BIOLOGY OF REPRODUCTION 54, 264-269 (1996)
Residence Half-Life of IgG Administered Topically to the Mouse Vagina'
Jill K. Sherwood, 3 Larry Zeitlin,4 Xiaolei Chen,4 Kevin J. Whaley, 4 Richard A. Cone,4 and W. Mark Saltzman 2 3
Departments of Chemical Engineering3 and Biophysics4
The Johns Hopkins University, Baltimore, Maryland 21218
ABSTRACT
Antibodies delivered directly to the vagina can provide passive immunoprotection against pregnancy and sexually transmitted disease.
The duration of protection is limited by the residence time of the antibodies inthe vagina; to our knowledge such residence times have
not been reported. We investigated the time-course of disappearance of IgG delivered to the mouse vagina using three different methods
to monitor the amount of administered IgG remaining inthe vagina: gamma counting of '251-lgG, viral neutralization of unlabeled monoclonal anti-herpes virus IgG2a, and ELISA of biotinylated IgG. The test IgG was delivered to the vagina insaline and recovered by lavage.
All three methods yielded similar results, suggesting that the residence half-life is not significantly affected by the volume administered,
phase of the estrous cycle, or labeling of IgG. In awake mice, a significant fraction of IgG disappeared with a relatively short half-life, (t,,2),,
of 0.7 0.1 h; but this rapid (a phase) decrease did not occur inanesthetized mice, suggesting that the movements of awake mice expel
some of the test IgG-saline solution from the vagina. Over the next 25 h,the test IgG disappeared with a residence half-life, (t1n2),, of 5 +
2 h.We believe this slow elimination of IgG may depend on the rate that mucus secretions are shed from the vagina.
INTRODUCTION
mice [4], and vaginal delivery of anti-rabbit sperm mAbs was
shown to prevent pregnancy in rabbits [5]. Furthermore, intravaginal polymeric devices have been demonstrated to release biologically active antibodies directly to the vaginal lumen for thirty days [6]. Despite these advantages and
demonstrated efficacy, the pharmacokinetics of intravaginal
antibody administration have not been well studied.
To develop effective methods of passive immunoprotection in the vagina, it is essential to know the rate of antibody
clearance from the vaginal lumen. This rate of antibody
clearance can be characterized by the residence half-life
(t 1,2), which is the time necessary for half of the administered antibody to be eliminated from the vagina. Any strategy for passive immunoprotection is only effective for as
long as the antibody concentration is maintained above a
minimum effective level; therefore, the residence half-life is
an important parameter because it enables prediction of the
dose of antibody required to provide protection for a specific interval of time after administration. To our knowledge,
no measurements of the residence half-life of antibodies in
the vaginal lumen have been reported.
The results reported in this paper were obtained by three
different investigators working independently; each used a
different technique to investigate the residence half-life of
antibodies delivered topically to the mouse vagina. The
concentration of IgG in the mouse vaginal lumen was studied after administration of a single intravaginal dose of either 125I-IgG, an anti-HSV2 IgG2a mAb, or biotinylated IgG.
All three methods gave similar results, and since each
method acted as a control for the others, the results are
combined in this report. In order to obtain the best value
for the residence half-life, the time-course of IgG in the vaginal lumen was modeled mathematically by optimizing parameters to best represent the experimental measurements.
Sexually transmitted diseases (STDs) and unwanted
pregnancies in humans are two escalating worldwide concerns. Every day approximately 685 000 new transmissions
of STDs occur worldwide [1]. This is a sobering statistic,
since many STDs cannot be cured, and several can be fatal.
Prevention of unwanted pregnancy, through contraception,
would decrease the incidence of abortion, which occurs
about 150 000 times a day [2]. Since it is unlikely that any
public health measure would significantly decrease the incidence of sexual intercourse, which occurs 100 million
times a day worldwide [2], increasing the number and desirability of available methods of prophylaxis is a reasonable
approach to resolving these dilemmas. Specifically, the use
of topically applied monoclonal antibodies (mAbs) to the
vagina for passive local immunoprotection may provide
new and more appealing methods of protection against disease as well as unwanted pregnancy [3].
