Note: Descriptions are shown in the official language in which they were submitted.
1~50~Z~
DESCRIPTION OF THE PRIOR ART
The infection caused by the organism Neisseria
gonorrhoeae commonly known as Gonorrhea is a venereal
disease of an extremely wide spread nature in humans. The
disease usually manifests itself by a visible discharge in
males but frequently is undetected and undetectable by ex-
texnal symptoms in females infected therewith. Heretofore,
the only reliable mode of detection of infection has been
by culturing discharges or mucus fluids believed to contain
the organism. Such cultures take a period of more than one
day to grow. Because of the social opprobrium attached to
the disease and the reluctance of many persons infected there-
with to return to the test clinic, it has long been desirable
to provide a screening method which can give a reliable in
dication of possible infection or noninfection during a time
for which it is reasonable to require the test subject to
remain in the clinic.
Furthermore, heretofore there has been no develop-
ment of any method of immunization agalnst infection with
the organism o~ N. gonorrhoeae in humans. One of the
greatest problems associated with research in this area has
besn the fact that the organism only appears to infect
humans and chimpanzees and, while there is a reasonable
degree of correlation between results in chimpanzees and
results in humans, such a correlation is not absolute.
Chimpanzees, although reasonably satis~actory as research
models, are extremely expensive as research subjects.
, ' ~
0236
~OSO~Z3
Four distinct colonial variants of Neisseria
gonorrhoeae have been characterized. These four variants fall
into two distinct categories. ~lariants T1 and T2 produce
experimental lnfection in human volunteers whereas Type T3
and T~ are not known to cause infection. The first group
may be distinguished from the second group in the observation
that the first group possess filamentous structures on the
surface of the colonial variants whereas the second group
are devoid of these filamentous structures. These filaments
are designated as Gonococcal pili thereinafter G. C. pili).
In 1973 two papers were published purporting to show the
isolation of Tl and T2 pili from N. gonorrhoeae and further
purporting show the formation of antibody response thereto
(Buchanan, et al., J.Clin.Invest. 52, 2896-2909 (1973) and
Punsalang and Sawyer, Infect.Immun. 8, 255-263 (1973)). See
also Buchanan, et al., J.Clin.Invest. 51 17A (1972). The
basic method utilized by Buchanan is acknowledged as Reference
41 in the 1973 paper as having been developed by Charles C.
Brinton, one of the inventors herein ~C.C. Brinton, Trans.
N.Y. Acad. of Sci. 27, 1003 (1965)), as well as by Punsalang
_.
and Sawyer.
The Brinton method was developed for the study of
; the pili of E. coli. Applicants herein have attempted to
repeat the work of Buchanan, et al. and have found that
the procedures set forth therein to all intents and purposes
do not ~ield pili of N. gonorrhoeae. A detailed study
setting forth the comparison of the work of Buchanan with
the work of Applicants as disclosed and claimed herein has
been prepared and is being readied for publication.
--3--
0568
lO~V4Z3
It will be seen from a comparison of the methods
utilized by Buchanan, et al as compared to the methods used
by Applicants herein that suchanan's methods and those of
Punsalang and Sawyer are in fact excellen~ly designed to
remo~e G. C. pili from the product obtained by said pro-
cedure and that if perchance any immunological response to
the alleged pilus material is achie~ed by suchanan~s product,
this must be cons.idered as being due either to artifacts or
defects in the method as intended to be carried out.
It is AppIicant's understanding that at the time ~f
filing this application Buchanan has acknowledged the defects
of his work, although at this time ~o retraction has appeared
in print.
'
SUMMARY OF THJ3 INVENTION
.:
The invention relates to the provision of purified
pili of type Tl and T2 Neisseria gonorrhoeae organisms as
well as the TR subvariants thereof if desired, in crystalline
form (hereinafter GC pilus crystals).
GC pili are isolated from either surface culture
or deep liquid culture of the corresponding organism. The
method of surface and deep culture of N. ~onorrhoeae cells
are substantially conventional. In a modification of the
- deep culture method, a high surface area, reaction inert,
medium, is added to the culture to aid in the purification.
A diatomaceous earth such as Celite~ has been found suitable.
In the case of the sur~ace growth, the entire
growth, that is to say, the cells and pili are removed from
6721(30~
-4-
105~4Z3
the culture medium and suspended in an aqueous medium at
a predetermined pH below 9.2. Where it is desired to iso-
late T2 pili only any pH above pH 5.5 and below pH 9.2 is
suitable. Where it is desired to isolate T1 pili either
per se or in the presence of T2 pili, a pH below p~ 7,7
suitably around pH 7.0 is required. In the case of deep
culture growth, such a suspending step is not necessary.
It has also been found that pilus crystals are
soluble below 4.5 and are substantially reconstitutable when
the pH is again raised above this volume provided it had not
be~n r~duced below about pH 2.5.
The portions of said suspensions which are soluble
are then separated from the portions of the suspension which
are insoluble. While filtration may be utilized for this
separation it is generally preferred to utilize centrifugation.
The supernate of the centrifugation (and the filtrate in the
case of filtration) is discarded and the residues retained.
In the next step of the purification procedure, the constituent
portions of the Gonococcal pili are brought into solution and
separated from the remaining material. In the case of the
surface culture this material will include whole cells and
debris and in the case of the deep culture, will additionally
include the high surface area material such as the Celite .
.,
The solution of the Gonococcal pilus crystals may
be achieved by two (2) different but closely related methods.
The solution of the material of the gonococcal pilus crystals
depends upon the breaking of inter pilus rod non-covalent
bonds involving the peptide material which constitutes a
major portion of the pili, while leaving the covalent bonds
intact. That is to say, the use of a solubilizing agent
6721
6712 (20) -5-
~OS0423
which will not denature the peptide, but merely disagregate
the crystals into single pilus rods. Such agents may be
independent of p~ s~ch as aqueous urea, suff icient water to
lower the ionic concentration of an aqueous suspending
medium below 0.002M, sufficient salt, suitably salts of
alkali or alkaline earth metals and the anions of mineral
acids to raise the ionic strength above 4.4, uxea to a con-
centration of between about 3M and about 5M and sufficient
sucrose to raise the concentration above about 50% weight
per volume~ The pilus crystal~ are re~recipitated by raisin~
the ionic strength above about 0.05 by the addition of salts
of mineral acid anions and alkali and alkaline earth metals;
the addition of sufficient water to reduce the salt concen-
tration below an ionic strength of 0.5, sufficient buffer
suitably tris buffered saline to provide a medium of ionic
strength about 0.05 to about 0.5 at a pH of about 4.5 to
about 9.2, and sufficient water to reduce the sucrose concen-
tration below 40% by weight respectively. ~he agents may
also be pH dependent such as basic buffers such as a tris
buffer which will raise the pH to a level of from about
pH 9.3 to about pH 11 for T2 pili or from about pH 7 to about
pH 8.6 for Tl pili. Said range being determined by the
commmencement of solubility at the lower end and the com-
mencement of danger of denaturation at the upper end. It
has been observed however that even where the pilus structure
is denatured by these or other methods to the extend that
the pili will not recrystallize, their antigenic characteristics
are apparently substantially unaffected.
After addition of the solvating medium to the solid
residues mentioned above, the soluble and insoluble portion of
said second susp~nsion are again separated. As hefore, this
separation may be by filtration or centrifugation, suitably
6712
~(~S0~23
centri~u~ation.
The centrifu~ation method may be either simple
or modifiecl. In the simple centrif~gati~n method, the sus-
pension is run in a low speed centrifuge, the supernate re-
tained and the residue set aside. Where i~ is desired to
raise the yield, the residue is re-suspended, re-centrifuged,
the residue discarded, and the supernate combined with the
immediately previous supernate. The combined supernates are
then subjected to high speed centrifugati{)n to remove the last
traces of small debris in the residue and the supe~nate then
set aside for ~se in the succ~eding precipitation step.
In the modified form of the centrifugation process,
the solubilized pili, that is to say, either those suspensions
at elevateA pH or those in an aqueous noncovalent bond
breaking medium, such as urea, are mixed with aqueous caesium
chloride. Since the caesium chloride gradient method involves
centrifugation, complete separation of the debris is not
necessary, however, a cleaner result is obtained by the use
of either prefiltration or precentrifugation. The mixture
in caesium chloride is then subjected to centrifugation in
the conventional manner for caesium chloride separations and
the absorption at various density gradient levels measured.
The location of the major peak, suitably measured at 280 nm
indicates the location of the pilus solution.
~ tilizing either of the methods of centrifugation,
the aqueous fractions containing the pilus solutions are then
treated in a manner conducive to the precipitation of the pilus
crystals. This may be done by the lowering of the pH where
the pH has been raised, or by removal of the noncovalent bond
breaking agent, the caesium chloride or, alternatively the
_ 7 _
6710
6705 (1)
.
~ 0504Z3
addition of a precipitating agent such as ammonium s-llfate.
Precipitating conditions may be achieved either by dialysis
or by direct addition.
upon lowering of the pH and removal of the bond
breaking agent, GC pilus crystals will form. The GC pilus
crystals are then removed from the aqueous medium either by
filtration, or, more suitably low speed centrifugation. The
supernate is separated and the thus obtained GC pilus
crystals are driea under reduced pressure, if desired, or
stored in a suitable aqueous medium. While it is desirable
to ~eep the crystals at reduced temperatures in a sterile
medium, this does not appear to be essential for their
stability in the ahsence of bacterial contamination.
It should b~ noted that the pilus crystals are in
fact agglomerations of single pilus rods having a high
molecular weight. Thus, where the medium containing the
solubilized pili has been purified by high speed centrifuga-
tion with, if desired, sterilization, thru a millipore
(suitably circa 0.45 micron) filter, the individual pilus
rods may be precipitated by ultra high speed centrifu~ation
suitably above 40 KG.
It has also been found that where pili of the very
highest purity are not re~uired, a convenient and rapid
abbreviated purification process is quite satisfactory.
~n this procedure the entire gonnococcal growth is trans-
ferred into a high pEI buffer, suitably an ethanolamine
buffer at a pH above the solution point of the variant
(Tl or T2) in question preferably about pH 10.0 to ensure
total solution, the solids removed by filtration or centri-
fugation and the pH dropped - suitably but not essentially
by dialysis.
