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. 2019 Jun 1;202(11):3234-3245.
doi: 10.4049/jimmunol.1900050. Epub 2019 Apr 19.

Vaccination with a Single-Cycle Respiratory Syncytial Virus Is Immunogenic and Protective in Mice

Megan E Schmidt  1 , Antonius G P Oomens  2 , Steven M Varga  3   4   5
Affiliations

Vaccination with a Single-Cycle Respiratory Syncytial Virus Is Immunogenic and Protective in Mice

Megan E Schmidt et al. J Immunol. .

Abstract

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children, but no vaccine is currently available. Live-attenuated vaccines represent an attractive immunization approach; however, balancing attenuation while retaining sufficient immunogenicity and efficacy has prevented the successful development of such a vaccine. Recently, a recombinant RSV strain lacking the gene that encodes the matrix (M) protein (RSV M-null) was developed. The M protein is required for virion assembly following infection of a host cell but is not necessary for either genome replication or gene expression. Therefore, infection with RSV M-null produces all viral proteins except M but does not generate infectious virus progeny, resulting in a single-cycle infection. We evaluated RSV M-null as a potential vaccine candidate by determining its pathogenicity, immunogenicity, and protective capacity in BALB/c mice compared with its recombinant wild-type control virus (RSV recWT). RSV M-null-infected mice exhibited significantly reduced lung viral titers, weight loss, and pulmonary dysfunction compared with mice infected with RSV recWT. Despite its attenuation, RSV M-null infection induced robust immune responses of similar magnitude to that elicited by RSV recWT. Additionally, RSV M-null infection generated serum Ab and memory T cell responses that were similar to those induced by RSV recWT. Importantly, RSV M-null immunization provided protection against secondary viral challenge by reducing lung viral titers as efficiently as immunization with RSV recWT. Overall, our results indicate that RSV M-null combines attenuation with high immunogenicity and efficacy and represents a promising novel live-attenuated RSV vaccine candidate.

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Figures

Figure 1.
Figure 1.. No infectious progeny is detected following RSV M-null infection in vivo.
(A) Diagram depicting the genome content of RSV recWT and RSV M-null. (B-E) BALB/c mice were infected with either RSV recWT or RSV M-null, and lungs were harvested on days 1, 2, 4, and 7 p.i. (B) EGFP+ focus-forming units and (C) infectious PFU were quantified in the lung by plaque assay using H2-M helper cells. Open (recWT) and closed (M-null) circles represent values for individual mice and lines indicate mean ± SEM of 2 independent experiments (n=8). (D) RSV N gene and (E) RSV M gene copy numbers per lung were determined by real-time PCR. Data are presented as mean ± SEM of representative results from 1 of 2 independent experiments (n=4). Dashed lines denote the limit of detection for each assay. Groups were compared using one-way ANOVA, ** p<0.01, *** p<0.001.
Figure 2.
Figure 2.. RSV M-null infection reduces weight loss and pulmonary dysfunction.
BALB/c mice were infected with either RSV recWT or RSV M-null and monitored daily for (A) weight loss and respiratory parameters including (B) Penh, (C) EF50, and (D) respiratory rate by whole body plethysmography. Data are presented as mean ± SEM of 6 independent experiments (n=35 for recWT and n=36 for M-null). Groups were compared using Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.
Figure 3.
Figure 3.. RSV M-null infection induces T cell responses of similar magnitude to recWT infection.
BALB/c mice were infected with either RSV recWT or RSV M-null, and lungs were harvested on days 6, 8, and 10 p.i. Total numbers of (A) CD11ahiCD49d+ CD4 T cells, (B) CD11ahiCD8lo CD8 T cells, and (C) M282-, (D) M2127-, (E) F85-, and (F) F249-tetramer+ CD8 T cells were quantified. Total numbers of (G) CD4 T cells and (H) CD8 T cells producing IFN-γ after re-stimulation with G183 and M282 peptides, respectively, were determined by intracellular cytokine staining. Data are presented as mean ± SEM of 2–3 independent experiments (n=8 for days 6 and 10 and n=12 for day 8). Groups were compared using Student’s t test, ** p<0.01.
Figure 4.
Figure 4.. T follicular helper cells are generated by RSV M-null infection.
BALB/c mice were infected with either RSV recWT or RSV M-null, and mLN and lungs were harvested on days 14 and 21 p.i. Cells were gated on CD4 T cells, and gates were placed using fluorescence minus one controls. Representative staining panels of Bcl6+CXCR5+ Tfh cells in the (A) mLN and (C) lung. Total numbers of Bcl6+CXCR5+ Tfh cells in the (B) mLN and (D) lung. Data are presented as mean ± SEM of 2 independent experiments (n=8). Groups were compared using Student’s t test.
Figure 5.
Figure 5.. RSV M-null infection induces similar numbers of germinal center B cells as recWT infection.
BALB/c mice were infected with either RSV recWT or RSV M-null, and mLN and lungs were harvested on days 14 and 21 p.i. Total numbers of CD19+ B cells in the (A) mLN and (B) lung. Representative staining panels of Fas+GL-7+ GC B cells in the (C) mLN and (E) lung. Total numbers of Fas+GL-7+ GC B cells in the (D) mLN and (F) lung. Data are presented as mean ± SEM of 2 independent experiments (n=8). Groups were compared using Student’s t test, * p<0.05.
Figure 6.
Figure 6.. RSV M-null infection generates long-lasting RSV-specific serum antibody responses.
BALB/c mice were infected with either RSV recWT or RSV M-null, and serum was collected on days 14, 28, and 84 p.i. Serum was assessed for levels of RSV-specific (A) total Ig, (B) IgG1, and (C) IgG2a by ELISA. Data are presented as mean ± SEM of representative results from 1 of 2 independent experiments (n=5). Groups were compared using Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.
Figure 7.
Figure 7.. RSV M-null infection induces RSV-specific tissue-resident memory CD8 T cells.
BALB/c mice were infected with either RSV recWT or RSV M-null and administered anti-CD45 antibody intravascularly 3 minutes prior to euthanasia on days 30 and 100 p.i. Cells negative for staining with the CD45 intravascular antibody (IV−) were gated, and total numbers of (A) CD11ahiCD49d+ CD4 T cells, (B) CD11ahiCD8lo CD8 T cells, and (C) M282-tetramer+ CD8 T cells within the lung parenchyma are shown. (D) Representative staining panels and (E) total numbers of CD69+CD103+ IV− M282-tetramer+ CD8 T cells. Data are presented as mean ± SEM of 2 independent experiments (n=10 for day 30 and n=9 for day 100). Groups were compared using Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.
Figure 8.
Figure 8.. RSV M-null infection provides protection against secondary viral challenge.
(A/B) Naive BALB/c mice were infected with either RSV recWT or RSV M-null and challenged with RSV either (A) 6 or (B) 12 weeks p.i. Lung RSV titers on day 4 p.i. were determined by plaque assay using VERO cells. (C/D) Naive BALB/c mice were infected with either RSV recWT or RSV M-null and challenged with IAV-M282 either (C) 6 or (D) 12 weeks p.i. Lung IAV titers on day 6 p.i. were determined by plaque assay using MDCK cells. Data are presented as mean ± SEM of 2 independent experiments (n=8). Groups were compared using one-way ANOVA, *** p<0.001.
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