Development of a challenge-protective vaccine concept by modification of the viral RNA-dependent RNA polymerase of canine distemper virus

Abstract

We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a "quick response" in vaccine development against well-known and "new" viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.

FIG. 1.

Construction of recombinant viruses. The positions of the T7 promoter (T7), the internal ribosome entry site (IRES), three domains of the RdRp (D1 to D3), the hinge regions of the RdRp (H1 and H2), the hepatitis delta ribozyme (δ), and the T7 terminator (Tφ) are indicated (not to scale). Bold lines indicate the positions of the intergenic trinucleotide spacers. (A) Schematic representation of rCDV^5804P and rCDV^5804P-L(CCegfpC) full-length genome plasmids. Three conservative domains and two hinge regions of the L protein are shown. The EGFP ORF was cloned into the MluI site. (B) Schematic representation of plasmids expressing rCDV^5804P and rCDV^5804P-L(CCegfpC) L proteins. (C) Relative activities of unmodified and EGFP-modified CDV^5804P polymerases. Various amounts of pEMC-L (CCC)5804P (diamonds) and pEMC-L(CCegfpC)5804P (squares) were used in minigenome transfection assays. The amount of DsRed2 produced was determined by flow cytometry and expressed in relative fluorescence units (RFU). Percentages represent the relative activities of the modified RdRp compared to the unmodified form of the protein.

FIG. 2.

Rescue of recombinant viruses. (A) RT-PCR products generated with primers spanning the MluI site in the L protein gene. Lanes: 1, no-RT control with RNA extracted from CDV^5804P-infected cells; 2, RNA extracted from CDV^5804P-infected cells (926-bp product); M, DNA marker (λ DNA digested with PstI); 3, RNA extracted from rCDV^5804P-L(CCegfpC)-infected cells (1,646-bp product); 4, no-RT control with RNA extracted from rCDV^5804P-L(CCegfpC)-infected cells. (B) Sequence analysis of RT-PCR product from lane 4 in panel A indicating that the EGFP ORF is inserted in frame and in the correct orientation. (C) Western blot developed with polyclonal anti-GFP antibodies. SDS-PAGE was carried out under reducing conditions. Left lane, VeroDogSLAM cell lysate; right lane, lysate of VeroDogSLAM cells infected with rCDV^5804P-L(CCegfpC). (D) Autofluorescence of rCDV^5804P-L(CCegfpC)-infected VeroDogSLAM cells (left) and ferret lymphocytes (right). The nuclei were stained with propidium iodide, and the signal in the green channel was produced by autofluorescence of the L protein. (E) Growth curves of viruses on VeroDogSLAM cells. Cells were infected at an MOI of 0.01. Viral titers in the supernatant (left panel) and cell-associated viral titers (right panel) are shown.

FIG. 3.

Disease development in animals infected with rCDV^5804P only (A, C to F, and I to K) or infected with rCDV^5804P-L(CCegfpC) (B) and then challenged with rCDV^5804P (G and H). (A and B) Ferret body weight dynamics after primary infection. (C) Abdominal skin rash. (D) Mucopurulent nasal and ocular discharge. (E and G) Individual data on rash onset, recovery, and terminal stage of disease. (F and H) Individual body temperature curves postinfection (F) and postchallenge (H). †, ferrets were severely moribund and were euthanized. (I and J) Histopathology of lung tissue showing acute purulent bronchopneumonia. Hematoxylin-eosin staining (I) and immunohistochemical staining with monoclonal antibodies (VMRD, Inc.) against the CDV N protein (J) were performed. (K and L) Histology structure of spleens from animals infected with rCDV^5804P (K) or infected with rCDV^5804P-L(CCegfpC) and then challenged with rCDV^5804P (L). Note the lymphoid depletion and absence of lymphoid follicles in the spleen of the moribund animal.

FIG. 4.

Hematological changes during infection. (A) WBC counts in peripheral blood. The curves show individual data for the group primarily infected with rCDV^5804P. (B to F) Differential counts of lymphocytes. The curves show individual data for the group primarily infected with rCDV^5804P (B) and the groups infected with rCDV^5804P-L(CCegfpC) (C) and rCDV^OP (E) and then challenged with rCDV^5804P (D and F, respectively). Legends display animal numbers.

FIG. 5.

Nonspecific cellular immunity assessment. DTH reaction profiles of animals infected with rCDV^5804P (A) or rCDV^5804P-L(CCegfpC) (B) are shown. Proliferation potencies of PHA-stimulated PBMCs from individual animals in the group primarily infected with rCDV^5804P (C) and in the groups infected with rCDV^5804P-L(CCegfpC) (D) and rCDV^OP (E) and then challenged with rCDV^5804P are also shown. Legends display animal numbers. Group averages are shown by bold horizontal lines. BRDU, bromodeoxyuridine.

FIG. 6.

Development of specific anti-CDV neutralizing antibodies. Plaque neutralization titers for individual animals infected with rCDV^5804P-L(CCegfpC) (A) and rCDV^OP (B) followed by challenge with rCDV^5804P are shown as reciprocal dilutions.