EP0216841A1

EP0216841A1 - Antigenically active protein and its use in the diagnosis of gonorrhoea

Info

Publication number: EP0216841A1
Application number: EP86901939A
Authority: EP
Inventors: Nicholas John Parsons; Harry Smith
Original assignee: BTG International Ltd; National Research Development Corp UK
Current assignee: BTG International Ltd
Priority date: 1985-03-18
Filing date: 1986-03-13
Publication date: 1987-04-08
Also published as: WO1986005592A1; GB2172704B; GB2172704A; GB8606293D0

Abstract

Un matériau protéinique ayant une masse moléculaire d'environ 20kDal isolé d'un extrait de cholate de sodium des vésicules de la membrane extérieure d'une souche de Neisseria gonorrhoeae comprend un déterminant de la résistance des N. gonorrhoeae aux attaques mortelles par des phagocytes. Il neutralise l'antisérum de la souche résistante aux phagocytes dont il est isolé, et s'est révélé utile dans des tests de dépistage de blennorragie clinique humaine. Il est également utile comme vaccin.A proteinaceous material having a molecular mass of approximately 20kDal isolated from a sodium cholate extract of the outer membrane vesicles of a strain of Neisseria gonorrhoeae comprises a determinant of the resistance of N. gonorrhoeae to lethal attack by phagocytes. It neutralizes the antiserum of the phagocyte-resistant strain from which it is isolated, and has been found useful in screening tests for human clinical gonorrhea. It is also useful as a vaccine.

Description

ANTIGENICALLY ACTIVE PROTEIN AND ITS USE IN THE DIAGNOSIS OF GONORRHOEA Background of the invention

1. Field of the invention

This invention relates to an antigenically active protein which can be extracted from Neisseria gonorrhoeae, and to its use in a method of diagnosis of clinical gonorrhoea in human patients.

2. Description of prior art

The causative agent of gonorrhoea is the bacterium Neisseria gonorrhoeae. The genus Neisseria are diplococci which attach themselves to mucosal cells. Two species, N_. gonorrhoeae and II. meningitidis, the agent of meningococcal meningitis, also known as epidemic cerebrospinal fever, are important pathogens. There are other, non-pathogenic, species of the genus.

The diagnosis of gonorrhoea is not always straightforward, particularly in women, and the examination of stains of cells or the use of fluorescent antibody methods does not lead to reliable results.

N.J. Parsons et al. , Journal of General Microbio¬ logy 118, 523-527 (1980) have attempted to assess the diversity of immunotypes of strains of N_. gonorrhoeae. In 21 strains assessed there appeared to be at least 8 different immunotypes. A problem in improving the diagnosis of N_. gonorrhoeae is therefore to find an antigen or antibody which is common to most clinically occur¬ ring strains.

The N_. gonorrhoeae bacteria invading the mucosal tissue are rapidly attached by leucocytes which attempt to kill them by phagocytosis. However, some gonococci survive and grow intra- cellularly within the cells of the phagocytes. Studies have been in progress at the University of Birmingham, England, for some years to identify the determinants responsible for this ability of gonococci to resist killing by phagocytes. At first there was thought to be an association of the degree of pilation (amount of hairlike pile on the bacterial cell surface) with this resistance to killing by phagocytes. Subsequently, the determinant of the resistance of a strain of Neisseria gonorrhoeae, BS4 (agar), to killing by human phagocytes was shown to be not pili but to reside in purified outer membrane vesicles (OMV). This conclusion was reached on the basis that the OMV neutralised an antiserum raised against whole, live gonococci of the strain BS4 (agar), which abolished the resistance of this strain to intracellular killing by human phagocytes; and also by the capacity of an antiserum raised against the OMV to similarly nullify the intracellular resistance of strain BS4 (agar), see N.J. Parsons et_^ al., Journal of General Microbiology 127, 103-112 (1981). Three proteins, A, B and C, in the purified OMV from BS4 (agar) were thought to be associated with its resistance to phagocyte killing, since they were not present in the OMV from a closely related, phagocyte- susceptible strain, BSSH, See N.J. Parsons et al., Journal of General Microbiology 128, 3077-3081 (1982). The three proteins had molecular masses of about 35, 47 and 74 kDal.

