Inclusion of the Viral Pentamer Complex in a Vaccine Design Greatly Improves Protection against Congenital Cytomegalovirus in the Guinea Pig Model

K Yeon Choi; Nadia S El-Hamdi; Alistair McGregor

doi:10.1128/JVI.01442-19

. 2019 Oct 29;93(22):e01442-19. doi: 10.1128/JVI.01442-19

Inclusion of the Viral Pentamer Complex in a Vaccine Design Greatly Improves Protection against Congenital Cytomegalovirus in the Guinea Pig Model

K Yeon Choi ^a, Nadia S El-Hamdi ^a, Alistair McGregor ^a,^✉

Editor: Rozanne M Sandri-Goldin^b

PMCID: PMC6819920 PMID: 31484753

Cytomegalovirus (CMV) is a leading cause of congenital disease in newborns, and an effective vaccine remains an elusive goal. The guinea pig is the only small-animal model for cCMV. Guinea pig cytomegalovirus (GPCMV) encodes a glycoprotein pentamer complex (PC) for entry into non-fibroblast cells, including placental trophoblasts, to enable cCMV. As with human cytomegalovirus (HCMV), GPCMV uses a specific cell receptor (PDGFRA) for fibroblast entry, but other receptors are required for non-fibroblast cells. A disabled infectious single-cycle (DISC) GPCMV vaccine strain induced an antibody immune response to the viral pentamer to enhance virus neutralization on non-fibroblast cells, and vaccinated animals were fully protected against cCMV. Inclusion of the PC as part of a vaccine design dramatically improved vaccine efficacy, and this finding underlines the importance of the immune response to the PC in contributing toward protection against cCMV. This vaccine represents an important milestone in the development of a vaccine against cCMV.

KEYWORDS: CRISPR, PDGFRA, animal model, cell receptor, congenital infection, cytomegalovirus, glycoprotein, guinea pig, pentamer, vaccine

ABSTRACT

A vaccine against congenital cytomegalovirus (cCMV) is a high priority. The guinea pig is a small-animal model for cCMV. A disabled infectious single-cycle (DISC) viral vaccine strain based on a guinea pig cytomegalovirus (GPCMV) capsid mutant was evaluated. A previous version of this vaccine did not express the gH/gL-based pentamer complex (PC) and failed to fully protect against cCMV. The PC is necessary for GPCMV epithelial cell/trophoblast tropism and congenital infection and is a potentially important neutralizing antigen. Here, we show that a second-generation PC-positive (PC⁺) DISC (DISCII) vaccine induces neutralizing antibodies to the PC and other glycoproteins and a cell-mediated response to pp65 (GP83). Additionally, a CRISPR/Cas9 strategy identified guinea pig platelet-derived growth factor receptor alpha (PDGFRA) to be the receptor for PC-independent infection of fibroblast cells. Importantly, PDGFRA was absent in epithelial and trophoblast cells, which were dependent upon the viral PC for infection. Virus neutralization by DISCII antibodies on epithelial and trophoblast cells was similar to that in sera from wild-type virus-infected animals and dependent in part on PC-specific antibodies. In contrast, sera from PC-negative virus-infected animals poorly neutralized virus on non-fibroblast cells. DISCII-vaccinated animals were protected against congenital infection, in contrast to a nonvaccinated group. The target organs of pups in the vaccine group were negative for wild-type virus, unlike those of pups in the control group, with GPCMV transmission being approximately 80%. Overall, the DISCII vaccine had 97% efficacy against cCMV. The complete protection provided by this PC⁺ DISC vaccine makes the possibility of the use of this approach against human cCMV attractive.

IMPORTANCE Cytomegalovirus (CMV) is a leading cause of congenital disease in newborns, and an effective vaccine remains an elusive goal. The guinea pig is the only small-animal model for cCMV. Guinea pig cytomegalovirus (GPCMV) encodes a glycoprotein pentamer complex (PC) for entry into non-fibroblast cells, including placental trophoblasts, to enable cCMV. As with human cytomegalovirus (HCMV), GPCMV uses a specific cell receptor (PDGFRA) for fibroblast entry, but other receptors are required for non-fibroblast cells. A disabled infectious single-cycle (DISC) GPCMV vaccine strain induced an antibody immune response to the viral pentamer to enhance virus neutralization on non-fibroblast cells, and vaccinated animals were fully protected against cCMV. Inclusion of the PC as part of a vaccine design dramatically improved vaccine efficacy, and this finding underlines the importance of the immune response to the PC in contributing toward protection against cCMV. This vaccine represents an important milestone in the development of a vaccine against cCMV.

INTRODUCTION

Cytomegalovirus (CMV) is a member of the Betaherpesvirinae subfamily and is a leading cause of congenital disease. In the United States, approximately 8,000 newborns each year have permanent disabilities associated with congenital CMV (cCMV) (1). Indeed, approximately 25 to 30% of cases of hearing loss in children are attributed to cCMV infection (2). The greatest risk of congenital infection is to the children of mothers who acquire a primary infection during pregnancy, for whom there is a 1:3 chance of vertical transmission (3, 4). Prior convalescent immunity can substantially reduce the risk of cCMV (5). Maternal protection against cCMV is considered to be based on the antibody response to neutralizing viral glycoprotein complexes and the cell-mediated response to viral antigens. Consequently, an impaired T cell response, poor antibody avidity, or a neutralizing response is a potential risk factor associated with impaired protection against cCMV (6 –9). Since cCMV does not occur in the mouse or rat, the guinea pig is unique, insofar as it is the only small-animal model for cCMV (10). Both human and guinea pig placentas are hemomonochorial, containing a homogeneous layer of trophoblast cells separating the maternal and fetal circulation (11 –13). Congenital infection in the guinea pig causes disease in utero and sensorineural hearing loss (SNHL) in newborn pups (14 –16). Consequently, the guinea pig model is well suited for evaluation of intervention strategies against cCMV.

In HCMV, six glycoproteins (gB, gH, gL, gM, gN, gO) are required for fibroblast cell entry, and they form specific glycoprotein complexes, gCI (gB), gCII (gM/gN), and gCIII (gH/gL/gO), on the viral membrane (17 –19). These complexes are important neutralizing antibody targets and vaccine candidates (20 –24). Guinea pig cytomegalovirus (GPCMV) forms functionally similar glycoprotein complexes, which are essential for cell entry, as well as important target antigens (25, 26). Human cytomegalovirus (HCMV) encodes another gH/gL-based complex known as the pentamer or pentameric complex (gH/gL/UL128/UL130/131) that is necessary for epithelial, endothelial, and myeloid cell tropism (27). GPCMV encodes a similar pentameric complex (gH/gL/GP129/GP131/GP133), which is necessary for virus renal epithelial cell, trophoblast, and macrophage tropism (28 –30). The pentamer complex (PC) is also highly important for GPCMV dissemination in the animal and significantly enhances congenital transmission of the virus (28, 29). In contrast, murine CMV (MCMV) does not encode a PC but instead encodes a second gH-based trimer, gH/gL/MCK-2, which is more similar to that of Epstein-Barr virus and which complicates studies in the mouse model that may have a focus on the PC (31). The cellular receptor for HCMV infection of fibroblast cells has been identified to be platelet-derived growth factor receptor alpha (PDGFRA) and is dependent upon the gH trimer, in conjunction with gB, and is independent of the PC (32, 33). PDGFRA has not been identified to be a cell receptor for any animal CMV. Knockout of the PDGFRA in human fibroblast cells renders HCMV dependent upon the PC route of entry (32, 33). Similarly, the absence of PDGFRA or the low-level expression of PDGFRA on epithelial cells and other non-fibroblast cell types renders the virus dependent on the PC route of cell entry, and potential cell receptors, e.g., neuropilin-2, have been identified (34). However, the underlying mechanism of viral PC cell entry is only partially understood.

The HCMV gB protein is essential for fusion of the viral envelope and plasma membrane in all cell types. A recombinant gB has been investigated as a subunit vaccine, but despite high antibody titers, it provided only approximately 50% efficacy in phase II clinical trials (21, 35). Separate studies of serum from gB-vaccinated individuals demonstrated that it is less effective at neutralizing virus infection on endothelial and epithelial cells than convalescent-phase serum from HCMV-infected individuals (36 –38). Even though GPCMV gB is also essential for infection on all cell types (25, 28), gB-based vaccine strategies fail to fully protect against cCMV (providing approximately 50% protection) (39, 40). This strongly suggests that additional target antigens should be included in any vaccine strategy (39). The gH/gL-based glycoprotein complexes, especially PC, have been identified to be potentially important candidates in vaccines against HCMV (41). Epidemiology studies of patients convalescing from HCMV infection indicate that the antibody response to PC is highly important in protecting against cCMV (4). Consequently, an immune response to gB, gH/gL, and PC is likely needed for a successful vaccine against cCMV. Patients convalescing from HCMV infection also have a robust T cell response to the tegument protein pp65, the major T cell target antigen (42). The pp65 antigen has been explored as a T cell target in a vaccine strategy in animal models and in clinical trials (43), including in vaccines against GPCMV.

A successful vaccine strategy against cCMV may require the induction of both antibody and T cells; thus, the use of live-attenuated CMV represents an attractive approach. However, given that a live-attenuated vaccine may preserve potential links of CMV-related cancer and vascular disease (44, 45), the alternative approaches include the use of a replication-incompetent or disabled infectious single-cycle (DISC) virus as a vaccine candidate like that employed with the HCMV-based V160 vaccine (46). In contrast to HCMV V160, we have targeted the knockout of a capsid gene (UL85 homolog) which is essential for virus assembly but which is relatively unimportant as a vaccine target (26) and which does not interfere with viral dense body production (47). CMV capsid genes are highly conserved between HCMV and animal CMV, and the process of capsid assembly in HCMV is well studied and defined (48). Previously, we reported on a phase I DISC (DISCI) vaccine which had high efficacy against cCMV with a reduction of cCMV transmission to 23% from 80% (26). In the current study, we added the PC to the phase II DISC (DISCII) vaccine design. Additionally, a CRISPR/Cas9 gene knockout strategy was used to identify guinea pig PDGFRA (gpPDGFRA) as the receptor for PC-independent GPCMV fibroblast cell infection. The immune response of DISCII-vaccinated animals was compared to that of animals hyperimmune to wild-type (wt) (PC-positive [PC⁺]) virus or lab-adapted (PC-negative [PC⁻]) virus. The neutralizing capability of sera was evaluated on guinea pig fibroblast, epithelial, and trophoblast cell lines. Complete serum or serum depleted of antibodies to specific glycoproteins complexes (gB, gH/gL, or PC) was evaluated to demonstrate the importance of specific neutralizing antibodies. Comparison of pooled historical sera from DISCI (PC⁻)-vaccinated animals (26) with pooled sera from DISCII-vaccinated animals under the same vaccine regime demonstrated that serum from DISCII-vaccinated animals was more effective in the neutralization of virus on all cell types. Additionally, a DISCII vaccine preconception protection study against cCMV by wild-type GPCMV was evaluated. Overall, the DISCII vaccine strategy induced a comprehensive immune response against GPCMV and fully protected pups against cCMV when protection was compared to that achieved in a control nonvaccinated (NOV) group.

