A Single Human Papillomavirus Vaccine Dose Improves B Cell Memory in Previously Infected Subjects

Abstract

Although licensed human papillomavirus (HPV) vaccines are most efficacious in persons never infected with HPV, they also reduce infection and disease in previously infected subjects, indicating natural immunity is not entirely protective against HPV re-infection. The aim of this exploratory study was to examine the B cell memory elicited by HPV infection and evaluate whether vaccination merely boosts antibody (Ab) levels in previously infected subjects or also improves the quality of B cell memory. Toward this end, the memory B cells (Bmem) of five unvaccinated, HPV-seropositive subjects were isolated and characterized, and subject recall responses to a single HPV vaccine dose were analyzed. Vaccination boosted Ab levels 24- to 930-fold (median 77-fold) and Bmem numbers 3- to 27-fold (median 6-fold). In addition, Abs cloned from naturally elicited Bmem were generally non-neutralizing, whereas all those isolated following vaccination were neutralizing. Moreover, Ab and plasmablast responses indicative of memory recall responses were only observed in two subjects. These results suggest HPV vaccination augments both the magnitude and quality of natural immunity and demonstrate that sexually active persons could also benefit from HPV vaccination. This study may have important public policy implications, especially for the older ‘catch-up’ group within the vaccine's target population.

Full text

Research Paper
A Single Human Papillomavirus Vaccine Dose Improves B Cell Memory in
Previously Infected Subjects
Erin M. Scherer
a
, Robin A. Smith
a
, Daniel F. Gallego
a
, Joseph J. Carter
a
, Gregory C. Wipf
a
, Manuela Hoyos
a
,
Michael Stern
b
, Tate Thurston
c
,NathanD.Trinklein
c,1
,AnnaWald
b,d,e,f
, Denise A. Galloway
a,g,
⁎
a
Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
b
Department of Medicine,University of Washington, Seattle, WA 98195, USA
c
Panoply Bio, a division of Active Motif, Carlsbad, CA 92008, USA
d
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA
e
Department of Epidemiology, University of Washington, Seattle, WA 98195, USA
f
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
g
Department of Microbiology, University of Washington, Seattle, WA 98195, USA
abstractarticle info
Article history:
Received 14 March 2016
Received in revised form 24 June 2016
Accepted 27 June 2016
Available online 29 June 2016
Although licensed human papillomavirus (HPV) vaccines are most efficacious in persons never infected with
HPV, they alsoreduce infection and disease in previously infected subjects, indicating naturalimmunity is not en-
tirely protective against HPV re-infection. The aim of this exploratory study was to examine the B cell memory
elicited by HPV infection and evaluate whether vaccination merely boosts antibody (Ab) levels in previously in-
fected subjects or also improvesthe quality of B cell memory.Toward this end, the memory B cells(Bmem) of five
unvaccinated, HPV-seropositive subjects were isolated and characterized, and subject recall responses to a single
HPV vaccine dose were analyzed. Vaccination boosted Ab levels 24- to 930-fold (median 77-fold) and Bmem
numbers 3- to 27-fold (median 6-fold). In addition, Abs cloned from naturally elicited Bmem were generally
non-neutralizing, whereas all those isolated following vaccination were neutralizing. Moreover, Ab and
plasmablast responses indicative of memory recall responses were only observed in two subjects. These results
suggest HPV vaccination augments both the magnitude and quality of natural immunity and demonstrate that
sexuallyactive persons could also benefit from HPVvaccination. This study may have important public policy im-
plications, especially for the older ‘catch-up’group within the vaccine's target population.
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords:
Human papillomavirus
Vaccine
Memory B cells
1. Introduction
Approximately 5% of the global cancer burden, including cervical,
anal, oropharyngeal, vaginal, vulvar, andpenile cancers, are attributable
to human papilloma virus (HPV) infections (de Martel et al., 2012).
There are three approved HPV vaccines that protect against those HPV
types that most commonly cause cancer, HPV 16 and 18 (de Sanjose
et al., 2010): the bivalent HPV (bHPV) vaccine against types 16 and
18; quadrivalent HPV (qHPV) vaccine against types 16, 18, 6, and 11;
and the recently licensed nine-valent HPV (nHPV) vaccine against
types 16, 18, 6, 11, 31, 33, 45, 52, and 58. In the US, a three-dose HPV
vaccine schedule is recommended for adolescents but approved for a
larger age range of women (9–26 years of age) and men (9–21 years
of age; qHPV and nHPV vaccines only).
The HPV vaccines are most efficacious in persons who have not been
infected with the vaccine types (Garland et al., 2007, FUTURE II Study
Group, 2007, Paavonen et al., 2009, Joura etal., 2015). Thus, they are pri-
marily targeted to adolescents in an effort to immunize prior to HPV ex-
posure through sexual activity. The putative mechanism of protection is
via type-restricted anti-L1 neutralizing antibodies (Abs) that block viral
entry (Kwak et al., 2011). Likely as a result of these type-restricted Ab
responses, the vaccines provide little cross-protection against heterolo-
gous HPV types and only limited protection against types that share
high L1 nucleotide sequence homology with vaccine types, e.g., HPV
31 and 16 or HPV 45 and 18 (Bernard et al., 2010, Kwak et al., 2011).
