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J Virol. 2013 February; 87(3): 1322–1332.
PMCID: PMC3554163

A Vaccine Based on the Rhesus Cytomegalovirus UL128 Complex Induces Broadly Neutralizing Antibodies in Rhesus Macaques


Neutralizing antibodies (NAb) are important for interfering with horizontal transmission of human cytomegalovirus (HCMV) leading to primary and congenital HCMV infection. Recent findings have shown that a pentameric virion complex formed by the glycoproteins gH/gL, UL128, UL130, and UL131A (UL128C) is required for HCMV entry into epithelial/endothelial cells (Epi/EC) and is the target of potent NAb in HCMV-seropositive individuals. Using bacterial artificial chromosome technology, we have generated a modified vaccinia Ankara virus (MVA) that stably coexpresses all 5 rhesus CMV (RhCMV) proteins homologous to HCMV UL128C, termed MVA-RhUL128C. Coimmunoprecipitation confirmed the interaction of RhgH with the other 4 RhCMV subunits of the pentameric complex. All 8 RhCMV-naïve rhesus macaques (RM) vaccinated with MVA-RhUL128C developed NAb that blocked infection of monkey kidney epithelial cells (MKE) and rhesus fibroblasts. NAb titers induced by MVA-RhUL128C measured on both cell types at 2 to 6 weeks postvaccination were comparable to levels observed in naturally infected RM. In contrast, MVA expressing a subset of RhUL128C proteins or RhgB glycoprotein only minimally stimulated NAb that inhibited infection of MKE. In addition, following subcutaneous RhCMV challenge at 8 weeks postvaccination, animals vaccinated with MVA-RhUL128C showed reduced plasma viral loads. These results indicate that MVA expressing the RhUL128C induces NAb inhibiting RhCMV entry into both Epi/EC and fibroblasts and limits RhCMV replication in RM. This novel approach is the first step in developing a prophylactic HCMV vaccine designed to interfere with virus entry into major cell types permissive for viral replication, a required property of an effective vaccine.


A vaccine strategy to prevent congenital human cytomegalovirus (HCMV) infection remains an unsolved public health priority despite several decades of effort (1, 2). Progress has been made in developing a subunit vaccine based on glycoprotein B (gB), the major envelope glycoprotein and dominant target of neutralizing antibodies (NAb) (3, 4). A phase II trial evaluating recombinant gB admixed in the adjuvant MF59 showed 50% efficacy to prevent primary HCMV infection of seronegative women who gave birth within the previous year (5, 6). In contrast, the live attenuated Towne strain failed in an earlier trial to protect seronegative mothers with at least one HCMV-shedding child from acquiring primary HCMV infection (7). The absence of complete protection in both trials argues that vaccine optimization is critical to eliminate the risk of primary infection in the mother and congenital infection in the fetus.

NAb inhibiting HCMV entry into host cells play an important role in prevention of horizontal and vertical virus transmission (6, 8). Studies based on neutralization of fibroblast infection with laboratory strain AD169 or Towne have defined gB, gH, and gM/gN complexes as major NAb targets (913). These studies have also demonstrated that gB/MF59- and Towne-induced NAb titers are comparable to those observed following natural infection (5, 9). However, recent findings indicate that fibroblast-based neutralization studies incompletely define NAb responses to HCMV infection. HCMV infects a wide variety of cell types, and viral entry into different cell types requires distinct gH/gL envelope glycoprotein complexes (14, 15). While HCMV entry into fibroblasts depends on gB and gM/gN and gH/gL/gO complexes, entry into epithelial/endothelial cells (Epi/EC) requires three additional proteins, designated UL128, UL130, and UL131A, that form a pentameric virion protein complex with gH/gL (UL128C) (1622). AD169 and Towne viruses have lost the ability to infect Epi/EC due to mutations in the UL128-UL131A locus (23, 24). Consequently, their restricted cell tropism makes these viruses unsuitable for detection of NAb that inhibit Epi/EC infection. The use of HCMV strains with intact cell tropism has shown that HCMV-infected individuals develop NAb to UL128C that potently block infection of Epi/EC, but these NAb are incapable of blocking infection of fibroblasts (25, 26). In addition, studies with AD169 repaired for UL128-131A have shown that gB/MF59 and Towne fail to induce Epi/EC-specific NAb titers comparable to those observed during natural infection (27). These results provide strong evidence that UL128C is an important determinant of NAb activity specific for Epi/EC (27, 28).

Here we report the construction of a modified vaccinia Ankara virus (MVA) expressing the UL128C of rhesus CMV (RhCMV), termed MVA-RhUL128C, and the induction of NAb in vaccinated rhesus macaques (RM) (2932). Taking advantage of bacterial artificial chromosome (BAC) technology (33), MVA stably coexpressing RhgH/gL/UL128-UL131A was generated. Interaction of RhgH with the other 4 subunits of the five-protein complex was demonstrated by coimmunoprecipitation (co-IP). Vaccinated RM developed NAb that prevented RhCMV infection of Epi/EC as anticipated and, remarkably, fibroblasts as well. NAb titers for RhCMV infection of both cell types of MVA-RhUL128C-vaccinated monkeys were comparable to those of RhCMV-seropositive animals. In addition, the vaccinees showed reduced viral loads (VL) in plasma compared to control groups following RhCMV challenge. These results support the use of the UL128C pentamer as an essential component of an HCMV subunit vaccine to induce broader neutralization activities than vaccines solely targeting gB, which may lead to higher protective efficacy against horizontal transmission of HCMV, thereby preventing congenital infection.


Viruses and cells.

