CA1050423A - Gonococcal pili, processes for the preparation thereof - Google Patents

Abstract

GONOCOCCAL PILI PROCESSES FOR THE PREPARATION
THEREOF AND THE USE THEREOF FOR THE DETECTION
OF AND PREVENTION OF INFECTIONS CAUSED BY
NEISSERIA GONORRHOEAE

CHARLES C. BRINTON
JOHN McMICHAEL

ABSTRACT OF THE DISCLOSURE

There are provided a crystalline and single rod produces derivable from the Pili of Type 1 and Type 2 Neisseria gonorrhoeae organisms. There are provided methods of growing said organisms to produce the maximum yield of Pili and procedures for purifying said Pili to produce said crystalline material. There are further provided methods of utilizing said Pili to determine the presence, in a system infectable by N. gonorrhoeae organisms, of antibodies to the Pili of said organisms, and methods of serotyping said Pili. There is also provided a mode of utilizing said crystalline material to provide a substantial degree of immunization to infection by N. gonorrhoeae in mammalian systems susceptible to such infection.

Description

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.

, ' ~

~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.

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.

' .:

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~

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 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 ~(~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 _ 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.

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 ~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
.

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) 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.

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.

3583 (14) 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, 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.

-:~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~) 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 ~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 0570 (7) ~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) ~()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 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 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 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 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 0558 (28) ~()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 ~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) ~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.
', 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 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.

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.

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.

3589 ~29) ~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 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).

6717 (20) 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 6953 (19) ~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.

2879 t24) 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 3587 (Z5) ~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 0240 (17) ~ 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.

:

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) 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 3586 (12) , , 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.
' 0242 (3) 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) ~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.

0241(28) ~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]

~ -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.

~)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 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.

3709 (11) 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

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

1~2~ o 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

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.

Claims (19)

CLAIMS:

1. A process for the preparation of pilus crystals from N. gonorrhoeae comprising the steps of:
a) suspending the culture of piliated N gonorrhoeae cells in an aqueous medium at a pH below pH 9.2 and above pH 5.5, b) separating the soluble and the insoluble portions of said suspensions from each other, c) suspending the insoluble portion from step (b) in an aqueous medium having a pH range of between pH 7.7 to pH 11.0, d) separating the insoluble and the soluble portions of the suspension of step (c) from each other, e) lowering the pH of the soluble portion derived in step (d) to below pH 9.2 and above pH 4 to form pilus crystals, and f) separating the thus formed pilus crystals from the said aqueous medium.

2. A process according to Claim 1 wherein the method of separation of steps (b), (d) and (f) comprises fil-tration wherein the insoluble portion of the suspension is maintained upon the surface of the filtering medium and the soluble portion of the suspension passes through the filtering medium as the filtrate.

3. A process in accordance with Claim 1 wherein the method of separation in steps (b), (d) and (f) is centri-fugation in which the insoluble portion of the suspension is compressed as a pellet and the soluble portion of the sus-pension is a supernatant liquid over said pellet which is re-movable therefrom.

4. A process according to Claim 3 wherein the suspension of step (a) contains culture of type T2 and is b) centrifuged in a low speed centrifuge to yield a pellet and a supernate, c) the pellet of step (b) is suspended in an aqueous medium having a pH of between pH 9.3 and pH 11.0, d) the suspension of step (c) is centrifuged in a low speed centrifuge to yield a pellet and a supernate, e) the pH of the supernate of step (d) is lowered to below pH 9.2, f) the product of step (e) is subjected to low speed centrifugation and the supernate thus formed is discarded to yield the pilus crystals in the residual thus formed pellet.

5. A process in accordance with Claim 4 wherein the pellet of step (d) is i) re-suspended in the medium of step (c), ii) the re-suspension is subjected to low speed centrifugation to yield a supernate and a pellet, and the supernate combined with the supernate of the first centrifugation of step (d), and the combined supernates subjected to high speed centrifugation to yield a high speed centrifugation supernate which is subjected to the procedures of step (e).

6. A process in accordance with Claim 5 wherein the N. gonorrhoeae cells are of the type T2 a) the piliated cell culture is suspended in a medium of pH 8.0 to pH 8.9, b) the centrifugation of step (b) is run at between 1 KG and 12 KG, c) the re-suspension of step (c) is carried out in an aqueous medium having a pH of between pH 10.0 and pH 10.4, d) the low speed centrifugation is run at between 1 KG and 12 KG, I) the re-suspendate of step (d) has a pH of between pH 10.0 and pH 10.4, II) the centrifugation of the re-suspendate is run at between 1 KG and 12 KG, and III) the combined supernates are high speed centrifuged at between 12 KG and 70 KG
e) the combined supernates from the high speed centrifugation are lowered in pH by dialysis against a buffer having a pH at 20°C of pH 8.2 to pH 8.7, f) subjecting the dialysate to centrifugation at between 1 KG to 10 KG to yield a supernate and a pellet of pilus crystals.

