Neonatal nasal vaccination with N+LT or N+LT+CpG augmented cellular infiltration in lung tissue upon RSV challenge.

<p>Mice vaccinated as neonates as indicated or non vaccinated littermates were sacrificed 8 days after the hRSV-A2 challenge. Lung were dissected out, fixed, embedded in paraffin and sectioned at 5 µm. Lung sections were then stained with hematoxylin-eosin-saffron and photographed using a Nanozoomer (Hamamatsu). One representative section per group is shown (A–C: original magnification 2.5×) with red asterix figuring eosinophils in the enlarged selected area (D–F: enlargement 20×). (G) Total iBALT area was measured using the NDPview software (Hamamatsu) for one representative lung section of each mouse (n≥2 mice per group). Data are mean±SEM.</p

Neonatal nasal vaccination with N+LT conferred viral protection but exacerbated airway disease upon RSV challenge.

<p>Male and female pups (5–7 day-old) were vaccinated by intranasal instillation of 10 µL saline containing or not 3 µg N and 2 µg LT as indicated. At 5 weeks of age, mice were challenged by intranasal instillation of 50 µL (5.10⁶ pfu) hRSV A2. Controls included unvaccinated infected (C+) and uninfected (C−) littermates. Animals were killed 5 days post challenge (d5 p.i.). (A) Individual viral load assessed by qRT-PCR: R.Q. of N transcripts, normalized to HPRT, are expressed as % of the unvaccinated infected control group (C+) (R.Q. = 100×2^−ΔΔCt). Four independent experiments combining different treatment groups are shown with ≥5 mice per group (C−: n = 7, C+: n = 10, LT: n = 5, N: n = 16, N+LT: n = 20). Mann-Whitney U-test was used for comparison between treatments (* p<0.05; *** p<0.001 and **** p<0.0001). (B) Mice were weighed daily from d0 till d5 p.i. and individual weight loss/gain was calculated as % of initial weight. Data are mean±SEM from n≥5 mice per group. Statistical analysis was performed to compare growth over the period d2 to 5 p.i. using the Tukey's multiple comparison test, repeated measures one way ANOVA (** p<0.01 and *** p<0.001).</p

CpG added to the neonatal vaccine reduced weight loss, increased secondary IgG2a response and maintained viral protection upon RSV challenge.

<p>Mice vaccinated as neonates as indicated or non vaccinated littermates (C+) were sacrificed 5 days after the hRSV-A2 challenge. Data from 2 independent experiments combining different treatment groups are shown with ≥5 mice per group (C+: n = 7, CpG: n = 5, N+CpG: n = 6, LT+CpG: n = 5, N+LT: n = 10, N+LT+CpG: n = 11). (A) Mice were weighed daily from d0 till d5 p.i. and individual weight loss/gain was calculated as % of initial weight. Statistical analysis was performed to compare growth over the period d2 to d5 p.i. using the Tukey's multiple comparison test, repeated measures one way ANOVA (* p<0.05; ** p<0.01 and *** p<0.001). (B) IgG1 and IgG2a anti-N antibodies were titrated by ELISA in sera collected 5 days after the challenge. Data are mean±SEM from n≥5 mice per group. (C) Individual viral load in lungs: R.Q. of N transcripts, normalized to HPRT, are expressed as % of the mean of unvaccinated infected control group (C+) (R.Q. = 100×2^−ΔΔCt). Data from 2 independent experiments combining different treatment groups are shown. Mann-Whitney U-test was used for comparison between treatments (* p<0.05; ** p<0.01; *** p<0.001 and **** p<0.0001).</p

Neonatal N+LT nasal immunization primed for an early anti-N Ab response after the RSV challenge.

<p>Mice vaccinated as neonates as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037722#pone-0037722-g001" target="_blank">Fig.1</a> were bled before (day 0) and 5 or 8 days after the viral challenge with hRSV-A2 (d5 or d8 p.i.). Serum anti-N antibody titers were assessed by an endpoint dilution ELISA assay on plates coated with N using HRPO-conjugated rabbit anti-mouse Ig(H+L) Abs. Individual titers and the mean titer for day 0 (white circles, dotted line), d5 p.i. (grey circles, plain line) and d8 p.i. (black circles, plain line) are figured. Man-Whitney U-test was used for comparison of titers at day 0, 5 and 8 p.i. for each group (** p<0.01; *** p<0.001).</p

LT and N contributed to the inflammatory responses recorded in BAL after the RSV challenge.

