Abstract

DNA vaccines expressing the guinea pig cytomegalovirus (GPCMV) homologs of the glycoprotein B (gB) and UL83 proteins were evaluated for protection against congenital GPCMV infection. After 4 doses of DNA administered by epidermal (gene gun) route, all guinea pigs developed enzyme-linked immunosorbent assay (ELISA) antibody and, for gB-vaccine recipients, neutralizing antibody. Dams were challenged with 1×104 plaque-forming units of GPCMV in the third trimester. Preconception vaccination with gB did not decrease overall pup mortality, although, within the gB-vaccine group, pup mortality was lower among dams with high ELISA responses. Preconception maternal vaccination with gB vaccine significantly reduced congenital transmission in liveborn pups. In contrast, UL83 vaccine had no significant effect on pup mortality or vertical transmission of GPCMV. Virus load was significantly lower in infected pups born to gB- and UL83-vaccinated dams than in infected pups born to control dams. These data support the concept that subunit gB vaccination may be useful in protecting against CMV-induced disease

Human cytomegalovirus (HCMV) infection, although generally asymptomatic in normal hosts, can result in severe disease in immunocompromised hosts, including newborn infants [1]. Acquisition of infection in utero can produce particularly devastating consequences, including neurodevelopmental sequelae and sensorineural deafness [2, 3]. Congenital HCMV infection occurs in 1%–2% of all pregnancies, complicating an estimated 40,000 pregnancies annually in the United States [4]. Since preconception maternal immunity to HCMV provides some degree of protection against vertical transmission of infection [5, 6], there has been considerable interest in development of CMV vaccines. HCMV vaccines capable of protecting newborns from the neurodevelopmental sequelae caused by congenital infection would be highly cost-effective, and development of such vaccines was recently identified as a “level 1” (most cost-effective category) priority by the Institute of Medicine [7]

Since the correlates of the maternal immune response to HCMV that are critical to protection of the fetus are unknown, it is unclear which vaccine strategy would be optimal. A variety of HCMV vaccines have been evaluated in clinical trials [8–11]. These have included live attenuated vaccines, as well as subunit and vectored vaccines, targeting the HCMV proteins that appear to be most important in protective immunity. Protein targets include the major envelope glycoprotein gB (gpUL55) and the tegument phosphoprotein pp65 (ppUL83) [12]. The majority of virus-neutralizing antibody responses after HCMV infection recognize gB [13], whereas pp65 elicits the majority of the CD8+ cytotoxic T lymphocyte (CTL) response to infection [14, 15]. Recombinant HCMV gB expressed in CHO cells is safe and well tolerated and elicits virus-neutralizing antibody responses after intramuscular (im) administration [16, 17]. Both gB and UL83 have been expressed in a recombinant canarypox (ALVAC) system and have undergone limited evaluation in phase 1 studies; these vaccines, too, are immunogenic and well tolerated [18–21]. However, at present, there has not been an evaluation of the efficacy of any HCMV vaccine for prevention of congenital infection

Ideally, preclinical evaluations of the immunogenicity and efficacy of HCMV vaccines would be performed in animal models of congenital infection. Unfortunately, the species specificity of CMV precludes evaluation of HCMV vaccines in animals and necessitates the study of animal CMVs [22]. In contrast to the CMVs of most small animals, guinea pig CMV (GPCMV) is unique in its ability to cross the placenta, causing infection in utero [23]. This feature makes the GPCMV model useful for evaluation of vaccine strategies. Previous studies have indicated that preconception vaccination of guinea pigs, using native GPCMV proteins administered with potent adjuvants, provides protection against GPCMV-induced pup mortality and infection, after viral challenge of pregnant guinea pigs [24–26]. However, the lack of detailed molecular characterization of GPCMV proteins has, at present, precluded any studies in this model using cloned, recombinant expression technologies. In recent studies, the GPCMV homologs of gB and UL83 (GP83) have been cloned [27, 28], and the immunogenicity of candidate subunit vaccines based on these genes, expressed as DNA plasmid vaccines, has been characterized [29]. The advantage of using DNA vaccine for such studies is the ease with which such vaccines can be generated and the usefulness they offer as “proof of concept” for the protective roles of specific viral genes in vaccine/challenge studies. The present study was undertaken to test the protective efficacy of DNA vaccines that target the GPCMV homologs of gB and UL83 (GP83), administered by epidermal (gene gun) route, in the guinea pig model of congenital CMV infection

Materials and Methods

Animal studiesYoung female and proven-breeder male Hartley guinea pigs were obtained from Harlan Laboratories. Inbred adult strain-2 guinea pigs were purchased from the Children’s Hospital Research Foundation (Cincinnati, OH). Guinea pigs were confirmed to be GPCMV seronegative by ELISA before vaccination. Animals were housed under conditions approved by the American Association of Accreditation of Laboratory Animal Care, in accordance with institutional animal-use committee policies

