A highly attenuated recombinant human respiratory syncytial virus lacking the G protein induces long-lasting protection in cotton rats

Abstract

Background: Respiratory syncytial virus (RSV) is a primary cause of serious lower respiratory tract illness for which there is still no safe and effective vaccine available. Using reverse genetics, recombinant (r)RSV and an rRSV lacking the G gene (DeltaG) were constructed based on a clinical RSV isolate (strain 98-25147-X).

Results: Growth of both recombinant viruses was equivalent to that of wild type virus in Vero cells, but was reduced in human epithelial cells like Hep-2. Replication in cotton rat lungs could not be detected for DeltaG, while rRSV was 100-fold attenuated compared to wild type virus. Upon single dose intranasal administration in cotton rats, both recombinant viruses developed high levels of neutralizing antibodies and conferred comparable long-lasting protection against RSV challenge; protection against replication in the lungs lasted at least 147 days and protection against pulmonary inflammation lasted at least 75 days.

Conclusion: Collectively, the data indicate that a single dose immunization with the highly attenuated DeltaG as well as the attenuated rRSV conferred long term protection in the cotton rat against subsequent RSV challenge, without inducing vaccine enhanced pathology. Since DeltaG is not likely to revert to a less attenuated phenotype, we plan to evaluate this deletion mutant further and to investigate its potential as a vaccine candidate against RSV infection.

Figure 1

Generation of recombinant RSV-X virus. A) Schematic diagram of the RSV-X genome (genome length 15213 nt) and positions of the genetic tags inserted in the cDNA copy of the rRSV-X and ΔG constructs. The SexA I, Xma I, BssH II, Bsiw I, and Mlu I sites were introduced to facilitate construction. ΔG was recovered by excision of the fragment BssH II and BsiW I and subsequent religation of the vector. B) Expression of RSV proteins by rRSV and ΔG deletion recombinant viruses. Vero cells were infected at an m.o.i of 0.1 TCID₅₀/ml. At 72 hr post infection cell monolayers was harvested and subjected to Western blotting using antiserum against RSV. The molecular weight size markers are depicted on the left and the position of the major RSV proteins are indicated at the right [29].

Figure 2

Growth of (recombinant) RSV in Vero and Hep-2 cells. Vero (A) and Hep-2 (B) cell monolayers were infected with wild type (wt) RSV, rRSV or ΔG with an MOI of 0.1 and incubated at 37°C. Cells were harvested at the indicated time points and virus TCID₅₀ titers were determined in Vero cells.

Figure 3

RSV replication in cell lines. Growth of RSV was tested in human lung mucoepidermoid carcinoma cells NCI-H292, human bronchial epithelial cell line 16HBE140, human lung epithelial carcinoma cells A549, human kidney epithelial cells 293T, human epithelial Hep-2 cells and monkey kidney Vero cells. Cells were infected with virus with an MOI of 0.1, harvested after 72 hr and virus CID₅₀ titers were determined in Vero cells.

Figure 4

Long term protection against RSV challenge lung histopathology in cotton rats. Cotton rats were immunized i.n. at day 0 with 10⁵ TCID₅₀ rRSV or ΔG. Challenge was performed at day 70 and 142 with 10⁶ TCID₅₀ RSV-X (i.n) and the animals were sacrificed 5 days later, at day 75 and 147, respectively. Groups consisted of 6 animals. Mean histopathological scores of following histopathological parameters: peribronchiolitis (black bars), hypertrophied mucous cells (brown bars), peribronchitis (white bars) and alveolitis (gray bars). Ctrl: control animals; mock: mock infected, challenged animals. *: statistically significant different (P < 0.05) compared to mock infected group based on the Wilxocon test.