Antibody in tears following intranasal
vaccination with inactivated virus
II. Enhancement of tear antibody production by
the use of polyinosinic: polycytidilic acid (poly I:C)
Harry L. S. Knopf, Neil R. Blacklow, Morris I. Classman, Walter L. Cline,
and Vernon G. Wong
Rabbits were immunized with a formalin-treated vaccinia virus vaccine by means of intranasal or intradermal administration. A comparison was made between animals receiving
vaccine with or without the adjuvant poly I:C. Two additional animals received live virus intradermally and were used as controls. The immunoglobulins of the tears and serum were analyzed,
and it was found that: (1) IgA (IIS type) was the predominant immuno globulin in the tears. (2)
IgA appeared to be correlated with the antibody activity in tears of intranasally vaccinated
animals. (3) The antiviral activity in the tears of intradermally vaccinated animals appeared
to be IgG. (4) Poly I:C enhanced both the serum and tear antibody liters. (5) The predominant immuno globulin and antibody in the serum was IgG. The indirect fluorescent antibody procedure was found to be a useful adjunctive method for assaying tear antibody.
The possible mechanisms involved in the induction of tear antibodies and the effects of
adjuvants are discussed.
Key words: tear antibody, poly I:C adjuvant, formalin inactivation, vaccinia virus,
intranasal vaccination, fluorescent antibody test
L
been fully elucidated.-"1 If it parallels that
in other external secretions, e.g., those of
the gastrointestinal or respiratory tracts,
tear IgA should play a role in protecting
the surface of the eye from invasion by
microorganisms.la"s
In order to investigate this proposed protective function for tear antibody, we
turned to the rabbit, because it had been
used in previous experiments by our group
and by others, and it appeared to have a
lacrimal secretory system being similar to
the human being.3'5> 6 We then posed three
questions: (1) Which route of vaccine administration would most readily stimulate
lacrimal gland and conjunctival antibodyproducing cells? (2) Would the use of the
like all other mucosal tissues which are
exposed to the environment, the conjunctiva and its associated lacrimal glands are
endowed with substantial collections of
lymphoid elements.la~e Although IIS IgA
antibody is synthesized in this tissue and
secreted into the tears, its function has not
From the Laboratory of Vision Research, National Eye Institute, National Institute of Allergy and Infectious Diseases, and National
Cancer Institute, National Institutes of Health,
United States Department of Health, Education, and Welfare, Bethesda, Md. 20014.
Manuscript submitted July 15, 1971; manuscript
accepted Aug. 3, 1971.
750
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Volume 10
Number 10
adjuvant poly I:C enhance the production
of tear antibody? (3) If immunoglobulin
production were successfully stimulated,
would this antibody have a protective role?
By answering these questions, this report
and the subsequent paper attempt to ascertain the nature and function of tear antibody.
Materials and methods
Animals. Fifteen male albino rabbits, weighing
2 to 3 kilograms, were used in the experiment.
All animals were seronegative for vaccinia antibody as determined by complement fixation test.
Two animals which received intradermal live
virus were maintained in isolation during the
vaccination period.
During the four-week vaccination period, the
animals were treated intermittently with tetracycline hydrochloride (Alliance Labs, Inc., Columbus, Ohio) in their drinking water (0.5
mg. per milliliter). This therapy decreased the
signs of mild upper respiratory infection ("snuffles") which was endemic to these rabbits.7 (In
previous experiments, when no therapy was used,
several rabbits developed Pasteurella multocida
pneumonia.)
Tear collections. The method employed was
similar to that already described for collection
of tears from human subjects.4 Tears were collected once or twice daily for periods of no
longer than five days. One or two sterile Schirmer
strips were placed in the unanesthetized lower
fornix of both eyes for less than ten minutes.
The saturated strips were then dropped into
cold phosphate-buffered saline (PBS), pH 7.2,
and stored at 4° C. All the strips taken from
a single animal throughout any collection period
were pooled. When the collections were completed, the saline was decanted and the strips
washed with 1 ml. of fresh PBS. The original
and second washing were combined, centrifuged
to remove debris, and concentrated by centrifugation in Centriflo XM 50 cones (Amicon Corporation, Lexington, Mass.) to approximately 20 to
30 per cent of original volume. After qualitatively
testing each concentrate and eluate by double immunodiffusion to ensure that no immunoglobulin
had been lost through the Centriflo filter, the
concentrated samples were stored at -20° C.