The propagation of MVA in baby hamster kidney (BHK) cells and the preparation and storage of viral stocks were performed according to our published protocols (34). Chicken embryo fibroblasts (CEF) for MVA propagation were maintained in virus production serum-free medium (VP-SFM) (Invitrogen). MVA-RhUL128C, MVA-RhUL128CΔ, or MVA expressing RhUL128-UL131A subunits was generated by the BAC technology as described below. MVA expressing either RhUL128 or RhUL130 alone was generated by the conventional manipulation strategy in eukaryotic cells as described previously (35). The construction of MVA-RhgB having a deleted transmembrane domain (TM) is described elsewhere (36). The epithelial cell-tropic UCD59 strain of RhCMV, which contains a full-length UL/b′ region, including an intact RhUL128-UL131A locus (GenBank accession number EU130540: originally annotated as strain 22659) (31), was serially passaged four times on monkey kidney epithelial (MKE) cells for these studies. RhCMV strain 68.1 (ATCC) was propagated on telomerized rhesus fibroblasts (Telo-RF) (37). MKE cells were maintained in Dulbecco's modified Eagle's medium–F-12 medium (DMEM-F12) (Invitrogen) supplemented with epithelial cell growth supplement (ScienCell), 1 mM sodium pyruvate, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine (Invitrogen), and 2% fetal bovine serum/SuperSerum (Gemini Bio-Products). Telo-RF cells were maintained as described previously (38). All cells were grown at 37°C, 5% CO2, and 95% humidity.

Transfer plasmids.

Transfer plasmids to insert gene expression cassettes for the individual RhCMV genes into the MVA BAC by en passant mutagenesis were generated as follows. First, synthetic intron-free coding sequences for RhUL128, RhUL130, or RhUL131A (GenScript), as well as PCR-amplified coding sequences for RhgL and RhgH of RhCMV strain UCD59 (GenBank accession numbers EU130540.1, HQ667932.1, and HQ667933.1), were individually inserted via PmlI and AscI restriction sites between the vaccinia virus modified H5 (mH5) promoter and the poly(T) (5TAT) transcription termination signal of plasmid pZero2-mH5 (34). The coding sequence for RhUL130 was synthesized with two C-to-T nucleotide changes at positions 99 and 102 of the open reading frame (ORF) in comparison to the published sequence. For the generation of RhgHΔTM, the first 690 codons of RhgH were amplified via PCR with a reverse primer providing a 3′-terminal coding sequence (GAG CAG AAA CTG ATA TCT GAA GAG GAC CTC TGA) for the myc tag epitope EQK LIS EED L. In contrast to RhUL128, the RhUL130, RhUL131A, RhgL, and RhgH ORFs were inserted with 5′-terminal Kozak sequences (GCC ACC ACC [RhUL130 and RhUL131A], GCC GCC GCC [RhgL], or GCC GCC ACC [RhgH]) preceding the ATG start codons. In the next cloning step, the kanamycin resistance (Kanr) marker aphAI and the homing endonuclease restriction site I-SceI of plasmid pEPkan-S2 (39) were PCR amplified with primers providing 50-bp gene duplications and inserted into unique restriction sites of the cloned genes. The primer sequences used to amplify the aphAI-I-SceI cassette and the restriction site used to clone the PCR product are given in Table 1. In the resulting constructs, the aphAI-I-SceI cassettes within the genes were flanked by 50-bp gene duplications (Fig. 1A). All cloned inserts were confirmed by sequencing. The complete sequences of all transfer plasmids are available upon request.

Table 1
Primers for cloning of aphAI-I-SceI and gene duplication into RhCMV genes
Fig 1
Insertion of RhCMV genes into MVA BAC. (A) Insertion of a vaccinia virus gene expression cassette into the MVA BAC by en passant mutagenesis. First, a transfer construct was generated that comprises the gene sequence with upstream vaccinia virus mH5 promoter, ...

En passant mutagenesis.

The cloned RhCMV genes were inserted into the MVA BAC by two-step Red recombination-based en passant mutagenesis in Escherichia coli strain GS1783 according to the published protocol (40). Briefly, the gene sequences with the upstream mH5 promoter, the downstream vaccinia virus termination signal, and the introduced aphAI-I-SceI cassette flanked by a 50-bp gene duplication were amplified via PCR from the pZero2-mH5 transfer plasmids with primers containing 50-bp extensions for homologous recombination and introduced into the viral genome by a first Red recombination (Fig. 1A). Following that, the Kanr selection marker was seamlessly excised from the inserted genes by the introduction of a DNA double-strand break at the I-SceI site via expression of the homing enzyme and a subsequent second Red recombination of the 50-bp gene duplications (Fig. 1A). By serial application of these reactions, the five RhCMV genes were successively inserted into four traditional MVA insertion sites as given in Fig. 1B. Primer sequences used to amplify the expression cassettes as well as the gene insertion sites are given in Table 2.

Table 2
Primers for Red recombination of RhCMV gene expression cassettes into MVA BAC

Virus reconstitution.

Virus reconstitution from the MVA BACs was performed in BHK cells using Fugene HD transfection reagent according to the manufacturer's instructions (Roche), similar to the procedures described before (33, 41). First, the BAC DNA was purified from GS1783 E. coli cells with the Plasmid Maxikit (Qiagen). Approximately 1 × 105 BHK cells were seeded in a six-well format and transfected 16 to 20 h later with 2 μg of purified BAC DNA via the Fugene HD lipid complexes. The cells were infected 4 h later with fowlpox virus (FPV) HP1.441 (42) (kindly provided by Bernard Moss, NIAID) at a multiplicity of infection (MOI) of 0.1. FPV is required to initiate the transcription machinery from the “naked” MVA DNA, but it does not undergo recombination with the MVA genome or establish a productive infection in BHK cells (33, 41). After 2 days of incubation, the cells were diluted in a ratio of 1 to 2, and virus reconstitution was monitored by green fluorescent protein (GFP) expression and plaque formation. If necessary, the dilution step was repeated until more than 90% of the cell monolayer was infected.