With human mAb production becoming more common
and less expensive, the use of antibody-based prophylaxis is
increasingly attractive. Furthermore, the vagina is a propitious delivery site because it is easily accessible, and local
treatment reduces side effects that frequently occur with systemic delivery. Recently, vaginal delivery of anti-herpes simplex virus 2 (HSV2) mAbs was shown to provide complete
protection against vaginal transmission of HSV2 infections in
Accepted September 1, 1995.
Received June 7. 1995.
'This work was supported by grant number GM43873 from the National Institutes of
Health. Larry Zeitlin is a Howard Hughes Medical Institute Predoctoral Fellow.
2Correspondence: Professor W. Mark Saltzman, Department of Chemical Engineering,
The Johns Hopkins University, 3400 N. Charles Street. Room 42 New Engineering Building,
Baltimore, MD 21218. FAX: (410) 516-5510.
264
RESIDENCE HALF-LIFE OF IgG IN THE MOUSE VAGINA
MATERIALS AND METHODS
Animals and Antibodies
Three different antibody preparations were used to determine the residence half-life of IgG in the mouse vaginal lumen: 12 5I-labeled polyclonal mouse IgG (Rockland, Gilbertsville, PA), biotin-labeled polyclonal mouse IgG (Jackson,
West Grove, PA), and an unlabeled anti-herpes virus monoclonal mouse IgG2a (III-174; produced in culture from a cell
line from Patricia G. Spear, Northwestern University, Evanston, IL). All procedures used 8- to 12-wk-old female C57BL/
6 mice (Harlan, Indianapolis, IN) and were approved by the
Johns Hopkins Animal Care and Use Committee.
Antibody Iodination
The purity of the antibody to be iodinated was confirmed
by SDS-PAGE. One millicurie of Na125I (Amersham, Arlington Heights, IL) was used to iodinate 100 plg of antibody in
50 pl of PBS (Sigma, St. Louis, MO) by use of an iodogen
tube, as described by Salacinski et al. [7]. Next, the solution
was passed through a Dowex column with PBS/10% horse
serum or a desalting column (Econo-Pac 10ODG; Bio-Rad,
Hercules, CA) with PBS to remove any unbound 1251.
Progestin Treatment
To eliminate differences due to the estrous cycle, in some
experiments mice were treated with a long-acting progestin
(Depo-Provera; Upjohn, Kalamazoo, MI) to induce an acyclic diestrus-like state [8]. Mice were administered a 2.5-mg
dose of progestin in 0.1 ml of PBS s.c. in the back of the
neck with a 26-gauge needle 7 days prior to experiments.
GeneralProcedure
Anesthetized mice (Metophane; Mallinckrodt, Mundelein,
IL), either cycling or progestin-treated, received a vaginal bolus of antibody in saline. The mice were then placed into
cages with free access to food and water. At the desired time
after vaginal administration, the mice were anesthetized and
vaginally lavaged by pipetting PBS in and out of the vaginal
lumen several times. The amount of antibody in the mucus
was then determined from these lavage samples. Three different procedures and detection methods were used.
1. Iodinated mouse IgG. Ten microliters or 2 Pll of 1 2 5IIgG in PBS was collected in a pipette tip, which was then
gently removed from the pipette, and the amount of radioactivity was determined by using a gamma counter (G5500;
Beckman, Fullerton, CA). The antibody solution was then
pipetted into the mouse vaginal lumen, and the amount of
radioactivity remaining in the tip was determined in a
gamma counter. The delivered dose was determined by
subtracting the residual amount of radioactivity in the tip
from the initial amount in the tip. The mice were then
housed individually with grates in the bottom of the cages.
To determine the amount of 125I-IgG remaining at various
265
times following administration, the vaginal lumen was lavaged with 20 lI of PBS by pipetting in and out 20 times; this
was repeated three times with additional 20-l aliquots of
PBS for each lavage. The four samples along with the pipette
tips (it was difficult to expel all of the mucus from the tip)
were each analyzed for radioactivity in a gamma counter.
This protocol was used on both normal cycling mice with
horse serum in the antibody solution (10 ll), and progestintreated mice without horse serum in the antibody solution (2
Pl). All experiments were performed in triplicate. Nine additional cycling mice also underwent the aforementioned
procedure (10 Il) but were kept anesthetized up to 2 h.
