Journal of Immune Based Therapies
and Vaccines

BioMed Central

Original research

Open Access

Kinetics and isotype profile of antibody responses in rhesus
macaques induced following vaccination with HPV 6, 11, 16 and 18
L1-virus-like particles formulated with or without Merck aluminum
adjuvant
Wanda Ruiz1, William L McClements2, Kathrin U Jansen2 and Mark T Esser*1
Address: 1Vaccine and Biologics Research Merck Research Laboratories 466 Devon Park Dr. Wayne, PA 19087-8630 USA and 2Vaccine andBiologics Research Merck Research Laboratories West Point, PA 19486 USA
Email: Wanda Ruiz - wanda_ruiz@merck.com; William L McClements - william_mcclements@merck.com;
Kathrin U Jansen - jansen@mac.com; Mark T Esser* - mark_esser@merck.com* Corresponding author

Published: 20 April 2005
Journal of Immune Based Therapies and Vaccines 2005, 3:2

doi:10.1186/1476-8518-3-2

Received: 03 March 2005
Accepted: 20 April 2005

This article is available from: http://www.jibtherapies.com/content/3/1/2© 2005 Ruiz et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

human papillomavirusvaccineneutralizing antibodyLuminex

Abstract
Background: Human papillomaviruses (HPV) are the most common sexually transmitted viruses.
Infection of the cervical epithelium by HPVs can lead to the development of cervical cancer. Recent
advances in vaccine research have shown that immunization with papillomavirus-like particles(VLPs) containing the major structural viral protein, L1 from HPV 16 can provide protection fromthe establishment of a chronic HPV 16 infection and related cervical intraepithelial neoplasia (CIN)in baseline HPV 16 naïve women.
Methods: To better understand the quantitative and qualitative effects of aluminum adjuvant on
the immunogenic properties of an HPV 6, 11, 16 and 18L1 VLP vaccine, we used an HPV-specific,
antibody isotyping assay and a competitive immunoassay that measures antibodies to neutralizing
epitopes to profile sera from rhesus macaques immunized with the HPV L1 VLP vaccine formulatedwith or without aluminum adjuvant.
Results: Immunization with VLPs formulated with the aluminum adjuvant elicited a significantly
stronger immune response with higher peak antibody titers both at four weeks post vaccination(12.7 to 41.9-fold higher) as well as in the persistent phase at week 52 (4.3 to 26.7-fold higher) thanthat of VLPs alone. Furthermore, the aluminum adjuvant formulated HPV VLP vaccine elicited a
predominantly T helper type 2 response, with high levels of IgG1 and IgG4 and low levels of IgG2.
The vaccine also elicited high levels of serum IgA, which may be important in providing mucosal
immunity to impart protection in the anogenital tract.
Conclusion: These results show that the HPV 6, 11, 16 and 18 L1-VLP vaccine formulated withMerck aluminum adjuvant elicits a robust and durable immune response and holds promise as a
vaccine for preventing cervical cancer.

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Journal of Immune Based Therapies and Vaccines 2005, 3:2

Background
Cervical cancer remains a leading cause of cancer-related
deaths in women. HPV infection is the obligate first stepin the development of cervical cancer [1]. Nearly a quarterof a million women die from cervical cancer and about
half a million are diagnosed with this disease each year[2]. Cervical cancer accounts for 12% of all cancers inwomen and is the second most frequent gynecologicalmalignancy in the world [3]. A large portion of this healthburden is in the developing world, where women do nothave access to good healthcare and Papanicolaou (Pap)screens. Although the widespread use of Pap screening in
the developed world has increased the early detection of
cervical dysplasia and cancer, thereby improving treatment outcomes for cervical cancer, it would be far more
preferable to have a vaccine that blocks HPV infection,
thereby preventing initiation of the disease process. Also,
developing countries that usually do not have access to
Pap screening and other preventive measures would further benefit from a vaccine that blocks HPV infection andits subsequent disease consequences. Therefore, there is agreat need for an effective and generally well-tolerated
HPV vaccine, having a low rate of occurrence of adverse
events during administration.
Human papillomaviruses are small double-stranded DNA
viruses. Infection with HPV is the most common viral sexually transmitted diseases worldwide [4]. HPV infects
cutaneous and mucosal epithelial cells and causes benign
and malignant hyperproliferative lesions, which includes
genital warts and cervical cancer [5]. To date, more than
100 HPV types have been identified. Of these, 35 infect
the genital tract [6,7]. Genital HPV types can be divided
into two broad categories: low-risk types which cause genital warts, cervical dysplasia, but little cancer, and highrisk types, which cause dysplasia that can progress to cancer. HPV type 16 and 18 infection cause 70% of cancer
cases and 25% of low grade cervical dysplasia [8]. HPV
types 6 and 11, on the other hand, cause approximately
95% of genital warts (condylomata acuminata or venereal
warts) and 25% of low grade cervical dysplasia (CIN1)[9,10]. Thus, a vaccine targeting HPV 6, 11, 16 and 18 will
target the majority of HPV-related clinical disease.
Recently, we reported a proof-of-concept efficacy study fora prophylactic vaccine composed of HPV 16 L1 virus-likeparticles (VLP) formulated on Merck Aluminum Adjuvant(MAA) [11]. The double-blinded study randomized 2,391
women (16–23 years old) to receive either placebo or
three doses of 40 µg of the HPV 16 VLPs in a 0, 2, and 6
month regimen. The primary efficacy endpoint was persistent HPV 16 infection, including HPV 16 related cervical dysplasia. Since the vaccine is being developed forprophylaxis against infection, the primary analysis was
conducted in women who were naïve to HPV 16 at enroll-

