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Characteristics of memory B cells elicited by a highly efficacious HPV vaccine in subjects with no pre-existing immunity.

Scherer EM, Smith RA, Simonich CA, Niyonzima N, Carter JJ, Galloway DA - PLoS Pathog. (2014)

Bottom Line: These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies.Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells.Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation.

View Article: PubMed Central - PubMed

Affiliation: Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

ABSTRACT
Licensed human papillomavirus (HPV) vaccines provide near complete protection against the types of HPV that most commonly cause anogenital and oropharyngeal cancers (HPV 16 and 18) when administered to individuals naive to these types. These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies. However, almost nothing is known about the immunological memory that forms following HPV vaccination, which is required for long-term immunity. Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells. Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation. These findings suggest that the quadrivalent HPV vaccine provides an excellent model for studying the development of B cell memory; and, in the context of what is known about memory B cells elicited by influenza vaccination/infection, HIV-1 infection, or tetanus toxoid vaccination, indicates that extensive somatic hypermutation is not required to achieve potent vaccine-specific neutralizing antibody responses.

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IgG cloned from HPV 16-specific Bmem are potently neutralizing.(A) Serial dilutions of human mAbs cloned from AF488-HPV 16+ Bmem (Ab 1, 2, 3, 5, 6, 8, 12, 13, 16, 17 and 18) were separately incubated in triplicate with a predetermined titer of unconjugated HPV 16 psV (solid pink lines) or BPV psV (negative control, solid black lines). All human mAbs were initially tested at a starting concentration of 50 nM (Fig. S8); however, if Abs neutralized HPV 16 at all dilution points (e.g., Ab 1–8), they were re-tested at a lower starting concentration to obtain precise IC50 values. The anti-HPV 16 murine mAb, H16.V5, and anti-BPV murine mAb, 5B6, served as positive controls for HPV 16 and BPV psV neutralization. HPV 16 and BPV psV are comprised of major and minor capsid proteins encompassing a secreted alkaline phosphatase (SEAP) reporter plasmid. After one hour at room temperature, the Ab-psV mixtures were transferred to 293TT cells and incubated for three additional days at 37°C. The amount of AP activity in the supernatant was then quantified, corrected for background, and expressed as percent neutralization, or the inverse of the AP signal reduction observed in the presence of Ab: [(meansignal without Ab−meansignal with Ab)/(meansignal without Ab)]×100. A dotted line indicates 50% neutralization; however, IC50 values shown in the adjacent table were derived from nonlinear regression analyses and represent the mean value (± SD) of two or more replicate experiments. (B) Table summarizing the characteristics of human mAbs cloned from HPV 16-specific Bmem.
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ppat-1004461-g003: IgG cloned from HPV 16-specific Bmem are potently neutralizing.(A) Serial dilutions of human mAbs cloned from AF488-HPV 16+ Bmem (Ab 1, 2, 3, 5, 6, 8, 12, 13, 16, 17 and 18) were separately incubated in triplicate with a predetermined titer of unconjugated HPV 16 psV (solid pink lines) or BPV psV (negative control, solid black lines). All human mAbs were initially tested at a starting concentration of 50 nM (Fig. S8); however, if Abs neutralized HPV 16 at all dilution points (e.g., Ab 1–8), they were re-tested at a lower starting concentration to obtain precise IC50 values. The anti-HPV 16 murine mAb, H16.V5, and anti-BPV murine mAb, 5B6, served as positive controls for HPV 16 and BPV psV neutralization. HPV 16 and BPV psV are comprised of major and minor capsid proteins encompassing a secreted alkaline phosphatase (SEAP) reporter plasmid. After one hour at room temperature, the Ab-psV mixtures were transferred to 293TT cells and incubated for three additional days at 37°C. The amount of AP activity in the supernatant was then quantified, corrected for background, and expressed as percent neutralization, or the inverse of the AP signal reduction observed in the presence of Ab: [(meansignal without Ab−meansignal with Ab)/(meansignal without Ab)]×100. A dotted line indicates 50% neutralization; however, IC50 values shown in the adjacent table were derived from nonlinear regression analyses and represent the mean value (± SD) of two or more replicate experiments. (B) Table summarizing the characteristics of human mAbs cloned from HPV 16-specific Bmem.

Mentions: To demonstrate that the low number of AF488-HPV 16+ Bmem found in the subjects' samples expressed functional Abs that were, in fact, HPV 16-specific, we evaluated our human mAbs in the aforementioned neutralization assay. We also included murine mAbs H16.V5 and 5B6 as well-characterized positive controls for HPV 16 and BPV neutralization, respectively [25]–[27]. A series of dilutions of each Ab were tested against both unconjugated HPV 16 psV (pink solid lines) and BPV psV (black solid lines) (Fig. 3A). From the neutralization curves it is immediately apparent that none of our human Abs neutralized BPV psV. In contrast, most of the human Abs neutralized HPV 16 psV with potent IC50 values ranging from 0.44 pM–2.98 nM (Fig. 3B). This result thus demonstrates the utility of our Ag-labeling approach for identifying HPV 16-specific Bmem.


