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. 2017 Jan 5;24(1):e00300-16.
doi: 10.1128/CVI.00300-16. Print 2017 Jan.

Additive Protection Against Congenital Cytomegalovirus Conferred by Combined Glycoprotein B/pp65 Vaccination Using a Lymphocytic Choriomeningitis Virus Vector

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Additive Protection Against Congenital Cytomegalovirus Conferred by Combined Glycoprotein B/pp65 Vaccination Using a Lymphocytic Choriomeningitis Virus Vector

Mark R Schleiss et al. Clin Vaccine Immunol. .
Free PMC article

Abstract

Subunit vaccines for prevention of congenital cytomegalovirus (CMV) infection based on glycoprotein B (gB) and pp65 are in clinical trials, but it is unclear whether simultaneous vaccination with both antigens enhances protection. We undertook evaluation of a novel bivalent vaccine based on nonreplicating lymphocytic choriomeningitis virus (rLCMV) vectors expressing a cytoplasmic tail-deleted gB [gB(dCt)] and full-length pp65 from human CMV in mice. Immunization with the gB(dCt) vector alone elicited a comparable gB-binding antibody response and a superior neutralizing response to that elicited by adjuvanted subunit gB. Immunization with the pp65 vector alone elicited robust T cell responses. Comparable immunogenicity of the combined gB(dCt) and pp65 vectors with the individual monovalent formulations was demonstrated. To demonstrate proof of principle for a bivalent rLCMV-based HCMV vaccine, the congenital guinea pig cytomegalovirus (GPCMV) infection model was used to compare rLCMV vectors encoding homologs of pp65 (GP83) and gB(dCt), alone and in combination versus Freund's adjuvanted recombinant gB. Both vectors elicited significant immune responses, and no loss of gB immunogenicity was noted with the bivalent formulation. Combined vaccination with rLCMV-vectored GPCMV gB(dCt) and pp65 (GP83) conferred better protection against maternal viremia than subunit or either monovalent rLCMV vaccine. The bivalent vaccine also was significantly more effective in reducing pup mortality than the monovalent vaccines. In summary, bivalent vaccines with rLCMV vectors expressing gB and pp65 elicited potent humoral and cellular responses and conferred protection in the GPCMV model. Further clinical trials of LCMV-vectored HCMV vaccines are warranted.

Keywords: congenital cytomegalovirus; cytomegalovirus; fetal infection; live vector vaccines; lymphocytic choriomeningitis virus; placental immunology.

