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, 87 (20), 11107-20

Cytomegalovirus Vaccine Strain Towne-Derived Dense Bodies Induce Broad Cellular Immune Responses and Neutralizing Antibodies That Prevent Infection of Fibroblasts and Epithelial Cells

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Cytomegalovirus Vaccine Strain Towne-Derived Dense Bodies Induce Broad Cellular Immune Responses and Neutralizing Antibodies That Prevent Infection of Fibroblasts and Epithelial Cells

Corinne Cayatte et al. J Virol.

Abstract

Human cytomegalovirus (HCMV), a betaherpesvirus, can cause severe disease in immunosuppressed patients and following congenital infection. A vaccine that induces both humoral and cellular immunity may be required to prevent congenital infection. Dense bodies (DBs) are complex, noninfectious particles produced by HCMV-infected cells and may represent a vaccine option. As knowledge of the antigenicity and immunogenicity of DB is incomplete, we explored characterization methods and defined DB production methods, followed by systematic evaluation of neutralization and cell-mediated immune responses to the DB material in BALB/c mice. DBs purified from Towne-infected cultures treated with the viral terminase inhibitor 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) were characterized by nanoparticle tracking analysis (NTA), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), immunoblotting, quantitative enzyme-linked immunosorbent assay, and other methods. The humoral and cellular immune responses to DBs were compared to the immunogenicity of glycoprotein B (gB) administered with the adjuvant AddaVax (gB/AddaVax). DBs induced neutralizing antibodies that prevented viral infection of cultured fibroblasts and epithelial cells and robust cell-mediated immune responses to multiple viral proteins, including pp65, gB, and UL48. In contrast, gB/AddaVax failed to induce neutralizing antibodies that prevented infection of epithelial cells, highlighting a critical difference in the humoral responses induced by these vaccine candidates. Our data advance the potential for the DB vaccine approach, demonstrate important immunogenicity properties, and strongly support the further evaluation of DBs as a CMV vaccine candidate.

