Limits...
Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway.

Sui J, Aird DR, Tamin A, Murakami A, Yan M, Yammanuru A, Jing H, Kan B, Liu X, Zhu Q, Yuan QA, Adams GP, Bellini WJ, Xu J, Anderson LJ, Marasco WA - PLoS Pathog. (2008)

Bottom Line: Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection.Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs.These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. Jianhua_sui@dfci.harvard.edu

ABSTRACT
Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.

Show MeSH

Related in: MedlinePlus

Serologic analysis.A, 11 of convalescent serum samples were obtained from SARS patients of the 2002/03 outbreak in China. Neutralizing activities of these serum samples were analyzed against pseudotyped viruses bearing the S protein of Tor2 or GD03 at indicated dilutions. Data were shown in a box and whiskers graph. The box extends from 25th percentile to the 75th percentile, with a line at the median. The whiskers above and below the box indicate the 95th and 5th percentiles. The dots above and below the box showed highest and lowest data points. A symbol of “*” indicates the data points of a SARS-CoV negative healthy human serum sample. IC90 for each patient serum was calculated and the statistic analysis was done with IC90 value using one way ANOVA for correlated samples. The same data presentation and the statistic analysis methods were used for the following panels B and D. B, Neutralization assay of Tor2 and GD03 pseudotyped viruses with six civet serum samples collected from animal market in Guangzhou in Jan. 2004. Similarly as Fig. 1A, “*” indicates the data points for a SARS-CoV negative civet cat serum sample. C, Neutralization assay of Tor2 and GD03 pseudotyped viruses with four serum samples from 2003/04 outbreak. The samples labeled in the graph as patient 1–4 that were the same patients described before [5]. D. Ten serum samples from civet cat farmers collected in June 2003in Guangdong Province were analyzed against Tor2 and GD03 pseudotyped viruses. E, Tor2 -RBD binding activity of 2002/03, 2003/04 outbreak, and Civet cat farmers' serum samples were analyzed by ELISA. These serum samples were the same as those used in Panel A, C, and D, respectively. Dilution of serum was 1∶180. The data was presented the same way as panel A and unpaired Student t-test was used for statistical analysis. The average and standard deviation (SD) of background binding of two SARS-CoV negative human serum samples (Ctrl.HS) were also shown. F, A comparison of the competition ability of different serum samples for 80R's binding to Tor2-RBD were evaluated by ELISA. Dilution for all samples was 1∶20. The non-specific competition for 80R's binding to Tor-RBD by two Ctrl. HS or one control civet cat serum (Ctrl.CS) was also shown. Serum samples were corresponding to those used in panel A, B, C or D. The data presentation and statistic analysis were done the same way as panel E.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2572002&req=5

ppat-1000197-g001: Serologic analysis.A, 11 of convalescent serum samples were obtained from SARS patients of the 2002/03 outbreak in China. Neutralizing activities of these serum samples were analyzed against pseudotyped viruses bearing the S protein of Tor2 or GD03 at indicated dilutions. Data were shown in a box and whiskers graph. The box extends from 25th percentile to the 75th percentile, with a line at the median. The whiskers above and below the box indicate the 95th and 5th percentiles. The dots above and below the box showed highest and lowest data points. A symbol of “*” indicates the data points of a SARS-CoV negative healthy human serum sample. IC90 for each patient serum was calculated and the statistic analysis was done with IC90 value using one way ANOVA for correlated samples. The same data presentation and the statistic analysis methods were used for the following panels B and D. B, Neutralization assay of Tor2 and GD03 pseudotyped viruses with six civet serum samples collected from animal market in Guangzhou in Jan. 2004. Similarly as Fig. 1A, “*” indicates the data points for a SARS-CoV negative civet cat serum sample. C, Neutralization assay of Tor2 and GD03 pseudotyped viruses with four serum samples from 2003/04 outbreak. The samples labeled in the graph as patient 1–4 that were the same patients described before [5]. D. Ten serum samples from civet cat farmers collected in June 2003in Guangdong Province were analyzed against Tor2 and GD03 pseudotyped viruses. E, Tor2 -RBD binding activity of 2002/03, 2003/04 outbreak, and Civet cat farmers' serum samples were analyzed by ELISA. These serum samples were the same as those used in Panel A, C, and D, respectively. Dilution of serum was 1∶180. The data was presented the same way as panel A and unpaired Student t-test was used for statistical analysis. The average and standard deviation (SD) of background binding of two SARS-CoV negative human serum samples (Ctrl.HS) were also shown. F, A comparison of the competition ability of different serum samples for 80R's binding to Tor2-RBD were evaluated by ELISA. Dilution for all samples was 1∶20. The non-specific competition for 80R's binding to Tor-RBD by two Ctrl. HS or one control civet cat serum (Ctrl.CS) was also shown. Serum samples were corresponding to those used in panel A, B, C or D. The data presentation and statistic analysis were done the same way as panel E.