RESULTS

PDGFRA is the receptor for PC-negative GPCMV infection of fibroblast cells.

Studies in HCMV have demonstrated that PDGFRA is the receptor for HCMV trimer (gH/gL/gO)-dependent infection of fibroblast cells (32, 33) and that this receptor is absent in epithelial cells, which require the PC for cell entry (49). Human and guinea pig PDGFRA are highly conserved, with 95% identity, based on an alignment determined by BLAST analysis, over the length of the predicted mature protein (UniProt accession number H0VJZ3 [https://www.uniprot.org/uniprot/H0VJZ3]). Guinea pig cell lines generated by our laboratory and fibroblasts available from ATCC were evaluated for PDGFRA protein expression. Cell lines included fibroblasts, renal epithelial cells (28), and trophoblasts (29). PDGFRA was detected in guinea pig lung (GPL) fibroblast cells but not in epithelial or trophoblast cells (Fig. 1a). β-Actin served as a control for lane loading (Fig. 1b). We employed the CRISPR/Cas9 strategy to knock out the PDGFRA gene by targeting exon 2 in guinea pig cells, as described in Materials and Methods. Western blot analysis of the PDGFRA knockout cell line (GPKO fibroblasts) failed to detect endogenous PDGFRA expression, whereas it was detected in control GPL fibroblasts (Fig. 1c), and β-actin was detected as a control protein in cell lysates (Fig. 1d). PCR cloning of the exon 2 locus of GPKO and GPL fibroblasts verified a targeted deletion (64 bases) in exon 2 of the PDGFRA gene in a clonally isolated cell line (Fig. 1e). A comparative growth curve of PC⁺ and PC⁻ GPCMV on confluent monolayers of GPL and GPKO fibroblasts in six-well plates demonstrated that the PC⁻ virus was dependent upon PDGFRA for infection of fibroblast cells. The absence of the receptor resulted in a lack of PC⁻ virus growth (Fig. 1f). In contrast, the PC⁺ virus was unaffected by the knockout of the PDGFRA receptor (Fig. 1g). This result was similar to that of HCMV infection of human fibroblast cells (33), where the PC-specific receptor also remains to be identified, demonstrating that PDGFRA is the likely receptor for PC-negative or PC-independent virus infection of fibroblast cells. Specific interactions of guinea pig PDGFRA (gpPDGFRA) and the GPCMV gH trimer are the subject of additional work (N. S. El-Hamdi, K. Y. Choi, and A. McGregor, unpublished data). An additional experiment with herpes simplex virus 1 (HSV-1), which does not require PDGFRA for cell entry, demonstrated that infection of confluent monolayers of GPL or GPKO cells resulted in similar progeny viral yields on both cell lines (Fig. 1h). This further confirmed that the restriction on GPKO cells was specific for PC-negative GPCMV and the absence of PDGFRA.

FIG 1 — Characterization of guinea pig PDGFRA as the receptor for PC-independent GPCMV infection of fibroblast cells. (a) Western blot analysis for the detection of endogenous PDGFRA in various guinea pig cell types. Lanes (cell lysates): 1, renal epithelial (REPI) cells; 2, trophoblast epithelial (TEPI) cells; 3, fibroblasts (GPL); 4, loading dye-only control; 5, GPL fibroblasts plus transient expression plasmid for gpPDGFRA (GenScript). (b) β-Actin as a Western blotting control for cell lysate lane loading in panel a. (c) CRISPR/Cas9 knockout of PDGFRA from a guinea pig fibroblast cell line verified by Western blot analysis. Lanes (cell lysates): 1, GPL fibroblasts; 2, GPL fibroblasts expressing Cas9; 3, GPL PDGFRA knockout (GPKO) cells; 4, GPL fibroblasts plus transient gpPDGFRA expression plasmid. (d) β-Actin as Western blotting control for cell lysate lane loading for panel c. The arrows in panels a and c indicate the location of gpPDGFRA. (e) CRISPR guide RNA (gRNA) targets (boxed). CRISPR A gRNA targets bases 214 to 233; CRISPR C gRNA targets bases 149 to 168. The locus in both the wild type and the clonal mutant cell line was PCR cloned and sequenced. The alignment shows part of exon 2 with the deletion of nucleotides 153 to 217 in CRISPR-treated cells. The sequences of the wild type (wt_exon2_PDGFRA) and the knockout mutant (PCR_CRISPR_KO 1) are shown. The alignment was performed using MacVector software. (f) GPCMV PC⁻ growth curve on GPL or GPKO fibroblasts. (g) GPCMV PC⁺ growth curve on GPL or GPKO fibroblasts. (h) HSV-1 infection on GPL and GPKO fibroblasts at 60 h postinfection. The MOI was 1 PFU/cell for growth curves (f to h).

Generation of a PC⁺ DISC (DISCII) virus strain.

The first-generation GPCMV DISCI vaccine was based on a lethal knockout of the GP85 small capsid gene to render the virus incapable of productive infection, unless the mutant virus was grown on a complementing cell line (26). In order to generate a PC⁺ DISC GPCMV mutant, the second-generation GPCMV bacterial artificial chromosome (BAC) (50), which encodes a defective UL128 homolog (GP129), was modified to encode a wild-type GP129 cDNA ectopically in the GP25-GP26 intergenic locus under simian virus 40 (SV40) promoter control, as previously described (28). Subsequently, the GP85 gene was placed under tetracycline-repressible (tet-off) promoter control as in the DISCI construct to create a DISCII GPCMV BAC (26) (Fig. 2a). A DISCII GPCMV BAC clone was characterized by HindIII restriction profile analysis (Fig. 2b), and the modified loci were sequenced as described in Materials and Methods to verify the modification. Subsequently, DISCII GPCMV BAC DNA was cotransfected with a Cre recombinase expression plasmid into the GPL tet-off cell line to excise both green fluorescent protein (GFP) and the BAC insertion and generate a recombinant virus (designated DISCII virus) (50). The DISCII virus had growth kinetics similar to those of the first-generation DISC virus on GP85-complemented cells for conditional replication but failed to replicate on noncomplemented GPL cells (Fig. 2c) (26). Ectopic expression of the GP129 Myc epitope-tagged protein encoded in the GP25-GP26 intergenic locus, which would enable PC formation, was confirmed by Western blot analysis of virus-infected cells (Fig. 2d) as previously described (28). Attenuation of the DISCII virus in vivo was evaluated by subcutaneous (SQ) inoculation of animals with the DISCII virus (10⁶ PFU). Animals failed to become infected when infection in blood was monitored at 4, 8, 12, and 27 days postinfection (dpi) (Fig. 2e). In comparison, a control group of animals inoculated with a similar dose of PC⁺ wild-type GPCMV generated on GPL tet-off fibroblast cells had detectable viremia at 4 and 8 dpi (Fig. 2e), demonstrating that the DISCII virus was severely attenuated.

FIG 2 — GPCMV DISCII BAC and virus characterization and evaluation for animal viremia. (a) GPCMV DISCII BAC. Schematic diagram of the GPCMV genome with the location of the BAC insertion, which was removed by Cre *loxP* recombination upon the generation of virus (50). The *GP85* locus was modified as previously described (26) to place gene expression under tet-off transactivator control. Full-length GP129 was restored by ectopic expression of a Myc-tagged ORF in the *GP25-GP25* intergenic locus (28) to enable PC (gH/gL/GP129/GP131/GP133) expression in GPCMV DISCII. (b) HindIII restriction profile analysis of GPCMV BAC DNA. Lanes: 1, DISCII BAC clone 1; 2, DISCII BAC clone 2; 3, second-generation GPCMV BAC. The second-generation GPCMV BAC (50) was modified in the *GP25-GP26* and *GP85* loci so that it had modifications identical to those described previously (26, 28). Two independent DISCII clones (clones 1 and 2) were evaluated by restriction profile analysis. Green circles indicate the original wt HindIII-A fragment (bases 102380 to 146446) for the *GP85* locus. Introduction of a novel HindIII site in the *GP85* locus resulted in a fragment size modification to generate two new HindIII subgenomic fragments, indicated by red circles (as originally described by Choi et al. [26]). The wt *GP25-GP26* locus encoded in the 17-kb HindIII subgenomic fragment, indicated by the green square (bases 26294 to 43382), was modified to introduce an ectopic GP129 expression cassette, which also introduced a novel HindIII restriction site (28). The modified subgenomic HindIII fragment is indicated by red squares. (c) DISCII virus growth curve on tet-off GPL cells or GPL cells. The MOI of the DISCII virus was 1 PFU/cell. DPI, day postinfection. (d) Western blot analysis for the detection of Myc-tagged GP129 in DISCII-infected GPL cells. (Top) Anti-Myc. Lanes: 1, DISCII-infected GPL cell lysate; 2, wt GPCMV-infected GPL cell lysate; 3, mock-infected GPL cell lysate. (Bottom) Lane loading control for β-actin. Lanes are as described for the blot in the top panel. Monolayers were harvested at 48 h postinfection. The MOI was 1 PFU/cell. (e) Real-time PCR evaluation of virus dissemination in the blood of infected animals. Animals were infected (10⁶ PFU of wt GPCMV or DISCII SQ) at day 0, and at 4, 8, 12, and 27 dpi, the animals were bled to evaluate the viral load, as described in Materials and Methods. The viral loads for DISCII-vaccinated animals are compared to those for wt GPCMV-infected animals at the bottom. Data are the means ± SD from a minimum of three replicates. Results are given as the number of genome copies per milliliter of blood (c/ml blood).

