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Animal models of congenital cytomegalovirus infection: an overview of progress in the characterization of guinea pig cytomegalovirus (GPCMV)
Journal of Clinical Virology 25 (2002) S37 /S49 www.elsevier.com/locate/jcv
Review article
Animal models of congenital cytomegalovirus infection: an overview of progress in the characterization of guinea pig cytomegalovirus (GPCMV) Mark R. Schleiss * Division of Infectious Diseases, Children’s Hospital Research Foundation, 3333 Burnet Ave, Cincinnati, OH 45229, USA
Abstract Background: The strict species-specificity of cytomegalovirus (CMV) precludes preclinical evaluation of human CMV (HCMV) vaccines in animal models and necessitates the study of nonhuman CMVs. Among the CMVs of small mammals, the guinea pig cytomegalovirus (GPCMV) has unique advantages, due to its ability to cross the placenta, causing infection in utero. Objective and study designs: Progress in GPCMV studies has been hampered by a lack of detailed molecular characterization of the viral genome. Therefore, recent efforts have been undertaken to characterize the GPCMV genome, and apply this information to in vivo subunit vaccine studies. Results: Progress in the sequencing of the GPCMV genome has revealed the presence of both highly conserved as well as novel open reading frames (ORFs). Cloning of GPCMV vaccine candidates, such as the glycoprotein B (gB) and UL83 proteins, has facilitated subunit vaccine evaluation. Protein vaccines and DNA vaccines have shown evidence of protection in pregnancy/ challenge experiments. In addition, the GPCMV genome has proved amenable to cloning as a bacterial artificial chromosome (BAC) in Escherichia coli , and BAC-derived recombinants retain the ability to replicate in vivo. Conclusions: Progress has been made in molecular characterization of GPCMV. Insights from these studies should prove germane to the understanding of the correlates of protective immunity for the fetus in vaccine studies, and should assist in prioritization of vaccine strategies in HCMV vaccine trials. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytomegalovirus (CMV); Guinea pig; CMV vaccines
1. Introduction Infection with human cytomegalovirus (HCMV), although ubiquitous in nature, can lead to severe disease manifestations in immunocompromised patients, including newborn infants (Alford and Britt, 1993). Acquisition of HCMV in
* Fax: /1-513-636-7655 E-mail address: [email protected] (M.R. Schleiss).
utero may cause significant neurodevelopmental handicap, including sensorineural deafness (Demmler, 1994). Although maternal vaccination against HCMV may be useful in preventing such damage (Adler, 1996; Pass, 1996; Plotkin, 1999), currently there are no licensed CMV vaccines available. CMV infections are highly speciesspecific, and, therefore, HCMV cannot be studied in animal models of infection (Staczek, 1990). Hence, the species-specific cytomegaloviruses of animals have been studied to provide in vivo
1386-6532/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 2 ) 0 0 1 0 0 - 2
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models for the study of vaccines and viral pathogenesis. Although mouse and rat cytomegalo viruses have been studied as models of CMV disease, these CMVs do not cross the placenta, and are, therefore, less useful for vaccine studies which target prevention of congenital infection (Medearis, 1964; Bruggeman et al., 1985; Ho, 1992). The rhesus macaque CMV (RhCMV) causes placental infection and fetal injury (Tarantal et al., 1998; Lockridge et al., 1999), but the expense of these primates, as well as the rarity of RhCMV seronegative animals, makes this model impractical for large-scale vaccine studies. Thus, each of these models has limited usefulness for the study of CMV vaccines. In contrast to the CMVs of other small mammals, the guinea pig cytomegalovirus (GPCMV) is unique in its ability to cross the placenta and cause fetal infection. GPCMV was first recognized following histopathologic identification of inclusions in guinea pig salivary glands in 1920 (Jackson, 1920). Although the ability of this virus to infect the guinea pig placenta was described in the 1930s (Markham and Hudson, 1936), further description of a guinea pig model of congenital CMV infection would not occur until the late 1970s, when three groups independently described studies of fetal infection following GPCMV inoculation of pregnant dams (Choi and Hsuing, 1978; Kumar and Nankervis, 1978; Johnson and Connor, 1979). Since these initial reports, several groups have studied the pathogenesis and prevention of transplacental CMV infection using the GPCMV model. A number of aspects of guinea pig reproductive biology have contributed to the usefulness of this model. Guinea pig gestational periods are fairly lengthy, ranging from 65 to 70 days, and can conveniently be divided into trimesters (Bia et al., 1983). The guinea pig placenta, like the human placenta, is hemochorial, with a single trophoblast layer separating maternal and fetal circulation, similar histologically to human placenta (Griffith et al., 1985). This aspect of guinea pig reproductive biology provides the greatest driving force justifying the use of this model for vaccine experiments. The maternal and fetal outcomes which may be monitored in the GPCMV model are dependent
upon a number of experimental variables, including the timing and route of GPCMV inoculation, the strain of guinea pig utilized and the source of virus. Viral inoculation in early pregnancy tends to lead to pup resorption, whereas challenge in late pregnancy tends to lead to pup mortality (stillborn pups). Inbred strains, such as strain 2 and JY-9, tend to have greater susceptibility to GPCMV infection than outbred strains, such as the Hartley strain. Accordingly, inoculation of these highly susceptible inbred animals with GPCMV during pregnancy may produce maternal mortality in addition to fetal demise. Salivary gland-adapted virus stocks produce more severe disease than tissue culture virus-attenuated virus, and inoculation via intravascular, intraperitoneal, or subcutaneous routes generates a more severe infection than inoculation at a mucosal surface. Irrespective of these differences, a number of vaccine endpoints are easily quantifiable in congenital infection studies. These include infection rate, pup mortality, maternal and pup weight, fetal resorption rate, viral load by PCR, and extent of placental infection. A number of studies have examined vaccines in the GPCMV model. In a study in outbred guinea pigs, a noninfectious, a live-attenuated vaccine GPCMV vaccine was shown to have efficacy against congenital GPCMV infection (Bia et al., 1980). This study also examined a partially purified, soluble envelope vaccine, administered with Freund’s adjuvant, and found this vaccine protective as well. Although these studies confirmed the importance of pre-existing maternal immunity to GPCMV in protection against congenital infection, there was limited data concerning the qualitative aspects of the immune responses in these studies, in part because of the lack of any characterization of immunogenic GPCMV structural proteins. In a later study using an inbred model of congenital GPCMV infection, an immunoaffinity-purified glycoprotein was found to induce strong antibody and CMI responses when administered with Freund’s adjuvant, and newborn pups were protected against congenital infection and disease (Harrison et al., 1995), providing more direct evidence for the importance of immune responses to specific viral proteins in
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protection of the fetus. However, although this and other GPCMV structural proteins had been characterized through the use of monoclonal antibodies (Tsutsui et al., 1986; Nogami-Satake and Tsutsui, 1988; Jones et al., 1994; Britt and Harrison, 1994), the molecular identity of GPCMV proteins at the genetic level had largely been unexplored. This overview reports the extensive effort which has been undertaken in recent years to better characterize the molecular biology of GPCMV and describes how this information has been applied to in vivo studies of vaccines and pathogenesis.
2. Materials and methods 2.1. Virus and cells Guinea pig CMV (strain no. 22122, ATC VR682) was propagated on guinea pig fibroblast lung cells (GPL, ATCC CCL 158) and maintained in F-12 medium supplemented with 10% fetal calf serum (FCS, HyClone Laboratories), 10 000 IU/l penicillin, 10 mg/l streptomycin (Gibco-BRL) and 7.5% NaHCO3 (Gibco-BRL). Recombinant viruses were selected by the addition of mycophenolic acid (MPA) and xanthine (Gibco-BRL) into the medium at 200 and 10 mm, respectively (McGregor and Schleiss, 2001b). 2.2. Cloning and sequence analyses Restriction enzymes Hin dIII and Eco RI were used to subclone fragments of the GPCMV genome, using strandard cloning techniques, for sequence analyses, using vectors pUC and pBR322 (Schleiss, 1994). Details of cloning of DNA vaccine construct and GPCMV gB-GST fusion construct have been summarized elsewhere (Schleiss et al., 2000; Bourne et al., 2001). DNA sequencing was performed using 33S-dATP and Sequenase† (United States Biochemical) according to the manufacturer’s specification, and by use of automated sequencing using an ABI-Prism automated sequencer. Nucleotide sequences and the deduced protein coding sequences of candidate open reading frames (ORFs) were compared with
published
HCMV sequences software package.
