Limits...
Chimeric rabies virus-like particles containing membrane-anchored GM-CSF enhances the immune response against rabies virus.

Kang H, Qi Y, Wang H, Zheng X, Gao Y, Li N, Yang S, Xia X - Viruses (2015)

Bottom Line: The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively.EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M).The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, South China Agricultural University, 483 Wushan Road, Guangzhou 510642, China. kang1989462@sina.com.

ABSTRACT
Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP), which containing glycoprotein (G) and matrix protein (M) of rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF), and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against RABV were evaluated by intramuscular administration in a mouse model. The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively. The membrane-anchored GM-CSF possesses a strong adjuvant activity. More B cells and dendritic cells (DCs) were recruited and/or activated in inguinal lymph nodes in mice immunized with EVLP-G. EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M). The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP. Moreover, the immune responses induced by EVLP-G protect all vaccinated mice from lethal challenge with RABV. These results suggest that EVLP-G has the potential to be developed as a novel vaccine candidate for the prevention and control of animal rabies.

Show MeSH

Related in: MedlinePlus

Construction and production of cRVLP containing membrane-anchored GM-CSF. (A) Schematic diagram of the construction about membrane-anchored GM-CSF containing mellitin SP and TM-CT of RABV G; (B) Schematic diagram of the recombinant plasmid pFBD-2GMCSF; (C) Detection of the expression of G, M and GM-CSF by rBVs. Sf9 cells were infected with rBVs rpFBD-2COG, rpFBD-2COM and rpFBD-2GMCSF, respectively. Additionally, the cells treated with PBS as mock. The infected cells were incubated at 27 °C for 48 h, and then examined the expression by using IFA with mouse anti-G antibody, rabbit serum against RABV M or mouse anti-GM-CSF antibody; (D) Western blot assays of EVLP-G. We analyzed purified EVLP-G for the incorporation of G, M and GM-CSF probed with mouse anti-rabies G antibody, rabbit serum against RABV M or mouse anti-GM-CSF antibody; (E) Electron microscopy of EVLP-G. The EVLP-G were stained with 1% sodium phosphotungstate and then observed by a transmission electron microscopy; (F) Immunoelectron microscopy of EVLP-G. The EVLP-G and EVLP (as mock) were incubated with mouse anti-GM-CSF antibody, and then with gold-labeled goat anti-mouse IgG antibody.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4379564&req=5

viruses-07-01134-f001: Construction and production of cRVLP containing membrane-anchored GM-CSF. (A) Schematic diagram of the construction about membrane-anchored GM-CSF containing mellitin SP and TM-CT of RABV G; (B) Schematic diagram of the recombinant plasmid pFBD-2GMCSF; (C) Detection of the expression of G, M and GM-CSF by rBVs. Sf9 cells were infected with rBVs rpFBD-2COG, rpFBD-2COM and rpFBD-2GMCSF, respectively. Additionally, the cells treated with PBS as mock. The infected cells were incubated at 27 °C for 48 h, and then examined the expression by using IFA with mouse anti-G antibody, rabbit serum against RABV M or mouse anti-GM-CSF antibody; (D) Western blot assays of EVLP-G. We analyzed purified EVLP-G for the incorporation of G, M and GM-CSF probed with mouse anti-rabies G antibody, rabbit serum against RABV M or mouse anti-GM-CSF antibody; (E) Electron microscopy of EVLP-G. The EVLP-G were stained with 1% sodium phosphotungstate and then observed by a transmission electron microscopy; (F) Immunoelectron microscopy of EVLP-G. The EVLP-G and EVLP (as mock) were incubated with mouse anti-GM-CSF antibody, and then with gold-labeled goat anti-mouse IgG antibody.

Mentions: As depicted in Figure 1A, chimeric GM-CSF was constructed by fusing mellitin SP and TM-CT from the ERA G gene at the N terminus and the C terminus of full-length GM-CSF, respectively. A schematic of the recombination plasmid pFBD-2GMCSF containing two GM-CSF genes is shown in Figure 1B. The resultant rBVs, rpFBD-2COG, rpFBD-2COM, and rpFBD-2GMCSF, were rescued successfully in sf9 insect cells. Expression of G, M, and GM-CSF by the rBVs was detected by immunostaining. As expected, cells infected with rpFBD-2COG, rpFBD-2COM, or rpFBD-2GMCSF were stained by antibody against G, mouse serum against M, or antibody against GM-CSF, respectively (Figure 1C).