Virus and cellsGPCMV (strain no. 22122; ATC VR682) was propagated on guinea pig fibroblast lung cells (GPL; ATCC CCL 158) and was maintained in F-12 medium supplemented with 10% fetal calf serum (HyClone Laboratories), 10,000 IU/L penicillin, 10 mg/L streptomycin (Gibco-BRL), and 7.5% NaHCO3 (Gibco-BRL). Salivary gland–passaged stocks (SG virus) were prepared by sequential passage in strain-2 guinea pigs, as described elsewhere [30]

Recombinant plasmids, DNA vaccine preparation, and experimental designThe cloning details used for generation of gB and GP83 plasmid constructs are described elsewhere [27–29]. In brief, the GPCMV gB homolog was expressed in a truncated, secreted form, spanning amino acid residues 1–692, in a plasmid designated pKTS 404. The GP83 protein was expressed as a full-length construct (aa 1–565) in a plasmid designated pKTS 437. Both plasmids use the HCMV major immediate-early promotor and a polyadenylation signal derived from the bovine growth hormone gene (pCDNA 3.0; Invitrogen). Plasmid DNA was purified by use of Qiagen column and was conjugated to gold particles, as described elsewhere [29]

To examine the protective efficacy of the DNA vaccines, young female Hartley guinea pigs were vaccinated with a series of 4 epidermal inoculations of gB (pKTS 404) or GP83 (pKTS 437) plasmid at monthly intervals, as described elsewhere [29]. For each vaccination, animals were inoculated with 6 doses (2 μg) of plasmid conjugated to 0.5 mg of gold “carrier” particles. Approximately 30 days after the fourth vaccination, blood was obtained for immunogenicity analyses, and the animals were placed with breeder males and were examined weekly by palpation for evidence of pregnancy. In the third trimester of pregnancy, animals were inoculated subcutaneously with 1 × 104 pfu of GPCMV and were observed daily until delivery to determine the outcome of pregnancy

ELISA, neutralization assays, and radioimmunoprecipitation (RIP)–PAGE analysesAntibody responses in vaccinated animals were monitored by ELISA. ELISA was performed using GPCMV antigen, as described elsewhere [31]. ELISA titers were defined as the reciprocal of the highest dilution that produced an absorbance of at least 0.10 and twice the absorbance against control antigen. Neutralization assays were performed by use of serum samples from guinea pigs vaccinated with pKTS 404, as described elsewhere [29], except that 5% rabbit serum was used as a source of exogenous complement. Neutralization assays were performed by use of an isolate of GPCMV tagged with green fluorescent protein, and plaques were enumerated by fluorescence microscopy [32]. Neutralization titers were defined as the highest dilution of serum that resulted in a ⩾50% reduction in plaques, compared with that in control (preimmune) serum samples. Serum samples from vaccinated animals were further examined by RIP-PAGE for immunoreactivity with GPCMV proteins. GPL cells were inoculated with GPCMV (MOI, ∼5 pfu/cell), and, at 96 h after infection, cells were incubated in media supplemented with 35S-cysteine and methionine (35S-Translabel; ICN Radiochemicals) at a specific activity of 50 μCi/mL, for 4 h. Proteins were immunoprecipitated with antibody from vaccinated guinea pigs (volume, 20 μL), as well as hyperimmune, polyclonal anti-GPCMV antibody and Staphylococcus aureus protein A, and were subjected to SDS-PAGE and autoradiography, as described elsewhere [28]

Viral culture and quantitative competitive PCR (qcPCR) analysesLiveborn pups were killed within 72 h of delivery, and tissue homogenates (10% wt/vol) of liver and spleen were prepared for viral culture on GPL cells. DNA from homogenates was extracted by use of the Qiagen QIAamp DNA mini kit DNA extraction system, according to the manufacturer’s specifications. Eluted DNA (1% of sample) was subjected to qcPCR analysis, as described elsewhere [33]. In brief, the primer pair UL83F6 (5′-CGACGACGACGATGACGAAAAC-3′) and UL83B11 (5′-TCCTCGGTCTCAACGAAGGGTC-3′) amplifies a 225-bp region, corresponding to Asp402 through Ser473 of GP83. This plasmid was modified by engineering a 68-bp internal deletion. The resultant clone served as an internal standard (IS) for qcPCR. A standard curve was generated by measuring the ratio of relative signal intensity of amplification products on ethidium bromide–stained gels for increasing amounts of full-length plasmid with IS. The signal intensity of the experimental standard was compared with this standard curve to quantify the total copy number (GPCMV genome equivalents) per milligrams of tissue extracted

