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A novel high-throughput vaccinia virus neutralization assay and preexisting immunity in populations from different geographic regions in China.

Liu Q, Huang W, Nie J, Zhu R, Gao D, Song A, Meng S, Xu X, Wang Y - PLoS ONE (2012)

Bottom Line: We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT).Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low.There was no significant difference observed for titer or prevalence by gender, age range and geographic origin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing, China.

ABSTRACT

Background: Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China.

Methodology/principal findings: A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30-55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin.

Conclusion: A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui provinces of China.

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Related in: MedlinePlus

Neutralizing antibodies in mouse, rabbit, and human serum samples.(A) Sera from two rTV-HIVgp 145-vaccinated mice (▪, ▴) and two naïve mice (□,△). (B) An anti-VTT rabbit serum (▪), together with an unrelated rabbit control serum (□). (C) Sera from two rMVA-HIVgpe-vaccinated human (▪, ▴) and two naïve human (□, △) were tested using the rTV-Fluc neutralization assay. The x-axis indicates serial dilutions of sera, ranging from 1∶30 to 1∶21870. (D) The dynamics of anti-rMVA-HIVgpe antibody titers. Two months after the first survey at 15 days after immunization, anti-rMVA-HIVgpe antibody titers of 27 participants were measured again. The x-axis indicates months after immunization. The y-axis indicates the MVA specific neutralization antibody titers. The dotted line represents the detection limit of the assay. Each data point in A-D represents the average of three experiments.
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pone-0033392-g002: Neutralizing antibodies in mouse, rabbit, and human serum samples.(A) Sera from two rTV-HIVgp 145-vaccinated mice (▪, ▴) and two naïve mice (□,△). (B) An anti-VTT rabbit serum (▪), together with an unrelated rabbit control serum (□). (C) Sera from two rMVA-HIVgpe-vaccinated human (▪, ▴) and two naïve human (□, △) were tested using the rTV-Fluc neutralization assay. The x-axis indicates serial dilutions of sera, ranging from 1∶30 to 1∶21870. (D) The dynamics of anti-rMVA-HIVgpe antibody titers. Two months after the first survey at 15 days after immunization, anti-rMVA-HIVgpe antibody titers of 27 participants were measured again. The x-axis indicates months after immunization. The y-axis indicates the MVA specific neutralization antibody titers. The dotted line represents the detection limit of the assay. Each data point in A-D represents the average of three experiments.

Mentions: BALB/c mice were vaccinated twice with recombinant rTV-HIVgp 145 vaccine, containing the env gene from B’/C recombinant HIV-1 S939, and at three weeks after the second administration, sera were collected and analyzed using the rTV-Fluc neutralization assay (Figure 2A). Neutralizing activity was detected for sera from two vaccinated mice (50% neutralization titer (NT50) of 126 and 583), while the two naïve mouse sera showed no significant neutralizing activity. No significant neutralizing activity was observed for the naïve rabbit serum; however, rabbit serum collected at two weeks after the second VTT inoculation was able to neutralize rTV-Fluc with an NT50 of 3468 (Figure 2B). Finally, sera obtained from two humans immunized with an experimental HIV vaccinia vaccine (recombinant modified vaccinia Ankara, rMVA-HIVgpe) from the Guangxi Center for Disease Control (CDC) were used to detect neutralizing activity against the heterologous rTV-Fluc virus (NT50 of 98 and 226) and no detectable neutralizing activity was shown in two naïve human sera (Figure 2C). An additional 27 rMVA-HIVgpe immunized individuals’ sera were analyzed for neutralizing antibodies. As shown in Figure 2D, the peak levels of specific anti-MVA NAbs were detected as early as two weeks after immunization, then declined rapidly to undetectable levels in most vaccinees after three months. Taken together, these data showed that homologous and heterologous anti-VACV NAbs from various species could be detected using the developed assay.


A novel high-throughput vaccinia virus neutralization assay and preexisting immunity in populations from different geographic regions in China.

Liu Q, Huang W, Nie J, Zhu R, Gao D, Song A, Meng S, Xu X, Wang Y - PLoS ONE (2012)

Neutralizing antibodies in mouse, rabbit, and human serum samples.(A) Sera from two rTV-HIVgp 145-vaccinated mice (▪, ▴) and two naïve mice (□,△). (B) An anti-VTT rabbit serum (▪), together with an unrelated rabbit control serum (□). (C) Sera from two rMVA-HIVgpe-vaccinated human (▪, ▴) and two naïve human (□, △) were tested using the rTV-Fluc neutralization assay. The x-axis indicates serial dilutions of sera, ranging from 1∶30 to 1∶21870. (D) The dynamics of anti-rMVA-HIVgpe antibody titers. Two months after the first survey at 15 days after immunization, anti-rMVA-HIVgpe antibody titers of 27 participants were measured again. The x-axis indicates months after immunization. The y-axis indicates the MVA specific neutralization antibody titers. The dotted line represents the detection limit of the assay. Each data point in A-D represents the average of three experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3306400&req=5

pone-0033392-g002: Neutralizing antibodies in mouse, rabbit, and human serum samples.(A) Sera from two rTV-HIVgp 145-vaccinated mice (▪, ▴) and two naïve mice (□,△). (B) An anti-VTT rabbit serum (▪), together with an unrelated rabbit control serum (□). (C) Sera from two rMVA-HIVgpe-vaccinated human (▪, ▴) and two naïve human (□, △) were tested using the rTV-Fluc neutralization assay. The x-axis indicates serial dilutions of sera, ranging from 1∶30 to 1∶21870. (D) The dynamics of anti-rMVA-HIVgpe antibody titers. Two months after the first survey at 15 days after immunization, anti-rMVA-HIVgpe antibody titers of 27 participants were measured again. The x-axis indicates months after immunization. The y-axis indicates the MVA specific neutralization antibody titers. The dotted line represents the detection limit of the assay. Each data point in A-D represents the average of three experiments.
Mentions: BALB/c mice were vaccinated twice with recombinant rTV-HIVgp 145 vaccine, containing the env gene from B’/C recombinant HIV-1 S939, and at three weeks after the second administration, sera were collected and analyzed using the rTV-Fluc neutralization assay (Figure 2A). Neutralizing activity was detected for sera from two vaccinated mice (50% neutralization titer (NT50) of 126 and 583), while the two naïve mouse sera showed no significant neutralizing activity. No significant neutralizing activity was observed for the naïve rabbit serum; however, rabbit serum collected at two weeks after the second VTT inoculation was able to neutralize rTV-Fluc with an NT50 of 3468 (Figure 2B). Finally, sera obtained from two humans immunized with an experimental HIV vaccinia vaccine (recombinant modified vaccinia Ankara, rMVA-HIVgpe) from the Guangxi Center for Disease Control (CDC) were used to detect neutralizing activity against the heterologous rTV-Fluc virus (NT50 of 98 and 226) and no detectable neutralizing activity was shown in two naïve human sera (Figure 2C). An additional 27 rMVA-HIVgpe immunized individuals’ sera were analyzed for neutralizing antibodies. As shown in Figure 2D, the peak levels of specific anti-MVA NAbs were detected as early as two weeks after immunization, then declined rapidly to undetectable levels in most vaccinees after three months. Taken together, these data showed that homologous and heterologous anti-VACV NAbs from various species could be detected using the developed assay.

Bottom Line: We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT).Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low.There was no significant difference observed for titer or prevalence by gender, age range and geographic origin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing, China.

ABSTRACT

Background: Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China.

Methodology/principal findings: A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30-55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin.

Conclusion: A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui provinces of China.

Show MeSH
Related in: MedlinePlus