Characteristics of memory B cells elicited by a highly efficacious HPV vaccine in subjects with no pre-existing immunity.

Scherer EM, Smith RA, Simonich CA, Niyonzima N, Carter JJ, Galloway DA - PLoS Pathog. (2014)

IgG cloned from HPV 16-specific Bmem are potently neutralizing.(A) Serial dilutions of human mAbs cloned from AF488-HPV 16+ Bmem (Ab 1, 2, 3, 5, 6, 8, 12, 13, 16, 17 and 18) were separately incubated in triplicate with a predetermined titer of unconjugated HPV 16 psV (solid pink lines) or BPV psV (negative control, solid black lines). All human mAbs were initially tested at a starting concentration of 50 nM (Fig. S8); however, if Abs neutralized HPV 16 at all dilution points (e.g., Ab 1–8), they were re-tested at a lower starting concentration to obtain precise IC50 values. The anti-HPV 16 murine mAb, H16.V5, and anti-BPV murine mAb, 5B6, served as positive controls for HPV 16 and BPV psV neutralization. HPV 16 and BPV psV are comprised of major and minor capsid proteins encompassing a secreted alkaline phosphatase (SEAP) reporter plasmid. After one hour at room temperature, the Ab-psV mixtures were transferred to 293TT cells and incubated for three additional days at 37°C. The amount of AP activity in the supernatant was then quantified, corrected for background, and expressed as percent neutralization, or the inverse of the AP signal reduction observed in the presence of Ab: [(meansignal without Ab−meansignal with Ab)/(meansignal without Ab)]×100. A dotted line indicates 50% neutralization; however, IC50 values shown in the adjacent table were derived from nonlinear regression analyses and represent the mean value (± SD) of two or more replicate experiments. (B) Table summarizing the characteristics of human mAbs cloned from HPV 16-specific Bmem.
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ppat-1004461-g003: IgG cloned from HPV 16-specific Bmem are potently neutralizing.(A) Serial dilutions of human mAbs cloned from AF488-HPV 16+ Bmem (Ab 1, 2, 3, 5, 6, 8, 12, 13, 16, 17 and 18) were separately incubated in triplicate with a predetermined titer of unconjugated HPV 16 psV (solid pink lines) or BPV psV (negative control, solid black lines). All human mAbs were initially tested at a starting concentration of 50 nM (Fig. S8); however, if Abs neutralized HPV 16 at all dilution points (e.g., Ab 1–8), they were re-tested at a lower starting concentration to obtain precise IC50 values. The anti-HPV 16 murine mAb, H16.V5, and anti-BPV murine mAb, 5B6, served as positive controls for HPV 16 and BPV psV neutralization. HPV 16 and BPV psV are comprised of major and minor capsid proteins encompassing a secreted alkaline phosphatase (SEAP) reporter plasmid. After one hour at room temperature, the Ab-psV mixtures were transferred to 293TT cells and incubated for three additional days at 37°C. The amount of AP activity in the supernatant was then quantified, corrected for background, and expressed as percent neutralization, or the inverse of the AP signal reduction observed in the presence of Ab: [(meansignal without Ab−meansignal with Ab)/(meansignal without Ab)]×100. A dotted line indicates 50% neutralization; however, IC50 values shown in the adjacent table were derived from nonlinear regression analyses and represent the mean value (± SD) of two or more replicate experiments. (B) Table summarizing the characteristics of human mAbs cloned from HPV 16-specific Bmem.
Mentions: To demonstrate that the low number of AF488-HPV 16+ Bmem found in the subjects' samples expressed functional Abs that were, in fact, HPV 16-specific, we evaluated our human mAbs in the aforementioned neutralization assay. We also included murine mAbs H16.V5 and 5B6 as well-characterized positive controls for HPV 16 and BPV neutralization, respectively [25]–[27]. A series of dilutions of each Ab were tested against both unconjugated HPV 16 psV (pink solid lines) and BPV psV (black solid lines) (Fig. 3A). From the neutralization curves it is immediately apparent that none of our human Abs neutralized BPV psV. In contrast, most of the human Abs neutralized HPV 16 psV with potent IC50 values ranging from 0.44 pM–2.98 nM (Fig. 3B). This result thus demonstrates the utility of our Ag-labeling approach for identifying HPV 16-specific Bmem.

Bottom Line: These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies.Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells.Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation.

View Article: PubMed Central - PubMed

Affiliation: Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

ABSTRACT
Licensed human papillomavirus (HPV) vaccines provide near complete protection against the types of HPV that most commonly cause anogenital and oropharyngeal cancers (HPV 16 and 18) when administered to individuals naive to these types. These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies. However, almost nothing is known about the immunological memory that forms following HPV vaccination, which is required for long-term immunity. Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells. Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation. These findings suggest that the quadrivalent HPV vaccine provides an excellent model for studying the development of B cell memory; and, in the context of what is known about memory B cells elicited by influenza vaccination/infection, HIV-1 infection, or tetanus toxoid vaccination, indicates that extensive somatic hypermutation is not required to achieve potent vaccine-specific neutralizing antibody responses.

Show MeSH
Related in: MedlinePlus