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using
the
MACVECTOR†
2.3. Immunization with subunit vaccines and outcome analyses in guinea pigs Young female and proven breeder male Hartley guinea pigs were obtained from Camm Research Institute (Wayne, NJ). Inbred adult strain-2 guinea pigs were purchased from the Children’s Hospital Research Foundation (Cincinnati). All animal experiments were performed in accordance with guidelines approved by the American Association of Accreditation of Laboratory Animal Care and were approved by the Animal Use Care Committee of Children’s Hospital Research Foundation. Glycoprotein immunogen was administered with Freund’s adjuvant as previously described (Bourne et al., 2001). For DNA vaccine studies, plasmid DNA encoding the GPCMV glycoprotein B ORF was administered by epidermal inoculation via gene gun as previously described (Schleiss et al., 2000). A total of four inoculations were administered. Antibody titers were determined using ELISA assay. The ELISA titer was defined as the reciprocal of the highest dilution that produced an absorbence of /0.10 and twice the absorbence against control antigen (Bratcher et al., 1995). Glycoprotein B (gB)specific antibodies in guinea pig serum were determined by ELISA, using a gB fusion protein with glutathione S-transferase (GST) as the antigen. Avidity index (AI) of antibody was determined by calculating the ratio of the OD450 of vaccine-derived antibodies in the presence and absence of 4 M urea denaturation reagent (Lazzarotto et al., 1997; Schleiss et al., 2001). Western blot assays were performed as described previously (Paglino et al., 1999). For congenital infection experiments, protocols for pregnancy/challenge studies are summarized in detail elsewhere (Bratcher et al., 1995; Bourne et al., 2001). Briefly, immunized animals (glycoprotein or DNA vaccine) were placed with GPCMVseronegative proven breeder male Hartley guinea pigs. The female animals were examined weekly by palpation for evidence of pregnancy. When they were estimated to be entering the third trimester of
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pregnancy, they were inoculated by subcutaneous injection with virulent salivary gland-passaged GPCMV. In some experiments, animals were inoculated with tissue culture adapted recombinant virus containing the eGFP reporter gene cassette (see below). After virus challenge, the animals were observed daily until delivery, to determine pregnancy outcome. Stillborn animals were dissected on the day of delivery, and liveborn pups were sacrificed within 48 h of birth. Minced explant cultures were inoculated onto guinea pig lung cell monolayers for identification of characteristic cytopathic effect (CPE). Outcomes in vaccinated and unvaccinated groups were compared statistically using Fisher’s exact test. In some experiments, at various times points post inoculation animals were humanely sacrificed and organs were removed for histopathological analyses. Selected tissues were formalin-fixed and paraffin-embedded for subsequent sectioning for staining with hematoxylin and eosin. 2.4. Isolation of recombinant viruses and BAC clone Viral DNA and plasmid DNA (containing guanosylphosphoribosyltransferase [gpt] and green fluorescent protein [eGFP] cassettes) were used to co-transfect confluent monolayers of GPL cells in 60 mm dishes using Lipofectin† (GibcoBRL) following the manufacturer’s specifications. After overnight incubation at 37 8C the cells were
washed with F-12 medium and incubated for a further 20/30 days post-transfection, or until significant CPE was observed. At this stage the medium was replaced with F-12 medium containing MPA and xanthine (McVoy et al., 1997; McGregor and Schleiss, 2001b). Additional passages were performed under continuous selection. Viral titrations were replated on a series of 24 well plates under limiting dilution to generate clonal viral stocks, which were further verified by Southern blot analysis. A recombinant virus carrying the BAC plasmid in Hin dIII ‘N’ was generated by cotransfection of wild-type viral DNA with pACYC177NKGG onto GPL cells and selection of gpt /eGFP-positive virus. Isolates cloned by limiting dilution were verified to be clonal by Hin dIII restriction profile and Southern blot analysis, which demonstrated an 8.8-kbp insertion into the Hin dIII ‘N’ region of the viral genome, corresponding to the BAC plasmid. Circular viral DNA was then purified and used to transform Escherichia coli to generate stable BAC clones (McGregor and Schleiss, 2001b).
3. Results 3.1. Molecular characterization of the GPCMV genome The strategy of low-stringency Southern blot hybridization of a HCMV gB probe with GPCMV
Fig. 1. Schematic of GPCMV genome identifying map positions of sequenced homolog genes characterized to date. Hin dIII restriction map based upon original map described by Isom (Gao and Isom, 1984). Homolog genes are identified based upon correlation with HCMV ORFs. Gene order and map positions are relatively well conserved from UL32 region (pp 150 homolog) through region of putative MIEP, but ORFs identified at genome termini are divergent relative to HCMV.
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restriction digests was used to investigate which region in the GPCMV genome contained the gB homolog. This homolog was found to map to the Hin dIII ‘K’ and ‘P’ fragments. This data allowed orientation of the GPCMV genome relative to other CMVs, and facilitated the identification of other homolog genes (Fig. 1). Northern blot revealed that the GPCMV gB mRNA is transcribed with ‘early’ kinetics as a 6.8 kb message, and encodes a 901 aa protein with 42% homology to HCMV gB (Schleiss, 1994). The protein was found to be cleaved into amino and carboxy terminal subunits of approximately 90 and 58 kDa. Immunological investigations revealed that GPCMV gB is a major target of the neutralizing
Fig. 2. Placental injury in GPCMV inoculated dams. Following third trimester challenge with virulent, SG-passaged GPCMV, pup mortality ranges from 30 to 70% and placental injury is noted. Top panel (a), healthy near-term placenta showing normal syncytiotrophoblast (upper portion of micrograph) layer. Lower panel (b), placenta from stillborn pup with extensive GPCMV infection, 21 days-post maternal inoculation. Placenta shows significant infarction, peri-vascular inflammatory cell infiltrate, and viral inclusions. Hematoxylin and eosin stains, 120 /magnification.
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antibody response following natural GPCMV infection, and antibodies to gB are uniformly found in GPCMV seropositive animals (Schleiss, 1994; Bourne et al., 2001). Characterization of the GPCMV gB homolog has provided a framework for study of the organization of the viral genome. Clones of GPCMV Hin dIII and Eco RI restriction fragments and, more recently, the genomic clone maintained in E. coli as a BAC (McGregor and Schleiss, 2001b) have been utilized for DNA sequencing. Following the identification of gB, other highly conserved structural proteins were mapped in the GPCMV genome. The glycoprotein H (gH; UL75 homolog) was found to map to the Xba I ‘T’ region within the Hin dIII ‘A’ fragment of the genome. This ORF is 723 aa in length, and the mature glycoprotein migrates at Mr /85 kDa in SDSPAGE (Brady and Schleiss, 1996). The gH chaperone, glycoprotein L (gL; UL115 homolog) maps to a GPCMV genome region collinear with HCMV UL115, within the Hin dIII ‘B’ region (Paglino et al., 1999). Both gH and gL of GPCMV are immunoreactive with anti-GPCMV antibodies, confirming that these conserved glycoprotein homologs are targets of immune response in natural GPCMV infection. An ORF with identity to HCMV UL74, the glycoprotein O member of the HCMV gCIII complex (Huber and Compton, 1998), has also been identified, although identification of the protein has not yet been confirmed in GPCMV. In addition to conserved structural glycoproteins, other GPCMV genes, both structural and nonstructural, were characterized at the molecular level. Sequence analysis of regions adjacent to the gB (UL55) homolog successfully identified the conserved DNA polymerase. GPCMV pol is transcribed as a 3.9 kb message with ‘early’ kinetics, and encodes an ORF of 1094 amino acids (Schleiss, 1995). The UL97 phosphotransferase homolog has been mapped to Hin dIII ‘C’ (Fox and Schleiss, 1997). A promoter with strong similarities, both in structure and cis -recognition sequences, to the HCMV major immediate early promoter (MIEP) has been identified in the Hin dIII ‘E’ region. Tegument phosphoprotein homologs have also been characterized in the
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Fig. 3
GPCMV genome. Within the Hin dIII ‘A’ region of the GPCMV genome, an ORF with homology to the HCMV UL83 tegument phosphoprotein has recently been identified. This ORF was predicted to encode a 565-amino-acid (aa) protein with a
molecular mass of 62.3 kDa. Western blot and immunoprecipitation analyses identified a 70 kDa phosphoprotein corresponding to the GPCMV UL83 homolog in both virion and dense body fractions (Schleiss et al., 1999). Other conserved,
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sequenced phosphoprotein homologs have been identified in the GPCMV genome, including homologs of UL32, the pp150 protein, and UL115, the pp28 protein. Of interest, an ORF with identity to the CC chemokine, MIP1-a, has been identified in the Hin dIII ‘D’ region of the genome, and is not present in other CMVs (Schleiss, unpublished data).