http://www.jibtherapies.com/content/3/1/2

ment and remained free of HPV 16 infection through thecompletion of the vaccination series. Among placebo
recipients within this cohort, 41 cases of persistent HPV
16 infection were detected. None of the women who
received HPV 16 L1 VLP vaccine in this cohort developed
an endpoint case [11].
Currently, we are investigating a quadrivalent vaccine,composed of HPV 6, 11, 16, and 18 L1 VLPs formulatedwith MAA. This vaccine has also been shown to be effective in preventing persistent infection and HPV 6, 11, 16
and 18 related cervical dysplasia [12]. To better understand the quantitative and qualitative effects of Merck aluminum adjuvant on the immunogenicity of the VLPs weused a novel HPV type-specific, antibody isotyping assay
and a competitive Luminex immunoassay (cLIA) to measure HPV type-specific antibody responses in rhesus
macaques. The results presented here show that formulation with Merck aluminum adjuvant increased the VLP's
immunogenicity without affecting the isotype profile.

Methods
Vaccines
Virus-like particles were prepared as previously described(with modifications) from individual lysates of types- Saccharomyces cerevisiae expressing the L1 genes of HPV 6, 11,
16 and 18, respectively [13]. Equal concentrations of all
four HPV VLPs were combined and used either directly, or
adsorbed to MAA. A standard vaccine dose was composedof 2 µg each of the four VLP types, with or without 225 µgof MAA. The Merck Aluminum Adjuvant is a proprietaryaluminum hydroxyphosphate sulfate based adjuvant usedin other vaccines manufactured by Merck & Co., [14].
Vaccine study on Rhesus MacaquesGroups of male and female Rhesus macaque (n = 5) were
immunized at weeks 0, 8 and 24 with the two experimental vaccines described above. Serum was collected at weeks
0, 2, 4, 8, 10, 12, 16, 20, 24, 26, 28 and 52. The animals
were maintained in accordance with the Institutional Animal Care and Use Committees of Merck Research Laboratories (West Point, PA).
Antibodies
The monoclonal antibodies used in the HPV cLIA
included H6.M48 [15] for HPV 6, K11.B2 for HPV 11,
H16.V5 [16] for HPV 16 and H18.J4 [16] for HPV 18.
These antibodies have been shown to be HPV type-specific and to bind to neutralizing epitopes [17]. The mAbs
were conjugated to phycoerythrin (Chromaprobe, Aptos,
CA) and used at a final concentration of 0.1 µg/mL each.
HPV-specific immunoglobulin isotyping assay
A multiplexed antibody isotyping Luminex assay wasdeveloped to characterize the qualitative aspects of the

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Table 1: Isotype specific antibodies and streptavidin-PE used in the HPV antibody isotyping assays

Isotype Reagent

Type or MAb Identity Specificity

SourceSupplier

Concentration

Primary
IgM
IgA
IgE

polyclonal
polyclonal
HP-6029

Monkey IgM (mu chain specific)
Monkey IgA (alpha chain specific)
Human IgE (epsilon chain specific)

Goat
Goat
Mouse

50 µg/mL
25 µg/mL
25 µg/mL

HP-6091or 8c/639
HP-6014
HP-6025
polyclonal

Human IgG1
Human IgG2
Human IgG4
Monkey IgG (gamma chain specific)

Mouse
Mouse
Mouse
Goat

Rockland, Gilbertsville, PA
Rockland, Gilbertsville, PA
Southern Biotech,
Birmingham, AL
Sigma, St. Louis, MO
Sigma, St. Louis, MO
Sigma, St. Louis, MO
Rockland, Gilbertsville, PA

