesvirus reactivations, individuals with impaired CMI often are at risk for more severe infections, either with wild-type or vaccine strains. Because mild immunocompromise may be undetected in children prior to vaccination, it is important for vaccine strains to be attenuated adequately. On the other hand, many candidate herpesvirus vaccines (including some early varicella vaccines) proved to be too attenuated to induce adequate immune responses. Moreover, the issue of latency and reactivation is particularly important in live-

HERPESVIRUS VACCINES

63

attenuated vaccine development, because their ability to reactivate may limit the long-term safety of the vaccine. The difficulty in adjusting vaccine attenuation so that the virus is sufficiently immunogenic but not virulent for compromised patients led to several alternative strategies using noninfectious reagents. Although safe, these in turn have been bedeviled by insufficient immunogenicity. Several herpesvirus vaccine strategies are summarized in Table 1, and their current and potential roles in preventing herpesvirus diseases are reviewed next. We begin with and emphasize VZV because a vaccine is licensed for prevention of varicella.

The first infection with VZV leads to chickenpox (varicella), a common childhood disease characterized by widespread vesicular skin lesions and fever.74Although varicella is usually a benign process, the lesions may be superinfected with bacteria, complicating about 10% of cases (of particular concern with invasive group A streptococcus6*). Other complications include viral pneumonia, encephalitis, hemorrhagic varicella, and death. In the United States, about 100 people die from varicella each year.19 The incidence of severe varicella is relatively high in children less than 1 month of age (generally those born to mothers experiencing varicella themselves near delivery, and thus lacking in maternal antibody-derived immunity) and in adults.= Immunocompromised hosts, including otherwise normal pregnant women in their second to third trimester, are also at risk for more severe infections. Individuals generally experience only one episode of chickenpox per lifetime. It is presumed that after inhalation of aerosolized infected respiratory secretions, VZV replicates in the naso- or oropharynx and in the regional lymph nodes. This infection is believed to engender a primary viremia,7 which subsequently seeds the reticuloendothelial system, followed by a secondary viremia, which by 10 to 21 days after exposure leads to the typical varicella exanthem. Sensory nerve ganglia, which m the sites of latency for VZV, are also seeded during this time. The requirement for several rounds of replication over a 2- to 3-week period before skin disease becomes apparent may make VZV an easier target for vaccine-induced immunity than HSV-1 and -2, which replicate and cause disease at or near the site of inoculation after an incubation period as short as 2 days. When VZV reactivates, it causes zoster (shingles), a painful, dermatomal skin rash. The incidence of zoster is about 15% of the population, with older and immunocompromised individuals at highest risk.25,37, It is unusual, however, for an individual to experience more than one episode of zoster. The most common complication of zoster is chronic, occasionally debilitating pain called postherpetic neuralgia, which also becomes more common with advanced age.d3Zoster may rarely present

VZV HSV

Live-attenuated

HSV

CMV HSV EBV CMV

Subunit

DNA vaccine Peptide Poxvirus

CMV

Target

Strategy

Chiron Apollon Queensland Institute Pasteur-Merieux Connaught

Aviron SKB Chiron

T o m e + missing genes gD2t/MPL gB/gD/MF59

gB CMV-gD HLA-B3/EBNA3 ALVAC-CMV

Merck Pasteur-Meneux Aviron Cantab MA

Manufacturer

Oka R7020 RAV 9395 DISC gHTome

Designation

Table 1. HERPESVIRUS VACCINE STRATEGIES AND STATUS OF CLINICAL DEVELOPMENT ~

Licensed product Avirulent Unreported Phase I studies Low immunogenicity, studies continue Unreported Phase III studies Terminated for lack of efficacy Phase I1 studies Phase I studies Phase I studies Unreported

Status

~~

53 11 33

54

40 48 76

44 18,55 69 14 2

Reference

HERPESVIRUS VACCINES

r

65

without as dermatomal pain in the absence of skin lesions, retinal necrosis, or as viscerally disseminated zoster in the severely immunocompromised. Other complications of zoster include cerebral vasculopathies, and cranial and peripheral sensory and motor neuropathies. In patients with diminished CMI, zoster may disseminate widely to involve not only the skin, but also the liver, lungs, and central nervous system, leading to severe morbidity or death. A recent survey of VZV infections in HIV-positive children did not demonstrate increased severity of varicella in children with low CD4 counts, but zoster rates were elevated.31 Although high antibody levels to VZV are sufficient to protect an individual against chickenpox (as evidenced by the efficacy of immunoglobulin in postexposure pr0phylaxis),6~the precise correlates of immunity to zoster are less well understood. Because the incidence of zoster increases with age and declining CMI, it is believed that CMI plays an important role in preventing zoster. The rate of viral reactivation from the sensory ganglia is not well-understood. One hypothesis is that fairly frequent asymptomatic reactivations occur, periodically boosting and sustaining immunity throughout life.37 Such reactivations have been documented in immunocompromised hosts.51,88 From a public health standpoint, there are several possible goals of vaccination against VZV. One is to prevent disease in the vaccinees upon subsequent exposure. Even the modest morbidity of varicella in healthy children leads to significant economic loss when one considers parent time taken off work to care for sick children.50A second goal is to reduce the frequency at which varicella circulates in the community, thus protecting unvaccinated individuals as well as immunocompromised patients, most of whom are not acceptable candidates for vaccination with a live attenuated virus vaccine. A third goal is to reduce the incidence or complications of zoster by preventing the accumulation of latent virus during natural varicella infection. A fourth possible goal is to reduce the frequency of zoster by boosting immunity through vaccination of older adults. The Oka strain of varicella vaccine that is used today in several nations was isolated from a healthy Japanese child with chickenpox, and attenuated by repeated passage in tissue culture.77,78 An Oka-strain Since varicella vaccine was developed in Japan about 25 years then, varicella vaccines have been licensed in many other countries, including South Korea, Europe, and the United States. The vaccine strain expresses nearly all virus antigens and is capable of establishing latency and reactivating. In the United States, the vaccine is currently indicated for immunocompetent persons above 12 months of age without a history of varicella.lg, 56 It is contraindicated in people with a history of hypersensitivity to vaccine components (including gelatin and n e o m y ~ i n )Because .~~ of the theoretical risk of Reye’s syndrome, salicylates should be avoided in vaccinees for 6 weeks after vaccination. Vaccine efficacy may be compromised by administration of immunoglobulin products between 5