Polyclonal antisera.

Rabbit polyclonal antisera to the RhCMV proteins were generated via the Express complete peptide polyclonal antibody package from GenScript against the following peptide sequences: CID SDS YPY EED IDG was used for the RhUL128 antiserum, CTP RSA PAK QVA PKP for the RhUL130 antiserum, CVR PGE IDE CLY RQQ for the RhUL131 antiserum, CFT GET FSP EDD SW for the RhgL antiserum, and HNS TKC NNN GTR RNC for the RhgH antiserum.


Western blotting (WB) was accomplished by a method similar to published standard protocols (43). Briefly, 80 to 90% confluent BHK cells seeded in 6-well plates were infected with MVA at an MOI of 0.1. After 36 to 40 h, the cells were harvested and centrifuged at 300 × g, and total cell lysates were prepared in 200 μl of SDS sample buffer (2% SDS, 100 mM dithiothreitol [DTT] or 10% β-mercaptoethanol, and 125 mM Tris-HCl [pH 8.8]). To detect secreted proteins in the medium, confluent monolayers of CEF cells in 6-well plates were infected at an MOI of 0.1 and grown for 36 to 40 h in 2 ml of VP-SFM. The medium was harvested, cleared by centrifugation at 300 × g, and concentrated ~20-fold using Amicon ultracentrifugal filter devices (10-molecular-weight cutoff [MWCO]; Millipore). The concentrated medium was then prepared for WB by mixing with 5-fold-concentrated SDS sample buffer. Lysates of infected CEF cells were prepared as described for BHK cells. Samples were boiled, and 10- to 20-μl portions of the denatured proteins were electrophoretically separated on 10% SDS-polyacrylamide gels and then transferred onto a polyvinylidene fluoride (PVDF) membrane. Rabbit polyclonal antisera were applied in a dilution of 1/5,000. Secondary anti-rabbit antibody coupled to horseradish peroxidase (HRP) was employed in a dilution of 1/50,000 (GE Healthcare). Protein bands were finally visualized via chemiluminescent detection (Pierce).


BHK cells (80 to 90% confluent) in a 100-cm2 tissue culture dish were infected with MVA-RhUL128CΔ at an MOI of 5 and incubated for 16 to 20 h. The cells were harvested in 1 ml ice-cold cell lysis buffer containing 1% (wt/vol) Triton X-100, 50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 4 mM EDTA, 0.02% (wt/vol) sodium azide, 1 mM phenylmethylsulfonyl fluoride (PMSF), and Complete mini-protease inhibitor cocktail tablets (Roche). After incubation for 30 min on ice, cell debris was removed by centrifugation at ~10,000 × g for 10 min at 4°C. The cell lysate was precleared for 30 min at 4°C with protein A/G Plus-agarose beads and mouse IgG (Santa Cruz Biotechnology). In parallel, protein A/G Plus-agarose beads and 1 to 2 μg of mouse anti-c-myc tag antibody clone 4A6 (Millipore) or mouse IgG irrelevant control antibody were incubated for 2 h in ice-cold phosphate-buffered saline (PBS), washed 2 times in PBS, and then combined with 500 μl of precleared cell lysate. The mixture was incubated for 2 h or overnight at 4°C. Following that, the agarose beads were washed 3 times in PBS and boiled in 50 μl of SDS sample buffer. The samples (10 to 20 μl) were analyzed via WB as described above.


Genetically outbred RM (Macaca mulatta) from the California National Primate Research Center (CNPRC), which were repeatedly confirmed to be RhCMV seronegative, were used for these studies. Their age was ~1 to 2 years at the time of RhCMV inoculation. The animals were cohoused in pairs for at least 2 weeks before vaccination and remained cohoused toward the end of the study at 7 weeks after challenge. The Institutional Animal Care and Use Committee of the University of California, Davis (UC Davis), which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, approved all animal protocols in advance of any procedures.

Vaccination and challenge.

Groups of 4 RM (3 RM for MVA-RhUL130) were vaccinated by intramuscular injection with ~5 × 108 PFU of purified MVA 6 weeks apart, as previously described (36). Eight weeks after the second vaccination, the animals were challenged via subcutaneous injection with 1 × 103 PFU of RhCMV UCD59 according to our published protocols (36). Blood, oral swabs, and urine samples for determination of NAb titers and viral loads were prepared as previously described (36).

Real-time PCR.

DNA was extracted from plasma and oral swabs using the QIASymphony automated DNA processor (Qiagen) according to the manufacturer's instructions and published protocols (44). The final elution volume was 300 μl. Extracted DNA was stored at −80°C until real-time PCR analysis was performed. RhCMV UCD59 DNA copies in plasma and oral swabs were detected by a previously described real-time PCR assay (45).

Neutralization assays.

NAb titers of monkey plasma (with EDTA anticoagulant) on fibroblasts were assayed by the use of Telo-RF cells and RhCMV strain 68.1 for infection as described previously (38, 46). NAb on MKE cells were determined as follows. Briefly, 25 PFU of UCD59 was incubated with serial half-log dilutions (1:31 to 1:100) of heat-inactivated (56°C, 30 min) plasma in a final volume of 500 μl of DMEM-F12 with 10% fetal bovine serum. A pooled mixture of plasma from eight RhCMV-uninfected rhesus monkeys was included as a negative control. The virus-plasma mixture was incubated for 2 h at 37°C and then added in triplicate to monolayers of MKE cells in 24-well plates (500 μl/well), which had been seeded the day before at a density of 6 × 104 cells/well. Cells in three wells were incubated in growth medium only. After 4 h of incubation, the virus-plasma mixture was removed, and the cells were washed twice with DMEM-F12 and then overlaid with 0.5% agarose and 2 ml of growth medium. After 10 to 12 days, the plaques were counted. The percent neutralization titer (NT) for each dilution was calculated as follows: NT = [1 − (plaque number with immune plasma)/(plaque number with negative-control plasma)] × 100. The titer that gave 50% plaque reduction (NT50) was calculated by determining the linear slope of the graph plotting NT versus plasma dilution.