10 µg/mL
10 µg/mL
25 µg/mL
5 µg/mL

NA

NA

NA

Rockland, Gilbertsville, PA

5 µg/mL

IgG1
IgG2
IgG4
Total IgG
SecondaryPE- Streptavidin

HPV-specific humoral immune response. The multiplexed, isotyping assay was used to classify HPV-specificantibodies as IgM, IgA, IgE, IgG1, IgG2, IgG4, or total IgG.
IgG3 was not evaluated since rhesus macaques do notmake the IgG3 subclass of antibodies [18,19]. A panel ofisotype-specific monoclonal antibodies to IgE, IgG1,
IgG2, and IgG4 were purchased from either Sigma (St.
Louis, MO) or Southern Biotechnology (Birmingham, AL)
and isotype-specific polyclonal antibodies to IgA, IgM,
and total IgG were purchased from Rockland Inc. (Gilbertsville, PA). These antibodies (Table 1) were evaluatedfor reactivity to human, rhesus and African green monkey
immunoglobulins. Specificity of the antibodies to theirrespective immunoglobulin isotypes was established by a
Luminex assay using purified human IgM, IgA, IgE, IgG1,
IgG2, IgG3 and IgG4 covalently conjugated to Luminex
microspheres. Microspheres were incubated overnightwith Antibody Depleted Human Serum (ADHS), washedand detected using the isotype specific antibodies at theconcentrations shown in Table 1. Optimum antibody
concentrations, shown in Table 1, were established bycomparing differing concentrations of the detection Abs
in titrations of sera prior to immunization and positive
sera from HPV 6, 11, 16 and 18L1 VLP vaccinated African
green monkeys. Ab concentrations were chosen that gave
the most sensitivity and the greatest signal to noise ratio
between pre-immune and sera from vaccinated animals.
Cross-reactivity between isotype detection antibodies was
examined by conjugating purified human Igs to Luminex
microspheres and incubating these microspheres witheach of the isotyping antibodies at their chosen assay concentrations. In these experiments we detected some crossreactivity of the anti IgG1 MAb towards IgG2 (71%) andthe anti IgG2 towards IgG1 (58%). This cross-reactivity
was the rationale for decreasing the concentration of theanti-IgG1 and IgG2 MAbs to 10 µg/mL (Table 1).

For the multiplexed, HPV-specific antibody isotyping
assay, HPV 6, 11, 16, and 18 VLPs were covalently conjugated to four distinct Luminex microspheres identified as
microsphere 6, 11, 16 and 18, respectively. Test sera fromvaccinated rhesus macaques were serially diluted 5-foldstarting from a dilution of 1:10 with ADHS and incubatedovernight with the VLPs conjugated to microspheres(5,000 VLP-microspheres for each type in PBS + 1% Triton
X-100 (PBST) in a total volume of 100 µl in a 1.2 µm
hydrophilic, low protein binding, Durapore® membranefilter plate (Millipore, Bedford, MA). The plate contents
were washed 3 times with 200 µl of PBST and resuspended
in PBST. HPV type-specific antibodies bound to the VLPs
were incubated for 2 hours with biotinylated, isotype-specific secondary detection antibodies at the concentrations
shown in Table 1. The microspheres were washed threetimes, and resuspended in PBST. The biotinylated antibodies were detected by incubation with streptavidin conjugated to phycoerythrin (Strep-PE) for 30 min at a final
concentration of 5 µg/mL. The plates were washed 3 times
and read on a Bio-plex analyzer purchased from Bio-Rad
Laboratories, Inc. (Hercules, CA). The Bio-plex analyzer,
based on Luminex xMAP technology, is a modified flow
cytometer that allows for simultaneous quantitation of up
to 100 analytes in a single well and reports Median Fluorescence Intensity (MFI) signals from the Strep-PE detection reagent [20]. End-point dilution titers were definedby comparing signals to those of ADHS, the negative control. For positivity, an MFI value had to be above theaverage of ADHS + 10 standard deviations and the endpoint dilution had to demonstrate positivity at the previous lower dilution.
HPV type-specific competitive Luminex Immunoassay(cLIA)
An optimized and previously validated HPV competitive
Luminex Immunoassay (cLIA) first described by Opalka etPage 3 of 11(page number not for citation purposes)

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al. [21] was used to quantify HPV 6, 11, 16, and 18 specific antibodies to known-neutralizing epitopes on VLPs.
Using the same VLP-conjugated microspheres describedabove, antibody titers were determined using a competitive format, where HPV type-specific phycoerythrin (PE)labeled monoclonal antibodies (MAbs) to known neutralizing epitopes compete with serum antibodies forbinding to conformationally dependent, neutralizing
epitopes. The fluorescent signals from bound HPV-specific MAbs are inversely proportional to the subject's neutralizing antibody titers. Relative inhibition of MAb
binding in test serum was compared to a standard reference serum using a four-parameter logistic curve fit [22].
The reference sera for HPV 6, 11, 16 and 18 used for the
standard curve were assigned arbitrary values expressed inmilli-Merck Units per milliliter (mMU/mL). An antibody
titer of >200 mMU/mL for HPV 11 has been shown toneutralize ~108 virions in the athymic mouse xenograft
assay [23]. The titers for HPV 6 and 11 reference sera werepreviously determined in a pseudoneutralization assay[17]. The lower limit of quantitation for HPV6, 11 and 18
cLIAs is 8 mMU/mL and for HPV 16 is 12 mMU/mL.