Strain BS4, referred to herein, is resistant to killing by human phagocytes. It was obtained from a laboratory strain BS (Kellogg type 2, small colony-forming, pilated) by selecting gonococci thereof which survived and grew within phagocytes, growing colonies of these gonococci, passaging them four times in plastic chambers implanted subcutaneously in guinea pigs and then growing them by minimal culture on agar. BS4 has long been made available by Professor H. Smith, Department of Microbiology,

University of Birmingham, England and cultures have accordingly been supplied to researchers. In order to ensure long-term availa¬ bility of the strain it has been deposited, on 7th October 1985 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the National Collection of Type Cultures, 175 Colindale Avenue, London, NW9 5HT, England. The deposit number is NCTC 11922.

Strain BSSH, referred to herein, is susceptible to killing by human phagocytes. It was obtained from the same laboratory strain BS by selecting gonococci thereof which were killed by phagocytes (these being the majority and recognisable from the survivors by an optical characteristic known as single highlight - the survivors gave a double highlight) and growing colonies on agar. Recently, J.G. Cannon et al. , Infection and Immunity 43,

994-999 (1984), extracted outer membrane factions from II. gonorrhoeae using "Sarkosyl" (a surfactant) or lithium acetate and raised hybridomas against the fractions. A monoclonal antibody "H8" bound to the pathogenic Neisseria species, II. gonorrhoea ΪI. meningitidis, and II. lactamica, but not to non-pathogenic species of Neisseria. It was shown that the H8 antibody bound to an outer membrane protein of molecular mass about 20 kDal. Summary of the invention

The present inventors have isolated a proteinaceous material from the outer membrane vesicles of the phagocyte-resistant strain BS4 and have shown it to react strongly with an antiserum to the phagocyte-resistant organism.

For the purposes of the present invention, the proteinaceous material can be defined as that obtainable from the strain of Neisseria gonorrhoeae BS4 (NCTC 11922), which strain is resistant to being killed by human phagocytes, said proteinaceous material being obtainable by (but not necessarily obtained by) extraction of the outer membrane vesicles (OMV) in 10 g/litre sodium cholate in an aqueous buffer at pH 9.5 and removing undissolved OMV, fractionating the extract and selecting a fraction comprising (consisting of or including) a proteinaceous material having a relative molecular mass of about 20 kDal and which neutralises antiserum to the phagocyte killing-resistant strain BS4 of Neisseria gonorrhoeae. This is a definition of the proteinaceous material per se, regardless of how it is actually obtained, e.g. by extraction with a different surface active material from sodium cholate. Indeed, it is believed adequate to define the material as a protein of said BS4 strain of relative molecular mass about 20 kDal and which neutralises antiserum to BS4. The above-defined proteinaceous material is useful as an antigenically active component in an immunological test for the diagnosis of clinical gonorrhoea. (For the purposes of the invention any infection by Neisseria gonorrhoeae which gives rise to symptoms associated with the disease is regarded as clinical gonorrhoea). Tests have shown that the reaction of the protein- acous material with patients' sera gives a very high degree of specific neutralisation of sera from patients having clinical gonorrhoea, with virtually no false positives or false negatives. Accordingly the invention further provides a method of of assay of^" human clinical gonorrhoea which comprises (1) incubating a sample, suspected of containing N. gonorrhoea or antibodies to N. gonorrhoea, from a body fluid of a human patient, (a) with the above-defined proteinaceous material or (b) with antibodies to said proteinaceous material, respectively, and (2) detecting whether neutralisation between the proteinaceous material and the antibodies has taken place. Preferably an ELISA is used.

The invention includes, of course, a kit for use in such a method of diagnosis comprising (a) the proteinaceous material or (b) antibodies thereto as one component, usually insolubilised, and labelled antibodies as a second component. The kit may also include materials for use as a solid phase or in conjunction with the labelling and so forth, i.e. any materials normally present in EIA, fluorescent, or RIA diagnostic kits. A surprising element in the present invention is that the proteinaceous material interacted with sera from many different patients, giving no false readings. This indicates that the 20 kDal protein is an antigen common to many different strains of N_. gonorrhoeae, as distinct from being merely an antigen specific to BS4. This common antigenicity could not have been predicted.