2. Monoclonal mouse IgG2a. Two microliters of antiherpes simplex virus 2 (anti-HSV2) monoclonal antibody
III-174 in PBS (0.5 mg/ml) was pipetted into the mouse vaginal lumen. At 1 min, 1 h, 5 h, or 25 h, each of three mice
was lavaged (by pipetting fluid in and out 20 times) with
one 20-pl aliquot of medium (Tissue Culture Refeeding Medium; Baxter, McGaw Park, IL). The samples were analyzed
by a neutralization assay. Serial dilutions were made from
each sample, and 75 Pl of each dilution was mixed with 25
P1 of 500 TCID 50 (that is, 500 times the amount of tissue
culture infectious dose required to infect 50% of the wells)
of HSV2 (Virotech, Rockville, MD). These samples were incubated for 1 h at 37°C and then placed on target cells (human diploid foreskin fibroblasts; Baxter) and incubated at
37°C for 2 days. The target cells were then visually inspected
for cytopathic effect. This procedure was used on normal
cycling mice and progestin-treated mice.
3. Biotinylated mouse IgG. Two microliters of biotinylated polyclonal mouse IgG in PBS (1 mg/ml) was pipetted
into the vaginal lumen of progestin-treated mice. Lavage
samples were taken from six mice at 0.5, 2, 6, and 24 h by
lavaging six times with 25-pl aliquots of PBS. Each lavage
consisted of pipetting PBS in and out of the vagina 10 times.
The six samples were pooled for each mouse, and the
amount of antibody was determined by an ELISA. Briefly,
the ELISA consisted of coating with 10 gg/ml of avidin (Vector, Burlingame, CA) and blocking with 2.5% hydrolyzed
casein. Next, the mucus in the lavage samples was sheared
by a 1/2-cc Lo-Dose insulin syringe (Becton Dickinson,
Cockeysville, MD) and diluted in blocking buffer before incubation. Next, the avidin coating was saturated with a
1:1000 dilution of free biotin (ABC kit; Vector), and the plate
was incubated with a 1:3000 dilution of ABC. o-Phenylenediamine/hydrogen peroxide in citrate buffer was used as
the substrate, and the absorbance at 490 nm of each sample
was determined after 15 min.
Calculation of Residence Half-Life
The residence half-life of antibody within the vaginal lumen was defined as the amount of time it takes for half of
the administered antibody to be eliminated; it is related to
the first-order elimination constant, k, by t/ 2 = (In 2)/k. A
266
SHERWOOD ET AL.
TABLE 1. Model parameters.
100
Type of IgG
0
c_
125
1-lgG
125l9-gG
Anti-HSV2
Anti-HSV2
Biotin-lgG
10
a,)
C
n
Dose
(u
1 L)
Ra
(%)
(t112),,
(h)
(t/ 2),
(h)
None
Progestin
Progestin
None
Progestin
10
2
2
2
2
90
70
60
N/A
N/A
0.7
0.7
0.6
0.8
N/A
5.0
4.6
7.0
N/A
3.7
aAmount eliminated in the a phase.
of 10 Al of
0.1
12
5I-IgG, 74 ± 9% of the bolus dose was recov-
ered in the vaoinal lavaoes 0
0
5
10
15
Time (hours)
20
25
h after
idministratin
(Fie
1A). Antibody elimination from the vaginal lumen had two
distinct phases (Table 1): 90% of the antibody was elimi..-..... ~~ _ Jl_ /'a1
_':
1
n/
.1 _ .1
.1_.1:L=
. / n), while
/o or me anUDouy
was eliminated more slowly ((tl/2) = 5.0 h). In contrast,
nateo rapully tt,2)
1 UU
co
C
._
0
Mouse treatment
A
0
o
1
C
a)
Co
0.1
0
5
10
15
Time (hours)
20
25
FIG. 1. Percentage of vaginal dose of 25l-lgG remaining in me
house
vaginal lumen
quantified by gamma counting. Squares denote measureme
nts
from
individual
mice; dotted line is lavage efficiency. A) Normal cycling mice tthat received 10 pl of
25
' 1-lgG
solution containing horse serum. B)Progestin-treated rnice administered 2
lI of 251-gG.
simple exponential function did not adequa tely represent
tely
represent
ocall of the data. Therefore, we assumed that e elimination
curred in two first-order phases and used a biexponential
function to characterize the data:
C,,,ode(t) = Rexp(-k.'t) + (100-R)exp(-k
where C,,ode(t) was the predicted value, k= arnd ki were the
elimination constants, and R was the percentaige of the substance that was eliminated by the initial (a) eliimination rate.
