Vaccine 39 (2021) 5295–5301

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Increases in HPV-16/18 antibody avidity and HPV-specific memory

B-cell response in mid-adult aged men post-dose three of the
quadrivalent HPV vaccine
Cheryl N. Miller a,1, Troy J. Kemp a,1, Martha Abrahamsen b, Kimberly Isaacs-Soriano b, Kim Dunham a,
Bradley Sirak b, Yuanji Pan a, Eduardo Lazcano-Ponce c, Jorge Salmeron c, Ligia A. Pinto a,2,⇑,
Anna R. Giuliano b,2
a
Vaccine, Immunity, and Cancer Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, National Cancer Institute, Frederick, MD, USA
b
Center for Immunization and Infection Research in Cancer (CIIRC), Moffitt Cancer Center, Tampa, FL, USA
c
National Institute of Public Health, Cuernavaca, Morelos, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Strong quantitative and functional antibody responses to the quadrivalent human papillomavirus (HPV)
Received 12 February 2021 vaccine were reported in mid-adult aged men, but there are limited data on the avidity of the antibody
Received in revised form 28 June 2021 response and the memory B-cell response following vaccination. Although circulating antibodies induced
Accepted 23 July 2021
by vaccination are believed to be the main mediators of protection against infection, evaluation of avidity
Available online 6 August 2021
of antibodies and memory B cell responses are critical for a better understanding of the vaccine immuno-
genicity mechanisms. Both the modified enzyme-linked immunosorbent assay (ELISA) and the enzyme-
linked immunosorbent spot (ELISpot) assay are tools to measure the humoral and cellular immune
responses post vaccination to characterize vaccine immunogenicity. The avidity of HPV-16 and HPV-18
specific IgG in the serum of mid-adult aged men (N = 126) who received three quadrivalent HPV vaccine
doses was examined using a modified ELISA. HPV-16 memory B-cell responses were assessed via ELISpot
at month 0 (prior to vaccination) and 1-month post-dose three of the vaccine (month 7). The quadrivalent
vaccine induced an increase in HPV-16 and HPV-18 antibody avidity at month 7. HPV-18 avidity levels
moderately correlated with anti-HPV-18 antibody titers, but no association was observed for HPV-16
antibody titers and avidity levels. The HPV-16-specific memory B-cell response was induced following
three vaccine doses, however, no association with anti-HPV-16 antibody avidity was observed. Three
doses of quadrivalent HPV vaccine increased antibody affinity maturation for HPV-16/18 and increased
the frequency of anti-HPV-16 memory B-cells in mid-adult aged men.
Ó 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).

1. Introduction HPV types, accounting for approximately 60% of oropharyngeal

cancers, and approximately 90% of anal cancers [1]. The quadriva-
Human papillomavirus (HPV) is one of the most common sexu- lent HPV vaccine, Gardasil, has been shown to be highly immuno-
ally transmitted viruses, and a cause of several cancers in both men genic and provides excellent protection against HPV-6/11/16/18
and women. HPV-16 and HPV-18 are the most common oncogenic [2,3]. While HPV vaccination holds promise to reduce the incidence
and mortality of HPV-related cancers, vaccine uptake, particularly
in males remains low and sub-optimal in most countries. This is
Abbreviations: AB, antibody; CI, confidence interval; ELISA, enzyme-linked particularly problematic as men have a 4-fold higher incidence of
immunosorbent assay; ELISpot, enzyme-linked immunosorbent spot; EU, ELISA oropharyngeal cancers compared to females [4], and a significantly
units; GMA, geometric mean avidity; GuHCl, guanidine hydrochloride; HPV, human
higher prevalence of oral HPV than do women. Unlike cervical HPV
papillomavirus; IU, international units; M, molar; N, number; VLP, virus-like
particle. infection, the prevalence of oral HPV increases with age among
⇑ Corresponding author at: Frederick National Laboratory for Cancer Research, men with the highest oral HPV prevalence in men aged 55 to
Building 469, Room 111, Frederick MD 21702, USA. 74 years [5].
E-mail address: [email protected] (L.A. Pinto). Routine vaccination in mid-adult aged men could help reduce
1
These authors contributed equally.
2
the incidence of oropharyngeal cancers, by preventing oral HPV
These authors contributed equally.