Figures

Fig 1
Fig 1
Fractions and particle sizes from glycerol-tartrate gradients monitored by NTA. (A) Image of a representative gradient, with brackets on the right indicating the relative positions of fractions 1 to 4 recovered for evaluation of particle size, as determined by NTA. Indicated on the left are the relative positions of DB and virion fractions selected for imaging and composition analyses. (B) Composite image of NTA results from five independent particle size and number evaluations for each of the fractions 1 to 4 recovered from glycerol-tartrate gradients. The particle counts graphed correspond to the arithmetic mean values calculated from five repeated measurements.
Fig 2
Fig 2
DB and virion preparations for immunoblot and 2D-DIGE analyses of composition. (A) Composite image of NTA results produced from five independent assessments of virion and DB fractions recovered from glycerol-tartrate gradients, as indicated in the legend to Fig. 1A. Graphed particle counts were determined as indicated in the legend to Fig. 1B. (B and C) Representative TEM images acquired from the virion (B) or DB (C) fractions recovered from glycerol-tartrate gradients, as indicated in the legend to Fig. 1A.
Fig 3
Fig 3
DB fractions evaluated with specific monoclonal antibodies or Cytogam. (A) Image of silver-stained DBs or BSA separated by denaturing gel electrophoresis, with the arrowhead indicating the expected migration of pp65 in DBs. (B) Immunoblot analysis of DBs reacted with pp65-specific antibodies. Arrowhead, expected migration of pp65; MM, molecular mass marker. (C) Immunoblot analysis of DBs reacted with gB-specific antibodies. Arrow, expected migration of the mature 55-kDa gB. (D) 2D immunoblot analysis of DBs reacted with pp65 antibodies. (E) 2D immunoblot analysis of DBs reacted with gB antibodies. (F) 2D gel and immunoblot analysis of DBs reacted with Cytogam. Regions of the 2D gel selected for mass spectrometry are indicated by rectangles 1 to 6. Viral peptides identified within the region marked by rectangles 1 to 6: 1, gB (UL55); 2, tegument protein pp71 (UL82); 3, UL51; 4, pp65 (UL83); 5, tegument protein (UL88); 6, myristylated tegument protein (UL99). (G) 2D gel and immunoblot analysis of virions reacted with Cytogam. Rectangles indicating gel regions selected for mass spectrometry and identification are as described in the legend to panel F.
Fig 4
Fig 4
2D-DIGE of DB and virion fractions recovered from glycerol-tartrate gradients. (A) Image from 2D-DIGE analysis of virion lysates labeled with Cy3 (green) in comparison to DB lysates labeled with Cy5 (red). (B) PCA of purification and DIGE replicates to evaluate the consistency in differences between virion (light and dark green) and DB (pink and red) fractions. Virion-1 and DB-1, purification replicate for preparation 1; Virion-2 and DB-2 purification replicate for preparation 2. 2D-DIGE replicates are indicated by symbol color changes (pink versus red and light green versus dark green). (C) Abundant and differentially abundant proteins identified by 2D-DIGE and selected for identification by mass spectrometry. DB/virion fold change differences determined by 2D-DIGE are indicated by the color of the rectangles placed over the gel regions selected for mass spectrometry. For fold changes of <1, the negative of the inverse is reported. Red rectangles, fold change of >2; green rectangles fold change of <−2; gray rectangles, fold change with an absolute value of <2.
Fig 5
Fig 5
DBs induce high-titer broadly protective complement-independent neutralizing antibodies. (A) gB-specific total IgG titers induced by gB/Addavax following three inoculations in BALB/c mice. AU, absorbance units. (B and C) Comparisons of neutralizing antibody (NAb) titers induced following vaccination with three different DB preparations (DB-3, DB-4, and DB-5) in two different animal experiments (closed or open symbols). Neutralizing antibody titers (IC50s) in mouse serum collected after three inoculations were determined in vitro from neutralization of Toledo-GFP infection of MRC-5 fibroblast cells. Neutralization assays whose results are reported in panels B and C were completed at the same time. For panel B, heat-inactivated serum samples were evaluated without addition of complement. *, P < 0.05. For DB-3, DB-4, and DB-5 in comparison to PBS, all P values were <0.0001, except for DB-2 experiment 2 (P = 0.0003). For panel C, 1% guinea pig complement was added to the heat-inactivated serum samples during the neutralization assay. For experiments 1 and 2, P values in comparison to the results obtained with PBS were <0.0009 for gB/AddaVax, DB-3, DB-4, and DB-5. ND, not determined.
Fig 6
Fig 6
DBs induce complement-independent neutralizing antibody titers that prevent infection of ARPE-19 epithelial cells. Neutralizing antibody titers (IC50s) in mouse sera collected after three inoculations were determined by in vitro neutralization of VR1814 infection. Neutralization assays whose results are reported in panels A and B were completed at the same time. (A) Neutralizing antibody titers were determined from infections of MRC-5 fibroblast cells following incubation of heat-inactivated mouse serum and VR1814 virus in the presence or absence of 1% guinea pig complement, as indicated. *, P < 0.05; **, P < 0.01. P values were <0.0001 for DB-3 and DB-4 in comparison to PBS in the presence or absence of added complement. (B) Neutralizing antibody titers were determined from infections of ARPE-19 epithelial cells, as reported for panel A. ***, P < 0.0005.
Fig 7
Fig 7
DBs induce lower gB-specific total IgG and IgG1 than gB/AddaVax. ELISA titers of total IgG (A and B) or IgG1 (C and D) and IgG2a (E and F) isotypes in mouse sera obtained following inoculations with gB/AddaVax or DBs are shown. The protein used to determine binding by ELISA was gB for the titers reported in panels A, C, and E and denatured gB for the titers reported in panels B, D, and F.
Fig 8
Fig 8
DB-induced gB-specific antibodies contribute to neutralization of VR1814 infection of epithelial cells. (A) Total IgG ELISA titer results before and after depletion of pooled sera from mice immunized with DBs with gB or denatured gB from Cytogam. The gB or denatured gB protein used to deplete sera was subsequently used to determine binding in ELISA, as indicated in the depletion and detection lines beneath the graph. (B) Neutralizing antibody titers (IC50s) determined by Towne infection of MRC-5 fibroblasts following depletion of gB-binding or denatured gB-binding antibodies and graphed as the percentage of the neutralization titer in nondepleted sera. (C) Neutralizing antibody titers determined from neutralization of VR1814 virus infection of MRC-5 fibroblast cells. Sera were depleted with gB or denatured gB, and results are graphed as reported for panel B. (D) Neutralizing antibody titers determined from neutralization of VR1814 virus infection of ARPE-19 epithelial cells. Sera were depleted with gB or denatured gB, and results are graphed as reported for panel B.
Fig 9
Fig 9
DBs induce a broad T cell response. IFN-γ ELISPOT assay results from splenocytes of mice analyzed after immunization with DB (DB-4) or gB/AddaVax, as indicated. (A) Responses to a pp65-specific peptide pool and to a putative immunodominant MHC class I H2-Dd pp65 T cell epitope. Responses were determined following three immunizations and are graphed as the number of SFC per 106 splenocytes. ***, P ≤0.0006. (B) Responses to a gB-specific peptide pool determined and graphed as reported for panel A. Solid symbols, results from experiment 1; open symbols, results from experiment 2. **, P = 0.004; ***, P = 0.0002; ****, P < 0.0001. (C) Responses determined in triplicate by IFN-γ ELISPOT assay with peptides spanning 17 HCMV ORFs. Black bars, proteins expected to be components of DBs (44); white bars, proteins not reported as DB components. (D) IFN-γ responses to IE1-, pp65-, or gB-specific peptide pools in splenocytes recovered from mice following two immunizations with UV-treated DBs or PBS. Responses were determined and results are graphed as reported for panel A. ***, P = 0.0004; ****, P < 0.0001; ns, not significant.

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