Further, since no false positives were obtained, it is unlikely that the 20 kDal protein could be common to II. meningitidis. Antibodies to the latter organism are very widely carried by many people who show no symptoms of the meningitis disease and it is therefore predicted that antibodies to N. meningitidis would be present in patients of the control group tested for II. gonorrhoeae using the proteinaceous material of the present invention. Since the control group gave no such interaction, it is considered that the 20 kDal protein is specific to N. gonorrhoeae. It follows, therefore, that it must differ from the 20 kDal protein located on a gel by Cannon e__ al. , loc. cit. , since their monoclonal antibody (H8) to that protein interacted with _N. meningitidis as well as with N_. gonorrhoeae. Brief description of the drawings

Figure 1 is a graph relating material obtained from fractiona- tion of the cholate extract to a measure of the amount of protein thereby obtained; and

Figure 2 is a photograph of SDS-PAGE in five lanes, on OMV, sodium cholate extract and various fractions of sodium cholate extract. Description of the preferred embodiments

Preferably the fraction selected comprising proteinaceous material is that hereinafter designated 1b, defined as the fraction which gives a shoulder to the low molecular mass side of the UV absorption peak at 279 nm during continuous fractionation of the cholate extract by gel filtration. Fraction 1b contains proteins of approximately 35 and 60 kDal and lipopolysaccharide as well as the desired 20 kDal product. The 20 kDal product can be further purified by gel filtration and, if necessary, ion-exchange and the invention therefore includes fraction 1b when thus further purified and components of fraction 1b which also show the antiserum neutra¬ lising activity.

Inasmuch as it has been shown that only the 20 kDal protein comprises the phagocyte killing-resistant antigen, it is preferable to exclude other proteins so that such other proteins constitute no more than (say) 5 or 10% by weight of total proteinaceous material and this is achievable by gel filtration(s) .

The proteinaceous material or antibodies thereto can be used in any convenient method of assay (detection or measurement) of gonorrhoea diagnosis. Conveniently sandwich assay is used with the proteinaceous material as solid phase antigen, but competitive and inhibition assays are, of course, alternatives. Labelling is . . .

- 6 - preferably by any of the enzymes commonly used for this purpose such as horseradish peroxidase or alkaline phosphatase, but radiolabelling and fluorescent labelling are also possible in appropriate cases. Monoclonal antibodies can be raised against the proteinaceous material of the invention by the now well known methods using for example, mice spleen cells to fuse with a myeloma, for example mouse myeloma cells. Hybridomas from II. gonorrhoeae are not novel, but it is believed that hybridomas secreting antibodies to the proteinaceous material of the invention are novel and they form part of the present invention. Such hybridomas can of course readily be obtained by the usual screening methodology, using the proteinaceous material of the present invention to detect the desired antibodies by for example, Western blotting. A preferred form of assay of the invention is the sandwich assay wherein either the proteinaceous material or antibodies thereto are insolubilised and after incubation with the liquid sample, the product of the interaction is incubated with labelled anti-human immunoglobulin, unbound label is separated from the insoluble material and any labelled insoluble material is detected or measured. The sandwich assay is illustrated schematically below

(PR = above-defined proteinaceous material,

MCA = monoclonal antibodies thereto raised in mice,

* = enzyme, radio-, fluorescent or luminescent label)

h-PR — anti-human IgG

antibody insolubilised labelled second to be antigen antibody detected

anti-human IgG

insolubilised antigen to labelled antibody be detected antibody

(This assay has the advantage that it is not necessary to prepare the protein itself, merely antibody to it.)

Another form of assay is a competitive or displacement assay wherein either (1) the proteinaceous material or (2) antibodies thereto are insolubilised and incubated (a) with the liquid sample and (b) (1) with an insufficiency of labelled antibody or (2) with an insufficiency of labelled proteinaceous material, respectively, the unbound label is separated from the insoluble material and the label is detected or measured. The term "competitive" denotes simultaneous incubation of the competing partners, "displacement" that one is reacted before the other, but that the equilibrium between them is displaced by subsequent addition of the other. This type of assay is illustrated schematically below using the same symbols as above:

MCA to PR - anti-mouse IgG

sample)

Insolubilised antigen Limited amount of a and antigen to be labelled double antibody detected in competition (preferably pre-prepared as such)

in sample)

Insolubilised antibody Limited amount of a and antibody to be labelled antigen,

*_ detected in competition e.g. PR-biotin-streptavidin Many variants on the above-illustrated schemes well known to those in the immunoassay art can be employed.