https://doi.org/10.1016/j.vaccine.2021.07.069
0264-410X/Ó 2021 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
C.N. Miller, T.J. Kemp, M. Abrahamsen et al. Vaccine 39 (2021) 5295–5301

infection. The quadrivalent HPV vaccine is safe and effective in month 0 and month 7 serum and PBMC specimens were collected
reducing HPV-6/11/16/18 anogenital infections in young males and stored frozen at 80 °C until tested.
ages 16–26 years [6]. We previously demonstrated men ages 27–
45 years mount a strong and specific antibody response compara- 2.2. HPV ELISA
ble to levels in young males after three doses of quadrivalent HPV
vaccine where clinical efficacy of the vaccine was demonstrated Specimens were tested for anti-HPV-16 and HPV-18 IgG levels
[7,8]. Further, the quadrivalent HPV vaccine administered to HIV- by the L1 VLP an enzyme-linked immunosorbent assay (ELISA),
infected men was also effective at eliciting a strong specific as previously performed [8,18,19]. Flat-bottom high-binding
immune response [9], a strong indication that protective immunity microtiter plates (MaxiSorp, Nunc, Thermo Fisher Scientific, Wal-
occurs among mid-adult aged men. tham, MA) were coated with HPV-16 or HPV-18 L1 VLP at a con-
To further evaluate the quality of the antibody response post centration of 2.7 mg mL 1 in 100 ml for at least 72 h at 4 °C. The
vaccination among mid-adult aged men, antibody avidity and plates were then washed three times with 350 ml of phosphate-
number of antigen specific memory B-cells were measured. Circu- buffered saline (PBS) containing 0.2% Tween 20 and blocked in
lating antibodies against HPV induced by vaccination play an 300 ml of PBS with 4% skim milk and 0.2% Tween-20 (blocking buf-
essential role in protection against repeated exposures and subse- fer) for 90 min at room temperature. The plates were washed three
quent HPV infections. The antibody levels that are induced by vac- times. Serial dilutions of serum (starting dilution 1:100) in block-
cination are often much higher than that observed during natural ing buffer in 2-fold increments were added to the plate in a final
HPV infection and more detailed analysis of this robust immune volume of 100 ml. The plates were sealed and incubated at room
response will uncover key factors in long-term immune protection temperature for 60 min on a plate shaker at 250 rpm. After wash-
against HPV infection [10–12]. The avidity index is a measure of ing the plate three times, peroxidase-labeled goat anti-human IgG
the relative strength of the interaction between the antibody with (KPL, Inc.) was added and the plates were sealed and incubated at
the antigen complex and is a method to evaluate the quality of room temperature for 60 min on a plate shaker at 250 rpm. Plates
antibody responses and protection against disease [13]. There are were then developed with tetramethylbenzidine substrate solution
limited studies investigating the role of avidity in protection (KPL, Inc.). After 25 min of incubation in the dark at room temper-
against HPV infections because the HPV vaccines have demon- ature, the reaction was stopped by the addition of 100 ml of 0.36 N
strated remarkably high efficacy [14]. However, avidity of HPV H2SO4. The absorbance at 450 nm and 620 nm were measured with
antibodies may be associated with the strong vaccine efficacy a microtiter plate reader (Spectramax M5; Molecular Devices, Sun-