While most sexually active adults have already been infected with
some HPV types, it remains unclear whether natural immunity is suffi-
cient to protect these persons against re-infection, i.e., autoinoculation
or new infections of the same type. Natural history studies have
shown that high levels of infection-elicited serum Abs are associated
with protection against same-type re-infections (Ho et al., 2002,
Safaeian et al., 2010, Wentzensen et al., 2011, Beachler et al., 2016). A
prior study also found that both previously infected and previously
EBioMedicine 10 (2016) 55–64
⁎Corresponding author at: Human Biology Division, Fred Hutchinson Cancer Research
Center, Seattle, WA 98109, USA.
E-mail address: dgallowa@fredhutch.org (D.A. Galloway).
1
Present address: TeneoBio, Menlo Park, CA 94025, USA.
http://dx.doi.org/10.1016/j.ebiom.2016.06.042
2352-3964/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Contents lists available at ScienceDirect
EBioMedicine
journal homepage: www.ebiomedicine.com

vaccinated subjects generated anamnestic Ab responses following a sin-
gle qHPV vaccine dose, indicating that both natural infection and vacci-
nation elicit memory B cells (Bmem) (Olsson et al., 2007). At the same
time, a number of independent studies have shown reduced incidence
of same-type re-infection or disease in seropositive subjects that re-
ceived an HPV vaccine compared to those that received placebo, sug-
gesting that natural immunity is not entirely protective (Castellsague
et al., 2011, Olsson et al., 2009, Szarewski et al., 2012). Moreover,
there is no information as to what level of Abs is protective and little in-
formation as to whether qualitative differences exist between vaccine-
and infection-elicited immunity. Therefore, to understand more about
the immunity natural HPV infection imparts, HPV 16-specific Bmem
responses were examined in subjects with serological evidence of HPV
16 infection before and after they received a single HPV vaccine dose.
2. Materials and Methods
2.1. Research Subjects and Study Design
To characterize the Bmem elicited by natural HPV 16 infection and
test binding Ab, neutralizing Ab, plasmablast, and Bmem responses to
a single HPV vaccine dose, ten healthy women, aged 27–45 years,
were enrolled into an unblinded pilot study. At entry, the women
reported ≥five heterosexual lifetime partners and were HPV 16
seropositive.
Participants of this group were randomized at day 0 by block design
to either receive a single qHPV vaccine dose (n= 5) or serve as non-im-
munized controls (n = 5). Blood samples (~60 ml) were collected from
these subjects at month −6; day 0 (± 1 month); week 1 (± 1 day);
month 1 (±1 week); and month 6 (±1 month). On the day of vaccina-
tion, the blood draw preceded the immunization. Institutional Review
Boards at both the University of Washington and Fred Hutchinson Can-
cer Research Center approved study protocol and the participants pro-
vided written informed consent.
2.2. Screen for HPV 16 Seropositivity
An anti-L1 IgG binding assay using glutathione-S-transferase
(GST)-HPV L1 fusion proteins was performed on a BioPlex (Bio-
Rad Laboratories, Inc.) with magnetic beads as previously described
(Katzenellenbogen et al., 2015). Controls included the titration of
serum from a high titer vaccinee that had been previously identified
and the international standard HPV 16 serum (10 Units/ml, U/ml).
To convert median fluorescent intensity (MFI) values to U/ml a stan-
dard curve was generated using the net MFIs (after subtracting the
MFI of beads coated with GST) of the control using the sigmodal
dose response program (GraphPad Prism) with weighting (1/Y
2
).
Interpolated values were calculated from the standard curve and inter-
polated values were converted to U/ml using the formula: U/ml
(test) = 10 U/ml × interpolated value (standard)/interpolated value
(test).
To screen subjects, sera were tested at dilutions of 1:200, 1:400 and
1:800. A concentration was calculated for each dilution and the results
averaged. A cutoff of 3 U/ml was selected on the basis that it was
three standard deviations above the mean value obtained for a panel
of sera from virgins. Following vaccination, sera were initially tested at
1:200, 1:2000 and 1:20,000. Based upon the results of that test sera
were retested using 1:2 dilutions (3) starting at 1:200, 1:2000 or
1:10,000.
2.3. Flow Cytometry
To identify HPV 16-specific Bmem, Alexa Fluor 488 (AF488)-labeled
HPV 16 pseudoviruses (psV) or negative control AF488-labeled bovine
papillomavirus (BPV) psV were generated as previously described
(Scherer et al., 2014). Samples were also enriched, stained, and sorted
the same as before, except that only 1/10th of each enriched sample
was used for AF488-BPV labeling.
To identify and isolate single plasmablasts, peripheral blood mono-
nuclear cell (PBMC) samples were rapidly thawed in pre-warmed,
heat-inactivated FBS. These samples were then washed and counted
in PBS and stained with Live/Dead Violet viability dye (Life Technolo-
gies) for 30 min. To stain cell surface receptors, samples were washed
and re-suspended in 2% FBS-PBS and incubated with anti-CD3 V500
(clone UCHT1; RRID:AB_10612021), anti-CD19 APC-Cy7 (SJ25-C1;
RRID:AB_396873), anti-CD20 PerCP-Cy5.5 (clone 2H7; RRID:AB_
1727451), anti-CD27 PE-Cy7 (clone M-T271; RRID:AB_1727456), anti-
CD38 APC (clone HIT2; RRID:AB_398599), and anti-IgD PE (clone IA6-
2; RRID:AB_396114) for 30 min. Samples were again washed and re-
suspended in 2% FBS-PBS, placed on ice, and protected from light until
fluorescence-activated cell sorting (FACS). All staining was conducted
with pre-optimized amounts of reagents and with samples on ice and
protected from light. All Abs, including those used for Bmem staining,
were purchased from BD Biosciences. Samples were analyzed with a
FACSAria II cell sorter (BD Biosciences), and plasmablasts were isolated
in single cell sort mode. Cells were sorted into PCR plates containing 8 μl
of ice-cold lysis buffer [0.425 × RNase-free PBS (Life Technologies),
10 mM dithiothreitol, and 16 U RNasin (Promega)] and stored at
−80 °C. For kinetic analysis, 1 × 10
6
PBMC per sample were used for
staining.