Mentions: Eleven randomly selected serum samples from patients who developed SARS during the 2002/03 outbreak were analyzed for their neutralizing activities against S protein pseudotyped viruses. The S protein from Tor2 and GD03 viral strains that were used are representative of the late phase of the 2002/03 outbreak and 2003/04 human cases, respectively. Phylogenetic analysis of different viral isolates from human patients and civets of the two epidemics demonstrated a close relationship of Tor2 and SZ3 (2003 Civets); while GD03 is closer to PC04 (2004 Civets) [8]. Though all 11 serum samples from 2002/03 outbreak were able to neutralize Tor2 and GD03 pseudotyped viruses, the potency was quite different (Fig. 1A). The 2002/03 patient serum samples are statistically significantly more potent in neutralizing Tor2 than GD03. In contrast, statistical analysis of 2004 civet cat sera showed higher neutralizing activity against GD03 compared to Tor2 (Fig 1B). In addition, neutralization activity was found in three of the four 2003/04 outbreak patient sera to be slightly higher against GD03 strain compared to Tor2 (Fig. 1C). Interestingly, surveillance sera samples collected from civet cat farmers in June 2003, a period between the 2002/03 and 2003/04 outbreaks, showed the same neutralizing titers to Tor2 and GD03, likely owing to their chronic exposure to the SARS-like-CoVs (Fig. 1D). The Tor2-RBD binding activity of these serum samples was also tested. Unexpectedly, there was higher binding activity of Tor2-RBD with 2003 civet cat farmers' serum than was seen with 2002/03 patient sera (Fig. 1E). A further comparison of the ability of different serum samples to compete for 80R binding to Tor2-RBD showed that the 2002/03 patient sera competed for 80R's binding significantly stronger than did each of the 2003/04 serum samples (Fig. 1F). Taken together, these two latter observations suggest that a larger percentage of Tor2-RBD directed Abs in 2002/03 serum samples are “80R-like” nAbs than in 2003/04 serum samples. These results also demonstrate that contemporaneous-strain and cross-strain nAb are produced in both humans and animals following natural SARS-CoV/SARS-like-CoV infection.


Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway.

Sui J, Aird DR, Tamin A, Murakami A, Yan M, Yammanuru A, Jing H, Kan B, Liu X, Zhu Q, Yuan QA, Adams GP, Bellini WJ, Xu J, Anderson LJ, Marasco WA - PLoS Pathog. (2008)

Serologic analysis.A, 11 of convalescent serum samples were obtained from SARS patients of the 2002/03 outbreak in China. Neutralizing activities of these serum samples were analyzed against pseudotyped viruses bearing the S protein of Tor2 or GD03 at indicated dilutions. Data were shown in a box and whiskers graph. The box extends from 25th percentile to the 75th percentile, with a line at the median. The whiskers above and below the box indicate the 95th and 5th percentiles. The dots above and below the box showed highest and lowest data points. A symbol of “*” indicates the data points of a SARS-CoV negative healthy human serum sample. IC90 for each patient serum was calculated and the statistic analysis was done with IC90 value using one way ANOVA for correlated samples. The same data presentation and the statistic analysis methods were used for the following panels B and D. B, Neutralization assay of Tor2 and GD03 pseudotyped viruses with six civet serum samples collected from animal market in Guangzhou in Jan. 2004. Similarly as Fig. 1A, “*” indicates the data points for a SARS-CoV negative civet cat serum sample. C, Neutralization assay of Tor2 and GD03 pseudotyped viruses with four serum samples from 2003/04 outbreak. The samples labeled in the graph as patient 1–4 that were the same patients described before [5]. D. Ten serum samples from civet cat farmers collected in June 2003in Guangdong Province were analyzed against Tor2 and GD03 pseudotyped viruses. E, Tor2 -RBD binding activity of 2002/03, 2003/04 outbreak, and Civet cat farmers' serum samples were analyzed by ELISA. These serum samples were the same as those used in Panel A, C, and D, respectively. Dilution of serum was 1∶180. The data was presented the same way as panel A and unpaired Student t-test was used for statistical analysis. The average and standard deviation (SD) of background binding of two SARS-CoV negative human serum samples (Ctrl.HS) were also shown. F, A comparison of the competition ability of different serum samples for 80R's binding to Tor2-RBD were evaluated by ELISA. Dilution for all samples was 1∶20. The non-specific competition for 80R's binding to Tor-RBD by two Ctrl. HS or one control civet cat serum (Ctrl.CS) was also shown. Serum samples were corresponding to those used in panel A, B, C or D. The data presentation and statistic analysis were done the same way as panel E.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572002&req=5