Statistical analysesIncidence data were compared by use of Fisher’s exact test. Continuous variables were compared by use of Student’s t test. All comparisons were 2-tailed

Results

Immune response to DNA vaccinationPrevious analyses of the immunogenicity of GPCMV gB and GP83 expression plasmids indicated that 4 doses of vaccine are required for optimal immunogenicity by epidermal inoculation [29]. ELISA indicated that all animals (n=17 with gB and n=14 with GP83) seroconverted to GPCMV antigen after 4 doses of plasmid inoculation (figure 1). As observed elsewhere, ELISA titers were consistently higher after vaccination with gB plasmid, compared with those after vaccination with GP83 plasmid [29]. The mean ELISA titer in gB-vaccinated animals was 3.3 log10, compared with 1.8 log10 in GP83-vaccinated animals (figure 1; P<.001). In serum from gB-vaccinated guinea pigs that became pregnant, complement-dependent neutralizing titers were determined by plaque-reduction methods. Neutralizing titers ranged from 1:80 to 1:1280, with a mean neutralizing titer of 2.55 log10 (figure 1B)

Figure 1

A ELISA antibody responses in guinea pigs vaccinated with gB or GP83 DNA vaccines. ELISA was performed against guinea pig cytomegalovirus (GPCMV) antigen, as described elsewhere [31]. Animals were bled after the fourth dose of DNA vaccine. Mean log10 titer for gB-vaccine recipients was 3.3; for GP83-vaccine recipients, mean log10 titer was 1.8, which is significantly lower than that for gB-vaccine recipients (P<.0001). B Neutralizing antibody titers in 13 dams that received gB vaccine and became pregnant. Mean complement-dependent neutralizing titer was 2.55 log10

Figure 1

A ELISA antibody responses in guinea pigs vaccinated with gB or GP83 DNA vaccines. ELISA was performed against guinea pig cytomegalovirus (GPCMV) antigen, as described elsewhere [31]. Animals were bled after the fourth dose of DNA vaccine. Mean log10 titer for gB-vaccine recipients was 3.3; for GP83-vaccine recipients, mean log10 titer was 1.8, which is significantly lower than that for gB-vaccine recipients (P<.0001). B Neutralizing antibody titers in 13 dams that received gB vaccine and became pregnant. Mean complement-dependent neutralizing titer was 2.55 log10

Immunoprecipitation analyses using serum samples from vaccinated guinea pigsTo confirm that vaccination with recombinant gB plasmid induced antibodies capable of immunoprecipitating the native gB complex, RIP-PAGE analyses were performed (figure 2). All vaccinated animals made antibodies capable of immunoprecipitating species of ∼150 kDa (representing the uncleaved intracellular gB precursor protein), ∼90 kDa (representing the amino-terminal moiety), and ∼58 kDa (representing the carboxy-terminal moiety) from 35S-labeled, GPCMV-inoculated tissue culture lysates

Figure 2

Immunoprecipitation analysis of serum from gB–vaccinated animals. Serum (volume, 20 μL) from vaccinated guinea pigs and Staphylococcus aureus protein A were used to immunoprecipitate 35S-labeled lysates from guinea pig cytomegalovirus (GPCMV)–infected guinea pig fibroblast lung cells. Representative results observed using serum samples from 7 gB-vaccinated animals (including serum samples from dams with both high-mortality and low-mortality litters) are indicated (lanes 1–7). All animals engendered antibody capable of immunoprecipitating the ∼90 and ∼58 kDa subunits of the gB complex. In addition, an ∼150 kDa polypeptide representing the intracellular gB precursor was identified (arrows). Immunopreciptation with preimmune serum samples did not demonstrate any GPCMV polypeptides (data not shown). Position of molecular weight markers is indicated. Immunoprecipitation profile of polypeptides immunoreactive with polyclonal anti-GPCMV antisera is also indicated (lane P)

Figure 2

Immunoprecipitation analysis of serum from gB–vaccinated animals. Serum (volume, 20 μL) from vaccinated guinea pigs and Staphylococcus aureus protein A were used to immunoprecipitate 35S-labeled lysates from guinea pig cytomegalovirus (GPCMV)–infected guinea pig fibroblast lung cells. Representative results observed using serum samples from 7 gB-vaccinated animals (including serum samples from dams with both high-mortality and low-mortality litters) are indicated (lanes 1–7). All animals engendered antibody capable of immunoprecipitating the ∼90 and ∼58 kDa subunits of the gB complex. In addition, an ∼150 kDa polypeptide representing the intracellular gB precursor was identified (arrows). Immunopreciptation with preimmune serum samples did not demonstrate any GPCMV polypeptides (data not shown). Position of molecular weight markers is indicated. Immunoprecipitation profile of polypeptides immunoreactive with polyclonal anti-GPCMV antisera is also indicated (lane P)