Kits of the invention will usually include all the above- mentioned components except the sample together with appropriate standards. Polyclonal antibodies can of course be substituted for monoclonal antibodies with the usual disadvantages.

The 20 kDal proteinaceous material is also useful as a component of a vaccine. It can therefore be formulated, for this purpose, with an adjuvant or carrier for example of aluminium hydroxide, especially "Alhydrogel" (Registered Trade Mark), or aluminium phosphate. A dosage in the range of 5 to 500 micrograms of 20 kDal protein is likely to be employed.

The following Examples illustrate the invention. Example 1 shows the preparation and properties of the proteinaceous material "Fraction 1b". Example 2 illustrates its use in diagnosis.

Example 3 shows that the monoclonal antibody (H8) of Cannon ejt_^ al. , supra is not anti to a determinant of resistance to killing by phagocytes. Example 4 describes further purification of fraction 1b. Example 5 provides further evidence that the 20 kDal protein of the invention comprises the determinant of phagocyte killing- resistance. Example 6 describes the preparation of hybridoma cell lines. EXAMPLE 1 METHODS Extraction of crude outer membrane vesicles (OMV) with sodium cholate

11

OMV were extracted from strain BS4 (agar) (100 ml; 10 bacteria ml ) in 1 M lithium chloride as described previously (Parsons et al. , loc. cit. 1982). Sedimented OMV were resuspended in glycine/NaOH buffer, 0.1 M, pH 9.5 (approximately 20 ml) contain¬ ing sodium cholate (1% w/v) and agitated in 50 ml Erlenmeyer flasks with glass beads (3 mm diameter; 2 beads ml ) on an orbital shaker (60 cycles rnin^" ) for 1 hour at 37°C. Undissolved OMV were removed by centrifugation (100,000 g, 1 hour, 4 C) and the extract was fractionated immediately. Fractionation of cholate extract

The extract (3-4 ml) containing about 1000 yg protein ml and derived from 5 x 10 11 gonococci ml-1, was applied to a column (1 m long, 15 mm diameter) of "Sephadex G75" (Pharmacia) and eluted with the same buffer as that used for extraction (flow rate, 10 ml h ). Elution was followed by monitoring the absorbance at 279 nm, A„__q. Appropriate fractions (each of 2 ml) were pooled and concentrated by negative pressure dialysis to the original volume of the extract applied to the column. Extraction of lipopolysaccharide (LPS)

The method of 0. Westphal and K. Jann, Methods in Carbohydrate

Chemistry 5_, 83-89 (1965) was used. Approximately 4 g wet weight

13 of strain BS4 (agar) (approximately 1 x 10 organisms) were suspended in 20 ml 4.8 M phenol, vortex-mixed with 20 glass beads for 10 minutes at room temperature and then heated to 60 C for 10 minutes. The mixture was cooled to 4 C and the bacteria removed by centrifugation (10,000 g, 10 minutes, 4 C) . The top aqueous layer was retained and the middle and lower layers were re-extracted with distilled water, volume equal to the aqueous layer removed. The combined aqueous phases were pooled, centri- fuged (20,000 g, 10 minutes, 4 C) and dialysed against running tap water at room temperature for 2-3 days. The retentate was treated with 3 volumes of ice-cold dry acetone. The LPS precipitate (approxi¬ mately 50 mg) was collected by centrifugation (4000 g, 10 minues, room temperature) , redissolved in distilled water at a concentra- tion equivalent to 2 x 10 11 gonococci ml-1 and dialysed against distilled water for 14-15 hours at room temperature. The product was not purified further; it contained some protein and probably small amounts of nucleic acid and peptidoglycan. SDS-PAGE

The method of U.K. Laemmli, Nature 227, 680-685 (1970) was used, with modifications described by Parsons et al. , loc. cit.

