A live guinea pig cytomegalovirus vaccine deleted of three putative immune evasion genes is highly attenuated but remains immunogenic in a vaccine/challenge model of congenital cytomegalovirus infection

Abstract

Live attenuated vaccines for prevention of congenital cytomegalovirus infections encode numerous immune evasion genes. Their removal could potentially improve vaccine safety and efficacy. To test this hypothesis, three genes encoding MHC class I homologs (presumed NK evasins) were deleted from the guinea pig cytomegalovirus genome and the resulting virus, 3DX, was evaluated as a live attenuated vaccine in the guinea pig congenital infection model. 3DX was attenuated in vivo but not in vitro. Vaccination with 3DX produced elevated cytokine levels and higher antibody titers than wild type (WT) virus while avidity and neutralizing titers were similar. Protection, assessed by maternal viral loads and pup mortality following pathogenic viral challenge during pregnancy, was comparable between 3DX and WT and significant compared to naïve animals. These results suggest that the safety and perhaps efficacy of live attenuated human cytomegalovirus vaccines could be enhanced by deletion of viral immunomodulatory genes.

Fig. 1

(a) Hind III map of the GPCMV genome (top) with the Hind III L - M region enlarged below to show the locations of ORFs encoding MHC I homologs gp147, gp148 and gp149 in the WT genome and their replacement with kan^r/lacZ in virus 3DX. Thick bars indicate sequences used as hybridization probes. (b) Southern hybridizations of Hind III- and EcoR I-restricted BAC DNAs hybridized with kan^r/lacZ or gp148 probes. Positions of DNA size markers are shown on the left. (c) Hind III- and Nhe I-restricted WT and 3DX virion DNAs separated on 0.6% agarose and visualized with ethidium bromide and UV light. Arrows indicate diagnostic fragments; sizes of DNA markers are shown on the right.

Fig. 2

Structure-based alignment of gp147 and gp149 with Cavia porcellus MHC I predicting α1, α2, and α3 domains in the GPCMV MHC I homologs. ClustalW alignments of gp147 (ACJ35890) and gp149 (ACJ35892) with Cavia porcellus MHC I (gp MHC I, Ensemble protein # ENSCPOP00000002441). Secondary structure of (a) gp147 and (b) gp149 was predicted using the Jpred online server. Predicted β-strands are depicted by arrows and α-helices are depicted by solid line. The conserved cysteines are bold and underlined, conserved tyrosines are marked with an asterisk, and potential N-linked glycosylation sites are underlined. The single N-linked glycosylation site in gp MHC I is underlined.

Fig. 3

ORFS encoding gp147, gp148 and gp149 do not appear to be involved in MHC-I down-regulation. GPL cells were infected with WT-BAC (a) or 3DX (b) at an MOI of 0.1 for 72 h. Control in panel (c), mouse IgG isotype control for B640 antibody. Cells were analyzed by FACSCAN for surface expression of MHC-I (PE) and GFP. No impact of deletion of the gp147-149 region is noted on MHC-1 down-regulation.

Fig. 4

Sequences encoding gp148, gp147 and gp149 are not required for efficient viral growth in vitro. Cells were infected with WT (25CF) or 3DX (25EF) at an MOI of 0.01. Viral titers in the culture supernatants were determined on the days indicated post infection. Results represent the means of triplicate titers and error bars indicate ± one standard deviation.

Fig. 5

Genes encoding gp147, gp148 and gp149 are important for DNAemia. Seronegative outbred Hartley strain female guinea pigs were inoculated IP and SC with 1×10⁸ total pfus of WT or 3DX. Blood viral loads were measured by real-time PCR and DNAemia expressed as mean genome copies per ml. Data points are means of data from duplicate blood samples drawn from 7 WT-infected animals or 8 3DX-infected animals. Error bars indicate ± one standard deviation. DL, detection limit.

Fig. 6

Deletion of gp147, gp148 and gp149 alters IFN-γ and IL-12 responses. Levels of IFN-γ, IL-12, and GAPDH mRNA in whole blood from 3DX- and WT- inoculated animals were measured by RT-PCR. Relative units were calculated as ratios of IFN-γ (a) or IL-12 (b) sum intensities to GAPDH sum intensities. Data shown represent means ± SEM (standard error of the mean) (uninfected, n=5; WT, n = 7; 3DX, n=8). *p < 0.05 or **p < 0.005, Mann-Whitney U test.

Fig. 7

Anti-GPCMV IgG ELISA titers (a), avidity (b), or neutralizing titers (c) were measured in sera obtained from 3DX- and WT-inoculated animals on the indicated days post inoculation. **p<0.02, Mann-Whitney U test.