6705 (34) -8-
~0504Z3
Indeed pH control to, say. pH 8.6 in the first in-
stance will crystallize out T2 pilus crystals and a further
drop to below pH 7.7, say to pH 7 will crystallize out Tl
pilus crystals. Thus indicating in one step the nature of
the variant growth in question. This latter is merely con-
firmatory since a competent bacteriologist can differentiate
between the two forms by inspection of their colonies.
Alternatively, pilus crystals may be precipitated
by the addition of ammonium sulfate. An anionic concentra-
tion of between about 4% and 7% of ~aturation ~at room tempera-
ture~ will precipitate T2 pili as crystals, while Tl pili are
precipitated by betwe~n 5% and 10% of saturation.
The GC pili have been s~bjected to SDS poly-
acrylamide gel electrophoresis and show a ma~or and a minor
b~nd. The major band, designated GC pilin shows it to com-
prise phosphoglycoprotein material.
The T2 GC pilin has further been shown to comprise
a peptide portion of 200 + 9 amino acids, between 2 and 3
phosphate groups, and between 1 and 2 hexose sugars, and is
3ubstantially 601uble in aqueous media at a pH greater
than 10.1 and substantially insoluble in aqueous media at
a pH of less than 8.6 both pH's bein~ measured at 20C.
The major portion of T2 GC pili, namely T2
GC pilin has a molecular weight, as determined by SDS
acrylamide gel electrophoresis of 21,500 + 1000 daltons.
The GC pili isolated from type Tl N. ~onorrhoeae
growth aPPear to be extremely similar immunologically to those
isolated from type Tz N. gonorrhoeae.
6705 -9-
2432 (17)
l~SQ4Z3
By comparison Type Tl GC pili are substantially
soluble in aqueous media above about pH 8.5 and substantially
insoluble in aqueous media at a pH of less than 7.7, both
pH's being measured at 20C. Type Tl GC pilin has a molecular
weight of 22,000 + 1000 as de~ermined by SDS acrylamide gel
electrophoresis.
GC pili have been isolated from cultures of more
than 20 different strains of N gonorrhoeae. When injected
into test animals GC pilus crystals as well as single rod
pili and the eluate from SDS acrylamide gel electrophoresis
will cause the formation of antibodies in the serum ~f the
test animals.
When GC pilus crystals are treated, either ~ se or
in a 9uitable suspending medium, with serum containing anti-
bodies thereto, the crystals will agglutinate. This agglu-
tination i9 readily observable most suitably in a dark field
microscope but also by other means, and provides a simple
and immediate test for the presence of the pilus antibodies
in a test serum.
Amon~ the uses of the test may be mentioned
screening for gonnorhea to select individuals for culture
testing, identification of high risk individuals distinguishing
new, from old infections in a particular individual, identi-
f ying the strain responsible for a local epidemic and strains
responsible for particular symptoms.
It should be noted that while this test is useful
for the determination of the presence of antibodies to GC
pili in a test sample~ it is not possible to determine
--10--
2432
~706 ~11)
~)5~423
directly whether the subject from whom the serum was drawn
has active gonorrhoeae or has ~een infected in the past and
is merely a carrier of antibodies. It should be further
noted that a very recently infected (i.e. in the previous
2 or 3 days) subject may nvt give a positive response since
there may not have been sufficient time for the body to
create a sufficient concentration of antibodies to give a
detectable titre.
It has been noted that the pili of N. ~onorrhoeae
organisms contain one or more immunological determinants
~elected from a group of at least four such determinants.
Thus, the antibody agglutination reaction will occur between
pili and a serum containing antibodies against at least one
of such immunological determinants. The strength of the
response will depend upon the concentration of antibodies
in the test serum sample and equally the number of inter-
~cting immunological determinants on the pili and in the serum.
It is thus possible, given say, four different GC
pilu5 samples known to contain at least one of ~he aforesaid
determinants to rapidly test for the presence of the
corresponding antibodies to GC pili in ~ test serum.
Similarly, where a source of organisms is available
and may be readily cultuxed the pili from said organisms are
isolatable. Sinoe standardized sera containing antibodies
against any predetermined one of the four antigenic determinants
of GC pili are available as a result of the present invention,
said pili from the unknown test source are serotypable as
to the identity and number of these determinants thereon.
This pxocedure will greatly assist the epidemiological work
6706 ~44)
~0504Z3
connected with infection tracking of Gonorrhea.
Pili may be absorbed on various carriers known
to immunological testing such as latex, washed red blood
cells, charcoal, polyacrylamide, agarose, and the like to
provide the substrates for serum or plasma agglutination
tests.
The availability of pili also provides the basis
for haemagglutination and haemagglutination inhibition
tests. Both tests depend upon the principle that pili
contain speci~ic combining sites which will interact with
red blood cells. Thus, where pili and blood cells are
incubated together the red blood cells will give a diffuse
agglutinated pellet by gravity settling. If no pili a~e
presen~ in the test medium the red blood cells settle to
give a clearly defined pellet by gravity settling. This
provides a means of testing for the presence of pili in a
solution.
In the haemagglutination inhibition test, a test
serum believed to contain antibodies to GC pili is added
to a solution~containing a predetermined quantity of pili
and the mixture incubated and centrifuged. Pili interacting
with antibodies thereto will be precipitated. The supernate
is then added to red blood cells. When all the pili have
been reacted with the antibodies in the test material, a
sharp pellet (i.e. no agglutination) will result. It will
be understood by those skilled in the art that such a test
has significance when run against controls (i.e. no anti-
bodies) and at predetermined dilutionsD
.
6706 -12-
3707 t30~
~ILOS0423
The a~curacy of this test is raised if the serum
is first txeated with washed red blood cells (i.e. prior
to addition to pili). This procedu~e removes factors in
the serum which would cause agglutination of the red blood
cells regardless of the presence of pili.
Heretofore no mode of immunization against N.
gonorrhoeae in humans has in any way been possible. It has
been found that when huma~ volunteer subjects were injected
with a sufficient quantity of GC pilus crystals, suitably
of the order of from about 2 to about 100 micrograms per
kilogram of body weight of said pili to raise the antibody
level of their serum to a PAT (Pilus Agglutination Test)
titer of at least 100, a degree of protection of at least
1.6 log cycles was obtained. That is to say, that the
subject was able to resist infection by a counted number
of organisms of the strain from which the injected GC pilus
crystals were derived, of approximately 1.6 orders of magni-
tude greater than that required to bring about infection in
control subjects in the un-immunized state. No toxic
effects attributable to the pili have been observed from
the injection of GC pilus crystals. Human subjects having
a titer of up to 200 appear to be unaffected and the test
primates (Rhesus monkeys) have been subjected to titer
levels of about 10,000 in the PAT Tes~ without any ill effects
whatsoever being noted. It has further been noted that it
appears to be advisable that the injections of GC pilus
cr~stal~, in a suitable carrier medium, be made over a
~omewhat extended period suitably a period of up to about
S weeks. Ad~ini~tration may be in between 1 to 5 aliquots
of GC pilus crystals,single rod pili, or any ~uitable source
of G.C. pilin. A spreadout rate of administration while
helpful i~ not essential. This rate of administration
67~7 ~32)
13-
lQ5~4;~;~
permits the gradual huild-up of antibodies in the system.
It should further be noted that no local adverse
reaction against the crystals at the point of injection has
been noted in injections in a subject to whom the crystals
had already been administered at a previous point in time.
In view of the existence of several antibody deter-
minants as mentioned hereinabove, it is desirable to adminis-
ter pilus crystals, single rod pili or other sources of G.C.
pilin containing each of the known determinants in order to
obtain maximum protection.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Preparation of GC Pilus Crystals
Growth of N. ~onorrhoeae Organisms
Surface Culture
Neisseria gonorrhoeae is ~ound in four (4) colonial
forms arbitrarily designated types Tl, T2, T3 and T4. This
designation however is generally accepted. Type Tl and T2
organisms are the causative organisms of the disease Gonorrhe~
in humans and only these forms possess pili. The procedures set
forth below are applicable to the growth of type Tl and type
T2 organisms. It should be noted that strains of N. gonorrhoeae
are isolatable from body secretions taken from human patients.
6707
3582 (2~) -14-
~504Z3
Such secretions will usually contain not only the desired
Tl or T2 types but also the unpiliated T3 and T4 types.
Further, it should be noted that a culture which commences
as, say, a fairly pure T2 ~ype will in due course, upon
subculture, produce nonpiliated T3 and T4 types as well
as Tl types.
In the culture of Tl and T2 colonial variants a
third piliated variant, arbitrarily designated TR is noted.
Thi~ variant has a rough ~ppearance, and while is completely
characterized is believed to be closely related to the Tl
and T2 types as these types are yielded upon subculture of
TR colonies. The yield of pili from TR types i~ the same as
from T2 cultures.
In order to maximize production of either T2 or
Tl pili, certain preliminary procedures should be followed.
The original samples are cultured on Thayer Martin
(T-M) plates which allow the growth of N. gonorrhoeae inhibiting
the growth of most other bacteria. Since T-M plates are not
suitable for distinguishing colonial types, colonies from
the T-M plates are streaked onto a suitable growth medium
(GC medium, Ca~alog No. 0289-1 Difco) for example). A suc-
~ cession of subcultures are then prepared from single
; oolonieY on the medium (hereinafter GC medium) until the
colonie~ comprise greater th~n about 90% of the desired type.
While the procedure may be employed equally well for type
Tl as for type T2~ and, a~ has been shown, type Tl and
type T2 are immunologically similar, it is preferred to
ma~ntain separation of the types. ~ereinbelow T2 will be
di~cussed. Except where the difference~ are specifically
noted, growth procedures for T2 are equally applicable to
~582
0249 (12)
; ~15-
~)5(~Z3
Tl. When the growth on a plate shows greater than 90% of,
say, T2 colonies, the growth is removed from the plate and
suspended in a suitable freezing medium,for example, BSA-
glutamine, divided into aliquots, and stored at reduced
temperatures, suitably of the order of -70C to -196C.
It should be noted that, as stated heretofore, samples
with an initially high proportion of T2 have a tendency, upon
subcultures, to become unstable and yield lower amounts of T2.
