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Quantification of Lyssavirus -Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles

View Article: PubMed Central - PubMed

ABSTRACT

Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV) although other members of the genus Lyssavirus can cause rabies as well. As yet, 14 serologically and genetically diverse lyssaviruses have been identified, mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations, virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work, a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV) from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT). Importantly, pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together, a pseudotype virus system has been successfully developed which allows the safe, fast, and sensitive detection of neutralizing antibodies directed against different lyssaviruses.

No MeSH data available.


Related in: MedlinePlus

Pseudotype virus neutralization (PVN) test using the GFP reporter for detection. VSV*∆G(FLuc) was trans-complemented with G protein from either VSV or CVS-11. (a) The trans-complemented viruses (100 f.f.u.) were incubated for 60 min with either anti-RABV reference serum (2 IU/mL, 1:80), anti-VSV serum (1:100) or without (w/o) any serum and subsequently inoculated with either T-Rex™-CHO-SAD-G helper cells (if virus was trans-complemented with VSV G protein) or T-Rex™-CHO-VSV-G helper cells (if virus was trans-complemented with CVS-11 G protein) in the presence or absence of DOX. Expression of GFP was visualized 24 h post inoculation by fluorescence microscopy; (b) Comparison of the GFP and luciferase reporter read-outs. Serial two-fold dilutions of human anti-RABV reference serum (2 IU/mL) were incubated in quadruplicates for 60 min with 100 focus-forming units (f.f.u.) of VSV*∆G(sNLuc) that was trans-complemented with the CVS-11 G protein. Vero cells were added to the wells and incubated at 37 °C for 20 h. GFP expression was monitored by fluorescence microscopy. In parallel, the secreted Nano luciferase (sNLuc) activity was determined in the cell culture supernatant. The luciferase endpoint titers were calculated according to the method by Spearman–Kärber using a cutoff value of 200 RLU (see Figure 2e). The mean values of 11 independent experiments are shown; (c) The luciferase data were also used to calculate the serum dilution causing a reduction of luciferase by 50% (red dashed line). The luminescence recorded following infection of the cells in the absence of antibody was set to 100%. Mean values (n = 11) and standard deviations are shown.
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viruses-08-00254-f003: Pseudotype virus neutralization (PVN) test using the GFP reporter for detection. VSV*∆G(FLuc) was trans-complemented with G protein from either VSV or CVS-11. (a) The trans-complemented viruses (100 f.f.u.) were incubated for 60 min with either anti-RABV reference serum (2 IU/mL, 1:80), anti-VSV serum (1:100) or without (w/o) any serum and subsequently inoculated with either T-Rex™-CHO-SAD-G helper cells (if virus was trans-complemented with VSV G protein) or T-Rex™-CHO-VSV-G helper cells (if virus was trans-complemented with CVS-11 G protein) in the presence or absence of DOX. Expression of GFP was visualized 24 h post inoculation by fluorescence microscopy; (b) Comparison of the GFP and luciferase reporter read-outs. Serial two-fold dilutions of human anti-RABV reference serum (2 IU/mL) were incubated in quadruplicates for 60 min with 100 focus-forming units (f.f.u.) of VSV*∆G(sNLuc) that was trans-complemented with the CVS-11 G protein. Vero cells were added to the wells and incubated at 37 °C for 20 h. GFP expression was monitored by fluorescence microscopy. In parallel, the secreted Nano luciferase (sNLuc) activity was determined in the cell culture supernatant. The luciferase endpoint titers were calculated according to the method by Spearman–Kärber using a cutoff value of 200 RLU (see Figure 2e). The mean values of 11 independent experiments are shown; (c) The luciferase data were also used to calculate the serum dilution causing a reduction of luciferase by 50% (red dashed line). The luminescence recorded following infection of the cells in the absence of antibody was set to 100%. Mean values (n = 11) and standard deviations are shown.

Mentions: In order to see whether CVS-11 G protein-pseudotyped VSV*∆G(Luc) would be sensitive to neutralization by immune sera directed to the RABV G protein, the virus (100 f.f.u.) was incubated for 60 min with either human anti-RABV serum, rabbit anti-VSV serum, or no serum at all. Subsequently, the pseudotype virus-serum mixtures were incubated with T-Rex™-CHO(VSV-G) or T-Rex™-CHO(SAD-G) helper cells, either in the presence or absence of doxycycline. At 18 h p.i., GFP expression was visualized by fluorescence microscopy (Figure 3a). In non-induced helper cells, GFP expression was restricted to single cells whereas in doxycycline-induced cells, virus was able to propagate, which led to an increased number of GFP-positive cells, thereby facilitating the detection of non-neutralized virus. Anti-RABV serum (1:80) completely neutralized CVS-11 G protein-pseudotyped VSV*∆G(FLuc) but had no effect if the virus had been trans-complemented with VSV G protein. Vice versa, anti-VSV immune serum (1:100) efficiently neutralized VSV G protein-pseudotyped but not CVS-11 G protein-pseudotyped VSV*∆G(FLuc), indicating that the trans-complementing envelope glycoprotein specifically determined the susceptibility of the virus to antibody-mediated neutralization.


