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

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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.
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 amplified from AF488-HPV 16 + Bmem is shown. These Bmem were single cell sorted from D0 and M1 samples of the vaccinated group. The ratio below each graph indicates the number of paired Ab sequences obtained out of the total heavy chain sequences (both paired and unpaired). Only those heavy chains with high quality sequences 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 with 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 the Control group as a point of comparison.
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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 efcacious 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 ve
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 benet from HPVvaccination. This study may have important public policy im-
plications, especially for the older catch-upgroup 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 (926 years of age) and men (921 years
of age; qHPV and nHPV vaccines only).
The HPV vaccines are most efcacious 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 suf-
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) 5564
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-specic 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 2745 years,
were enrolled into an unblinded pilot study. At entry, the women
reported ve 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 identied
and the international standard HPV 16 serum (10 Units/ml, U/ml).
To convert median uorescent 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-specic 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
uorescence-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 nnucleotides 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-
ications (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 nal 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 nal 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) 5564
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 nal dilution of 1:3.3. Secondly, su-
pernatants that tested positive in the rst 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-specic reactivity) at a starting
concentration of 100 nM with ve 1:10 dilutions (100 nM1 pM)versus
HPV 16 GST-L1 and GST as described above. The median uorescent 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 signicant 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 2745 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-
ned 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 identied 14 who were seropositive
by this denition, 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 ve women served as unimmunized controls. The
qHPV vaccine has been shown to be safe, efcacious, and immunogenic
in women of this age compared to 1826 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 ow 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-specic. In fact, the plasmablast phenotype was originally
based on activation status, not antigen (Ag) specicity per se
(Wrammert et al., 2008). To conrm that the peak plasmablast re-
sponses were HPV 16 specic, 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-specic (Fig. S4). Moreover, as the immunoglobulin
sequences amplied 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-specic Bmem Responses to Natural Infection and Vaccination
Bmem responses were then evaluated, and HPV 16-specicBmem
isolated for further analysis. We previously found HPV 16-specic
Bmem in the blood of HPV vaccinees using a combination of Ag labeling
with AF488-HPV 16 psV and ow 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-specic Bmem from day 0 and month 1 samples
by FACS (Fig. 3a). To improve the efciency of nding such rare Ag-
specic cells, PBMC were rst enriched for B cells. As before, AF488-
BPV psV were used as a negative control to conrm the specicity 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 signicant 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-specic Bmem (p = 0.04) (Fig. S5a and b) and a 3- to 27-fold change
in the numbers of Ag-specic Bmem (p = 0.026) (Fig. 3bandc).
57E.M. Scherer et al. / EBioMedicine 10 (2016) 5564
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 amplied 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 (323
and 333). 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 reect 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 dened 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-specic 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-specic 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;
n2 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 gure legend and remain constant between gures.
58 E.M. Scherer et al. / EBioMedicine 10 (2016) 5564
3.7. Binding Properties of Abs Cloned From AF488-HPV 16
+
Bmem
In order to compare the binding afnities 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 nal 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 tted 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 nding
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 ow 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 nding 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 6070% 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 reect 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 ve vaccinees; and elicited HPV 16-specic
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 ve vac-
cinees. The study that rst 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) 5564
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-specic plasma cells. We assume that
these subjects also elicited HPV 16-specic Bmem. If this assumption
is true, there are two possible reasons for the nding 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-specic,
but resulted from noise in the method. Therefore, we did not efciently
nd their rare HPV 16-specic 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-specic 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 nd 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 sufcient to bind uorescently labeled
HPV 16 psV,but their corresponding afnities as soluble Abs were insuf-
cient 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 nding
that the avidities of naturally elicited HPV-specic 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 specicities 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-specic Bmem atday 0 and month 1
in a representative subjectfrom the vaccinated and control groups (subjects 2 and 7, respectively). Flow cytometry data were rst 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 rst 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-specic 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) 5564
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 ampliedfrom 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 amplied from subject 2s 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) 5564
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 uorescence intensity (MFI) of a uorescently 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) 5564
a small fraction of Bmem and plasmablast clonotypes identied 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 benet 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 efcacy 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 benet from a parallel next gen-
eration sequencing analysis to improve our sampling depth for nding
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 nding that all mAbs isolated from HPV
16-specic 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 sufcient
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.