Hence, in growing the cultures, care must be taken on the one
hand to ensure a high initial proportion of T2 organisms and
on the other hand, care must be taken to utilize a sufficiently
"young" strain, that is to say, one that has not been overly
often subcultured, to guard against the occurrence of in-
stability. Where it is desired to produce Tl pili, it is even
more important than in the T2 case to use an inoculum con-
taining over 90% Tl.
The inoculum for pilus production is prepared by
streaking the primary, suitably but not necessarily, frozen,
aliquots on a GC medium petri dish and incubating for between
12 and 24 hours. Twelve to 15 hours for strains with more
unstable T2 types and 12 to 24 hours for strains with more
stable T2 types~ It is preferred to grow the inoculum at
a temperature of between 35 thru 37C, although 35C i5
deemed preferable. High humidity conditions are also
; deemed desirable. At humidities of less than 70% the
pilus yield has been noted to be lower than at higher
humidities~ It is therefore deemed desirable to operate
at a humidity of between 70% and 90%. While the effect of
the atmosphere of growth is incompletely understood, and
older N onorrhoeae cultures will grow without the addition
of carbon dioxide, an atmosphere of between 5 and 10% carbon
02~9
~577 ~8~ -16-
105~4Z3
dioxide together with ~0 to 95% air has been found highly
suitable.
After the initial inoculation, the plate is covered
with from about 50 to about 75% growth, the growth is removed
therefrom. In a suitable procedure, a small amount of
sterile casamino acids solution is added to the inoculum plate
and the growth scraped off with a sterile glass spreader. It
has been found suitable t~ utilize between 5 and 6 ml. of
solution per plate and from 2 to 3 ml. of the thus prepared
suspension is sufficient to inoculate the larger growth pans of
GC medium. The pans are then incubated for the same order of
time under the same conditions as the inoculum petri dishes
and the pili harvested therefrom.
At this point of the procedure it is no longer
necessary to utilize sterile techniques although of course as in
all procedures, it is desirable to use clean equipment, pure
reagents, and to carry out all operations at as low a tempera-
ture as possible to inhibit undesired bacterial growth.
The gonococcal growth is harvested using a suitable
buffer. While the chemical nature of the buffer is not
critical, the pH range is important. For reasons which will
become apparent, when purifying T2 pili the buffer may not be
utilized at a range exceeding pH 9.3. It is preferred to
operate in a pH range between 5.5 and 9.2, most suitably in
a range of 7.0 to 8.6. Where a predominantly Tl culture
has been employed the pH should not exceed 7.7, but should
lie in the range of pH 5.5-7.5, suitably pH 7.0-7.2.
These ranges will ensure the maintainence of all piliated ~-
material in the aggregated state. As the most suitable
buffer, may be mentioned tris buffered saline.
3577
3583 (14)
-17-
gL050~23
In the preferred procedure the washing buffer is
placed on the surface of the growth medium, the growth scraped
off the medium with a suitable instrument, and the aqueous
suspension removed in a suitable manner, for example, with
a pipette or vacuum aspirator. If desired, a second washing
may be carried out in the same manner and the liquid sus-
pensions pooled.
In order to raise the yield a third washing may be
carried out with a ~ pH buffer. That is to say, a buffer
having a pH above 9.3, suitably between 10.1 and 10.3. The
use of such a buffer will cause dissolution of all remaining
pilic material. Where Tl pilus isolation is in view, the
pH need only exceed about pH 8.6, but there is no disadvantage
in the higher values. This basic suspension is not pooled
with the first washes but held aside for a later stage of the
purification. It should be noted that where there has been a
~ good growth of pili, the growth has a characteristic orange/pink
; or warm pink color and the growth medium has an odor resembling
cooking food. The growth is noted to be clumped together in
sticky ropey aggregates and slides easily off the growth
medium when pushed with a suitable instrument, such as a
glass spreader.
While the foregoing procedurès are desirable where
high yields of high purity pili are in view, quite acceptable
results are obtainable by a substantially abbreviated pro-
cedure. In this procedure the first and second wash pro-
cedures set forth above are not utilized. The entire growth
i8 treated with substantially elevated pH buffer suitably
an ethanolamine buffer in accordance with the procedures of
the third washing. The wash medium will contain, in addi-
tion to the dissolved pili, many impurities otherwise removed,
3583
0244 (13) -18-
1~50~Z3
however, it has been found that these impurities may be held
in solution upon precipitation of the pilic material in the
manner discussed below.
Deep Culture of N. gonorrhoeae Organisms
The deep culture of the organisms of type Tl and T2
in liquid medium is carried out in a conventional manner util-
izing a medium and environment identical to that used for
the surface culture except that the medium does not contain
agar as a solidifying agent. It has been noted that the
organisms grow and shuck pili continuously. Thus, a deep
culture medium will contain much suspended pilic material.
Where type T2 cultures are being grown the pH, being normally
below pH 9.3, there is no undesired solubilization of the
pili. In the culture of Tl organisms the pH may rise above
pH 7.7, hence prior to work-up as described hereinbelow,
the pH should be adjusted down into the range of 5.5-7.7,
preferably to about pH 7.0-pH 7.2. While it is not critical,
; it is preferred to make such an adjustment a few hours,say
8-20 hours before work-up to ensure crystallization of
partially solubilized Tl pili.
,
In both Tl and T2 cultures, it is helpful, but
by no means essential to carry out the growth in a mildly
agitated medium in the presence of a small amount of diatom-
aceous earth such as Celite . The amount of, say, Celite
should suitably be between about 0.1 and about 0.5~, suitably
about 0.3~ by weight of the growth medium.
0244 -19-
-:~050g~3
Purification of Pili
Separation of pilus crystals from ~rowth mediu~
It should be noted that the wash from the surface
growth of the N. ~onorrhoeae or~anisms contains material soluble
therein which is of no interest in the isolation of GC pilus
crystals. Similarly, the same is true of the deep growth
liquid cultures. In the case of the surface growth wash
~first two washes only), the amount of li~uid relative to
the amount of growth is relatively small. It is preferred
to centrifuge the entire wash material at relatively low
speeds. The speed of centrifugation and the time of spin
down is by no means critical. However, it has been found
helpful to spin at between about 1000 ~ and 12,000 G
(hereinafter written as 1 KG and 12 KG), for from about 5 to
about 30 minutes, preferably at about 3 KG for from about
10 to about 15 minutes. The residue in the pellet contains
both cellular, and pilic material, both of which are retained
at this stage and the supernatant is discarded. The
supernatant contains substantial amounts of impurities as
well as small amounts of pili which are not worth recovering.
Where deep growth is the mode of the culture utilized,
the volume of liquid is rather substantial and therefore
centrifugation may be somewhat cumbersome. The use of a
diatomaceous earth sandwich filter has been found useful in
concentrating the growth from deep cultures. In this pro-
aedure a ~ery coarse filter paper is laid on the filter pad,
suitably a sintered glass or Buchner type surface, a layer of
diatomaceous earth, suitably Celite of about 2 to about 5 mm
thickness is charged thereon and covered with a second coarse
filter paper. The Celite ser~es as the actual filtration
0243 ~3~)
-20-
1(3504'~3
medium while the upper filter paper serves merely to preserve
the surface. The culture broth, havinq been checked for pH
to ensure the pili are present in crystalline form ls filtered
through the filter pad and the filtrate discarded. The com-
bined residues are then taken up in say, a high pH huffer
similar to that utilized for the third wash of the surface
~rowth culture, and this suspension is centrifuged at from
about 1 KG to about 12 KG. It should be noted of course
that in this case the pellet will contain the diatomaceous
earth carrier and cell debris, and the pilic material will
be in the supernate.
It is desirable at this point to separate the GC
pilus material from the cell debris and, in the case of
the deep growth culture, the diatomaceous earth as well.
ln the case of the surface culture pellet centrifuged
from the low pH wash, this may be done by adding an aqueous
medium which will break the noncovalent bonds between pilus
rods in the system while leaving the covalent bonds intact
thus dissolving the pilus crystals to give solubilized single
rod pili. Such a medium may require raising the pH or may
permit the pH to be unchanged. Where solubilization is to
be carried out by pH change, there is added to the residue
a suitable, moderately high pH buffer. It is desirable that
the buffer have a pH of between 9.3 and 11, preferably be-
tween pH 10.0 and pH 10.4. With higher pH's there is a risk
of denaturation of the peptides. This level of pH will
solubilize Tl and T2 pilus crystals.
'
Where it is desired to separate Tl from T2 pili
in the original solid pellet, the pH of the buffer is
; initially provided to be greater than about pH 7.7 but less
0243
0571 -21-
~0504Z3
than about pH 9.3. The suspension is then centrifuged and
the supernate therefrom can then be set aside or discarded
according to the needs of the procedure. If the original
solid pellet was believed to have contained substantial
amounts of type T2 pili, fresh buffer of higher pH/ namely
above about pH 9.3 is added whlch will provide a solvent
phase containing said T2 pili but free of type Tl pili.
The actual composition of the buffer utilized at this stage
is not critical, however, a tris-saline buffer is
especially preferred.
In both of the foregoing modifications, there is
added to the total solids a volume of buffer approximately
equal to 3 times the volume of the solids. Again, this
amount is not critical but has been found to be sufficient
to dissolve the pil.ic material without utilizing excessive
volumes of the aqueous medium. If the pili are from a
suxace culture and said surface culture was subjected to
a third stage high pH wash with a similar buffer, this wash
may be added at this point. The pellet is then suspended
in the aqueous medium. The method of bringing the pellet
into suspension is not critical, short gentle sonication,
long magnetic stirring, hand pipetting, hand mixing,
vortexing or mechanical stirring may be used.
It has been found preferable to utilize mechanical
stirring for a few seconds. While the mode of suspension is
not critical, it is important that whatever mode is utilized
the cells are not ruptured since cell rupture will introduce
undesired material into the aqueous layer. The occurrence of
cell rupture is noted as a layered pellet of pink above white
in the subsequent centrifugation. The suspended material i5
0571
0570 (7)
-22-
~05~Z3
then centrifuged. The manner of centrlfugation is not critical,
however, the conditions set forth above for the first centri-
fugation step have been found suitable. Filtration may be
used in place of centrifugation.