Vaccine-induced antibody immune response to glycoprotein complexes.

Using a three-dose vaccination regime (26), shown in Fig. 3a, the DISCII-vaccinated group of seronegative female animals (n = 19) matched for age were vaccinated SQ with the DISCII vaccine (10³ PFU) on days 0, 26, and 49. The animals were bled at 24 and 48 days after initial vaccination (which was considered day 0) to determine the anti-GPCMV antibody titer. On day 70, a final bleed was used to evaluate the immune response to specific glycoprotein complexes (gB, gH/gL, gM/gN, and PC) (25, 26). A comprehensive evaluation of antibody responses against DISCII was carried out by enzyme-linked immunosorbent assay (ELISA) for all animals prior to mating (Fig. 3). Hyperimmune sera pooled from animals infected with wild-type GPCMV (PC⁺) or lab-adapted GPCMV (PC⁻) were also evaluated (Fig. 3b and c). Animals were made hyperimmune to GPCMV (PC⁺ or PC⁻) following a protocol similar to that described below in “Ethics” in Materials and Methods.

Figures 3b to e compare the mean DISCII virus serum ELISA titers and individual serum results for the various assays: anti-GPCMV (mean values and individual results are shown in Fig. 3b and d, respectively) and specific glycoprotein complexes (mean values and individual results are shown in Fig. 3c and e, respectively). In Fig. 3b and c, comparisons of sera from the DISCII-vaccinated group were also made to pooled sera from hyperimmune animals infected with wt (PC⁺) GPCMV or lab-adapted (PC⁻) virus. All assays were carried out concurrently. The mean anti-GPCMV ELISA titer (titer, 8,623) of the DISCII-vaccinated animals was significantly higher (P < 0.05) than the anti-GPCMV titer (titer, 5,120) of pooled sera from animals hyperimmune to GPCMV (PC⁺/PC⁻) (Fig. 3b). An evaluation of individual anti-GPCMV titers demonstrated that 7 animals had titers more similar to those of animals in the other groups and 11 animals had titers close to 10,000 (Fig. 3d). When the anti-GPCMV antibody avidity in serum from individual DISCII-vaccinated animals was compared to that in pooled serum from GPCMV (PC⁺)-hyperimmune animals, there was a significantly higher avidity for antibodies from pooled hyperimmune (PC⁺ GPCMV) serum than the mean value for the DISCII-vaccinated group (Fig. 3f). However, the anti-GPCMV response would include antibodies to neutralizing target proteins as well as nonneutralizing target antigens, and consequently, the anti-GPCMV results serve as a rough indicator for the vaccine strategy.

Serum ELISA titers of antibodies to specific viral glycoprotein complexes (Fig. 3c) revealed significant differences between groups. DISCII-vaccinated animals had the lowest anti-gB titer (mean titer, 427), whereas sera from GPCMV (PC⁺) hyperimmune animals had a mean titer of 1,600 and GPCMV (PC⁻) hyperimmune animals had a mean titer of 2,240. In contrast, the mean avidity of anti-gB antibodies for DISCII-vaccinated animals was equivalent to that of antibodies in the pooled hyperimmune GPCMV (PC⁺) sera (Fig. 3g). This was despite an almost 4-fold higher anti-gB titer (mean titer, 1,600) in the hyperimmune sera (Fig. 3c). There was no difference in the anti-gH/gL titer between DISCII-vaccinated animals and hyperimmune (PC⁺) animals, but the anti-gH/gL titer was significantly higher for the hyperimmune GPCMV (PC⁻) sera than for the sera from animals in the other groups (Fig. 3c). The anti-PC ELISA titer was similar between hyperimmune GPCMV (PC⁺) and DISCII-vaccinated animals (mean titer, 1,757 versus 1,920) and significantly lower for GPCMV (PC⁻) hyperimmune sera, which was presumably because this virus lacked the ability to make PC and therefore mounted a response only to gH/gL. The response to gM/gN in the DISCII-vaccinated group was low overall (Fig. 3e). However, the presence of PC in the DISCII-vaccinated group, like the GPCMV (PC⁺)-hyperimmune group, potentially negatively impacted the immunogenicity of the gM/gN complex, which was significantly higher in pooled sera from GPCMV (PC⁻)-hyperimmune animals than in sera from the other groups (Fig. 3c).

Individual immune responses to gH/gL and also PC were variable in DISCII-vaccinated animals (Fig. 3e). However, there was no difference in the mean anti-gH/gL or anti-PC titers between sera from DISCII-vaccinated animals and pooled sera from GPCMV (PC⁺)-hyperimmune animals (Fig. 3c). In DISCII-vaccinated animals, anti-PC antibody titers had the greatest spread in range (320 for one animal and 2,560 for five animals). However, a low gH/gL response did not correspond to a low anti-PC titer. For example, two animals with the lowest anti-gH/gL response (titers, 480 and 373) had anti-PC titers closer to the average titer of 1,757 (1,280 and 1,920, respectively) (Fig. 3e).

Anti-gB, anti-gH/gL, or anti-PC antibody depletion and impact on virus neutralization.

The impact on virus neutralization of specific antibody depletion (anti-gB, anti-gH/gL, or anti-PC) was evaluated for pooled sera from DISCII-vaccinated animals and compared to that for pooled sera from hyperimmune (PC⁺ or PC⁻ virus) animals. Pooled sera from DISCII-vaccinated animals were used in various antibody depletion experiments, and the results were confirmed by assessment of the responses in anti-GPCMV and glycoprotein complex-specific ELISAs before and after the antibody depletion experiments. Figure 4 shows the depletion results for pooled sera from DISCII-vaccinated animals: anti-gB depletion (Fig. 4a and b), anti-gH/gL depletion (Fig. 4c to e), and anti-PC depletion (Fig. 4f to h). GPCMV (PC⁺)-hyperimmune sera (Fig. 5) and GPCMV (PC⁻)-hyperimmune sera (Fig. 6) were also subjected to specific antibody depletions. Anti-gB (Fig. 5a and b), anti-gH/gL (Fig. 5c to e), and anti-PC (Fig. 5f to h) depletions were carried out for GPCMV (PC⁺)-hyperimmune sera. In the case of GPCMV (PC⁻)-hyperimmune sera, only anti-gB (Fig. 6a and b) and anti-gH/gL (Fig. 6c to e) depletions were carried out. Antibody depletion was carried out as previously described, and neutralization assays were performed concurrently with the same virus stocks (25). After anti-gH/gL depletion (Fig. 4d), pooled sera from the DISCII-vaccinated animals retained anti-PC-specific antibodies (Fig. 4e), confirming that the DISCII-vaccinated group, like the GPCMV (PC⁺)-hyperimmune group, generated PC-specific antibodies rather than antibodies only to gH/gL (Fig. 5e). In contrast, GPCMV (PC⁻)-hyperimmune sera depleted of anti-gH/gL (Fig. 6d) did not retain anti-PC-specific antibodies when evaluated by the anti-PC ELISA (Fig. 6e). The depletion of anti-PC antibody from sera from DISCII-vaccinated or hyperimmune (PC⁺) animals also removed anti-gH/gL antibodies (Fig. 4h and 5h).

FIG 4 — Results for pooled sera from DISCII-vaccinated animals depleted of antibodies to specific glycoprotein complexes. (a and b) Verification of anti-gB depletion by ELISA of pre-ΔgB-depleted DISCII and post-ΔgB-depleted DISCII vaccination sera. (a) Anti-GPCMV ELISA; (b) anti-gB ELISA. (c to e) Verification of anti-gH/gL depletion by ELISA of pre-ΔgH/gL-depleted DISCII and post-ΔgH/gL-depleted DISCII vaccination sera. (c) Anti-GPCMV ELISA; (d) anti-gH/gL ELISA; (e) anti-PC ELISA. (f to h) Verification of anti-PC depletion by ELISA of pre-ΔPC DISCII and post-ΔPC DISCII vaccination sera. (f) Anti-GPCMV ELISA; (g) anti-PC ELISA; (h) anti-gH/gL ELISA. Red dashed lines, detection level limit; Abs, absorbance.

FIG 5 — Results for pooled sera from GPCMV (PC⁺)-infected animals depleted of antibodies to specific glycoprotein complexes. (a and b) Verification of anti-gB depletion by ELISA of pre-ΔgB-depleted GPCMV PC⁺ and post-ΔgB-depleted GPCMV PC⁺ hyperimmune sera. (a) Anti-GPCMV ELISA; (b) anti-gB ELISA. (c to e) Verification of anti-gH/gL depletion by ELISA of pre-ΔgH/gL-depleted GPCMV PC⁺ and post-ΔgH/gL-depleted GPCMV PC⁺ hyperimmune sera. (c) Anti-GPCMV ELISA; (d) anti-gH/gL ELISA; (e) anti-PC ELISA. (f to h) Verification of anti-PC depletion by ELSA of pre-ΔPC-depleted GPCMV PC⁺ and post-ΔPC-depleted GPCMV PC⁺ hyperimmune sera. (f) Anti-GPCMV ELISA; (g) anti-PC ELISA; (h) anti-gH/gL ELISA. Red dashed lines, detection level limit.

FIG 6 — Results for pooled sera from GPCMV (PC⁻)-infected animals depleted of antibodies to specific glycoprotein complexes. (a and b) Verification of anti-gB depletion by ELISA of pre-ΔgB-depleted GPCMV PC⁻ and post-ΔgB-depleted GPCMV PC⁻ hyperimmune sera. (a) Anti-GPCMV ELISA; (b) anti-gB ELISA. (c to e) Verification of anti-gH/gL depletion by ELISA of pre-ΔgH/gL-depleted GPCMV PC⁻ and post-ΔgH/gL-depleted GPCMV PC⁻ hyperimmune sera. (c) Anti-GPCMV ELISA; (d) anti-gH/gL ELISA; (e) anti-PC ELISA. Red dashed lines, detection level limit.