When a line was detected in the eluate, the
sample was recombined and concentrated again.
Virus vaccine preparation. Preparation of the
formalin-inactivated vaccine0 was modified from
"Lyophilized, calf lymph—derived vaccinia virus, Lot
274701, identical to that used for human smallpox vaccination, was kindly provided by Allen Bernstein, Ph.D.,
Wyeth Laboratories.
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Antibody in tears. II 751
the method described by McNeill.s Virus was mixed
with an equal volume of a solution F (0.04 per cent
formalin and 0.02M DL-glycine in phosphate-buffered saline, pH 7.1) and incubated for two hours
at 37° C , 70 hours at 25° C , and finally 70 hours
at 4° C. Samples taken during this time showed
a decreasing titer of virus. Removal of virus
from solution F was accomplished by first diluting the virus/formalin mixture with Hanks'
balanced salt solution plus 0.5 per cent gelatin
and 0.1 per cent neomycin, then centrifuging the
diluted material at 100,000 x g for 30 minutes,
and finally suspending the pellet in Hanks' solution with gelatin and neomycin. The final 142
hour "inactivated" material was stored at -20°
C.
The original and the "inactivated" virus were
titered on: (1) WLs (human diploid cell strain)
monolayers (HEM Research, Rockville, Md.)
and (2) CAM (chick chorioallantoic membrane,
DBS standard reference system, performed by
Dr. V. J. Fuller, Division of Biologic Standards,
National Institutes of Health). The virus titer
on WLs was 106-5 TCD3o (50 per cent tissue
culture infective doses) per milliliter initially and
100-9 TCDso per milliliter after inactivation. The
assay on CAM gave a lower titer than that on
WLs:104-85 PFU (plaque-forming units) per milliliter for the untreated virus and 10°-° PFU per
milliliter (average value less than 20 plaques
per 0.1 ml. of inoculum in five replicate eggs)
for the "inactivated" virus. The difference between this value and that obtained in WLs presumably represents a difference in sensitivities
of two assay systems. Incubation of a second
lot of virus in PBS for 142 hours did not significantly alter the virus titer.
Vaccination. Formalin-inactivated virus was
added to an equal volume of 0.1 per cent
poly I:C solution (poly inosinic: polycytidilic
acid in normal saline, kindly provided by Dr.
Samuel Baron, National Institute of Allergy and
Infectious Diseases) to produce a vaccine of
neomycin (500 ng per milliter), poly I:C
(500 Mg per milliliter), and a theoretical titer
of inactivated virus about 10G-2 TCDso/mi.. Vaccine without adjuvant was prepared by mixing
an equal volume of PBS and formalin-inactivated
virus. Vaccines were administered twice during
the first week (three days apart) and then weekly
for two more doses (see Fig. 1). Tears and sera
were collected throughout this period.
For intranasal instillation, a total dose of 1
ml. of vaccine was dropped into both nares of
unanesthetized animals over 1 to 2 minutes, as
tolerated; no adverse effects were noted.
Intradermal vaccination was carried out by
injecting 0.2 to 0.25 ml. into four or five contiguous sites on the shaved back of the animal.
Although there were no injections into blood
752
Knopf et al.
vessels and no apparent systemic reactions,
erythema and induration were noted at the sites
of injection in two of the three animals.
Live virus was given on two occasions, two
weeks apart (Fig. 1), as a YIQ dilution of the
original Wyeth vaccine in Hanks' solution with
gelatin and neomycin; estimated titer was, therefore, 105-5 TCDso/mi.. A volume of 0.5 ml. was
divided among four to five injection sites similar to the intradermal injections described above.
After primary vaccination, mild erythema and
induration developed. One animal exhibited an
eschar after ten days. Following a second injection of live material, the skin sites became
mildly indurated, but no necrosis was noted.
No vesicles appeared in either animal.
Serologic tests. In all of the tests, the samples
were initially inactivated at 56° C. for 30 minutes.
The vims strains used in these tests were not
always the same as the Wyeth vaccine strain.
However, since there are no significant antigenic
differences between various strains of vaccinia,9
all tests were considered to be comparable.