6, 11, 16 and 18 also revealed statistically significant Pvalues of 0.045, 0.026, 0.039 and 0.008 respectively. Student's t-Tests of log-transformed antibody titers fromweeks 2–52 of the VLP+MAA versus the VLP alone groupfor HPV 6, 11, 16 and 18 also revealed statistically significant P-values of 0.0022, 0.0026, 0.0021, and 0.0026
respectively (Fig. 1).

Statistical Analysis
Antibody titers obtained from the HPV cLIA were logtransformed and analyzed using a one-tailed, non-paired,
Student's t-Test. P-values were obtained by comparing the
average log-transformed titers of each of the 5 monkeys
from week 2 to 52 within the two groups. The existence ofa statistically significant difference between antibody titers from rhesus macaques vaccinated with VLP + MAA versus VLP alone was shown by a P-value of less than 0.05.
Error bars represent the standard error of log-transformed
titers.

Results
Vaccination with HPV VLPs formulated with aluminum
adjuvant induces significantly higher titers than VLPs alone
The purpose of this study was to characterize the quantitative and qualitative effects of including MAA to the HPV6, 11, 16 and 18 VLP vaccine. First we wanted to measure
antibody titers to neutralizing epitopes on HPV L1
induced by a quadrivalent vaccine formulated with orwithout aluminum adjuvant using the HPV cLIA. Antibody titers for all four HPV types in both vaccine groups
developed after the first vaccination, declined, and thenincreased again following each boost (Fig. 1). The data areconsistent with a typical prime boost response. Peak antibody titers four weeks post dose 3, at week 28, for all four
HPV types were 12.7 to 41.9-fold higher for the group vaccinated with VLPs + MAA compared to the group vaccinated with VLPs alone, and between 4.3 to 26.7-foldhigher in the persistence phase at week 52 (Table 2A). Student's t-Tests of log-transformed antibody titers at week
52 of the VLP + MAA versus the VLP alone group for HPV

We also wanted to measure the total HPV-specific IgG levels to determine whether the formulation with MAA
increased the total HPV-specific Ab titers. The total HPVspecific IgG peak titers, and titers seven months post dose
three were consistently higher in the VLP+MAA groupcompared to the VLP group alone (Fig. 2). An anamnestic
response was observed after each vaccine boost and total
IgG levels were detectable through one year. The peak
antibody titers four weeks post dose 3, at week 28, in the
VLP+MAA group were between 65.7 to 125.0-fold higher
than those in the VLP alone group throughout the vaccination series and between 18.1 to 90.6-fold higher in thepersistence phase, at week 52 (Table 2B).
HPV-specific antibody isotype responses
To characterize the antibody isotypes elicited by VLPs formulated with MAA or VLPs, alone we modified a multiplexed Ab isotyping assay first developed to measure Ab
isotypes in humans [24]. These changes included using
detection mAbs that react with human, rhesus and African
green monkey Igs, where possible (Table 1). In addition
we used biotinylated Abs so that we could use a common
streptavidin-PE detection reagent. This novel HPV-specificantibody isotyping assay was found to be specific and sensitive for the detection of rhesus macaque IgM, IgA, IgE,
IgG4 and total IgG. Low cross reactivity was seen with theanti-IgG1 and IgG2 antibodies to purified human IgG2
and IgG1 respectively. Responses to these antibody isotypes were measured separately for HPV 6, 11, 16 and 18
and were similar to each other. Results for HPV16
responses which are representative of all four types areshown in figure 3.

We first examined the HPV-specific IgM responses because
IgM antibodies are the first to be produced following vaccination and would indicate a primary immune responseto the vaccine. As expected, an IgM response for all fourtypes was measured after the first immunization at week 0
and is shown for the representative type HPV 16 (Fig. 3A).
Interestingly, two additional primary IgM responses wereobserved after each of the booster vaccinations given at
weeks 8 and 24 in both the VLP and VLP+MAA groups ofthe study (Fig. 3A). IgM responses in the VLP alone groupreturned to baseline by week 52, which was seven months
post dose 3, while an IgM antibody response in the VLP +MAA group was detectable at week 52.

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A.

http://www.jibtherapies.com/content/3/1/2

B.