and protection against newly detected HPV infections observed nyvale, CA). Antibody levels were expressed as ELISA units (EU
in vaccine recipients [14,15]. Recall immune responses are rapid mL 1) which were interpolated from the standard curve. Antibody
and produce high affinity antibodies because long lived memory levels were then normalized for total IgG in each specimen and
B cells differentiate into the antibody producing plasma cells expressed as EU mg 1. Both the HPV-16 and HPV-18 ELISA were
[16]. Long-lived plasma cells secrete affinity matured antibodies, calibrated with the World Health Organizations International Stan-
which can mediate protection form HPV infection [17]. Here, we dards. The International Units (IU) conversion factor for anti-HPV-
measured these humoral and cellular immune responses to deter- 16 antibodies is 1 IU mL 1 = 5.29 EU mL 1, and for anti-HPV-18
mine quality of the immune response following three doses of the antibodies is 1 IU mL 1 = 6.81 EU mL 1.
quadrivalent HPV vaccine among mid-adult aged men. Further-
more, we optimized an established ELISpot assay for the use with
HPV-16 and assessed the associations between antibody avidity 2.3. Avidity assay
and memory B-cell responses in this population of men.
To approximate the binding strength of IgG antibodies to HPV,
the avidity of antibodies was determined as previously described,
2. Material and methods using a modified ELISA-based method with the chaotropic agent
guanidine hydrochloride (GuHCl; Sigma, St. Louis, MO) [20,21].
2.1. Participants and serum samples The avidity index is the concentration of GuHCl, expressed in Molar
(M), that reduce the optical density by 50% compared to sample
2.1.1. Study population wells without GuHCl treatment. Flat-bottom microtiter plates
Sera were collected at months 0 and 7 from 150 men, ages 27– were coated with HPV antigen, and blocked, as previously
45 who received quadrivalent HPV vaccine at 0, 2, and 6 months. described above in the HPV ELISA method. Serum samples were
This study was conducted in compliance with regulations by an diluted based on previous evaluation in the HPV-16 or HPV-18
ethical committee review and was monitored by the Moffitt Cancer ELISA to yield an absorbance reading of 1.0 ± 0.5. Samples were
Center Protocol Support Office. The study population consisted of excluded if their reading was below 1.0 ± 0.5, or the sample could
mid-adult aged males from two clinics located in Cuernavaca, Mex- not be diluted down to 1.0 ± 0.5 with a 1:100 dilution. The diluted
ico and Tampa, Florida (the MAM Trial; www.clinicaltrials.gov, serum was incubated for 1 h at room temperature. Buffer alone, or
NCT01432574). A total of 145 men had specimens contributing 0.5 to 3.5 M GuHCl was added for 15 min at room temperature.
to both month 0 and month 7 serum HPV antibody evaluations After washing the plate, the secondary goat anti-human IgG was
[7]. Here we evaluated anti-HPV-16 and anti-HPV-18 antibody added for one hour at room temperature, and the plate was devel-
avidity for 126 men. A subset of participants had peripheral blood oped with TMB and the absorbance was read as previously
mononuclear cells (PBMC) collected prior (N = 47) and post vacci- described for the HPV ELISA.
nation (N = 46), which were used for evaluating HPV-16 memory
B-cell response. 2.4. Enzyme-linked immunosorbent spot (ELISpot) assay

