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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.


Comparison of the PVN test with the modified rapid fluorescent focus inhibition test (mRFFIT). Serum samples (not treated at 56 °C) from mice immunized with the indicated vaccine doses were analyzed with either the modified RFFIT or the PVN test. The PVN test was performed with CVS-11 G protein pseudotyped VSV*∆G(sNLuc) using the GFP reporter read-out. The neutralization dose 50% (ND50) values for individual animals (represented by different symbols) and the mean values for each vaccine group (n = 10) are shown.
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viruses-08-00254-f004: Comparison of the PVN test with the modified rapid fluorescent focus inhibition test (mRFFIT). Serum samples (not treated at 56 °C) from mice immunized with the indicated vaccine doses were analyzed with either the modified RFFIT or the PVN test. The PVN test was performed with CVS-11 G protein pseudotyped VSV*∆G(sNLuc) using the GFP reporter read-out. The neutralization dose 50% (ND50) values for individual animals (represented by different symbols) and the mean values for each vaccine group (n = 10) are shown.

Mentions: In order to compare the PVN test with a commonly used RABV neutralization test, the assay was run with immune sera collected from mice that had been immunized with decreasing doses of RABV vaccine. The ND50 values of these sera were calculated and compared with the ND50 values determined by the modified RFFIT [29,30]. With both methods, the antibody responses of the immunized mice were found to depend on the vaccine dose with the highest ND50 values associated with the 1/10 vaccine group and the lowest with the 1/250 vaccine group (Figure 4). No significant differences were observed between the mean ND50 values calculated with either of the two methods.


Quantification of Lyssavirus -Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles
Comparison of the PVN test with the modified rapid fluorescent focus inhibition test (mRFFIT). Serum samples (not treated at 56 °C) from mice immunized with the indicated vaccine doses were analyzed with either the modified RFFIT or the PVN test. The PVN test was performed with CVS-11 G protein pseudotyped VSV*∆G(sNLuc) using the GFP reporter read-out. The neutralization dose 50% (ND50) values for individual animals (represented by different symbols) and the mean values for each vaccine group (n = 10) are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035968&req=5

viruses-08-00254-f004: Comparison of the PVN test with the modified rapid fluorescent focus inhibition test (mRFFIT). Serum samples (not treated at 56 °C) from mice immunized with the indicated vaccine doses were analyzed with either the modified RFFIT or the PVN test. The PVN test was performed with CVS-11 G protein pseudotyped VSV*∆G(sNLuc) using the GFP reporter read-out. The neutralization dose 50% (ND50) values for individual animals (represented by different symbols) and the mean values for each vaccine group (n = 10) are shown.
Mentions: In order to compare the PVN test with a commonly used RABV neutralization test, the assay was run with immune sera collected from mice that had been immunized with decreasing doses of RABV vaccine. The ND50 values of these sera were calculated and compared with the ND50 values determined by the modified RFFIT [29,30]. With both methods, the antibody responses of the immunized mice were found to depend on the vaccine dose with the highest ND50 values associated with the 1/10 vaccine group and the lowest with the 1/250 vaccine group (Figure 4). No significant differences were observed between the mean ND50 values calculated with either of the two methods.

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.