Outcomes of pregnancy in vaccine/challenge studyTo assess the protective efficacy of GPCMV subunit DNA vaccines against congenital CMV infection, a challenge experiment was performed after animals were bred. A total of 17 guinea pigs were vaccinated with gB plasmid, and 14 were vaccinated with GP83 plasmid. Among gB-vaccinated animals, 13 became pregnant and underwent GPCMV challenge; 1 was excluded from the analysis because she gave birth <7 days after challenge. Among GP83-vaccinated animals, 11 became pregnant and underwent GPCMV challenge; 1 was excluded from the analysis because she gave birth <7 days after challenge. Pregnancy was established in animals vaccinated with gB, GP83, or negative control plasmid. After challenge in the third trimester, pup mortality was compared. Outcomes of pregnancy were compared among animals for which there was a window of at least 7 days between GPCMV challenge and delivery of pups (table 1). For control dams (n=10 litters), pup mortality was 33% (13/39). In pups born to gB-vaccinated (n=12 litters) and GP83-vaccinated (n=11 litters) dams, pup mortality was similar: 34% (14/41) for the gB-vaccine group and 34% (13/38) for the GP83-vaccine group. Although overall mortality in the gB-vaccine group was similar to that in the control group, significant differences in pup mortality were observed within the gB-vaccine group as a function of the magnitude of the ELISA titer. In litters born to dams with an ELISA titer <3.4 log10 (n=8), pup mortality was 50% (14/28), a rate not statistically significantly different from that of the negative control group. In contrast, in pups born to dams with a mean log10 ELISA titer >3.4 log10 (n=4), no pup mortality was observed (0/13; P<.005 vs. dams with ⩽3.4 log10 ELISA titer; P<.0001 vs. negative controls) (table 1)

Table 1

Pup mortality after maternal inoculation with guinea pig cytomegalovirus

Table 1

Pup mortality after maternal inoculation with guinea pig cytomegalovirus

Effect of DNA vaccination on congenital GPCMV infection ratesTo analyze the effect of DNA vaccination on congenital GPCMV infection, liveborn pups were killed within 72 h of delivery and then dissected, and organs (liver and spleen) were homogenized for tissue culture and PCR detection of viral genome. Among liveborn pups, preconception vaccination with gB vaccine, but not UL83 vaccine, resulted in a significant decrease in the incidence of congenital CMV infection (table 2). Of 26 liveborn pups in the control group, 17 had evidence of GPCMV infection by culture, and an additional 3 pups with negative cultures had viral DNA demonstrated by PCR, for a total congenital infection rate of 77% (20/26). In contrast, only 7 (26%) of 27 pups in the gB-vaccine group had evidence of GPCMV infection by viral culture, and an additional 4 pups had viral DNA demonstrated by PCR, for a total congenital infection rate of 41% (11/27; P<.05 vs. control group). Of 25 pups born to dams vaccinated with GP83, 13 had evidence of GPCMV infection by viral culture, and an additional 4 pups had viral DNA demonstrated by PCR, for a total congenital infection rate of 68% (17/25; P is not significant vs. control)

Table 2

Vertical transmission rates of guinea pig cytomegalovirus (GPCMV) and virus load analyses of infected pups, after third-trimester maternal GPCMV challenge

Table 2

Vertical transmission rates of guinea pig cytomegalovirus (GPCMV) and virus load analyses of infected pups, after third-trimester maternal GPCMV challenge

Analysis of virus load in GPCMV-infected pupsTo further evaluate the effect of DNA vaccine on congenital infection, virus loads were compared among infected pups in the gB- and GP83-vaccine groups and the control group by qcPCR (table 2). Mean virus loads in infected pups born to gB- and GP83-vaccinated dams were significantly lower than those in control pups. In infected pups born to gB-vaccinated dams, the mean virus load in the liver was 1.8 log10 genomes/mg and in the spleen was 1.3 log10 genomes/mg. In infected pups born to GP83-vaccinated dams, the mean virus load in the liver was 1.4 log10 genomes/mg and in the spleen was 1.3 log10 genomes/mg. In contrast, in infected pups born to negative-control dams, the mean virus load in the liver was 3.8 log10 genomes/mg and in the spleen was 4.0 log10 genomes/mg (table 2)