ppat-1000197-g001: Serologic analysis.A, 11 of convalescent serum samples were obtained from SARS patients of the 2002/03 outbreak in China. Neutralizing activities of these serum samples were analyzed against pseudotyped viruses bearing the S protein of Tor2 or GD03 at indicated dilutions. Data were shown in a box and whiskers graph. The box extends from 25th percentile to the 75th percentile, with a line at the median. The whiskers above and below the box indicate the 95th and 5th percentiles. The dots above and below the box showed highest and lowest data points. A symbol of “*” indicates the data points of a SARS-CoV negative healthy human serum sample. IC90 for each patient serum was calculated and the statistic analysis was done with IC90 value using one way ANOVA for correlated samples. The same data presentation and the statistic analysis methods were used for the following panels B and D. B, Neutralization assay of Tor2 and GD03 pseudotyped viruses with six civet serum samples collected from animal market in Guangzhou in Jan. 2004. Similarly as Fig. 1A, “*” indicates the data points for a SARS-CoV negative civet cat serum sample. C, Neutralization assay of Tor2 and GD03 pseudotyped viruses with four serum samples from 2003/04 outbreak. The samples labeled in the graph as patient 1–4 that were the same patients described before [5]. D. Ten serum samples from civet cat farmers collected in June 2003in Guangdong Province were analyzed against Tor2 and GD03 pseudotyped viruses. E, Tor2 -RBD binding activity of 2002/03, 2003/04 outbreak, and Civet cat farmers' serum samples were analyzed by ELISA. These serum samples were the same as those used in Panel A, C, and D, respectively. Dilution of serum was 1∶180. The data was presented the same way as panel A and unpaired Student t-test was used for statistical analysis. The average and standard deviation (SD) of background binding of two SARS-CoV negative human serum samples (Ctrl.HS) were also shown. F, A comparison of the competition ability of different serum samples for 80R's binding to Tor2-RBD were evaluated by ELISA. Dilution for all samples was 1∶20. The non-specific competition for 80R's binding to Tor-RBD by two Ctrl. HS or one control civet cat serum (Ctrl.CS) was also shown. Serum samples were corresponding to those used in panel A, B, C or D. The data presentation and statistic analysis were done the same way as panel E.
Mentions: Eleven randomly selected serum samples from patients who developed SARS during the 2002/03 outbreak were analyzed for their neutralizing activities against S protein pseudotyped viruses. The S protein from Tor2 and GD03 viral strains that were used are representative of the late phase of the 2002/03 outbreak and 2003/04 human cases, respectively. Phylogenetic analysis of different viral isolates from human patients and civets of the two epidemics demonstrated a close relationship of Tor2 and SZ3 (2003 Civets); while GD03 is closer to PC04 (2004 Civets) [8]. Though all 11 serum samples from 2002/03 outbreak were able to neutralize Tor2 and GD03 pseudotyped viruses, the potency was quite different (Fig. 1A). The 2002/03 patient serum samples are statistically significantly more potent in neutralizing Tor2 than GD03. In contrast, statistical analysis of 2004 civet cat sera showed higher neutralizing activity against GD03 compared to Tor2 (Fig 1B). In addition, neutralization activity was found in three of the four 2003/04 outbreak patient sera to be slightly higher against GD03 strain compared to Tor2 (Fig. 1C). Interestingly, surveillance sera samples collected from civet cat farmers in June 2003, a period between the 2002/03 and 2003/04 outbreaks, showed the same neutralizing titers to Tor2 and GD03, likely owing to their chronic exposure to the SARS-like-CoVs (Fig. 1D). The Tor2-RBD binding activity of these serum samples was also tested. Unexpectedly, there was higher binding activity of Tor2-RBD with 2003 civet cat farmers' serum than was seen with 2002/03 patient sera (Fig. 1E). A further comparison of the ability of different serum samples to compete for 80R binding to Tor2-RBD showed that the 2002/03 patient sera competed for 80R's binding significantly stronger than did each of the 2003/04 serum samples (Fig. 1F). Taken together, these two latter observations suggest that a larger percentage of Tor2-RBD directed Abs in 2002/03 serum samples are “80R-like” nAbs than in 2003/04 serum samples. These results also demonstrate that contemporaneous-strain and cross-strain nAb are produced in both humans and animals following natural SARS-CoV/SARS-like-CoV infection.

Bottom Line: Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection.Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs.These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. Jianhua_sui@dfci.harvard.edu

ABSTRACT
Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.

Show MeSH
Related in: MedlinePlus