Conict 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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... HPV vaccination among previously exposed cohorts It is also unclear why vaccine efficacy should differ so substantially between SPDN women (and indeed among women who have undergone ablative surgical procedures [26,27]) compared to women with detectable DNA infection, among whom vaccination has been demonstrated to be ineffective [6,[22][23][24][25], but whose classification as DNA positive would, under the supposition of latency, simply reflect an isolated measurement in an infection whose natural history is characterised by oscillation between periods of active viral shedding and periods of latency/immune control [70][71][72]. One possible answer may be that the immune response observed with L1-based vaccination differs both quantitatively [38], and qualitatively [73], to immune responses occurring as a consequence of natural HPV infection, particularly with respect to the generation of highly-avid neutralising antibodies [73,74]. Such differences may be sufficient to explain the maintenance or prolongation of periods of viral suppression among subjects who have already mounted a competent immune response, or to provide greater protection against HPV infections occurring as a consequence of re-exposure, despite being insufficient to control or reverse active infections. ...
... HPV vaccination among previously exposed cohorts It is also unclear why vaccine efficacy should differ so substantially between SPDN women (and indeed among women who have undergone ablative surgical procedures [26,27]) compared to women with detectable DNA infection, among whom vaccination has been demonstrated to be ineffective [6,[22][23][24][25], but whose classification as DNA positive would, under the supposition of latency, simply reflect an isolated measurement in an infection whose natural history is characterised by oscillation between periods of active viral shedding and periods of latency/immune control [70][71][72]. One possible answer may be that the immune response observed with L1-based vaccination differs both quantitatively [38], and qualitatively [73], to immune responses occurring as a consequence of natural HPV infection, particularly with respect to the generation of highly-avid neutralising antibodies [73,74]. Such differences may be sufficient to explain the maintenance or prolongation of periods of viral suppression among subjects who have already mounted a competent immune response, or to provide greater protection against HPV infections occurring as a consequence of re-exposure, despite being insufficient to control or reverse active infections. ...
Article
Objective: Vaccine efficacy among previously exposed, but currently uninfected women, i.e., those who have serological evidence of a prior human papillomavirus (HPV) infection without corresponding detectable HPV DNA, remains incompletely defined. This meta-analysis assessed the serotype-specific efficacy of prophylactic HPV vaccination against HPV16/18 persistent infection (PI) and cervical intraepithelial neoplasia (CIN) among seropositive, DNA negative (SPDN) women enrolled to randomized controlled trials (RCTs) of HPV L1-based vaccines. Methods: Searches were conducted on 08/16/20 on MEDLINE, Embase, Scopus and CENTRAL. RCTs of L1-based prophylactic bivalent or quadrivalent HPV vaccines, reporting serotype-specific clinical efficacy endpoints in the HPV16/18 seropositive, DNA-negative populations were included. Relative risks (RRs) of 6-month PI (6mPI), 12-month PI (12mPI), CIN1+ and CIN2+ were pooled using a random-effects model. Results: A total of 1,727 citations were reviewed. 8 studies, with a total of 9,569 SPDN participants, met all eligibility criteria. The RR of 6mPI (RR=0.22; 95% confidence interval [CI]=0.08-0.61; p=0.018), 12mPI (RR=0.20; 95% CI=0.05-0.80; p=0.035), CIN1+ (RR=0.13; 95% CI=0.05-0.30; p=0.003) and CIN2+ (RR=0.15; 95% CI=0.04-0.59; p=0.022) was significantly reduced in the vaccinated compared to the unvaccinated group. Conclusion: Our findings suggest high serotype-specific efficacy for HPV vaccination among cohorts of women with evidence of prior HPV16/18 infections, including 87% efficacy (95% CI=70%-95%; p=0.003) against HPV16/18 cervical dysplasia. HPV vaccination is highly effective among uninfected women, regardless of prior exposure history. Trial registration: PROSPERO Identifier: CRD42020206888.
... In a randomized pilot study, 10 women aged 27-45 years who tested positive for HPV 16 were divided into the vaccinated and non-vaccinated groups. In the vaccination group, antibody titers increased by 24-930-fold, and the number of memory B cells increased by 3-27-fold [35]. ...
... This must be interpreted carefully, as it can be misunderstood as the vaccine leading to HPV clearance in women after infection. Clinical studies have demonstrated that vaccination does not eliminate HPV already present in women [12,35,36]. ...
Article
Since the human papillomavirus (HPV) vaccine guidelines were developed by the Korean Society of Gynecologic Oncology (KSGO) in 2011, 2016, and 2019, several recent studies on the efficacy and safety of HPV vaccines in middle-aged women and men have been reported. Furthermore, there has been an ongoing debate regarding the efficacy of the HPV vaccine in women with prior HPV infection or who have undergone conization for cervical intraepithelial neoplasia (CIN). We searched and reviewed studies on the efficacy and safety of the HPV vaccine in middle-aged women and men and the efficacy of the HPV vaccine in patients infected with HPV and those who underwent conization for CIN. The KSGO updated their guidelines based on the results of the studies included in this review.