(1982). For protein profiles, the samples (50 μl) applied were of concentrations equivalent to 5 x 10 11 gonococci ml-1, thus relating the results to the biological tests which were done on the same concentrations or simple multiples of them. Cytochrome c, horse myoglobin, bovine chymotrypsinogen A, ovalbumin, BSA and ovotrans- ferrin (12, 17, 26, 45, 66 and 77 kDal respectively) were used as standards to indicate approximate molecular weights of protein. For separating the components of fraction 1b, 50 μl aliquots of the

12 fraction, equivalent to 2 x 10 organisms, were electrophoresed in each of seven channels on a 12.5% (w/v) polyacrylamide gel as described by Parsons et al., loc. cit. (1982). Two regions

(approximately 2 cm) of the gel corresponding roughly to proteins of 20 and 60 kDal, and the region corresponding to the principal outer membrane proteins (around 35 kDal) , were located by reference to a previous analytical gel; also two channels of the gel were stained in retrospect to confirm the location of the three regions. These regions from the remaining five channels were excised and each was macerated in an equal volume (2-3 ml) of phosphate buffered saline (PBS) with a pestle and mortar. The gel was removed by centrifugation (2000 g, 5 minutes, room temperature) and the extract concentrated by negative pressure dialysis to the original volume (5 x 50 μl) that had been applied to the gel. Thus, the whole volume was equivalent to 1 x 10 13 organisms and 1/10th was used to absorb the antiserum against whole gonococci (see below). For controls, 2 cm portions of the gel preceding the marker dye were treated similarly.

Assays of 2-keto-3-deoxyoctonate (KDO) , protein and carbohydrate Assays for KDO and protein (with BSA as standard) were as described by Parsons et al. , loc. cit. (1982). Carbohydrate was estimated by the anthrone method using glucose as standard. Indirect tests for the determinant of resistance to intracellular killing; pretreatment of strain BS4 (agar) with unabsorbed and absorbed antisera before phagocytosis tests

In summary, an antiserum against live organisms of strain BS4 (agar) drastically reduced the capacity of this strain to survive intracellularly in human phagocytes but the relevant antibody could be neutralised by prior absorption with extracts or with OMV containing the putative determinant of resistance to intracellular killing (Parsons t_ al. , loc. cit. 1981, 1982).

Rabbit antiserum to strain BS4 (agar) was raised by multiple intravenous injections of live organisms in Trypticase Soy Broth. For absorption with crude OMV (Table 1), equal volumes of antiserum and suspension of OMV (equivalent to 5 x 10 11 gonococci ml-1) were mixed and allowed to stand overnight at 4°C. Absorption with the

LPS preparations was done similarly with concentrations equivalent

12 -1 to 2 x 10 gonococci ml . The cholate extract of OMV and its fractions from the "Sephadex" column (Figure 1, Table 1) were

11 -1 mixed at a concentration equivalent to 5 x 10 gonococci ml with an equal volume of antiserum, kept at 4 C for 5 hours and dialysed overnight at 4 C against PBS to remove free sodium

12 cholate. The concentrated eluates (25 μl, equivalent to 1 x 10 gonococci) from each area of SDS-PAGE gels (Table 2) were mixed with antiserum (100 μl) and allowed to stand overnight at 4 C.

7 -1 Gonococci [BS4 (agar), 2 x 10 ml ] were incubated

(1 hour, 37°C) in Parker medium 199 containing 10% (v/v) heated (56 C, 30 minutes) human serum (HHS) obtained from defribinated blood and 1% (v/v) rabbit antiserum against live strain BS4 (agar) or the corresponding concentration of absorbed antiserum (see above). Antiserum was omitted from control samples. After incubation, the medium was then removed by centrifuga¬ tion (2000 g, 3 minutes, room temperature) and after washing three times with fresh medium (1 ml) without antiserum, the gonococci were suspended at a concentration of 2 x 10 ml^" in Parker medium 199 containing HHS (10%, v/v) for mixing with phagocytes in the Leighton tubes.