2.1.2. Vaccination and clinical procedure Frozen PBMC were thawed, washed and resuspended in assay
The quadrivalent HPV vaccine, Gardasil (Merck & Co., Inc) con- media containing RPMI 1640 (Gibco, Carlsbad, CA), 10% fetal
taining HPV 6/11/16/18 virus-like particles (VLP) was used to vac- bovine serum (HyClone, Logan, UT) and 1x penicillin–streptomy
cinate men at month 0, months 2 and 6 in a volume of 0.5 mL by cin-glutamine (Gibco). Cell counts and viabilities were obtained
intramuscular administration in the deltoid or thigh muscle. At with a MUSE Cell Analyzer (Millipore).
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Approximately, 1  106 viable cells per well were plated into vaccination timepoints were run simultaneously for each partici-
24-well tissue culture plates (Costar, Corning Inc.) in 2 mL stimu- pant and a positive and negative donor control were run with each
lation media containing RPMI 1640 (Gibco), 10% fetal bovine serum batch.
(HyClone), 1x penicillin–streptomycin-glutamine (Gibco), 50 mM
2-mercaptoethanol (Sigma), 1 mM CpG ODN-2006 (Invitrogen),
1 mg mL 1 Protein A from Staphylococcus aureus Cowan (Sigma), 2.5. Statistical analysis
1 mg mL 1 pokeweed mitogen (Sigma). The stimulated PBMC were
incubated in a 37 °C humidified 5% CO2 atmosphere for 6 days. Geometric mean avidity responses were measured, and their
We adapted a previously established method for the HPV-16- associated 95% confidence intervals were calculated using Graph-
specific memory B-cell ELISpot assay [22]. The day before assay Pad Prism Version 8.4.3. Statistical significance was determined
setup, 96-well polyvinylidene fluoride (PVDF) membrane, HTS opa- using either a Mann-Whitney test or a Wilcoxon Matched pairs
que plates (Millipore) were pre-wetted with 27% ethanol signed rank test between time points. For determining statistical
for < 1 min and washed four times with PBS (Lonza). HPV-16 VLP significance between two clinical sites an unpaired t-test was used.
and negative control keyhole limpet hemocyanin (KLH) (Millipore) Correlations between anti-HPV-specific IgG and anti-HPV-specific
coating antigens were diluted to 5 mg per well and anti-IgG capture avidity indices were determined by Spearman correlation coeffi-
antibody (Mabtech) was diluted to 1.5 mg per well in PBS. The cients (q). Correlation between memory B-cell responses and avid-
plates were then incubated overnight at 4 °C, washed four times ity of anti-HPV-16 antibodies were determined by Spearman
with PBS and blocked with 200 ml per well with assay media for correlation coefficients (q). A p value of  0.05 was considered
at least 30 min at room temperature. The 6-day stimulated PBMC significant.
were washed, counted and resuspended to plate 300,000 and
100,000 cells in the HPV-16 VLP and KLH wells and 10,000 cells
in the total IgG wells. All conditions were plated in triplicate and 3. Results
incubated for 2 h at 37 °C and 5% CO2. After the two-hour incuba-
tion, the plates were washed six times with 0.05% Tween 20 3.1. Men who received three doses of the quadrivalent HPV vaccine
(Sigma) in DPBS, followed by a 2-hour incubation at room temper- had an increase in avidity for both HPV-16 and HPV-18
ature with a 1:500 dilution of biotinylated anti-IgG (Mabtech) in
DPBS with 1% bovine serum albumin (Thermo Scientific) and All men that received three doses of the quadrivalent HPV vac-
0.05% Tween 20. After incubation and four washes in DPBS to cine developed HPV-16 and HPV-18 antibodies [7]. The three doses
remove excess antibody, a 1:1000 dilution of streptavidin alkaline of the quadrivalent vaccine also induced a significant increase in
phosphatase (Mabtech) in DPBS with 1% bovine serum albumin antibody avidity and thus, antibody affinity maturation at month
(Thermo Scientific), was added to each well for 1 h at room tem- 7 (Fig. 1). The avidity assay was not performed on the majority
perature followed by 4 washes in DPBS. One hundred ml per well of month 0 samples because the antibody titers were below or
of BCIP/NBT substrate (KPL Inc., Gaithersburg, MD), was filtered close to the lowest detection levels. HPV vaccination induced an
and added for 7–10 min, resulting in the development of spots. increase in HPV-16 antibody avidity from 0.9 M, at month 0 to
The reaction was stopped by washing three times in distilled 1.9 M at month 7 (Table 1). HPV-18 antibody avidity also increased
water. Plates were dried overnight, and the spots were visualized from 0.8 M, at month 0 to 1.6 M at month 7 (Table 1). There was an
and counted using the ImmunoSpot Imaging Analyzer system (Cel- overall 2-fold increase in both HPV-16 and HPV-18 avidity levels
lular Technology Ltd.) and ImmunoSpot software v5.1. All wells post vaccination.
were counted with set parameters and then each count was veri- Of the 150 participants, 24 were seropositive for HPV-16 and/or
fied. The spot counts were adjusted to reflect the average number HPV-18 antibodies on day 1, and thus, had a previous history of
of spots per 1  106 cells and then the frequency of antigen-specific infection with HPV-16 and/or HPV-18 before vaccination.
B-cells was calculated by dividing the number of antigen-specific Twenty-three seropositive participants also had an increase in
spots by the estimated number of total B-cells. Pre- and Post- antibody avidity after receiving three doses of vaccine (Fig. 2 and