3.2. Experimental vaccine evaluations in the guinea pig congenital infection model Studies of vaccine-induced protection against congenital CMV infection were designed to test the importance of immune responses to GPCMV envelope glycoproteins in protective immunity. In a late (third trimester) challenge model in pregnant outbred Hartley strain guinea pigs, inoculation of GPCMV subcutaneously during the third trimester results in significant injury to pups (Bratcher et al., 1995; Bourne et al., 2001). Pup mortality is manifest either as stillbirth, or death soon after birth. Depending upon the dose of virus used in the maternal inoculation, mortality rates are observed which may range from 30 to 70%. Higher doses which result in greater degrees of pup mortality often cause maternal mortality as well. Infected pups show widespread evidence of disease, with viral inclusions and inflammatory cell infiltrates in liver, spleen, lung, and occasionally brain. These studies confirmed that the placenta is a significant target of virus-induced injury. In dams sacrificed following such third-trimester challenge, placentas corresponding to stillborn pups show extensive damage, including viral
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inclusions, and areas of infarction and inflammatory cell infiltrates (Fig. 2). Subunit glycoprotein vaccines for GPCMV were used to test whether preconceptual immunization can protect the maternal-placenta-fetal unit against this virally-induced damage. In a protein subunit vaccine study, immunity conferred by immunization with envelope glycoprotein resulted in substantial protection against pup mortality, with a reduction in pup mortality from 56 to 14% in the glycoprotein immunized group (P B/0.001). In addition, among live-born pups, glycoprotein immunization substantially reduced infection rates. GPCMV was isolated from 24 of 54 liveborn pups born to immunized mothers, compared with 16 of 20 live-born pups born to controls, indicating that immunization significantly reduced in utero transmission in surviving animals (P B/ 0.01). The dominant immunogen in the glycoprotein vaccine was the gB homolog (Bourne et al., 2001). Although dams in this study generated high-titer antibodies as measured by ELISA assay of sonicated GPCMV coating antigen (Bratcher et al., 1995), more detailed analyses of the qualitative nature of the antibody response were undertaken to test whether antibody responses to key domains of GPCMV gB correlated with protection against congenital infection. When antibody titers were assessed against a series of GST fusion proteins spanning the GPCMV gB, the highest levels of antibody were found targeting a region of GPCMV gB collinear with HCMV gB antigenic domain 1 (AD-1). This domain, corresponding to amino acid residues 546 /626 of GPCMV gB,
Fig. 3. Antibody responses to glycoprotein vaccine and outcome correlations in GPCMV congenital infection model. (A) Comparison of amino acid sequence between HCMV (top line) and GPCMV gB (lower line). Although overall amino acid similarity is approximately 40%, amino acid identity is strikingly high in region corresponding to AD-1 domain. Within a 38 amino acid region bracketed by two conserved cysteine residues and a conserved N-linked glycosylation site, identity is greater than 70% (shaded). GSTgB fusion proteins map strong antibody responses following glycoprotein immunization to this region. Western blot: lanes 1 and 2, preimmune sera nonreactive with GST carrier protein (lane 1) and GST-AD1 fusion (lane 2). Lanes 3 and 4, sera from immunized dam is not reactive with GST (lane 3) but strongly immunoreactive with GST-AD1 fusion (42 kDa protein, arrow). In vaccine/transmission study, pups free of congenital infection (n /9) had statistically significantly higher ELISA titers to GST-AD1 than pups bom with congenital infection (n /12). (B) IgG AI and outcome in congenital GPCMV infection. Urea-based avidity determination identifies range of AI following glycoprotein immunization, pregnancy, and delivery. In pups born to dams with high AI, only 3/18 had congenital GPCMV infection. In contrast, pups born to dams with low or intermediate AI had congenital infection in 7/11 cases (P B/ 0.02 compared with high avidity).
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includes a 38 amino acid region bracketed by two conserved cysteine residues and a conserved Nlinked glycosylation site (Britt and Mach, 1996). Although the overall amino acid similarity of the HCMV and GPCMV gB proteins is approximately 42%, amino acid identity between the two species is particularly striking in this region, at 71% (Fig. 3a). When antibody titers against a GST-GPCMV AD1 fusion protein were compared among pups in which transplacental infection occurred (n /12) and pups which were uninfected (n /9), titers against this domain were significantly correlated with protection against infection (P B/ 0.005). Similar observations correlating the importance of the qualitative aspects of the vaccineinduced antibody response with protection against transplacental transmission were made when the IgG AI was examined. The AI was determined by measuring the OD in ELISA assay in the presence and absence of urea. Among pups in which transmission of GPCMV occurred, an AI of greater than 0.45 was likewise found to correlate with protection against infection (Fig. 3b). More recently, DNA vaccines have been evaluated for protective efficacy in the congenital infection model. A GPCMV gB vaccine was engineered which expresses the GPCMV gB ORF under control of the HCMV MIEP. This vaccine was found to induce significant ELISA and neutralizing antibody levels when administered epidermally via ‘gene gun’. To evaluate the protective efficacy of the vaccine in a congenital Table 1 GPCMV DNA vaccine study: outcome
Control gB Vaccine
Litters
Pups
Infected pups
10 12
26 27
17 (70%) 7 (26%)*
Analysis of protection conferred against congenital GPCMV infection by DNA vaccination. Plasmid construct encoding GPCMV gB (pKTS404) was administered by gene gun inoculation. DNA immunization induces high titer ELISA and neutralizing antibody (Schleiss et al., 2000). Immunized dams were mated, then challenged with GPCMV. In pups born to gB immunized dams, vertical transmission of virus only occurred in 26% of pups, compared with 70% of pups born to dams immunized with vector plasmid alone (P B/0.01, Fisher’s exact test). *, P B/0.01 vs. control.
infection model, female Hartley guinea pigs were immunized with gB plasmid prior to establishment of pregnancy, and pup outcomes were compared, following third trimester viral challenge, between the vaccine group (pKTS404) and the control vector group (pCDNA). Pre-existing immunity conferred by the DNA vaccine resulted in significant reductions in transmission of CMV to the pup (Table 1), with an overall infection rate of 26% (7/27) in the vaccine group, compared with 70% (17/26) in the control group (P B/0.01, Fisher’s exact test). 3.3. Generation of recombinant GPCMV and BAC cloning of viral genome Efforts were undertaken to generate recombinant viruses expressing reporter genes to aid in the detection of virus in vivo, and to develop a strategy for generation of targeted viral mutants to facilitate the study of the role of specific viral gene products in pathogenesis. The gpt selection system was utilized to test whether metabolic selection could be used to generate such GPCMV recombinants (McVoy et al., 1997). Selection with gpt was successful in generating a mutant GPCMV with a eGFP cassette inserted into the nonessential Hin dIII ‘N’ region of the viral genome. Next, attempts were made to clone the GPCMV genome as a BAC in E. coli . To clone the GPCMV genome as a bacterial artificial chromosome (BAC), a BAC plasmid was first cloned into a unique Pac I site introduced into the Hin dIII ‘N’ locus of the GPCMV genome, on a shuttle plasmid which contained the gpt and eGFP cassettes (pACYC177NKGG) to provide sufficient sequence for homologous recombination (Fig. 4). A recombinant virus carrying a BAC plasmid in Hin dIII ‘N’ was generated by co-transfection of wild type viral DNA with pACYC177NKGG onto GPL cells and selection of gpt /eGFP positive virus. Isolates were purified by limiting dilution and verified to be clonal by Southern blot analysis. Next, in order to clone the BAC GPCMV genome as a plasmid in E. coli , GPL cells were infected with recombinant virus, and circular viral DNA intermediates purified. The circular viral DNA was transformed by electroporation into
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Fig. 4. Scheme used to clone GPCMV genome as BAC in E. coli . (A) Protocol for generation of BAC recombinant HindIII ‘N’ region of viral genome selected as site of insertion of BAC plasmid pKGG (modified BAC plasmid pKSO with gpt /eGTP cassettes added). Following gpt selection, recombinant virus selected and purified circular DNA obtained from tissue culture prep. Circular viral DNA in turn is used to transform E. coli . Following selection of transformants on chloramphenicol containing media, miniprep DNA confirmed successful cloning of GPCMV genome as BAC. (B) Expression of eGFP in GPCMV recombinant virus. Cell culture expression of eGFP reporter gene is easily visualized in this plaque photographed using GFP filter (140 /magnification).
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recA DH10B cells, and chloramphenicol drug resistant colonies selected (McGregor and Schleiss, 2001a,b). In vivo analyses of recombinants indicate, depending upon the locus of insertion, that eGFP-expressing viruses retain the capability to replicate and cause disease in guinea pigs. The replication kinetics of Hin dIII ‘N’ recombinant viruses generated both by gpt selection and BAC technology were comparable to that of wild type. The ability of recombinant virus to replicate in cell culture was further analyzed using fluorescence microscopy. Expression of eGFP protein could be detected as early as 12 h post inoculation, maximizing by 48 h post-inoculation. In strain 2 guinea pigs immunocompromised by pregnancy or pretreatment with cyclophosphamide, recombinant was found to be fully infectious, and replicated to levels identical to wild type GPCMV in lung, spleen, liver and salivary gland. In pregnant outbred Hartley guinea pigs, this virus was further found to be capable of dissemination, placental infection, and passage to the fetus. BAC technology and gpt selection have been used to select for additional mutant GPCMVs, including mutants generated by random transposon insertion and a mutant with a deletion in the UL83 homolog of GPCMV.