As formerly (Parsons ___ ____ _t loc. cit. 1981, 1982), phago- cytosis tests used strain BS4 (agar) pretreated with antiserum absorbed with a single concentration of the fraction containing the putative determinant. This concentration was equivalent to a

11 standard number (5 x 10 ) of organisms extracted. The requiremer for many replicates in the phagocytosis tests and the need to repeat each experiment several times to establish a single point (Parsons et al. , loc. cit. 1981), precluded routine examination of several dilutions of the standard concentrations. Infrequently, dilutions were tested, and sometimes when loss of material could have occurred in a procedure, such as in the preparation of the LPS and gel eluates, materials were examined at concentra¬ tions 2-4 times the standard level (see above) .

The other test for the determinant of resistance to intra- celluar killing was to raise an antiserum against the putative determinant and see if it abolished the capacity of strain BS4 (agar) to survive intracellularly, as did the antiserum against whole organisms (Parsons et_ al. , loc. cit. 1981, 1982). Antisera for this purpose were raised against fraction 1b from the Sephadex column (Figure 1) as follows. Rabbits received three subcutaneous injections of fraction 1b (each equivalent to 10 gonococci) at 10-day intervals. The first two were given emulsified with an equal volume of Freund's complete adjuvant. Blood was collected and sera were prepared 14 days after the third injection. This antiserum was used to pretreat strain BS4 (agar) as described above for the antiserum raised against whole gonococci.

Test for the resistance of gonococci to intracellular killing

After treatment with antisera as described above, the treated

6 —1 and control of suspensions of gonococci (0.5 ml, 2 x 10 ml ,

>60% viable) were mixed in Leighton tubes with an equal volume of suspensions of human phagocytes (2 x 10 ml , about 80% PMN phagocytes) prepared from fresh human peripheral blood as described by K. Witt et al., Journal of Medical Microbio¬ logy 9_, 1-12 (1976) and suspended in Parker medium 199 with HHS (10% v/v). After incubation for 1 hour at 37°C, the medium was discarded and the infected phagocyte deposit washed three times with PBS containing BSA (0.1% w/v) . The following determinations were made on the infected phagocytes. The number of gonococci per phagocyte (phagocytic index) seen microscopically on stained films on coverslips placed on the flat surface of the Leighton tubes was determined. The number of phagocytes in the deposits from Leighton tubes was determined from their content of DNA (Parsons et_ al_. , loc. cit. 1981). The washed deposits were scraped off with a rubber-tipped glass rod and suspended in 75 μl 0.1% (w/v) BSA in PBS. Suspensions from three replicate tubes were pooled and 20 μl removed for an immediate viable count of gonococci (see below). The remaining pooled phagocyte suspen¬ sion was treated with 5 M NaOH (50 μl) for 1 hour at 37°C and then with indole reagent (indole, 0.02% w/v, in 5 M HC1, 250 μl) for 10 minutes at 100 C. Cooled samples were extracted with chloroform (1 ml) by vortex mixing (30 s) and the absorbance at 490 nm, ,__n (in a Pye-Unicam SP 1800 spectrophotometer) of the aqueous phase obtained by centrifugation (3000 g, 10 minutes, room temperature) was read against a blank of 0.1% (w/v) BSA in PBS after reaction with the indole reagent. The number of phagocytes on the surface of the Leighton tube (4-20 x 10 in most tests) was obtained from a standard curve relating A, _ to numbers of phagocytes obtained from 12 different donors (Parsons et al. , loc. cit. 1981).

The number of viable gonococci in the phagocyte deposits was obtained by plating the 20 μl sample of the suspension used for DNA assay (see above). The viable counts were corrected for settled non-cell-associated gonococci by deducting the low viable counts (<10% of those in tubes containing phagocytes) found in control tubes without phagocytes. The results (Tables 1 and 2) were expressed as the percentage of the total cell-associated gonococci (the product of the phagocytic index and the total number of phagocytes) that were viable.

These indirect phagocytosis tests for the determinants of intracellular survival are not rigidly quantitative for the reasons stated previously by K. Witt et_^ al. , loc. cit. , but they demonstrate valid differences in intracellular survival of strain BS4 (agar) after various treatments. The tests give variable results and only by repeating them on many batches of material were unequivocal results achieved. The results presented are from representative experiments; the variable viability of gonococci in phagocyte deposits from different donors on different days precluded the presentation of mean values and standard errors.