The parameters k., k,, and R were adjusted u ntil the model
best represented the data by minimizing th.e sum of the
squared errors, X2:
2.
En
]2
X2 =
[(Cmast.red)i
-
Cmodel(ti)
where ti and (Cneatred)i were the x and y comlponents of the
data, respectively, and Cmode(ti) was the value of the model
evaluated at the independent variable ti.
when mice were maintained under anesthesia, 75 + 10%
of the dose was recovered in the lavages at 0.5 h; over the
next 1.5 h there was no detectable decrease in the amount
of 125I-IgG recovered (78 + 8%, Fig. 2).
Progestin-treatedmice. In progestin-treated mice that
received 2 1of 1'2 I-IgG, 78
19% of the dose given was
recovered from the vaginal lumen by lavage 1 min after
administration (Fig. 1B). Antibody elimination in progestintreated mice that received a 2-pl bolus with horse serum
was similar to that in cycling mice that received a 10-11 bolus
without horse serum, except that the amount of antibody
eliminated rapidly by the initial elimination rate was 70%
instead of 90%, respectively. The initial residence half-life
of
I-IgG in progestin-treated mice, (tl/2),, was 0.7 h, and
the subsequent slow half-life, (t/,2)f, was 4.6 h (Table 1).
Monoclonal Mouse IgG2a
Normal cycling mice. In normal cycling mice that received 1000 ng of anti-herpes IgG2a, 600
400 ng was
recovered in the vaginal lavages 1 min after administration.
By 25 h, neutralizing activity was not detected in the lavage
samples, suggesting that the antibody recovered from the
lumen was less than 1 ng. Since there were not any measurements after 5 h, only the initial residence half-life (tl/,),,
could be calculated, which was 0.8 h (Table 1).
Progestin-treatedmice. The amount of antibody recovered from the vaginal lumen of progestin-treated mice 1 min
after administration of anti-HSV2 IgG2a was 130 ng (Fig. 3).
After 25 h, 0-16 ng of anti-HSV2 antibody was recovered
in the vaginal lavages. The percentage of dose eliminated
by the initial elimination rate, R, was 60%. The residence
half-lives, (t,/,2) and (t,/2), were determined to be 0.6 h and
7.0 h, respectively (Table 1).
RESULTS
lodinated Mouse IgG
Normal cycling mice. In cycling mice that were allowed
to awake from anesthesia immediately after administration
BiotinylatedMouse IgG
Of the 2 ptg of biotinylated IgG administered to progestintreated mice, 0.6
0.4 fig of the IgG was recovered by
267
RESIDENCE HALF-LIFE OF IgG IN THE MOUSE VAGINA
vaginal lavage at 0.5 h after administration. After 24 h, 0.01
+ 0.01 jig remained in the vaginal lumen. Only the residence half-life (tl/ 2 ), could be calculated for these results; it
was 3.7 h (Table 1).
100
o,
a)
DISCUSSION
The pattern of IgG disappearance from the vaginal lumen following vaginal administration of a bolus was studied
with 125I-IgG, HSV2-specific mAb, or biotinylated IgG. The
method using 125I-IgG had several advantages. First, gamma
counting to determine 125I-IgG concentration was at least 10
times more sensitive than either the neutralization assay
used to detect the anti-HSV2 IgG2a or the ELISA to determine biotinylated IgG concentration. With the dose and
specific activity that was used, the gamma counter could
detect as little as 0.01% of the dose given to the mice. Second, using the 125I-IgG permitted accurate determination of
the delivered dose, since it was possible to determine the
residual radioactivity of the intended dose remaining in the
pipette tip. In our experience, as much as half of the intended dose was not delivered. The difference between intended dose and delivered dose could not be determined
in the other two procedures. Third, the 125I-IgG lavage samples were read directly in a gamma counter; no manipulation of the samples was necessary. In contrast, the antiHSV2 IgG2a and the biotinylated IgG samples had to be
diluted, and then the amount of IgG in the samples was
assayed indirectly. On the other hand, the procedure using
mAb against HSV2 had two important advantages: the antibody was unlabeled, and the method of detection was
based on the functional activity of the mAb. All three methods gave similar results (Table 1), suggesting that IgG was
eliminated from the vagina without being detectably denatured and that the 125I and biotin labels did not significantly alter the residence half-life.