2.4. Ab Sequence Analysis
To analyze the number of potential nucleotide mutations and amino
acid changes from germline, as well as variable gene usage of each
human monoclonal Ab (mAb) cloned in this study, heavy and light
chain variable region sequences obtained by Ab cloning weresubmitted
to V-QUEST (Brochet et al., 2008). Default parameters were used except
that insertions and deletions were also searched and nucleotide muta-
tions and amino acid changes that overlapped with ‘n’nucleotides or a
given diversity or joining gene were not counted.
2.5. Pseudovirus Neutralization Assay
Experiments to generate HPV 16, HPV 31, and BPV psV comprised of
the L1 and L2 (major and minor) capsid proteins of these respective vi-
ruses encapsidating a secreted alkaline phosphatase (SEAP) reporter
gene were conducted as previously described, as was the psV neutrali-
zation assay in 293TTcells (RRID: CVCL_1D85), with the following spec-
ifications (Scherer et al., 2014, Buck et al., 2005):
To evaluate the levels of circulating neutralizing Abs in each subject
over the course of the study, plasma samples collected at each time
point were tested in monoplicate against HPV 16 and BPV psV, starting
at a final plasma dilution of 1:100 in PBS and continuing as a four-fold
dilution series in assay media. Sample controls included psV only in
media and media only, and psV neutralization controls included dilu-
tion series of H16·V5 and 5B6 supernatants for HPV 16 and BPV psV, re-
spectively. The monoplicate experiments were repeated at least twice
for each subject time point. The signal obtained from the media only
controls was subtracted from the signals of other controls and samples,
and the percent neutralization was calculated using the following
formula: (Abs 405nm
psV only
−Abs 405nm
Ab/plasma + psV
)/(Abs
405nm
psV only
) * 100. The theoretical dilution at which each plasma
sample exhibited 50% neutralization, or IC
50
, was determined by
nonlinear regression analysis of the neutralization curve using the log
(inhibitor) vs. response formula (GraphPad Prism).
Each of the mAbs was screened for neutralization activity against
HPV 16 psV and BPV psV in monoplicate, once, starting at a final mAb
concentration of 50 μg/ml in PBS and continuing as a three-fold dilution
series in assay media. The same controls were used. If a mAb did not
reach 50% neutralization at a concentration of 50 μg/ml, it was regarded
as non-neutralizing. For any mAb with ≥50% neutralization, the assay
56 E.M. Scherer et al. / EBioMedicine 10 (2016) 55–64

was repeated in triplicate starting at an appropriate mAb concentration
(typically 200 pM and continuing over a three-fold dilution series). For
those mAbs that exhibited binding to HPV 31 L1, neutralization activity
to HPV 31 psV was examined in parallel with HPV 16 psV, using H31.A6
as a positive control for HPV 31 psV neutralization.
2.6. HPV L1 Binding Assay
The test of tissue culture supernatants from Panoply was conducted
in two parts. The standard HPV 16 L1 binding assay (see above, under
Screen for HPV 16 seropositivity) was conducted using tissue culture
supernatant without blocking at a final dilution of 1:3.3. Secondly, su-
pernatants that tested positive in the first part were titrated (dilutions
1:4.4 to 1:1.06 × 10
5
) and tested against HPV 16, 31, 33, 35, 52, 56, 67
and 18 GST-L1 proteins.
Human mAbs were tested in monoplicate (no blocking was required
as these Abs have extremely low non-specific reactivity) at a starting
concentration of 100 nM with five 1:10 dilutions (100 nM–1 pM)versus
HPV 16 GST-L1 and GST as described above. The median fluorescent in-
tensity (MFI) values for GST binding were subtracted from the MFI
values for L1 binding for each well.
2.7. Statistical Analyses
An unpaired, two-tailed student's t-test was used to determine if
there was a significant difference between the mean increase of
Bmem responses ± standard deviation (SD) in the vaccinated versus
control group in Fig. 3.
2.8. Accession Numbers
All mAbs that were expressed and characterized or whose sequences
contributed to data in the Figures or Supplemental data were submitted
to GenBank (Table S1).
3. Results
3.1. Study Design
In order to investigate the B cell memory elicited by natural infec-
tion, ten healthy women, aged 27–45 years, were enrolled in an explor-
atory, unblinded pilot study on the basis of having serum Abs against
HPV 16. This age group was chosen because it falls outside those cur-
rently recommended toreceive the vaccine. Since natural HPV infection
is assumed to mainly elicit type-restricted Ab responses (Stanley et al.,
2012), women were only screened for serum Abs to HPV 16 L1. Howev-
er, multiple HPV infections are possible and occur frequently in healthy,
sexually active women (Clifford et al., 2005). Here, a woman was de-
fined as being seropositive if her anti-HPV 16-L1 serum Ab levels were
three standard deviations above the mean Ab level of uninfected sub-
jects. We screened 52 women and identified 14 who were seropositive
by this definition, corresponding to a HPV 16 seroprevalence rate of 27%.