RESULTS

Extraction of antiserum-neutralising components from OMV by sodium cholate (1% w/v)

The results of experiments 1 and 2 in Table 1 show that substantial material with antiserum-neutralising activity was extracted from crude OMV of BS4 (agar) by the procedures described in Methods. The protein, carbohydrate and KDO contents of the cholate extract indicated almost complete solubilisation of the

OMVs. The high figure for extractable protein relative to that of the crude OMV may have been due to the fact that not all the protein of the latter reacted in the assay. Fractionation of the sodium cholate extract on Sephadex G75 Figure 1 depicts the profile of the separation of sodium cholate (1% w/v) extract on "Sephadex G75"; this profile is typical of 17 similar experiments. Protein concentration is represented on the ordinate by the absorbance AJ -J_Q ^an<^ ^t-^e fractions are shown on the abscissa. The antiserum-neutralising activities of the cholate extract and fractions 1a, 1b, 2 and 3 for two experiments typical of 17 are shown in Table 1 (experi¬ ments 3 and 4) . The results of all 17 experiments indicated that the antiserum-neutralising activities of fractions 1a and 1b were roughly equal. Thus, both fractions were active in all experi- ments and the number of times (9) when fraction 1b appeared slightly more active than fraction 1a (as in Table 1) was almost equal to that (8) when the position was reversed. Also, in a few tests four- and 5-fold dilutions of the standard concentrations of both fractions 1a and 1b were inactive, indicating the absence of gross differences between them. Fractions 2 and 3 contained little activity. Fraction 1b contained much less protein than fraction 1a but the amounts of carbohydrate in the two fractions were similar and usually less than those found in the cholate extract and fractions 2 and 3. Experiments in which only the whole cholate extract and fraction 1b were examined (experiments 5

TABLE 1

Intracellular killing of strain BS4 (agar) by human phagocytes; effect of antiserum (against whole organisms) absorbed with crude OMV of BS4 (agar) , their sodium cholate (1% w/v) extract and fractions of the latter

The results of experiments 1 and 2, 3 and 4, and 5 and 6 are representative of a total of 4, 17, and 4 experiments respectively with different batches of materials.

Viable gonococci in

Analyε ;is of absorbent ml^"1

Λ phagocyte deposits

1

Exp no. Protein Carbohydrate after 1 h at 37°C

(phagocyte (μg BSA (μg glucose (% of total micro-

^•fc donor) Antiserum Absorbed with 4* equiv. equiv. KDO (μg) scopic count) t

1

1. (CP) 11.1

+ - <0.2 1

+ crude OMV 500 447 ND 4.9

+ cholate ext. 584 370 ND 4.0

2.(CP) - 7.8

+ <0.2

+ crude OMV 920 715 360 8.0

+ cholate ext. 680 525 324 4.4

TABLE 1 (continued)

-1 Viable gonococci in

Analysis of absorbent ml phagocyte deposits f Λ

Exp no . Protein Carbohydrate after 1 h at 37°C (phagocyte (μg BSA (μg glucose (% of total micro- ι_ donor) Antiserum Absorbed with V equiv. equiv. KDO (μg) scopic count)

3. (HL) 34.0

+ 1.0

+ cholate ext. 800 702 ND 19.7 + fraction 1a 700 177 ND 10.3 + fraction 1b 95 105 ND 15.1 + fraction 2 ND 310 ND 4.0 + fraction 3 ND 218 ND 0.8

I

4.(CP) 17.0

+ "* 1.0

+ cholate ext. 990 625 ND 17.5

+ fraction 1a 535 73 ND 11.0

+ fraction 1b 58 109 ND 13.0

+ fraction 2 20 185 ND 2.3

+ fraction 3 20 106 ND 2.0

TABLE 1 (continued)

Exp no.

(phagocyte (μg BSA (μg glucose (% of total micro¬

^• donor) Antiserum Absorbed with 4 equiv. equiv. KDO (μg) scopic count) -V

5. (RH) 34.3

+ - 1.5

+ cholate ext. 1400 ND 720 28.1

+ fraction 1b 251 ND 155 13.4

6 (NP) - - 25.2 oo

+ - 0.9 I

+ cholate ext. 664 ND 460 12.0

+ fraction 1b 190 ND 216 22.1

ND. Not determined.