4. Discussion Small animal models of congenital CMV infection are useful tools for the study of pathogenesis and immunity. Although GPCMV provides a wellcharacterized model of transplacental viral infection, studies in this system have been hampered by a lack of molecular characterization of the genome and the lack of availability of subunit vaccine candidates. This overview describes recent progress in the development of the GPCMV model. Cloning of the viral genome, both as a library of restriction fragments as well as a BAC in E. coli , has facilitated the sequence analysis of much of the viral genome. Detailed molecular characterization of specific subunit vaccine candidates, such as the gB and UL83 homologs, has allowed for testing of the role of immunity to these proteins in protec-
tion against transplacental infection. The recent development of strategies for generation of recombinant GPCMV will further facilitate the evaluation of viral determinants of fetal infection and injury. The GPCMV genome is similar in structure to that of MCMV, in that it does not isomerize (Ho, 1992). GPCMV does have two genomic configurations, one in which there is a single DNA repeat segment at one end of the genome, and a second configuration in which the terminal repeat is duplicated (Gao and Isom, 1984; McVoy et al., 1997). In all betaherpesviruses, highly conserved gene blocks are present in the central portion of the respective genomes, and the GPCMV genome is no exception. Although earlier descriptions of the organization of the GPCMV genome provided useful information, including detailed restriction maps, the orientation of the GPCMV genome relative to other CMVs was unknown. Indeed, cross-hybridization studies between the HCMV AD 169 Hin dIII ‘E’ fragment and the GPCMV Hin dIII ‘D’ region suggested that the putative GPCMV MIEPr mapped to the leftward portion of the genome, and that the arbitrarily assigned orientation of the GPCMV genome was ‘inverted’ relative to HCMV (Isom and Yin, 1990). In retrospect this interpretation was incorrect. The orientation of the GPCMV genome as originally described is, in fact, the same as that designated for the HCMV genome, and conserved, collinear ORFs are present in the regions spanning the UL32 homolog through the MIEPr region (map units 0.2 /0.6, Fig. 1). In contrast, there is considerable heterogeneity of sequence near the termini, where few ORFs are identified which share other betaherpesvirus homologs. One unique aspect of the organization of the GPCMV genome compared with other CMVs is the presence of an immediate early locus near the left terminus of the genome, within the Hin dIII ‘D’ region. Sequencing of this region identifies ORFs not conserved in other CMVs, for example, the presence of a CC chemokine in the Hin dIII ‘D’ region with identity to MIP1-a. Although such chemokine homologs are well described in other cytomegaloviruses (Murphy, 2001), based upon comparison of the map position of this ORF to other CMVs, the
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GPCMV MIP1-a homolog appears to not have positional homology to the other betaherpesvirus chemokines. GCR homologs, however, are present in the GPCMV genome, including a recently described homolog of the HCMV UL33 ORF, and a homolog of the UL78 ORF (Liu and Biegalke, 2001; Schleiss, unpublished data). Completion of the DNA sequence of the GPCMV genome should facilitate the phylogenetic analysis of this virus, and its relationship to other sequenced betaherpesviruses. Vaccine studies in the GPCMV congenital infection model will be greatly aided by the detailed molecular characterization of immunogenic GPCMV proteins. As with HCMV, the most important target of humoral immune responses following GPCMV infection appears to be the gB homolog. Antibodies to HCMV gB are responsible for the majority of the virus-neutralizing capacity of convalescent sera, and subunit vaccine studies in the GPCMV model suggest that gB, either administered as a protein vaccine or as a DNA vaccine, is capable of eliciting antibody responses which confer protection to the developing fetus. The qualitative aspects of the antibody response, including responses to the immunodominant AD-1 domain and antibody avidity, also appear to be critical determinants of transmission to the fetus (Boppana and Britt, 1995; Britt and Mach, 1996; Lazzarotto et al., 1997). Future subunit protein vaccine studies, utilizing gB expressed in other recombinant systems such as baculovirus, and with clinically relevant adjuvants more suitable for human use, should be useful in clarifying which vaccine strategies currently undergoing evaluation in clinical trials are most likely to be of value in preventing congenital infection. It is less clear in the GPCMV model what the role of cytotoxic Tlymphocyte (CTL) mediated immunity is in prevention of congenital CMV. Although GPCMV encodes a highly related homolog of the dominant HCMV CTL target, UL83 (Schleiss et al., 1999), the major targets of CTL immunity in the setting of GPCMV infection remain to be determined. Studies with recombinant UL83 protein are unlikely to be relevant to natural immunity, and vectored approaches, such as with recombinant vaccinia virus, are more likely to provide mean-
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ingful analyses of the contribution of CTL to fetal protection against CMV disease in utero. In addition, the role of related ORFs which are also potential CTL targets, such as the UL84 homolog, needs to be further examined (Morello et al., 2000). Ultimately, an understanding of viral and host factors involved in the pathogenesis of congenital CMV infection will require a genetic approach for analysis of the role of specific viral gene products in fetal injury. To this end, the successful generation of GPCMV recombinants using gpt selection and, more recently, BAC technology, will be tremendously useful in elucidating mechanisms of placental and fetal injury. Since packaging constraints play a major role in the viability and stability of recombinant CMVs, there was concern that recombinant viral genomes may exceed the packaging limits of the virion (Messerle et al., 1997; Borst et al., 1999). Hence it was of considerable interest to examine the replication kinetics and stability of recombinant BAC GPCMV constructs. The BAC plasmid insert (8.8 kb) in the GPCMV recombinant is stable in tissue culture, as reflected by normal replication kinetics compared with wild type virus and persistent, robust eGFP expression after multiple rounds of passage, including in vivo passage in guinea pigs. Mutagenesis of the genome using the BAC construct has been successfully accomplished both by conventional plasmid homologous recombination techniques as well as with the use of a modified Tn7 transposon-based mutagenesis system (McGregor and Schleiss, 1999, 2001a,b). Specific mutations in domains of known replication proteins or vaccine targets could aid in the development of antiviral therapies and the elucidation of critical aspects of protective immune responses. The availability of the cloned GPCMV BAC genome will allow for rapid completion of the DNA sequence of the virus. In addition, the prospect of using BACs to generate recombinant viruses for use as gene therapy vectors may be promising. Finally, BACs should prove useful in vaccine development, not only in generation of live attenuated recombinant vaccines, but also as a DNA vaccine in its own right. Application of these technologies to the GPCMV model should yield important insights
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into the pathogenesis and prevention of congenital infection.
Acknowledgements I acknowledge the contributions of many skilled technicians, in particular Debbie Fox and Nancy Jensen, to this work. I gratefully acknowledge the effort of my principle collaborators in animal studies, Dr Nigel Bourne, and molecular studies, Dr Alistair McGregor. I thank Fernando Bravo, Mike McVoy, Jeff Vieira, Martin Messerle and Ulrich Koszinowski for helpful discussions, plasmids, and protocols. I acknowledge the advice and assistance of the Director of the Division of Infectious Diseases at Children’s Hospital, Cincinnati, David Bernstein. Finally, I am indebted to Collett Mack Schleiss for helpful discussions and support. These studies were supported by NIH HD38416-01 and AI65289 and March of Dimes Basic Research Grants 6-FY98/99-0416 and FY01-226.
References Adler SP. Current prospects for immunization against cytomegaloviral disease. Inf Agents Dis 1996;5:29 /35. Alford CA, Britt WJ. Cytomegalovirus. In: Roizman B, Whitley RJ, Lopez C, editors. The Human Herpesviruses. New York: Raven Press, 1993:227 /55. Bratcher DF, Bourne N, Bravo FJ, Schleiss MR, Slaoui M, Myers MG, Bernstein DI. Effect of passive antibody on congenital cytomegalovirus infection in guinea pigs. J Infect Dis 1995;172:944 /50. 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;142:732 /8. Bia FJ, Griffith BP, Fong CKY, Hsuing GD. Cytomegaloviral infections in the guinea pig: experimental models for human disease. Rev Infect Dis 1983;5:177 /95. Boppana SB, Britt WJ. Antiviral antibody responses and intrauterine transmission after primary maternal cytomegalovirus infection. J Infect Dis 1995;17:1115 /21. Borst EM, Hahn G, Koszinowski UH, Messerle M. Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli : a new approach for construction of HCMV mutants. Virology 1999;73:8320 /9. 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;183: 59 /64. Brady RC, Schleiss MR. Identification and characterization of the guinea-pig cytomegalovirus glycoprotein H gene. Arch Virol 1996;141:2409 /24. Britt WJ, Harrison C. Identification of an abundant disulfidelinked complex of glycoproteins in the envelope of guinea pig cytomegalovirus. Virology 1994;201:294 /302. Britt WJ, Mach M. Human cytomegalovirus glycoproteins. Intervirology 1996;39:401 /12. Bruggeman CA, Meijer H, Bosnian F, van Boven CPA. Biology of rat cytomegalovirus. Intervirology 1985;24:1 /9. Choi YC, Hsuing GD. Cytomegalovirus infection in guinea pigs. II. Transplacental and horizontal transmission. J Infect Dis 1978;138:197 /202. Demmler GJ. Congenital cytomegalovirus infection. Semin Pediatr Neurol 1994;1:36 /42. Fox DS, Schleiss MR. Sequence and transcriptional analysis of the guinea pig cytomegalovirus UL97 homolog. Virus Genes 1997;15:255 /64. Gao M, Isom HC. Characterization of the guinea pig cytomegalovirus genome by molecular cloning and physical mapping. J Virol 1984;52:436 /47. Griffith BP, McCormick SR, Fong CKY, Lavellee JT, Lucia HL, Goff E. The placenta as a site of cytomegalovirus infection in guinea pigs. J Virol 1985;55:402 /9. 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;172:1212 /20. Ho M. Cytomegalovirus: Biology and Infection, second ed.. New York: Plenum Publishing, 1992. Huber MT, Compton T. The human cytomegalovirus UL74 gene encoded the third component of the glycoprotein Hglycoprotein L-containing envelope complex. J Virol 1998;72:8191 /7. Isom HC, Yin CY. Guinea pig cytomegalovirus gene expression. Curr Top Microbiol Immunol 1990;154:101 /21. Jackson L. An intracellular protozoan parasite of the ducts of the salivary glands of the guinea pig. J Infect Dis 1920;26:347 /50. Johnson KP, Connor WS. Guinea pig cytomegalovirus: transplacental transmission. J Exp Med 1979;59:263 /7. Jones CT, Keay SK, Swoveland PT. Identification of GPCMV infected cells in vitro and in vivo with a monoclonal antibody. J Virol Methods 1994;48:133 /44. Kumar ML, Nankervis GA. Experimental congenital infection with cytomegalovirus: a guinea pig model. J Infect Dis 1978;138:650 /4. Lazzarotto T, Spezzacatena P, Pradelli P, Abate DA, Varani S, Landini MP. Avidity of immunoglobulin G directed against human cytomegalovirus during primary and secondary infections in immunocompetent and immunocompromised subjects. Clin Lab Diagn Lab Immunol 1997;4:469 /73.