The dissolved pili are found in the supernate or
filtrate from which they may be precipitated upon lowering
of the pH. The degree of pH lowering will of course depend
on whether type Tl or T2 pili are in process. A]ternately
precipitation may be achieved by adding sufficient ammonium
salt, suitably a mineral acid salt such as the sulfate, pre-
ferably as an aqueous solution, to provide an ammonium
sulfate, concentration of between about 4% saturation to
about 10~ saturation. However, in order to increase the
yield and increase the degree of purity, it has been found
desirable to introduce intermediate steps prior to the
precipitation.
In carrying out these additional yield and puri-
fication steps, the supernatant and the pellet from the high
pH centrifugation or filtration are both retained. The pellet
from the high pH centrifugation stage is re-suspended,
suitably in th~e same aqueous medium at the same pH in the
same manner and the suspension centrifuged again in the same
manner. After this centrifugation, the pellet is discarded
and the supernates from both high pH centrifugations are com-
bined and recentrifuged.
The purpose of the recentrifugation is to remove
-~ residual suspended impurities. Centrifugation is therefore
carried out at a higher speed than heretofore. Speeds from
0570 (35)
-23-
~()50~3
between 12 KG and 70 KG for from about 30 to about 60
minutes are operative. It is generally preferred however to
spin at from about 27 KG to about 40 KG for about 60 minutes.
The pellets are discarded and the supernate retained.
At this stage it is usually desirable to sterilize
the pilus solution. This is required by certain FDA rules
for certain purposes. Such sterilization may be readily
achieved by passing the pilus solution, immediately before
precipitation, thru a millipore fllter, a 0.45 micron filter
has been found especially suitable. Thereafter of course
the materials must be handled in an aseptic manner, if it is
desired to maintain sterility.
Under certain circumstances it may be desirable to
isolate the pili in individual rod form rather than in
crystal form. In this case the supernate is respun in an
ultra high speed centrifuge at between 60 and 166, suit-
ably 106 KG for between 2 and 4 hours, whereby the pili
are pelleted in individual rod form.
The pili are re-precipitated by lowering the pH
of the supernate to below 9.1. It has been found that the
best results in terms of the nature of the crystalline
material have been obtained by dialysis against a suitable
low pH buffer. It has been found desirable to utilize a
buffer havin~ an initial pH of between 8.3 and 8.6,for T2
pili, while the chemical nature of the buffer is not critical,
tris buffered saline has been found suitable. Dialysis is
suitably carried out utilizing an excess of between about
30 and about 60 fold, suitably about 40 fold. The dialysis
0570
2467
0569 (12) 24-
~0504Z3
is carried out with magnetic stirring of the external
dialyzing medium for a period of from about 12 to ahout
18 hours. It is preferred to carry out the dialysis at
a reduced temperature, that is to say, an amhient temperature
of from about 0 to about 10C. This lower temperature range
lowers the incidence of undesired bacterial contamination.
It should also be noted that the buffer pH is temperature
dependent. Hence, if the temperature of the buffer is
measured after the system has cooled down to its operating
temperature, the pH may be found to have risen as high as
p~ 9.1. ~owe~er, satisfactory results are still obtained.
It should further be noted that it is not generally
necessary to change the buffer where excess buffer in the
range stated herein is utilized. The crystal suspension
is then processed to separate the pilus crystals. Most
suitably separation is carried out by moderate speed centri-
fugation. Centrifugation at from about 3 KG to about 8 KG
for about 60 minutes has been found suitable. The supernate
is then discarded.
Where it is desired to purify the pilus crystals
further, the cycle of solution-high speed centrifugation-
dialysis-recen~trifugation may be repeated two or three times.
If the pilus crystals are not to be utilized
immediately, notwithstanding sterilization by filtration,
the addition of preservative has been found helpful. It
is desirable that the preservative be added not to the
: pilus crystals themselves or to a solution containing them, but
rather to the dialysis buffer utilizad to lower the pH of the
solution. In the event that the added preservative is in~
compatible with the buffer, then, after crystallization of the
0569
0572 (8)
-25~
~L(I 504Z3
pilus crystals has occurred, the incompatible buffer may
be removed by dialysis against a compatible buffer and a
further dialysis carried out utilizing the preservative plus
the new buffer. Among the preservatives ~hat may be used are
formaldehyde, merthiolate and azide. There are used be-
tween about 0.02 and about 0. 05~ of these preservatives.
Each of the named preservatives has certain detri-
mental effects. Formaldehyde causes cross-linking between
the pilus rods. They may thus not be redissolved as before.
Merthiolate has no cross-linking effect and the crystals may
be reformed, however, such reformed crystals have a decreased
ability to agglutinate in the presence of antibodies to the
pili. Nevertheless, the antigenicity is not affected. That
is to say, when injected into test subjects they cause the
formation of apparently normal antibodies to the pili.
Azide is a very satisfactory preservative in that it affects
neither crystal structure nor antigenicity nor agglutination.
Unfortunately, it is toxic and cannot be employed where in-
jection of the pilus crystals into human subjects is contem-
plated. The crystal preparation is suitably stored at low
temperatures, i.e. at about 1C to 4C. However, where long
storage is con~templated it is preferable to dissolve the
crystals in an appropriate high pH buffer, filter thru a
millipore filter and store in solution under sterile condi-
tions. When the pili are required in crystalline form it
is preferable to reconstitute them by lowering the pH to
the appropriate crystallization value for Tl or T2 pili
as the case may be. Both sterilization and a preservative
can be employed when it is desired to achieve the best possible
conditions of preservation
0572 (36~
-2~-
lOS~Z3
Purification of Pili by constant pH Dissolution
The technique utilized to purify pili at constant
pH is substantially similar to that utilized hereinabove
using di~ferent pH le~els.
As solubilizing agents there may be utiliæed aqueous
solutions of, for example, salts, suitably salts of alkali,
and alkaline earth metals with the anions of mineral acids,
at an ionic strength above 0.5, suitably between about
4.0 and about 5.0, preferably about 4.4, urea at a concen-
tration of between 3M and a~out 5M sucr~se abo~e a concen-
tration of 50% by weight and finally water itself where the
` ionic strength of the solution is reduced below 0.002 M.
:,
In one modification of this embodiment of the
invention instead of suspending the first centrifugate pellet
which comprises cells, pili, and debris, in a high pH medium,
there iæ utiliæed in place thereof any of the foregoing
agents in the environments aforesaid. The suspension is
then low speed centrifuged as before. If higher yield is
desired the sùpernate from the low speed centxifugation is
~ set aside, peliets resuspended in a similar medium, re-
; centrifuged and the thus produced supernate combined with
the previous supernate and high speed centrifiuged. Pellets
from high speed centrifugation are discarded and the supernate
dialyzed against a suitable buffer to remove the solvating
medium.
Thus, where solvating agent is a high concentra-
tion salt or sucrose, the ionic strength is reduced to below
0~5 for the salt and below 40% for the sucrose. In the
case of urea, the urea is dialyzed against a suitable
2464
0573 (10) -27-
6951 (5)
lOS~Z3
buffer, say, tris buffered saline, to provide ionic strength
of between . 05 and O . 3 at pH 7 . O or pH 8 . 3, depending
on whether Tl or T2 pili are being processed, simi]arly when
the solvating agent is water similar procedures are used to
raise the ionic strength to at least 0.05.
The buffers ~tilized for this purpose are the same
as the buffers utilized in the differential pH purification
method and, moreover, are utilized in the same manner.
Purification of GC Pili by Density Gradient Centr~fugation
Density gradient centrifugation is carried out by
subjecting a mixture of pili and aqueous caesium chloride to
centrifugation together and the optical density at a given wave
length utilized to indicate the portion of the tube containing
the pili.
While centrifugation may be carried out at a pH
under pH 9, better results are obtained by carrying out the
centrifugation in the pH range of 10.0 to 10.4, suitably at pH
10.1. In this procedure, the crude pellet containing cellular
materials, pili, and debris, is suspended in a high pH buffer,
as set forth hereinabove, and a suitable quantity of caesium
chloride added thereto. For example, it has been found suita~le
to prepare a medium containing from about 2 to about 5 grams of
dry caesium chloride per 10 ml. of aqueous medium. Thus,
it has been found most suitable to utilize about 7.5 grams of
caesium chloride to 20 ml. of aqueous medium.
0573 (31
-28-
10504Z3
The mixture is then spun at from about 110 to about
250 KG, suitably about 200 KG for from about 30 to about 60
hours, suitably for about 42 hours and the optical density
of fractions at a given point in the tube measured. Optical
density measurements at 280 nm show a single peak. The density
fractions under this peak are seDarated and dialyzed against
a low pH buffer to form the pilus crystals in the same manner
as that set forth hereinabove. The actual density range of the
solution fractions collected at this point at 20C is between
1.35 to 1.33 at pH 10.1.
PAT Test for N. Gonorrhoeae Antibodies
...... _ ._ . _ . . .. .
~ he basic ability of GC pilus crystals or single
pilus rods to agglutinate in the presence of antibodies to
N. gonorrhoeae is the basis of the PAT Test. In this test,
. _
sera from the blood of subjects suspected of having been
exposed to N. gonorrhoeae are mixed with GC pilus crystals
or single pilus rods and the mixture observed for agglutina-
tion of the crystals or the rods.
In order for this test to be evaluated in its true
light, three important factors must be considered. First,
the test is nok intended to replace the standard "culture"
test but may serve as a screen to determine exposure to
N. gonorrhoeae. The test will therefore show positive
for both subjects who have had an active infection for more
than a few days and also for subjects who have been exposed
tQ the disease but have since been cured. The third
caveat is that very recently infected subjects may not
have developed enough antibodies to give a positive reading.
5ubjects showing a positive result i~ the test should be
subject to the traditional culture test. It has been found
0S73
0558 (28)
-29-
~()S04Z3
as will be discussed further hereinbelow, that the pili ~f
infectious forms of N. gonorrhoeae possess a number of
specific immunologic determinants. Pili of certain strains
will possess one or more of these determinants. Hence,
for a screen to be effective, it must be carried out using
pilus crystals which cover th~ spectrum of immunologic
determinants.
The GC pilus crystals utilized in the test are pre-
pared in the manner set forth hereinabove.
