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. 2016 Nov:135:108-119.
doi: 10.1016/j.antiviral.2016.10.008. Epub 2016 Oct 19.

Intranasal treatment with a novel immunomodulator mediates innate immune protection against lethal pneumonia virus of mice

Elisa C Martinez  1 , Ravendra Garg  2 , Pratima Shrivastava  2 , Susantha Gomis  3 , Sylvia van Drunen Littel-van den Hurk  4
Affiliations

Intranasal treatment with a novel immunomodulator mediates innate immune protection against lethal pneumonia virus of mice

Elisa C Martinez et al. Antiviral Res. 2016 Nov.

Abstract

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in infants and young children. There are no licensed RSV vaccines available, and the few treatment options for high-risk individuals are either extremely costly or cause severe side effects and toxicity. Immunomodulation mediated by a novel formulation consisting of the toll-like receptor 3 agonist poly(I:C), an innate defense regulator peptide and a polyphosphazene (P-I-P) was evaluated in the context of lethal infection with pneumonia virus of mice (PVM). Intranasal delivery of a single dose of P-I-P protected adult mice against PVM when given 24 h prior to challenge. These animals experienced minimal weight loss, no clinical disease, 100% survival, and reduced lung pathology. Similar clinical outcomes were observed in mice treated up to 3 days prior to infection. P-I-P pre-treatment induced early mRNA and protein expression of key chemokine and cytokine genes, reduced the recruitment of neutrophils and eosinophils, decreased virus titers in the lungs, and modulated the delayed exacerbated nature of PVM disease without any short-term side effects. On day 14 post-infection, P-I-P-treated mice were confirmed to be PVM-free. These results demonstrate the capacity of this formulation to prevent PVM and possibly other viral respiratory infections.

Keywords: Immunomodulators; Innate immunity; PVM; Protection; RSV.