Quantification of Lyssavirus -Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles
Pseudotype virus neutralization (PVN) test using the GFP reporter for detection. VSV*∆G(FLuc) was trans-complemented with G protein from either VSV or CVS-11. (a) The trans-complemented viruses (100 f.f.u.) were incubated for 60 min with either anti-RABV reference serum (2 IU/mL, 1:80), anti-VSV serum (1:100) or without (w/o) any serum and subsequently inoculated with either T-Rex™-CHO-SAD-G helper cells (if virus was trans-complemented with VSV G protein) or T-Rex™-CHO-VSV-G helper cells (if virus was trans-complemented with CVS-11 G protein) in the presence or absence of DOX. Expression of GFP was visualized 24 h post inoculation by fluorescence microscopy; (b) Comparison of the GFP and luciferase reporter read-outs. Serial two-fold dilutions of human anti-RABV reference serum (2 IU/mL) were incubated in quadruplicates for 60 min with 100 focus-forming units (f.f.u.) of VSV*∆G(sNLuc) that was trans-complemented with the CVS-11 G protein. Vero cells were added to the wells and incubated at 37 °C for 20 h. GFP expression was monitored by fluorescence microscopy. In parallel, the secreted Nano luciferase (sNLuc) activity was determined in the cell culture supernatant. The luciferase endpoint titers were calculated according to the method by Spearman–Kärber using a cutoff value of 200 RLU (see Figure 2e). The mean values of 11 independent experiments are shown; (c) The luciferase data were also used to calculate the serum dilution causing a reduction of luciferase by 50% (red dashed line). The luminescence recorded following infection of the cells in the absence of antibody was set to 100%. Mean values (n = 11) and standard deviations are shown.
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Related In: Results  -  Collection

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viruses-08-00254-f003: Pseudotype virus neutralization (PVN) test using the GFP reporter for detection. VSV*∆G(FLuc) was trans-complemented with G protein from either VSV or CVS-11. (a) The trans-complemented viruses (100 f.f.u.) were incubated for 60 min with either anti-RABV reference serum (2 IU/mL, 1:80), anti-VSV serum (1:100) or without (w/o) any serum and subsequently inoculated with either T-Rex™-CHO-SAD-G helper cells (if virus was trans-complemented with VSV G protein) or T-Rex™-CHO-VSV-G helper cells (if virus was trans-complemented with CVS-11 G protein) in the presence or absence of DOX. Expression of GFP was visualized 24 h post inoculation by fluorescence microscopy; (b) Comparison of the GFP and luciferase reporter read-outs. Serial two-fold dilutions of human anti-RABV reference serum (2 IU/mL) were incubated in quadruplicates for 60 min with 100 focus-forming units (f.f.u.) of VSV*∆G(sNLuc) that was trans-complemented with the CVS-11 G protein. Vero cells were added to the wells and incubated at 37 °C for 20 h. GFP expression was monitored by fluorescence microscopy. In parallel, the secreted Nano luciferase (sNLuc) activity was determined in the cell culture supernatant. The luciferase endpoint titers were calculated according to the method by Spearman–Kärber using a cutoff value of 200 RLU (see Figure 2e). The mean values of 11 independent experiments are shown; (c) The luciferase data were also used to calculate the serum dilution causing a reduction of luciferase by 50% (red dashed line). The luminescence recorded following infection of the cells in the absence of antibody was set to 100%. Mean values (n = 11) and standard deviations are shown.
Mentions: In order to see whether CVS-11 G protein-pseudotyped VSV*∆G(Luc) would be sensitive to neutralization by immune sera directed to the RABV G protein, the virus (100 f.f.u.) was incubated for 60 min with either human anti-RABV serum, rabbit anti-VSV serum, or no serum at all. Subsequently, the pseudotype virus-serum mixtures were incubated with T-Rex™-CHO(VSV-G) or T-Rex™-CHO(SAD-G) helper cells, either in the presence or absence of doxycycline. At 18 h p.i., GFP expression was visualized by fluorescence microscopy (Figure 3a). In non-induced helper cells, GFP expression was restricted to single cells whereas in doxycycline-induced cells, virus was able to propagate, which led to an increased number of GFP-positive cells, thereby facilitating the detection of non-neutralized virus. Anti-RABV serum (1:80) completely neutralized CVS-11 G protein-pseudotyped VSV*∆G(FLuc) but had no effect if the virus had been trans-complemented with VSV G protein. Vice versa, anti-VSV immune serum (1:100) efficiently neutralized VSV G protein-pseudotyped but not CVS-11 G protein-pseudotyped VSV*∆G(FLuc), indicating that the trans-complementing envelope glycoprotein specifically determined the susceptibility of the virus to antibody-mediated neutralization.

View Article: PubMed Central - PubMed

ABSTRACT

Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV) although other members of the genus Lyssavirus can cause rabies as well. As yet, 14 serologically and genetically diverse lyssaviruses have been identified, mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations, virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work, a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV) from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT). Importantly, pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together, a pseudotype virus system has been successfully developed which allows the safe, fast, and sensitive detection of neutralizing antibodies directed against different lyssaviruses.

No MeSH data available.


Related in: MedlinePlus