M.R. Schleiss / Journal of Clinical Virology 25 (2002) S37 /S49 Liu Y, Biegalke B. Characterization of a cluster of late genes of guinea pig cytomegalovirus. Virus Genes 2001;23:247 /56. Lockridge KM, Sequar G, Zhou SS, Yue Y, Mandell CP, Barry PA. Pathogenesis of experimental rhesus cytomegalovirus infection. J Virol 1999;73:9576 /83. Markham FS, Hudson NP. Susceptibility of the guinea pig fetus to the submaxillary gland virus of guinea pigs. Am J Pathol 1936;12:175 /81. McGregor A, Schleiss MR. Generation of guinea pig cytomegalovirus mutants via the use of a novel transposon based in vitro insertion system. Twentyfourth International Herpesvirus workshop, 1999. McGregor A, Schleiss MR. Molecular cloning of the guinea pig cytomegalovirus (GPCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli . Mol Gen Metab 2001;72:15 /26. McGregor A, Schleiss MR. Recent advances in herpesvirus genetics using bacterial artificial chromosomes. Mol Gen Metab 2001;72:8 /14. McVoy MA, Nixon DE, Adler SP. Circularization and cleavage of guinea pig cytomegalovirus genomes. J Virol 1997;71:4209 /17. Medearis DN. Mouse cytomegalovirus infection. III. Attempts to produce intrauterine infections. Am J Hyg 1964;80:113 / 20. Messerle M, Crnkovic I, Hammerschmidt W, Ziegler H, Koszinowski UH. Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci USA 1997;94:14759 /63. 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;74:3696 /708. Murphy PM. Viral exploitation and subversion of the immune system through chemokine mimicry. Nat Immunol 2001;2:116 /22.
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Nogami-Satake T, Tsutsui Y. Identification and characterization of a 50K DNA-binding protein of guinea-pig cytomegalovirus. J Gen Virol 1988;69:2267 /76. Paglino JC, Brady RC, Schleiss MR. Molecular characterization of the guinea-pig cytomegalovirus glycoprotein L gene. Arch Virol 1999;144:447 /62. Pass RF. Immunization strategy for prevention of congenital cytomegalovirus infection. Infect Agents Dis 1996;5:240 /4. Plotkin SA. Vaccination against cytomegalovirus, the changeling demon. Pediatr Infect Dis J 1999;16:313 /25. Schleiss MR. Cloning and characterization of the guinea pig cytomegalovirus glycoprotein B gene. Virology 1994;202:173 /85. Schleiss MR. Sequence and transcriptional analysis of the guinea-pig cytomegalovirus DNA polymerase gene. J Gen Virol 1995;76:1827 /33. Schleiss MR, McGregor A, Jensen NJ, Erdem G, Aktan L. Molecular characterization of the guinea pig cytomegalovirus UL83 (pp65) protein homolog. Virus Genes 1999;19:205 /21. 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. Virol Immunol 2000;13:155 /67. Schleiss MR, Bernstein DI, Bourne N. Anti cytomegalovirus (anti-CMV) IgG avidity following glycoprotein immunization predicts neonatal outcome in the guinea pig model of congenital CMV infection. Pediatr Res 2001;49:1768. Staczek J. Animal cytomegaloviruses. Microbiol Rev 1990;54:247 /65. Tarantal AF, Salamat MS, Britt WJ, Luciw PA, Hendrickx AG, Barry PA. Neuropathogenesis induced by rhesus cytomegalovirus in fetal rhesus monkeys (Macacamulatta ). J Infect Dis 1998;177:446 /50. Tsutsui Y, Yamazaki Y, Kashiwai A, Mizutani A, Furukawa T. Monoclonal antibodies to guinea-pig cytomegalovirus: an immunoeiectron microscopic study. J Gen Virol 1986;67:107 /18.
Review article
Animal models of congenital cytomegalovirus infection: an overview of progress in the characterization of guinea pig cytomegalovirus (GPCMV) Mark R. Schleiss * Division of Infectious Diseases, Children’s Hospital Research Foundation, 3333 Burnet Ave, Cincinnati, OH 45229, USA
Abstract Background: The strict species-specificity of cytomegalovirus (CMV) precludes preclinical evaluation of human CMV (HCMV) vaccines in animal models and necessitates the study of nonhuman CMVs. Among the CMVs of small mammals, the guinea pig cytomegalovirus (GPCMV) has unique advantages, due to its ability to cross the placenta, causing infection in utero. Objective and study designs: Progress in GPCMV studies has been hampered by a lack of detailed molecular characterization of the viral genome. Therefore, recent efforts have been undertaken to characterize the GPCMV genome, and apply this information to in vivo subunit vaccine studies. Results: Progress in the sequencing of the GPCMV genome has revealed the presence of both highly conserved as well as novel open reading frames (ORFs). Cloning of GPCMV vaccine candidates, such as the glycoprotein B (gB) and UL83 proteins, has facilitated subunit vaccine evaluation. Protein vaccines and DNA vaccines have shown evidence of protection in pregnancy/ challenge experiments. In addition, the GPCMV genome has proved amenable to cloning as a bacterial artificial chromosome (BAC) in Escherichia coli , and BAC-derived recombinants retain the ability to replicate in vivo. Conclusions: Progress has been made in molecular characterization of GPCMV. Insights from these studies should prove germane to the understanding of the correlates of protective immunity for the fetus in vaccine studies, and should assist in prioritization of vaccine strategies in HCMV vaccine trials. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytomegalovirus (CMV); Guinea pig; CMV vaccines
1. Introduction Infection with human cytomegalovirus (HCMV), although ubiquitous in nature, can lead to severe disease manifestations in immunocompromised patients, including newborn infants (Alford and Britt, 1993). Acquisition of HCMV in
* Fax: /1-513-636-7655 E-mail address: [email protected] (M.R. Schleiss).
utero may cause significant neurodevelopmental handicap, including sensorineural deafness (Demmler, 1994). Although maternal vaccination against HCMV may be useful in preventing such damage (Adler, 1996; Pass, 1996; Plotkin, 1999), currently there are no licensed CMV vaccines available. CMV infections are highly speciesspecific, and, therefore, HCMV cannot be studied in animal models of infection (Staczek, 1990). Hence, the species-specific cytomegaloviruses of animals have been studied to provide in vivo
1386-6532/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 2 ) 0 0 1 0 0 - 2
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models for the study of vaccines and viral pathogenesis. Although mouse and rat cytomegalo viruses have been studied as models of CMV disease, these CMVs do not cross the placenta, and are, therefore, less useful for vaccine studies which target prevention of congenital infection (Medearis, 1964; Bruggeman et al., 1985; Ho, 1992). The rhesus macaque CMV (RhCMV) causes placental infection and fetal injury (Tarantal et al., 1998; Lockridge et al., 1999), but the expense of these primates, as well as the rarity of RhCMV seronegative animals, makes this model impractical for large-scale vaccine studies. Thus, each of these models has limited usefulness for the study of CMV vaccines. In contrast to the CMVs of other small mammals, the guinea pig cytomegalovirus (GPCMV) is unique in its ability to cross the placenta and cause fetal infection. GPCMV was first recognized following histopathologic identification of inclusions in guinea pig salivary glands in 1920 (Jackson, 1920). Although the ability of this virus to infect the guinea pig placenta was described in the 1930s (Markham and Hudson, 1936), further description of a guinea pig model of congenital CMV infection would not occur until the late 1970s, when three groups independently described studies of fetal infection following GPCMV inoculation of pregnant dams (Choi and Hsuing, 1978; Kumar and Nankervis, 1978; Johnson and Connor, 1979). Since these initial reports, several groups have studied the pathogenesis and prevention of transplacental CMV infection using the GPCMV model. A number of aspects of guinea pig reproductive biology have contributed to the usefulness of this model. Guinea pig gestational periods are fairly lengthy, ranging from 65 to 70 days, and can conveniently be divided into trimesters (Bia et al., 1983). The guinea pig placenta, like the human placenta, is hemochorial, with a single trophoblast layer separating maternal and fetal circulation, similar histologically to human placenta (Griffith et al., 1985). This aspect of guinea pig reproductive biology provides the greatest driving force justifying the use of this model for vaccine experiments. The maternal and fetal outcomes which may be monitored in the GPCMV model are dependent
upon a number of experimental variables, including the timing and route of GPCMV inoculation, the strain of guinea pig utilized and the source of virus. Viral inoculation in early pregnancy tends to lead to pup resorption, whereas challenge in late pregnancy tends to lead to pup mortality (stillborn pups). Inbred strains, such as strain 2 and JY-9, tend to have greater susceptibility to GPCMV infection than outbred strains, such as the Hartley strain. Accordingly, inoculation of these highly susceptible inbred animals with GPCMV during pregnancy may produce maternal mortality in addition to fetal demise. Salivary gland-adapted virus stocks produce more severe disease than tissue culture virus-attenuated virus, and inoculation via intravascular, intraperitoneal, or subcutaneous routes generates a more severe infection than inoculation at a mucosal surface. Irrespective of these differences, a number of vaccine endpoints are easily quantifiable in congenital infection studies. These include infection rate, pup mortality, maternal and pup weight, fetal resorption rate, viral load by PCR, and extent of placental infection. A number of studies have examined vaccines in the GPCMV model. In a study in outbred guinea pigs, a noninfectious, a live-attenuated vaccine GPCMV vaccine was shown to have efficacy against congenital GPCMV infection (Bia et al., 1980). This study also examined a partially purified, soluble envelope vaccine, administered with Freund’s adjuvant, and found this vaccine protective as well. Although these studies confirmed the importance of pre-existing maternal immunity to GPCMV in protection against congenital infection, there was limited data concerning the qualitative aspects of the immune responses in these studies, in part because of the lack of any characterization of immunogenic GPCMV structural proteins. In a later study using an inbred model of congenital GPCMV infection, an immunoaffinity-purified glycoprotein was found to induce strong antibody and CMI responses when administered with Freund’s adjuvant, and newborn pups were protected against congenital infection and disease (Harrison et al., 1995), providing more direct evidence for the importance of immune responses to specific viral proteins in
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protection of the fetus. However, although this and other GPCMV structural proteins had been characterized through the use of monoclonal antibodies (Tsutsui et al., 1986; Nogami-Satake and Tsutsui, 1988; Jones et al., 1994; Britt and Harrison, 1994), the molecular identity of GPCMV proteins at the genetic level had largely been unexplored. This overview reports the extensive effort which has been undertaken in recent years to better characterize the molecular biology of GPCMV and describes how this information has been applied to in vivo studies of vaccines and pathogenesis.
