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A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus.

Frank GM, Angeletti D, Ince WL, Gibbs JS, Khurana S, Wheatley AK, Max EE, McDermott AB, Golding H, Stevens J, Bennink JR, Yewdell JW - MBio (2015)

Bottom Line: As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate.Using this method, we showed that the route and form of immunization significantly impacts the quality and quantity of B cell antibody responses.This method provides a relatively simple yet powerful tool for better understanding the contribution of affinity maturation to viral immunity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

No MeSH data available.


Related in: MedlinePlus

Intramuscular vaccination with inactivated PR8 and adjuvant produces an inferior HA-specific GC B cell response. We injected mice bilaterally with a total dose of 2.5 × 107 TCID50/mouse (pre-UV inactivation) diluted 1:1 with Titermax gold adjuvant or infected mice i.n. with 50 TCID50 per mouse. At 14 and 28 dpi, we removed lymphoid organs draining the site of injection, the inguinal LN, and/or popliteal LN (i.m. vaccination) or mediastinal LN (i.n. infection), and spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. (A) Representative flow plots depict total GC B cell response (top) as well as HA binding (bottom). Red dot plots indicate no HA control staining, while blue dot plots represent HA staining at 66 nM rHAPR8. The table depicts the frequency with which an HA-specific GC B cell response was seen in the lymphoid organ following i.m. vaccination. (B and C) Bar graphs depicting numbers of HA-specific GC B cells and frequency of HA-specific GC B cells per tissue. Mice vaccinated i.m. have sporadic distal LN involvement and no splenic HA response, while productively infected mice have large HA-specific populations in the draining MLN as well as the distal spleen. (D) AC50 for each condition. GC B cells responding to i.m. vaccination have ~4-fold-higher AC50 than those formed to productive i.n. infection. Scatter plots of HA binding signal (E) and Ab off-rate (F). Serum from 14 and 28 days after i.m. PR8-vaccinated and i.n. PR8-infected mice were tested by surface plasmon resonance (SPR) analysis to determine Ab signal and off-rates. Serum from i.m.-vaccinated mice had an amount of HA binding antibody and had significantly higher off-rates at both 14 and 28 dpi. Data are representative of 2 to 4 independent experiments.
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fig6: Intramuscular vaccination with inactivated PR8 and adjuvant produces an inferior HA-specific GC B cell response. We injected mice bilaterally with a total dose of 2.5 × 107 TCID50/mouse (pre-UV inactivation) diluted 1:1 with Titermax gold adjuvant or infected mice i.n. with 50 TCID50 per mouse. At 14 and 28 dpi, we removed lymphoid organs draining the site of injection, the inguinal LN, and/or popliteal LN (i.m. vaccination) or mediastinal LN (i.n. infection), and spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. (A) Representative flow plots depict total GC B cell response (top) as well as HA binding (bottom). Red dot plots indicate no HA control staining, while blue dot plots represent HA staining at 66 nM rHAPR8. The table depicts the frequency with which an HA-specific GC B cell response was seen in the lymphoid organ following i.m. vaccination. (B and C) Bar graphs depicting numbers of HA-specific GC B cells and frequency of HA-specific GC B cells per tissue. Mice vaccinated i.m. have sporadic distal LN involvement and no splenic HA response, while productively infected mice have large HA-specific populations in the draining MLN as well as the distal spleen. (D) AC50 for each condition. GC B cells responding to i.m. vaccination have ~4-fold-higher AC50 than those formed to productive i.n. infection. Scatter plots of HA binding signal (E) and Ab off-rate (F). Serum from 14 and 28 days after i.m. PR8-vaccinated and i.n. PR8-infected mice were tested by surface plasmon resonance (SPR) analysis to determine Ab signal and off-rates. Serum from i.m.-vaccinated mice had an amount of HA binding antibody and had significantly higher off-rates at both 14 and 28 dpi. Data are representative of 2 to 4 independent experiments.

Mentions: As proof of principle of the utility of AC50 for characterizing immune responses, we compared i.n. infection with infectious PR8 to a single intramuscular (i.m.) injection of UV-inactivated PR8 in Titermax adjuvant. The latter is known to be less protective against lethal PR8 challenge (27). Following i.m. immunization, serum anti-HA Abs were always detected, confirming successful vaccination (data not shown). HA-specific GC B cell responses were uniformly observed in the most proximal popliteal draining lymph nodes, 33% of the time in the more distant inguinal nodes, but never within the spleen (Fig. 6A), indicating only localized generation of HA-reactive GC B cells.