All previously reported GPCMV neutralization studies by various investigators were performed only on fibroblast cells. Therefore, previous studies lacked a complete evaluation of the contribution of antibodies to specific complexes for virus neutralization on non-fibroblast cells. Comparison of the virus neutralization titers for various cell types (fibroblasts, epithelial cells, and trophoblasts) for pooled nondepleted (native) sera from DISCII-vaccinated animals against those for pooled sera from hyperimmune (PC⁺ or PC⁻) animals are shown in Fig. 7. Additionally, the results of a comparison of sera from the different groups depleted of antibodies for different glycoprotein complexes (gB, gH/gL, and PC) are shown in Fig. 7. The sera from DISCII-vaccinated animals had 50% neutralizing activity (NA₅₀) values on fibroblasts comparable to those of the sera from the GPCMV (PC⁺/PC⁻)-hyperimmune animals (Fig. 7a). The sera from DISCII-vaccinated animals had NA₅₀ values more similar to those of sera from hyperimmune (PC⁺) animals for both epithelial and trophoblast cells (Fig. 7b and c). However, for both of these groups, the titers were 50% lower than the NA₅₀ values for fibroblast cells. Pooled sera from GPCMV (PC⁻)-hyperimmune animals were poorly neutralizing on renal epithelial and trophoblast cells compared to sera from the other groups, with a roughly 3-fold lower neutralizing titer (Fig. 7b and c).

Depletion of anti-gB from sera from the different groups generally reduced the NA₅₀ values on various cell types. On fibroblast cells, the reduction in NA₅₀ was similar for all groups (Fig. 7a). However, anti-gB depletion resulted in a more extensive reduction in NA₅₀ for renal epithelial and trophoblast cells for sera from the GPCMV (PC⁻)-hyperimmune groups than for sera from the other groups (mean NA₅₀, 180 for epithelial cells and 240 for trophoblasts) (Fig. 7b and c). After anti-gB antibody depletion, the NA₅₀ for the DISCII-vaccinated group was reduced from 1,627 to 427 for epithelial cells and from 1,440 to 960 for trophoblasts (Fig. 7b and c). Anti-gB depletion of sera from the GPCMV (PC⁺)-hyperimmune group also reduced the NA₅₀ values but to a lesser degree than that of sera from the DISCII-vaccinated group (Fig. 7b and c).

Depletion of anti-gH/gL antibodies had a roughly similar impact on sera from both DISCII-vaccinated and GPCMV (PC⁺)-hyperimmune animals (Fig. 7), with NA₅₀ values being more severely impacted on epithelial and trophoblast cells than on fibroblast cells. After anti-gH/gL antibody depletion, the NA₅₀ for DISCII-vaccinated animals was reduced from 1,627 to 240 for epithelial cells and from 1,440 to 240 for trophoblasts. The NA₅₀ for fibroblast cells in pooled sera from GPCMV (PC⁻)-hyperimmune animals depleted of anti-gH/gL was roughly similar to that in sera from the other groups after depletion (Fig. 7a). However, pooled sera from GPCMV (PC⁻)-hyperimmune animals poorly neutralized virus on non-fibroblast cells prior to depletion of anti-gH/gL antibodies (Fig. 7b and c).

When anti-PC antibody depletion was carried out for sera from GPCMV (PC⁺)-hyperimmune and DISCII-vaccinated animals, NA₅₀ values were further reduced on epithelial or trophoblast cells compared to those for anti-gH/gL-depleted sera (Fig. 7b and c). Anti-PC depletion had an impact on the NA₅₀ for fibroblast cells, as anti-PC depletion also depleted antibodies to gH/gL (Fig. 4h and 5h). Anti-PC depletion in DISCII-vaccinated animals had the greatest impact on activity for renal epithelial cells, with a reduction in the NA₅₀ of from 1,627 to 40, compared to an NA₅₀ of 240 with anti-gH/gL depletion (Fig. 7b). For trophoblasts from DISCII-vaccinated animals, the NA₅₀ was also reduced (from 1,440 to 160). GPCMV (PC⁺)-hyperimmune sera depleted of anti-PC antibodies (Fig. 7b and c) also had reduced NA₅₀ values: from 1,920 to 160 for epithelial cells and 1,707 to 240 for trophoblasts. GPCMV (PC⁻)-hyperimmune pooled sera depleted of anti-PC or anti-gH/gL had similar results (Fig. 7), presumably because the PC⁻ virus did not encode the pentamer; therefore, additional pentamer-specific antibodies could not be generated in infected animals (Fig. 6e).

Although anti-gB antibodies have the potential to neutralize GPCMV infection (25, 39, 51), depletion of anti-gB demonstrated that the neutralization of virus was also achieved through antibodies against other viral glycoprotein complexes. Depletion of anti-gH/gL had a greater impact on decreasing the NA₅₀ for both fibroblast and epithelial cells for sera from both hyperimmune and DISCII-vaccinated animals (Fig. 7). This likely indicates the importance of the anti-gH/gL immune response, which presumably targets all gH-containing complexes, including the PC, necessary for cell entry. Depletion of anti-PC antibodies (anti-gH/gL and anti-PC) potentially demonstrated the importance of neutralizing antibodies to the PC within sera from the DISCII-vaccinated group in comparison to that of the gB immune response, but the overall neutralization of virus was likely attained by antibodies to the various glycoprotein complexes.

Virus neutralization in sera from DISCI (PC⁻)- versus DISCII (PC⁺)-vaccinated animals for various cell types.

We previously evaluated the efficacy of a PC-negative GPCMV DISC (DISCI) vaccine, which provided encouraging results but failed to fully protect against cCMV (26). In the original study, neutralization titers in sera from DISCI-vaccinated animals were evaluated only with fibroblast cells, as other cell types had not been established at that time. The original study demonstrated that the neutralizing titer for fibroblast cells in sera from DISCI-vaccinated animals was lower than that for fibroblast cells in sera from hyperimmune wild-type-infected animals (26). Using historical pooled sera from DISCI-vaccinated animals that were vaccinated under a vaccine regime identical to that used in the DISCII study, we compared the neutralization titers for various cell types in concurrent assays. Figure 8 compares the neutralization titers for sera from DISCI- and DISCII-vaccinated animals for fibroblast, epithelial, and trophoblast cell lines. The results demonstrated that pooled sera from DISCII-vaccinated animals was significantly better for virus neutralization for all cell types than pooled sera from DISCI-vaccinated animals. Consequently, it was likely that the improved neutralizing antibody response of the DISCII vaccine compared to that of the DISCI vaccine was an important basis for the success of the DISCII vaccine.

FIG 8 — Neutralizing antibody titers (NA₅₀) on different cell types of sera from DISCII- and DISCI-vaccinated animals. Pooled sera from DISCII-vaccinated animals or pooled historical sera from DISCI-vaccinated animals receiving an identical vaccine regime (26) were tested for NA₅₀ against wt GPCMV (PC⁺) on guinea pig fibroblast (GPL), epithelial (REPI), and trophoblast (TEPI) cell lines. *, P < 0.05, Student's t test.

DISCII vaccine-induced cell-mediated immune response to the pp65 homolog protein GP83.

The DISC vaccine strategy is designed to mimic natural virus infection and was therefore expected to induce a cell-mediated immune response to important T cell target antigens. Previously, we demonstrated that the GPCMV pp65 tegument protein homolog GP83 is a T cell target (26, 52). The cell-mediated response to the DISCII virus vaccine was evaluated by a guinea pig-specific gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay (26). Results demonstrated that the DISCII vaccine induced a cell-mediated response in animals similar to that in wild-type GPCMV-seropositive animals when evaluated against the same GP83 peptide pools (Fig. 9) (26). Evaluation of other potential T cell target antigens, such as IE1 (53), awaits the development of guinea pig-specific IFN-γ ELISPOT assays for other viral antigens and is a limitation of the guinea pig model but was beyond the scope of the current study.

FIG 9 — T cell response to the DISCII vaccine determined by IFN-γ ELISPOT assay. The guinea pig-specific IFN-γ response to GPCMV GP83 peptide pools in DISCII-vaccinated and GPCMV PC⁺-infected animals are shown. Two reactive GP83 peptide pools, peptide pools X (yellow) and XX (red), previously identified (26), responded with splenocytes isolated from DISCII-vaccinated animals or seropositive wt GPCMV-infected animals. Blue, ConA positive control; orange, DMSO control; gray, unstimulated (unstim) control; green, unresponsive negative (neg) peptide pool II; purple, unresponsive negative peptide pool IV. Final counts were calculated based on the number of SFC per 10⁶ cells after the values for background spots (cells only, without any stimulation) were subtracted. ^{a, b}, P < 0.05, Student's t test.

Wild-type virus dissemination in DISCII-vaccinated versus seronegative animals.

In an effort to evaluate the impact of the immune response in DISCII-vaccinated animals against wild-type virus dissemination, a pathogenicity study was carried out in vaccinated and nonvaccinated dams. In group 1 (n = 12), seronegative dams matched for age were vaccinated with the DISCII vaccine as described in Fig. 3a and evaluated for anti-GPCMV titers. Subsequently, group 1 animals and a control seronegative group of dams (group 2, n = 12) were challenged with 10⁵ PFU of salivary gland (SG) stock virus. At days 4, 8, 12, and 27 dpi, three animals per group were euthanized and the viral load was evaluated in the target organs (liver, lung, spleen) and blood. At 27 dpi, the salivary gland tissue was additionally evaluated for the viral load. The results demonstrated that in seronegative animals, the wild-type virus disseminated to all target organs and induced viremia (Fig. 10a and b). In contrast, virus failed to be detected in the target organs of DISCII-vaccinated animals, and there was a complete lack of viremia (Fig. 10c and d). Possibly, virus dissemination occurred at a level below the limit of detection of the real-time PCR assay. Figures 10e and f demonstrate that the real-time PCR assay for the amplification target GP44 gene (range, 10⁷ to 10 copies) generated a standard curve with an efficiency of 1.929. While further analysis demonstrated a detection level of 2.5 copies, the detection level cutoff was set at 5 copies with a 95% efficiency. The contrast between the viral load in the seronegative and the vaccinated animal groups (Fig. 10) demonstrated the potent inhibition of wild-type virus dissemination in vaccinated animals, and it is likely that neutralizing antibodies were a significant contributing component of the immune response to prevent virus dissemination.