Discussion

Because of the ability of GPCMV to cross the placenta and cause infection and disease in utero, the guinea pig provides a uniquely useful model for study of CMV vaccine strategies. A number of studies have examined the protective efficacy of vaccines consisting of native GPCMV proteins purified by immunoaffinity column, lectin column chromatography, or detergent solubilization of virus and dense body fractions. All were administered with Freund’s adjuvant [24–26], an adjuvant not approved for human use. All of these strategies have proven useful in protecting against congenital GPCMV infection and/or disease, with the extent of protection dependent on the strain of guinea pig used, timing of viral challenge, and study end points examined. However, the general lack of detailed molecular characterization of the GPCMV genome has precluded subunit-vaccine studies using recombinant expression strategies. With the recent successful cloning and expression of the GPCMV homologs of the vaccine targets gB and GP83 [27–29], the present study was undertaken to evaluate the protective efficacy of these gene products against congenital CMV infection

Previous studies using the murine CMV (MCMV) model have supported the concept that subunit vaccines using cloned, recombinant expression technologies are capable of conferring protection against CMV disease. A vaccinia recombinant expressing MCMV gB provided protection against lethal MCMV challenge in BALB/c mice when administered as a vaccine [34]. Studies have also examined protection against MCMV disease after vaccination with DNA vaccines against CTL targets; interestingly, in this model, homologs of immediate-early proteins and the MCMV UL84 homolog conferred the best protection, whereas the MCMV UL83 vaccine was generally ineffective [35–38]. Since MCMV does not cross the placenta or cause congenital infection, extrapolating these results to the prevention of congenital HCMV infection is problematic [39]. The present study has examined the value of maternal DNA vaccination against congenital CMV infection and disease. In liveborn pups, gB vaccine was found to provide protection against congenital infection, and, when congenital infection did occur, viral DNA load was lower in pups born to gB-vaccinated guinea pigs, compared with that in controls. These observations suggest that immune responses to gB play a key role in protective maternal immunity during pregnancy

One observation of interest in gB-vaccinated dams was the important influence of the magnitude of the antibody response on GPCMV-induced pup mortality. Thus, there was no pup mortality (0/13) in pups born to gB-vaccinated dams with a high-titer ELISA antibody response (>3.4 log10). In contrast, 50% overall pup mortality was noted in pups born to gB-vaccinated dams with ELISA titers ⩽3.4 log10 (table 1). Although differences in the magnitude of the gB response correlated with outcome, all vaccinated dams made antibodies capable of immunoprecipitating the full gB complex, including the carboxy-terminal moiety, gp58, the region of the gB molecule that appears to encode neutralization-related epitopes [27]. The observation of the critical importance of antibody response in fetal protection is consistent with previous reports in the guinea pig model using passive antibody transfer studies and warrants additional study of alternative gB-expression strategies [31, 40]. Future studies of DNA vaccine designed to optimize the immunogenicity of the gB plasmid may therefore be warranted. In addition to the effect of vaccination on pup mortality in a subset of gB-vaccinated dams, these data clearly indicate an effect of gB vaccine on the congenital CMV infection rate in liveborn pups and on the magnitude of virus load in infected pups. These data are of particular relevance to HCMV-vaccine studies, because congenital infection, and not mortality or disease, is likely to be the end point that is most relevant for congenital HCMV infection, a disease that rarely causes mortality. The data indicating reduction of fetal virus load in congenitally infected pups is also relevant to HCMV disease, since evidence in HCMV-infected infants suggests that virus load may be predictive of neurodevelopmental sequelae [41]

In contrast to gB vaccine, DNA vaccine based on the GPCMV UL83 homolog, GP83, provided no significant protection against infection. Several possibilities could explain the lack of efficacy of the GP83 vaccine. The epidermal (gene gun) administration of DNA vaccine, although a more efficient route for induction of strong neutralizing-antibody responses [42], may be less useful for CTL targets, because of the Th-2 cytokine bias induced by this approach. Alternatively, the GP83 homolog, like the MCMV M83 homolog, may not represent the dominant CTL target in the setting of GPCMV infection. Since a DNA vaccine based on the MCMV UL84 homolog, M84, appears to be more strongly protective than M83 DNA vaccine [36], testing a DNA vaccine based on the GPCMV UL84 homolog may prove of interest. Detailed analyses of T cell responses to GPCMV infection may provide useful insights into logical future subunit-vaccine targets. Interestingly, however, although preconception vaccination with GP83 did not decrease either pup mortality or the rate of congenital infection, there were statistically significant reductions in virus load among liveborn, congenitally infected pups, compared with those in liveborn, infected pups born to negative-control dams (table 2). Thus, although GP83 vaccine did not reduce the incidence of vertical transmission, it did appear to modify the magnitude of viral transmission in newborn pups, suggesting the need for future evaluation of GP83-vaccine approaches in this model. Evaluation of alternative vaccine approaches, such as in administration of DNA vaccine, or vectored approaches, such as vaccinia virus, may be useful in clarifying the role that the UL83 homolog plays in protective immunity against congenital GPCMV infection