HPV 6

HPV 11

10000

10000

GMT (mMU / mL)

100000

GMT (mMU / mL)

100000

1000
100
10

VLP (+MAA)

P-value 0.0022

1000
100
10

1

VLP (-MAA)

1
0

10

20

C.

30

Time (weeks)

40

50

60

0

10

10000

GMT (mMU / mL)

10000
1000
100
10

VLP (+MAA)

P-value 0.0028
20

30Time (weeks)

40

50

60

50

60

100
10

VLP (+MAA)

P-value 0.0026

1
10

40

1000

VLP (-MAA)

130Time (weeks)

HPV 18

100000020

D.

HPV 16

100000

GMT (mMU / mL)

VLP (+MAA)

P-value 0.0026

VLP (-MAA)

0

10

20

30

Time (weeks)

VLP (-MAA)
40

50

60

FigureHPV6, 11,
1 16 and 18 type-specific antibody titers measured in the competitive Luminex Immunoassay (cLIA)
HPV 6, 11, 16 and 18 type-specific antibody titers measured in the competitive Luminex Immunoassay (cLIA).
Sera from rhesus macaques immunized with 2 µg each of HPV 6, 11, 16 and 18L1-VLPs formulated with Merck Aluminum
Adjuvant (MAA) (-◆-) or 2 µg each of HPV 6, 11, 16 and 18L1-VLPs alone (- -) were collected a t the indicated time points
and tested for Abs to neutralizing epitopes on HPV 6, 11, 16 and 18 using the HPV cLIA. Responses are reported as GMTs in
milli-Merck Units per milliliter (mMU/mL) (n = 5 animals per group) for HPV 6, 11, 16 and 18 (Fig 1A, 1B. 1C. and 1D respectively). Arrows indicate vaccination boosts at weeks 8 and 24. A one-tailed, un-paired t-Test analysis was conducted on logtransformed antibody titers obtained from the cLIA. Error bars represent the standard error of the titers within each group.

Since HPVs are sexually transmitted viruses that cross the
mucosal barrier in the anogenital tract, we next examined
whether the VLPs induced an IgA response and what effectformulation with MAA would have on an IgA response. In
theory, the induction of IgA would greatly enhance theeffectiveness of an HPV vaccine. Both vaccine groups demonstrated type-specific IgA responses to the VLPs that weremaintained through one year (Fig. 3B). Each vaccine
boost elicited secondary IgA responses in both vaccination study groups (Fig. 3B). Higher IgA end-point dilution
titers were observed in the rhesus macaques vaccinatedwith VLP + MAA compared to those vaccinated with VLP
alone.
We also examined whether there was an IgE response
induced following vaccination. The geometric mean titers(GMTs) of IgE responses for the two vaccine groups were
not above background, however one animal in the VLP
group had a detectable IgE response at week 4 for HPV 6
and 16, and at week 24 for HPV 16 (data not shown). This
response was just barely above the limit of detection andPage 5 of 11(page number not for citation purposes)

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Table 2: Fold difference in geometric mean titers of VLP+MAA-immunized group over VLP alone-immunized group.