The complement fixation (CF) test was performed by the microtiter technique as described
by Sever.10 The antigen was a sixth passage
in WLs cultures of a laboratory strain of vaccinia virus which behaved similarly to the Wyeth
strain vaccinia in CF. Positive complement fixation was judged as less than 25 per cent hemolysis of indicator cells, i.e., > 7 5 per cent fixation.
The hemagglutination inhibition (HI) test was
that described by Downie and Kempe,11 modified for microtiter by Sever.10 Samples to be
tested were absorbed with chick cells, heated,
and serially diluted in dextrose-gelatin-veronal
buffer (DGV), pH 7.3. The hemagglutination
antigen was prepared on monolayers of primary
rhesus monkey kidney cells (RMK)i seeded with
vaccinia virus (DBS reference strain No. 2, previously adapted to RMK). When maximum virus
growth was attained (24 to 48 hours), the
cells were harvested by freeze-thaw and lowspeed centrifugation, suspended in distilled water,
and sonicated for 60 seconds in a Heat Systems
Ultrasonic Sonifier (Plaineville, N. Y.). This
preparation was then diluted in DGV and assayed at room temperature for agglutinating activity; four to eight agglutinating units were
used in the test. The final HI titers were
determined after one hour of incubation at room
temperature and 16 to 20 hours incubation at
4° C; >50 per cent inhibition was scored as
positive.
The neutralization titers of tears and sera were
determined by the plaque reduction technique11
(PRT) modified for special tissue culture dishes
by Baron and associates.12 (All neutralization
tests were performed in the laboratory of Dr.
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Investigative Ophthalmology
October 1971
Robert Rafajko, North American Biologicals, Inc.,
Rockville, Md.) The virus used in this test was
a laboratory strain of vaccinia, passaged in vero
cells and diluted to produce approximately 80
to 100 plaques per dish. The per cent plaque
reduction was calculated from the ratio of the
number of plaques at each dilution, divided by
the average number of plaques in the virus
controls or known negative samples. These results were assayed by a standard regression
curve (log dilution versus per cent reduction),
and the 50 per cent plaque reduction titer
(PRT50) was determined for serum samples. If
the correlation coefficient of the regression curve
was less than 0.9, the sample was tested again.
Each serum or tear sample was tested in at least
two dilutions (two- to fourfold), except one
group of "pre" samples, which was screened
at a single dilution. Tear neutralization titers
were usually lower than those of sera, and,
therefore, the 40 per cent plaque reduction titer
(PRTio) was used. To make all tests comparable,
a standard reference serum was employed, and
the observed titers were adjusted to agree with
the PRT.,o of the standard.
Tear immunoglobulin determinations. A modification of the radial immunodiffusion technique
(RID) described previously13 was used to determine the immunoglobulin content in tears.
A commercial goat antiserum to purified rabbit
7S IgG (Meloy Laboratories, Springfield, Va.)
was tested for reactivity to rabbit tears. Two
lines which developed in double diffusion tests
were identified as IgA and IgG with the use of
purified standards. Following absorption of the
antisera with pure IgG, both lines disappeared;
indicating that the IgA line was most likely due
to L chain reactivity, while the IgG line probably represented both H and L chain reactivity.
The antiserum was appropriately diluted with
liquid agarose at 56° C. and poured into plastic forms. Wells were then cut from the hardened
agar and filled with approximately 10 fA of
sample. Standard dilutions of purified secretory
IgA (kindly provided by Rose Mage, National
Institute of Allergy and Infectious Diseases) and
IgG were tested simultaneously in serial dilution.
On incubation, the usual pattern which developed
with tear samples was two concentric rings of
precipitation. The outer, less dense circle, developed at about 24 hours and corresponded
to the IgA; the inner, darker ring, developed at
about 4 hours and correlated with IgG.
To determine the immunoglobulin concentration of the samples, the diameters of the
standard immunoglobulin precipitin rings were
measured and compared to those of the samples.