months prior to and 2 months after A dose of vaccine costs approximately $39 (not including the delivery costs). Pregnant women should not receive the vaccine because of the theoretical risk of congenital varicella syndrome (in early pregnancy) and the immunocompromise associated with late pregnancy. A registry of pregnant women exposed to VZV vaccine is being maintained. Women who ieceive vaccine while pregnant or within 3 months of becoming pregnant should be reported to the registry at US telephone number 800-986-8999.19,22 To date, most reported exposures have been among individuals who took vaccine prior to realizing that they were pregnant or about to become pregnant, and there is no evidence as yet of vaccine-associated teratogenicity. In immunocompromised individuals, there is significant risk associated with the administration of a live vaccine, and these individuals should not be vaccinated except as a part of clinical trials,56which are currently underway for children with several immunocompromising conditions. The major risk to these children is that of severe vaccineinduced varicella infections, which may require administration of antivirals.16,46 Low doses of steroids (inhaled steroids, or less than 2 mg/kg/ d of prednisone, up to a maximum of 20 mg/d) are not considered sufficiently immunocompromising to contraindicate v a ~ c i n a t i o n22, ~but ~~ because sufficient data are lacking, these patients should be carefully observed after vaccination. In all cases, it is prudent to consider the risks of vaccination in comparison with the risks of contracting wild-type chickenpox. In immunocompetent individuals, the Oka vaccine is safe. The most common side effects are summarized in Table 2. They include injectionsite discomfort, fever, and rashes that appear about 2 weeks after vaccination, either at the site of injection or a mild, disseminated, varicellalike rash? Because there is a low risk that the virus could be transmitted from vaccinees with rashes (or theoretically even 1 or 2 days before the rash occurs), it is advisable to avoid close contact between vaccinees and individuals at high risk (e.g., pregnancy, severe immunocompromise) for severe varicella. In a controlled US clinical trial of a high dose of vaccine, the Table 2. VARICELLA VACCINE SIDE EFFECTS IN HEALTHY VACCINEES Observation

Incidence (%)

Injection site complaints Varicella-lie rash Injection site Generalized Fever

-20 3 4-6

-10

Adapted from Merck & Co: Varivax (Varicella Virus Vaccine Live [Oka & Merck]) Package circular. In Physician’s Desk Reference, ed 52. Montvale, NJ, Medical Economics Co, 1998, p 1762; with permission.

HERPESVIRUS VACCINES

67

Oka strain was 100% effective over 1 year in preventing chickenpox in vaccinated children.84In a controlled trial of different preparations of Oka-strain vaccine in Finland, vaccine efficacy was reported at 64% to 77y0.~' The vaccine currently marketed in the United States is about 70% to 90% effective in preventing any manifestations of chickenpox. Children who experience chickenpox after vaccination generally have a mild illness, as measured by fewer lesions and a decreased likelihood of fever.12,86 These studies are summarized in Figure 1. Because children over 12 years of age and adults respond less well immunologically to a single dose of vaccine, two doses administered 4 to 8 weeks apart are recommended in those age groups. Commerically available assays for anti-VZV antibody are less sensitive than the special gpELISA test used to monitor immune responses in the vaccine trials.= Thus, up to 20% to 30% of vaccinees may not mount antibody responses to vaccination that are detectable by the commercial tests. Whatever assay is used, however, higher titers of antibody are correlated with greater protection from infections6although even vaccinees with undetectable antibody levels appear to be significantly protected against severe infection. Thus, antibody testing after vaccination does not yield definitive information regarding the immunity of the individual. Although a positive result indicates some level of immunity, it does not guarantee protection from varicella. Likewise a negative result, although implying a greater risk of infection, does not imply complete lack of protection from varicella. Guidelines for vaccination of health care providers have been a source of debate. This is because (as is the case with unvaccinated health care workers) there is no strategy using the vaccine that can provide 70

8

60

7 6

50

5

40

4

30

3

20

2

10

1

0

0 T>100"F (% cases)

Median # of lesions

Duration (days)

Figure 1. Breakthrough versus natural varicella. The severities of breakthrough varicella (defined as varicella in vaccinees) and natural varicella were compared. The percent of varicella cases with a temperature greater than lOO"F, the median maximal number of lesions, and the duration of lesions are shown from the results of a controlled study1* for vaccinees (solid bar) and unvaccinated controls (hatched bar).

68

KRAUSE & STRAUS

assurance that patients will not contract varicella from health care workers, although the vaccination can substantially reduce the risk. The Center for Disease Control and Prevention's Advisory Committee on Immunization Practices (ACIP) and the American Academy of Pediatrics Committee on Infectious Diseases (COID) recommend ensuring the immunity of all health care workers by history, serologic testing, or vaccination.19,22 Moreover, induction of primary immunity to varicella among adults and adolescents is worthwhile, because of the susceptibility of this group to more severe disease. One possible implementation of these recommendations is depicted in Table 3. A positive history of varicella is considered reliable for the purpose of deciding whom not to vaccinate; however, it is considered cost-effective serologically to test those with a negative history19,22 to determine who among them are truly susceptible. After vaccination, vaccinees who develop a rash have a low risk of transmitting vaccine-strain virus to contacts; therefore, contact should be avoided between vaccinees with rash and pregnant or immunocompromised persons who may be susceptible to complications from vaccine strain exposure. In cases of caregivers to severely immunocompromised patients (e.g., bone marrow transplant patients) for whom the risk of exposure to virus must be minimized, reassignment of vaccinated staff to other duties for the 1 to 3 week period after vaccination should be considered. Because not all adults are protected by vaccination, when a vaccinated health care provider is exposed to varicella, it is still necessary to decide whether it is appropriate to furlough them (the most conservative approach); or reassign them to care for individuals not at severe risk for varicella (the preferred approach in most settings); or simply to observe them. Some hospitals use antibody testing at the time of exposure to select between these options, because seropositive individuals have a 22 In some cases, it may be lower (but still positive) risk of chickenp~x.'~, reasonable to attempt postexposure prophylaxis of health care workers with acyclovir? Because varicella-zoster immunoglobulin may prolong Table 3. IMPLEMENTATION OF THE ACIP-COID RECOMMENDATION TO ENSURE VARICELLA IMMUNITY OF ALL HEALTH CARE WORKERS

I. Ensure immunity of health care workers by vaccinating (two doses) those without immunity Use history + / - serology to identify vaccination candidates After vaccination, avoid contact with pregnant and immunocompromised individuals Routine postvaccination serology is not recommended 11. Options after known exposure 1. Furlough 2. Reassign and observe 3. No reassignment and observe (Some hospitals use antibody levels to decide among these options) 111. If varicella occurs in a vaccine, furlough and trace contacts Datafrom references 19 and 22.