FIG 10 — Comparative dissemination of GPCMV to target organs in seronegative and DISCII-vaccinated animals. Seronegative animals (a and b) and DISCII-seropositive animals (c and d) (n = 12 per group) were infected with wt GPCMV (10⁵ PFU). (a, c) At various days (4, 8, 12, and 27 dpi), 3 animals per group were evaluated for the viral load in the target organs by real-time PCR of DNA extracted from tissue. The viral load is plotted as the number of viral genome copies per milligram of tissue. Salivary gland (sal gland) tissue was evaluated only at day 27. (b, d) Blood viremia at 4, 8, 12, and 27 dpi plotted as the number of genome copies per milliliter of blood. The target organs were lung, liver, spleen, salivary gland, and blood. (e and f) Real-time PCR standard amplification. A known concentration of GPCMV *GP44* plasmid DNA was diluted 10-fold and run in triplicate, as stated in Materials and Methods, to generate the amplification curve (e) and the standard curve (f). The red lines indicate positive amplification; the green lines indicate the negative no-template control.

Congenital GPCMV protection study.

The DISCII vaccine strategy was evaluated for its ability to protect against congenital GPCMV (Fig. 3). Initially, 34 female guinea pigs seronegative for GPCMV were randomly assigned to two groups: group 1 (DISCII, n = 19), vaccinated with DISCII virus at 10³ PFU SQ, and group 2, control nonvaccinated (NOV) animals (n = 15). At days 26 and 49 postvaccination, the DISCII-vaccinated group of animals received an additional booster dose. Next, these animals were mated with seronegative male guinea pigs. The dams were confirmed to be pregnant by palpation (20 to 25 days of gestation). At approximately the late 2nd trimester (days 30 to 35 gestation), the dams were challenged SQ with 10⁵ PFU of salivary gland stock wild-type virus and the animals were allowed to go to term. After the pups were delivered, the viral load in the target organs (liver, lung, spleen, brain) of live or stillborn pups was evaluated by real-time PCR. Table 1 summarizes the pregnancy outcomes in the two animal groups. Overall, the DISCII-vaccinated group had a higher proportion of live pups than the NOV group (96.8% versus 56.25%) (Table 1). All stillborn pups in the control NOV group that were evaluated for viral load were positive for GPCMV in at least one organ, with 80% of NOV stillborn pups being positive for GPCMV in two or more tissues (liver, lung, spleen, or brain) (Table 1). Based on the outcome (live versus stillborn pups) between the groups, the DISCII vaccine was considered to be highly effective in protecting against congenital CMV mortality (P = 0.0001). The mortality rate for the pup control group was 43.8%. The DISCII vaccine group had a higher average pup weight than the NOV group (Fig. 11), indicating that the vaccine also prevented growth retardation in utero. The results fall within the range of values in previously published studies of congenital GPCMV with a challenge virus inoculum of 10⁵ PFU, where the pup mortality rate ranged from 34 to 81% (26, 39, 54 –59).

TABLE 1.

Congenital infection outcomes for live versus dead pups

Characteristic	Value for the following animals:
Characteristic	DISCII vaccinated	NOV
No. of guinea pigs pregnant/total no. of guineas pigs (%)	19/19 (100)	15/15 (100)
No. of litters delivered	19	14^a
No. of litters with only live pups	18	7
No. of litters with a mix of live and dead pups	1	3
No. of litters with only dead pups	0	4
Total no. (%) of pups alive^d	60 (96.8)	27 (56.25)
No. of live pups evaluated by PCR	60	24^b
Total no. (%) of pups dead^d	2 (3.2)	21 (43.75)
No. of dead pups evaluated by PCR	2	11^c

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One dam died during pregnancy at day 8 postchallenge.

Three live pups were not evaluated by PCR due to premature birth (before day 12 postchallenge).

Ten dead pups were not evaluated by PCR due to premature birth (before day 12 postchallenge).

P = 0.0001, Fisher’s exact test.

FIG 11 — Pup weight at birth. DISCII-vaccinated or nonvaccinated (NOV) dams were challenged with wt GPCMV during the early second trimester and pregnancy was allowed to go to term. The weights of the pups from the DISCII group (open circles) and the NOV group (open squares) were measured. Red lines indicate the mean weight. *, P < 0.05, Student's t test.

The timing of birth and the lack of availability of a complete uncontaminated pup carcass (stillborn animals) were limiting factors in the evaluation of the pups in the NOV group. Consequently, only 11 out of 21 stillborn pups in the NOV group could be evaluated for the viral load by PCR. Additionally, 24 out of 27 liveborn pups in the NOV group were evaluated for the viral load (Table 1). In the DISCII-vaccinated group, all animals, including two stillborn pups, were evaluated for the viral load (Table 1). The congenital GPCMV transmission rate in the control group (80%) was based on detectable virus in 28 out of 35 pups (Table 2). Virus was present in the brains of 51% (18/35) of these animals (Table 2). The transmission rate in the control group was roughly similar to that observed previously in control groups (50 to 85% transmission) when challenged with 10⁵ PFU of salivary gland stock wild-type virus (average cCMV transmission rate, 74.4%) (39, 54 –57, 59). All pups in the DISCII vaccine group, including the two stillborn pups, were negative for virus in all organs tested. A comparison of the viral load in the target tissues of pups in the different groups is shown in Table 3. The viral load in the pups in the no-vaccine control group was roughly similar to that found in a previous vaccine study (26). The previous DISCI-vaccinated animals showed a transmission rate that was reduced to 23.5% (26), suggesting that the current DISCII vaccine had a higher efficacy, which was likely because of the inclusion of the pentamer antigen.

TABLE 2.

Congenital infection outcome, based on viral load in target tissues of pups

Organ or group	No. of infected pups/total no. of pups (%)		P value^a
Organ or group	DISCII-vaccinated group	NOV group	P value^a
Lung	0/62 (0)	21/35 (60.00)	0.0001
Liver	0/62 (0)	15/35 (42.86)	0.0001
Spleen	0/62 (0)	19/35 (54.29)	0.0001
Brain	0/62 (0)	18/35 (51.43)	0.0001
CMV-positive pups	0/62 (0)	28/35 (80.00)	0.0001

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Statistics were determined by Fisher’s exact test.

TABLE 3.

Congenital infection outcome determined by viral load in target tissue of pups

Vaccine group	Viral load (no. of log₁₀ genome copies/mg tissue)
Vaccine group	Lung	Liver	Spleen	Brain
DISCII vaccinated	No viral load	No viral load	No viral load	No viral load
NOV	2.7 ± 2.1	2.6 ± 2.1	3.2 ± 2.9	3.3 ± 2.6

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DISCUSSION

The guinea pig is the only small-animal model for cCMV, as transplacental infection of the fetus does not occur in the mouse model. Additionally, MCMV does not encode a PC but instead encodes a second gH-based trimer, gH/gL/MCK-2 (31), which further complicates translational vaccine studies in the mouse model. Although a rhesus macaque nonhuman primate (NHP) model with rhesus macaque CMV (RhCMV) has recently been developed for cCMV, no vaccine protection studies against cCMV have been evaluated, and the focus in studies with the NHP model has been on horizontal infection, and these studies have had various levels of success (60). In the current study, we evaluated a replication-incompetent virus-based vaccine approach in guinea pigs, and the term DISC virus vaccine (61) was adopted for the type of virus vaccine studied (26). This is the first report of a vaccine strategy in the guinea pig model that includes the PC. Additionally, this is the first CMV vaccine in any model to fully protect against congenital CMV. Although the gB glycoprotein is considered an immunodominant antigen and an important neutralizing target, it is most effective on fibroblast cells (39, 51), and in phase II clinical trials, a gB subunit vaccine had about 50% efficacy (21). Potentially, a failing of the gB vaccine was that the recombinant gB could form only a monomeric gB and not the multimeric triplex gB complex found in the virion (62). The gB expressed in the DISC vaccine strategy has the capacity to be both monomeric and multimeric. Although the anti-gB antibody titer evoked by the DISC vaccine was lower than that in wild-type virus-infected animals, the antibodies generated were of similar neutralizing titers. Indeed, the DISC vaccine anti-gB on both epithelial and fibroblast cells contributed to GPCMV neutralization (Fig. 7b). This would be expected, given the essential nature of gB for GPCMV infection of cells (28). Depletion of the anti-gB response in GPCMV-hyperimmune animals or DISC-vaccinated animals reduced the neutralizing response by approximately 50% when evaluated on fibroblast or epithelial cells, demonstrating the importance of additional viral target antigens. An additional impact of nonneutralizing anti-gB antibodies was recently identified for the HCMV gB vaccine (63). However, this aspect has not been evaluated in the guinea pig animal model.

In MCMV, a spread-deficient M94 mutant virus was safe in innate immune knockout mice and highly protective against challenge with a pathogenic strain of MCMV because of an induced T cell response (CD4⁺ and CD8⁺) as well as effective neutralizing antibodies (64). The DISCII GPCMV study induced both antibody and T cell responses, and overall, these responses likely contributed to protection. However, the previous DISC vaccine (26) does not encode the PC like the present DISCII vaccine does, and the antibody response to the latter complex was likely an important factor for protection against cCMV since the previous DISC vaccine lacking a PC failed to fully protect against cCMV. One concern related to an MCMV replication-defective vaccine was the potential for long-term T cell inflation (65), and this might be a similar problem for any CMV DISC vaccine. A more recent strategy for creation of a replication-defective virus was explored for HCMV, where viral protein expression was made conditional by use of the shield1 ligand to enable propagation of the vaccine strain (46). This HCMV vaccine induced antibody and a T cell response in an NHP model and is currently in phase II clinical trials (46). A significant difference in our DISC vaccine strategy is that we disabled the virus by targeting the ability to assemble viral capsids, a late-stage process. In contrast, the HCMV V160 vaccine encodes a general destabilization of the virus by mutation of multiple genes affecting various stages of virus replication, which potentially alters virus immunogenicity compared to that of the wild type. Importantly, the DISC vaccine retains the ability to produce viral dense bodies, which is separate from capsid assembly (47). Potentially, dense bodies are important for induction of an overall immune response for both the antibody response to PC and other glycoprotein complexes and the T cell response to pp65 (66).