The test may be carried out using either serum
or plasma from the test subject. References herein to serum
or plasma therefore can be considered interchangeable for pur-
poses of the test. The amount of ser~n or plasma required is
extremely small. It has been found satisfactory to puncture
the subjects finger to obtain a few drops of blood and to
spin these down in a small (circa 250 yl) centrifuge tube
to yield between 10 and 20 ~1 of plasma which are ade-
quate for carrying out the test.
It is customary to carry out tests of this nature
at various levels of dilution. The serum is therefore diluted
in predetermined (usually serial dilutions) with a predetermined
suitable diluent. The nature of the diluent is not critical
provided that it does not interfere with the operation of the
test. Any aqueous buffer such as phosphate buffered saline
or tris buffered saline having a pH of between 7.0 and 7.5
may be utilized. Tris buffered saline having a pH of
7.2 is preferred. In the operation of the test, pilus
0558
0559
-30-
~050423
suspension is added to the diluted serum to give a final
concentration of between lO and 50 micrograms per ml. of
cry~tals in the suspension. It has been found that the best
results are obtained at the lower concentration of pilus
crystals, hence, for purposes ~f standardiæation of pilus
antibody levels in test sera, the arbitrary concentration
for compari60n purposes of 20 micrograms of pilus crystals
per ml. of diluted serum (or plasma) have been taken as the
~tandard.
After mixing the pilus crystals with the diluted
sera, the mixture is incuhated. The time of incubation is not
critical and may be from as little as 15 minutes to as long as
48 hours with negligible change in the reading. In a rapid
form of the PAT Test the serum-pilus mixture is agitated on
a slide by hand for l to 3 minutes, similar results are ob-
tained but with some loss in sensitivity. The temperature
at which the mixture is maintained is also not significant
as long as it is maintained between 0 and about 45
except that it appears preferable to maintain the mixture at
a temperature of ~etween 22C and 40C, suitably at about
22 for at least 15 minutes. Storage of the mixture thereafter
- at temperatures as low as 4C for up to 24 hours do not appear
~o give significant changes of titer. Satisfactory results
have been obtained by incubation for 30 minutes at 37C.
At the expiration of the incubation period an aliquot is placed
under a dark field microscope and the agglutination noted and
scored. The scoring is done in the usual arbitrary relative
way for tests of this nature, namely, 4+, 3+, 2+, l+, - + and -.
In determining the titer of a given sample the last dilution
which gives a l+ agglutination is taken as the final reading.
0560 (27)
-31-
~0S(~4'~3
The l+ score is that score which shows the minimal noticeable
agglutination over a standard control sample.
Since the titer readings may vary from batch to
batch of pilus crystals according to their condition it is
advisable to monitor the test, when carried out, by running
the crystals against antisera of known PAT titer in addition to
the usual controls against diluent without serum, and diluent
with normal serum.
It has been found that under the conditions
utilized, the test is reproduciable within a titer reading
of a times 2 (X 2) factor.
Serotyping of N Gonorrhoeae
Heretofore, it has not been possible to serotype
N. gonorrhoeae organisms into a useful and meaningful system.
It is not unusual for organisms of different strains of the
same species to call forth different antibody responses.
These antibody responses characterize the stralns and
identification of these characteristics constitutes a sub-
stantial aid in epidemiological studies of the progress
and origin of a particular outbreak of the disease. It is
particularly of interest in the venereal diseases since
the elimination of the disease very often depends upon person-
to-person contact tracking. Thus, if the source of an infection
can be identified by serotyping, this type of tracking can be
greàtly assisted.
',
0560
0566 (19) -32-
~)5()4Z3
It has been found that by examination of 21 strains
derlved from sources well distributed throughout the United
States that there are at least four (4) immunological deter-
minants present in the pili of these strains. One or more of
these determinants are present in each strain. Hence, if
the determinant characteristics of a particular sample can
be identified the origin of the infection can be more readily
discovered.
Where N gonorrhoeae organisms are derived
from an infected subject and grown to produce pilus crystals
the immunologic determinants present in the pilus crystals from
the subject may be readily discovered by subjecting them to
the PAT Test with the aforementioned typing sera which are
known to contain antibodies for but one determinant. Thus,
the serological profile of any given sample organism may
be established.
-
Gonorrhoeae Vaccine - Safety and Potency
. . _
G. C. Pilus crystals and single rod pili have been
injected into test subjects 1n vivo and found to have no
toxic effect whatsoever. The only negative effects were
noted with intravenously injected chick embryoes. Test
rabbits were given three (3) injections subcutaneously ~f
100 to 200 micrograms per Kg, giving a total dosage of 300
to 600 micrograms per Kg, rats were similarly injected two
(2~ times at 8,000 micrograms per Kg, giving a total dosage
of 16,000 micrograms per Rg. Rhesus monkeys received three
(3) injections of 100 micrograms per Kg intramuscularly,
and humans received injections of 2-10 micrograms per Kg,
followed ~y one (1) injection of 50 micrograms per Kg
intramuscularly. None of the test animals died nor showed
0566
2875 -33-
l()S04Z3
any local or systemic toxic effects, the chick embryos
showed an LD-5G of S0 micrograms per pound. The reacti~ns
in human subjects varied from no systemic effects whatsoever
to transient chills and fever in one of the subjects tested.
The PAT titer in rabbits reached l,000 to 8,000, and in
Rhesus monkeys reached lO,000+. The PAT titer in humans
varied between 100 and 200.
The dose required to infect 50~ of test subjects
tID50) is of the order of 5.0 x io2 organisms. Preliminary
experiments indicate that the ID50 of a human suhject havin~
a PAT titer of 100-200 is 2.0 X 104 organisms. This
represents 1.6 log cycles of protection, or stated another
way, a human subject having a PAT titer of lQ0-200 has only
- about a 0.86~ chance of being infected after l contact
whereas an unimmunized person runs about a 30% risk.
In view of these findings, in order to provide an
acceptable level of protection in humans, the human subject
should have administered a sufficient quantity of GC pilus
crystals suitably against all known determinant factors to
raise the PAT level against each of these determinants to
at least lO0, preferably to at least 200. Such levels are ob-
tainable by administering between from about 2 to about lO0
micrograms per kilogram body weight of GC pilus crystals of
each determinant. It should be noted however, that a titre
of lO0 is achievable with as little as l ~g/Kg. The mode
of administration will depend upon the sensitivity, if any,
of the subject, but may suitably be administered in between
1 and 5 doses over a time period of up to 8 weeks.
The GC pilus crystals, single pil~s rods, or other
sources of GC pilin may be administered in any suitable
2875 medium for intramuscular injection.
2878
-34-
105~4~3
EXPERIMENTAL
Sources of N. Gonorrhoeae IJtilized
21 strains of N. gonorrhoeae were isolated
from humans with Gonorrhoeae and are designated as follows:
Pittsburgh 1-2 CDC M~2 Seattle 1-2
Pittsburgh 3-2 CDC T-2 Seattle 3-2
Pittsburgh 4-2 CDC F62-2 Seattle 9-2
Pittsburgh 6-2 Atlanta 4-2 Norfolk 2-2
Pittsburgh 7-2 Atlanta 6-2 Norfolk 7-2
CDC B-2 Atlanta 9-2 Dayton 8-2
CDC C-2 Atlanta 10-2 CDC 005-2
In all of the following Examples, specific
reference is made to the Pittsburgh 3-2 strain. When the
other named organisms are subjected to the same procedures,
as the Pittsburgh 3-2 strain, similar results are obtained.
EXAMPLE I
Strain Purification
.
The primary cultures of the Pittsburgh 3-2 strain
were plated out on Thayer-Martin plates containing Thayer-
Martin Agar (Manual of Clinical Microbiology, 2nd. Ed., Amer.
Soc. Microbiol, Lenette, et al. (Ed.) 1974, p. 920). The
plates are incubated for about 18 hours at 3-5C in a
humidity of 90~ in an atmosphere comprising 95~ air and
5% carbon dioxide. The plates are inspected and colonies
resembling the highly piliated T2 form are re-streaked on
GC m~dium. After incubation under the above conditions the
T2 type colonies are picked and restreaked again on GC medium.
3599
3589 (4) -35-
~)S04Z3
In accordance with the above procedure, but where
the culture of the Tl type of this or~anism is desired
colonies having a preponderance of the less piliated T
type are similarly restreaked.
EXAMPLE II
_eparation of GC Growth Medium
a) Preparation of DSF Supplement
An aqueous solution of cocarboxylase (0.2% by
weight) in distilled water is prepared at ambient tempera-
ture and sterilized by filtration through a 0.45 micron
millipore filter. An aqueous solution comprising glucose
(40 g.), glutamine (1.0 g.), ferric nitrate (0.5~ by weight,
10 ml. in distilled water), and distilled water (90 ml.)
are heated in an autoclave at 121C with 16 psi pressure for
10 minutes and the solution cooled. To this autoclaved
solution is added 1 ml. of the pxeviously prepared cocar-
boxylase solution to provide the DSF solution.
;
b) Preparation of Growth Medium
Bacto GC Medium Base (Difco Mannual l9th. Ed.,
p. 122) (Difco I,aboratories, Detroit, Michigan) (10.8 g.) and
distilled water (300 ml.) are gently agitated in a suitable
container and the mixture in the container autoclaved at
121C with 16 psi pressure or 15 minutes. The container
is removed from the autoclave, cooled to between 50C and
60C and the DSF supplement, prepared as above, (3 ml.)
is added thereto.
6952
3589 ~29)
-36-
~S04Z3
Preparation of Inoculum Plates and Growth Dishes
Pyrex or aluminum growth dishes and petri type
inoculum dishcs are washed, rinsed in distilled water, covered
with aluminum foil, and autoclaved for 30 minutes at 121C
with 16 psi pressure. Into the thus prepared dishes is
poured the molten growth medium prepared as above in
Example II. The plates should be poured with care in a
closed dust free room utiIizing aseptic techniques to prevent
contamination with undesired bacteria.
EXAMPLE III
Growth of Inoculum
. _
The T-2 colonies from the Thayer-Martin plates
(Example I) are re-streaked on inoculum plates prepared as
above and cultured at 35,90~ humidity, in an atmosphere of
5% carbon dioxide and 95~ air. The plates are progressively
subcultured until more than 90% of the growth is the piliated
T2 colonial type. Depending upon the stability of the strain
the growth time is between 12 and 24 hours, at which time
the plate will be covered with between 50% and 75~ of growth.