HERPESVIRUS VACCINES

69

the incubation period of chickenpox by about 1 week,19 it is generally not helpful or appropriate in this situation. If chickenpox develops in a vaccinated health care worker, the worker should be furloughed, and all contacts for 2 days prior to the rash should be traced so appropriate measures may be taken. The ACIP19 and the COIDZ2recommend universal vaccination of American children with Oka strain vaccine. This recommendation remains controversial. Because vaccinees in long-term follow-up studies were re-exposed to circulating wild-type virus, the relative contributions of the vaccine and re-exposure to sustaining immunity to VZV over the long-term are not known.8 Antibody titers of vaccinees who were followed increased over time, presumably as a result of this wild-type exposure.M Should a successful vaccination program eliminate most circulating wild-type virus, it is feared that waning immunity might leave some vaccinees susceptible to more severe infections as adults.70 Mathematic modeling suggests that with achievable vaccination rates, universal vaccination may increase the rate of adult varicella, mostly in the population of unvaccinated children who grow into adults without getting chickenpox as children, even in the absence of waning immunity.35To prevent this, it may be necessary to achieve higher than traditional vaccination rates, or to mandate subsequent boosting, as is done for measles. Postlicensure studies are currently being performed to evaluate the long-term efficacy of vaccine, permitting appropriate measures to be taken if immunity wanes.@ As a live herpesvirus, the Oka strain vaccine establishes latency and can reactivate, although zoster develops less often following vaccination than following wild-type varicella in immunocompromised hosts.17,36, 47 Moreover, in short-term follow-up studies, zoster rates reported in healthy vaccinated children did not exceed those observed in naturally infected Studies examining zoster incidence in vaccinees are summarized in Figure 2. Moreover, because vaccine-induced immunity is also weaker than that achieved after wild-type infection, long-term zoster rates among vaccinees cannot now be predicted. Because periodic re-exposure to wild-type virus may boost immunity of individuals infected with wild-type varicella, and varicella, in turn, reducing susceptibility to z0ster,3~it is not known what effect the elimination of most wildtype boosting will have on eventual zoster rates in the unimmunized. Several small studies4,6, 39 suggested that administration of Oka strain vaccine within 5 days of an exposure to VZV may reduce the likelihood of clinical disease. Efficacy rates for the vaccine in this setting have been reported between 67% and 100%. Larger confirmatory studies need to be done before the vaccine can be recommended for this purpose. Ultimately, the efficacy and expense of vaccine postexposure prophylaxis need to be compared with that of varicella-zoster immunoglobulin and acyclovir. Studies are underway to determine whether a dose of Oka strain vaccine administered to elderly individuals will boost their existing VZV-specific immunity and reduce the incidence or severity of zoster.

70

KRAUSE&STRAUS 218

7

6

F

610

1

.

118

I

I

0 -

Hardy (ALL)

Brunell (ALL)

J

Healthy

Figure 2. Zoster incidence in varicella vaccine recipients. The incidence of zoster in 3 prelicensure studiesi7. 36,58 for vaccinees (solid bar) and non-vaccinees with previous natural varicella (hatched bar). For each group, the number of person-years of follow-up is shown above each bar. In the Hardy study,% the non-vaccinated controls were age-matched to the vaccinees. The asteriskdenotesthat the completeness of reporting was not ascertained. No contemporary controls were avaitable for the healthy children.

This hypothesis is based on the assumption that repeated exposure to virus, either through exposure to children with chickenpox or through exposure to internal reactivations, reduces the likelihood of zoster in the naturally infected population. Because zoster typically occurs only once, if at all, in most people, it is assumed that an episode of zoster enhances immune responses to levels that are sufficient to prevent further recurrences. A single dose of Oka strain vaccine improves CMI to VZV to a level comparable with that of an episode of zoster by some in vitro measures, although the immunologic correlates of protection from subsequent zoster are not Immunocompromised children with more than one dose of vaccine or with household exposures to varicella following vaccination had a lower incidence of zoster than children with one dose or those without household exposures, suggesting that immunologic boosting may improve zoster outcomes.3° The half-life of the vaccine-induced immunologic response was calculated at 4 to 5 years, suggesting that a dose of vaccine has a long-term immunologic effect that might reduce the subsequent rate of VZV reactivation^.^^ HSV

HSV-1 and HSV-2 cause oral and genital he~pes.2~ Less common but serious complications include encephalitis, meningitis, erythema multiforme, and visceral infections in the severely compromised host.