A 4 p < 0.0001 B 3 p < 0.0001

Avidity Index (M)
Avidity Index (M)

3
Anti-HPV-18
Anti-HPV-16

2
1
1

0
0
Month 0 Month 7
Month 0 Month 7

Fig. 1. Avidity of HPV antibodies in serum pre and post vaccination with the quadrivalent HPV vaccine. A) Avidity of anti-HPV-16 antibodies in mid-adult aged men at month
0 before vaccination and month 7 post-dose three of vaccine. The avidity was measured in individuals with detectable HPV-16 antibody levels. B) Avidity of anti-HPV-18
antibodies in mid-adult aged men at month 0 before vaccination and month 7 post-dose three of vaccine. The avidity was measured in individuals with detectable HPV-18
antibody levels. The bars depict the 95% confidence intervals and the geometric mean. Statistical significance was calculated using a Mann-Whitney test between time points
(p < 0.0001).

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Table 1
Avidity of anti-HPV-16 and HPV-18 antibodies prior and post vaccination in serum.

Month 0 Month 7
a b
HPV type Specimen Type Clinic N AB positive (%) GMA (95% CI) AB positive (%)a GMAb (95% CI)
16 Serum U.S. 51 2 (3.9) 0.8 (0.6, 0.9) 51 (1 0 0) 1.9 (1.7, 2.0)
Mexico 75 10 (13.3) 1.0 (0.8, 1.3) 75 (1 0 0) 1.9 (1.8, 2.0)
Total 126 12 (9.5) 0.9 (0.8, 1.2) 126 (1 0 0) 1.9 (1.8, 2.0)
18 Serum U.S. 51 6 (11.8) 0.6 (0.3, 1.0) 51 (1 0 0) 1.6 (1.5, 1.7)
Mexico 75 8 (10.7) 1.1 (0.9, 1.4) 75 (1 0 0) 1.6 (1.5, 1.7)
Total 126 14 (11.1) 0.8 (0.6, 1.1) 126 (1 0 0) 1.6 (1.5, 1.6)
a
AB positive represents the number (%) of subjects whom met the criteria for measuring avidity of anti-HPV-16 and HPV-18 antibodies.
b
GMA represents the geometric mean avidity response amongst subjects who met the criteria for measuring avidity of anti-HPV-16 and HPV-18 antibodies.

A 4 p < 0.0005 B 3 p < 0.0001

Avidity Index (M)

Avidity Index (M)

3
Anti-HPV-16

Anti-HPV-18
2

1
1

0 0
Month 0 Month 7 Month 0 Month 7

Fig. 2. Paired analysis among measurable avidity levels at month 0 before vaccine versus month 7 post-dose three of vaccine. A) Anti-HPV-16 avidity index increased
between month 0 and month 7. B) Anti-HPV-18 avidity index increased between month 0 and month 7. The circles represent individuals with a positive, detectable antibody
response to HPV-16 or HPV-18, in which avidity can be measured. Statistical significance was determined using a Wilcoxon matched pairs signed rank test between time
points.

Table 2
Avidity levels in a subset of individuals with an increase in avidity between month 0 before vaccine versus month 7.

Participant HPV-16 Avidity HPV-18 Avidity

Month 0 Month 7 Month 0 Month 7
1 0.51 0.57 – –
2 0.67 2.08 0.75 1.45
3 0.76 2.22 – –
4 0.78 1.12 – –
5 0.83 1.41 – –
6 0.91 1.70 – –
7 0.94 2.62 – –
8 1.11 1.85 – –
9 1.19 1.92 – –
10 1.22 2.05 1.35 1.57
11 1.30 1.50 1.28 2.31
12 1.69 1.73 – –
13 – – 0.19 1.61
14 – – 0.53 1.27
15 – – 0.65 1.66
16 – – 0.74 1.78
17 – – 0.75 1.55
18 – – 0.94 1.01
19 – – 0.95 1.16
20 – – 1.01 1.93
21 – – 1.08 1.74
22 – – 1.11 1.62
23 – – 1.64 1.94

Table 2). There was an average increase in the anti-HPV-16 and avidity (Fig. 2A). Of the 14 with a natural history of HPV-18 infec-
anti-HPV-18 avidity index from month 0 to month 7 of 1.8 and tion, 4 individuals had a minimal increase of  1.2-fold in avidity
2.2, respectively. Of the 12 with a natural history of HPV-16 infec- (Fig. 2B). The quadrivalent HPV vaccine increased avidity above
tion, only 3 individuals had a minimal increase of  1.2-fold in what is seen with a natural history of infection with HPV.