Although the guinea pig provides a useful model for a variety of congenital infections—including CMV, toxoplasmosis, and syphilis [43–45]—there have been few evaluations of subunit-vaccine strategies using this small-animal model. In the guinea pig model of congenital toxoplasmosis, a subunit vaccine based on the SAG protein was found to provide protection against vertical transmission [45]. As noted, purified native GPCMV proteins [34–36] have shown efficacy as vaccines against congenital GPCMV infection and disease. The present study, however, represents the first report of the efficacy of a subunit vaccine using recombinant expression technology for congenital CMV infection. Furthermore, these data represent the first report of the efficacy of any DNA vaccine for a congenital infection. In addition to confirming the potential value of gB subunit vaccines for prevention of congenital CMV infection, these data provide support for the continued development of CMV DNA vaccines for human use [46, 47]. Continued investigation of subunit vaccines in the GPCMV model should help to prioritize which strategies are likely to be most useful in development of HCMV vaccine

Acknowledgments

The technical assistance of Greg Stroup, Nancy Jensen, Amber Hickson, and Fernando Bravo is acknowledged

References

1
Demmler
GJ
Congenital cytomegaloviral infection and disease
Semin Pediatr Infect Dis
 , 
1999
, vol. 
10
 (pg. 
195
-
200
)
2
Bale
JF
Miner
L
Petheram
SJ
Congenital cytomegalovirus infection
Curr Treat Options Neurol
 , 
2002
, vol. 
4
 (pg. 
225
-
30
)
3
Dahle
AJ
Fowler
KB
Wright
JD
Boppana
SB
Britt
WJ
Longitudinal investigation of hearing disorders in children with congenital cytomegalovirus
J Am Acad Audiol
 , 
2000
, vol. 
11
 (pg. 
283
-
90
)
4
Gaytant
MA
Steegers
EA
Semmekrot
BA
Merkus
HM
Galama
JM
Congenital cytomegalovirus infection: review of the epidemiology and outcome
Obstet Gynecol Surv
 , 
2002
, vol. 
57
 (pg. 
245
-
56
)
5
Adler
SP
Starr
SE
Plotkin
SA
, et al.  . 
Immunity induced by primary human cytomegalovirus infection protects against secondary infection among women of childbearing age
J Infect Dis
 , 
1995
, vol. 
171
 (pg. 
26
-
32
)
6
Fowler
KB
Stagno
S
Pass
RF
Britt
WJ
Boll
TJ
The outcome of congenital cytomegalovirus infection in relation to maternal antibody status
N Engl J Med
 , 
1992
, vol. 
326
 (pg. 
663
-
7
)
7
Stratton
KR
Durch
JS
Lawrence
RS
Vaccines for the 21st century: a tool for decision making Committee to Study Priorities for Vaccine Development, Division of Health Promotion and Disease Prevention, Institute of Medicine
1999
Washington, DC
8
Plotkin
SA
Vaccination against cytomegalovirus, the changeling demon
Pediatr Infect Dis J
 , 
1999
, vol. 
18
 (pg. 
313
-
25
)
9
Adler
SP
Current prospects for immunization against cytomegaloviral disease
Infect Agents Dis
 , 
1996
, vol. 
5
 (pg. 
29
-
35
)
10
Pass
RF
Immunization strategy for prevention of congenital cytomegalovirus infection
Infect Agents Dis
 , 
1996
, vol. 
5
 (pg. 
240
-
4
)
11
Plotkin
SA
Is there a formula for an effective CMV vaccine
J Clin Virol
 , 
2002
, vol. 
25
 