A.
HPV type

HPV cLIA time (weeks)
0
2
4
8

10

12

16

20

24

26

28

52

HPV 6
HPV 11
HPV 16
HPV 18

1.1
0.8
1.0
1.0

10.0
23.5
21.4
70.5

12.6
18.1
15.7
18.6

36.3
16.2
16.1
27.1

10.9
8.2
11.4
35.4

6.3
6.1
17.7
26.3

22.7
18.4
4.8
6.7

41.9
36.8
12.7
26.8

4.3
5.3
6.4
26.7

B.
HPV type

HPV Total IgG antibodies time (weeks)
0
2
4
8
10

12

16

20

24

26

28

52

HPV 6
HPV 11
HPV 16
HPV 18

1.9
1.9
0.9
0.7

328.3
90.6
65.7
47.6

125.0
125.0
90.6
65.7

65.7
47.6
65.7
65.7

34.4
34.5
13.1
25.0

34.5
34.5
13.1
47.6

65.7
90.6
65.7
125.0

18.1
18.1
25.0
90.6

3.2
2.9
5.2
4.8

9.5
13.1
5.0
9.5

2.4
2.2
8.8
13.7

9.5
25.0
9.5
13.1

4.3
5.5
8.4
19.4

13.1
25.0
6.9
25.0

125.0
125.0
65.7
125.0

was not detected following the booster doses given at
weeks 8 and 24.
We next measured the IgG subtypes to determine whether
formulation with Merck aluminum adjuvant affected theIg isotype profile. Characterizing the IgG isotype profile
would also provide insight into whether formulation with
MAA altered the TH1 or TH2 profile of the immune
response. The main IgG subclass observed in both vaccination groups was IgG1 (Fig. 3C). Similar to responses
observed for total IgG, secondary IgG1 responses wereobserved after each boost, and titers were maintained
through one year in the VLP + MAA group (Fig. 3C). However, the group vaccinated with VLP alone had detectable
titers only after the second immunization and these titers
approached baseline values at one year (Fig 3C). We also
observed high levels of IgG4 and Ab titers in the VLP +MAA group were detected early after each vaccination, butwere undetectable by week 52 (Fig. 3D). The monkeys
vaccinated with VLPs alone only demonstrated IgG4
responses after each vaccination boost, but the titers fell
below the limit of detection soon after (Fig. 3D). We did
not detect a strong IgG2 response in the VLP-alone group
and IgG2 levels in the VLP + MAA group were observed
only immediately following vaccination (data notshown). However, because we observed some cross-reactivity of the anti-IgG2 MAb to human IgG1, we cannot
rule out the possibility that the IgG2 signals observed were
due to cross-reactivity of the anti-IgG2 MAb to the highlevels of rhesus IgG1 Ab (Fig. 3A). HPV-specific IgG3
responses were not measured because rhesus macaques
do not make the IgG3 subclass of antibodies [18,19]. Insummary, higher total IgG, IgG1, and IgG4 Ab titers wereobserved in the rhesus macaques vaccinated with VLP +MAA compared to those vaccinated with VLPs alone.

Discussion
This is the first study to directly examine the effects of the
Merck aluminum adjuvant on immune responses to aquadrivalent HPV 6, 11, 16 and 18 L1 vaccine. The results
here were generated using a novel HPV-type specific antibody isotyping assay to characterize the immunoglobulin
subclasses to HPV 6, 11, 16 and 18 in rhesus macaques,
and a competitive Luminex immunoassay (cLIA) thatmeasures HPV-type specific antibodies to known neutralizing epitopes. The quadrivalent HPV L1 VLP vaccine formulated with MAA induced higher titers for total IgG (Fig.
2), as well as for all Ig isotypes measured (Fig. 3) compared to the vaccine without MAA. Also the MAA-formulated vaccine induced higher overall antibody titers toneutralizing epitopes compared to one without MAA (Fig.
1). Strong immune responses were elicited against all four
HPV types and high antibody titers persisted through one
year.
Results from the HPV cLIA showed that the vaccine
induced antibodies that competed with MAbs to known
neutralizing epitopes, and are, thus, theoretically able toneutralize HPV. It is worth noting that we have previously
shown that a competitive radioimmunoassay (cRIA) [25]and the cLIA correlate well with an HPV 11 neutralization
assay. Antibody titers in the cLIA also followed a "primeboost" response wherein antibody titers were higher after
every vaccine dose. Furthermore, cLIA titers show that formulation of the vaccine with MAA significantly increased
peak neutralizing titers, and, more importantly, thedifferences in the responses were durable, persisting
through one year.
Previously, we reported that the Merck HPV 16 L1 VLPvaccine induced a predominantly TH2 immune responsewith IL-4-producing T-helper cells, high levels of neutralizing Abs and low levels of IFN-γ secreting CD8+ or CD4+
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A.

http://www.jibtherapies.com/content/3/1/2

B.

HPV 6

HPV 11

1000000

1000000

100000100000

10000

10000

GMT

10000000

GMT

10000000

1000
100

1000
100

Total IgG (+MAA)

10

Total IgG (-MAA)

Total IgG (-MAA)

1

1
0

10

20

C.

30

Time (weeks)

40

50

0

60

10

20

D.

HPV 16

1000000

1000000

100000100000

10000

10000

GMT

10000000

1000
100

40

60

Total IgG (+MAA)

10

Total IgG (-MAA)

50

1000
100

Total IgG (+MAA)

1030

Time (weeks)

HPV 18

10000000

GMT

Total IgG (+MAA)

10

Total IgG (-MAA)

1

1
0

10

20

30

Time (weeks)

40

50

60

0

10

20

30

Time (weeks)

40

50

60

FigureHPV6, 11,
2 16 and 18 L1 VLP-specific total IgG antibody titers
HPV 6, 11, 16 and 18 L1 VLP-specific total IgG antibody titers. Sera from rhesus macaques immunized at week 0, 8
and 24 with 2 µg each of HPV 6, 11, 16 and 18L1-VLP formulated with Merck Aluminum Adjuvant (+MAA) (-◆-) or 2 µg eachof HPV 6, 11, 16 and 18L1-VLPs alone (-MAA) (- -) were collected at the indicated time points and tested for HPV 6, 11, 16
and 18 L1 VLP specific total IgG (-◆-, - -) titers in a multiplexed detection assay. Responses are reported as the geometricmeans of end point dilution titers (n = 5 animals per group) for HPV 6, 11, 16 and 18 (2A, B, C, and D respectively). Arrows
indicate vaccination boosts at weeks 8 and 24. The starting dilution for each sample was 1:10 and responses above an end point
dilution of 10 were considered positive.

cells [26]. Given that the HPV quadrivalent vaccine also
contains the L1 capsid proteins, the response was also
expected to be TH2-like [27]. Using the HPV isotyping
assay, which is a direct binding assay detecting antibodies
to conserved epitopes shared across multiple HPV types,
we showed that the isotype profile of the vaccinated rhesus macaques was consistent with a TH2-like immune
response. Interestingly, the formulation with MAA did notaffect the overall isotype profile of the vaccinatedmonkeys.

