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

Etude des interactions moléculaires au sein du complexe polymérase du virus respiratoire syncytial

Le virus respiratoire syncytitial (VRS) est le principal agent causal des bronchiolites chez le jeune veau et les enfants. Actuellement, il n existe ni vaccin, ni traitements anti-viraux efficaces permettant de lutter contre le VRSH. Ce virus enveloppé dont le génome est composé d une seule molécule d ARN de polarité négative, possède sa propre machinerie permettant de répliquer et transcrire: le complexe ARN-polymérase. Mon travail de thèse a consisté a cartographier et caractériser des domaines d interactions sur différentes protéines faisant partie du complexe ARN-polymérase du VRS. Celui-ci se compose de la nucléoprotéine (N), la phosphoprotéine (P), la polymérase (L pour large ) et le co-facteur de transcription M2-1. Ce sont des cibles potentielles pour le développement de drogues antivirales. J ai cartographié le domaine nécessaire et suffisant pour l interaction entre P et N sur P. Ceci m a permis d isoler de façon inattendue un complexe N-ARN structuré en anneaux. À partir de ces objets, j ai pu caractériser la taille des ARNs encapsidés par la nucléoprotéine et déterminer le nombre de bases en contact avec chaque monomère de N. Ce matériel a également été utilisé dans des essais de vaccination en collaboration. Des essais d expression de la grosse sous-unité de la polymérase du VRS n ont pas abouti. Enfin, l étude du facteur de transcription viral M2-1 nous a permis d identifier un domaine globulaire capable d interagir à la fois avec P, de l ARN ou de l ADN. J ai pu montrer que la région N-terminale est bien un zinc finger . De façon étonnante, celui-ci fixe la tubuline. Le rôle exact de cette interaction dans le cycle viral reste à déterminer.The respiratory syncytitial virus (RSV) is the main causal agent of bronchiolites at the young calf and the children. There is no antiviral treatment, nor vaccine against the RSV for the humain species for the moment. The genome of the RSV is a single negative stranded RNA molecule. The virus possesses its own machinery allowing to replicate and to transcribe: the RNA-polymerase complex. My phD work consisted in mapping and in characterizing domains of interactions on various proteins being a part of the RNA-polymerase complex of the RSV. This one consists of the nucleoproteine (N), the phosphoproteine (P), the polymerase (L for "large") and the co-factor of transcription M2-1. They are potential targets for the development of antiviral drugs.I mapped the necessary domain for the interaction between P and N on P protein. This allowed me to isolate in an unexpected way a complex N-RNA structured in rings. From these objects, I was able to characterize the size of the RNA encapsidated by the nucleoproteine and to determine the number of nucleotide contacts by monomer of N. This material was also used in vaccination trials in collaboration.The expression of the RSV polymerase L did not succeed. Finally, the study of the viral factor of transcription M2-1 allowed us to identify a globular domain interacting with P, the ARN or the DNA. I was able to show that the N-terminal region is indeed a zinc binding domain. In a surprising way, this one fixes the tubulin. The exact role of this interaction in the viral cycle remains to identify.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

N-GFP fusion protein assembles into fluorescent SRS that are internalized by murine macrophage and dendritic cell lines.

<p>(A) SDS-PAGE analysis of purified GST-PCT+N-GFP complex. Sample (1) was denatured in Laemmli buffer, run on a 12% polyacrylamide gel and detected with Coomassie brilliant blue staining. (2) protein molecular size standards (kDa). (B) Observation of the different complexes adsorbed on glutathione-Sepharose 4B beads under UV light. (C) Electron micrographs of ring-like structures produced by heterologous expression of N-GFP (left) and N (right) purified by GST-PCT. Bars, 50 nm. (D) N-GFP SRS (thin line) adsorption by RAW and D2SC/1 cell lines after 1 hour incubation at 4°C (controls: PBS in grey or GFP bold line). Green fluorescence associated with living cells was analyzed by flow cytometry (the gate was set up excluding dead cells stained with propidium iodide in FL3). The data (100,000 events) were acquired with a FACScalibur and analyzed with Cell Quest-Pro. (E) Confocal microscopy analysis showing entry of N-GFP SRS (green fluorescence) within cells. Filamentous actin was stained with phalloidin Rhodamin (red). Images of individual cells are either z sections through confocal images taken at sequential focal planes or xy views. Stacks of confocal images were acquired at 0.37 µm intervals (bars 10 µm).</p

T cell-mediated immune response to N SRS.