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In this study, the participants seropositive for HPV-16 differed 3.3. Memory B-cell responses was induced by the quadrivalent HPV
by location, with a higher prevalence of positivity (13.3%) at month vaccine
0 in Mexico compared to 3.9% positivity in the US (Table 1). The
participants seropositive for HPV-18 were similar with 11.8% and HPV-16 specific memory B-cells were enumerated from PBMC
10.7% positivity at month 0, for specimen from the US and Mexico, using the ELISpot assay. The antigen-specific memory B-cells were
respectively (Table 1). After three doses of vaccine, all individuals calculated as a percentage of the total IgG positive memory B-cells.
mounted a robust immune response against HPV-16 and HPV-18. Up to 4.4 % of IgG positive B-cells were HPV-16-specific post vacci-
The avidity was also consistent across clinic locations for both nation (Median = 0.43% of IgG positive B-cells) (Fig. 4A). Antigen-
HPV-16 and HPV-18; even though, their initial seropositivity dif- specific memory B-cells at month 0 were low (Median = 0.006%
fered. (Table 1). of IgG positive B-cells). There was a significant, 72-fold increase
in the percent of memory B-cells that were HPV-16-specific
3.2. Anti-HPV-18 avidity levels were moderately associated with the between month 0 and month 7 (Table 3). However, there was no
corresponding anti-HPV-18 antibody levels significant correlation between the frequency of anti-HPV-16-
specific memory B-cells and the antibody avidity in men post-
The avidity levels of anti-HPV-16 antibodies post vaccination at dose three of the quadrivalent HPV vaccine (Spearman q = 0.03,
month 7 did not correlate with the total anti-HPV-16 IgG levels p = 0.87) (Fig. 5).
(Fig. 3A). There were 11 individuals with high total anti-HPV-16
IgG levels (500 EU mg 1) with avidity indices below the geomet- 4. Discussion
ric mean and 63 individuals with avidity indices at or above the
geometric mean with total anti-HPV-16 IgG levels below 500 EU Tracking antibody affinity and memory B-cells were two inde-
per mg 1. The concentration of circulating antibodies to HPV-16 pendent parameters to evaluate the humoral and B cellular
does not appear to correlate with antibody affinity maturation immune response to HPV vaccination. The avidity assay measured
for HPV-16. However, there was a moderate but significant corre- the binding strength of antibodies to HPV and thus, an important
lation between anti-HPV-18 avidity levels and total HPV-18- immunogenicity marker. The ELISpot provided quantification of
specific IgG concentration (Spearman q = 0.43, p < 0.0001). memory B-cells that were antigen specific and was another inde-

A B

Fig. 3. Correlation between total anti-HPV IgG antibodies and avidity at month 7 post vaccination. A) Anti-HPV-16 total IgG expressed as ELISA units per mg (EU mg 1) from
serum compared to avidity levels of anti-HPV-16 antibodies, B) Anti-HPV-18 total IgG expressed as ELISA units per mg (EU mg 1) from serum compared to avidity levels of
anti-HPV-18 antibodies. The Spearman correlation coefficient (q) and p-values are indicated on each graph.

A p < 0.0001 B p < 0.0001

800 5
# of HPV-16-Specific

memory B cells (%)

Spots/1x106 cells

4
HPV-16-specific

600

3
400
2
200
1

0 0
Month 0 Month 7 Month 0 Month 7
Fig. 4. HPV-16-specific memory B-cell response pre and post vaccination with the quadrivalent HPV vaccine. A) Number of HPV-16-specific spots per 106 cells in mid-adult
aged men at month 0 before vaccination and month 7 post vaccination. B) The percent of HPV-16-specific memory B-cells in mid-adult aged men at month 0 before
vaccination and month 7 post vaccination. The bars depict the 95% confidence intervals and the median. Statistical significance was calculated using a Mann-Whitney test
between time points (p < 0.0001).

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Table 3
HPV-16 memory B-cell responses prior and post vaccination in serum.