Suppl 2
(pg. 
S13
-
21
)
12
Spaete
RR
Gehrz
RC
Landini
MP
Human cytomegalovirus structural proteins
J Gen Virol
 , 
1994
, vol. 
75
 (pg. 
3287
-
308
)
13
Britt
WJ
Vugler
L
Butfiloski
EJ
Stephens
EB
Cell surface expression of human cytomegalovirus (HCMV) gp55–116 (gB): use of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody response
J Virol
 , 
1990
, vol. 
64
 (pg. 
1079
-
85
)
14
McLaughlin-Taylor
E
Pande
H
Forman
SJ
, et al.  . 
Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes
J Med Virol
 , 
1994
, vol. 
43
 (pg. 
103
-
10
)
15
Wills
MR
Carmichael
AJ
Mynard
K
, et al.  . 
The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL
J Virol
 , 
1996
, vol. 
70
 (pg. 
7569
-
79
)
16
Pass
RF
Duliege
AM
Boppana
S
A subunit cytomegalovirus vaccine based on recombinant envelope glycoprotein B and a new adjuvant
J Infect Dis
 , 
1999
, vol. 
180
 (pg. 
970
-
5
)
17
Frey
SE
Harrison
C
Pass
RF
, et al.  . 
Effects of antigen dose and immunization regimens on antibody responses to a cytomegalovirus glycoprotein B subunit vaccine
J Infect Dis
 , 
1999
, vol. 
180
 (pg. 
1700
-
3
)
18
Gonczol
E
Berensci
K
Pincus
S
, et al.  . 
Preclinical evaluation of an ALVAC (canarypox) human cytomegalovirus glycoprotein B vaccine candidate
Vaccine
 , 
1995
, vol. 
13
 (pg. 
1080
-
5
)
19
Adler
SP
Plotkin
SA
Gonczol
E
, et al.  . 
A canarypox vector expressing cytomegalovirus (CMV) glycoprotein B primes for antibody responses to a live attenuated CMV vaccine (Towne)
J Infect Dis
 , 
1999
, vol. 
180
 (pg. 
843
-
6
)
20
Bernstein
DI
Schleiss
MR
Berencsi
K
, et al.  . 
Effect of previous or simultaneous immunization with canarypox expressing cytomegalovirus (CMV) glycoprotein B (gB) on response to subunit gB vaccine plus MF59 in healthy CMV-seronegative adults
J Infect Dis
 , 
2002
, vol. 
185
 (pg. 
686
-
690
)
21
Berencsi
K
Gyulai
Z
Gonczol
E
, et al.  . 
A canarypox vector-expressing cytomegalovirus (CMV) phosphoprotein 65 induces long-lasting cytotoxic T cell responses in human CMV-seronegative subjects
J Infect Dis
 , 
2001
, vol. 
183
 (pg. 
1171
-
9
)
22
Bernstein
DI
Bourne
N
Zak
O
Sande
M
Animal models for cytomegalovirus infection: guinea pig CMV
Experimental models in antimicrobial chemotherapy
 , 
1999
London
Academic Press
23
Griffith
BP
McCormick
SR
Fong
CKY
Lavellee
JT
Lucia
HL
The placenta as a site of cytomegalovirus infection in guinea pigs
J Virol
 , 
1985
, vol. 
55
 (pg. 
402
-
9
)
24
Bia
FJ
Griffith
BP
Tarsio
M
Hsuing
GD
Vaccination for the prevention of maternal and fetal infection with guinea pig cytomegalovirus
J Infect Dis
 , 
1980
, vol. 
142
 (pg. 
732
-
8
)
25
Harrison
CJ
Britt
WJ
Chapman
NM
Mullican
J
Tracy
S
Reduced congenital cytomegalovirus (CMV) infection after maternal immunization with a guinea pig CMV glycoprotein before gestational primary CMV infection in the guinea pig model
J Infect Dis
 , 
1995
, vol. 
172
 (pg. 
1212
-
20
)
26
Bourne
N
Schleiss
MR
Bravo
FJ
Bernstein
DI
Preconception immunization with a cytomegalovirus (CMV) glycoprotein vaccine improves pregnancy outcome in a guinea pig model of congenital CMV infection
J Infect Dis
 , 
2001
, vol. 
183
 (pg. 
59
-
64
)
27
Schleiss
MR
Cloning and characterization of the guinea pig cytomegalovirus glycoprotein B gene
Virology
 , 
1994
, vol. 
202
 (pg. 
173
-
85
)
28
Schleiss
MR
McGregor
A
Jensen
NJ
Erdem
G
Aktan
L
Molecular characterization of the guinea pig cytomegalovirus UL83 (pp65) protein homolog
Virus Genes
 , 
1999
, vol. 
19
 (pg. 
205
-
21
)
29
Schleiss
MR
Bourne
N
Jensen
NJ
Bravo
F
Bernstein
DI
Immunogenicity evaluation of DNA vaccines that target guinea pig cytomegalovirus proteins glycoprotein B and UL83
Viral Immunol
 , 
2000
, vol. 
13
 (pg. 
155
-
67
)
30
Harrison
CJ
Myers
MG