All tear specimens were first measured in single
RID test, and the values obtained were adjusted
with PBS to be approximately within five- to
Volume 10
Number 10
Antibody in tears. II 753
VACCINE
ROUTE POLY I X
O——O Inoct ive
Dermal
A
Nosol
A Inoct ve
+
+
• • - - « Live
Dermol
—
•
Nasol
—
• Inoct ive
VACCINE
J
ADMINISTRATION
I
I
2
3
WEEKS
Fig. 1. Serum complement-fixing antibody in
vaccinated animals. Each point represents the
geometric mean reciprocal titer for the group.
tenfold of each other. Then, each of 77 specimens was measured, in duplicate, in two large
inclusive tests; and 59 of the 77 samples were
assayed additionally on one or more occasions
in several smaller tests. During the experiment,
the procedure was modified somewhat, so the
early tests differed from the later ones in several
technical aspects: antiserum concentration (1 to
7 versus 1 to 8); amount of agar per plate
(3 ml. versus 3.5 ml.); well diameter (3 mm.
versus 4 mm.), and incubation temperature (37°
C. versus 25° C). However, the same standards
were used in all determinations, and these
standards provided the bases for concentration
determinations.
Sucrose gradient analysis. This method has
been described in detail elsewhere.13 Aliquots
of 0.05 ml. of sera or 0.4 to 0.5 ml. of tear
pools were placed on the top of a five to 22
per cent gradient and centrifuged at 100,000 x g
for 16 hours. The tubes were then punctured from
the bottom and the fractions were collected and
analyzed for immunoglobulin content by radial
immunodiffusion and/or double diffusion. Specific
antisera for rabbit secretory (colostrol) IgA
(kindly provided by Dr. John Johnson, National
Institute of Allergy and Infectious Diseases), antisera against rabbit IgM and IgG (Capell Laboratories, Downingtown, Pa.), and H chain-specific
rabbit y-globulin (CBDS, Woodbury, N. Y.)
were used in double diffusion analyses. After
the patterns had developed, the fractions were
pooled for testing by neutralization or CF.
Immunofluorescence tests. WI3S cell cultures
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(Flow Laboratories, Inc., Rockville, Md.) were
trypsinized, grown on coverslips in plastic Petri
dishes, and then used in fluorescent antibody
(FA) tests as previously described.14 WI3s coverslip preparations were infected with vaccinia
virus and fixed in cold acetone 24 hours later,
at a time when 25 per cent of the cell sheet
was involved with characteristic focal cytopathic
effects. FA tests were done by the indirect
method, using undiluted rabbit tears or rabbit
sera at a 1:10 dilution and fluorescein isothiocyanate-conjugated antirabbit globulin prepared in
sheep (Progressive Laboratories, Baltimore, Md.,
Lot No. 409). All readings were performed
without knowledge of the sample ("blind").
Vaccinia FA-stainable antigen was of the characteristic morphology described elsewhere.15-1G
Statistical analysis. Statistical tests for significance were performed where appropriate. These
were limited to correlated t test, t test for unrelated samples, correlation coefficient determination (r), and one-way analysis of variance.
Results
Measurement of immunoglobulins. In the
77 tear samples assayed, the geometric
mean IgA concentration for all tests (407
determinations) was 137.3 /xg per milliliter.
IgG concentrations were measured in 59
of the 77 samples in only two large tests
(236 determinations). The average value
was 43.2 /xg per milliliter, representing the
average of the geometric means for each
test. The mean IgA/IgG ratios calculated
for the 59 samples were 3.47 in the first
test and 4.62 in the second. These ratios
are both in reasonable agreement with the
A/G ratios obtained for tears and other
external secretions in human beings and
mammals.2'1S The IgA value of 137.3 /.ig
per milliliter was used to adjust all of the
observed tear neutralization titers to a
standard protein concentration.
Antiviral activity in serum and tears.
Serum antibody. It is clear that the administration of adjuvant exerted a significant enhancing effect on serum antibody
levels (Table I, Fig. 1). In addition, it
appeared that parenteral injection of vaccine produced a faster appearance and
slightly higher peak serum level than did
intranasal instillation of inactivated vaccine
with adjuvant (Group A versus Group
754
Investigative Ophthalmology
October 1971
Knopf et al.
Table I. Geometric mean serum neutralization titers from vaccinated animals'*
Week ||
Poll!
Group
No.f
Vaccinei
Routed
5
5
2
3
5
K
K
L
K
(K + L)
IN
IN
ID
ID
ID
A
B
C
D
EU
+
+
(+/-)
Pre
2
<1.0
<1.0
<1.0
<1.0
<1.0
1.51
<1.0
1.0**
2.21
1.73
Significance levels]]
4
2.60
1.61
2.57ft
2.92
2.78
test
A vs. B
B vs. C
<S vs. D
A vs. D
A vs. E
V
0.01#
0.10
0.50
0.40
0.05#
"Expressed as the logio of the reciprocal 50 per cent plaque reduction titer.
f Number of animals in the group.