Figures

FIG 1
FIG 1
Schematic drawing of rLCMV vector design and production. (A) LCMV vector particle and encapsidated genomic segments (GP, surface glycoprotein; NP, nucleoprotein; Z, ring finger protein; L, polymerase) and genetic organization of the two encapsidated genomic segments (S, short; L, long). (B) Representation of the full-length HCMV gB open reading frame HgB(FL), the truncated isoform HgB(dCt), and the corresponding design for guinea pig CMV gB [GPgB(dCt)] expressed in vaccine vectors. Numbers represent amino acid positions, and shaded boxes represent the proposed transmembrane region of the protein. Position 773 in HgB(dCt) is an extraneous arginine residue. (C) Schematic drawing of rLCMV vaccine vector rescue, stock production, and the single-round infectious character of rLCMV-vectored vaccination.
FIG 2
FIG 2
rLCMV vector characterization. (A) Growth kinetics of rLCMV vectors and LCMV wild-type virus in the production cell line 293-GP. Titers represent means of 2 independent experiments, with standard deviations shown as error bars. (B) rLCMV-gB(dCt) was selected as the representative vector to demonstrate deficiency in formation of infectious progeny in cells that do not provide LCMV GP protein in trans (293F). All cell types were infected with 0.001 FFU/cell. Samples for individual time points were analyzed by means of a FFU assay based on LCMV GP-complementing 293T cells. Dotted lines indicate detection limits of the FFU assay. (C) gB(dCt) and pp65 vaccine antigen expression levels were analyzed by Western blotting. HCMV gB and HCMV pp65-specific MAbs were used to detect vaccine antigen expression, and a polyclonal anti-LCMV serum reactive with LCMV NP was used for the infection/loading control.
FIG 3
FIG 3
Immunogenicity analyses of rLCMV-gB and pp65 vectors. Error bars represent 95% confidence intervals for serological assays and standard deviations for T cell analyses. (A) Immunogenicity in C57BL/6 mice. A total of 1 × 105 FFU per dose of rLCMV or 5 μg per dose of adjuvanted recombinant gB were administered i.m. on days 0 and 21. (Left) Antibody induction and persistence were measured by ELISA. (Right) Levels of neutralizing antibodies in sera collected on day 42 after two administrations of rLCMV-gB(dCT) or adjuvanted recombinant gB, in comparison to 5 human sera from CMV-infected subjects which covered a typical range of titers for naturally induced humoral immunity. (B) Immunogenicity in New Zealand White rabbits; 1 × 107 FFU, 1 × 105 FFU, or 1 × 103 FFU of rLCMV-gB(dCt) was administered i.m. on days 0, 28, and 56. (Left) Antibody induction and persistence measured by ELISA. (Right) Levels of neutralizing antibodies in sera collected on day 69 after three administrations of rLCMV-gB(dCt). (C) T cell responses induced by pp65 vector in C57BL/6 mice; a dose of 1 × 105 or 1 × 103 FFU was administered i.m. on days 0 and 28. T cell analysis of splenocytes was performed at day 10 (d10 postprime) and day 38 (day 10 postboost) by ICS. Frequencies of pp65-specific CD8 T cells expressing at least one of the cytokines IFN-γ, TNF-α, or IL-2 are shown. (D) Immunogenicity of monovalent and bivalent rLCMV formulations in C57BL/6 mice; 1 × 105 FFU of rLCMV vector were administered i.m. on days 0 and 28; gB-specific antibody induction and pp65-specific CD8 T cell responses were measured at day 49 by ELISA and ICS.
FIG 4
FIG 4
Immunogenicity analyses of rLCMV GPCMV vectors in guinea pigs. Error bars represent 95% confidence intervals. A total of 8 × 105 FFU per dose of each rLCMV vector were administered i.m., or 50 μg per dose of adjuvanted recombinant GPgB was administered s.c. on days 0, 30, and 60. Animals were challenged with 1 × 105 PFU GPCMV after day 155. (A) Antibody induction measured by ELISA. (B) Neutralizing titers in sera at day 103. The titers in individual sera are shown with geometric means and 95% CI indicated. Sera without neutralizing effects at the lowest dilution were assigned a titer of half the limit of detection (LOD). Hyperimmune serum was derived from an animal immunized with adjuvanted recombinant gB protein (50 μg in IFA) and subsequently infected with GPCMV. Statistical comparisons were made by ANOVA on log-transformed values.
FIG 5
FIG 5
Reduction of maternal viral load following rLCMV-vectored vaccination. (A) Mean (± the standard error of the mean) maternal viral loads were analyzed by qPCR in vaccine and control groups at 7 days following SG-GPCMV challenge. All vaccine groups had significantly lower viral loads at day 7 compared to the rLCMV-GFP control (*, P < 0.05). In addition, maternal viral load at day 7 was significantly reduced by the combination of rLCMV-vectored gB(dCt) and GPpp65, compared with rLCMV gB(dCt) vaccine administered alone (**, P = 0.0386). (B) Immunization with both gB and pp65 (GP83) conferred significantly improved protection than either antigen administered alone. Combined analysis of single-antigen vaccine groups rLCMV-GPgB(dCt) and rLCMV-GPpp65 identified a mean day 7 magnitude of DNAemia of (1.65 ± 0.3) × 106 copies/ml, compared to (5.8 ± 3) × 105 copies/ml in the bivalent rLCMV-GPgB(dCt)/GPpp65 group (P = 0.03, t test).
FIG 6
FIG 6
Improved pup weights and reduced pup mortality conferred by preconception vaccination. Pup weights were significantly improved in all vaccine groups compared to the rLCMV-GFP control group (P < 0.0001, Kruskal-Wallis and Dunn's multiple-comparison tests). Open circles, live-born pups; closed circles, stillborn pups.

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