A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus.

Frank GM, Angeletti D, Ince WL, Gibbs JS, Khurana S, Wheatley AK, Max EE, McDermott AB, Golding H, Stevens J, Bennink JR, Yewdell JW - MBio (2015)

Intramuscular vaccination with inactivated PR8 and adjuvant produces an inferior HA-specific GC B cell response. We injected mice bilaterally with a total dose of 2.5 × 107 TCID50/mouse (pre-UV inactivation) diluted 1:1 with Titermax gold adjuvant or infected mice i.n. with 50 TCID50 per mouse. At 14 and 28 dpi, we removed lymphoid organs draining the site of injection, the inguinal LN, and/or popliteal LN (i.m. vaccination) or mediastinal LN (i.n. infection), and spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. (A) Representative flow plots depict total GC B cell response (top) as well as HA binding (bottom). Red dot plots indicate no HA control staining, while blue dot plots represent HA staining at 66 nM rHAPR8. The table depicts the frequency with which an HA-specific GC B cell response was seen in the lymphoid organ following i.m. vaccination. (B and C) Bar graphs depicting numbers of HA-specific GC B cells and frequency of HA-specific GC B cells per tissue. Mice vaccinated i.m. have sporadic distal LN involvement and no splenic HA response, while productively infected mice have large HA-specific populations in the draining MLN as well as the distal spleen. (D) AC50 for each condition. GC B cells responding to i.m. vaccination have ~4-fold-higher AC50 than those formed to productive i.n. infection. Scatter plots of HA binding signal (E) and Ab off-rate (F). Serum from 14 and 28 days after i.m. PR8-vaccinated and i.n. PR8-infected mice were tested by surface plasmon resonance (SPR) analysis to determine Ab signal and off-rates. Serum from i.m.-vaccinated mice had an amount of HA binding antibody and had significantly higher off-rates at both 14 and 28 dpi. Data are representative of 2 to 4 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Intramuscular vaccination with inactivated PR8 and adjuvant produces an inferior HA-specific GC B cell response. We injected mice bilaterally with a total dose of 2.5 × 107 TCID50/mouse (pre-UV inactivation) diluted 1:1 with Titermax gold adjuvant or infected mice i.n. with 50 TCID50 per mouse. At 14 and 28 dpi, we removed lymphoid organs draining the site of injection, the inguinal LN, and/or popliteal LN (i.m. vaccination) or mediastinal LN (i.n. infection), and spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. (A) Representative flow plots depict total GC B cell response (top) as well as HA binding (bottom). Red dot plots indicate no HA control staining, while blue dot plots represent HA staining at 66 nM rHAPR8. The table depicts the frequency with which an HA-specific GC B cell response was seen in the lymphoid organ following i.m. vaccination. (B and C) Bar graphs depicting numbers of HA-specific GC B cells and frequency of HA-specific GC B cells per tissue. Mice vaccinated i.m. have sporadic distal LN involvement and no splenic HA response, while productively infected mice have large HA-specific populations in the draining MLN as well as the distal spleen. (D) AC50 for each condition. GC B cells responding to i.m. vaccination have ~4-fold-higher AC50 than those formed to productive i.n. infection. Scatter plots of HA binding signal (E) and Ab off-rate (F). Serum from 14 and 28 days after i.m. PR8-vaccinated and i.n. PR8-infected mice were tested by surface plasmon resonance (SPR) analysis to determine Ab signal and off-rates. Serum from i.m.-vaccinated mice had an amount of HA binding antibody and had significantly higher off-rates at both 14 and 28 dpi. Data are representative of 2 to 4 independent experiments.
Mentions: As proof of principle of the utility of AC50 for characterizing immune responses, we compared i.n. infection with infectious PR8 to a single intramuscular (i.m.) injection of UV-inactivated PR8 in Titermax adjuvant. The latter is known to be less protective against lethal PR8 challenge (27). Following i.m. immunization, serum anti-HA Abs were always detected, confirming successful vaccination (data not shown). HA-specific GC B cell responses were uniformly observed in the most proximal popliteal draining lymph nodes, 33% of the time in the more distant inguinal nodes, but never within the spleen (Fig. 6A), indicating only localized generation of HA-reactive GC B cells.

Bottom Line: As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate.Using this method, we showed that the route and form of immunization significantly impacts the quality and quantity of B cell antibody responses.This method provides a relatively simple yet powerful tool for better understanding the contribution of affinity maturation to viral immunity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

No MeSH data available.


Related in: MedlinePlus