JK = formalin-inactivated vaccine; L = live virus.
§IN = intranasal; ID = intradermal.
II Week number designates serum drawn before (pre) or one week after the second and fourth doses of inactivated vaccine.
fit Tests between groups at Week 4; significance expressed as less than the next highest p value.
# p Values which are significant.
• "Serum drawn two weeks after the first dose of live vaccine.
H Serum drawn one week after the last dose of live vaccine.
\ \ E = combined group of animals receiving intradermal vaccine (C+D).
2 0 --
VACCINE ROUTE POLY I=C
O Inoctive
A inactive
• Live
• Inactive
O
A
•
•
Dermal
Nasal
Dermal
Nasal
+
P
4-
It
J
10 -
7/
•
w
5 -
y
/
r
/
.
/
/
/
A
pre
1
/
-^
t
t
ilnactive
Live
VACCINE ADMINISTRATION
1
1
2
WEEKS
Fig. 2. Neutralizing antibody in the tears of
vaccinated animals. Each point represents the
geometric mean reciprocal 40 per cent plaque
reduction titer for the group.
E). There was no significant difference
between the mean serum-neutralizing antibody titers of any of the other groups.
The CF data shown in Fig. 1 corroborate
the neutralization results and demonstrate
the excellent agreement between the two
tests. These data also suggest that there
may have been only a short-lived adjuvant
effect, similar to that described by Braun
and co-workers.19 By two weeks after the
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last dose of vaccine and adjuvant, the
titers had begun to fall. However, since
this was also true of the nonadjuvant
groups, it may reflect simply the slowing
down of antibody production after a hyperimmunizing regimen.
Hemmagglutination inhibition (HI)
titers measured in these sera reflected the
same general pattern as did the other tests,
even though the HI test is judged to be the
least sensitive of the measurements of antipoxvirus antibody.20
Tear antibody. Fig. 2 depicts the titers
of neutralizing antibody observed in the
tears of vaccinated animals during the four
weeks of the vaccination period. Again,
there is a clear difference between the
titers obtained in animals given inactivated
vaccine with adjuvant and those given the
vaccine alone. When the average, peak
fourth week titer of the eight "adjuvant"
animals (Table II, Groups A and D) was
compared to the mean peak titer of the
group which received intranasal inactivated
vaccine without adjuvant (Table II,
Group B), the difference was significant
(14.15 versus 5.5, p < 0.05). When only
the intranasal groups were compared (adjuvant-treated versus nonadjuvant), the
difference in the mean peak titers was more
than twofold and approached significance
(12.6 versus 5.5, p = 0.08). The same
value, p = 0.08, was also obtained by a
one-way analysis of variance, which mea-
Antibody in tears. II 755
Volume 10
Number 10
Table II. Relationship offluorescenceto neutralizing antibody titer in tears
Poly
A
X
IN
1
2
3
4
5
1.037
1.341
1.236
1.369
0.544
B
X
IN
1
2
3
4
5
0.838
0.100
1.193
0.422
0.428
C
L
ID
-
1
2
1.140
1.081
D
X
ID
+
1
2
3
1.330
0.940
1.403
Fluorescence^
i
Adjusted titer
(Week 4)t
i
No.
i
Route f
i
Vaccine0
i
Group
+
_
+
°K = formalin-inactivated; L = live virus.
fIN = intranasal; ID = intradermal.
| Adjusted to 137.3 US Per milliliter IgA; expressed as logio reciprocal 40 per cent plaque reduction titer.
^Indicates presence of vaccinia fluorescent-stainable antigen.
sured the difference between the intranasal
groups over the period of Week 2 through
Week 4. As with the serum data described
above, comparisons between the other
groups displayed no statistical difference in
the mean antibody titers for Week 4. Attempts to measure tear antiviral activity by
means of CF and HI assays were technically unsatisfactory.
Fluorescent antibody studies. In order to
provide some other method for confirmation of the neutralization test in tears, we
used indirect fluorescence. There were no
false positives in 7 of 17 "pie" samples
which were tested, and two control animals, which had been given live virus into
the conjunctival sac and developed conjunctivitis, were strongly positive. Table II
illustrates the relationship of positive fluorescence to the final, adjusted neutralization titer at Week 4. Although the FA test
was performed on samples which were unadjusted for protein concentration, there
was a rough correlation between FA activity and antibody titer: (1) All but one
of the FA positives had titers of > 1-10.