In HCMV, the PC is a potent neutralizing target antigen in convalescent-phase patients and in vaccine studies (41, 67, 68). In this report, we demonstrated that inclusion of the PC dramatically improved the NA₅₀ values on epithelial and trophoblast cells, as well as protection against congenital infection, compared to those achieved with a PC-negative DISC vaccine (26) or in control NOV animals. Surprisingly, the anti-gH/gL neutralizing antibodies appeared to be more important than gB on both fibroblast and epithelial cells, with a greater decrease in neutralization being seen in depleted sera from both vaccinated and hyperimmune animals (Fig. 7). It is likely that anti-gH/gL neutralizing antibodies would target all gH-containing complexes, including the PC (25, 28). Depletion of the anti-gH/gL antibodies in pooled sera from DISCII-vaccinated animals and also from GPCMV (PC⁺ and PC⁻)-hyperimmune sera demonstrated that a specific anti-PC antibody response is mounted to the complex and not just to gH/gL. The anti-PC-specific response contributes to virus neutralization on epithelial and trophoblast cells. Depletion of a full anti-PC response greatly impacted the neutralizing titer in sera from DISCII-vaccinated animals on epithelial and trophoblast cells. Consequently, the antibody response to PC is likely a key component to protection against cCMV in the DISCII vaccine study, especially since trophoblasts in the placenta represent the barrier between the maternal and fetal units and neutralization would prevent infection of these cells. Furthermore, the PC is essential for effective dissemination in the guinea pig model and anti-PC antibodies would reduce virus dissemination. However, other immune responses also likely contributed to the high vaccine efficacy. A more focused PC antigen vaccine study in combination with gB is under way in our laboratory to evaluate efficacy in the guinea pig cCMV model. Notably, a recent study with HCMV suggested that gH trimer- and PC-specific antibodies synergize for virus neutralization but in themselves do not fully protect against congenital infection (69). Undoubtedly, a response against gB remains an important factor, more especially since a novel gH/gL/gB complex was recently identified (70).

In RhCMV, the PC has been demonstrated to be an important virulence factor as well as a neutralizing target antigen (71). In the NHP model, a hyperimmune antibody (hyperimmune globulin [HIG]) preparation from RhCMV-seropositive rhesus macaques demonstrated that both anti-gB and anti-PC antibodies contributed to neutralizing activity (72). A recombinant modified vaccinia virus Ankara (MVA)-based vaccine strategy against the PC in RhCMV failed to protect against natural infection in a horizontal transmission model (71), a finding that likely indicates that a PC-only immune response is insufficient to protect against CMV infection of the host. A PC-based vaccine strategy or any other vaccine against cCMV in the NHP model has not been evaluated. However, a HIG therapy strategy in the NHP applied to pregnant CD4⁺-depleted animals resulted in protection against congenital RhCMV, which implies that neutralizing antibodies are sufficient to protect against cCMV but that protection requires inclusion of potent antibodies to PC, in addition to other target antigens (72). The efficacy of the HIG strategy against cCMV in a human clinical trial did not reach statistical significance (73). Potentially, protection against congenital RhCMV may have requirements different from those for protection against congenital HCMV. It is important to recognize the failure of RhCMV-seropositive status in preventing reinfection with the same virus used as a simian immunodeficiency virus vector (60, 74). In contrast, DISCII vaccination was highly effective in preventing the dissemination of the challenge wild-type GPCMV in vaccinated animals compared to seronegative animals (Fig. 10). Potentially, a current limitation of the guinea pig model is the use of only the 22122 strain of GPCMV, and infection studies with a new strain of GPCMV may provide more limited protection, but this awaits future study.

Unfortunately, previous GPCMV vaccine studies did not perform specific glycoprotein complex ELISAs or neutralization assays with antibody-depleted sera or utilize non-fibroblast cell lines (e.g., guinea pig renal epithelial or trophoblast cells), making it difficult to evaluate previous reports in light of our current research results. A brief summary of some vaccine strategies evaluated in the guinea pig model for cCMV is represented in Table 4. The DISC vaccine was, perhaps unsurprisingly, more successful than previous gB-based vaccine strategies (54, 55, 57, 75) (Table 4). Various live-attenuated GPCMV variants have been explored as vaccines but have not achieved the efficacy of the DISC strategy (56, 58, 59, 76). The most effective live-attenuated GPCMV vaccine was a GP83 knockout mutant, which had a reduction in transmission from 70% to 17% (Table 4) and potentially indicated that an immune response to the pp65 homolog antigen was less important in protecting against cCMV than an immune response to other antigens (76). A cell-mediated response to the GP83 antigen was demonstrated for the DISCII vaccine, but the response was slightly weaker than that in wild-type GPCMV-infected animals. The importance of the immune response to pp65 antigen for protection against cCMV compared to that to other T cell target antigens has not been resolved because specific assays for IE1 and other target antigens remain to be developed. A recombinant defective alphavirus vaccine strategy for GP83 did demonstrate some efficacy against cCMV, with a reduction of cCMV transmission from 85% to 47%. It is worth noting that a strategy with a recombinant lymphocytic choriomeningitis virus (rLCMV) encoding gB and pp65, despite inducing neutralizing antibodies to gB and a T cell response to GP83 (pp65), did not prevent cCMV, with the rate of transmission of GPCMV to pups being 53% (77). This was slightly better than the cCMV transmission rate for a gB-only rLCMV-based vaccine, which was 60% (Table 4), but the impact of the response to GP83 was limited. It is important to note that all of the live GPCMV attenuated vaccine strategies evaluated to date did not encode a PC, and so modification to these strain variants could potentially enhance the efficacy of these candidate vaccine strategies.

TABLE 4.

Comparison of GPCMV vaccine studies and cCMV transmission rates and pup mortality

Type of vaccine (reference)	Congenital transmission rate (%)		Mortality (%)
Type of vaccine (reference)	Vaccine	No vaccine	Vaccine	No vaccine
DISC PC⁻ (26)	23	76	6	36
gB/GSK adjuvant (53)	44	63	10	65
gB DNA vaccine (52)	41	77	34	33
MVA-gB (55)	59	79	11	63
rLCMV-gB (72)	60	83	23	49
Protein kinase R mutant GPCMV (54)	33	65	14	81
GP83 knockout GPCMV (73)	17	70	10	70
GP83 alphavirus (56)	47	85	13	57

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In our previous DISCI (PC⁻) vaccine study, congenital infection occurred in the vaccine group, albeit to a substantially lower level than in the control no-vaccine group (26). In this original study, the anti-GPCMV vaccine ELISA antibody titer was not a successful predictor of protection against cCMV since congenitally infected pups occurred in litters of animals from vaccinated dams with low, medium, or high anti-GPCMV titers (26). In our current study, where the DISCII vaccine encodes the PC, no congenital infection was detected in any of the pups. However, there was variability in the immune response to antibody complexes. Importantly, all dams evoked an immune response to the PC and additionally had high-avidity antibodies to gB. Furthermore, the virus-neutralizing antibody titers on non-fibroblast cells was similar to that in sera from GPCMV (PC⁺)-hyperimmune animals and better than that in sera from GPCMV (PC⁻)-hyperimmune animals. Consequently, our conclusion is that these factors contributed significantly to limiting virus dissemination in the host and the complete protection against cCMV.

The identification by use of the CRISPR gene knockout strategy that guinea pig PDGFRA is the cell receptor for PC-negative GPCMV infection of fibroblast cells and that this protein is not expressed by other guinea pig cell lines that require the PC for entry is an important finding for the translational aspect of the GPCMV studies. Further studies related to the guinea pig PDGFRA and GPCMV gH trimer are part of a more focused analysis which also demonstrates that epidermal growth factor receptor is not the cell receptor (El-Hamdi et al, unpublished). PC-specific receptors await to be identified for GPCMV. Currently, no cell receptor has been specifically identified for other relevant animal CMV models. In the future, the CRISPR/Cas9 gene editing strategy not only will enable the identification of additional potential cell receptors for GPCMV but also will allow an ability to evaluate the cellular innate immune response of guinea pig cells. The innate immune pathways in guinea pig cells are poorly explored not only for GPCMV infection but also for infection with other viral pathogens, such as influenza virus, Ebola virus, or herpes simplex virus, where the guinea pig represents important human disease models.

Overall, inclusion of the PC in the DISCII vaccine design increased the vaccine efficacy in comparison to that of the original PC⁻ DISC strain. The vaccine strategy was successful in fully preventing congenital CMV, which was not prevented in the NOV control group, and was more effective than any previous vaccine strategy evaluated in this model. This is the first report of a GPCMV vaccine to include PC and is also the first report of a vaccine that provides complete protection against cCMV in any animal model. The outcome of this study represents a significant milestone for the development of a vaccine against cCMV in a preclinical model. The increased safety of a non-replication-competent virus vaccine makes this approach very attractive as a basis for a vaccine against congenital HCMV going forward into clinical trials.

MATERIALS AND METHODS

Cells, viruses, and oligonucleotides.

GPCMV (strain 22122; ATCC VR682) and GPCMV BAC (50, 78)-derived viruses were propagated on guinea pig fibroblast lung (GPL) cells (ATCC CCL 158) or tet-off GPL cells (26). Additionally, PC⁺ virus was propagated on renal epithelial and trophoblast cells as previously described (28, 29). Wild-type GPCMV used in congenital CMV animal challenge studies were serially maintained as pooled salivary gland (SG) GPCMV stocks from infected guinea pigs. The salivary gland virus was originally provided by M. R. Schleiss and Cincinnati Children’s Research Foundation (Cincinnati, OH), and subsequently, stocks were serially maintained in guinea pigs by the A. McGregor lab. PC-negative GPCMV was generated by continued passage of virus on fibroblast cells to generate virus in which the GP129 to GP133 locus was deleted (28). High-titer recombinant defective adenovirus (Ad) stocks were generated by Welgen Inc. (MA). HSV-1 was a gift from J. R. Lokensgard (University of Minnesota). All oligonucleotides were synthesized by Sigma-Genosys (The Woodlands, TX). A FLAG epitope-tagged synthetic cDNA of the predicted guinea pig PDGFRA open reading frame (ORF) (GenBank accession number XM_003471663) was generated by GenScript, cloned into mammalian expression vector pcDNA3.1 (pgpPDGFRAflag), and used in anti-PDGFRA antibody verification studies.

CRISPR/Cas9 mutagenesis knockout strategy of guinea pig PDGFRA gene.