7. A process according to Claim 3 wherein the sus-pension of step (a) contains a culture of Type T1 and is b) centrifuged in a low speed centrifuge to yield a pellet and a supernate, c) the pellet of step (b) is suspended in an aqueous medium having a pH of between pH 8.6 and pH 11.0, d) the suspension of step (c) is centrifuged in a low speed centrifuge to yield a pellet and a supernate, e) the pH of the supernate of step (d) is lowered to below pH 7.7, f) the product of step (a) is subjected to low speed centrifugation and the supernate thus formed is discarded to yield the pilus crystals in the residual thus formed pellet.

8. A process in accordance with Claim 7 wherein the pellet of step (d) is i) re-suspended in the medium of step (c), ii) the re-suspension is subjected to low speed centrifugation to yield a supernate and a pellet, and the supernate combined with the supernate of the first centrifugation of step (d), and the combined supernates subjected to high speed centrifugation to yield a high speed centrifugation supernate which is subjected to the procedures of step (e).

9. A process in accordance with Claim 8 wherein a) the piliated cell culture is suspended in a medium of pH 7.0 to pH 7.7, b) the centrifugation of step (b) is run at between 1 KG and 12 KG, c) the re-suspension of step (c) is in an aqueous medium having a pH of between pH 8.6 and pH 10.4, d) the low speed centrifugation is run at between 1 KG and 12 KG, I) the re-suspendate of step (d) has a pH of between pH 8.6 and pH 10.4, II) the centrifugation of the re-suspendate is run at between 1 KG, and 12 KG, and III) the combined supernates are high speed centrifuged at between 12 KG and 70 KG, e) the combined supernates from the high speed centrifugation are lowered in pH by dialysis against a buffer having a pH at 20°C of pH 5.5 to pH 7.7, f) subjecting the dialysate to centrifuga-tion at between 3 Krpm to 10 Krpm to yield a supernate and a pellet of pilus crystals.

10. A process according to Claim 1 wherein in step (a) the pH is lowered to below pH 7.7 but above 5.5 whereby pilus crystals derived from Type T1 and Type T2 organisms are maintained in the insoluble phase.

11. A method of testing for the presence of anti-bodies to N. gonorrhoeae pili which comprises:
a) treating a member selected from the group consisting of G.C. pilus crystals and single rod G.C. pili with a sample of serum suspected of con-taining said antibodies thereto, and b) observing the occurrence of agglutination of said crystals or said rods, the presence of said agglutination indicating the presence of the antibodies in the serum.

12. A method of Claim 11 wherein the occurrence of the agglutination is observed with a dark field microscope.

13. A method of Claim 11 for determining the rela-tive concentrations of antibodies in said test serum which comprises diluting said test serum with predetermined amounts of an immunologically inactive diluent and determining the dilution at which no observable agglutination occurs.

14. A method of Claim 13 in which the diluent is tris or phosphate-buffered saline of pH between pH 7.0 and pH 8Ø

15. A method of testing for the presence of pre-determined determinants on the antibodies against the pili of N. gonorrhoeae in blood fluids suspected of containing such antibodies, said blood fluids being free of red blood cells by means of agglutination inhibition comprising the steps of:
a) charging a predetermined amount of said blood fluid to an aqueous medium containing pili of N, gonorrhoeae bearing predetermined antigenic determinants, b) incubating the mixture of step (a) to form a precipitate and a supernate, c) separating the precipitate from the supernate, d) mixing said supernate with a charge of washed red blood cells and incubating said mixture wherein the presence of unagglutinated sedimented red blood cells indicates the presence of antibody determinants in the blood fluids corresponding to correlated antigenic determinants on the pili.

16. A method of serotyping a strain of N. gonorrhoeae comprising the steps of:

a) culturing the species and isolating the pili therefrom, b) treating said pili with predetermined sera containing antibodies against a single member of the group of antigens of N. gonorrhoeae pili carrying the antigens determinants designated as a,b,c and d, wherein resultant agglutination of the pili by a specific pre-determined serum indicates that said strain from which the test pili are derived carries the particular antigenic determinant against which said serum is designated as con-taining antibodies wherein the antigenic determinants are those whose carriers would be agglutinated by contact with antibodies against pili derived from strains of N. gonorrhoeae designated Pittsburgh 3-2 (ATCC31149), CDC-M-2 (ATCC31148), CDC-T-2 (ATCC31150), and Pittsburgh 4-2 (ATCC31151).

17. G.C. pilin, being a phosphoglycoprotein isolat-able from the pili of cultures of Type T2 N. gonorrhoeae, said phosphoglycoprotein being a peptide chain of 200 ? 9 amino acids having between 2 and 3 phosphate groups and between 1 and 2 hexose sugars covalently bound to said chain, said chain having a molecular weight of 21,500 ? 1000 daltons, said protein being substantially soluble in aqueous media at pH
greater than 10.1 and substantially insoluble in aqueous media at pH values less than 8.3 at 20°C, when prepared by the process defined in Claim 4 or by an obvious chemical equiva-lent.

18. A composition of Claim 17, wherein the compo-sition is a crystalline composition, when prepared by the process defined in Claim 4 or by an obvious chemical equiva-lent.

19. A composition of Claim 17, wherein the ratio of amino acids in said composition is:

when prepared by the process defined in Claim 4 or by an obvious chemical equivalent.