Predictably, abundant amounts of IgG antibody isotype
were found, and of all the IgG subclasses, IgG1 was most
readily detected. The abundant presence of IgG1 elicitedin both vaccination groups affirms that the vaccines did
induce immune responses through a TH2 pathway. It isalso interesting to note that the next abundant IgG subclass detected, primarily detected in the VLP+MAA group,
was IgG4 (Fig. 3D). IgG4 is more characteristic of a TH1
response and, by inducing this IgG subclass, the vaccine isshown to be also effective in eliciting a cellular (TH1)immune response, albeit, predictably not long lasting. It is

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1000000

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FigureHPV
L1-VLP
3 specific IgM, IgA, IgG1 and IgG4 antibody titers
HPV L1-VLP specific IgM, IgA, IgG1 and IgG4 antibody titers. Sera from rhesus macaques immunized at week 0, 8 and24 with 2 µg each of HPV 6, 11, 16 and 18 L1-VLPs formulated with Merck Aluminum Adjuvant (+MAA) (-◆-) or 2 µg each of
HPV 6, 11, 16 and 18L1-VLPs alone (-MAA) (- -) were collected at the indicated time points and tested for HPV L1 VLP specific (A) IgM, (B) IgA, (C) IgG1 and (D) IgG4 titers in a multiplexed detection assay. Responses are reported as GMTs (n = 5monkeys per group) for HPV 16. Arrows indicate vaccination boosts at weeks 8 and 24. The starting dilution for each sample
was 1:10 and responses above an end point dilution of 10 were considered above background. Graphs shown are for HPV 16
and are representative of HPV 6, 11, and 18.

worth noting that we previously observed low levels of
IFN-γ secreting CD8+ and CD4+ T-cells [26], suggesting thevaccine induces a low level cellular immune response.
Similar to what we observed in rhesus macaques, isotype
analysis of sera from women vaccinated with the HPV 11
VLP vaccine formulated with MAA also detected little tono levels of IgG2 [24]. Isotype analysis performed on an
epidemiological study by Wang et al. also found no IgG2
antibody, which they attributed to the unfavorable

cytokine profile of HPV infection for the induction of
IgG2 [28]. Detection of the IgG3 subclass was not performed, because rhesus macaques do not make IgG3
[18,19].
We also measured high levels of serum IgA observed in
vaccinated animals. IgA, the secretory immunoglobulin, isthought to be very important in blocking viral entry into
mucosal tissues [29]. These findings are consistent with

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what we have observe in the sera from women vaccinatedwith the HPV 11 VLP vaccine [24]. The elicitation of IgAshould be advantageous for a vaccine that prevents HPV
infection in defending against entry of HPV, which commonly enters through the urogenital tract. The high levelsof IgA elicited by the vaccine may also be one reason why
the vaccine has been efficacious in human clinical trials[11]. IgE responses were measured because IgE antibodies
are implicated in allergic symptoms. Consistent IgE antibody responses were not observed in either group. Only
one animal in the VLP alone vaccine group had detectable
IgE, but the response was barely above the limit of detection and transient as it was not detected following thebooster immunizations. This is consistent with the observation of no allergic reactions or severe adverse events inthe monkeys following vaccination. These observations
are consistent with our clinical findings that a VLP + MAA
vaccine was safe and well-tolerated [30]. In summary, theisotype analysis data are consistent with the VLP+MAA
vaccine inducing primarily a TH2-like immune responsewith high titers of IgA and IgG1 antibodies.

For this reason, we used Antibody Depleted Human
Serum (ADHS) as a negative control rather than sera fromrhesus macaques prior to vaccination.