<p>Nasal vaccination with N SRS generated antigen-specific CD8⁺ T cells and IFN-γ producing CD4⁺ T cells. (A) Antigen-specific proliferation of CD4⁺ and CD8⁺ T splenocytes after 7 days restimulation with RSV-A2 (1 PFU/cell), N (10 µg/ml) or medium. Pooled splenocytes from non immunized (grey lines), LT(R192G) (black lines) or N SRS+LT(R192G) (red lines) immunized groups were stained with CFSE, cultured for 7 days and then labeled with anti-CD8-biot and anti-CD4-PE for flow-cytometry analysis. The data (100,000 events) were acquired with a FACScalibur and analyzed with Cell Quest-Pro. The CD8⁺ or CD4⁺ lymphocyte population was gated according to SSC/FCS and FL4 (CD8) or FL2 (CD4) fluorescence criteria and the fluorescence corresponding to CFSE was monitored in FL1. The percentage of proliferating cells (low CFSE staining) is indicated on the plot with the color corresponding to the immunization condition. (Data from one out of two experiments with similar results). (B) Two weeks after the booster immunization with LT(R192G) or N SRS+LT(R192G), spleen (white bars) and draining LN (cervical and sub-maxilliary LN, black bars) were dissected out and cell suspensions prepared. Splenocytes from individual mice and pooled draining LN cells from each group of mice were re-stimulated for 72 hr with N SRS (10 µg/ml) or medium (mock). IFN-γ secretion was measured in cell culture supernatant with a standardized specific sandwich ELISA assay (white bars represent the mean and SEM of 5 individual spleens, black bars represent the pool of LN, data from one out of three experiments). (C) The frequency of IFN-γ secreting splenocytes after 20 hr restimulation with N (10 µg/ml) was monitored by ELISPOT. Spleen cells from LT(R192G) or N SRS+LT(R192G) immunized mice were assayed for each mouse (each bar represents the mean and SEM of 5 mice). Depletion of CD4⁺ or CD8⁺ T cells was done by immuno-magnetic separation of pooled splenocytes from either LT(R192G) or N SRS+LT(R192G) groups. (Data from one out of two experiments with similar results).</p

Nasal vaccination with N SRS and LT(R192G) augments cellular infiltration in lung tissue.

<p>BALB/c mice were administered i.n. 10 µg N SRS and/or 5 µg LT(R192G), twice at two weeks interval. Two weeks after the second immunization, all animals were challenged with 10⁷ PFU hRSV strain A2, together with a control group of non-immunized mice. One group of mice was neither immunized nor infected. Lung were dissected out 5 days post challenge, embedded in paraffin, sectionned at 7 µm and stained with eosin-hematoxylin. One representative section per group is shown (original magnification 20×, bars 100 µm). (A) control group (no vaccine, no virus), (B) primary infection group (no vaccine, RSV); (C) adjuvant only group (LT(R192G), RSV), (D) N SRS vaccinated group (N SRS+LT(R192G), RSV). Areas with an infiltration of inflammatory cells are indicated with a white arrow. (E) Enlargement showing that the immune infiltrate in N SRS vaccinated group was composed predominantly of lymphocytes and some neutrophils (black arrows) (original magnification 63x, bars 20 µm).</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

Nasal vaccination with N SRS induces neutrophil and lymphocyte recruitment in BAL.

<p>BALB/c mice were administered i.n. 10 µg N SRS and/or 5 µg LT(R192G) twice at two weeks interval. One group was not immunized. Two weeks after the second immunization, all animals were challenged with 10⁷ PFU hRSV strain A2. Cellular composition of BAL sampled at 0, 5 and 10 days after RSV challenge as determined by May-Grünwald-Giemsa staining: lymphocytes (dotted bars), macrophages (white bars) and neutrophils (grey bars). Results are expressed as mean and SEM percentages from 5–8 individual mice (Data from two experiments with similar results).</p

Systemic and mucosal antibody response to N SRS administered intra-nasally.

<p>BALB/c mice were administred 10 µg N SRS and/or 5 µg LT(R192G) twice i.n. at two weeks interval. Individual sera were collected at day 0, 14 and 28 and individual BAL supernatants at day 28. (A) Total Ig, (B) IgG1 (white bars) and IgG2a (black bars) anti N antibodies in serum. (C) total Ig (white bars) and IgA (black bars) in BAL. Titers were determined in an endpoint dilution ELISA assay on plates coated with N SRS. Each bar represents the mean and SEM of 5–6 mice. Titers are shown with logarithmic scale (data from one out of three experiments with similar results). (D) Systemic and mucosal antibodies raised against N SRS recognize RSV-A2. Total Ig, IgG1, IgG2a and IgA in BAL or serum were titrated by ELISA on plates coated with RSV infected HEp-2 versus non infected HEp-2 cells. Data shown are ODx1000 at dilution 270 for sera samples and dilution 27 for BAL samples, after subtracting OD value of non infected HEp2. Each bar represents the mean and SEM of 5–6 mice (Data from one out of two experiments with similar results).</p