2. Materials and methods 2.1. Virus and cells Guinea pig CMV (strain no. 22122, ATC VR682) was propagated on guinea pig fibroblast lung cells (GPL, ATCC CCL 158) and maintained in F-12 medium supplemented with 10% fetal calf serum (FCS, HyClone Laboratories), 10 000 IU/l penicillin, 10 mg/l streptomycin (Gibco-BRL) and 7.5% NaHCO3 (Gibco-BRL). Recombinant viruses were selected by the addition of mycophenolic acid (MPA) and xanthine (Gibco-BRL) into the medium at 200 and 10 mm, respectively (McGregor and Schleiss, 2001b). 2.2. Cloning and sequence analyses Restriction enzymes Hin dIII and Eco RI were used to subclone fragments of the GPCMV genome, using strandard cloning techniques, for sequence analyses, using vectors pUC and pBR322 (Schleiss, 1994). Details of cloning of DNA vaccine construct and GPCMV gB-GST fusion construct have been summarized elsewhere (Schleiss et al., 2000; Bourne et al., 2001). DNA sequencing was performed using 33S-dATP and Sequenase† (United States Biochemical) according to the manufacturer’s specification, and by use of automated sequencing using an ABI-Prism automated sequencer. Nucleotide sequences and the deduced protein coding sequences of candidate open reading frames (ORFs) were compared with
published
HCMV sequences software package.
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using
the
MACVECTOR†
2.3. Immunization with subunit vaccines and outcome analyses in guinea pigs Young female and proven breeder male Hartley guinea pigs were obtained from Camm Research Institute (Wayne, NJ). Inbred adult strain-2 guinea pigs were purchased from the Children’s Hospital Research Foundation (Cincinnati). All animal experiments were performed in accordance with guidelines approved by the American Association of Accreditation of Laboratory Animal Care and were approved by the Animal Use Care Committee of Children’s Hospital Research Foundation. Glycoprotein immunogen was administered with Freund’s adjuvant as previously described (Bourne et al., 2001). For DNA vaccine studies, plasmid DNA encoding the GPCMV glycoprotein B ORF was administered by epidermal inoculation via gene gun as previously described (Schleiss et al., 2000). A total of four inoculations were administered. Antibody titers were determined using ELISA assay. The ELISA titer was defined as the reciprocal of the highest dilution that produced an absorbence of /0.10 and twice the absorbence against control antigen (Bratcher et al., 1995). Glycoprotein B (gB)specific antibodies in guinea pig serum were determined by ELISA, using a gB fusion protein with glutathione S-transferase (GST) as the antigen. Avidity index (AI) of antibody was determined by calculating the ratio of the OD450 of vaccine-derived antibodies in the presence and absence of 4 M urea denaturation reagent (Lazzarotto et al., 1997; Schleiss et al., 2001). Western blot assays were performed as described previously (Paglino et al., 1999). For congenital infection experiments, protocols for pregnancy/challenge studies are summarized in detail elsewhere (Bratcher et al., 1995; Bourne et al., 2001). Briefly, immunized animals (glycoprotein or DNA vaccine) were placed with GPCMVseronegative proven breeder male Hartley guinea pigs. The female animals were examined weekly by palpation for evidence of pregnancy. When they were estimated to be entering the third trimester of
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pregnancy, they were inoculated by subcutaneous injection with virulent salivary gland-passaged GPCMV. In some experiments, animals were inoculated with tissue culture adapted recombinant virus containing the eGFP reporter gene cassette (see below). After virus challenge, the animals were observed daily until delivery, to determine pregnancy outcome. Stillborn animals were dissected on the day of delivery, and liveborn pups were sacrificed within 48 h of birth. Minced explant cultures were inoculated onto guinea pig lung cell monolayers for identification of characteristic cytopathic effect (CPE). Outcomes in vaccinated and unvaccinated groups were compared statistically using Fisher’s exact test. In some experiments, at various times points post inoculation animals were humanely sacrificed and organs were removed for histopathological analyses. Selected tissues were formalin-fixed and paraffin-embedded for subsequent sectioning for staining with hematoxylin and eosin. 2.4. Isolation of recombinant viruses and BAC clone Viral DNA and plasmid DNA (containing guanosylphosphoribosyltransferase [gpt] and green fluorescent protein [eGFP] cassettes) were used to co-transfect confluent monolayers of GPL cells in 60 mm dishes using Lipofectin† (GibcoBRL) following the manufacturer’s specifications. After overnight incubation at 37 8C the cells were
washed with F-12 medium and incubated for a further 20/30 days post-transfection, or until significant CPE was observed. At this stage the medium was replaced with F-12 medium containing MPA and xanthine (McVoy et al., 1997; McGregor and Schleiss, 2001b). Additional passages were performed under continuous selection. Viral titrations were replated on a series of 24 well plates under limiting dilution to generate clonal viral stocks, which were further verified by Southern blot analysis. A recombinant virus carrying the BAC plasmid in Hin dIII ‘N’ was generated by cotransfection of wild-type viral DNA with pACYC177NKGG onto GPL cells and selection of gpt /eGFP-positive virus. Isolates cloned by limiting dilution were verified to be clonal by Hin dIII restriction profile and Southern blot analysis, which demonstrated an 8.8-kbp insertion into the Hin dIII ‘N’ region of the viral genome, corresponding to the BAC plasmid. Circular viral DNA was then purified and used to transform Escherichia coli to generate stable BAC clones (McGregor and Schleiss, 2001b).
3. Results 3.1. Molecular characterization of the GPCMV genome The strategy of low-stringency Southern blot hybridization of a HCMV gB probe with GPCMV
Fig. 1. Schematic of GPCMV genome identifying map positions of sequenced homolog genes characterized to date. Hin dIII restriction map based upon original map described by Isom (Gao and Isom, 1984). Homolog genes are identified based upon correlation with HCMV ORFs. Gene order and map positions are relatively well conserved from UL32 region (pp 150 homolog) through region of putative MIEP, but ORFs identified at genome termini are divergent relative to HCMV.
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restriction digests was used to investigate which region in the GPCMV genome contained the gB homolog. This homolog was found to map to the Hin dIII ‘K’ and ‘P’ fragments. This data allowed orientation of the GPCMV genome relative to other CMVs, and facilitated the identification of other homolog genes (Fig. 1). Northern blot revealed that the GPCMV gB mRNA is transcribed with ‘early’ kinetics as a 6.8 kb message, and encodes a 901 aa protein with 42% homology to HCMV gB (Schleiss, 1994). The protein was found to be cleaved into amino and carboxy terminal subunits of approximately 90 and 58 kDa. Immunological investigations revealed that GPCMV gB is a major target of the neutralizing
Fig. 2. Placental injury in GPCMV inoculated dams. Following third trimester challenge with virulent, SG-passaged GPCMV, pup mortality ranges from 30 to 70% and placental injury is noted. Top panel (a), healthy near-term placenta showing normal syncytiotrophoblast (upper portion of micrograph) layer. Lower panel (b), placenta from stillborn pup with extensive GPCMV infection, 21 days-post maternal inoculation. Placenta shows significant infarction, peri-vascular inflammatory cell infiltrate, and viral inclusions. Hematoxylin and eosin stains, 120 /magnification.