Data analysis.

Viral load copy numbers were summarized for each animal as the total area under the curve (AUC) for 16 weeks postchallenge. The AUC between two successive time points (T1 and T2, in weeks) was calculated as the area of the trapezoid formed by the VL at those two time points, according to the following formula: AUC (between T1 and T2) = 1/2(VLT1 + VLT2) × (T2 − T1). The sum of individual AUC measurements represented the total AUC for each animal.

Statistical analysis.

One- and two-sided Wilcoxon rank sum tests were used for calculation of statistical differences between groups.


Assembly of RhgH/gL/UL128-UL131A in a single MVA genome.

We took advantage of MVA BAC technology to assemble all five RhCMV genes within a single MVA genome (33). Using markerless sequence insertion by en passant mutagenesis (39, 40), vaccinia virus expression cassettes for the individual RhCMV genes were serially introduced into 4 known MVA insertion sites (Fig. 1A). Expression cassettes for RhUL128, RhUL131A, or RhgH, either as a full-length or a C-terminal TM-deleted form (RhgHΔTM) with a myc tag epitope, were inserted into deletion II site (Del2), intergenic region 3 (IGR3) (47), and the insertion site between the essential ORFs G1L and I8R (48), respectively (Fig. 1B). Further, expression cassettes for RhUL130 and RhgL were introduced into the deletion III site (Del3) site at both ends of the BAC vector (Fig. 1B). The TM-deleted construct was generated with the rationale that expression of a soluble RhUL128C may enhance immunogenicity (43, 49). The individual gene expression cassettes were inserted into separate insertion sites in opposite transcription orientations to allow comparable transgene expression while reducing the risk of homologous recombination between the promoter elements (Fig. 1B) (48). The integrity of the cloned MVA genome, as well as markerless insertion of the RhCMV genes, was confirmed by restriction fragment analysis, PCR, and sequencing (data not shown). These results demonstrated that en passant mutagenesis allowed rapid insertion of all five RhUL128C subunits into the cloned MVA genome.

Reconstitution of MVA expressing RhgH/gL/UL128-UL131A.

Virus reconstitution from the assembled MVA BACs was performed in BHK cells in the presence of fowlpox virus (FPV) as a helper virus (33, 41). MVA coexpressing RhgL/gH/UL128-UL131A, either with or without the TM of gH, was termed MVA-RhUL128C and MVA-RhUL128CΔ, respectively. Reconstituted virus was propagated, and infected BHK cells were analyzed via WB. Using rabbit polyclonal antisera to the individual RhUL128C subunits, the expression of all 5 inserted RhCMV genes was confirmed for both MVA vectors (Fig. 2A). The detected protein sizes were ~55 kDa for RhUL128, ~43 kDa for RhUL130, ~23 kDa for RhUL131, ~32 kDa for RhgL, and ~95 kDa for RhgH. All protein sizes were larger than the theoretically calculated protein sizes based on amino acid composition, suggesting that all RhUL128C subunits are posttranslationally modified in BHK cells (30). The two faster-migrating forms of RhgL can likely be explained by unprocessed proteins that require O-glycosylation as a late posttranslational maturation process, as has been shown for herpes simplex virus (HSV) (Fig. 2A) (50). As expected, deletion of the TM resulted in expression of RhgH with a lower molecular weight than full-length RhgH (Fig. 2A). MVA expressing only one, two, or all three of the RhUL128-UL131A subunit proteins was also generated with assistance of BAC technology (Fig. 2B). Coexpression of all RhUL128C protein subunits resulted in an increase of cellular amounts of RhUL128-UL131A, which could be explained by either increased stability or increased expression (Fig. 2B). MVA with single-gene insertions was generated within 2 to 3 weeks, and MVA-RhUL128C and MVA-RhUL128CΔ were generated in 4 to 5 months. In conclusion, we have successfully used BAC technology to rapidly generate MVA vaccines expressing subsets of RhUL128C genes or a full complement of all five genes.

Fig 2
Coexpression of RhUL128C subunits from MVA. Shown are WBs for the detection of RhUL128C subunits expressed from MVA using rabbit polyclonal antisera. (A) WB analysis of total cell lysates of BHK cells infected with MVA-RhUL128C or MVA-RhUL128CΔ. ...

Formation of RhUL128C pentamer expressed from MVA.

We investigated conditions to detect evidence for the formation of the RhUL128C pentamer by conducting co-IP studies of RhgH/gL/UL128-UL131A proteins expressed from MVA. We took advantage of the C-terminal myc tag incorporated into RhgHΔTM to utilize anti-c-myc tag monoclonal antibody to immunoprecipitate complexes that interact with RhgH from BHK cells infected with MVA-RhUL128CΔ. The precipitated proteins were analyzed by WB using polyclonal antisera for the detection of the individual RhUL128C subunits. IP of RhgHΔTM resulted in co-IP of RhgL, RhUL128, RhUL130, and RhUL131A (Fig. 3A). The detected protein sizes were consistent with the expected protein sizes based on previous WB results (Fig. 2). In contrast to previous results (Fig. 2A), only two bands were detectable for gL, possibly indicating that only more highly glycosylated mature forms of gL are incorporated into RhUL128C. These results provide evidence that RhgH/gL/UL128-UL131A proteins expressed from MVA interact with each other and likely form a pentameric complex similar to the HCMV counterparts.