The different methods used in these combined studies
also provide practical details for future work. For example,
in the procedure that used 125I-IgG, each of the four lavages
taken from an individual mouse was analyzed separately in
the gamma counter; this allowed us to determine the lavage
efficiency. The first lavage removed approximately 70 -+5
10% of the 125I-IgG from the lumen, and the amount of 125IIgG in each subsequent lavage decreased by a factor of ten;
therefore, ravaging more than four times did not significantly increase the amount of 125I-IgG recovered. Lavaging
four times, as was done in this procedure, was sufficient for
obtaining all of the 125I-IgG that could be removed from the
vaginal lumen by lavage, which was 80
10% of the dose.
This suggests that as much as 20% of the delivered antibody
adhered to the vaginal surfaces in a way that resisted repeated ravaging. We verified this by removing the reproductive organs from some of the animals that received 125IIgG; most of the residual activity remained associated with
10
Un
0o
n
1
1
Time (hours)
0
2
FIG. 2. Percentage of vaginal dose of 251-lgG remaining in mouse vaginal lumen
in awake mice versus those remaining under anesthesia. These mice were in a
natural estrous cycle and received 10 i of IgG. Circles indicate measurements from
mice that remained anesthetized; squares denote measurements from mice that
were awake after administration of bolus (Fig. 1A). Error bars represent standard
deviation from three mice. Dashed lines represent range of half-lives for P phase;
dotted line is lavage efficiency.
the vaginal tissue (data not shown). In all of the figures, a
dotted line at 80% of the administered dose represents the
lavage efficiency. Studies using biotinylated IgG also
showed that adequate recovery required four lavages, but
that more than four lavages was not significantly beneficial.
The elimination of 25I-IgG from the vaginal lumen was
best described mathematically by a biexponential function,
suggesting that '2 5 I-IgG solution was eliminated from the vaginal lumen by two different processes. We hypothesize that
a large portion of the administered solution was eliminated
in the first few hours (a-phase) by fluid leakage after awakening, a process that was facilitated by the mouse's movements and grooming. The remaining '2 5I-IgG was eliminated
more slowly (3-phase), probably because it had diffused into
the mucus that lines the vaginal epithelium and therefore was
not entirely expelled by the initial fluid flow. In mice that
received the largest volume of 125I-IgG in PBS (10 pil), more
1UUU .... ....... ........... .......... .......................................................
0
D
13
0)
ci
100O
(,
I
c
:
Z
10 L
0
0
E
I
I
.
0
5
_ I
10
15
I
-.
20
.
.
I.~
i
25
Time (hours)
FIG. 3. Amount of vaginal dose of anti-HSV2 lgG2a remaining in mouse vaginal
lumen as determined by a neutralization assay. Each progestin-treated mouse received 1 g of anti-HSV2 IgG2a in 2 1of PBS. Squares indicate measurements from
individual mice; dotted line is lavage efficiency.
268
SHERWOOD ET AL.
than 90% of the dose was eliminated during the a-phase; only
70% of the dose was eliminated during the a-phase from the
mice that received a much smaller volume of 125I-IgG solution (2 l). This suggests that the percentage of the dose
eliminated by the initial elimination rate, R, increases with
increasing volume of liquid administered intravaginally;
however, other variables may also have been involved such
as the horse serum in the 10-gl dose and progestin treatment
of the mice that received the 2-1l dose. In addition, this aphase elimination probably depends on the properties of the
liquid, such as viscosity. A more viscous solution would presumably have a longer (t,/2),.
