Production of Cytomegalovirus Dense Bodies by Scalable Bioprocess Methods Maintains Immunogenicity and Improves Neutralizing Antibody Titers

Abstract

With the goal of developing a virus-like particle-based vaccine based on dense bodies (DB) produced by human cytomegalovirus (HCMV) infections, we evaluated scalable culture, isolation, and inactivation methods and applied technically advanced assays to determine the relative purity, composition, and immunogenicity of DB particles. Our results increase our understanding of the benefits and disadvantages of methods to recover immunogenic DB and inactivate contaminating viral particles. Our results indicate that (i) HCMV strain Towne replicates in MRC-5 fibroblasts grown on microcarriers, (ii) DB particles recovered from 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB)-treated cultures and purified by tangential flow filtration (TFF-DB) or glycerol tartrate gradient sedimentation (GT-DB) constitute 92% or 98%, respectively, of all particles in the final product, (iii) epithelial cell-tropic DB particles are recovered from a single round of coinfection by AD169 and Towne strain viruses, consistent with complementation between the UL130 and UL131A expressed by these strains and restoration of gH/gL/UL128-UL131A (gH pentamer), (iv) equivalent neutralizing antibody titers are induced in mice following immunization with epithelial cell-tropic DB or gH pentamer-deficient DB preparations, (v) UV-inactivated residual virus in GT-DB or TFF-DB preparations retained immunogenicity and induced neutralizing antibody, preventing viral entry into epithelial cells, and (vi) GT-DB and TFF-DB induced cellular immune responses to multiple HCMV peptides. Collectively, this work provides a foundation for future development of DB as an HCMV-based particle vaccine.

Importance: Development of a vaccine to prevent congenital HCMV infection remains a high priority. Vaccination with human cytomegalovirus-derived noninfectious particles, or dense bodies, may constitute a safe vaccination strategy that mimics natural infection. The standard approach for purification of virus particles has been to use a multiple-step, complex gradient that presents a potential barrier to production scale-up and commercialization. In the study described here, we employed an approach that combines treatment with an antiviral terminase inhibitor and purification by a simplified process to produce a vaccine candidate providing broad antiviral humoral and cellular immunity as a foundation for future development.

FIG 1

Infectious HCMV produced by cultures adaptable to a scalable bioprocess can be controlled by addition of the terminase inhibitor BDCRB as late as 48 h postinfection. (A) Image of MRC-5 cells grown on Cytodex-1 microcarriers under conditions optimized to minimize seed cell number and promote growth to confluence by 4 days. (B) Production of HCMV strain Towne evaluated over multiple days from MRC-5 cells grown on Cytodex-1 microcarriers and infected at a low multiplicity (MOI, 0.1). The time of addition of 30 μM BDCRB (in hours postinfection) is indicated. (C) Viral yield and impact of BDCRB following infection (MOI, 1) of MRC-5 cells grown in conventional tissue culture flasks with addition of 15 μM BDCRB. NA, not applicable.

FIG 2

Comparison of the purification schema for HCMV particles recovered by the conventional glycerol tartrate gradient purification method or a bioprocess-adaptable method. (Process A) The glycerol tartrate gradient purification method separates virions, DB, and other noninfectious particles produced by HCMV. The image shown was obtained from cultures without BDCRB in order to illustrate the separation of all particles. The method can be applied to the purification of dense bodies from infected cultures treated with BDCRB. (Process B) Schematic representation of a scalable process to recover and purify TFF-DB particles produced from BDCRB-treated cultures.

FIG 3

Size and composition of particles purified by process A (glycerol tartrate gradient-purified dense bodies [GT-DB]) and process B (TFF-purified dense bodies [TFF-DB]). (A to C) TEM cryo-imaging of virions, GT-DB, and TFF-DB purified from BDCRB-treated HCMV-infected MRC-5 cells in conventional tissue culture flasks. (D) Composite image of results of nanoparticle tracking analysis (NTA) from five independent evaluations of virions, GT-DB, and TFF-DB. (E and F) Anti-pp65 (E) and anti-gB (F) immunoblot analyses of 5-μg GT-DB and TFF-DB protein lysates. Lanes: 1, protein standard; 2, GT-DB proteins; 3, TFF-DB proteins. Arrowheads, gB and pp65 major bands. (G) Image of Coomassie blue-stained gels following denaturing polyacrylamide gel electrophoresis of GT-DB and TFF-DB proteins. The lanes are as described in the legend to panels E and F. (H and I) Images from GT-DB and TFF-DB proteins labeled with Cy3 and Cy5 dyes and separated by two-dimensional gel electrophoresis. Areas 1 and 2 indicate the main spots corresponding to gB and pp65, respectively.