In these isotyping studies, we used polyclonal goat antimonkey IgM, IgA, and total IgG; because monkey-specificreagents are not available for the IgG subtypes and IgE, weused anti-human mouse MAbs to measure IgG1, IgG2,
IgG4, and IgE responses. The polyclonal anti-IgA, IgM andtotal IgG reagents were selected for their specificity andtheir ability to broadly detect African Green monkey, rhesus macaque and human antibody isotypes. The various
differences between human, rhesus macaque and African
green monkey sera could account for changes in the sensitivity and specificity of the antibody-isotyping reagentsand so the appropriate antibody reagent concentrations
used in the HPV type-specific antibody isotyping assay
were optimized for use with rhesus macaque serum.
Due to the unavailability of purified rhesus Igs, we usedpurified human Igs to determine the specificities of thetype-specific antibody isotyping reagents. There was someobserved reactivity of the anti- human IgG2 antibody topurified human IgG1, and vice versa, but these data wereobtained using purified human Igs and the differences
between human and rhesus antibodies may account for
why we saw a strong IgG1 response with low IgG2 titers(Fig. 3). However, because we cannot rule out that the
IgG2 titers were merely a reflection of the cross-reactivity
to the high IgG1 titers, we cannot definitively say that
there were any IgG2 responses observed.
In some cases, the test animals had low Ab titers to the
VLPs prior to vaccination. This was not unexpected sincemore than 100 papillomaviruses have been identified[31] and many of these share conserved structural
epitopes that would cross react in a direct binding assay.

Currently, the only FDA approved adjuvants for use withprophylactic vaccines are aluminum based [32]. The purpose of this study was to evaluate the effect of the Merck
Aluminum Adjuvant on the immunogenicity of the HPV
L1 VLP 6, 11, 16 and 18 vaccine and our results show thatthe addition of the MAA significantly increases the immunogenicity of the vaccine. In sharp contrast to our results,
Harro et al. have reported that formulation with adjuvant
did not significantly enhance the immunogenicity of an
HPV VLP vaccine [33]. In that study, 72 human volunteers
were randomized to receive placebo or an HPV 16 L1 VLPvaccine produced in insect cells. Vaccine recipients were
given a dose of 10 µg or 50 µg of VLPs formulated without
adjuvant, with an aluminum adjuvant, or with MF59
adjuvant. Since no significant differences in neutralizing
antibody titers and ELISA titers were found in subjects inall three groups, the authors concluded adjuvant was not
required. Our results were obtained using the quadrivalent Merck HPV-L1 VLP vaccine, which is formulated withMerck aluminum adjuvant. While the mechanisms bywhich aluminum adjuvants enhance immunogenicity arepoorly understood, they may act to form an antigen
depot, allowing the vaccine to linger at the site of administration and perhaps, more importantly, stimulate theimmune system by inducing higher neutralizing Ab titers[34]. The inclusion of MAA to the vaccine did not induce
a qualitatively different humoral immune response in thatthe isotype profile was similar between animals vaccinated with the two quadrivalent vaccines (Fig. 3). However, data from both the antibody isotyping assays and thecLIA showed that the inclusion of MAA to the VLP vaccine
induced significantly higher Ab titers (Fig. 1, 2, 3). These
data clearly show a benefit to the formulation of the quadrivalent HPV-L1 VLPs with MAA.
The differences observed in the immune responses elicited by the vaccine reported by Harro et al. and the Merck
quadrivalent HPV vaccine may be attributed to several factors. First, would be the differences in the formulation andpurification processes of the vaccines. The vaccine
reported by Harro et al. was produced in insect cells as
opposed to yeast for the Merck vaccine. Second, the differences in vaccine recipients, humans and rhesus macaques
may respond differently to adjuvanted HPV VLP vaccines.
Lastly, differences in formulation and composition of thetwo aluminum adjuvants used could have profound
impacts on immunogenicity. Different aluminum salt
adjuvants were used in these two studies; our study
adsorbed VLPs to a proprietary formulation of aluminumhydroxyphosphate sulfate (MAA), while the Harro et al.
study used aluminum potassium sulfate. Without a head

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to head comparison of the two vaccines, it is difficult toestablish why the results are different. However, the magnitude and durability of antibody responses found in thequadrivalent VLP+MAA vaccinated rhesus macaques
reported in this study are consistent with those found in
human clinical trials of the HPV 11 or HPV 16 monovalent vaccines [30].

HIV groups (Merck, MRL), for their assistance and support; R. Charbonneau and S. Macmullen for previous isotyping development, P. Boerckel forher expert laboratory assistance, T. Green, for expert statistical advice andassistance, R. Marchese (Merck MRL Wayne), for her critical reading of themanuscript and expert guidance, and T. Fujiwara for his continued motivation and encouragement.

References
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The isotyping and cLIA assays described in this study areuseful in characterizing the immune responses to HPV virions after natural infection or following vaccination. The
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Competing interests
W. Ruiz, W. McClements, K. Jansen and M. Esser were all
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W. McClements and K. Jansen designed the immunization study. M. Esser and W. Ruiz developed thecompetitive Luminex immunoassay and the new isotyping assays. W. Ruiz performed all the laboratory assays. W.
Ruiz and M. Esser wrote the manuscript and all authors
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