HERPESVIRUS VACCINES

71

After a primary infection, usually at an oral or genital site, the virus establishes latency in the sensory nerve ganglia innervating that site. From time to time stimuli, including menses, fever, stress, and ultraviolet light, may provoke virus reactivation. Although recurrences are generally less severe than initial infections, it is through asymptomatic recurrences that most disease is transmitted.&, Along with other ulcerating genital diseases, HSV infection is considered to be an important cofactor in the transmission of HIV.72Although approximately 60% to 70% of American adults are seropositive for, and hence latently infected with HSV-1, infection rates with HSV-2 now average 23%and still are increasiWZ6, 38 The immunology of HSV infections is not well-understood. The observation that individuals who were previously infected with either HSV-1 or HSV-2 are less likely to get infected with the other HSV type suggests that previous infections confer some protective immunity against subsequent infections, implying that it may be possible to design a vaccine that induces protective immunity. Because antibodies to virion surface glycoprotein D (gD) neutralize the virus, many vaccine efforts have targeted gD. The facts that agammaglobulinemic patients do not experience severe or frequent herpetic outbreaks, and that patients with AIDS, cytotoxic chemotherapy, and organ transplants do experience severe and frequent outbreaks, indicate that CMI plays the most important role in containing recurrent disease, and suggest that a vaccine that primarily induces only humoral responses is unlikely to succeed in reducing the frequency of recurrence in individuals who are already infected. As compared with VZV, HSV recurs to cause clinical disease much more frequently, suggesting that HSV may be more successful at evading host-mediated cellular immunity. Several possible goals of vaccination against HSV may be considered. Clearly, a vaccine that protects against acquisition of genital herpes would have substantial benefit, both for recipients and in the prevention of neonatal herpes. A therapeutic vaccine that reduces recurrence rates of already established genital herpes would also be beneficial, particularly if it also reduces transmission rates from infected individuals. One important principle of HSV therapeutic vaccine development has been the absolute need for placebo-controlled, double-blind trials. Because stress and other factors induce recurrences, early trials may have been flawed by substantial placebo effects. In these uncontrolled trials, many therapies that subsequently proved useless originally appeared to be benefi~ial.~~ Another important issue in HSV vaccine development is related to the animal models used to study vaccines. Many prospective vaccines generated excellent immune responses and proved effective in animals, but ultimately failed in humans. This may be in part due to specific interactions of the virus with the human immune system that affect the presentation of viral antigens. Thus HSV, in evolving specifically to avoid human immune responses, may be more difficult for the human

72

KRAUSE&STRAUS

immune system to control, as compared with the immune system of animals. Several approaches have been used in attempts to construct vaccines against genital herpes. These include administration of killed virus, purified virus glycoproteins, viral glycoprotein subunit vaccines, liveattenuated vaccines, and DNA vaccines. Early on, killed-virus vaccines were demonstrated to be ineffe~tive.~ A purified glycoprotein vaccine also failed in a human trial to prevent genital h e ~ e s . 5 Evaluation ~ of the immunogenicity of this vaccine suggested that it did not generate antibody responses comparable with those in naturally infected humans. This led to the development of recombinant HSV glycoproteins and a search for stronger adjuvants. Despite the early promise of this approach in inducing strong humoral and cellular immune responses in vaccinees,73,75 a glycoprotein subunit vaccine consisting of two HSV surface glycoproteins, gD and gB, together with an oil-in-water adjuvant (MF59), recently failed both in preventive and t h e r a p e ~ t i cstudies. ~~ The results of the therapeutic study are summarized in Table 4, which shows that vaccination influences the severity of the first recurrence after vaccination, but not the frequency of recurrence. It is postulated that this protein-based vaccine failed to elicit protective cellular immune responses, whatever these might be. To ensure the development of such responses, antigens may require presentation directly by cells, either by their infection with live virus, or transfection with viral genes. Trials continue with another gD vaccine, formulated with a different adjuvant.48 In constructing live vaccine candidates, a balance must be struck between pathogenicity (safety) and immunogenicity. Viruses that are safest are typically not very immunogenic, and vice versa. A recent effort involving an engineered form of HSV-I, in which some genes associated with virulence were deleted and genes encoding HSV-2 glycoproteins were proved the vaccine to be too attenuated and to Table 4. STUDY OF gBUgDUMF59 AS A THERAPEUTIC VACCINE TO PREVENT RECURRENT GENITAL HERPES IN ALREADY INFECTED INDIVIDUALS Clinical End Point

Monthly recurrence rate Virus shedding (days) at 1st recurrence New lesion formation (days) at 1st recurrence Itching and pain (days) at 1st recurrence Complete healing (days) at 1st recurrence

MF59 Placebo Recipients

gBagD2NF59 Recipients

0.55 +- 0.04 3.8 k 0.5

0.49 & 0.04 2.7 ? 0.6

0.22

6.9 f 0.9

4.1 f 1

0.04

6.8 +- 0.4

4.9 & 0.5

0.003

0.3 k 0.5

7 k 0.5

0.002

p Value 0.16

Adapted from Straus SE, Wald A, Kost RG, et al: Immunotherapy of recurrent genital herpes simplex virus type 2 glycoproteins D and B Results of a placebo-controlled vaccine trial. J Infect Dis 176:1129-1134, 1997; with permission.

HERPESVIRUS VACCINES

73

lack adequate immunogenicity.*sAn alternative method currently under investigation in humans is to develop viruses that are capable of undergoing only one round of replication in vivo. In this strategy a critical gene, such as glycoprotein H, that is required for cell-to-cell spread of the virus is deleted from the genome, but is supplied by the cell culture system in which the vaccine is prepared.14Early animal studies of this disabled infectious single cycle vaccine are encouraging for prevention of infection, although not as impressive when used to prevent recurrence in already infected animals (Fig. 3). This strategy has the potential advantage of presenting virus antigens in a natural context, but by permitting only one round of virus replication may mean that the vaccine is also inadequately immunogenic. Other live-virus vaccine strategies include other types of deletions that affect tissue specificity and virulence69(particularly those affecting neurovirulence, as is the case with deletion of the HSV ICP34.5 gene.87)Vaccine strain stability is also an issue: An attenuated ICP34.5 mutant regained a neurovirulent phenotype after serial passage in mouse brain. (R. Kaiwar, personal communication.) Direct injection into tissues of plasmid DNAs encoding viral proteins has also been considered for HSV. Preliminary animal data show promise, and the encoded viral proteins do seem to elicit cytotoxic Tcell responses, although much more work is needed.I3,45, 53 Another strategy currently under investigation is immunization with viral L (light) particles,” which are incomplete viral particles that lack nucleocapsids or viral nucleic acids. Other investigators are attempting to’ induce high levels of mucosal responses, in an effort to target the immunity to HSV to the initial site of inoculation before an infection can be e~tablished.~~ This strategy is appealing, because it could permit clearance of the virus before it has an opportunity to enter cells, establish latency, and use its genetic tools to avoid host immune defenses.