Fig 3
Interaction of RhUL128C subunits expressed from MVA. (A) Coimmunoprecipitation. Shown are WBs for individual RhUL128C subunits using polyclonal antisera after IP of RhgHΔTM with anti-c-myc tag monoclonal antibody or IgG control antibody. (B) Secretion ...

Enhanced secretion of RhgHΔTM upon coexpression with RhgL and RhUL128-UL131A.

Ryckman et al. demonstrated using adenovirus-based expression vectors that secretion of a TM-deleted gH (gHΔTM) is enhanced by coexpression of all UL128C pentamer subunits (19). We characterized similar properties of an RhUL128C by comparing the secretion efficacies of RhgHΔTM expressed from MVA alone or in combination with RhgL or with RhgL and UL128-UL131A (MVA-RhUL128CΔ). As expected, coexpression of all RhUL128C subunits (MVA-RhUL128CΔ) led to the highest secretion levels of RhgHΔTM compared to its expression alone or in combination with gL (Fig. 3B). In addition, the amounts of secreted RhgHΔTM expressed alone, in combination with RhgL, or in combination with all other RhUL128C subunits were larger than those of full-length RhgH coexpressed together with all other RhUL128C subunits (MVA-RhUL128C) (Fig. 3B), suggesting that the presence of the TM of RhgH tethered the complexes to the cell surface. Interestingly, the size of RhgHΔTM or RhgH detectable in the medium was slightly larger than that observed in cell lysates (relative to the 95-kDa size marker), indicating that secreted forms of RhgH are posttranslationally modified differently from their cellular counterparts. These data show that coexpression of RhgHΔTM with RhgL and RhUL128-UL131A promotes secretion of RhgHΔTM, providing further evidence that RhUL128C has similarities to HCMV UL128C (19).

Stable coexpression of RhgH/gL/UL128-UL131A in MVA.

As a next step we investigated the expression stability of MVA-RhUL128C upon virus propagation. MVA-RhUL128C was passaged 5 times on BHK cells, and relative protein expression levels from all 5 inserted RhCMV genes were determined after each virus passage by WB. Constant amounts of all five RhCMV proteins were confirmed during the 5 virus passages of MVA-RhUL128C (Fig. 4). We also confirmed stable expression of the inserted RhCMV genes during 5 virus passages of MVA coexpressing RhUL128, RhUL130, and RhUL131A (data not shown). In addition, expression of the solitary RhUL128 gene from a vaccine vector generated by BAC technology or from a vaccine vector derived by the conventional MVA transfection/infection strategy showed comparable expression levels over 10 virus passages (data not shown) (51). These results demonstrated that MVA-RhUL128C stably coexpressed all RhCMV genes during serial virus passage and that BAC-derived MVA provided insert stability comparable to that with conventional derived recombinants. Accordingly, the pentamer and other RhCMV subunit MVA recombinants were judged suitable for large-scale expansion to prepare stocks for vaccination.

Fig 4
Expression of RhUL128C subunits upon propagation of MVA-RhUL128C. Shown are WBs for the detection of RhUL128, RhUL130, RhUL131A, RhgL, and RhgH during 5 virus passages of MVA-RhUL128C on BHK cells. Total cell lysates of the 5 different virus passages ...

Induction of Epi/EC-specific NAb in RM vaccinated with MVA-RhUL128C.

To achieve the central goal of the vaccine program, we investigated the capacity of MVA-RhUL128C or MVA-RhUL128CΔ to generate NAb in RM. Two groups of four RhCMV-negative monkeys were each vaccinated twice with either MVA-RhUL128C or MVA-RhUL128CΔ (n = 4 RM/vaccine). MVA expressing either RhUL128 or RhUL130 (n = 3 RM) alone or MVA coexpressing RhUL128-UL131A was also used for vaccination. NAb titers that gave 50% neutralization (NT50s) were determined on monkey kidney epithelial (MKE) cells using RhCMV strain UCD59 for infection. Plasma samples of monkeys vaccinated with RhgB or the bacterial marker gus in a DNA prime/double MVA boost procedure from a previous study were analyzed as additional controls (38). NT50s of MVA-RhUL128C- or MVA-RhUL128CΔ-vaccinated RM at 2 weeks postvaccination were comparable to the normative NT50 range of naturally infected RM and ranged from 108 to 402 (median, 146) or 88 to 513 (median, 209), respectively (Fig. 5A). The normative range of NT50 to UCD59 measured on MKE cells for 3- to 4-year-old corral-housed monkeys is 67 to 1,060 (median, 662) (Y. Yue et al., unpublished data). In contrast, NT50s of RM vaccinated with MVA expressing only RhUL128C subunits or RhgB remained under the detection limit of the assay (Fig. 5A). Only one animal of the MVA-RhgB group had an NT50 of 72 (Fig. 5A). Consequently, NAb titers measured on MKE cells of RM vaccinated with either MVA-RhUL128C or MVA-RhUL128CΔ were significantly higher than those of the MVA-venus control group as well as all other vaccine groups (Fig. 5A). At 6 weeks after the second vaccination, NT50s ranged from 70 to 101 (median, 79) for monkeys vaccinated with MVA-RhUL128C and from 36 to 94 (median, 56) for MVA-RhUL128CΔ-vaccinated animals. These NAb titers were still at the lower end of or slightly below the normative NT50 range determined on MKE cells (Fig. 5B). NAb titers determined for the MVA-RhUL128C and MVA-RhUL128CΔ vaccine groups were not significantly different (P = 0.69 at 2 weeks and 0.2 at 6 weeks) (Fig. 5). These results indicate that coexpression of all 5 RhUL128C subunits is needed to elicit epithelial cell-specific NAb.

