Development and preclinical evaluation of an alphavirus replicon particle vaccine for cytomegalovirus

Abstract

We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus (CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-gamma ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.

Figure 1

Maps of the replicon plasmids containing the gene for CMV gB (A) or pp65/IE1 (B). Starting at the T7 promoter and moving clockwise, the solid arrows represent the four alphavirus nonstructural protein (nsP) genes, the 342 bp spacer, the EV71 IRES, the CMV gene and the kanamycin resistance [KN (R)] gene, respectively.

Figure 2

SDS-PAGE analysis of process pools from a pilot lot of gB VRP. Lane 1, molecular weight markers; Lane 2, human serum albumin control; Lane 3, salt wash harvest pool; Lane 4, pool of material at end of tangential flow filtration; Lane 5, tangential flow filtration permeate; Lane 6, Cellufine Sulfate unbound fraction; Lane 7, Cellufine Sulfate wash fraction; Lane 8 VRP elution from Cellufine Sulfate, 2 × 10⁸ IU load; Lane 9 VRP elution from Cellufine Sulfate, 1 × 10⁸ IU load. The positions of molecular weight markers (kDa) are indicated on the left and the positions of the alphavirus envelope (E) and capsid (C) proteins are indicated on the right.

Figure 3

Southern blot analysis of process pools from a pilot lot of pp65/IE1 VRP. Lane 1, DNA size markers; Lane 2, 1 ng of AluI-digested Vero DNA loaded on the gel as a control that underwent no sample processing; Lane 3, Upstream process pool; Lane 4, pool of VRP taken after Benzonase treatment; Lane 5, pool of VRP taken after Benzonase treatment and spiked with 1 ng of AluI-digested Vero DNA; Lane 6, tangential flow filtration pool; Lane 7, tangential flow filtration pool spiked with 1 ng of AluI-digested Vero DNA; Lane 8 VRP elution from Cellufine Sulfate; Lane 9 VRP elution from Cellufine Sulfate spiked with 1 ng of AluI-digested Vero DNA. The positions of DNA size markers (kbp) are indicated on the left.

Figure 4

Antibody response in mice immunized with gB VRP from a pilot lot (open symbols) or a clinical lot (solid symbols) by IM injection on study days 1, 22 and 43. Each data point represents the antibody titer, as determined by gB ELISA or CMV neutralization assay, in serum obtained from individual mice on study day 64. Control values in serum obtained before immunization were <40 for ELISA and <50 for neutralization. The geometric mean titer in this assay was 635 (range 200–1600) for serum samples from nine CMV seropositive humans.

Figure 5

Anti-gB and anti-pp65 antibody titers measured by ELISA in rabbits immunized with placebo or with gB VRP and pp65/IE1 VRP by the subcutaneous (SC) or intramuscular (IM) route on study days 1, 15, 29 and 43. Each data point represents the geometric mean ± SEM titerin serum from 6 rabbits (or from 3 rabbits at study days 45 and 57).

Figure 6

IFN-γ ELISPOT responses in mice immunized with pp65/IE1 VRP from a pilot lot (open symbols) or a clinical lot (solid symbols) by IM injection on study days 1 and 22 and sacrificed on study day 29. Each data point represents the mean number of spot-forming cells (SFC) per 10⁶ spleen cells from individual mice after stimulation with pools of overlapping peptides spanning the amino- or carboxy-terminal half of pp65 (pp65-1 and pp65-2) or the amino- or carboxy-terminal half of IE1 (IE1-1 and IE1-2). Control values from cells stimulated with an irrelevant peptide were 8 ± 6 (mean ± SEM) SFC per 10⁶ spleen cells.