CMV infections usually cause little or no clinical disease in healthy people. In the United States, approximately half of the population is CMV seropositive, with wide ranges according to ages, socioeconomic status, and sexual habits. In some, CMV causes an infectious mononucleosis-like syndrome, manifested by lymphadenopathy, fever, and mild hepatitis. CMV is transmitted perinatally; by close contact (e.g., in day care centers); by sexual contact; or by contact with blood products, particularly in association with cardiac surgery (referred to as postperfusion syndrome). The virus reactivates frequently, with normal individuals shedding CMV in the urine as often as 15%of days.27 CMV causes considerable morbidity to the fetus. Congenital CMV infection may present as classic CMV inclusion disease, characterized by jaundice, hepatosplenomegaly, and petechial rash, and leave microcephaly, chorioretinitis, and motor dysfunction in its wake. Many congenital

74

KRAUSE & STRAUS

20

0

A

1

I

5

10

l !5

14

B

Days post challenge

24

34

44

54

1

I

Day post challenge

7 2

.-0

100%

6

-8 5 a

-3 5=

4

c

2

5

1

2

0

63.7%

3

0

17

c 4

27

4

37

47

57

67

Dayspostchallenge

10 2" Vaccinations

Figure 3. Efficacy of DISC HSV-2 vaccine as prophylaxis and therapy for acute and recurrent genital herpes in guinea pigs. gH negative disabled infectious single cycle (DISC) HSVQ vaccine, administered in two doses of lo7 pfu, given 3 weeks apart, with wild-type virus challenge 3 weeks later. The efficacy was tested against primary infection (mean number of lesions, A) and recurrent infection (mean cumulative lesions per animal, B ) in guinea pigs. C,The influence on recurrent disease of two doses of vaccine administered at days 17 and 30 after primary infection of guinea pigs with HSV. Triangle = mock circle = DISC HVS-2. (FromBoursnell MEG,et al: A genetically inactivated herpes simplex virus type 2 (HSV-2) vaccine provides effective protection against primary and recurrent HSV-2 disease. J Infect Dis 175:1&25, 1997; with permission.)

CMV cases are only appreciated later in childhood, when hearing loss and mental retardation become apparent. It is estimated that the annual economic cost of congenital CMV is approximately $1billion per year." The most severe congenital CMV infections occur when a previously uninfected mother develops primary infection during the first trimester 71 of

HERPESVIRUS VACCINES

75

CMV is also a serious problem among some immunocompromised individuals, in whom it causes a wide selection of disease ranging from prolonged fever to enterocolitis, pneumonia, retinitis, encephalitis, and death. CMV infections themselves are immunosuppressive, especially in transplant recipients. CMV-induced neutropenia appears to increase the susceptibility of these individuals to other opportunistic infections, including severe fungal and protozoal infections.@ Correlates of immunity to CMV are not well-understood. Studies in which CMV-immunoglobulin were given prophylactically to transplant patients showed that antibody protects against severe infections.68The CMV glycoprotein B is the major viral surface antigen, and an important target of neutralizing antibodies15and cellular immune responses. CMVseropositive individuals possess partial protection against exogenous reinfection; and reinfected pregnant women, or those in whom the virus reactivates, are less likely to transmit CMV to the fetus.28This suggests that complete immunity to CMV may not be required to have a substantial effect on the incidence of congenital CMV. Several major potential goals for vaccination should be considered. The first goal is to protect immunocompromised individuals against lifethreatening CMV infections; the second goal, and of the greater public health impact, is to prevent congenital CMV infection by inducing immunity in women before pregnancy. One special use of vaccine is to immunize plasma donors, to increase the anti-CMV-specific antibody titers in commercial immunoglobulin preparation^.^^ Because CMV causes little clinical disease in most healthy children and adults, and the incidence of congenital CMV is quite low, vaccine efficacy trials for this indication would be enormous and prohibitively expensive. A vaccine that is proved to prevent acquisition of infection in adults would presumably prevent congenital CMV. If the candidate vaccine merely attenuates disease in a particular adult setting, it might also reduce the morbidity of congenital CMV infection, but this is difficult to prove. Three major CMV vaccine strategies have emerged. One involves the development of live-attenuated vaccines. The first widely studied such vaccine involved the CMV Towne strain. This vaccine was safe and immunogenic, but provided only modest benefit to renal transplant recipients, reducing the severity of CMV disease in several studies, but In yet another study, it did not the incidence of infection (Table 5).'0,62,63 not reduce the rates of CMV transmission to young women.* Immunologic markers suggested that the anti-CMV immunity elicited by the Towne strain vaccine is weaker than that which follows natural infection. Consideration is now being given to administration of multiple doses of Towne strain vaccine' in order to achieve higher immune responses. Efforts are also underway to develop newer vaccines derived from Towne strain. It was recently shown that the Towne strain lacks several genes present in wild-type CMV.20Presumably, they were lost through serial passage of the original virus isolate. It is hoped that adding some

76

KRAUSE & STRAUS

Table 5. STUDIES OF TOWNE STRAIN CMV VACCINE IN PREVENTION OF ALL CMV DISEASE AND SEVERE CMV DISEASE IN RENAL TRANSPLANT RECIPIENTS= Rate of All CMV Disease

Trial

n

61 Multicenter Pennsylvania 67 Minnesota 35 163 All

Rate of Severe CMV Disease’

Vaccine (%) Placebo (%) Vaccine (%) Placebo (“YO)

38 39 33 37

59 55 43 54

0.0 6 5 3

17 35 36 29

Efficacy vs Severe Disease (%)

100 84

87 89

From Plotkin SA, Higgins R, Kurt2 JEi, et a1 Multicenter trial of Tome strain attenuated virus vaccine in seronegative renal transplant recipients. Transplantation581176-1178,1994; with permission.

of these genes back to the virus may improve its immunogenicity while not increasing its virulence.40 A second strategy has been the development of subunit CMV vaccines in which recombinant antigen is combined with strong adjuvants. One candidate vaccine of this type consists of CMV glycoprotein B coupled with a lipid emulsion adjuvant called MF59. This vaccine induces high titers of CMV-specific neutralizing antibodies and is currently being studied in clinical trials.%, A third strategy involves expression of CMV glycoprotein B in live, avirulent poxvirus vectors (e.g., canarypox) in an effort to improve cytotoxic lymphocytic (CTL) responses to gB.33 EBV