Fig 5
NAb in vaccinated RM. (A and B) NT50s measured on epithelial cells (MKE cells) at 2 (A) and 6 (B) weeks postvaccination (post Vx) for the indicated vaccine groups. (C and D) NT50s measured at 2 (C) and 6 (D) weeks postvaccination on fibroblasts (Telo-RF). ...

Generation of fibroblast-specific NAb in RM vaccinated with MVA-RhUL128C.

Plasma samples from RM vaccinated with MVA-RhUL128C or MVA-RhUL128CΔ vaccine were analyzed for their capacity to inhibit infection of telomerized rhesus fibroblasts (Telo-RF) with RhCMV strain 68-1. Surprisingly, both vaccine constructs induced strong NAb activity, preventing RhCMV infection of Telo-RF cells (Fig. 5C). At 2 weeks postvaccination, plasma samples from RM vaccinated with MVA-RhUL128C or MVA-RhUL128CΔ showed NT50s ranging from 590 to 785 (median, 608) or from 450 to 864 (median, 732), respectively. These NT50s were in the normative NT50 range for naturally infected RM measured on Telo-RF, which is from 231 to 3,348 for long-term-infected animals (median, 833) (Fig. 5C) (38). Interestingly, NAb measured on Telo-RF of MVA-RhUL128C- or MVA-RhUL128CΔ-vaccinated RM at week 2 postvaccination were significantly higher than those of RM vaccinated with MVA-RhgB (3-fold; P = 0.01) or control-vaccinated animals (P = 0.005) from a previous study (Fig. 5C) (38). NAb titers at 6 weeks postvaccination declined and ranged from 160 to 383 (median, 216) for MVA-RhUL128C-vaccinated RM and from 148 to 350 (median, 226) for animals of the MVA-RhUL128CΔ vaccine group. NAb at week 6 were still maintained at the lower end of the normative range of NT50 values measured on fibroblasts (Fig. 5D). As with the NAb titer measured on MKE cells, NAb titers determined on Telo-RF from the MVA-RhUL128C and MVA-RhUL128CΔ vaccine groups were not significantly different (P = 0.49 and 0.89) (Fig. 5). These unexpected results demonstrate that MVA expressing RhCMV gH/gL/UL128-UL131A induces NAb activity that not only inhibits entry into Epi/EC but, impressively, also blocks RhCMV infection of fibroblasts.

Reduced viral load in MVA-RhUL128C-vaccinated RM following RhCMV challenge.

The 2 MVA vaccine groups expressing different forms of RhUL128C were next evaluated for protective efficacy against challenge with RhCMV. RM vaccinated with MVA-RhUL128CΔ or MVA-RhUL128C were subcutaneously inoculated 8 weeks after the booster vaccination (at week 6) with RhCMV strain UCD59 (37). To serve as a control, unvaccinated animals were also challenged. Figure 6 presents the cumulative plasma viral load of UCD59 genome copies determined by computing the area under the curve (AUC) over a 16-week time interval beginning at the time of challenge (see Fig. S1 in the supplemental material for primary data). Whereas the plasma viral load of animals vaccinated with MVA-RhUL128C (median AUC = 584) was 21-fold lower than that of control RM (median AUC = 12,652), the viral load of animals vaccinated with MVA-RhUL128CΔ (median AUC = 5,247) was only 2.4-fold lower than that of control unvaccinated animals (Fig. 6). The viral load of the majority of monkeys vaccinated with MVA-RhUL128C was lower than that of nonvaccinated monkeys (P = 0.03). Three of four animals vaccinated with MVA-RhUL128C had no or only very few detectable RhCMV copies in plasma (Fig. 6). In contrast, two RM of the MVA-RhUL128CΔ vaccine group showed no viral load, but the other two had high viral loads (Fig. 6). Consequently, we cannot infer any improvement in viral load of MVA-RhUL128CΔ-vaccinated RM compared to controls (P = 0.33) or any difference from the MVA-RhUL128C group (P = 0.64). However, pooling the two vaccine groups and comparing viral loads to those of control nonvaccinated animals yield a significance probability of P = 0.07, which is very close to unequivocal statistical significance. These data indicate that vaccination of RhCMV-naïve RM with MVA-RhUL128C reduced plasma the viral load in the majority of RM given a virulent challenge virus. The NAb and challenge results are strong indicators that the UL128C pentamer will be a required component of a successful prophylactic vaccine for monkeys and, more importantly, for humans.

Fig 6
Viral load of RhCMV challenge virus in plasma samples from vaccinated RM. Shown is the area under the curve (AUC) of detectable genome copy numbers in plasma samples from monkeys vaccinated with MVA-RhUL128CΔ or MVA-RhUL128C or unvaccinated control ...

No reduced viral shedding after challenge of MVA-RhUL128C-vaccinated RM.

We also assessed the important property of viral shedding in MVA-RhUL128C- or MVA-RhUL128CΔ-vaccinated RM challenged with RhCMV UCD59 (38). Unexpectedly, there was no evidence of reduction of RhCMV genome copies in oral swabs obtained from all animals in either of the vaccine groups (data not shown). While this may indicate that a vaccine incorporating only UL128C subunits is insufficient to provide protection against shedding, it is equally likely that the delivery method was inadequate to achieve this goal, as we amplify in Discussion.


Epi/EC play a pivotal role for HCMV entry, dissemination, persistence, and host-to-host transmission (14). A vaccine strategy for effective prevention of HCMV infection will likely depend on the ability to induce potent NAb that inhibit virus entry into these cell types (52, 53). Previous vaccine strategies based on recombinant gB or Towne failed to elicit high-titer NAb that inhibit HCMV infection of Epi/EC (27). Since the UL128-UL131A proteins that form a pentameric virion complex with gH/gL are required for entry into Epi/EC and serve as targets of potent NAb in HCMV-seropositive individuals, these proteins have been proposed as prime vaccine targets (16, 21, 26, 28). Here, we have shown that MVA coexpressing all five RhCMV counterparts of HCMV UL128C induces NAb potently blocking virus infection of MKE cells as well as rhesus fibroblasts (Fig. 5). A vaccine expressing these proteins may be an effective candidate to inhibit multiple HCMV entry routes.