FIG 4

Towne, AD169, and VR1814 particles induce similar neutralizing antibody titers. (A) ARPE-19 cell/MRC-5 cell (ARPE/MRC-5) infectivity ratio determined for AD169, Towne, virus produced by coinfection with AD169 and Towne, or low-passage, epithelial cell-tropic strain VR1814. (B and C) Neutralizing antibody (NAb) titers in mouse serum following immunization with 100 μg total particles produced by AD169, Towne, 1:1 mixtures of AD169 and Towne, or coinfections. Total viral particles (virions, noninfectious particles, and dense bodies) were evaluated directly or following inactivation by UV (*). Neutralizing antibody titers were determined from VR1814 infection of MRC-5 fibroblasts (B) or ARPE-19 epithelial cells (C) and are compared to those for Cytogam, which is purified, concentrated human IgG (50 μg/ml IgG). (D and E) Neutralizing antibody titers in mouse serum following immunization with 10 μg total particles produced by VR1814 or Towne and formulated with GLA-SE adjuvant. Neutralizing antibody titers were determined from VR1814 infection of MRC-5 (D) or ARPE-19 (E) cells and are compared to those for Cytogam, as supplied (50 μg/ml IgG). Bars in panels B to D, geometric mean neutralizing titers.

FIG 5

Human serum includes predominantly complement-independent neutralization activity preventing HCMV infection of MRC-5 or ARPE-19 cells. Sera (n = 12 seronegative serum samples and n = 25 seropositive serum samples) were heat inactivated and evaluated for VR1814 neutralizing titers with 1% guinea pig complement (1% GC) and without guinea pig complement (no GC). The results for the concentrated IgG product Cytogam are included for comparison, and titers are reported relative to those for the concentrated IgG product (50 μg/ml IgG). For human serum, the results and the mean titer (bars) for individual donors are shown. For Cytogam, results from 5 replicate dilutions are reported. (A) MRC-5 fibroblasts; (B) ARPE-19 epithelial cells. Sero(−), seronegative; Sero(+), seropositive. *, P < 0.05.

FIG 6

Neutralizing antibody titers in mouse serum following immunization with GT-DB, TFF-DB, or UV-inactivated TFF-DB. BALB/c mice were immunized with 100 μg of GT-DB and TFF-DB derived from HCMV strain Towne. As a negative control, BALB/c mice were immunized with PBS (closed circles). At 2 weeks after dose 3, neutralization antibody titers in heat-inactivated mouse serum were determined in vitro by neutralization assays. (A and B) In vitro neutralization assays with VR1814 and infection of MRC-5 fibroblasts (A) or ARPE-19 cells (B) in the presence of 1% (final concentration) guinea pig complement (1% GC) or the absence of guinea pig complement (no GC) in mouse serum. Closed squares, GT-DB replicate 1; closed triangles, GT-DB replicate 2; open squares, TFF-DB replicate 1; open triangles, TFF-DB replicate 2. Group means and SDs are shown. *, P < 0.05; **, P < 0.005; ****, P < 0.0001 (Student t test, pairwise comparison). (C) In vitro neutralization assay with Toledo-GFP and infection of MRC-5 cells. The group means and SDs are shown.

FIG 7

GT-DB and TFF-DB induce comparable gB-specific antibody responses. The titers of HCMV gB-specific total IgG (A) or IgG1 and IgG2a (B) isotypes in BALB/c mouse serum following three inoculations with 100 μg of GT-DB or TFF-DB derived from HCMV strain Towne were determined by ELISA. As a negative control, BALB/c mice were immunized with PBS. The group mean is shown in panel A, and the group mean and SD are shown in panel B. AU, arbitrary units.

FIG 8

Cell-mediated immune responses to HCMV peptides of mouse splenocytes following TFF-DB or UV-inactivated TFF-DB immunization. (A) Responses to a pp65-specific overlapping peptide pool and to a putative immunodominant MHC class I H2-D^d pp65 T cell epitope. Responses were determined 2 weeks following three immunizations with 50, 100, or 200 μg of TFF-DB (TFF-DB replicate 1) and are graphed as the number of SFC per 10⁶ splenocytes. As a negative control, BALB/c mice were immunized with PBS. (B) Comparison of the breadth of the immune response between GT-DB (GT-DB replicate 1) and TFF-DB (TFF-DB replicate 1) immunization. Responses were determined in triplicate by an IFN-γ ELISPOT assay with peptides spanning 17 HCMV ORFs. Group means and responses from individual animals are shown in panel A. For panel B, splenocytes from 3 to 5 animals were pooled prior to analysis. (C) BALB/c mice were immunized with 100 μg of TFF-DB or UV-inactivated TFF-DB (TFF-DB + UV) derived from HCMV Towne. At 2 weeks after dose 3, IE1- and pp65-specific responses were assessed in IFN-γ ELISPOT analyses following restimulation of splenocytes with IE1 and pp65 overlapping peptide pools. Responses are graphed as the number of SFC per 1 × 10⁶ splenocytes.