... In addition, HPV vaccination may provide cross-protection to other HPV types not covered by the vaccine 10 and may also boost the immune response to HPV infection from the same type, thereby providing additional protection from reinfection with the same HPV type. 11,12 ...
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Objectives: Individuals treated for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) are at long-term risk of persistent or recurrent disease despite treatment. This committee opinion aims to summarize and provide evidence-based recommendations for adjuvant human papillomavirus (HPV) vaccination based on available, published literature. Methods: A task force from the ASCCP Practice Committee reviewed current Centers for Disease Control and Prevention (CDC) guidelines and previously published literature about the role of adjuvant HPV vaccination in previously unvaccinated individuals undergoing treatment for CIN2+ and other HPV-related diseases. Results: Current CDC guidelines recommend routine or catch-up HPV vaccination for individuals aged 9 to 26 years, and shared decision making regarding vaccination for individuals aged 27 to 45 years. Multiple published studies suggest a possible benefit for adjuvant HPV vaccination in previously unvaccinated individuals undergoing treatment for CIN2+. Conclusions: The American Society for Colposcopy and Cervical Pathology recommends adherence to current CDC recommendations for vaccination of individuals aged 9 to 26 years and consideration of the possible benefit of adjuvant HPV vaccination during shared decision making for previously unvaccinated individuals aged 27 to 45 years who are undergoing treatment for CIN2+.
... Our lab has in the past studied the quality of B-cell response among the previously infected women after a single dose of HPV vaccine. The study demonstrated that while antibodies cloned from naturally elicited Bmem cells were generally non-neutralizing, those produced after a single dose of vaccination were both neutralizing and of higher titer [71]; thus, suggesting that sexually active persons also could potentially benefit from HPV vaccination. Women, previously exposed to HPV with detectable anti-HPV antibodies prior to vaccination, when immunized with quadrivalent vaccine, had~12-26-fold higher titers of antibodies than HPV-naïve vaccine recipients [72], again suggesting the potential utility of vaccinating women who were HPV positive at baseline. ...
Article
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Infection with human papillomavirus (HPV) is the necessary cause of cervical cancer. Availability of vaccines against HPV makes it a highly preventable disease. HPV vaccines act through type-specific neutralizing antibodies produced by antigen-specific plasma cells known as long-lived plasma cells (LLPC). However, just as any other vaccine, success of HPV vaccine is attributed to the immunologic memory that it builds, which is largely attained through generation and maintenance of a class of B cells named memory B cells (Bmem). Both LLPCs and Bmems are important in inducing and maintaining immune memory and it is therefore necessary to understand their role after HPV vaccination to better predict outcomes. This review summarizes current knowledge of B-cell responses following HPV vaccination and natural infection, including molecular signatures associated with these responses.
... Evaluation of the antibody responses of individuals seropositive and DNA negative at entry into vaccine trials shows a similar anamnestic response following vaccination suggesting that naturally induced recall memory is also quite robust (20)(21)(22). In a seminal study of B cell clones derived following natural infection and after receiving a single vaccine dose, demonstrate differences in both magnitude but also key qualitative differences in the resulting neutralizing antibody capacity (23). Taken together, these data suggest some degree of commonality between specificities of antibodies elicited by natural infection and vaccination but in the latter case are induced to much higher levels. ...
Article
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Human papillomavirus (HPV) is the causative agent of cervical and other cancers and represents a significant global health burden. HPV vaccines demonstrate excellent efficacy in clinical trials and effectiveness in national immunization programmes against the most prevalent genotype, HPV16. It is unclear whether the greater protection conferred by vaccine-induced antibodies, compared to natural infection antibodies, is due to differences in antibody magnitude and/or specificity. We explore the contribution of the surface-exposed loops of the major capsid protein to antigenic domains recognized by vaccine and natural infection neutralizing antibodies. Chimeric pseudoviruses incorporating individual (BC, DE, EF, FG, HI) or combined (All: BC/DE/EF/FG/HI) loop swaps between the target (HPV16) and control (HPV35) genotypes were generated, purified by ultracentrifugation and characterized by SDS-PAGE and electron microscopy. Neutralizing antibody data were subjected to hierarchical clustering and outcomes modeled on the HPV16 capsomer crystal model. Vaccine antibodies exhibited an FG loop preference followed by the EF and HI loops while natural infection antibodies displayed a more diverse pattern, most frequently against the EF loop followed by BC and FG. Both vaccine and natural infection antibodies demonstrated a clear requirement for multiple loops. Crystal modeling of these neutralizing antibody patterns suggested natural infection antibodies typically target the outer rim of the capsomer while vaccine antibodies target the central ring around the capsomer lumen. Chimeric pseudoviruses are useful tools for probing vaccine and natural infection antibody specificity. These data add to the evidence base for the effectiveness of an important public health intervention. IMPORTANCE The human papillomavirus type 16 (HPV16) major virus coat (capsid) protein is a target for antibodies induced by both natural infection and vaccination. Vaccine-induced immunity is highly protective against HPV16-related infection and disease while natural infection associated immunity significantly less so. For this study, we created chimeric functional pseudoviruses based upon an antigenically distant HPV genotype (HPV35) resistant to HPV16-specific antibodies with inserted capsid surface fragments (external loops) from HPV16. By using these chimeric pseudoviruses in functional neutralization assays we were able to highlight specific and distinct areas on the capsid surface recognized by both natural infection and vaccine induced antibodies. These data improve our understanding of the difference between natural infection and vaccine induced HPV16-specific immunity.