Chimeric rabies virus-like particles containing membrane-anchored GM-CSF enhances the immune response against rabies virus.

Kang H, Qi Y, Wang H, Zheng X, Gao Y, Li N, Yang S, Xia X - Viruses (2015)

Construction and production of cRVLP containing membrane-anchored GM-CSF. (A) Schematic diagram of the construction about membrane-anchored GM-CSF containing mellitin SP and TM-CT of RABV G; (B) Schematic diagram of the recombinant plasmid pFBD-2GMCSF; (C) Detection of the expression of G, M and GM-CSF by rBVs. Sf9 cells were infected with rBVs rpFBD-2COG, rpFBD-2COM and rpFBD-2GMCSF, respectively. Additionally, the cells treated with PBS as mock. The infected cells were incubated at 27 °C for 48 h, and then examined the expression by using IFA with mouse anti-G antibody, rabbit serum against RABV M or mouse anti-GM-CSF antibody; (D) Western blot assays of EVLP-G. We analyzed purified EVLP-G for the incorporation of G, M and GM-CSF probed with mouse anti-rabies G antibody, rabbit serum against RABV M or mouse anti-GM-CSF antibody; (E) Electron microscopy of EVLP-G. The EVLP-G were stained with 1% sodium phosphotungstate and then observed by a transmission electron microscopy; (F) Immunoelectron microscopy of EVLP-G. The EVLP-G and EVLP (as mock) were incubated with mouse anti-GM-CSF antibody, and then with gold-labeled goat anti-mouse IgG antibody.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379564&req=5

viruses-07-01134-f001: Construction and production of cRVLP containing membrane-anchored GM-CSF. (A) Schematic diagram of the construction about membrane-anchored GM-CSF containing mellitin SP and TM-CT of RABV G; (B) Schematic diagram of the recombinant plasmid pFBD-2GMCSF; (C) Detection of the expression of G, M and GM-CSF by rBVs. Sf9 cells were infected with rBVs rpFBD-2COG, rpFBD-2COM and rpFBD-2GMCSF, respectively. Additionally, the cells treated with PBS as mock. The infected cells were incubated at 27 °C for 48 h, and then examined the expression by using IFA with mouse anti-G antibody, rabbit serum against RABV M or mouse anti-GM-CSF antibody; (D) Western blot assays of EVLP-G. We analyzed purified EVLP-G for the incorporation of G, M and GM-CSF probed with mouse anti-rabies G antibody, rabbit serum against RABV M or mouse anti-GM-CSF antibody; (E) Electron microscopy of EVLP-G. The EVLP-G were stained with 1% sodium phosphotungstate and then observed by a transmission electron microscopy; (F) Immunoelectron microscopy of EVLP-G. The EVLP-G and EVLP (as mock) were incubated with mouse anti-GM-CSF antibody, and then with gold-labeled goat anti-mouse IgG antibody.
Mentions: As depicted in Figure 1A, chimeric GM-CSF was constructed by fusing mellitin SP and TM-CT from the ERA G gene at the N terminus and the C terminus of full-length GM-CSF, respectively. A schematic of the recombination plasmid pFBD-2GMCSF containing two GM-CSF genes is shown in Figure 1B. The resultant rBVs, rpFBD-2COG, rpFBD-2COM, and rpFBD-2GMCSF, were rescued successfully in sf9 insect cells. Expression of G, M, and GM-CSF by the rBVs was detected by immunostaining. As expected, cells infected with rpFBD-2COG, rpFBD-2COM, or rpFBD-2GMCSF were stained by antibody against G, mouse serum against M, or antibody against GM-CSF, respectively (Figure 1C).

Bottom Line: The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively.EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M).The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, South China Agricultural University, 483 Wushan Road, Guangzhou 510642, China. kang1989462@sina.com.

ABSTRACT
Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP), which containing glycoprotein (G) and matrix protein (M) of rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF), and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against RABV were evaluated by intramuscular administration in a mouse model. The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively. The membrane-anchored GM-CSF possesses a strong adjuvant activity. More B cells and dendritic cells (DCs) were recruited and/or activated in inguinal lymph nodes in mice immunized with EVLP-G. EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M). The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP. Moreover, the immune responses induced by EVLP-G protect all vaccinated mice from lethal challenge with RABV. These results suggest that EVLP-G has the potential to be developed as a novel vaccine candidate for the prevention and control of animal rabies.

Show MeSH
Related in: MedlinePlus