The sequence of the guinea pig PDGFRA (gpPDGFRA) gene (NCBI gene accession number 100726209) was based on the current guinea pig NCBI genome sequence (Cavia porcellus annotation release 103 GCF_000151735.1), and predicted introns and exons were additionally identified via use of the genome with Ensembl accession number ENSCPOG00000011782. Exon 2 of gpPDGFRA was targeted for mutagenesis by use of the CRISPR/Cas9 strategy. Exon 2-specific guide RNA (gRNA) was designed via an online program (www.rgenome.net/be-designer/) to avoid off-target sites. DNA sequences were cloned under US6 promoter control in three separate gRNA expression plasmids (pCas-Guide-EF1a-GFP; OriGene): pR1 (5′-GGTGTGGGCCGCCGAGGCGT-3′), pR2 (5′-TCTGGGAGAGTTCCCCGACG-3′), and pR3 (5′-CGTTTCTGATGTCCACGTCG-3′). GPL cells in 6-well plates were transduced with defective lentivirus expressing Cas9 under HCMVIE enhancer control (Origene) to enable expression of Cas9 and subsequently transfected with the gRNA expression plasmids (pR1, pR2, and pR3). At 2 days posttransfection, the cells were reseeded in 96-well plates by limiting dilution to generate individual cell lines. PDGFRA gene knockout cell lines were screened by exon 2 PCR sequencing of extracted genomic DNA. Genomic extraction was carried out with a DNeasy extraction kit (Qiagen), and PCR was performed using primers Fpd (5′-CTGAGCCTAATCTGCTGCCAGCTTTCG-3′) and Rpd (5′-CGGCACGGTAGATGTAGATATGC-3′). Western blotting of total cell lysate for specific modified cell lines verified the loss of gpPDGFRA expression. Goat anti-human PDGFRA antibody (R&D Systems) was predicted to react with gpPDGFRA based on 100% conservation of the target sequence, and this was verified by Western blot analysis of cells transfected with a transient expression plasmid encoding a synthetic full-length gpPDGFRA with a C-terminal FLAG epitope tag (GenScript). The PCR products of the wild-type GPL and PDGFRA mutant knockout cell lines were cloned into the TA cloning vector (Invitrogen) and sequenced as previously described (28). The PDGFRA knockout cell line was designated GPKO. The alignment of the PDGFRA exon 2 DNA sequence for wild-type GPL and GPKO cells is shown in Fig. 1.

Generation of a DISC strain via GPCMV BAC mutagenesis.

The second-generation GPCMV BAC (50) was modified to encode an ectopic copy of GP129 (Myc tag) cDNA under SV40 promoter control in the GP25-GP26 intergenic locus to enable reconstituted virus to express the PC (28). This BAC, designated GP129FRT GPCMV (Fig. 2), retained an additional HindIII site introduced into the GP25-GP26 locus, which resulted in the 17-kb HindIII subgenomic fragment (bases 26294 to 43382) being modified from the unmodified second-generation BAC (Fig. 2b, lane 3) to produce two fragments of approximately 11.5 and 6.6 kb (indicated by squares in Fig. 2b) (28). The GP85 locus was modified in a fashion identical to that described previously (26). The modification removed the GP85 promoter and the intergenic sequence between GP85 and GP86, and these were replaced with an SV40 poly(A) sequence downstream of the GP86 ORF and a kanamycin (Km) resistance cassette for BAC mutant selection in bacteria. Additionally, a tet-off (Tre-tight advanced) promoter (Clontech) was inserted upstream of the GP85 ORF. These elements were contained in the previously described shuttle vector pGP8586TRESV40AKm (26). Modification of the GPCMV BAC was performed using a linearized shuttle vector (26). Insertion of the Km resistance cassette into the viral genome introduced a novel HindIII restriction enzyme site at the HindIII-A subgenomic fragment (bases 102380 to 146446) to result in a truncation in the 44-kb fragment to enable verification of locus modification (Fig. 2). Specific gene modifications were additionally confirmed by comparative PCR analysis between wild-type and mutant GPCMV BACs using common flanking primers for the GP85-GP86 locus and GP25-GP26 locus as described previously (26, 28). The gene knockout mutant was further verified by sequencing of the cloned PCR product.

Generation of mutant GPCMV.

For the generation of recombinant viruses, large-scale GPCMV BAC DNA was purified from the Escherichia coli DH10B strain using a plasmid maxikit (Qiagen). BAC DNA was transfected onto GPL or GPL tet-off cells in six-well dishes using the Lipofectamine 2000 reagent (Invitrogen) as previously described (26). GPCMV BAC transfections were carried out with two independent clones. Transfections were followed for at least 4 weeks for the production of viral plaques. GFP-positive viral plaques were detected via microscopy (26). A non-GFP-tagged version of the DISCII virus was generated by cotransfection of GPCMV BAC DNA with a Cre recombinase expression plasmid to excise the BAC plasmid insertion upon transfection into guinea pig fibroblast cells (GPL tet-off cells) and the generation of virus (28, 50). GFP-tagged virus was designated DISCIIGFP. The non-GFP-tagged virus (from which BAC was excised) was designated DISCII. The vaccine study was carried out with the DISCII virus from which BAC was excised.

Ethics.

Guinea pig (Hartley) animal studies were carried out under IACUC (Texas A&M University) permit 2013#013. All study procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council (79). Animals were observed daily by trained animal care staff, and animals that required care were referred to the attending veterinarian for immediate care or euthanasia. Terminal euthanasia was carried out by lethal CO₂ overdose, followed by cervical dislocation, in accordance with the IACUC protocol and the guidelines of the National Research Council. The animals were purchased from Charles River Laboratories and were verified to be seronegative for GPCMV by toenail clip bleed and anti-GPCMV ELISA of sera, as previously described (25). Animal studies were carried out to determine (i) the antibody immune response to the GPCMV DISC vaccine (DISCII) or GPCMV (PC⁺ or PC⁻) infection (in the case of hyperimmune animals), (ii) the cell-mediated response evoked by the DISCII vaccine, (iii) DISCII viremia, (iv) DISCII vaccine protection against virus dissemination, and (v) the protection against congenital GPCMV evoked by the DISCII vaccination strategy.

Congenital GPCMV DISC vaccine protection studies were carried out as described below in “Congenital GPCMV vaccine protection studies” and in Results. Additionally, animals hyperimmune to wild-type GPCMV (designated PC⁺ virus) or lab-adapted GPCMV (designated PC⁻ virus) were evaluated for the antibody response to GPCMV and specific glycoprotein complexes via ELISA as well as by determination of neutralizing antibody titers. Guinea pigs (n = 5 per group) were made hyperimmune to GPCMV by three injections of GPCMV (either PC⁺ or PC⁻ virus, depending on the assigned group) SQ (10⁵ PFU per injection). Each injection was separated by an interval of 4 weeks. Animals were evaluated after the first and the last injections for the anti-GPCMV titer. Animals were euthanized approximately 4 weeks after the last injection, and serum from all 5 animals in each group was pooled for evaluation of antibody titers (anti-GPCMV, anti-gB, anti-gH/gL, anti-gM/gN, and anti-PC). The DISCII vaccine cell-mediated response was evaluated by guinea pig-specific IFN-γ ELISPOT assay of DISCII-inoculated animals (n = 3) or wt GPCMV-infected animals (n = 3). Each animal received 3 injections SQ (10³ PFU for DISCII, 10⁵ PFU for wt GPCMV) 4 weeks apart, and splenocytes were harvested 7 to 10 days after the last injection (see “Guinea pig IFN-γ ELISPOT assay” below). DISCII viremia-seronegative animals (n = 3 per group) were divided into two groups: group 1 received 10³ PFU of DISCII SQ at day 0, and group 2 received 10³ PFU of wt GPCMV SQ. At days 4, 8, 12, and 27 dpi, the animals were bled by toenail clip to obtain blood for evaluation of GPCMV viremia by real-time PCR assay. For determination of DISCII vaccine protection against virus dissemination, seronegative animals were divided into two groups (n = 12 per group). Group 1 animals were vaccinated with DISCII following the vaccination regime described above, and the anti-GPCMV antibody titer was determined 4 weeks after the last vaccination. Subsequently, both groups were challenged with wt GPCMV (10⁵ PFU SQ). At days 4, 8, 12, and 27 dpi, 3 animals per group were euthanized to evaluate the viral load in the target organs and blood via DNA extraction from tissue and blood and real-time PCR assay (see “Real-time PCR for DISCII congenital infection study” below).

Congenital GPCMV vaccine protection studies.

Seronegative female guinea pigs were randomly assigned to two different groups. Group 1 (the DISCII-vaccinated group; n = 19) were vaccinated SQ with the GPCMV DISCII vaccine (10³ PFU) and boosted twice at day 26 and day 49 (10³ PFU). The animals were confirmed to have seroconverted for GPCMV (determined by an anti-GPCMV ELISA). The immune response to individual glycoprotein complexes was also determined for vaccinated animals prior to mating. Next, the dams were paired with seronegative males for mating. The dams were confirmed to be pregnant by palpation at approximately days 20 to 25 of gestation. A second control group of nonvaccinated (NOV) seronegative females (n = 15) was also paired for mating. At the late second trimester/early third trimester, pregnant animals in both groups were challenged with a salivary gland stock of wild-type GPCMV (10⁵ PFU) SQ, and the animals were allowed to go to term. The viral load in the target organs (liver, lung, spleen, brain) of liveborn or stillborn pups was evaluated by real-time PCR.

Real-time PCR for DISCII congenital infection study.

Tissues were collected from euthanized or stillborn guinea pigs to determine the viral load. For pups from the congenital infection studies, tissues (lung, liver, spleen, brain) were collected within 3 days postbirth. For viral load determination, DNA from tissue homogenates was extracted and the viral load was determined by real-time PCR on a LightCycler 480 instrument (Roche Life Science) using the primers and probe previously described (26, 28). Standard controls and no-template controls (NTC) were run with each assay for quantification. The data were analyzed with LightCycler data analysis software (version 1.5.1; Roche). Each sample was run a minimum of two times in triplicate. The viral load was expressed as the copy number per milligram of tissue. The results calculated were the mean value from triplicate PCR runs per sample.

ELISAs, antibody avidity, GPCMV neutralization, and Western blot analysis. (i) ELISAs.