... One possible explanation is that low-avidity memory B cells (mBCs), preferentially generated after mucosal infection, can be driven into LLPCs after secondary exposure to antigen by parenteral vaccination, but remain "imprinted" with a generally lower avidity B cell receptor, despite presumably having participated in a secondary GC reaction and therefore a second round of somatic hypermutation. A previous study found that most of the HPV16-VLP monoclonal antibodies (MoAb) generated from infection-induced mBCs were non-neutralizing, while most of the MoAb generated after vaccination were strongly neutralizing 14 . This observation suggests that the antibodies resulting from infection may generally be of lower avidity, despite the fact that HPV16-VLP-specific IgG genes from mBCs induced by infection and vaccination had similar numbers of somatic mutations 14 A second possibility is that the avidity levels measured after vaccination of HPV-exposed women reflect the sum of the lower avidities of the antibodies produced by LLPCs generated from naive B cells after infection and those of higher avidities generated after vaccination. ...
Article
Full-text available
The HPV vaccine has shown sustained efficacy and consistent stabilization of antibody levels, even after a single dose. We defined the HPV16-VLP antibody avidity patterns over 11 years among women who received one- or three doses of the bivalent HPV vaccine in the Costa Rica HPV Vaccine Trial. Absolute HPV16 avidity was lower in women who received one compared to three doses, although the patterns were similar (increased in years 2 and 3 and remained stable over the remaining 8 years). HPV16 avidity among women who were HPV16-seropositive women at HPV vaccination, a marker of natural immune response to HPV16 infection, was significantly lower than those of HPV16-seronegative women, a difference that was more pronounced among one-dose recipients. No differences in HPV16 avidity were observed by HPV18 serostatus at vaccination, confirming the specificity of the findings. Importantly, point estimates for vaccine efficacy against incident, six-month persistent HPV16 infections was similar between women who were HPV16 seronegative and seropositive at the time of initial HPV vaccination for both one-dose and three-dose participants. It is therefore likely that this lower avidity level is still sufficient to enable antibody-mediated protection. It is encouraging for long-term HPV-vaccine protection that HPV16 antibody avidity was maintained for over a decade, even after a single dose.
... However, a recent study has shown that vaccine-induced IgG levels are comparable between subjects that were HPV seropositive or seronegative at the time of vaccination 30 . This seems to be caused by low titers, avidity and neutralizing capacity of antibodies induced by natural infections 31,32 . It is thus very unlikely that the lower responses we observed in adults could be due to pre-existing IgGs that neutralize the VLPs and hamper vaccine immunogenicity. ...
Article
Full-text available
Adult vaccination programs are receiving increasing attention however, little is known regarding the impact of age on the maintenance of the immune response. We investigated this issue in the context of a human papillomavirus (HPV) vaccination program collecting real-world data on the durability of humoral immunity in 315 female subjects stratified according to vaccination age (adolescents and adults) and sampled at early or late time points after the last vaccine dose. HPV-specific IgGs, but not memory B cells, were induced and maintained at higher levels in subjects vaccinated during adolescence. Nonetheless, antibody functions waned over time to a similar degree in adolescents and adults. To shed light on this phenomena, we analyzed quantitative and qualitative properties of lymphocytes. Similar biochemical features were observed between B-cell subsets from individuals belonging to the two age groups. Long term humoral responses toward vaccines administered at an earlier age were comparably maintained between adolescents and adults. The percentages of naïve B and CD4+ T cells were significantly higher in adolescents, and the latter directly correlated with IgG titers against 3 out of 4 HPV types. Our results indicate that age-specific HPV vaccine responsiveness is mostly due to quantitative differences of immune cell precursors rather than qualitative defects in B cells. In addition, our results indicate that adults also have a good humoral immunogenic profile, suggesting that their inclusion in catch-up programmes is desirable.