EXA~IPLE IV
Production of GC Pili by Surface Culture
; The petri dishes of Example III containing the T2
growth are washed with aqueous casamino acid solution (5 ml,
0.7% by weight). The growth is scraped off the medium with a
3589
0561 (24)
~OS04Z3
sterile glass spreader, the suspension of the growth in the
casamino acid solution is pipetted from the plate and
divided between two (2) growth pans ca. (14 X 10 in). Said
pans having been prepared in the manner set forth above.
The suspension is spread evenly over the surface and the pans
incubated at 35C, 90% humidity, in 5~ carbon dioxide plus
95% air atmosphere for 20 hours.
Harvestin~ of Surface Culture Growth
-
A stock solution of tris buffered saline is pre-
pared by dissolving sodium chloride (510 g.), tris, also
known as tris(hydroxymethyl)aminomethane (363 g.) and con-
centrated hydrochloric acid (100 ml.), toqether with dis-
tilled water sufficient to produce a stock solution having
a volume of 10 liters. The pTI is adjusted to a standard
working pH of pH 8.5 by the addition of more concentrated
hydrochloric acid. Where an upward pH adjustment is required,
concentrated (10 N) aqueous sodium hydroxide is added. Prior
to use the stock solution is diluted to 1/6 of the original
concentration. The tris buffered saline (hereinafter TsS)
has an initial pH of 8.5. 10 ml. of the TBS solution is
placed on the growth surface of the production pan, the
growth scraped off with a glass scraper and the suspension
pipetted off into a reservoir. The washing and scraping
is repeated with a second batch of TBS (10 ml.), and both
washinss pooled. The growth surface is washed a third time
; with ethanolamine buffer (10 ml., pH 10.1) and the suspension
retained but not pooled. (The ethanolamine ~uffer is pre-
pared from liquid ethanolamine, 37.3 ml., aqueous hydro-
chloric acid, 1 N, 147.0 ml. and distilled water to 1 liter).
0561
6717 (20)
-38-
lOSO~Z3
In accordance with the above procedure, where Tl
rather than T2 pili are being cultured the pH of the TBS is
between pH 7.0 and 7.2.
In a good production run the growth has a character-
istic orange/pink or warm pink color and an odor resembling
cooking food. The growth clumps together in sticky ropy ag-
gregates and slides readily off the agar surface of the
medium when pushed with a glass spreader.
The pans containing the growth medium are then
cleaned, washed and sterilized in the manner set forth above,
and recharged w1th more growth medium.
EXAMPLE_V
Deep Culture Growth of G. C. Pili
In accordance with the procedures of Example
IV using the same growth medium and nutrient supplement
but excluding the agar and substituting soluble starch for
the insoluble starch, inoculum is charged to the growth med-
ium and incuba~ted in an atmosphere of 95% air and 5% C02
at 35-37C in the presence of up to 0.5% (based on volume of
liquid medium) of Celite, for 18 hours under gentle agitation.
Th~ culture medium is then filtered thru a sandwich
filter pad on a coarse sintered glass funnel. The filter
pad comprises a coarse filter paper, a 5 mm layer of Celite
and a further coarse filter paper.
The filtrate is discarded and the residue treated
6717
6953 (19)
-39-
~050423
in accordance with the third (ethanolamine buffer) wash
procedure of Example IV, which is then processed in
accordance with the procedures of Example VI, paragraph
iii, infra et seq.
EXAMPLE VI
Separation of Pili from Cells and Debris
i) The TBS suspensions produced in the foregoing
Examples are charged to centrifuge tubes tvolume of the
tubes depending on the number of production runs combined)
and centrifuged for 15 minutes at 3 KG. The supernatant
is discarded and the pellet retained.
ii) To the pellet is added the ethanolamine sus-
pension from the third wash of the growth pan and additional
ethanolamine buffer at pH 10.1 to a volume approximately
3 times the observed pellet volume in the centrifuge tube.
The liquid layer is stirred rapidly for 5 seconds with a
mechanical stirrer to bring the soluble portion of pelleted
material into suspension.
iii~ The suspension is then centrifuged for lS minutes
at 3 KG the supernate decanted from the pellet and pre-
served. The pellet is then resuspended in 3 times its
volume of ethanolamine buffer pH 10.1 as hereinabove, and
recentrifuged for 15 minutes at 3 KG. The supernate is
decanted and pooled with the ethanolamine buffer supernate
from the immediately prior step and the pellet discarded.
6953
2879 t24)
-40-
lOS(~kZ3
The combined ethanolamine buffer supernates are
centrifuged at 31 KG for 60 minutes. The supernate is de-
canted and preserved and the pellet discarded.
EXAMPLE VII
Crystallization of GC Pili
Preparation of Dialysis Tubing and
Tris Buffered Saline
A roll of dialysis tubing (lO0 foot, Fisher
Scientific Catalog Number 8-677 B) is boiled sequentially
in a~ distilled water (2X, 4 liters each time), b) aqueous
sod~um ~icarbonate (2X, distilled water, 4 liters, containing
sodium bicarbonate 2 teaspoons each wash), c) aqueous di-
sodium ethylene diamine tetraacetate (2X, distilled water,
4 litexs, Na EDTA 2 teaspoons; each time), d) aqueous
ethanol (2X, ethanol/water, l:l, 4 liters; each time),
e) distilled water (2X, 4 liters, each time). The dialysis
tubing is then stored in distilled water containing a trace
of benzoic acid (distilled water 4 liters, benzoic acid
l teaspoon). Tris buffered saline stock (TBS) prepared
in accordance with Example IV is diluted with distilled
water to provide the di~lysis solution (166 ml. stock solu-
tion diluted to l liter with distilled water).
D~alysis of GC Pili Solution
s
lO0 ml. of ethanolamine buffer containing GC
Pili in solution as produced in the foregoing Exampls are
2879
3587 (Z5)
-41-
~0504Z3
dialyzed against 4 liters of TBS (pH 8.5 measured at 20C)
utilizing dialysis tubing prepared as above and utilizing
diluted TBS as prepared above. The dialysis is carried out
in a cold room (ambient temperature circa 4C). The
external dialysis buffer solution is stirred magnetically.
Dialysis is carried out for 18 hours. A rise to pH circa
8.7 in the dialysis medium is noted. A cloudy blue/white
birefringent precipitate of GC pilus crystals is formed at
the end of the dialysis period. The thus precipitated
material is centrifuged at 7.5 Krpm for 60 minutes and
the supernatant discarded to leave GC pilus crystals of
Pittsburgh strain 3-2 N. gonorrhoeae as the pellet.
In accordance with the above procedures, but where Tl
; rather than T2 pili are to be isolated, the initial pH of
the TBS is pH 7.0-7.2.
EXAMP~E VIII
Further Purification of Pili
;
The pellet of Example VII is suspended in ap-
proximately 30 times its volume of ethano~amine buffer
(pH 10.1). The~tubes swirled gently to dissolve the pellet
and the suspension centrifuged for 60 minutes at 31 KG.
The supernate is decanted off and the pellet discarded. The
supernate is dialyzed against TBS in accordance with the
procedure of Example VII, and the thus obtained crystalline
material isolated by centrifugation also in accordance with
the procedures of Example VII. The pellet is once more
resuspended in ethanolamine in accordance with the fore-
going procedures, filtered through a 0.45 micron filter
3587
0240 (17)
-42-
~ 50~Z3
if sterilization is desired and s,Lmilarly recentrifuged
and redialyzed as heretofore, and, similarly, isolated
by centrifugation.
In accordance with the foregoing procedures,
where the thus formed GC pilus crystals are to be stored for
a moderate period of time, a preservative is added to the
dialyzing TsS solution where the preservative is compatible
therewith. Cornpatible preservatives which may be utilized
in accordance with this procedure are 0.05% weight per
volume of neutral aqueous formaldehyde, 0.01% weight per
volume merthiolate or 0.02% weight per volume sodium azide.
In accordance with the foregoing procedure where
the preservative is not compatible with TBS after the
Pili have crystallized, the tris buffer is removed by
dialysis against saline (0.15 M aqueous sodium chloride,
18 hours). The preservative is added to a fresh batch of
said saline, and dialyzed against the suspension of GC
pilus crystals for 18 hours. The crystals are stored in this
medium at 40C. Alternatively the medium containing the
crystals may be frozen and stored at -70C.
Yield
The yield of GC pilus crystals from the strains
o~ N. gonorrhoeae listed in Example I, where the inocula
contain at least 90~ T2 colonial types, lies in the range
of 5 to 15 micrograms/square centimeter of growth surface.
Similar but slightly lower yields are obtained
from Tl colonial types.
0240
3579
-43--
:
1.~5V4;~3
EXAM LE IX
Caesium_Chloride De~sity Gradient Isolation of GC Pili
1 ml. of the TBS (pH 8 . 5) wash containing GC
pilus crystal suspension from Example VI is diluted to
20 ml. with tris pH 8.5 buffer, the pH adjusted to pH
10.1 by the addition of aqueous sodium hydroxide (0.lN)
and 7.5 grams of dry caesium chloride added thereto. The
solution is spun at 200 KG in the SW41 Rotor of a
Beckman L-265 Centrifuge for 42 hours. F`ractions are
collected from the tube and the optical density at 280 nm
and the refractive index for each sample measured. The
reractive index is related to caesium chloride density
to which the refractive index readings are converted. The
fraction number is plotted on the X axis against solution
density on one Y axis and optical density on a second Y axis.
A single principal peaX corresponding to the GC pili is
p (buoYant dens~ty)
located at/ ~qua~s 1.3422 ~ .0038. The fractions whose
e~uals 1.35 to 1 33 are combined and dialyzed and purified
to yield GC pilus crys~als in accordance with the procedures
of Examples VII through VIII.
EXAMPLE X
Gel Electrophoresis of GC Pili
(Method of Ornstein and Davis - Disc Electrophoresis
1962 - Distillation Products Industries, ~ochester, N.Y.)
.