Expression cassettes for all 5 RhCMV genes could be rapidly inserted into separate insertion sites of a single MVA genome by combining BAC technology with markerless sequence insertion by en passant mutagenesis (Fig. 1) (33, 40). In contrast, an approach based on homologous recombination in eukaryotic cells with subsequent laborious screening procedures would have taken considerably more time to generate these recombinants (51). This is the first description of MVA with gene expression cassettes in 5 separate insertion sites, propelling MVA into a new category for multiantigenic vaccine design. MVA-RhUL128C maintained stable expression of all five RhCMV genes over 5 virus passages (Fig. 4), indicating that this vector construct is a feasible candidate for vaccine development. While each of the individual UL128C subunits could be expressed from separate vectors, simultaneous delivery into a single cell to enable assembly of the functional pentamer will be difficult to achieve in vivo rather than in an in vitro laboratory setting. We have identified an optimal approach for pentamer assembly and function that has profound translational consequences as a building block for an HCMV vaccine.

Some investigators have demonstrated that tagged fusion proteins or derivative peptides of UL128, UL130, or UL131A induce Epi/EC-specific NAb for HCMV in mice or rabbits (21, 54, 55), suggesting that these UL128C subunits might be sufficient to generate NAb activity in humans. However, generating immunity in small animals against a xeno-antigen may reflect only an immunologic property rather than targeted immunity against host-restricted CMV. This conclusion is based on the fact that the UL130 and UL131A peptide sequences used to generate NAb in rabbits did not bind serum antibodies from HCMV-seropositive individuals (55), strongly suggesting that these single linear epitopes would not be immunogenic in humans. In contrast, our results confirm the necessity of coexpression of all five RhUL128C subunits to elicit an NAb titer comparable to that in naturally infected RM (Fig. 5). A five-protein complex might be crucial to stabilize the formation of conformational epitopes (19, 56).

Our vaccine design initially focused on the RhUL128, RhUL130, and RhUL131A proteins, either alone, in combination with another, or as a pentamer with RhgH/gL, to elicit Epi/EC-specific NAb, since the HCMV counterparts of these proteins have been proposed as major NAb targets (16, 21, 26). With that focus, we did not set up a control group with a vaccine expressing only RhgH/gL. In hindsight, the result that only vaccines expressing RhUL128C induced NAb that prevented infection of Epi/EC, whereas none of the vaccines expressing individual RhUL128-UL131A subunits did so, made it logical to have included RhgH/gL as a single experimental group. Therefore, we must be cautious in our interpretation, as it is not ruled out that RhgH/gL is minimally necessary to generate Epi/EC-specific NAb. Preliminary observations obtained by mouse immunization studies indicate that MVA expressing HCMV gH/gL only minimally elicits NAb that inhibit HCMV entry into human epithelial cells (ARPE-19) (unpublished data). The functional and structural similarity of RhCMV UL128C and HCMV UL128C highlights that observations made for these proteins of the two viruses are likely applicable to each other (21, 2932) (Fig. 3). Our data support that UL128C pentamers should be a part of a comprehensive prophylactic HCMV vaccine.

An unexpected finding was the discovery that RM vaccinated with MVA-RhUL128C developed NAb activity that inhibited RhCMV infection of rhesus fibroblasts with efficacies comparable to that induced by naturally infected monkeys (Fig. 5). These observations suggest that the MVA-RhUL128C vaccine is capable of stimulating NAb simultaneously against both RhUL128C and RhgH/gL complexes (26, 57, 58). Of great significance, NAb raised in MVA-RhUL128C-vaccinated RM that inhibited RhCMV infection of rhesus fibroblasts had significantly (~3-fold) greater titers than NAb raised in MVA-RhgB-vaccinated animals from our previous study (Fig. 5) (38). We have additionally shown that MVA-RhgB-vaccinated RM developed only minimal levels of NAb that inhibited RhCMV infection of MKE cells (Fig. 5). These observations strongly support that MVA-RhUL128C is superior to MVA-RhgB for the induction of NAb inhibiting RhCMV entry into both fibroblasts and Epi/EC. Our results are consistent with the recent analysis of HCMV hyperimmune globulins, which contain predominantly NAb that are directed against UL128C and gH/gL complexes and only a small proportion that target gB (57). Our data support HCMV vaccine strategies based on UL128C and/or gH/gL as being more effective than strategies relying solely on gB to generate NAb blocking infection of multiple cell types. However, a combination of the two approaches may provide even higher and broader NAb activities than strategies based on only one of these important neutralizing determinants.

It is striking that the decline in MKE cell-specific-NAb titers from week 2 to 6 postvaccination is lower in the MVA-RhUL128C vaccine group than in the MVA-RhUL128CΔ group, and the difference in NAb titers between these two groups may be even greater at the time of challenge inoculation (at week 8 postvaccination) (Fig. 5). This could serve as an explanation why the MVA-RhUL128C vaccine group showed a significant reduction in plasma viral load compared to controls (P = 0.03), in contrast to the MVA-RhUL128CΔ group (P = 0.33) (Fig. 6). The difference in NAb decline may also indicate that membrane-tethered RhUL128C induces more durable Epi/EC-specific NAb than its soluble counterpart. However, since we could not detect a significant difference in NAb titers either on MKE cells (P = 0.2 at week 6) or on fibroblasts (P = 0.89 at week 6) or a significant difference in plasma viral load (P = 0.64) between the MVA-RhUL128C and MVA-RhUL128CΔ vaccine groups (Fig. 5 and and6),6), we could not support any inverse immune correlates by statistics. We also did not find a statistical correlation between NAb titers and plasma viral loads of individual animals within the vaccine groups.