... Neutralization assays for HPV16 and HPV18 utilized unconjugated PsV, described above, following previously established procedures (Scherer et al., 2014;Scherer et al., 2016;Buck et al., 2005). In brief, 293TT cells were plated in a 96-well plate and incubated for 4 to 6 h at 37 • C. The HPV PsV were incubated with diluted supernatant (1:3.2) in a separate 96-well plate for 1 h at RT. ...
Article
The isolation of human monoclonal antibodies (mAbs) arising from natural infection with human pathogens has proven to be a powerful technology, facilitating the understanding of the host response to infection at a molecular level. mAbs can reveal sites of vulnerability on pathogens and illuminate the biological function of the antigenic targets. Moreover, mAbs have the potential to be used directly for therapeutic applications such as passive delivery to prevent infection in susceptible target populations, and as treatment of established infection. The isolation of antigen-specific B cells from vaccine trials can also assist in deciphering whether the desired B cells are being targeted by a given vaccine. Several different processes have been developed to isolate mAbs, but all are generally labor-intensive and result in varying degrees of efficiency. Here, we describe the development of a cost-effective feeder cell line that stably expresses CD40-ligand, interleukin-2 and interleukin-21. Sorting of single B cells onto a layer of irradiated feeder cells sustained antibody production that permits functional screening of secreted antibodies in a manner that enables subsequent recovery of B cells for recombinant antibody cloning. As a proof of concept, we show that this approach can be used to isolate B cells that secrete antibodies that neutralize human papilloma virus (HPV) from participants of an HPV vaccine study.
Article
Aims: To analyze the consistency between HPV neutralizing antibodies and specific total IgG antibodies in unvaccinated females. Materials & methods: Serum samples from 978 unvaccinated Chinese females aged 9–26 years were measured for antibodies against HPV-16 and HPV-18 using simultaneous pseudovirus-based neutralization assay and ELISA. Results: There was a moderate level of consistency between HPV neutralizing antibodies and specific IgG in females aged 18–26 years (Cohen's κ coefficient for HPV-16 and HPV-18: 0.52 and 0.38) and poor consistency in those aged 9–17 years (Cohen's κ coefficient <0.05). However, Cohen's κ coefficient remained almost unchanged in sensitivity analysis when the IgG antibody cut-off value was raised. Conclusion: HPV neutralizing antibodies are a more specific indicator for the evaluation of HPV natural humoral immunity.
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Objectives Men who have sex with men (MSM) have an increased risk of human papillomavirus (HPV) infection and related diseases compared with men who have sex exclusively with women. From April 2018, there has been a phased roll-out of HPV vaccination offered to MSM aged up to 45 years old who are attending sexual health clinics and HIV clinics in England. The vaccine is most effective if delivered prior to HPV infection. We estimated the proportion of MSM with no current vaccine-type infection and no serological evidence of prior infection, in a study undertaken prior to vaccine introduction. Methods We conducted a cross-sectional study among 484 MSM aged 18–40 years old who attended a sexual health clinic in London between 2010 and 2012. We estimated the prevalence of current and past infection by testing for HPV DNA in anogenital samples and for serum antibodies to HPV16 and HPV18. Results The median age was 30 years (IQR 25–35). The prevalence of HPV16 and HPV18 DNA was 13.2% and 6.2%, respectively. Seropositivity for HPV16 and HPV18 was 28.5% and 17.1%, respectively, with 11.4% seropositive for both types. Seropositivity for the same HPV type was strongly associated with anogenital DNA detection. 279 MSM (57.6%) tested negative for both HPV16 and HPV18 serology and were DNA negative for these two types; only 5 MSM (1.0%) were seropositive and DNA positive for both HPV types. Conclusions This is the first study to determine both the prevalence of HPV DNA in anogenital samples and HPV seroprevalence among MSM attending a sexual health clinic in the UK. Over half of MSM in this study had no evidence of a previous or current infection with either of the high-risk HPV types included in the quadrivalent vaccine, which supports the rationale for opportunistic HPV vaccination of MSM attending sexual health clinics.