Standard cylinders o~ 10~ acrylamide gel (9.7~
acrylamide and 0.3% N,N'-methylene-bis-acrylamide are poly
merized with TEMED) and ammonium persul~ate were prepared
and set up between gel and reservoir buffers comprising
3578 (33)
-44-
1050423
tris hydrochloride at pH 8.0 and 0.1% SDS. The upper gel
surface was loaded with a charge comprising 50 micrograms
of T~ pilus cry~tals, 20 ug of Clelands Reagent (O.OlM)
1 mg. of SDS 20 ~ of glycol and 20~ of sromophenol
Blue (0.002%). Prior to charge the pili were heated with
-the SDS and the Cleland's reagent for 2 minutes at 100C.
The electrophoresis was run at 5 ma (at ca. 170 v) until
the Bromophenol slue had run 6 cm. The gels were removed
and cut thru the dye band and two gels stained with
Coomassie Blue Stain (0.2%) to give two bands - a major
band and a minor band.
The unstained gels were frozen and bands corres-
ponding in position to the stained bands were cut out.
The major band was extracted with reservoir
buffer at 37C in a rotator for 24 hours, the buffer drawn
off and evaporated almost to dryness. A rerun of the
product yielded a single band of the same Rf value.
This material is designated GC pilin.
Antigenicity Test of G.C. Pilin
The gel containing the major band was homogenized
with about 10 ml. of saline and injected into three test
rabbits subcutaneously. Test animals PI and PII received
3.1 ml suspension and PIII only 2 ml. of the suspension.
Second and third injections were made about 15
and about 30 days later. The materials of the second and
3578
3586 (12)
-45-
,
,
10'~1 ~Z3
third injections were prepared by extracting the major
protein into reservoir buffer (0.8 ml.) at 37C for 24
hours. The extracted buffer was then combined with an
equal volume of Freund's Incomplete adjuvant and one-third
of each mixture injected into each rabbit.
.,
One week after the last injection all rabbits
showed titres exceeding 1000 in the PAT Test against
Pittsburgh 3-2 Type T2 pili as shown in the Table below.
PCA TEST USING 50 y¦ml "3-2" PILI
Pre-
I~nune .
Rabblt PI Pr PI pIr prI PII PIII PIII PIII Rabb~t
Bled:Day# 1 15 29 1 15 29 1 15 29 -204
~est n 16 16 35 16 16 35 16 16 35 35
Dane
O~lution
`lJ2 + +~1+ }~ 3+ 3+ 4+ ~ ++ 3+ 0
l/4 + + `3~ 3~ 5+ 5+ 0 3~ 3* 0
l/8 0 + 3+ 3~ 5+ 5+ 0 3~ 4~ 0
1/16 o 0 3~ 3+ 5+ 4+ o +~ 4+ 0
1/32 0 0 3+ + 4+ 4+ 0 _ 4+ 0
l/64 0 O + 0 4+ 3~ 0 0 0
l/128 0 0 + 0 4+ 3~ 0 0 ++ O
1/Z5~ ~ 3~ +
1/512 + ~+ + O
1~'1024 ~ +~ ~ .
1/2048 ~
- 1140g6 ' O . + ~
e~d
point ~2 4 1024 32 ~128 ~4096 ~2 16 2048 ~2
:
3586 (22~ ~46-
.
~ ` .
~SQ423
Slab gel electrophoresis against myoglobin,
chymotrypsinogen and human gamma globulin give the major
fraction a M.W. of 20,500 to 21,500 and the minor pro-
tein a M.W. of about 28,000.
EXAMPLE XI
Carbohydrate Analysis - Phenol-sulfuri_ Acid Test
A standard curve was prepared by treating stock
glucose solution with O.lN aqueous sodium hydroxide and
measuring the W absorption at 485 nm. Runs on the
Pittsburgh 3-2 pili and the CDC B-2 pili indicates sub-
stantially the same amount of carbohydrate content, namely,
1.49 + 0.56% corresponding to 1-2 hexose residues per pro-
tein subunit.
EXAMPLE XII
Phosphorus Analysis
(Method of Chen, et al, Anal.Chem, 28 1756 (1956))
The pili were digested in sulfuric acid and assayed
against a potassium dihydrogen phosphate solution in water
by the ammonium molybdate-ascorbic acid assay. The mean
value for the Pittsburgh 3-2 pilus strains was 0.332 + 0.026%
and for the CDC B-2 strain pili, 0.366 + 0.048%, indicating
2.5 and 2.3 phosphorus atoms per protein subunit respectively.
'
3586
0242 (3)
-47-
10~04Z3
EXAMPLE XIII
Amino Acid Analysis of Type T2 Pilus Crystals
~ . . . ~
(Modified method of Spackman et al, Anal. Chem.
30, 1190 (195B)~.
The analysis was run on a Beckman Spinco Model
120B Amino Acid Analyzer utilizing as internal standard
norluceine and 2-amino 3-guanidino propionic acid. The
protein sample (10 mg.) was hydrolyzed with concentrated
hydrochloric acid at elevated temperatures (6N, 110C),
for 24 hours in evacuated vials (0.025 mm.Hg.) Trypto-
phan analysis was estimated by the spectral method of Bence
et al (Anal. Chem. 29, 1193, (1957)).
aspartic + asparagine 26 isoleucine 9
alanine 23 arginine 8
glutamic + glutamine 21 tyrosine 7
lysine 20 proline 6
glycine 17 tryptophan 4-5
valine 17 histidine 3
serine 14 1/2 cystine 2
leucine 12 methionine 2
threonine 9 phenylalanine 2
Number of amino acids - 200 + 9, m.w. 21, 500 + 1000 daltons.
EXAMPLE XIV
Physical Properties
Solubility
~ .
Pilus crystals, appearing in an electron micro-
! scope as bundles of pilus rods, exist in the crystalline
0242 (37)
-48-
~0504Z3
state about pH 5 . 5 and about pH 9.3. The crystals from
T2 variants start to separate into sin~le pilus rods be-
tween pH 9.3 and pH 10.1. Above pH 10.1 they exist as
single pilus rods. Similarly, the crystals of Tl pili
start to separate into single pilus rods at pH 7.7 and
exist as rods above pH 8.6- i.e. the crystals are entirely
disaggregated. A~ove ahout pH 11.0 T2 pilus rods dis-
assemble into smaller oligomeric units with a sedlmentation
constant of about 5.5.
The pilus crystals are soluble at pH 8.5 in
4 M. aqueous sodium chloride, 50% aqueous sucrose and 20%
saturated aqueous calcium chloride (both by weight). The
crystals are also soluble in urea at 3 M and above.
However, treatment with urea at 3.5 ~q or greater for more
than 2 days leads to denaturation of the GC pili.
Ultracentrifugation
A GC pilus preparation (1 mg./ml.) was prepared
in ethanolamine buffer ~0.147 ionic strength, pH 10.1).
The solution was run at 20 Krpm in a Beckman Spinco Model
E Ultracentrif~uge using an AN-D Rotor. Uncorrected sedi-
mentatin rate S equals 37 svedbergs.
Pilus Rod Dimensions
GC pilus crystals were taken up in ethanolamine
buffer at p~ 10.0 with stacked disc rods of TMV protein
negatively stained and examined in an electron microscope.
; The average diameter of GC pili is 83.4 + 2.3 A.
0242
0241(28)
-49-
~0504~3
EXAMPLE XV
PAT Test
Pilus crystals are suspended in T~S at pH 7.0
at a concentration of between 30 to 60 ug/ml. Mon cloudy
test serum is utilized. Where the test serum is cloudy
it is centrifuged at 30 KG for 30 minutes and the supèr-
nate utilized. Serial dilutions of the serum are prepared
and 0.025 ml. of the serum samples and 0.025 ml. of pilus
crystal suspension are each charged to each micro titre
plate well and the mixture agitated for 30 minutes at
ambient temperature. The wells are then read for crystal
clumping in a dark field microscope.
The wells are then scored on the basis of maximum
dilution which gives crystal agglutination observably
greater than control.
Sample Test
White, New Zealand, female rabbits weighing
from 4 to 6 pounds were injected subcutaneously with
purified pilus preparations from CDCM-2, Pittsburgh 3-2,
CDCT-2, CDC005-2 and Pittsburgh 4-2 strains, mixed 1:1 with
Freund's Incomplete adjuvant and emulsified by syringing
the mixture. About 100 to 200 ~g/Kg. of pili was given in
3 injections ahout 2 weeks apart and the rabbits were bled
1 to 2 weeks after the 3rd injection. Th~_ blood was al-
lowed ~o clot and the serum removed in the usual manner.
The first 3 test sera were then run against pili from 21
N. gonorrhoeae strains and the results set forth in the
Table below.
024]
6955
~ -50-
~ i` 1
~5~)4Z3 1
~o ~ ~ ~ c~ ~
~3 L~LL~
~ tn ~ t~ I t~ _ _ _ _ ~ : ~
~ ~1 .~ .~ t~a t~a~ ~ ~ t~ ~: ~
~1~ Ct~ ~D .D t`-~ .Q ~ ~ ~
t~ eZ _ __ .. . _ _ ~
a '~ tx~ I t t~ .,~ .
O ~ Q tl I tr~ t~ ~ ~ . ~:
tJ ~ ~ t-~J ot co ~o co .
UP:I . __ c~ z
~ ~ [~ a ¦ ; ~
~ c V ~0 CO V l ... _ ~ I ' V - .,, ,,,- .
z ~e _ ~ ~D ~ g ~
. ~ ~ o o u~ ~ _ O
S _ _ - jr __ .
D ~ ~ N ~ ~4 ~n / C .
~T~ o ~
.
~ .~ .
.. . . . .
., ~ ~ . .
. .
, . . ,
. ~ , . . .
.,.... , ..... . ~. , ,.. , ..... ... .. .,....... . . ,.
... . . .. . ; , . . .. ..
. . .
l~S~)4Z3
In accordance with the above procedure a 7 x 7 serotype
pilus agglutination test was run using the pili and the sera
derived from seven desig~ated strains. For ease of interpretation
of the results set forth in Table la below, the cross reaction
readings (pili against sera of the same strain) are normalized to
100 and the other readings adjusted accordingly.
TABLE la
Serum .
Strain Pitts- Pitts- CDC CDC CDC CDC Norfolk
Pilus burgh burghM-2 T-2 339-2 C-2 2-2
Strain 3-2 4-2 _ _
3-2 100 3.1 8.3 0.17 1.6 <0.008 0.52
.