EBV is the major cause of infectious mononucleosis. It is strongly associated with African Burkitt’s lymphoma; nasopharyngeal carcinoma (mostly in North Africa and Asia); and posttransplant lymphoproliferative disorder, among other syndromes. EBV also has been associated with other lymphocytic malignancies, in particular Hodgkin’s disease. In the United States, over 90% of the adult population is infected with this virus. Most infections occur during adolescence and young adulthood, and thereafter virus is shed (approximately 15% of the time) asymptomatically in the saliva. While EBV replicates in the oropharyngeal epithelium, and the virus is shed in the saliva, it persists in B lymphocytes. An association with some T-cell lymphomas implies that EBV can also sometimes infect T lymphocytes. Because the immune system is frequently restimulated by reactivating EBV, many individuals have high antibody titers directed against EBV.42 Antigens important in the immune response to EBV include the surface glycoproteins gp340 (the predominant envelope glycoprotein, and an important target of neutralizing a n t i b ~ d i e sand ) ~ ~ gp85 (a major

HERPESVIRUSVACCINES

77

target of the early humoral immune responses). In contrast, the major targets for CMI are EBV proteins that are latently expressed in the nuclei and on the surface of B cells. Because infectious mononucleosis is generally a mild disease, the major impetus for an EBV vaccine lies in the prevention of cancer. The belief, however, that a cancer-preventing vaccine would not be useful unless it prevents the establishment of EBV latency, an unlikely prospect, has limited commercial interest in developing EBV vaccines. Approaches for EBV vaccines have included the development of a gp340 subunit vaccine59 and the development of a live recombinant vaccinia virus engineered to express EBV gp340. Both of these approaches displayed some early promise in animals trials. The recombinant vaccinia vaccine was recently tested in humans in China and shown to induce EBV-specific immune resp0nses.3~A third approach is to develop a peptide-based EBV vaccine. A specific nonapeptide from EBNA3 (one of the EBV latent nuclear antigens that is expressed in some EBV-associated lymphoproliferative disorders), whose cellular immune recognition is restricted through human leukocyte antigen (HLA)-B8, has been tested in phase I trials in Australia.'l If effective, this peptide vaccine could potentially be used to induce or enhance CMI, but this approach would require HLA-specific vaccines for different individuals. Further studies are necessary. FUTURE TRENDS

The varicella vaccine represents the paradigm of a successful herpesvirus vaccine. This live-attenuated vaccine demonstrates unequivocally that it is possible to develop vaccines against herpesvirus. Each of the other herpesviruses, however, presents its own unique challenges to vaccine development and testing. In retrospect, it is curious that a successful vaccine for varicella has been achieved, whereas vaccines for the other herpesviruses remain unachieved. Additional information about the immunopathogenesis of herpesvirus infections will doubtless lead to improved strategies for immunization. Although in the past live-attenuated vaccines have been considered a low-technology approach, molecular biologic techniques now permit the rational engineering of live vaccine strains to have predictable effects on the virus' replication and virulence. Other new approaches to inducing cellular responses to herpesviruses, such as DNA vaccination, may well also yield effective vaccines. References 1. Adler SP: Current prospects for immunization against cytomegalovirus disease. Infectious Agents and Disease 5:29, 1996 2. Adler SP, Starr SE, Plotkin SA, et al: Immunity induced by primary human cytomegalo-

78

KRAUSE & STRAUS

virus infection protects against secondary infection among women of childbearing age. J Infect Dis 171:26, 1995 3. Anderson SG,Hamilton J, Williams S: An attempt to vaccinate against herpes simplex. Aust J Exp Biol Med Sci 28:579, 1950 4. Arbeter A, Starr SE, Plotkin SA: Varicella vaccine studies in healthy children and adults. Pediatrics 78(suppl):748, 1986 5. Asano Y Varicella vaccine: The Japanese experience. J Infect Dis 174(suppl3):S310,1996 6. Asano Y, Hirose S, Iwayama S, et al: Protective effect of immediate inoculation of a live varicella vaccine in household contacts in relation to the viral dose and interval between exposure and vaccination. Biken J 25:43, 1987 7. Asano Y, Itakura N, Hiroishi Y Viremia is present in incubation period in nonimmunccompromised children with varicella. J Pediatr 106:69, 1985 8. Asano Y, Suga S, Yoshikawa T, et al: Experience and reason: Twenty-year follow-up of protective immunity of the Oka strain live varicella vaccine. Pediatrics 94:524, 1994 9. Asano Y, Yoshikawa T, Suga S: Postexposure prophylaxis of varicella in family contacts by oral acyclovir. Pediatrics 92219, 1993 10. Balfour HH Jr, Welo PK, Sachs G W Cytomegalovirus vaccine trial in 400 renal transplant candidates. Transplant Proc 1781, 1985 11. Bames J: Peptide-based EBV vaccine in phase I trial. Pharma Weekly 1:11, 1995 12. Bemstein HH, Rothstein EP, Watson BM, et al: Clinical survey of natural varicella compared with breakthrough varicella after immunization with live attenuated Oka/ Merck varicella vaccine. Pediatrics 92:833, 1993 13. Bourne N, Milligan GN, Schleiss MR, et al: DNA immunization confers protective immunity on mice challenged intravaginally with herpes simplex virus type 2. Vaccine 141230, 1996 14. Boursnell MEG, Entwisle C, Blakeley D, et al: A genetically inactivated herpes simplex virus type 2 (HSV-2) vaccine provides effective protection against primary and recurrent HSV-2 disease. J Infect Dis 175:16, 1997 15. Britt WJ, Vugler L, Stephen EB: Induction of complement-dependent and -independent neutralizing antibodies by recombinant-derived human cytomegalovirus gp55-116 (gB). J Virol 62:3309, 1988 16. Brunell PA, Geiser CF, Novelli V, et al: Varicella-like illness caused by live varicella vaccine in children with acute lymphocytic leukemia. Pediatrics 79:922, 1987 17. Brunell PA, Taylor J, Geiser CF, et a1 Risk herpes zoster in children with leukemia: Varicella vaccine compared with history of chickenpox. Pediatrics 7753, 1981 18. Cadoz M, Micoud M, Seigneurin J M Phase I trial of R7020 A live-attenuated recombinant herpes simplex virus (HSV) candidate vaccine. Presented at The 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, CA, 1992 19. Centers for Disease Control and Preventions: Prevention of varicella: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 45(RRll):l,1996 20. Cha TA, Tom E, Kemble GW, et al: Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol 7078, 1996 21. Cohen J I Infection of cells with varicella-zoster virus down-regulates surface expression of class I major histocompatibility complex antigens. J Infect Dis 1771390, 1998 22. Committee on Infectious Diseases of the American Academy of Pediatrics: Recommendations for the use of live attenuated varicella vaccine. Pediatrics 95:791, 1995 23. Corey L, Spear PG: Infections with herpes simplex viruses. N Engl J Med 314686,1986 24. Dargan DJ, Subak-Sharpe J H The effect of herpes simplex virus type 1 L-particles on virus entry, replication, and the infectivity of naked herpesvirus DNA. Virology 239:378, 1997 25. De Moragas JM, Kierland RR: The outcome of patients with herpes zoster. Arch Dermatol 75:193, 1957 26. Fleming DT, McQuillan GM, Johnson RE, et al: Herpes simplex virus type 2 in the United States, 1976 to 1994. N Engl J Med 3371105, 1997 27. Ford-Jones EL, Kitai I, Davis L, et al: Cytomegalovirus infections in Toronto child-care

