A new subunit vaccine based on nucleoprotein nanoparticles confers partial clinical and virological protection in calves against bovine respiratory syncytial virus

Human and bovine respiratory syncytial viruses (HRSV and BRSV) are two closely related, worldwide prevalent viruses that are the leading cause of severe airway disease in children and calves, respectively. Efficacy of commercial bovine vaccines needs improvement and no human vaccine is licensed yet. We reported that nasal vaccination with the HRSV nucleoprotein produced as recombinant ringshaped nanoparticles (NSRS) protects mice against a viral challenge with HRSV. The aim of this work was to evaluate this new vaccine that uses a conserved viral antigen, in calves, natural hosts for BRSV. Calves, free of colostral or natural anti-BRSV antibodies, were vaccinated with NSRS either intramuscularly, or both intramuscularly and intranasally using MontanideTM ISA71 and IMS4132 as adjuvants and challenged with BRSV. All vaccinated calves developed anti-N antibodies in blood and nasal secretions and N-specific cellular immunity in local lymph nodes. Clinical monitoring post-challenge demonstrated moderate respiratory pathology with local lung tissue consolidations for the non vaccinated calves that were significantly reduced in the vaccinated calves. Vaccinated calves had lower viral loads than the nonvaccinated control calves. Thus NSRS vaccination in calves provided cross-protective immunity against BRSV infection without adverse inflammatory reaction

Nucleoprotein Nanostructures Combined with Adjuvants Adapted to the Neonatal Immune Context: A Candidate Mucosal RSV Vaccine

BACKGROUND: The human respiratory syncytial virus (hRSV) is the leading cause of severe bronchiolitis in infants worldwide. The most severe RSV diseases occur between 2 and 6 months-of-age, so pediatric vaccination will have to be started within the first weeks after birth, when the immune system is prone to Th2 responses that may turn deleterious upon exposure to the virus. So far, the high risk to prime for immunopathological responses in infants has hampered the development of vaccine. In the present study we investigated the safety and efficacy of ring-nanostructures formed by the recombinant nucleoprotein N of hRSV (N(SRS)) as a mucosal vaccine candidate against RSV in BALB/c neonates, which are highly sensitive to immunopathological Th2 imprinting. METHODOLOGY AND PRINCIPAL FINDINGS: A single intranasal administration of N(SRS) with detoxified E. coli enterotoxin LT(R192G) to 5-7 day old neonates provided a significant reduction of the viral load after an RSV challenge at five weeks of age. However, neonatal vaccination also generated an enhanced lung infiltration by neutrophils and eosinophils following the RSV challenge. Analysis of antibody subclasses and cytokines produced after an RSV challenge or a boost administration of the vaccine suggested that neonatal vaccination induced a Th2 biased local immune memory. This Th2 bias and the eosinophilic reaction could be prevented by adding CpG to the vaccine formulation, which, however did not prevent pulmonary inflammation and neutrophil infiltration upon viral challenge. CONCLUSIONS/SIGNIFICANCE: In conclusion, protective vaccination against RSV can be achieved in neonates but requires an appropriate combination of adjuvants to prevent harmful Th2 imprinting

Sub-Nucleocapsid Nanoparticles: A Nasal Vaccine against Respiratory Syncytial Virus

Background: Bronchiolitis caused by the respiratory syncytial virus (RSV) in infants less than two years old is a growing public health concern worldwide, and there is currently no safe and effective vaccine. A major component of RSV nucleocapsid, the nucleoprotein (N), has been so far poorly explored as a potential vaccine antigen, even though it is a target of protective anti-viral T cell responses and is remarkably conserved between human RSV A and B serotypes. We recently reported a method to produce recombinant N assembling in homogenous rings composed of 10–11 N subunits enclosing a bacterial RNA. These nanoparticles were named sub-nucleocapsid ring structure (N SRS). Methodology and Principal Findings: The vaccine potential of N SRS was evaluated in a well-characterized and widely acknowledged mouse model of RSV infection. BALB/c adult mice were immunized intranasally with N SRS adjuvanted with the detoxified E. coli enterotoxin LT(R192G). Upon RSV challenge, vaccinated mice were largely protected against virus replication in the lungs, with a mild inflammatory lymphocytic and neutrophilic reaction in their airways. Mucosal immunization with N SRS elicited strong local and systemic immunity characterized by high titers of IgG1, IgG2a and IgA anti-N antibodies, antigen-specific CD8+ T cells and IFN-c-producing CD4+ T cells. Conclusions/Significance: This is the first report of using nanoparticles formed by the recombinant nucleocapsid protein as an efficient and safe intra-nasal vaccine against RSV

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 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

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

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 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