<p>Mice vaccinated as neonates as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037722#pone-0037722-g001" target="_blank">Fig.1</a> were killed 5 days post challenge (d5 p.i.). BAL cells were numerated, cytocentrifuged and stained with May-Gründwald-Giemsa (MGG). Data are mean±SEM from n≥10 mice per group for total leukocytes (A), lymphocytes (B), neutrophils (C) and eosinophils (D) numbers. Mann-Whitney U-test was used for comparison between treatments (** p<0.01; *** p<0.001 and **** p<0.0001).</p

List of primers used for quantitative real time PCR.

<p>List of primers used for quantitative real time PCR.</p

Memory T cell responses primed by neonatal nasal vaccination shifted from Th2 to Th1 cytokine profile when CpG were added as adjuvant.

(a)<p>Neonates were immunised <i>i.n.</i> at age 5–7 days as indicated (prime) and received one intranasal boost with N (10 µg) at 5 weeks.</p>(b)<p>Spleen and cervical lymph node (LN) were collected 7 days after the boost (LN were pooled from 2 to 3 mice). Single cell suspensions were cultured with N, PMA-ionomycin or medium for 72 hours.</p>(c)<p>Culture supernatants were assayed by ELISA for IFNγ and IL5.</p>(d)<p>Cytokine concentrations in supernatants from cultures restimulated with N (mean pg/mL ±SEM for n≥2 for spleen and pool of 2 for LN). (All cell cultures responded to PMA, producing 39838±8542 pg/mL IFNγ).</p

CpG added to the neonatal vaccine decreased pulmonary CCL11 mRNA expression upon RSV challenge.

<p>The level of expression of genes encoding mCCL2 (A), mCCL3 (C), mCCL5 (D) and mCCL11 (B) was monitored by quantitative real time PCR of RNAs extracted from individual lungs collected at d5 p.i. Data were normalized to the mHPRT and expressed relative to the unvaccinated infected control group (C+) (R.Q. = 100×2^−ΔΔCt). Data are mean±SEM from n≥4 mice per group (Mann Whitney test, * p<0.05). Two independent experiments were done.</p

Anti-N antibody responses primed by neonatal nasal vaccination shifted from IgG1 to IgG2a and IgA isotypes when CpG were added as adjuvant.

(a)<p>Neonates were immunised <i>i.n.</i> at age 5–7 days as indicated (prime) and received one intranasal boost with N (10 µg) at 5 weeks. Sera and BALF were collected 7 days after the boost.</p>(b)<p>Antibody titers against N were determined by ELISA using endpoint dilution assay. Data are mean±SEM (≤30 or ≤3: not detected at the first tested dilution for sera or BALF).</p>(c)<p>Number of responders/number tested.</p

Nasal vaccination with N SRS protects against RSV replication in the lungs without causing disease exacerbation.

<p>BALB/c mice were administered twice at 2 weeks interval i.n. (A) or s.c (B) 10 µg N SRS and 5 µg LT(R192G) (black bars) or 5 µg LT(R192G) alone (grey bars). Two weeks after the second immunization, mice were challenged with 10⁷ PFU hRSV strain A2, together with a non-immunized control group of mice (white bars). Mice were monitored daily for body weight. Viral replication in lung was monitored by quantitative real-time RT-PCR. The number of N-specific RNA copies for 1 µg total reverse-transcribed lung RNA was determined against a standard curve using a plasmid encoding the viral N gene. (A) Intranasal immunization with N SRS reduced N-specific RNA in lungs 4 to 10 days after RSV challenge. (B) Sub-cutaneous vaccination induced moderate protection at 5 days after RSV challenge. Each bar represents the mean and SEM of 5–8 mice. (C) Weight loss in vaccinated versus non vaccinated groups is expressed as the percentage of initial weight (plot of mean±SEM, n = 4–5, day 0 is 100%). Data are representative from four independent experiments.</p