ECB-ART-46898
Nat Commun
2019 Jan 21;101:361. doi: 10.1038/s41467-018-08265-9.
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Efficient oral vaccination by bioengineering virus-like particles with protozoan surface proteins.
Serradell MC
,
Rupil LL
,
Martino RA
,
Prucca CG
,
Carranza PG
,
Saura A
,
Fernández EA
,
Gargantini PR
,
Tenaglia AH
,
Petiti JP
,
Tonelli RR
,
Reinoso-Vizcaino N
,
Echenique J
,
Berod L
,
Piaggio E
,
Bellier B
,
Sparwasser T
,
Klatzmann D
,
Luján HD
.
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Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host''s intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.
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Genes referenced: LOC100893907 LOC105441782 LOC115919910
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Fig. 1. Resistance to degradation of protozoan CXXC-rich proteins. a High magnification representative images of trophozoites from Giardia lamblia, Entamoeba histolytica, Tetrahymena thermophila, and Paramecium tetraurelia, and non-adherent mammalian cell (NS0) incubated for 90 min with high trypsin concentration (20 mg ml−1). The top panel shows phase contrast images. Bottom panel shows live (green cytoplasm) and dead (red nucleus) images of the same cells stained with fluorescein diacetate and propidium iodide. The bars represent 25 μm. b, c Recombinant proteins were expressed in insect cells and highly purified by affinity chromatography. Western blotting analysis of the effects of extreme pH, trypsin (T), intestinal extract (IE), and stomach extract (SE) on recombinant proteins. Proteolytic profile of ΔVSP1267 and HA. Representative images are on the left; densitometric measurements are on the right (mean ± s.e.m.) (b). Proteolytic profile of recombinant ΔVSPH7 and ΔVSP9B10 (c). d Proteolytic profile of native VSP1267 compared to an unrelated parasite protein, GRP78/BIP and native VSPs from Giardia lysates. e Trypsin digestion of ΔVSP1267 subjected to different pre-treatments to modify its structure. The ratio protein:trypsin (P:T) is expressed as w-w. Dilutions of IE and SE are indicated on top. *p < 0.05, **p < 0.01; Student’s t-test, n = 4 from two independent experiments. Source data are provided as a Source Data file | |
Fig. 3. VLP characterization. a Immunofluorescence microscopy of HEK cells used in VLPs production. The different DNA constructs used are indicated on top of each figure. In all immunofluorescence images, nuclei are labeled with DAPI (blue). The white signal represents colocalization. The bars represent 20 μm. b Electron micrographs of 100 nm-thick cryosections showing VLPs budding from the surface of HEK-1267 cells. Five nanometer immunogold labeling of HA (top panels) and VSP1267 (bottom panels) indicates their localization in cells and VLPs membranes (arrows). N, nucleus. The bars represent 100 nm. c Western blotting and hemagglutination assays showing the correct assembly of VLPs. d Representative TEM negative staining of VLPs (VLP-HA on the left and VLP-HA/VSP1267-G on the right). The bars represent 100 nm. e Immunofluorescence images of VSPs on the surface of Giardia trophozoites (left) and of VSP-G on the surface of VLPs (right). In both cases, mAb 7F5-FITC anti-VSP1267 extracellular domain was used. VSPs are labeled in green and nuclei stained with DAPI in blue. The bars represent 25 μm. Source data are provided as a Source Data file | |
Fig. 6. Protective efficacy of VLP-HA/VSP-G oral vaccination against influenza virus challenge and HA-expressing tumors. a, b Ten days after H5N1 VLP immunization, mice were challenged intranasally with a mouse-adapted influenza virus, n = 5. Kaplan–Meier life survival curve analysis was performed using the log-rank Mantel-Cox method for curve comparison analysis (a). Body weight is plotted as a percentage of the average initial weight taken at day 0. The body weight changes were evaluated for 2 weeks (b). c, d Mice immunized with H1N1 VLPs were injected 10 days after the last dose with AB1-HA tumor cells. Graph showing the tumor volume growth, n = 8 from two independent experiments (c). Thirty-one days after tumor inoculation the tumors were harvested and weighed, and representative tumor photographs are shown, n = 8 from two independent experiments (d). e Ten days after H1N1 VLPs immunization, the titer of neutralizing antibodies was measured in sera by a standard microneutralization assay, n = 8 from two independent experiments. f, g, Ten days after H1N1 VLPs immunization, mice were injected with AB1-HA cells and after 31 days the mice were sacrificed. IFN-γ was measured in HA re-stimulated splenocyte supernatants, n = 8 from two independent experiments (f). In vitro cytotoxicity assay using splenocytes and CFSE-labeled AB1-HA as target cells was performed. Dead cells quantification on CFSE + cells is shown, n = 6 from two independent experiments (g). h AB1-HA or 4T1-HA tumor cells were inoculated (day 0; n = 10) and once tumors were detected, half of the mice were therapeutically vaccinated (arrows). Data were analyzed by one-way ANOVA and Tukey’s multiple comparison test (d to g) or by two-way ANOVA and Bonferroni post-tests (c). Values represent mean ± s.e.m. **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file |
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