HPV type Time N ELISpot (% of IgG specific) N ELISpot (# of Spots/106 cells)

Median (95% CI) Median (95% CI)
HPV-16 Month 0 47 0.006 (0–0.01) 47 2.2 (0–4.4)
Month 7 46 0.43 (0.3–0.6) 46 120 (46–183)
a
Median (middle value) and 95% CI of the median for percent HPV-16 specific-IgG memory B-cells.
b
Median (middle value) and 95% CI of the median for number of HPV-16-specific spots per 106 cells.

vical cancer even though anti-HPV antibody levels eventually

dropped below detection, emphasizing the importance for immune
memory [28]. Three doses of quadrivalent HPV vaccine increased
antibody avidity and thus, affinity maturation for HPV-16/18 above
the levels observed after natural infection prior to vaccination in
mid-adult aged men. This vaccine regiment also increased the fre-
quency of anti-HPV-16 memory B-cells, critical for immune mem-
ory and long-term protection against viral infection. The avidity of
anti-HPV-16 antibodies does not correlate with the percent of
HPV-16-specific memory B-cells. This work provides evidence that
the ELISpot and avidity assay can be used to assess vaccine
immunogenicity, and that these assays could be used to measure
long term protection from HPV infections.

Declaration of Competing Interest

Fig. 5. Correlation between memory B-cell responses and avidity of anti-HPV-16
antibodies. A) Correlation between the percent of HPV-16-specific memory B-cells
and anti-HPV-16 avidity index (M). The Spearman correlation coefficient (q) and p-
A.R.G reports receiving grants from Merck during the conduct of
values are indicated on the graphs. the study and other grants from Merck outside the submitted work.
J.S. reports receiving grants from GlaxoSmithKline, Qiagen, and
pendent measurement for vaccine immunogenicity and potency. Merck during the conduct of the study and grants from
Three doses of the quadrivalent vaccine elicited antigen-specific GlaxoSmithKline, Qiagen, and Merck outside the submitted work.
memory B-cells similar to levels seen with the anthrax vaccine The other authors report no potential conflicts and declare that they
[23]. The frequency of HPV-16 specific memory B-cells did not have no known competing financial interests or personal relation-
appear to correlate with anti-HPV16 antibody avidity. In mice, ships that could have appeared to influence the work reported in
repeat immunization significantly increased somatic mutation in this paper.
GC not in memory B cells and had little effect on memory B cell
antibody affinity [16]. There has been evidence of cross- Acknowledgments
protective vaccine efficacy of the bivalent HPV vaccine against
HPV types that are not included in the vaccine, such as HPV-31, This work was supported in part by a research grant from
and this is associated with antibody responses [15]. The mainte- Investigator-Initiated Studies Program of Merck Sharp & Dohme
nance of systemic antibody levels is linked to memory B-cells, Corp. The opinions expressed in this paper are those of the authors
but this process did not appear to be correlated with antibody and do not necessarily represent those of Merck Sharp & Dohme
avidity for HPV-16. It would be interesting to determine if the anti- Corp. (research grant to A.R.G.); the Miles for Moffitt program, Mof-
body avidity for HPV-18 correlates with the frequency of antigen- fitt Cancer Center; the National Cancer Institute, National Institutes
specific memory B-cells, considering there was a modest correla- of Health Contract No. HHSN261200800001E; and the Moffitt Can-
tion between the amount of anti-HPV-18 antibody and the anti- cer Center. This project has been funded in part with Federal funds
body avidity to HPV-18. However, due to the limited number of from the National Cancer Institute, National Institutes of Health,
PBMC we were unable to analyze HPV-18 memory B-cell responses Department of Health and Human Services, under Contract No.
in this study. Previous work showed no correlation between HPV- 75N91019D00024. This project has been funded in part with Fed-
16 or HPV-18 specific avidity indices and the respective antibody eral funds from the National Cancer Institute, National Institutes of
titers for women at month 7 post vaccination with Cervarix [24]. Health, under Contract No. HHSN261201800001I. The content of
Three doses of the quadrivalent HPV vaccine provided both a this publication does not necessarily reflect the views or policies
high quality and high quantity antibody response against HPV in of the Department of Health and Human Services, nor does men-
mid-adult aged men. Vaccines against influenza and hepatitis B tion of trade names, commercial products, or organizations imply
virus also showed similar induction of antibody quantity and qual- endorsement by the U.S. Government.
ity. An increase in the concentration and avidity of influenza-
specific IgG was also detected after three doses of influenza vaccine
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