An anti-GPCMV ELISA and glycoprotein complex-specific (anti-gB, anti-gH/gL, anti-gM/gN, and anti-PC) ELISAs were carried out as previously described using a positive coating antigen derived from cell monolayers transduced with recombinant replication-defective adenovirus (Ad) vectors expressing specific glycoproteins or, in the case of gM/gN, by plasmid transfection (25, 26). Negative coating antigen was derived from cells transduced with a recombinant Ad vector expressing GFP or transfected with an empty plasmid (pcDNA3) for the gM/gN control (25, 26). All ELISAs were run a minimum of three times in duplicate. ELISA reactivity was considered positive if the net optical density (OD) was greater than or equal to 0.2, as determined with GPCMV-negative serum. Assays were carried out concurrently between different groups. The described approach is based on similar strategies for glycoprotein complex expression for HCMV and RhCMV (80, 81).

(ii) Antibody avidity.

IgG avidity was determined following a previously described ELISA protocol with the addition of 6 M urea as the dissociating agent (82). After incubation with diluted test sera, one set of wells was washed with regular wash buffer, whereas the other sets of wells were washed with wash buffer containing urea. The ELISA was completed as described above. The results were expressed as an avidity index (AI; in percent), which was calculated as AI = (OD of urea-washed well/OD of regular well) × 100.

(iii) Serum neutralization.

GPCMV neutralization assays (for determination of the NA₅₀) were performed on GPL fibroblast, renal epithelial, and trophoblast cells with PC⁺ GPCMV stocks generated on a matching cell line as previously described (25). Neutralization assays were carried out with pooled sera from a specific group. Sera were either complete (native) or depleted for antibodies to specific viral glycoprotein complexes. The final neutralizing antibody titer was the inverse of the highest dilution producing a 50% or greater reduction in plaques compared to that for the virus-only control. Neutralization assays were performed from each sample three times. Neutralization assays were performed concurrently with the same virus stocks between groups.

(iv) Western blot analysis.

Western blot analyses were carried out on cell lysates separated on 4 to 20% SDS-PAGE gradient gels (Bio-Rad) following a previously described protocol (25). Epitope (Myc)-tagged GP129 was detected with a primary mouse anti-Myc epitope tag monoclonal antibody (Novus) (28, 29). Western blot analyses for control β-actin protein lane loading were carried out with primary antibody mouse anti-β-actin (Cell Signaling). Goat anti-human PDGFRA (R&D Systems) was used to detect guinea pig PDGFRA. Antibody cross-reactivity to guinea pig PDGFRA was predicted based on a 100% match in sequence homology. Antibody reactivity was validated with transient expression studies with a synthetic guinea pig PDGFRA gene (GenScript) with a C-terminal FLAG epitope tag to enable the codetection of FLAG and PDGFRA in parallel Western blot analyses using anti-FLAG M2 (Sigma) (25). The secondary antibodies used for Western blot analysis were anti-mouse IgG-horseradish peroxidase (HRP), anti-goat IgG-HRP, and anti-guinea pig IgG-HRP from Sigma (25).

Antibody depletion from sera.

Immunodepletion of antibodies to specific glycoprotein complexes (gB, gH/gL, PC) was carried out as previously described (25). Briefly, guinea pig cells were transduced with recombinant defective adenovirus expressing specific complexes for depletion: (i) gB, (ii) gH and gL, or (iii) gH, gL, GP129, GP131, and GP133 (multiplicity of infection [MOI] = 20 transducing units/virus/cell). At 48 to 72 hpi, the cells were harvested, washed twice with phosphate-buffered saline (PBS), and then fixed with a 1:1 ratio of an acetone-methanol fixation mixture for 20 min at −20°C. Fixed cells were pelleted and then resuspended in 500 μl of PBS with 0.1% Tween 20. An equal volume of serum was used for depletion overnight at 4°C in a tube rotator. The cells were centrifuged at 10,000 × g for 20 min at 4°C to pellet the cells, and the serum was collected and then stored at −80°C until needed. Glycoprotein-depleted sera were used for ELISAs and neutralization assays as described above in “ELISAs, antibody avidity, GPCMV neutralization, and Western blot analysis.” The starting serum dilution of 1:80 was adjusted for the 1:2 dilution during the immunodepletion step. Therefore, to reach the starting dilution of 1:80, the depleted serum starting dilution was 1:40.

Guinea pig IFN-γ ELISPOT assay.

The anti-guinea pig interferon gamma (IFN-γ) monoclonal antibodies used in the assay were based on previously characterized monoclonal antibodies against guinea pig IFN-γ (83). The guinea pig IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assays were performed following a previously described protocol using freshly isolated splenocytes and GPCMV GP83 peptide pools (26). Final counts were determined using an ImmunoSpot S6 microanalyzer (CTL) and calculated on the basis of the number of spot-forming cells (SFC) per 10⁶ cells after the values for background spots were subtracted. IFN-γ ELISPOT assays were performed in polyvinylidene difluoride membrane 96-well plates coated with 0.5 μg guinea pig IFN-γ capture antibody (V-E4) and incubated overnight at 4°C. The membranes were washed and then blocked for 2 h at room temperature, before 1 × 10⁵ isolated splenocytes were added. GPCMV GP83 peptide pools were added to each well of cells at a final concentration of 5 μg/ml. Concanavalin A (ConA; 10 μg/ml) was used as a positive control, and other controls included a cell-only control, a dimethyl sulfoxide (DMSO) control (peptide background), and a GFP-only (nonspecific peptide control) and medium-only control. The plates were covered with foil and then incubated at 37°C in a 5% CO₂ cell culture incubator for 18 h. The membranes were washed before N-G3 biotinylated detection antibody was added and then incubated at room temperature for 2 h. Detection antibody was diluted to 1 μg/ml with diluent and filtered through a 0.2-μm-pore-size filter before use. The membranes were washed before streptavidin-alkaline phosphatase (R&D Systems) diluted 1:3,200 in diluent was added and then incubated for 1.5 h at room temperature. For detection, 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (Life Technologies) was added to the membranes before incubation for 30 min at room temperature with protection from light. The membranes were washed and dried before counting the spots on an ImmunoSpot S6 microanalyzer (CTL). Final counts were calculated based on the number of spot-forming cells (SFC) per 10⁶ cells after the counts for the background spots (cells only without any stimulation) were subtracted. As previously described (26), a total of 140 PEPscreen 9-amino-acid peptides overlapping by 5 amino acids were generated (Sigma-Aldrich, The Woodlands, TX) to create the GPCMV GP83 peptide library expanding the full-length gene. Nine amino acid peptides were used to target CD8⁺ T cell activation (67). Peptide pools were generated using a configuration matrix similar to that described previously (67, 68). The GP83 matrix consisted of 24 peptide pools in a 12-by-12 grid, with each pool containing 12 peptides. Pools IX, X, XI, and XII contained 11 peptides, and pool XXIV had 8 peptides. Additional DMSO was added to keep the concentration the same in all pools. All peptide pools were diluted to a 10-μg/ml working stock concentration in RPMI for stimulation. The matrix was designed for each peptide to be included in exactly 2 pools, keeping the number of pools at a minimum. The intersection of positive pools corresponds to the stimulating peptides. Peptide pools X and XX were the most reactive, while peptide pools II and IV (Fig. 9) did not respond to a level above the background. The sequences of pooled peptides with positive and negative responses are as previously described (26).

Statistical analysis.

All statistical analyses were conducted with GraphPad Prism (version 7) software. Replicate means were analyzed using one-way analysis of variance, Student’s t test (unpaired), or Fisher’s exact test, with significance taken as a P value of <0.05 or as specified in the figure legends.

ACKNOWLEDGMENTS

We thank Jim Choi and Darijana Horvat for their excellent technical assistance. We are grateful to M. A. McVoy (Virginia Commonwealth University) for supplying the second-generation GPCMV BAC. We are also grateful to M. R. Schleiss (University of Minnesota) for supplying the original GPCMV salivary gland seed stock. Hybridoma cell lines for the anti-IFN-γ monoclonal antibodies were a generous gift from H. Schäfer (Robert Koch Institute, Germany). We thank D. N. McMurray (Texas A&M University) for his constructive comments on the manuscript.

Research was supported by grants from the NIH (NIAID and NICHD) and the Texas A&M University HSC (Clinical Science & Translational Research Award). The funders did not participate in the design or analysis of research experiments.

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PERMALINK

Inclusion of the Viral Pentamer Complex in a Vaccine Design Greatly Improves Protection against Congenital Cytomegalovirus in the Guinea Pig Model

K Yeon Choi

Nadia S El-Hamdi

Alistair McGregor

Roles

ABSTRACT

INTRODUCTION

RESULTS

PDGFRA is the receptor for PC-negative GPCMV infection of fibroblast cells.

FIG 1.

Generation of a PC+ DISC (DISCII) virus strain.

FIG 2.

Vaccine-induced antibody immune response to glycoprotein complexes.

FIG 3.

Anti-gB, anti-gH/gL, or anti-PC antibody depletion and impact on virus neutralization.

FIG 4.

FIG 5.

FIG 6.

FIG 7.

Virus neutralization in sera from DISCI (PC−)- versus DISCII (PC+)-vaccinated animals for various cell types.

FIG 8.

DISCII vaccine-induced cell-mediated immune response to the pp65 homolog protein GP83.

FIG 9.

Wild-type virus dissemination in DISCII-vaccinated versus seronegative animals.

FIG 10.

Congenital GPCMV protection study.

TABLE 1.

FIG 11.

TABLE 2.

TABLE 3.

DISCUSSION

TABLE 4.

MATERIALS AND METHODS

Cells, viruses, and oligonucleotides.

CRISPR/Cas9 mutagenesis knockout strategy of guinea pig PDGFRA gene.

Generation of a DISC strain via GPCMV BAC mutagenesis.

Generation of mutant GPCMV.

Ethics.

Congenital GPCMV vaccine protection studies.

Real-time PCR for DISCII congenital infection study.

ELISAs, antibody avidity, GPCMV neutralization, and Western blot analysis. (i) ELISAs.

(ii) Antibody avidity.

(iii) Serum neutralization.

(iv) Western blot analysis.

Antibody depletion from sera.

Guinea pig IFN-γ ELISPOT assay.

Statistical analysis.

ACKNOWLEDGMENTS

REFERENCES

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Generation of a PC⁺ DISC (DISCII) virus strain.

Virus neutralization in sera from DISCI (PC⁻)- versus DISCII (PC⁺)-vaccinated animals for various cell types.