(2) The reciprocal mean titer for positive
samples was 14.2 and that for negative
samples was 6.2. The difference in these
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mean titers was not significant, (t = 1.859,
p < 0.1), but its proximity to the 0.05 level
indicated a possible trend toward significance. These results tended to confirm
those of the neutralization test.
Identification of immunoglobulins with
antiviral activity. Sucrose gradient studies
were performed on pooled tear samples
and sera from several groups of animals.
In addition, tears and sera from previous
studies5 were analyzed.
Fig. 3 shows the results of neutralization tests on the pools of sucrose gradient
fractions obtained from the tears of intranasally vaccinated animals. Although there
is some activity associated with the IgGrich fractions, the major portion of the neutralizing titer is correlated with the pool of
pure IgA. The average IgA sedimentation
coefficient in 19 sucrose gradient analyses
was 10.25 ± 1.05 (calculated according to
the method of Stanworth,21 assuming human IgG = 7S). This is in good agreement with the value of 10.8S obtained by
analytic ultracentrifugal determinations of
rabbit colostral IgA.22
In the serum (Fig. 4), the complement
fixation activity of both intranasal and
intradermal vaccinees is associated with
756
Investigative Ophthalmology
October 1971
Knopf et al.
"30
40
50
60
70
PERCENT EFFLUENT VOLUME
Fig. 3. Sucrose gradient separation of immunoglobulins in a pool of tears from intranasally
vaccinated animals. IgAs = secretory IgA. Relative intensities were subjectively graded from
1 to 4, based on brightness and sharpness of the lines and their distance from the center well.
Immunoglobulin concentrations were determined by the radial immunodiffusion technique in
pooled fractions. Neutralization titers represent the reciprocal 40 per cent plaque reduction
titer.
Pooled Froctions
20 30 40
50 60 70 80
PERCENT EFFLUENT VOLUME
90
100
Fig. 4. Sucrose gradient separation and quantitative measurement of IgG in the serum of an
animal vaccinated intradermally with inactivated virus plus poly I:C. IgM is depicted by its
position only. Radial immunodiffusion determinations were done on alternate gradient fractions.
Complement fixation activity is shown as the reciprocal of the highest dilution in pooled
fractions.
IgG only. In other studies, some antibody
in serum has been found associated with
the IgM fractions.13 However in the six
sera investigated for this study (including
three influenza B- and 3 vaccinia-exposed
animals) we did not observe complementfixing or neutralizing antibody (only one
serum tested) in the IgM region.
In an attempt to further characterize the
immunoglobulins in tears, a series of FA
blocking experiments was performed. Using
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a rabbit antivaccinia serum from a parenterally vaccinated animal, we demonstrated
that all of the fluorescence of either diluted
whole serum or a sucrose gradient-isolated
7S portion of the same serum (Fig. 4, Pool
III) could be eliminated by intermediate
treatment with unconjugated goat antirabbit 7S IgG. This same goat antiserum
also eliminated most of the fluorescence associated with the tear samples collected
from the intradermally vaccinated animals
Volume 10
Number 10
(inactive vaccine with poly I:C). However, it had no effect on the tears of the
animals which received intranasal vaccine
with poly I:C. Unfortunately, we were not
able to obtain enough blocking activity
from a goat anti-IgA serum to test its activity directly.
We concluded from the FA blocking experiments that: (1) IgG provided most or
all of the fluorescence in the serum and
tears of parenterally vaccinated animals,
but not in the tears of intranasal vaccinees,
and (2) that IgA was probably responsible
for the fluorescence in the tears of the intranasally vaccinated animals, particularly
in view of the sucrose gradient data (Fig. 3).
These experiments apparently answer
two disparate reports in the literature.
Sapse and associates3 had reputed that
parenteral vaccination of rabbits with various antigens produced only IgG-associated
antibody in tears. We had previously
shown that IgA-associated antibody was
present in the tears of human volunteers
who were given intranasal doses of inactivated rhinovirus vaccine.4 The fluorescent
antibody-blocking experiments and sucrose
gradient studies suggest that different
routes of administration can produce different antibody-associated activity in the
tears.