It should be noted that there is no clinically approved serology test for
HPV 16 Abs. Moreover, natural HPV infection elicits low serum Ab re-
sponses (Stanley et al.,2012) that approach the limit of detection in cur-
rent assays. Therefore, false positives cannot be ruled out.
Five women were randomized to receive a single qHPV vaccine dose
to evaluate recall responses i.e., the ability of memory immune cells to
elicit more rapid responses to antigen than naive cells during a primary
response. The other five women served as unimmunized controls. The
qHPV vaccine has been shown to be safe, efficacious, and immunogenic
in women of this age compared to 18–26 year old women (Einstein et
al., 2011, Munoz et al., 2009, Castellsague et al., 2011). Blood samples
were collected at enrollment (six months prior to vaccination, or
month -6), just prior to vaccination on the same day (day 0), at one-
week post-vaccination (week 1), at one-month post-vaccination
(month 1), and at six months post-vaccination (month 6). From each
blood sample, plasma and PBMC were isolated for Ab measurements
and cellular analyses.
3.2. Ab Responses to Natural Infection and Vaccination
Each of the subjects in the vaccinated group, save one (subject3, ◇),
elicited a robust Ab response to the single qHPV vaccine dose, whether
measured in terms of plasma Ab binding to HPV 16 L1 (Fig. 1a) or plas-
ma neutralization against HPV 16 (Fig. 1b). In contrast, the unvaccinated
control group showed no change in Ab levels or plasma neutralization
activity. It should be noted that subject 3 elicited a similarly weak re-
sponse against the other vaccine types (Fig. S1). Two of the vaccinated
subjects' anti-HPV 16 Ab levels increased 45- and 92-fold at one-week
post-vaccination, suggesting that they elicited anamnestic responses
to vaccination (Fig. 1a; subjects 2 (○)a
nd4(□), respectively). However,
these subjects also elicited neutralizing Ab responses to HPV 18 at one-
week and one-month post-vaccination (Fig. S2). Therefore, it is unclear
whether these subjects, which were HPV 18 seronegative at day 0, sim-
ply elicited rapid vaccine responses or whether they had been previous-
ly co-infected with HPV 16 and 18 and generated recall responses to
both types. The other vaccinated subjects' Ab levels to HPV 16 and 18 in-
creased at one-month post-vaccination (Fig. 1 and Fig. S2), indicative of
primary responses to vaccination.
3.3. Plasmablast Responses to Vaccination
When the magnitude of the plasmablast response was assessed
for the day 0, week 1, and month 1 PBMC samples using
immunophenotyping and flow cytometry (Fig. S3), subjects 2 (○)and
4(□) showed a 6- and 41-fold increase in plasmablast frequencies
and a 5- and 36-fold increase in plasmablast numbers at one-week
post-vaccination, respectively (Fig. 2 a and b). Subject 3 (◇) elicited
an unusual plasmablast response, in that it did not peak at one-week
post-vaccination. As this subject did not elicit a strong Ab (see above)
or Bmem response (see below), her plasmablast response was likely
not HPV 16-specific. In fact, the plasmablast phenotype was originally
based on activation status, not antigen (Ag) specificity per se
(Wrammert et al., 2008). To confirm that the peak plasmablast re-
sponses were HPV 16 specific, Abs were cloned and recombinantly
expressed from plasmablasts that were single cell sorted from a subject
with a robust plasmablast response (subject 2, (○). Out of 18 IgG, eight
(44%), wereHPV 16-specific (Fig. S4). Moreover, as the immunoglobulin
sequences amplified from the plasmablasts of subjects 2 and 4 are so-
matically mutated and class-switched (Table S2), it suggests they de-
rivedfromBmem(Chiu et al., 2013, Priyamvada et al., 2016).
3.4. HPV 16-specific Bmem Responses to Natural Infection and Vaccination
Bmem responses were then evaluated, and HPV 16-specificBmem
isolated for further analysis. We previously found HPV 16-specific
Bmem in the blood of HPV vaccinees using a combination of Ag labeling
with AF488-HPV 16 psV and flow cytometry-based immunophenotyping
to identify CD3
−
CD19
+
CD20
+
CD27
+
IgD
−
AF488-HPV 16-psV
+
Bmem
(Scherer et al., 2014). Similar methods were applied here to identify
and isolate single HPV 16-specific Bmem from day 0 and month 1 samples
by FACS (Fig. 3a). To improve the efficiency of finding such rare Ag-
specific cells, PBMC were first enriched for B cells. As before, AF488-
BPV psV were used as a negative control to confirm the specificity of
staining and establish sorting gates. PsV are comprised of L1 and L2
proteins self-assembled around a reporter plasmid (Pastrana et al., 2004).
Statistically significant Bmem responses were noted at one-month
post-vaccination in all subjects of the vaccinated group except subject
3(◇), corresponding to a 4- to 26-fold change in the frequencies of
Ag-specific Bmem (p = 0.04) (Fig. S5a and b) and a 3- to 27-fold change
in the numbers of Ag-specific Bmem (p = 0.026) (Fig. 3bandc).