Peripheral blood mononuclear cell–mediated cytolytic activity during cytomegalovirus (CMV) infection of guinea pigs
J Med Virol
 , 
1988
, vol. 
25
 (pg. 
441
-
53
)
31
Bratcher
DF
Bourne
N
Bravo
FJ
, et al.  . 
Effect of passive antibody on congenital cytomegalovirus infection in guinea pigs
J Infect Dis
 , 
1995
, vol. 
172
 (pg. 
944
-
50
)
32
McGregor
A
Schleiss
MR
Molecular cloning of the guinea pig cytomegalovirus (GPCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli
Mol Genet Metab
 , 
2001
, vol. 
72
 (pg. 
15
-
26
)
33
Schleiss
MR
Bourne
N
Bravo
F
Jensen
NJ
Bernstein
DI
Quantitative competitive PCR (qcPCR) analysis of viral load following experiment guinea pig cytomegalovirus (GPCMV) infection
J Virol Methods
 , 
2003
, vol. 
108
 (pg. 
103
-
10
)
34
Rapp
M
Messerle
M
Lucin
P
Koszinowski
UH
Michelson
S
Plotkin
SA
In vivo protection studies with mCMV glycoproteins gB and gH expressed by vaccinia virus
Multidisciplinary approach to understanding cytomegalovirus disease
 , 
1993
Amsterdam
Elsevier Science Publishers BV
(pg. 
327
-
32
)
35
Gonzalez Armas
JC
Morello
CS
Cranmer
LD
Spector
DH
DNA immunization confers protection against murine cytomegalovirus infection
J Virol
 , 
1996
, vol. 
70
 (pg. 
7921
-
8
)
36
Morello
CS
Cranmer
LD
Spector
DH
Suppression of murine cytomegalovirus (MCMV) replication with a DNA vaccine encoding MCMV M84 (a homolog of human cytomegalovirus pp65)
J Virol
 , 
2000
, vol. 
74
 (pg. 
3696
-
708
)
37
Morello
CS
Ye
M
Spector
DH
Development of a vaccine against murine cytomegalovirus (MCMV), consisting of plasmid DNA and formalin-inactivated MCMV, that provides long-term, complete protection against viral replication
J Virol
 , 
2002
, vol. 
76
 (pg. 
4822
-
35
)
38
Ye
M
Morello
CS
Spector
DH
Strong CD8 T-cell responses following coimmunization with plasmids expressing the dominant pp89 and subdominant M84 antigens of murine cytomegalovirus correlate with long-term protection against subsequent viral challenge
J Virol
 , 
2002
, vol. 
76
 (pg. 
2100
-
12
)
39
Johnson
KP
Mouse cytomegalovirus: placental infection
J Infect Dis
 , 
1969
, vol. 
120
 (pg. 
445
-
50
)
40
Chatterjee
A
Harrison
CJ
Britt
WJ
Bewtra
C
Modification of maternal and congenital cytomegalovirus infection by anti–glycoprotein b antibody transfer in guinea pigs
J Infect Dis
 , 
2001
, vol. 
183
 (pg. 
1547
-
53
)
41
Revello
MG
Gerna
G
Diagnosis and management of human cytomegalovirus infection in the mother, fetus, and newborn infant
Clin Microbiol Rev
 , 
2002
, vol. 
15
 (pg. 
680
-
715
)
42
Gurunathan
S
Klinman
DM
Seder
RA
DNA vaccines: immunology, application, and optimization
Annu Rev Immunol
 , 
2000
, vol. 
18
 (pg. 
927
-
74
)
43
Bia
FJ
Griffith
BP
Fong
CK
Hsiung
GD
Cytomegaloviral infections in the guinea pig: experimental models for human disease
Rev Infect Dis
 , 
1983
, vol. 
5
 (pg. 
177
-
95
)
44
Wicher
K
Baughn
RE
Abbruscato
F
Wicher
V
Vertical transmission of Treponema pallidum to various litters and generations of guinea pigs
J Infect Dis
 , 
1999
, vol. 
179
 (pg. 
1206
-
12
)
45
Haumont
M
Delhaye
L
Garcia
L
, et al.  . 
Protective immunity against congenital toxoplasmosis with recombinant SAG1 protein in a guinea pig model
Infect Immun
 , 
2000
, vol. 
68
 (pg. 
4948
-
53
)
46
Temperton
NJ
DNA vaccines against cytomegalovirus: current progress
Int J Antimicrob Agents
 , 
2002
, vol. 
19
 (pg. 
169
-
72
)
47
Endresz
V
Kari
L
Berencsi
K
, et al.  . 
Induction of human cytomegalovirus (HCMV)–glycoprotein B (gB)–specific neutralizing antibody and phosphoprotein 65 (pp65)–specific cytotoxic T lymphocyte responses by naked DNA immunization
Vaccine
 , 
1999
, vol. 
17
 (pg. 
50
-
8
)
Financial support: National Institutes of Health (grants AI-65289 and HD38416-01); March of Dimes (basic research grants 6-FY98/99-0416 and FY01-226)