The residence half-lives of IgG in the vagina obtained by
use of different techniques and types of IgG were remarkably similar (Table 1). For both sets of results involving the
elimination of 125I-IgG from the vaginal lumen, the initial
residence half-life (t,, 2), was 0.7 h. The subsequent residence half-life (t,2) was 5.0 h and 4.6 h for the 10-p1l dose
in cycling mice and the 2-1 l dose in progestin-treated mice,
respectively. In addition, the residence half-life (tl/2) obtained for the biotinylated IgG data was 3.7 h. These halflives were so similar it appears that horse serum, volume of
the dose, phase of the estrous cycle, and type of IgG were
not significant factors influencing the elimination rate, and
thus the residence half-life, of IgG in the vaginal lumen. For
progestin-treated mice that were administered anti-HSV2
IgG2a, the residence half-lives (t,1/2) and (t,, 2 ) were 0.6 h
and 7.0 h, respectively, which were similar to the half-lives
observed with 25SI-IgG and biotinylated IgG. Other experiments using anti-HSV2 IgG2a in cycling mice gave similar
results, suggesting that the phase of the estrous cycle does
not affect the elimination rate of IgG from the vaginal lumen.
We believe that the second half-life of IgG in the vaginal
lumen, (t,,/2) = 5
2 h, was dependent on the rate at
which mucus was shed from the lumen, with the maximum
residence time of IgG limited by the time necessary for the
mucus layer to be renewed. To our knowledge, no such
turnover times have been reported for the mucus layer lining the vaginal lumen. Other researchers have shown that
the turnover time of intestinal mucus in the rat in situ loop
is 0.8-4.5 h [9], and that of the mucus lining the jejunum in
weanling pigs is about 40 h [10].
In mice maintained under anesthesia after intravaginal
administration, the amount of 125I-IgG in the vaginal lumen
remained constant over 2 h (Fig. 2). The anesthesia was
used to prevent movement and grooming, and the mice
were kept on their backs to prevent the solution from leaking out, which occurs in the a phase. The only possible
method of elimination in this case was absorption through
the epithelium. No reduction in IgG concentration in the
vagina was detected, suggesting that only very small
amounts of the antibody could have been absorbed. In Figure 2, the dashed lines represent the (t1,,2) range, which is
less than the values for anesthetized mice; this suggests that
the {3phase was not a result of absorption. Since no elimination was detected, any absorption must have been less
than the deviation seen in the data; therefore, absorption
must have been less than 5% per hour. This is consistent
with measurements made by other researchers. With repeated high doses, sufficient amounts of bovine anti-LH antibodies were absorbed from the baboon vagina to inhibit
ovarian steroid synthesis [11]. Vaginal administration of a
synthetic LHRH decapeptide (leuprolide) was shown to
have a bioavailability of 0.6% in humans [12], 1-2% in immature rats [13], and 3.8% in diestrous rats [14].
Our data, obtained by different methods, indicate that
intravaginally administered IgG solution was eliminated by
two processes that had different elimination rates. Intravenously administered IgG is eliminated from the plasma in
two phases as well. In this well-known situation, the rapid
initial decay in serum IgG concentration corresponds to distribution between intravascular and extravascular compartments, while the slower intravascular decrease represents
actual catabolism. Likewise, our data followed a biexponential pattern of decay, suggesting elimination by two firstorder processes. Presumably, a large portion of the vaginal
bolus was eliminated initially by leaking out of the vaginal
canal. This initial residence half-life, (t,/ 2 ),, of IgG solution
0.1
in the mouse vaginal lumen was approximately 0.7
h. We believe that the elimination of the IgG that penetrated
the mucus layer lining the vaginal epithelium (-phase) was
primarily dependent on the shedding of the mucus from the
lumen, since only small amounts of antibody could have
been absorbed through the epithelium and this absorption
would have to be slow, (< 5%/h). The residence half-life
of this fraction of the IgG from the mouse vaginal lumen,
(tl/,2), was 5 + 2 h. This elimination may be similar to the
clearance of IgG normally found in the vaginal mucus, and
the maximum residence time of IgG in the vaginal lumen is
probably limited by the mucus turnover time. This long halflife suggests that vaginally administered IgG can provide
reasonably long immunoprotection. Since the mouse vagina
has about 20 times the surface area-to-volume ratio of the
human vagina, we expect that the residence half-life (t,, 2)
of vaginally delivered antibodies in humans may be much
longer than 5 h, suggesting that several days of immunoprotection may be provided by a single topical application
of antibody.
ACKNOWLEDGMENTS
We thank Dr. Douglas M. Farmbrough for generously performing the antibody iodina
tions and Dr. Patricia G. Spear for her gift of hybridoma II111-174.
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