57E.M. Scherer et al. / EBioMedicine 10 (2016) 55–64

3.5. Characteristics of Abs Cloned From AF488-HPV 16
+
Bmem
To learn more about the diversity of the Bmem responding to vacci-
nation, heavy and light chains from AF488-HPV 16
+
Bmem that had
been single cell sorted from the vaccinated group were amplified and
sequenced. Bmem expressed IgA, IgG and IgM Abs with IgM
+
Bmem
enriched in the month 1 response (Fig. 4a). Collectively, the paired
IgA, IgG and IgM sequences utilized 29 different immunoglobulin
heavy chain variable (IGHV) genes between the vaccinated and control
groups (Fig. 4a). Fourteen IGHV genes were shared among individuals;
thus approximately half were unique to a given individual. Only two
IGHV genes were observed in more than half of the subjects (3–23
and 3–33). Five IGHV genes were observed only at day 0, whereas near-
ly three-fold more IGHV genes were observed only at month 1 (Fig. 4c).
To understand if there was an increase in somatic mutations post-
vaccination that would be indicative of pre-existing Bmem re-entering
germinal center reactions, paired IgG sequences were examined for
the number of nucleotide mutations (Fig. 4d) and amino acid changes
(Fig. 4e) from germline. For two of the three subjects, we found little
change in the somatic hypermutation level of IgG sequences on day 0
compared to month 1. In contrast, IgG sequences cloned from Bmem
of subject 1 unexpectedly exhibited a decrease in somatic hypermutation
from day 0 to month 1, which may reflect naïve cells being recruited
into the response. The same results were observed when comparing
the number of amino acid changes from germline.
3.6. Clonally Related Bmem
To learn more about the diversity and dynamics of the B cell clones
that responded to vaccination in seropositive subjects, paired Ab se-
quences resulting from the original amplifying PCR reactions were
analyzed to identify those that potentially derived from the same B
cell lineage and thus shared the same clonotype (Table S2). Here, se-
quences are defined as sharing the same clonotype if they have the
same heavy and light chain variable and joining gene usages, as well
as identical CDR3 lengths. The following was found: First, the response
to vaccination was at least polyclonal, for there was more than one
clonotype responding at one-week or one-month post-vaccination
within a given subject that were HPV 16-specific based on available
neutralization and binding data. Second, the sequences representing
each lineage appeared to be somatic variants as most exhibited different
numbers of nucleotide mutations or amino acid changes from germline
and/or substitutions to CDR3 residues in the heavy and/or light
chains. Third, shared clonotypes were observed between the week 1
plasmablasts and month 1 Bmem in subjects 1, 2, and 4, implying that
pre-existing Bmem clones were being expanded as both plasmablasts
and Bmem in response to vaccination. In only one case was a shared
clonotype found between day 0 and month 1 likely due to low frequen-
cies of HPV 16-specific Bmem at day 0. In this case, both Bmem exhibit-
ed cross reactivity to HPV 16 and 31 (discussed below). Interestingly, as
for the IgG above, no overall trendtoward increasing variable gene mu-
tations with time was found for the clonotypes (Fig. S6). Instead, de-
pending on the clonotype, increasing, decreasing, or no change in
variable gene mutations was found. Thus, in addition to the afore-
mentioned suggestion that naïve B cells were being drawn into the
one-month post-vaccination response, these clonotype data imply
that month 1 Bmem may also derive from less somatically mutated
Bmem precursors that were not isolated as a result of their rarity or
our sorting approach, which excluded IgD
+
IgM
+
Bmem. Indeed,
IgD
+
IgM
+
Bmem have been shown to be less somatically mutated
and more long-lived than class-switched Bmem (Tangye and Good,
2007, Pape et al., 2011).
Fig. 1. A single vaccine dose boostedAb levels 77-fold (median) in the vaccinated group. (a) Anti-HPV 16 L1 plasma Ab levels (±SD; n ≥3 independent experiments) were measured for
different time points (month -6 (M-6), day 0 (D0), week 1 (W1), month 1 (M1), and month 6 (M6)) using a binding assay and expressed in U/ml relative to an international serum
standard. (b) Neutralizing HPV 16 Ab levels were measured using a 293TT psV assay and expressed as the plasma dilution at which half-maximal inhibition (IC
50
) occurred (±SD;
n≥2 independent experiments). Given that the dilution series started at a 100-fold plasma dilution, any neutralization curves that did not reach 50% neutralization or for which the
IC
50
was b100 are plotted below the dashed baseline. Symbols in (a) and (b) are linked to the subject ID as shown in the figure legend and remain constant between figures.
58 E.M. Scherer et al. / EBioMedicine 10 (2016) 55–64

3.7. Binding Properties of Abs Cloned From AF488-HPV 16
+
Bmem
In order to compare the binding affinities of Bmem elicited by natu-
ral immunity to those elicited in response to vaccination, paired heavy
and light chain variable region sequences were cloned into correspond-
ing heavy or light chain expression vectors from 21 IgG
+
Bmem sorted
at day 0 from the vaccinated and control groups and from 21 Bmem
sorted at month 1 from the vaccinated group. For the day 0 samples,
every IgG possible wascloned. For the month 1 samples, every IgG pos-
sible was cloned from every subject but subject 2, who had 33 paired
IgG. From this subject a representative sample of IgG was chosen to
bring the final count up to 21. The resulting vectors were then co-
transfected in mammalian cells according to their original pairing in
order to recombinantly express the IgG as mAbs.