Discussion
Administration of poxvirus via the intranasal route is not a new idea. In 1901,
Calumette and Guerin23 reported that intranasal inoculation of a powder of virulent
smallpox virus was capable of producing
immunity in the rabbit, without an apparent systemic dissemination of the virus.
These authors also noted that "oriental
people" vaccinated patients by introducing
"cotton balls powdered with crusts from
smallpox pustules" into the noses of susceptible individuals. In our own experiments,
intranasal instillation of an inactivated vaccine produced both a local and a systemic
antibody response. However, the levels of
both antibodies were enhanced by the use
of a polynucleotide adjuvant.
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Antibody in tears. II 757
In addition to their activity as interferon
inducers24 and their apparent antitumor effects,25 polynucleotides and other polyamnionic compounds are also capable of enhancing antibody production and cellular
immunity.19'2G Braun and associates19 and
others27'2S have suggested that this effect is
mediated by the same route as the more
classic adjuvants, namely by stimulating
macrophage function.
In previous experiments, we observed
that the simultaneous intranasal administration of inactivated rhinovirus or influenza virus with poly I:C produced a
definite enhancement of serum antibody
and a relative increase in local production
of antibody (nasal secretory type) to these
viruses.5 In addition, we noted an incidental rise in tear antibody to these organisms,
similar to our previous experiments in human volunteers.4
In the present experiments, we were able
to increase the antibody titer against a
relatively poor viral antigens by the simultaneous administration of poly I:C, either
intranasally or intradermally, without any
of the apparent toxic effects reported by
Ostler and associates.29 However, our results also suggested that the mechanism of
induction and the type of tear antibody
which was evoked may be different for different routes of immunization.
The nasal submucosa and lacrimal gland
stroma contain plasma cells which produce
mainly IgA.la> b> e> h It follows that intranasal administration of an antigen will
stimulate primarily IgA production in nasal
cells. In addition, it will enhance tear IgA
by either direct or indirect transfer of antigen or cells.16'4 (This was also suggested
in previous studies with human volunteers.4) The lymphatic pathways from the
skin lead to systemic lymph nodes which
produce IgG and IgM. Thus, intradermal
inoculation of antigen will usually induce
IgG and IgM antibodies and circulating
lymphoid memory cells with IgG specificity.111' g These "G" cells might then be
attracted to the eye (or other parts of the
body) by any mild inflammatory stimulus
758
Knopf et al.
and there begin to synthesize IgG. In addition, simple transudation of serum antibody, secondary to the mild inflammation
caused by the filter paper strips, may contribute to the IgG-associated antibody activity observed in both intranasally and
intradermally vaccinated animals. These
hypothetical explanations may account for
the difference in tear antibody classes observed by us4 and by Sapse and associates3
after different routes of immunization.
This theory for serum-associated local
antibody was originally offered by Fazekas
de St. Groth and Donnelly,30 who injected
mice intraperitoneally with influenza virus
and then applied tannic acid to the respiratory mucosa. This local irritation resulted in higher antibody titers in their
respiratory tract secretions than in those of
animals given the injections without tannic
acid treatment. They referred to this
phenomenon as "pathotopic immunity."
Similarly, Liotet and Rouchy31 reported increased local IgG in the conjunctival secretions of patients who had undergone curettage.
The possible function of tear antibody
will be pursued in the following paper.
The present set of experiments have shown
that in the rabbit: (1) the induction of tear
and serum antibody against vaccinia virus
can be accomplished by intranasal instillation of a formalin-inactivated preparation
of that virus; (2) that this response can
be stimulated or enhanced by application
of poly I:C at the same time; (3) that an
alternate route of inoculation can accomplish the same end result, but perhaps by
different mechanisms; and (4) that the
rabbit appears to behave very much like
the human being with respect to tear antibody production and therefore may be a
good model for investigating the workings
of the system.
We gratefully acknowledge the cooperation
of Dr. Robert M. Chanock, Dr. Albert Z. Kapikian, and the personnel of the Laboratory of
Infectious Disease, National Institutes of Health.
We also appreciate the help of Mr. Jerry Selig
and Mrs. Mitzi Sereno for technical assistance,
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Investigative Ophthalmology
October 1971
and Mrs. Ann Korade and Mrs. Sally Gogarty
for collation of data.
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