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Background Infection with human papillomavirus (HPV) 16 or HPV18 elicits an antibody response, but whether the elicited antibodies protect women against subsequent infection by a homologous HPV type compared with seronegative women is unknown. Methods Study participants were women aged 18–25 years at enrollment in the control group of the ongoing National Cancer Institute–sponsored, community-based, randomized HPV16/18 Costa Rica Vaccine Trial. At enrollment, 2813 participants were negative for cervical HPV16 DNA and 2950 for HPV18 DNA. Women were interviewed regarding sociodemographic data and medical and health history. Medical and pelvic examinations were conducted for all consenting sexually experienced women. Serum samples taken at enrollment were tested for total HPV16/18 antibodies with a polyclonal enzyme-linked immunosorbent assay, and cervical specimens were tested for type-specific HPV DNA over 4 years of follow-up. Using Poisson regression, we compared rate ratios of newly detected cervical HPV16 or HPV18 infection among homologous HPV-seropositive and HPV-seronegative women, adjusting for age, education, marital status, lifetime number of sexual partners, and smoking. Results There were 231 newly detected HPV16 infections during 5886 person-years among HPV16-seronegative women compared with 12 newly detected HPV16 infections during 581 person-years among HPV16-seropositive women with the highest HPV16 sero-levels. There were 136 newly detected HPV18 infections during 6352 person-years among HPV18-seronegative women compared with six new infections detected during 675 person-years among HPV18 seropositives with the highest sero-levels. After controlling for risk factors associated with newly detected HPV infection, having high HPV16 antibody titer at enrollment was associated with a reduced risk of subsequent HPV16 infection (women in the highest tertile of HPV16 antibody titers, adjusted rate ratio = 0.50, 95% confidence interval = 0.26 to 0.86 vs HPV16-seronegative women). Similarly, having high HPV18 antibody titer at enrollment was associated with a reduced risk of subsequent HPV18 infection (women in the highest tertile of HPV18 antibody titers, adjusted rate ratio = 0.36, 95% confidence interval = 0.14 to 0.76 vs HPV18-seronegative women). Conclusion In this study population, having high antibody levels against HPV16 and HPV18 following natural infection was associated with reduced risk of subsequent HPV16 and HPV18 infections.
Article
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Importance: In addition to their role in protection, antibody responses have been hypothesized to contribute to the pathology of dengue. Recent studies characterizing memory B cell (MBC)-derived mAbs have provided valuable insight into the targets and functions of B cell responses generated after DENV exposure. However in case of secondary infections, such MBC-based approaches fail to distinguish acutely induced cells from the pre-existing MBC pool. Our characterization of plasmablasts and plasmablast-derived mAbs provides a focused analysis of B cell responses activated during ongoing infection. Additionally, our studies describe evidence of OAS in the acute phase dengue immune response, providing basis for future work examining the impact of OAS-phenotype antibodies on protective immunity and disease severity in secondary infections.
Article
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BACKGROUND: The investigational 9-valent viruslike particle vaccine against human papillomavirus (HPV) includes the HPV types in the quadrivalent HPV (qHPV) vaccine (6, 11, 16, and 18) and five additional oncogenic types (31, 33, 45, 52, and 58). Here we present the results of a study of the efficacy and immunogenicity of the 9vHPV vaccine in women 16 to 26 years of age. METHODS: We performed a randomized, international, double-blind, phase 2b-3 study of the 9vHPV vaccine in 14,215 women. Participants received the 9vHPV vaccine or the qHPV vaccine in a series of three intramuscular injections on day 1 and at months 2 and 6. Serum was collected for analysis of antibody responses. Swabs of labial, vulvar, perineal, perianal, endocervical, and ectocervical tissue were obtained and used for HPV DNA testing, and liquid-based cytologic testing (Papanicolaou testing) was performed regularly. Tissue obtained by means of biopsy or as part of definitive therapy (including a loop electrosurgical excision procedure and conization) was tested for HPV. RESULTS: The rate of high-grade cervical, vulvar, or vaginal disease irrespective of HPV type (i.e., disease caused by HPV types included in the 9vHPV vaccine and those not included) in the modified intention-to-treat population (which included participants with and those without prevalent infection or disease) was 14.0 per 1000 person-years in both vaccine groups. The rate of high-grade cervical, vulvar, or vaginal disease related to HPV-31, 33, 45, 52, and 58 in a prespecified