Initially, each mAb was screened against HPV 16 L1 and other related
(e.g., HPV 31 and 33) or relevant (e.g., HPV 18) HPV types. If a mAb ex-
hibited reactivityto another type (e.g., HPV 31), this psV type was incor-
porated into our neutralization experiments. Remarkably, mAbs cloned
from pre-existing naturally elicited Bmem at day 0 generally bound very
poorly to HPV 16 L1 even when tested at 100 nM (except HPV16.19 and
HPV16.13), whereas most of the mAbs cloned from Bmem at one-
month post-vaccination exhibited high binding levels at this concentra-
tion (except HPV 16.47, HPV16.41, and HPV 16.35; Fig. 5a, note the
scales).
Each mAb was then tested against psV and BPV psV in monoplicate
alongside positive controls, using a high starting mAb concentration of
50 μg/ml (~333 nM). If 50% neutralization was not achieved at this con-
centration, the mAb wasconsidered non-neutralizing. If a mAb exhibit-
ed ≥50% neutralization, the assay was repeated in triplicate, and
neutralization curves were fitted by non-linear regression analysis
to obtain IC
50
values (Fig. 5b). MAbs cloned from naturally elicited
Bmem at day 0 were found to be generally non-neutralizing (except
HPV16.19), whereas all of the mAbs cloned from Bmem elicited one-
month post-vaccination were neutralizing. Even if mAbs from subject
2 are excluded, which were not randomly selected, as well as mAbs
from control subjects, so as eliminate any bias that may result from dif-
ferences between subjects in each group, only 10% (1/10) of the mAbs
isolated at day 0 were neutralizing, whereas 100% (14/14) of subject-
matched mAbs isolated one-month post-vaccination were. The finding
that the naturally elicited mAbs were generally non-neutralizing was
particularly surprising given that the same labeling methods, gating,
and lots of reagents were used to identify and isolate the AF488-HPV
16
+
Bmem, in addition to samples being batched together for flow cy-
tometry experiments. Furthermore, in the case of subjects 1 and 4, the
AF488-HPV 16 labeling was well separated from background (Fig. S7),
making it improbable that all Bmem sorted from these samples repre-
sented noise.
3.8. Cross-reactive Antibodies Cloned From Bmem
The exception among the otherwise non-neutralizing and non-bind-
ing day 0 mAbs, HPV16.19, is also notable in that it neutralizes HPV 31
psV at a concentration approximately an order of magnitude lower
than that with which it neutralizes HPV 16 (IC
50 HPV 31
= 1.9 pM; Fig.
S8). Furthermore, a Bmem from the same subject at month 1 yielded a
mAb, HPV16.47, which neutralizes HPV 31 psV 2200-fold more potently
than HPV 16 psV (IC
50HPV 31
0.22 pM vs. IC
50HPV 16
0.48 nM; Fig. S7).
Thus, this subject was suspected to have also had pre-existing HPV 31
Abs. To test this, her enrollment plasma was re-evaluated for binding
to HPV 16 L1 and HPV 31 L1 in an L1 binding assay and her anti-HPV
31 plasma Ab levels were found to be 4.5 fold higher than her HPV 16
Ab levels (Fig. S9). This finding indicated that subject 1 was either pre-
viously co-infected with HPV 16 and HPV 31, or that her HPV 31 serum
Abs cross-reacted with HPV 16 L1 in our assay. The fact that this subject
elicited an appreciable Bmem response to vaccination at month 1 (3-
fold that of her day 0 numbers), but a low plasmablast response at
week 1 (1.2-fold that of her day 0 numbers) and a slower Ab response
suggests the latter. Moreover, all of the remaining mAbs cloned from
subject 1's Bmem at month 1 bound and neutralized HPV 16 in the ab-
sence of HPV 31 reactivity. Taken together, these data indicate that sub-
ject 1 generated a primary response to vaccination. It will be interesting
to identify the cross-reactive epitopes.
Subject 5 also elicited a robust Bmem response to vaccination at
month 1 (5.2-fold thatof her day 0 numbers),but did not generaterecall
responses. However, her day 0 plasma Abs did not bind strongly to any
other type in the alpha 9 papillomavirus species, i.e., the species of types
with 60–70% nucleotide identity to the HPV 16 L1 open reading frame
(de Villiers et al., 2004). Therefore, it is possible that she experienced a
prior HPV 16 infection, but did not generate an effective Bmem re-
sponse. Subject 3 who did not elicit a strong Ab response to any vaccine
type or a Bmem response to HPV 16 may reflect the response of persons
who require more than one vaccine dose to seroconvert.
4. Discussion
In this study we investigated whether a single HPV vaccine dose im-
proved the B cell memory of persons with pre-existing Abs to HPV 16.
We found that a single qHPV dose administered to HPV 16-seropositive
women boosted Ab levels 24- to 930-fold (median 77-fold) at one-
month post-vaccination; generated what appeared to be primary re-
sponses in three of the five vaccinees; and elicited HPV 16-specific
Bmem that were somatically mutated, class-switched and expressed
neutralizing Abs.
Therefore, pre-existing, infection-elicited Ab levels, were lower than
vaccine-elicited Ab levels, which is known (Stanley et al., 2012). More-
over, we only observed potential recall responses in two of the five vac-
cinees. The study that first noted anamnestic Ab responses indicative of
Fig. 2. Two subjects elicited rapid plasmablast responses following a single vaccine dose.
Frequencies (a) and numbers (b) of plasmablasts per million PBMC at D0, W1, and M1
were normalized to D0 responses.
59E.M. Scherer et al. / EBioMedicine 10 (2016) 55–64

Bmem following a single vaccine dose in previously infected subjects
also reported a high degree of variability in subject responses (Olsson
et al., 2007). The quality of B cell responses generated by natural infec-
tion must thus vary from person-to-person.