Pittsburgh
: 4-2 0.16 100 <4.2 0.086 0.20<0.008 0.065
CDC M-2 <0.039 <0.024 100 <0.02 0.20 0.13 0.016
_ _ ~
CDC T-2 <0.31 ~0.024 <4.2 100 0.20 <0.008 0.032
CDC 339-2 2.5 <0.024 4.2 ~0.02 100 0.016 0.260
CDC C-2 <0.0039 <0.78 <4.2 0.17 0.10 100 0.065
Norfolk
2-2 <0.31 0.78 <4.2 0.04 0.78<0.008 100
- 50b -
~0504Z;~
F.XAMPLE XVI
Serotyping
The PAT tests of Example XV indicated 3
or 4 strains had pili carrying only one determinant~ This
was confirmed by running sera derived from pili of four
selected strains against the corresponding pili. The
results are set forth in the Table below. The maximum
response has been normalized to 100 to adjust for the
different titres.
Antisera Pittsburgh CDC CDC Pittsburgh Antigenic
Pili _ 3-2 M-2 T-2 4-2 Determinant
Pittsburgh 3-2 100 3 2 1 a
CDC M~2 3 100 1 2 b
CDC T-2 0.4 2 100 2 c
Pittsburgh 4-2 2 2 1 100 d
Serotyping of Strains of Unknown Determinant Com~ition
Pili are grown from the strain~ under test and
run against antisera against the pili carrying the single
determinants a, b, c!and 9. In the PAT Test the pili
showed agglutination with one or more single determinants
as shown in the Table below.
6954
~)504Z~
Strain Seroty~e ATCC
Pittsburgh 3-2 a - - - 3ll49
CDC M-2 - b - 3ll48
CDC T-2 - - c - 3ll50
Pittsburgh 4-2 - - - d 3ll5l
Norfolk 7-2 - - - d
CDC B-2 - b - _
Pittsburgh 6-2 - b - -
CDC C-~ - - - - e
CDC005-2 a b c d 31l52
Norfolk 2-2 - - - - - f
Seattle 1-2 - - c
Seattle 3-2 a - -
Dayton8-2 - b c d
Atlanta 4-2 - b - -
Atlanta 6-2 - b -
Atlanta 10-2 a b -
Atlanta 9-2 - b - d
CDC 339-2 - - - - - - 9
EX~MPLE XVII
~emagglutination by GC ~ili Preparations
General Methods
Type O human blood containing EDTA as anti-
coagulant was obtained from a blood bank. Red Blood cells
were freshly prepared hy washing an aliquot of blood 4 times
with 15 volumes of pH 7.3,0.01 M phosphate-buffered saline,
and making a 3% (by volume~ suspension in the same buffer.
.
Solutions of l.0 mg/ml Pittsburgh 3-2 and CDC-
M-2 pili were made up using the ultraviolet absorbance of
the preparations at 280 nm, corrected for scattering, as
a measure of concentration. Aliquots ~50~) of the l.0 mg/
ml solutions of pili are placed in the first well of a
U-bottom Cooke Microtitre plate, 25 ~ of Tris-buffered
saline ~ 0.02% azide in all other wells, and the pili diluted
with a 25 ~ hand operated micro diluter out through the
12th well.
3708
3709 (11)
-52-
10~ 23
Tris buffered saline or saline (25~) and 25~ 3~
red blood cell suspension is added to each test well, the
plate agitated gently to mix, and placed at 4C. Results
are read after 1-2 hours with the ai.d of a light box.
Results
The degrees of hemagglutination observed are:
5+ No cell pel].et or clumps; even red color
in well
4+ Trace of cell pellet
3+ Small cell pellet with very distinct
fringe of cell clumps
2+ Distinct cell pellet with a fringe of
; moderate number of clumps
+ Distinct cell pellet with a few clumps
up the sides of the well.
Titres for both CDCM-2 and Pittsburgh 3-2 pili were done
on two different occasions and are shown in the Table below
and constitute control tests.
3709 ~ -53-
105~)~23
I ~
,~, ~ u~ + + + + +l o o o o o O
o ~
~ ~ 6E E ~ EEi E
O ~ i~
r~ 1~ `J ~. o~ Cr~
~r~ o 1 ~1 C~l ~ ~,~ .. .. . .
14 c~ ~ 3 o~ o o o
a) ~) o~ rl
+ + + ~ + O O O OO O O
a) t:
.,
,~ ~1 ~I E ` ~1 ~~1 ~
~,~ ~ E ~ -- ~I E E E e
U~ ~ ~ ~, ~ ~ ~ ~~,
~ ~ ~ ~ e E E E3-- ~~ ;~ ~
ri ~J ~'7 O ~ ~ ~ ~ ~ Lr0~
F. F ~ ~ 0d` ~I
O ~ ~ r,~ ~ 0 ~ -
,~ r~ ~C` O O
z
¢
;S
o I ~ + + + + + + + +1
x æ ~
:~ o ~ U~
Q) ~
E~ aJ ,1
V~ ~ ~
H ~ +
,_, + + + ~')
+1 o o o o o o .
F. ~ E ~ -- -- -- -- -- -- -- ~ E
~1
r~ rl ~ . r~ O r~
~rl O ~r1 ~ ~ r~ ~D ~ ~i 0 0 ~ ~ O
5 ~ ~ ~ o:) ~;t ~ ~1 0
r~
~I r, '7 ~ u~ ~D1~ CO O~ O
~ ~1 ~ ~
~(~50423
Inhibition of Hemagglutination:
Removal of Ab-A~ Complex from Antiserum-Pili
Mixtures before Titering for Hema~glutination
Methods:
To eliminate background hemagglutination by sera
alone 0.1 ml of undiluted washed red blood cells (RBCs) were
added to 1 ml portions of antisera in glass round-bottom
centrifuge tubes. The tubes were capped tightly with para-
film and placed upright in a rack on a Yankee Rotator at 4C.
After 1 hour-10 minutes, the sera were spun at 2400 rpm in
a refrigerated International Centrifuge, and tested for
hemagglutination ability. The sera were further clarified
by centrifugation at 12,000 G in a Sorvall RC-2 centrifuge.
Absorption of background hemagglutination factors
at various dilutions pre and post RBC treatment of the anti-
serum is summarized in the following Table.
Serum Pre- Post-
Well Dilution Adsorption Adsorption
l 1/7 4+ 2+
~ 2 l/4 3+
`, 3 l/~3 3f +
4 l/16 3+ 0
S 1/32 2+ 0
6 1/64 + 0
7 1/12~ 0
8 1/256 0
9 1/512 o
1/1024 0
11 1/2048 0
12 1/4096 0
3115
10~04;~3
Removal of Ab-Ag Complexes:
_ _ . _ _ _
Serial dilutions of Rsc-adsorbed rabbit pre-
immune and anti-CDCM-2 sera prepared as above were made in
saline. Aliquots (0.1 ml) of each dilution were added to
0.1 ml of lOOr/ml M-2 pili in 1.5 ml Microfuge tubes. The
tubes were incubated upright on a Yankee Rotator for 1 hour
at 37C. They were then spun 1 minute in the Beckman
Microfuge. Slightly over half of the supernatant was re-
moved and 50~ of each antiserum dilution placed in each of
2 wells of a Microtitre plate. A 25~ aliquot of 3% RBCs in
phosphate buffered saline was added, the plate gently
agitated to mix and left at 4 for 1 hour.
The results are set forth in the Table below.
Where in place of CDC-M-2 pili other pili are utilized,
similar results are obtained with sera containing antibodies
against said pili.
;Conc. of Serum
A~_,~n~M-2 ~ili
During Incubati~n WithHemagglutination Titres In
50 y/ml M-2 Pili Duplicate Rows
_ G H
1/10
1~2~ o 0
1/40 0 0
1/80 + +
.; 1/1~ + +
l/3~0 2+ 2~
1/640 3+ 3+
1/1280 2+ 2+
l/2~60 2~ 2
1/10 P~e-Immune
Rab~it 2+~3+ 2+/3+
*Note that the Anti CDCM-2 Serum
Alone has a + Hemagglutination
at 1/8 Diluti~n
695Ç -55-
1()SO423
EXAMPLE XVIII
~luman Test
T2 Pilus crystal from Pittsburgh 3-2 organisms
were prepared, preserved with 0.01~ merthiolate and emulsi-
fied with Freund's incomplete adjuvant. Human male volun-
teer test subjects were injected with the pilus suspension.
Subject B recei~ed three injections of 2.2 ~g/kg, 2.2 ~g/Kg
and 55 ~g/Kg at intervals of 2 weeks. Subject R received
three injections of ll ~g/Kg, ll ~g/Kg and 55 ug/Kg at the
same intervals. The PAT titres of hoth subjects rose to
100 after the first injection and to over 200 over the next
4 months.
The subjects were challenged by the intraurethral in-
troduction of a predetermined number of organisms of the
same strain, the virulence of which had been previously tested.
The same organism was administered to three unimmunized con-
trol subjects - T, S and M.
The test results are set forth in the Table below:
Resisting ancl Infecting Doses of Pittsburgh 3-2
Gonococci for Pilus-Immunized and Non-Immunized
Human Subjects
4 Original Subjects *Immunized
Infectious Dose Subjects Infected Subjects ~esisting
8 x 101 T B*, R*, S
3 x 102 B*, R*, S
8 x 103 S B*, R*
3 x 104 B*, R*
l additional subject
8 x 10l M
3 x 102 M
8 x 102 M
l x 103 M
6957
-56-
1050423
In accordance with the above procedures, in
place of merely using pili with a single determinan-t a
composite dose of pili carrying all determinants may be
used.
Analysis of Results
Probability analysis of the foreqoing results
shows that the ID50 f a nonimmune subject is 50 x 102 or-
ganisms and 2.0 x 104 for immune subjects.
Other experimental work has shown the probability
of a male being infected by an infected female is about 30%
caused by about 250 organisms being introduced into the male
urethral tract during intercourse.
The results of the tests of the present invention
indicate that the probability of infection of a male by an
infected female during intercourse falls from 30% to 0.86
as e result of immunization to a PAT titer of 100 to 200.
-57-