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antibody response following natural GPCMV infection, and antibodies to gB are uniformly found in GPCMV seropositive animals (Schleiss, 1994; Bourne et al., 2001). Characterization of the GPCMV gB homolog has provided a framework for study of the organization of the viral genome. Clones of GPCMV Hin dIII and Eco RI restriction fragments and, more recently, the genomic clone maintained in E. coli as a BAC (McGregor and Schleiss, 2001b) have been utilized for DNA sequencing. Following the identification of gB, other highly conserved structural proteins were mapped in the GPCMV genome. The glycoprotein H (gH; UL75 homolog) was found to map to the Xba I ‘T’ region within the Hin dIII ‘A’ fragment of the genome. This ORF is 723 aa in length, and the mature glycoprotein migrates at Mr /85 kDa in SDSPAGE (Brady and Schleiss, 1996). The gH chaperone, glycoprotein L (gL; UL115 homolog) maps to a GPCMV genome region collinear with HCMV UL115, within the Hin dIII ‘B’ region (Paglino et al., 1999). Both gH and gL of GPCMV are immunoreactive with anti-GPCMV antibodies, confirming that these conserved glycoprotein homologs are targets of immune response in natural GPCMV infection. An ORF with identity to HCMV UL74, the glycoprotein O member of the HCMV gCIII complex (Huber and Compton, 1998), has also been identified, although identification of the protein has not yet been confirmed in GPCMV. In addition to conserved structural glycoproteins, other GPCMV genes, both structural and nonstructural, were characterized at the molecular level. Sequence analysis of regions adjacent to the gB (UL55) homolog successfully identified the conserved DNA polymerase. GPCMV pol is transcribed as a 3.9 kb message with ‘early’ kinetics, and encodes an ORF of 1094 amino acids (Schleiss, 1995). The UL97 phosphotransferase homolog has been mapped to Hin dIII ‘C’ (Fox and Schleiss, 1997). A promoter with strong similarities, both in structure and cis -recognition sequences, to the HCMV major immediate early promoter (MIEP) has been identified in the Hin dIII ‘E’ region. Tegument phosphoprotein homologs have also been characterized in the
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Fig. 3
GPCMV genome. Within the Hin dIII ‘A’ region of the GPCMV genome, an ORF with homology to the HCMV UL83 tegument phosphoprotein has recently been identified. This ORF was predicted to encode a 565-amino-acid (aa) protein with a
molecular mass of 62.3 kDa. Western blot and immunoprecipitation analyses identified a 70 kDa phosphoprotein corresponding to the GPCMV UL83 homolog in both virion and dense body fractions (Schleiss et al., 1999). Other conserved,
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sequenced phosphoprotein homologs have been identified in the GPCMV genome, including homologs of UL32, the pp150 protein, and UL115, the pp28 protein. Of interest, an ORF with identity to the CC chemokine, MIP1-a, has been identified in the Hin dIII ‘D’ region of the genome, and is not present in other CMVs (Schleiss, unpublished data).
3.2. Experimental vaccine evaluations in the guinea pig congenital infection model Studies of vaccine-induced protection against congenital CMV infection were designed to test the importance of immune responses to GPCMV envelope glycoproteins in protective immunity. In a late (third trimester) challenge model in pregnant outbred Hartley strain guinea pigs, inoculation of GPCMV subcutaneously during the third trimester results in significant injury to pups (Bratcher et al., 1995; Bourne et al., 2001). Pup mortality is manifest either as stillbirth, or death soon after birth. Depending upon the dose of virus used in the maternal inoculation, mortality rates are observed which may range from 30 to 70%. Higher doses which result in greater degrees of pup mortality often cause maternal mortality as well. Infected pups show widespread evidence of disease, with viral inclusions and inflammatory cell infiltrates in liver, spleen, lung, and occasionally brain. These studies confirmed that the placenta is a significant target of virus-induced injury. In dams sacrificed following such third-trimester challenge, placentas corresponding to stillborn pups show extensive damage, including viral
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inclusions, and areas of infarction and inflammatory cell infiltrates (Fig. 2). Subunit glycoprotein vaccines for GPCMV were used to test whether preconceptual immunization can protect the maternal-placenta-fetal unit against this virally-induced damage. In a protein subunit vaccine study, immunity conferred by immunization with envelope glycoprotein resulted in substantial protection against pup mortality, with a reduction in pup mortality from 56 to 14% in the glycoprotein immunized group (P B/0.001). In addition, among live-born pups, glycoprotein immunization substantially reduced infection rates. GPCMV was isolated from 24 of 54 liveborn pups born to immunized mothers, compared with 16 of 20 live-born pups born to controls, indicating that immunization significantly reduced in utero transmission in surviving animals (P B/ 0.01). The dominant immunogen in the glycoprotein vaccine was the gB homolog (Bourne et al., 2001). Although dams in this study generated high-titer antibodies as measured by ELISA assay of sonicated GPCMV coating antigen (Bratcher et al., 1995), more detailed analyses of the qualitative nature of the antibody response were undertaken to test whether antibody responses to key domains of GPCMV gB correlated with protection against congenital infection. When antibody titers were assessed against a series of GST fusion proteins spanning the GPCMV gB, the highest levels of antibody were found targeting a region of GPCMV gB collinear with HCMV gB antigenic domain 1 (AD-1). This domain, corresponding to amino acid residues 546 /626 of GPCMV gB,
Fig. 3. Antibody responses to glycoprotein vaccine and outcome correlations in GPCMV congenital infection model. (A) Comparison of amino acid sequence between HCMV (top line) and GPCMV gB (lower line). Although overall amino acid similarity is approximately 40%, amino acid identity is strikingly high in region corresponding to AD-1 domain. Within a 38 amino acid region bracketed by two conserved cysteine residues and a conserved N-linked glycosylation site, identity is greater than 70% (shaded). GSTgB fusion proteins map strong antibody responses following glycoprotein immunization to this region. Western blot: lanes 1 and 2, preimmune sera nonreactive with GST carrier protein (lane 1) and GST-AD1 fusion (lane 2). Lanes 3 and 4, sera from immunized dam is not reactive with GST (lane 3) but strongly immunoreactive with GST-AD1 fusion (42 kDa protein, arrow). In vaccine/transmission study, pups free of congenital infection (n /9) had statistically significantly higher ELISA titers to GST-AD1 than pups bom with congenital infection (n /12). (B) IgG AI and outcome in congenital GPCMV infection. Urea-based avidity determination identifies range of AI following glycoprotein immunization, pregnancy, and delivery. In pups born to dams with high AI, only 3/18 had congenital GPCMV infection. In contrast, pups born to dams with low or intermediate AI had congenital infection in 7/11 cases (P B/ 0.02 compared with high avidity).
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includes a 38 amino acid region bracketed by two conserved cysteine residues and a conserved Nlinked glycosylation site (Britt and Mach, 1996). Although the overall amino acid similarity of the HCMV and GPCMV gB proteins is approximately 42%, amino acid identity between the two species is particularly striking in this region, at 71% (Fig. 3a). When antibody titers against a GST-GPCMV AD1 fusion protein were compared among pups in which transplacental infection occurred (n /12) and pups which were uninfected (n /9), titers against this domain were significantly correlated with protection against infection (P B/ 0.005). Similar observations correlating the importance of the qualitative aspects of the vaccineinduced antibody response with protection against transplacental transmission were made when the IgG AI was examined. The AI was determined by measuring the OD in ELISA assay in the presence and absence of urea. Among pups in which transmission of GPCMV occurred, an AI of greater than 0.45 was likewise found to correlate with protection against infection (Fig. 3b). More recently, DNA vaccines have been evaluated for protective efficacy in the congenital infection model. A GPCMV gB vaccine was engineered which expresses the GPCMV gB ORF under control of the HCMV MIEP. This vaccine was found to induce significant ELISA and neutralizing antibody levels when administered epidermally via ‘gene gun’. To evaluate the protective efficacy of the vaccine in a congenital Table 1 GPCMV DNA vaccine study: outcome
Control gB Vaccine
Litters
Pups
Infected pups
10 12
26 27
17 (70%) 7 (26%)*
Analysis of protection conferred against congenital GPCMV infection by DNA vaccination. Plasmid construct encoding GPCMV gB (pKTS404) was administered by gene gun inoculation. DNA immunization induces high titer ELISA and neutralizing antibody (Schleiss et al., 2000). Immunized dams were mated, then challenged with GPCMV. In pups born to gB immunized dams, vertical transmission of virus only occurred in 26% of pups, compared with 70% of pups born to dams immunized with vector plasmid alone (P B/0.01, Fisher’s exact test). *, P B/0.01 vs. control.
infection model, female Hartley guinea pigs were immunized with gB plasmid prior to establishment of pregnancy, and pup outcomes were compared, following third trimester viral challenge, between the vaccine group (pKTS404) and the control vector group (pCDNA). Pre-existing immunity conferred by the DNA vaccine resulted in significant reductions in transmission of CMV to the pup (Table 1), with an overall infection rate of 26% (7/27) in the vaccine group, compared with 70% (17/26) in the control group (P B/0.01, Fisher’s exact test). 3.3. Generation of recombinant GPCMV and BAC cloning of viral genome Efforts were undertaken to generate recombinant viruses expressing reporter genes to aid in the detection of virus in vivo, and to develop a strategy for generation of targeted viral mutants to facilitate the study of the role of specific viral gene products in pathogenesis. The gpt selection system was utilized to test whether metabolic selection could be used to generate such GPCMV recombinants (McVoy et al., 1997). Selection with gpt was successful in generating a mutant GPCMV with a eGFP cassette inserted into the nonessential Hin dIII ‘N’ region of the viral genome. Next, attempts were made to clone the GPCMV genome as a BAC in E. coli . To clone the GPCMV genome as a bacterial artificial chromosome (BAC), a BAC plasmid was first cloned into a unique Pac I site introduced into the Hin dIII ‘N’ locus of the GPCMV genome, on a shuttle plasmid which contained the gpt and eGFP cassettes (pACYC177NKGG) to provide sufficient sequence for homologous recombination (Fig. 4). A recombinant virus carrying a BAC plasmid in Hin dIII ‘N’ was generated by co-transfection of wild type viral DNA with pACYC177NKGG onto GPL cells and selection of gpt /eGFP positive virus. Isolates were purified by limiting dilution and verified to be clonal by Southern blot analysis. Next, in order to clone the BAC GPCMV genome as a plasmid in E. coli , GPL cells were infected with recombinant virus, and circular viral DNA intermediates purified. The circular viral DNA was transformed by electroporation into
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Fig. 4. Scheme used to clone GPCMV genome as BAC in E. coli . (A) Protocol for generation of BAC recombinant HindIII ‘N’ region of viral genome selected as site of insertion of BAC plasmid pKGG (modified BAC plasmid pKSO with gpt /eGTP cassettes added). Following gpt selection, recombinant virus selected and purified circular DNA obtained from tissue culture prep. Circular viral DNA in turn is used to transform E. coli . Following selection of transformants on chloramphenicol containing media, miniprep DNA confirmed successful cloning of GPCMV genome as BAC. (B) Expression of eGFP in GPCMV recombinant virus. Cell culture expression of eGFP reporter gene is easily visualized in this plaque photographed using GFP filter (140 /magnification).