The induction of strong NAb responses by MVA-RhUL128C was insufficient to reduce virus shedding in any of the vaccinated monkeys (Fig. 5). This observation has similarities to results of our previous study, in which a vaccine based solely on gB reduced plasma viral load following RhCMV challenge but did not reduce shedding (36, 38). Only vaccination with MVA expressing RhgB and Rhpp65/RhIE1 provided protection against challenge virus shedding in 50% of the vaccinated animals (38). These observations may indicate that RhUL128C in the absence of other dominant neutralizing determinants, such as RhgB or RhgM/gN, and/or the major targets of cell-mediated immunity, such as Rhpp65 or RhIE1, is insufficient to provide protection. It may also be possible that UL128C antigens are sufficient to provide protection and that other, unexplored cofactors or different prime-boost procedures may be required to improve the immune responses to limit RhCMV replication in RM. An alternative explanation for the failure to reduce shedding may be the decline of protective NAb at the time of challenge virus inoculation (Fig. 5). B-cell stimulatory ligands, such as CD40L, should be considered to increase the titer or durability of the NAb (59). An additional possibility for the failure might be the use of direct parenteral inoculation of challenge virus into vaccinated animals, the standard method of viral challenge in all animal models of HCMV. Importantly, subcutaneous inoculation circumvents the natural infection route at mucosal membranes, where NAb are believed to play an important role as a primary defense barrier. A critical threshold for HCMV vaccine candidates is the induction of sustained protective immunity against repeated mucosal exposure to potential antigenic variant viruses. Future vaccine evaluation should incorporate horizontal transmission of challenge virus from virus-excreting animals or inoculation of challenge virus onto the oral mucosa (37, 60).

It is important to discuss the apparent dichotomy in the NAb results showing that both RhUL128C and RhUL128CΔ generate strong NAb against Epi/EC and fibroblast infection by cognate RhCMV strains (Fig. 5). However, the plasma viral load and shedding data are not congruent with the strong NAb results, mainly because there is no significant difference in plasma viral load between the RhUL128CΔ vaccine group and the control group (Fig. 6). Furthermore, neither RhUL128C vaccine group showed a demonstrable effect on mucosal viral shedding. We can speculate that the avidity of the NAb is sufficient to inhibit in vitro RhCMV infection in cognate cell lines yet insufficient to prevent reduction in viremia when virus is introduced systemically by subcutaneous administration. Since there is no absolute correlation of in vitro NAb measurements and protective function in women who are seroimmune but are susceptible to reinfection with an additional HCMV strain (6164), it is possible that a similar dichotomy is found in the RhCMV model in RM. Recently, it has been uncovered that Epi/EC NAb are predominant in seroimmune women and hyperimmune globulin preparations, and a reassessment of the relationship of NAb and protective immunity is ongoing in the clinical setting (25, 27, 57, 6567). Although the number of RM in our vaccine groups should be larger to make a definitive conclusion about the requirement of UL128C pentamer-specific NAb, the work still highlights that an important immunologic metric is elicited by RhUL128C. Future work may guide us to the right combination of antigens and delivery mechanisms to achieve the optimal combination for protection against a challenge virus.

In summary, we have explored MVA BAC technology in combination with markerless sequence insertion by en passant mutagenesis to generate MVA stably coexpressing all five RhCMV counterparts of HCMV UL128C, which are required for virus entry into Epi/EC. Co-IP results provide evidence that the five RhCMV proteins expressed from MVA form a pentameric complex similar to the HCMV UL128C. MVA-RhUL128C-vaccinated RM not only developed strong neutralization activity preventing RhCMV infection of epithelial cells but also developed strong NAb activity inhibiting infection of fibroblasts. In addition, NAb titers measured on both cell types were comparable to those of naturally infected monkeys. Furthermore, the vaccinated RM showed reduced viral loads in plasma. While it would have been a great success if a vaccine incorporating only UL128C subunits successfully prevented shedding, it is equally likely that the antigen targets are too few or the delivery is inadequate. Despite not meeting all of its endpoints, this study still has a significant impact on vaccine design for the following reasons: (i) we confirmed that all 5 RhUL128C subunits are sufficient to induce NAb that inhibit RhCMV infection on both Epi/EC and fibroblasts, though single RhUL128, RhUL130, or RhUL131A subunits or combinations are not; (ii) NAb titers are equally strong whether the infection substrate is Epi/EC or fibroblasts, suggesting that a single vaccine composed of UL128C subunits may bypass the need for a gB subunit vaccine; and (iii) finally, the current HCMV vaccines in clinical evaluation to prevent horizontal transmission need to incorporate UL128C components or they will risk being inadequate for the task of preventing HCMV infection of both main infection portals, thereby reducing their effectiveness (6, 6870).

Supplementary Material

Supplemental material:


We thank Matthew G. Cottingham (Oxford University, United Kingdom) for providing the MVA BAC. For the en passant plasmids and the GS1783 cells, we thank B. Karsten Tischer and Klaus Osterrieder (Freie Universität Berlin, Germany) as well as Gregory A. Smith (Northwestern University). We are grateful to Bernard Moss (NIAID) for providing the FPV strain HP1.441.

This work was partially supported by Public Health Service grants AI63356 from the National Institute of Allergy and Infectious Diseases and CA030206 and CA077544 from the National Cancer Institute. The City of Hope Cancer Center is supported by grant CA033572. This project was also supported by the National Center for Research Resources (P51 RR00169) and is currently supported by the Office of Research Infrastructure Programs/OD (P51 OD011107) to the California National Primate Research Center (


Published ahead of print 14 November 2012

Supplemental material for this article may be found at


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