HERPESVIRUS VACCINES

79

centers: A prospective study of viral excretion in children and seroconversion among day-care providers. Pediatr Infect Dis J 15:507, 1996 28. Fowler KB, Stagno S, Pass RF, et al: The outcome of congenital cytomegalovirus infection in relation to maternal antibody status. N Engl J Med 326:663, 1992 29. Gallichan WS, Rosenthal KL Long-term immunity and protection against herpes simplex virus type 2 in the murine female genital tract after mucosal but not systemic immunization. J Infect Dis 1771155, 1998 30. Gershon AA, LaRussa P, Steinberg S, et al: The protective effect of immunologic boosting against zoster: An analysis in leukemic children who were vaccinated against chickenpox. J Infect Dis 173:450, 1996 31. Gershon AA, Mervish N, LaRussa P, et al: Varicella-zoster virus infection in children with underlying human immunodeficiency virus infection. J Infect Dis 176:1496, 1997 32. Gilden DH, Dueland AN, Devlin ME, et a1 Varicella-zoster virus reactivation without rash. J Infect Dis 166(suppl 1):S30, 1992 33. Gonczol E, Berensci K, Pincus S, et al: Preclinical evaluation of an ALVAC (canarypox)-human cytomegalovirus glycoprotein B vaccine candidate. Vaccine 13: 1080, 1995 34. Gu SY, Huang TM, Ruan L, et al: First EBV vaccine trial in humans using recombinant vaccinia virus expressing the major membrane antigen. Dev Biol Stand 84171, 1995 35. Halloran ME, Cochi SL, Wharton M, Fehrs L Theoretical epidemiologic and morbidity effects of routine varicella immunization of preschool children. Am J Epidemiol 140231, 1994 36. Hardy 1, Gershon AA, Steinberg SP, LaRussa P: The incidence of zoster after immunization with live attenuated varicella vaccine. N Engl J Med 3251545, 1991 37. Hope-Simpson RE: The nature of herpes zoster: A long-term study and a new hypothesis. Proc R SOCLond B Biol Sci 58:9, 1965 38. Johnson RE, Nahmias AJ, Magder LS: A seroepidemiologic survey of the prevalence of herpes simplex virus type 2 infection in the United States. N Engl J Med 321:7, 1990 39. Katsushima N, Yazaki N, Sakamoto M, et al: Application of a live varicella vaccine to hospitalized children and its follow-up study. Biken J 25:29, 1982 40. Kemble G, Duke G, Winter R, Spaete R A new generation of live, attenuated cytomegalovirus (CMV) vaccine strains. Presented at 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA, 1996 41. Kern AB, Schiff BL Vaccine therapy in recurrent herpes simplex. Dermatology 89944,1964 42. Khanna R, Burrows SR, Moss DJ: Immune regulation in Epstein-Barr virus-associated diseases. Microbiol Rev 59:387, 1995 43. Kost RG, Straus SE: Postherpetic neuralgia-pathogenesis, treatment, and prevention. N Engl J Med 335:32, 1996 44. Krause PR, Klinman D M Efficacy, immunogenicity, safety, and use of live attenuated chickenpox vaccine. J Pediatr 127:518, 1995 45. Kriesel JD, Spruance SL, Daynes RA, Araneo BA: Nucleic acid vaccine encoding gD2 protects mice from herpes simplex virus type 2 disease. J Infect Dis 173:536, 1996 , ' l Steinberg S, Gershon A: Varicella vaccine for immunocompromised chil46. LaRussa dren: Results of collaborative studies in the United States and Canada. J Infect Dis 174(suppl 3):S320, 1996 47. Lawrence R, Gerson AA, Holzman R, Steinberg SP: The risk of zoster after varicella vaccination in children with leukemia. N Engl J Med 318:543, 1988 48. Leroux-Roels G, Moreau E, Desombere I, et al: Safety, humoral and cellular immune responses of three different doses of glycoprotein D(gD2t) in herpes simplex vaccine with aluminum and MPL. Presented at 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA, 1997 49. Levin MJ, Hayward A R The varicella vaccine: Prevention of herpes zoster. Infect Dis Clin North Am 10:657, 1996 50. Lieu TA, Cochi SL, Black SB: Cost-effectiveness of a routine varicella vaccination program for U.S. children. JAMA 271:375, 1994 51. Ljungman P, Lonnqvist B, Gahrton G, et a1 Clinical and subclinical reactivation of varicella-zoster virus in immunocompromised patients. J Infect Dis 153:840, 1986