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Figures

Fig. 1
Fig. 1
Heat map of chemokine and cytokine mRNA expression profiles in the single-lobed lungs of 5–6 week-old female Balb/c mice at 6, 24, 96 and 144 h p.t. with either P-I-P or PBS. Each treatment group contained 20 mice in total, with sampling of 5 animals at each time-point indicated above. All Ct values were normalized against β-actin levels of control animals. Normalized fold-change calculations were performed using the 2−ΔΔCt method. Data are shown as the median of five biological replicates.
Fig. 2
Fig. 2
Protein expression levels of chemokine and cytokine genes in lung homogenates at 6, 24, 96 and 144 h p.t. Mice were given intranasal treatment of PBS or P-I-P and the multi-lobed lungs of 5 mice per group were collected separately for each individual animal at the time points indicated. Protein expression was assayed by electrochemiluminescence-based multiplex ELISAs. Panels A to H represent protein expression data in pg/mL for CCL2, CXCL1, TNF-α, IL-1β, IL-6, total IL-12 (p70), IL-10, and IFN-γ, respectively. Data are shown as individual values representing single animals as well as median with interquartile range of five biological replicates. *p < 0.05, **p < 0.01.
Fig. 3
Fig. 3
Effects of P-I-P treatment on cellular recruitment of immune cells and histology of the lung tissue. Mice were given an intranasal treatment of PBS or P-I-P and the lungs of 3 mice per group were collected, pooled, and processed at the time points indicated. Total numbers of alveolar macrophages (A), DCs (B), neutrophils (C), eosinophils (D), and NK cells (E) in the lungs were analyzed by flow cytometry. Results are presented as cell number x 104 per million cells. For histology experiments, five mice per group were treated as previously described and at 24 h p.t., the multi-lobed lung was collected. Panels F & G represent lung histology sections at 10× and 20× magnifications, respectively, for the P-I-P group. Data are shown as the median with interquartile range of three technical replicates per cell type assayed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig. 4
Fig. 4
Outline of trial, weight change, clinical and survival scores of P-I-P- or PBS-treated Balb/c mice before and after intranasal PVM challenge. Mice were given intranasal treatment of P-I-P or PBS 24 h prior to challenge with 3000 pfu PVM-15. Animals were weighed and scored before treatment (i.e. day -1), on the day of challenge (i.e. day 0), and for 14 days after infection. Panel A highlights the events occurring in the animal trial. Collection of lung tissue for virus titrations, cell influx and mRNA/protein expression studies was scheduled on days 0 (prior to infection), 3 and 5 p.i. Lung samples for histopathology studies were collected on day 6 p.i. On day 14 p.i., qPCR was performed on survivor mice to determine their PVM status. Panel B illustrates weight loss of mice following P-I-P treatment and PVM infection as a median percentage of the starting weight with error bars indicating the interquartile range. The stars indicate the difference between P-I-P/PVM and PBS/PVM groups. Panel C represents survival rates as the median percentage of the total number of animals per group during the 14 days following infection. Panels D and E show clinical scores for weight loss and appearance represented by the median value for each group throughout the entire animal trial. Each experiment was performed twice, with an average of n = 5, and a total number of animals equal to 10 per group. **p < 0.01.
Fig. 5
Fig. 5
PVM titers and absolute virus copy numbers in the lungs of P-I-P pre-treated Balb/c mice on days 0 (prior to infection), 3 and 5 p.i. The multi-lobed lung was homogenized in DMEM and used for virus titrations. Panel A shows the log of PVM titers in the lung tissue expressed in pfu/g. The single-lobed lung was processed in TRIzol and used for qPCR. Panel B shows the log of absolute PVM mRNA copy numbers, which were determined as described in the Materials and Methods section. Data are represented as the median with interquartile range of five biological replicates per group. Each experiment was repeated twice. *p < 0.05, **p < 0.01.
Fig. 6
Fig. 6
Lung pathology of Balb/c mice pre-treated with P-I-P or PBS 24 h prior to intranasal PVM challenge. Four to five mice per group were treated as previously described and infected intranasally with 3000 pfu of PVM. All lung samples were collected on day 6 p.i. Panels A & B represent lung sections at 10× and 20× magnifications, respectively, for the PBS/Medium control group. Panels C & D show lung sections at 10× and 20× magnifications, respectively of mice treated with P-I-P. Panels E to G illustrate lung sections of PBS-treated mice at 10×, 20× and 60× magnifications, respectively. White arrows point to several areas of edema in the tissue, as well as PMNs within this region. Panel H shows the lung histopathology scores given on the basis of the severity and dissemination of the lesions visible in duplicate lung sections. Data are represented as both individual and median values for each group with error bars indicating the interquartile range. *p < 0.05.
Fig. 7
Fig. 7
Heat map of chemokine and cytokine mRNA expression profiles of P-I-P- or PBS-treated Balb/c mice before and after intranasal PVM challenge. Mice were given an intranasal treatment of PBS or P-I-P 24 h before challenge with 3000 pfu PVM. The single-lobed lungs of 5 mice per group were collected on days 0 (prior to infection), 3 and 5 p.i. All Ct values were normalized against β-actin levels of control animals. Normalized fold-change calculations were performed using the 2−ΔΔCt method. Data are shown as the median of five biological replicates.
Fig. 8
Fig. 8
Protein expression levels of chemokine and cytokine genes of P-I-P pre-treated PVM-infected Balb/c mice. Mice were given an intranasal treatment of PBS or P-I-P 24 h before challenge with 3000 pfu PVM. The multi-lobed lungs of 5 mice per group were collected separately for each individual animal on days 0 (prior to infection), 3 and 5 p.i. Protein expression was assayed by electrochemiluminescence-based multiplex ELISAs. Panels A to H represent protein expression data in pg/mL for CCL2, CXCL1, TNF-α, IL-1β, IL-6, total IL-12 (p70), IL-10, and IFN-γ, respectively. Data are shown as individual values representing single animals as well as median with interquartile range of five biological replicates. *p < 0.05, **p < 0.01.
Fig. 9
Fig. 9
Infiltration of immune cell populations into the lungs of P-I-P pre-treated Balb/c mice on days 0 (prior to infection), 3 and 5 following lethal PVM challenge. Mice were given an intranasal treatment of PBS or P-I-P 24 h prior to challenge with 3000 pfu PVM. The lungs of 3 mice per group were collected, pooled, and processed at the time points indicated. Following physical and chemical digestion of the tissue, single-cell suspensions were acquired. Total numbers of alveolar macrophages (A), DCs (B), neutrophils (C), eosinophils (D), and NK cells (E) in the lungs were analyzed by flow cytometry. Results are presented as cell number x 104 per million cells. Data are shown as the median with range of three technical replicates per cell type assayed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig. 10
Fig. 10
Percent weight loss, survival and external appearance clinical scores of Balb/c mice treated with P-I-P or PBS 3 days (panels A–C), 4 days (panels D–F), 5 days (panels G–I) and 6 days (panels J–L) prior to intranasal PVM challenge. Mice were given prophylactic intranasal treatment of PBS or P-I-P at the time points indicated above, followed by a 3000 pfu PVM challenge. Animals were weighed and scored daily after treatment and for 14 days following infection. Data for weight loss is represented as the median percentage of the starting weight with error bars indicating the interquartile range. Percent survival rates are expressed as the median percentage of the total number of animals per group during the 14 days following infection. Clinical scores for external appearance are presented as the median value for each group during the entirety of the animal trial. Each experiment was performed twice, with an average of n = 5, and a total number of animals equal to 10 per group.
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