Development and preclinical evaluation of an alphavirus replicon vaccine for influenza

Abstract

We used a propagation-defective, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the hemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/Wyoming/03/2003 (H3N2). Efficient production methods were scaled to produce pilot lots of HA VRP and NA VRP and clinical lots of HA VRP. HA VRP-induced high-titered antibody responses in mice, rabbits and rhesus macaques, as measured by ELISA or hemagglutination inhibition (HI) assays, and robust cellular immune responses in mice and rhesus macaques, as measured by IFN-gamma ELISPOT. NA VRP also induced cellular immune responses in mice. A toxicology study with HA VRP and NA VRP in rabbits showed no adverse effects in any parameter. These studies support clinical testing of alphavirus replicon vaccines for influenza.

Fig. 1

Characterization of replicon constructs by Western blot analysis. Proteins extracted from Vero cells that were left untreated (Ctrl) or infected with NA VRP or HA VRP were probed with antibodies specific for NA (left panel) or HA (right panel). The positions of molecular weight markers are indicated on the left and the positions of NA or HA proteins are indicated on the right.

Fig. 2

(A) Silver-stained SDS-PAGE analysis of process pools from a pilot lot of HA VRP. Lane 1, molecular weight markers; lane 2, salt wash harvest pool; lane 3, pool of material at end of tangential flow filtration; lane 4, tangential flow filtration permeate; lane 5, Cellufine Sulfate unbound fraction; lane 6, VRP eluted from Cellufine Sulfate column. (B) Western blot of HA VRP probed with antibodies to VEE virus envelope and capsid proteins. Lane 1, molecular weight markers; lane 2, VRP eluted from Cellufine Sulfate column (undiluted); lane 3, VRP eluted from Cellufine Sulfate column (1 × 10⁸ IU). The positions of molecular weight markers (kDa) are indicated on the left and the positions of the VEE virus envelope (E) and capsid (C) proteins are indicated on the right.

Fig. 3

Southern blot analysis of process pools from a pilot lot of HA 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, salt wash harvest 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, pool of material at end of tangential flow filtration (TFF); lane 7, TFF pool spiked with 1 ng of AluI-digested Vero DNA; lane 8, Cellufine Sulfate elution pool; lane 9, Cellufine Sulfate elution pool spiked with 1 ng of AluI-digested Vero DNA. The locations of nucleotide size markers (kbp) are indicated on the left.

Fig. 4

Anti-HA ELISA titers in rabbits immunized with placebo or with a mixture of HA VRP and NA VRP, formulated with 1% human serum albumin (HSA) or 1% rabbit serum albumin (RSA), by the subcutaneous (SC) or intramuscular (IM) route, on SD 1, 15, 29 and 43 (indicated by arrows). Each data point represents the geometric mean ± SEM for serum from 16 rabbits (or from eight rabbits at study days 45/46 and 57/58).

Fig. 5

IFN-γ ELISPOT responses in rhesus macaques immunized on SD 1, 29 and 163 (indicated by arrows) with HA VRP (top panel) or an irrelevant control VRP (bottom panel). Each data point represents the mean number of spot-forming cells (SFC) per 10⁶ PBMC after stimulation with a pool of overlapping peptides spanning the HA protein.