per-protocol efficacy population (susceptible population) was 0.1 per 1000 person-years in the 9vHPV group and 1.6 per 1000 person-years in the qHPV group (efficacy of the 9vHPV vaccine, 96.7%; 95% confidence interval, 80.9 to 99.8). Antibody responses to HPV-6, 11, 16, and 18 were noninferior to those generated by the qHPV vaccine. Adverse events related to injection site were more common in the 9vHPV group than in the qHPV group. CONCLUSIONS: The 9vHPV vaccine prevented infection and disease related to HPV-31, 33, 45, 52, and 58 in a susceptible population and generated an antibody response to HPV-6, 11, 16, and 18 that was noninferior to that generated by the qHPV vaccine. The 9vHPV vaccine did not prevent infection and disease related to HPV types beyond the nine types covered by the vaccine. (Funded by Merck; ClinicalTrials.gov number, NCT00543543). Comment in HPV "coverage". [N Engl J Med. 2015]
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Persons with Fanconi Anemia (FA) are at risk for HPV-associated cancers; however, their natural HPV exposure and infection rates are unknown as are the adequacy with which they mount antibodies to HPV vaccination. This study aimed to determine, in 62 persons with FA, seroprevalence of skin and mucosal HPV types, seroprevalence in individuals self-reporting a history of HPV vaccination, and factors associated with HPV seropositivity. Bead Luminex assay was used to determine seropositivity for HPV types 1, 2, 4 (low-risk skin); 6, 11 (low-risk mucosal, included in one HPV vaccine); 16, 18, (high-risk mucosal, included in both HPV vaccines); and 52, 58 (high-risk mucosal). Health- and behavior-related questionnaires were completed. Type-specific seroprevalence estimates and participant characteristics associated with seroprevalence were calculated. 48% reported HPV vaccination. Type-specific seropositivity in unvaccinated persons ranged from 7-21% for skin HPV types and 7-38% for mucosal HPV types. Among unvaccinated participants, adults versus children demonstrated increased HPV 1, 6, 16, and 58 seroprevalence: 45% vs. 6%, 64% vs. 22%, 64% vs. 17%, and 36% vs. 0% respectively (p-values all <0.05). Vaccinated participants versus non-vaccinated participants demonstrated increased seroprevalence for HPV 6, 11, 16, and 18: 92% vs. 38%, 92% vs. 24%, 96% vs. 34%, and 75% vs. 7% respectively (p-values all <0.0001). Our data demonstrate that unvaccinated participants had serologic evidence of prior skin and mucosal HPV infections, and that seroprevalence increased among adults; in self-reported vaccinees, seroprevalence for HPV vaccine-types was 75-96%. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Author Summary There is an urgent need to better understand how to reliably generate effective vaccines, particularly subunit vaccines, as certain pathogens are considered to pose too great of a safety risk to be developed as live, attenuated or killed vaccines (e.g., HIV-1). The human papillomavirus (HPV) vaccines are two of the most effective subunit vaccines ever developed and have continued to show protection against HPV associated disease up to and beyond five years post-vaccination. Moreover, the target population for these vaccines have essentially no pre-existing immunity to the HPV types covered by the vaccine; therefore, these vaccines provide an excellent model for studying the immunity elicited by a highly effective subunit vaccine. As the HPV vaccines, like most vaccines, protect by generating antibodies, we are interested in characterizing the memory B cells elicited by the HPV vaccine. Memory B cells help to sustain antibody levels over time by rapidly differentiating into antibody secreting cells upon pathogen re-exposure. Although previous studies have provided evidence that the HPV vaccines elicit memory B cells, they did not characterize these cells. Here, we have isolated HPV-specific memory B cells from adolescent females and women who received the quadrivalent HPV vaccine and have cloned antibodies from these cells. Importantly, we find that these antibodies potently inhibit HPV and that the memory B cells from which they derive exhibit hallmarks of long-lived memory B cells.
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Significance Most vaccines confer immunity by eliciting long-term production of antibodies that bind to and neutralize the vaccine antigen. Remarkably, very little is known regarding the identities, sequence diversity, relative concentrations, or binding functionalities of the mAbs that comprise the serum repertoire elicited by vaccination. Here, we have delineated the constituent antibodies of the human serum IgG repertoire after vaccination and examined their relationship to the antibody V gene repertoire encoded by circulating B cells. The results detail the molecular composition and characteristics of the vaccine-specific serum antibody repertoire and demonstrate differences between the end-point response (the serum antibodies) and the peripheral B cells responding to the vaccine.