We also found that most infection-elicited Bmem expressed non-
neutralizing Abs despite beingclass-switched and somatically mutated.
Given that vaccinated subjects possessed low levels of neutralizing ac-
tivity in their plasma at day 0 (Fig. 5b) and were included in the study
on the basis of having low levels of HPV 16 Abs in their serum, we
know they elicited HPV 16-specific plasma cells. We assume that
these subjects also elicited HPV 16-specific Bmem. If this assumption
is true, there are two possible reasons for the finding that the AF488-
HPV 16
+
Bmem expressed non-neutralizing mAbs: The non-neutraliz-
ing mAbs were cloned from Bmem that were not truly HPV 16-specific,
but resulted from noise in the method. Therefore, we did not efficiently
find their rare HPV 16-specific Bmem. The discovery of one day 0 Bmem
that expresses a potent neutralizingHPV 16/31 mAb supports this inter-
pretation. However, the explanation that the remaining AF488-HPV
16
+
Bmem at day 0 are all non-specific seems improbable based upon
the frequency of neutralizing mAbs cloned from Bmem at month 1
post-vaccination using the same methods and reagents, as well as the
frequency of neutralizing mAbs isolated from AF488-HPV 16
+
Bmem
at one-month post-vaccination in our previous study (7/11 mAbs
or 64%) (Scherer et al., 2014). Furthermore, we find that some non-
neutralizing mAbs do exhibit weak binding to L1 (e.g., HPV16.13, sub-
ject 2_C7, and potentially HPV16.14, HPV16.15, HPV16.25, and
HPV16.30).
An alternative explanation is that the B cell receptor avidities of the
naturally elicited Bmem were sufficient to bind fluorescently labeled
HPV 16 psV,but their corresponding affinities as soluble Abs were insuf-
ficient to bind appreciably to HPV 16 L1 or to neutralize HPV 16 psV. In
this scenario, the polyclonal response may have resulted from plasma
cells producing an aggregate of similarly weak Abs. If this were true, it
would indicate a fundamental difference in the quality of Bmem elicited
by natural infection versus those elicited by vaccination. The finding
that the avidities of naturally elicited HPV-specific serum Abs are signif-
icantly lower than those of bHPV vaccine-elicited serum Abs further
supports this interpretation (Scherpenisse et al., 2013).
It is unclear how much overlap exists between Bmem, plasmablast
and serum Ab repertoires, as studies examining this subject are scarce
in the literature (Purtha et al., 2011, Lavinder et al., 2014). Purtha and
colleagues found that Bmem repertoires responding to West Nile virus
infection in mice possessed additional specificities beyond the domi-
nant clonotype expressed by plasma cell and serum Ab repertoires, sug-
gesting that the Bmem repertoire is better adapted to respond to viral
variants (Purtha et al., 2011). Lavinder et al. found little overlap be-
tween the Bmem and serum Ab repertoires or plasmablast and serum
Ab repertoires elicited in response to tetanus toxoid boosting, as only
Fig. 3. A singlevaccine dose elicitsa robust Bmem response in HPV 16-Exposed subjects.(a) Dot plots show thegating strategy usedto identify HPV 16-specific Bmem atday 0 and month 1
in a representative subjectfrom the vaccinated and control groups (subjects 2 and 7, respectively). Flow cytometry data were first gated to include lymphocytes (FSC vs. SSC), to exclude
doublets and dead cells, and to include B cells (CD3
−
CD19
+
cells). The dot plots in the first and third columns showthe frequency of bulk Bmem(CD27
+
IgD
−
, red gate) in each sample,
whereas dot plots in the second and fourth columns show the frequency of those Bmem that bound to AF488-labeled HPV 16 psV. Numbers associated with the quadrants and insets
indicate the percentage of cells. (b and c) Graphs illustrate the change in HPV 16-specific Bmem numbers between day 0 and month 1 for subjects in each of the indicated groups (b),
as well as the increase in AF488-HPV 16
+
Bmem numbers between day 0 and month 1 (±SD, unpaired, two-tailed student's t-test) (c).
60 E.M. Scherer et al. / EBioMedicine 10 (2016) 55–64

Fig. 4. Abs cloned from both infection- and vaccine-elicited Bmem include class-switched Abs, utilize diverse variable genes, and are somatically mutated. (a) The isotype distribution of
paired Ab sequences amplifiedfrom AF488-HPV 16
+
Bmem is shown. These Bmem were singlecell sorted from D0 and M1 samplesof the vaccinated group. Theratio below each graph
indicatesthe number ofpaired Ab sequences obtained out of thetotal heavy chain sequences (both paired and unpaired). Onlythose heavy chains with high qualitysequences that are
predicted to be productive and do not contain large deletions (N20 nucleotides) were included. The two unpaired heavy chains amplified from subject 2′s D0 Bmem w ith IgA and IgM
reverse primers yielded either unproductive or indiscernible sequences and thus were not included in the total for this subject at this time point. (b and c) Immunoglobulin heavy
chain variable (IGHV) gene usage of paired Ab sequences isolated from IgA/IgG/IgM Bmem of all subjects (b) or vaccinated subjects only (c) were determined by sequence analysis.
Stacked bar graphs show the number of subjects whose D0 and/or M1 Bmem utilized that particular IGHV gene [e.g., the IGHV gene 2-70D was utilized by D0 Bmem in one subject
and M1 Bmem in two subjects]. (d and e) The number of nucleotide mutations (d) and amino acid changes (e) observed in the heavy chain variable gene, light chain variable gene, or
in both chains (total) of paired IgG Bmem (SD) at D0 and M1 are shown for each responding subject in the Vaccinated group and at D0 for theControl group as a point of comparison.