80

KRAUSE&STRAlJS

52. Malinoski FJ, Linden J, Smith J, et a1 Immunization of plasmapharesis donors with cytomegalovirusvaccine composed of gB glycoprotein and MF-59 adjuvant. Presented at 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997 53. Manickan E, Rouse RJ, Yu Z , et al: Genetic immunization against herpes simplex virus: Protection is mediated by CD4 + T lymphocytes. J Immunol 155259,1995 54. Marshall GS, Frey S, Pass R, et a1 Safety and immunogenicity of CMV gBMF59 vaccine in healthy seronegative adults. Presented at 37th Interscience Conference on Antimicrobial Agents and Chemotherapy,Toronto, Canada, 1997 55. Meignier B, Whitley R, Roizman B In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020. 11. Studies in immunocompetent and immunosuppressed owl monkeys (Aotu trivargatus).J Infect Dis 162:313, 1990 56. Merck & Co: Varivax (Varicella Virus Vaccine Live [Oka/Merck]) package circular. In Physicians’ Desk Reference, ed 52. Montvale, NJ:Medical Economics Co., 1998, p 1762 57. Mertz GJ, Ashley R, Burke RL, et a1 Double-blind, placebo-controlled trial of a herpes simplex virus type 2 glycoprotein vaccine in persons at high risk for genital herpes infection. J Infect Dis 161:653, 1990 58. Miller DM, Rahill BM, Boss JM, et al: Human cytomegalovirusinhibits major histocompatibility complex class I1 expression by disruption of the Jak/Stat pathway. J Exp Med 187675,1998 59. Morgan AJ: Epstein-Barr virus vaccines. Vaccine 10:563, 1992 60. Pass W,Duliege AM, Boppana SB: Immunogenicityof a recombinant CMV gB Vaccine. Pediatr Res 37185A, 1995 61. Ploegh HL: Vial strategies of immune evasion. Science 280248, 1998 62. Plotkin SA, Farquhar J, Hornberger E: Clinical trials of immunization with the Towne 125 strain of human cytomegalovirus.J Infect Dis 134:470, 1976 63. Plotkin SA, Higgins R, Kurtz JB, et a1 Multicenter trial of Towne strain attenuated virus vaccine in seronegative renal transplant recipients. Transplantation 581176, 1994 64. Porath A, McNutt RA, Smiley LM, Weigle KA: Effectiveness and cost benefit of a proposed live cytomegalovirus vaccine in the prevention of congenital disease. Rev Infect Dis 1231, 1990 65. Ragozzino W ,Melton LJD, Kurland LT, et a1 Population-based study of herpes zoster and its sequelae. Medicine (Baltimore) 61:310, 1982 66. Rooney JJ, Felser JM, Ostrove JM, Straus SE Acquisition of genital herpes from an asymptomatic sexual partner. N Engl J Med 314:1561, 1986 67. Ross AH Modification of chickenpox in family contacts by administration of gammaglobulin. N Engl J Med 267369, 1962 68. Snydman DR, Werner BG, Heinze-Lacy BH Use of cytomegalovirusimmune globulin to prevent cytomegalovirus disease in renal transplant recipients. N Engl J Med 3171049,1987 69. Spector FC, Kern ER, Palmer J, et a1 Evaluation of a live attenuated recombinant virus RAV 9395 as a herpes simplex virus type 2 vaccine in guinea pigs. J Infect Dis 177143, 1998 70. Spingam RW, Benjamin JA. Universal vaccination against varicella. N Engl J Med 338:683, 1998 71. Stagno S, Pass RF, Dworsky ME: Congenital cytomegalovirus infection: The relative importance of primary and recurrent maternal infection. N Engl J Med 306945,1982 72. Stamm WE, Handsfield HH, Rompalo AM, et al: The associationbetween genital ulcer disease and acquisition of HIV infection in homosexual men. JAMA 26031429, 1988 73. Straus SE, Corey L, Burke RL, et a1 Placebo-controlled trial of vaccination with recombinant glycoprotein D of herpes simplex virus type 2 for immunotherapy of genital herpes. Lancet 343:1460,1994 74. Straus SE, Ostrove JM, Inchauspe G, et al: NM conference. Varicella-zoster virus infections: Biology, natural history, treatment, and prevention. Ann Intern Med 108:221, 1988 75. Straus SE, Savarese B, Tigges M Induction and enhancement of immune responses to herpes simplex virus type 2 in humans by use of a recombinant glycoprotein D vaccine. J Infect Dis 167:1045, 1993

HERPESVIRUS VACCINES

81

76. Straus SE, Wald A, Kost RG, et al: Immunotherapy of recurrent genital herpes with recombinant herpes simplex virus type 2 glycoproteins D and B: Results of a placebocontrolled vaccine trial. J Infect Dis 1761129, 1997 77. Takahashi M: Clinical overview of varicella vaccine: Development and early studies. Pediatrics 78:736, 1986 78. Takahashi M, Otsuka T, Okuno Y: Live vaccine used to prevent the spread of varicella in children in hospital. Lancet 21288,1974 79. Thorley-Lawson DA, Geilinger K: Monoclonal antibodies against the major glycoprotein (gp350/220) of Epstein-Barr virus neutralize infectivity. Proc Natl Acad Sci U S A 77:5307, 1980

80. Varis T, Vesikari T Efficacy of high-titer live attenuated varicella vaccine in healthy young children. J Infect Dis 174(suppl3):S330, 1996 81. Vugia DJ, Peterson CL, Meyers HB, et al: Invasive group A streptococcal infections in children with varicella in southern California. Pediatr Infect Dis J 15:146, 1996 82. Wald A Virologic characteristics of subclinical and symptomatic genital herpes infections. N Engl J Med 333770, 1995 83. Wasmuth EH, Miller WJ: Sensitive ELISA for antibody to varicella-zoster virus using purified VZV glycoprotein antigen. J Med Virol32189, 1990 84. Weibel RE, Neff BJ, Kuter BJ et al: Live attenuated varicella vaccine: Efficacy trial in healthy children. N Engl J Med 310:1409,1984 85. Wharton M. The epidemiology of varicella-zoster virus infections. Infect Dis Clin North Am 10:571, 1996 86. White CJ, Kuter BJ, Ngai A, et al: Modified cases of chickenpox after varicella vaccination: Correlation of protection with antibody response. Pediatr Infect Dis J 11:19, 1992 87. Whiteley RJ, Kern ER, Chatterjee S, et al: Replication, establishment of latency, and induced reactivation of herpes simplex virus gamma 1 34.5 deletion mutants in rodent models. J Clin Invest 912837, 1993 88. Wilson A, Sharp M, Koropchak CM, et a1 Subclinical varicella-zoster virus viremia, herpes zoster, and T lymphocyte immunity to varicella-zoster viral antigens after bone marrow transplantation. J Infect Dis 165319, 1992 Address reprint requests to Philip R. Krause Laboratory of DNA Viruses FDA/CBER/OVRR 29A/lC16, HFM-457 29 Lincoln Drive Bethesda, MD 20892-4555