Article
Background: Studies have been mixed on whether naturally acquired HPV antibodies may protect against subsequent HPV infection. We performed a systematic review and meta-analysis to assess whether naturally acquired HPV antibodies protect against subsequent genital HPV infection (i.e. natural immunity). Methods: We searched the Medline and Embase databases for studies examining natural HPV immunity against subsequent genital type-specific HPV infection in females and males. We utilized random-effects models to derive pooled relative risk estimates for each HPV type. Results: We identified 14 eligible studies that included over 24,000 individuals from 18 countries that examined HPV natural immunity. We observed significant protection against subsequent infection with HPV16 (pooled RR=0.65, 95%CI=0.50-0.80) and HPV18 (pooled RR=0.70, 95%CI=0.43-0.98) in females but not in males (HPV16: pooled RR=1.22, 95%CI=0.67-1.77, p-heterogeneity=0.05; HPV18: pooled RR=1.50, 95%CI=0.46-2.55; p-heterogeneity=0.15). We also observed type-specific protection against subsequent infection for a combined measure of HPV6/11/31/33/35/45/52/58 in females (pooled RR=0.75, 95%CI=0.57-0.92). Natural immunity was also evident in females when restricting to studies that employed neutralizing assays, to those that used HPV persistence as an outcome, and to those that reported adjusted analyses (each p-value<0.05). Conclusions: HPV antibodies acquired through natural infection provide modest protection against subsequent cervical HPV infection in females.
Article
Antibody responses to viral infections are sustained for decades by long-lived plasma cells (LLPCs). However, LLPCs have yet to be characterized in humans. Here we used CD19, CD38, and CD138 to identify four PC subsets in human bone marrow (BM). We found that the CD19(-)CD38(hi)CD138(+) subset was morphologically distinct, differentially expressed PC-associated genes, and exclusively contained PCs specific for viral antigens to which the subjects had not been exposed for more than 40 years. Protein sequences of measles- and mumps-specific circulating antibodies were encoded for by CD19(-)CD38(hi)CD138(+) PCs in the BM. Finally, we found that CD19(-)CD38(hi)CD138(+) PCs had a distinct RNA transcriptome signature and human immunoglobulin heavy chain (VH) repertoire that was relatively uncoupled from other BM PC subsets and probably represents the B cell response's "historical record" of antigenic exposure. Thus, our studies define human LLPCs and provide a mechanism for the life-long maintenance of anti-viral antibodies in the serum. Copyright © 2015 Elsevier Inc. All rights reserved.
Article
There is some evidence to suggest that one or two doses of the HPV vaccine provides similar protection to the three-dose regimen. The main aim of the study was to ascertain HPV-16/18 vaccine efficacy in both full and naive cohorts and to explore protection conferred against non-vaccine HPV types, by number of doses received. Summary data from the Costa Rica Vaccine Trial (CVT; NCT00128661) and ~the PATRICIA trial (NCT001226810), two phase 3, double-blind, randomised controlled clinical trials of the HPV-16/18 AS04-adjuvanted vaccine in young women, were combined in a post-hoc analysis (GlaxoSmithKline [GSK] e-track number 202142) to investigate the efficacy of fewer than three doses of the HPV-16/18 vaccine after 4 years of follow-up. Women were randomly assigned to receive three doses of the HPV-16/18 vaccine or to a control vaccine; yet, some received fewer doses. After exclusion of women with less than 12 months of follow-up or those who were HPV-16/18 DNA-positive at enrolment (for the HPV-16/18 endpoint), we calculated vaccine efficacy against one-time detection of incident HPV infections after three, two, and one dose(s). The primary study endpoint was one-time detection of first incident HPV-16/18 infections accumulated during the follow-up phase. We assessed vaccine efficacy against incident HPV-16/18 infection in the modified total vaccinated cohort (22 327 received three doses, 1185 two doses, 543 one dose). Vaccine efficacy against incident HPV-16/18 infections for three doses was 77·0% (95% CI 74·7-79·1), two doses was 76·0% (62·0-85·3), and one dose was 85·7% (70·7-93·7). Vaccine efficacy against incident HPV-31/33/45 infections for three doses was 59·7% (56·0-63·0), two doses was 37·7% (12·4-55·9), and one dose was 36·6% (-5·4 to 62·2). Vaccine efficacy against incident HPV-16/18 infection for two-dose women who received their second dose at 1 month was 75·3% (54·2-87·5) and 82·6% (42·3-96·1) for those who received the second dose at 6 months (CVT data only). Vaccine efficacy against HPV-31/33/45 for two-dose women who received their second dose at 6 months (68·1%, 27·0-87·0; CVT data only), but not those receiving it at one month (10·1%, -42·0 to 43·3), was similar to the three-dose group. 4 years after vaccination of women aged 15-25 years, one and two doses of the HPV-16/18 vaccine seem to protect against cervical HPV-16/18 infections, similar to the protection provided by the three-dose schedule. Two doses separated by 6 months additionally provided some cross-protection. These data argue for a direct assessment of one-dose efficacy of the HPV-16/18 vaccine. US National Cancer Institute, National Institutes of Health Office of Research on Women's Health, and Ministry of Health of Costa Rica (CVT); GlaxoSmithKline Biologicals SA (PATRICIA). Copyright © 2015 Elsevier Ltd. All rights reserved.