61E.M. Scherer et al. / EBioMedicine 10 (2016) 55–64

Fig. 5. MAbs isolated from Bmem one-month post-vaccination exhibit markedly improved binding and neutralization activities compared to those from pre-existing naturally elicited
Bmem. (a) MAbs cloned from naturally elicited Bmem inthe vaccinated group at D0 (HPV16.13-HPV16.22); naturally elicited Bmem in the control group at D0 (HPV16.23-HPV16.33);
or from Bmem elicited one-month post-vaccination (HPV16.34-HPV16.54) exhibit differing levels of binding to HPV 16 L1 coated beads in a binding assay. The amount of mAb bound
is represented as the median fluorescence intensity (MFI) of a fluorescently conjugated secondary Ab. (b) Neutralization potencies of the subjects' plasmas at the time the mAbs were
cloned, as well as th e potencies of the mA bs themselves, are shown. The subject from whom each mAb was iso lated is also noted . Warm colored boxes represent increasing
neutralization potency and gray boxes indicate no neutralization (IC
50
N50 μg/ml or ~333 nM).
62 E.M. Scherer et al. / EBioMedicine 10 (2016) 55–64

a small fraction of Bmem and plasmablast clonotypes identified through
next generation sequencing overlapped with Ab clonotypes found in
serum by proteomics analysis (Lavinder et al., 2014). Here, we noted
highly expanded clonotypes shared between Bmem and plasmablast
repertoires using single-cell approaches, which Franz et al. also ob-
served in response to tetanus toxoid vaccination (Franz et al., 2011). It
would be interesting to see if the highly expanded clonotypes in our
study were among the predominant serum Ab clonotypes, given that
highly expanded plasmablast clonotypes have been previously found
at elevated levels in serum and in the long-lived plasma cell repertoire
of human bone marrow (Lavinder et al., 2014, Halliley et al., 2015).
Collectively, the results from this study indicate that vaccination
augmentsnatural HPV immunity by boosting Ab levels and Bmem num-
bers, as well as by potentially improving the quality of Bmem that are
elicited. Thus, persons previously infected with HPV vaccine types
could also benefit from vaccination. Whether vaccination will lessen
the recurrence of HPV infections in persons who have previously been
treated for neoplastic lesions is unknown. Analysis of both Bmem
and serum Abs in future vaccine trials in previously infected sub-
jects would be valuable for identifying correlates of protection
against HPV re-infection. The contribution of vaccine-elicited T
cells to efficacy is likely negligible, given that HPV infection of
basal epithelial cells is temporally and spatially separated from L1
protein expression in the highly differentiated epithelia at/near
the surface (Kwak et al., 2011).
The major strengths of this study are that we have described previ-
ously unknown characteristics of naturally elicited Bmem and shown
that vaccination improves the quality of B cell memory in previously
infected persons. In addition, we have sampled B cell populations
responding to vaccination longitudinally and interrogated the function-
al properties of the Abs expressed by these cells. The limitations of our
study include small cohort sizes, the omission of AF488-HPV 16
+
IgD
+
-
IgM
+
Bmem from our sorting approach, and the restriction to only a
single HPV type. Our study would also benefit from a parallel next gen-
eration sequencing analysis to improve our sampling depth for finding
clonotypes over time and in different B cell populations.
The high titers of neutralizing plasma Abs following a single dose of
qHPV vaccine in this study and finding that all mAbs isolated from HPV
16-specific Bmem were neutralizing speak to the extraordinary immu-
nogenicity of the HPV vaccines. It is perhaps not surprising then that
the WHO has approved a two-dose qHPV vaccine schedule for 9–
13 year old girls based on immunogenicity studies, or that recent evi-
dence has been presented to suggest that a single dose may be sufficient
for protection (Kreimer et al., 2015). If the serum Ab levels elicited by
such reduced dose schedules do not remain stable at levels near the
minimum Ab level associated with protection in clinical trials, it will
be important to investigate the durability, magnitude, and quality of
the B cell memory elicited by these schedules.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ebiom.2016.06.042.
Funding Sources
This study was supported by NIH/NIAID grant R01 (AI038382) to
D.A.G., K24 (AI 071113) to A.W. and a Walker Immunotherapy Fellow-
ship and STD/AIDS Research Training Fellowship (NIH T32 AI07140)
to E.M.S. These funding sources had no role in the study design; in the
collection, analysis and interpretation of data; in the writing of the man-
uscript; and in the decision to submit the paper for publication.
Conflict of Interest Statement
D.A.G. is a member of Merck's Global Advisory Board for HPV, and
A.W. serves on a Merck DSMB for a clinical trial not related to HPV.
Author Contributions
E.M.S., A.W., and D.A.G. conceived the project. E.M.S. and D.A.G. su-
pervised the project. A.W. supervised the clinic where subjects were re-
cruited and enrolled. E.M.S., J.J.C., N.D.T., A.W., and D.A.G. wrote and
edited the paper. E.M.S., R.A.S., D.F.G., J.J.C., G.C.W., and T.T. performed
experiments. E.M.S., R.A.S., D.F.G., J.J.C., G.C.W., T.T., N.D.T, and D.A.G.
analyzed data. M.S. enrolled and collected samples from study subjects.
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