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recA DH10B cells, and chloramphenicol drug resistant colonies selected (McGregor and Schleiss, 2001a,b). In vivo analyses of recombinants indicate, depending upon the locus of insertion, that eGFP-expressing viruses retain the capability to replicate and cause disease in guinea pigs. The replication kinetics of Hin dIII ‘N’ recombinant viruses generated both by gpt selection and BAC technology were comparable to that of wild type. The ability of recombinant virus to replicate in cell culture was further analyzed using fluorescence microscopy. Expression of eGFP protein could be detected as early as 12 h post inoculation, maximizing by 48 h post-inoculation. In strain 2 guinea pigs immunocompromised by pregnancy or pretreatment with cyclophosphamide, recombinant was found to be fully infectious, and replicated to levels identical to wild type GPCMV in lung, spleen, liver and salivary gland. In pregnant outbred Hartley guinea pigs, this virus was further found to be capable of dissemination, placental infection, and passage to the fetus. BAC technology and gpt selection have been used to select for additional mutant GPCMVs, including mutants generated by random transposon insertion and a mutant with a deletion in the UL83 homolog of GPCMV.
4. Discussion Small animal models of congenital CMV infection are useful tools for the study of pathogenesis and immunity. Although GPCMV provides a wellcharacterized model of transplacental viral infection, studies in this system have been hampered by a lack of molecular characterization of the genome and the lack of availability of subunit vaccine candidates. This overview describes recent progress in the development of the GPCMV model. Cloning of the viral genome, both as a library of restriction fragments as well as a BAC in E. coli , has facilitated the sequence analysis of much of the viral genome. Detailed molecular characterization of specific subunit vaccine candidates, such as the gB and UL83 homologs, has allowed for testing of the role of immunity to these proteins in protec-
tion against transplacental infection. The recent development of strategies for generation of recombinant GPCMV will further facilitate the evaluation of viral determinants of fetal infection and injury. The GPCMV genome is similar in structure to that of MCMV, in that it does not isomerize (Ho, 1992). GPCMV does have two genomic configurations, one in which there is a single DNA repeat segment at one end of the genome, and a second configuration in which the terminal repeat is duplicated (Gao and Isom, 1984; McVoy et al., 1997). In all betaherpesviruses, highly conserved gene blocks are present in the central portion of the respective genomes, and the GPCMV genome is no exception. Although earlier descriptions of the organization of the GPCMV genome provided useful information, including detailed restriction maps, the orientation of the GPCMV genome relative to other CMVs was unknown. Indeed, cross-hybridization studies between the HCMV AD 169 Hin dIII ‘E’ fragment and the GPCMV Hin dIII ‘D’ region suggested that the putative GPCMV MIEPr mapped to the leftward portion of the genome, and that the arbitrarily assigned orientation of the GPCMV genome was ‘inverted’ relative to HCMV (Isom and Yin, 1990). In retrospect this interpretation was incorrect. The orientation of the GPCMV genome as originally described is, in fact, the same as that designated for the HCMV genome, and conserved, collinear ORFs are present in the regions spanning the UL32 homolog through the MIEPr region (map units 0.2 /0.6, Fig. 1). In contrast, there is considerable heterogeneity of sequence near the termini, where few ORFs are identified which share other betaherpesvirus homologs. One unique aspect of the organization of the GPCMV genome compared with other CMVs is the presence of an immediate early locus near the left terminus of the genome, within the Hin dIII ‘D’ region. Sequencing of this region identifies ORFs not conserved in other CMVs, for example, the presence of a CC chemokine in the Hin dIII ‘D’ region with identity to MIP1-a. Although such chemokine homologs are well described in other cytomegaloviruses (Murphy, 2001), based upon comparison of the map position of this ORF to other CMVs, the
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GPCMV MIP1-a homolog appears to not have positional homology to the other betaherpesvirus chemokines. GCR homologs, however, are present in the GPCMV genome, including a recently described homolog of the HCMV UL33 ORF, and a homolog of the UL78 ORF (Liu and Biegalke, 2001; Schleiss, unpublished data). Completion of the DNA sequence of the GPCMV genome should facilitate the phylogenetic analysis of this virus, and its relationship to other sequenced betaherpesviruses. Vaccine studies in the GPCMV congenital infection model will be greatly aided by the detailed molecular characterization of immunogenic GPCMV proteins. As with HCMV, the most important target of humoral immune responses following GPCMV infection appears to be the gB homolog. Antibodies to HCMV gB are responsible for the majority of the virus-neutralizing capacity of convalescent sera, and subunit vaccine studies in the GPCMV model suggest that gB, either administered as a protein vaccine or as a DNA vaccine, is capable of eliciting antibody responses which confer protection to the developing fetus. The qualitative aspects of the antibody response, including responses to the immunodominant AD-1 domain and antibody avidity, also appear to be critical determinants of transmission to the fetus (Boppana and Britt, 1995; Britt and Mach, 1996; Lazzarotto et al., 1997). Future subunit protein vaccine studies, utilizing gB expressed in other recombinant systems such as baculovirus, and with clinically relevant adjuvants more suitable for human use, should be useful in clarifying which vaccine strategies currently undergoing evaluation in clinical trials are most likely to be of value in preventing congenital infection. It is less clear in the GPCMV model what the role of cytotoxic Tlymphocyte (CTL) mediated immunity is in prevention of congenital CMV. Although GPCMV encodes a highly related homolog of the dominant HCMV CTL target, UL83 (Schleiss et al., 1999), the major targets of CTL immunity in the setting of GPCMV infection remain to be determined. Studies with recombinant UL83 protein are unlikely to be relevant to natural immunity, and vectored approaches, such as with recombinant vaccinia virus, are more likely to provide mean-
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ingful analyses of the contribution of CTL to fetal protection against CMV disease in utero. In addition, the role of related ORFs which are also potential CTL targets, such as the UL84 homolog, needs to be further examined (Morello et al., 2000). Ultimately, an understanding of viral and host factors involved in the pathogenesis of congenital CMV infection will require a genetic approach for analysis of the role of specific viral gene products in fetal injury. To this end, the successful generation of GPCMV recombinants using gpt selection and, more recently, BAC technology, will be tremendously useful in elucidating mechanisms of placental and fetal injury. Since packaging constraints play a major role in the viability and stability of recombinant CMVs, there was concern that recombinant viral genomes may exceed the packaging limits of the virion (Messerle et al., 1997; Borst et al., 1999). Hence it was of considerable interest to examine the replication kinetics and stability of recombinant BAC GPCMV constructs. The BAC plasmid insert (8.8 kb) in the GPCMV recombinant is stable in tissue culture, as reflected by normal replication kinetics compared with wild type virus and persistent, robust eGFP expression after multiple rounds of passage, including in vivo passage in guinea pigs. Mutagenesis of the genome using the BAC construct has been successfully accomplished both by conventional plasmid homologous recombination techniques as well as with the use of a modified Tn7 transposon-based mutagenesis system (McGregor and Schleiss, 1999, 2001a,b). Specific mutations in domains of known replication proteins or vaccine targets could aid in the development of antiviral therapies and the elucidation of critical aspects of protective immune responses. The availability of the cloned GPCMV BAC genome will allow for rapid completion of the DNA sequence of the virus. In addition, the prospect of using BACs to generate recombinant viruses for use as gene therapy vectors may be promising. Finally, BACs should prove useful in vaccine development, not only in generation of live attenuated recombinant vaccines, but also as a DNA vaccine in its own right. Application of these technologies to the GPCMV model should yield important insights
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into the pathogenesis and prevention of congenital infection.
Acknowledgements I acknowledge the contributions of many skilled technicians, in particular Debbie Fox and Nancy Jensen, to this work. I gratefully acknowledge the effort of my principle collaborators in animal studies, Dr Nigel Bourne, and molecular studies, Dr Alistair McGregor. I thank Fernando Bravo, Mike McVoy, Jeff Vieira, Martin Messerle and Ulrich Koszinowski for helpful discussions, plasmids, and protocols. I acknowledge the advice and assistance of the Director of the Division of Infectious Diseases at Children’s Hospital, Cincinnati, David Bernstein. Finally, I am indebted to Collett Mack Schleiss for helpful discussions and support. These studies were supported by NIH HD38416-01 and AI65289 and March of Dimes Basic Research Grants 6-FY98/99-0416 and FY01-226.
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