•"■Treatment of gonococci with antiserum against BS4 (agar) before mixing with phagocytes as described by

Parsons et al., loc. cit. (1981).

•(■Absorption was done as described in the text; the concentration of all absorbents was equivalent

11 -1 to 5 x 10 gonococci ml and analyses for protein, carbohydrate and KDO were as described in the text.

♦The phagocytosis test was described in the text. Suspensions of human phagocytes (10 ; about 80% PMN phagocytes) were mixed with 10 BS4 (agar) organisms (>60% viable) in Leighton tubes and, after incubating (1 hour, 37 C), viable and visual counts of gonococci and counts of total numbers of phagocytes were made as described by Parsons ejt_^ al. , loc cit. (1981).

and 6, Table 1) support the above results. Analysis for KDO indicated that 20-40% of the original LPS was present in fraction 1b.

In summary, fraction 1b was as active as fraction 1a and

Q5 contained much less protein. Also, an antiserum raised against it reduced the resistance of BS4 (agar) to killing by phagocytes. Thus, in one phagocytosis test typical of five, the viable gonococci in the phagocyte deposits after 1 hour at 37 C, calculated as a percentage of the total visible organisms,

•JQ were 9.7, 1.7 and 2.8 for untreated BS4 (agar), BS4 (agar) treated with antiserum to whole organisms, and BS4 (agar) treated with antiserum against fraction 1b respectively. Fraction 1b was, therefore, the subject for further study. Absence of antiserum-neutralising activity in LPS prepared

T5 from BS4 (agar)

The presence of LPS in fraction 1b prompted an investiga¬ tion into whether LPS itself was the determinant of resistance. Accordingly, antiserum-neutralising activity of the cholate extracts and LPS preparations made from the same batches of BS4

20 (agar) . The latter were tested at four times the concentration of the cholate extract in relation to the standard concentration of

11 -1 gonococci (5 x 10 ml ) because the extraction process could not be expected to yield a full recovery of LPS. The LPS preparations contained a protein but far less than was in the cholate extracts.

25 No LPS preparation showed antiserum-neutralising activity, indicating that the protein component of fraction 1b contained the determinant of resistance to killing by phagocytes.

Comparison of the proteins in OMV, whole cholate extract and its fractions on SDS-PAGE

30 Figure 2 shows the protein patterns on SDS-PAGE for OMV,

(Lane 1) whole cholate (Lane 2) extract and fractions 1a (Lane 3) and 1b (Lane 4) from the "Sephadex G75" column. To relate to the biological test, the materials of Lanes 1-4 were examined in amounts equivalent to the same numbers of gonococci (50 μl

35 containing 2.5 x 10 /ml = 1.25 x 10 » 40-fold less than the standard number, 5 x 10 ); thus some channels were overloaded and others underloaded. In Lane 5 fraction 1b was loaded at 5x the amount of Lane 4. The OMV, the whole cholate extract and fraction 1a showed a full range of proteins including proteins A, B and C (of about 35, 47 and 74 kDal respectively; see Figure 2) previously thought to be associated with phagocyte killing-resistance (Parsons et al. , loc. cit. 1982). In contrast, fraction 1b showed much less total protein and fewer individual proteins even when five times the amount of material was examined. The principal membrane proteins of Neisseria gonorrhoeae (sizes around 35 kDal) were very much reduced in fraction 1b compared with the OMV, cholate extract and fraction 1a, thus indicating they were not associated with phagocyte resistance. Similarly, proteins A, B and C were much reduced or not detected in the biologically active fraction 1b. The proteins in fraction 1b that appeared to be present in similar amounts in the equally biologically active fraction 1a, and in OMV and the cholate extract, were a group of proteins in the 60 kDal region (marked X in Figure 2) and one or more proteins in a diffuse band around 20 kDal (marked Y in Figure 2) .

In two experiments in which a fraction 1b obtained from the phagocyte-susceptible strain BSSH by the same method as that described for BS4 (agar) was examined at the standard concen¬ tration, and at five times that concentration, the groups of proteins associated with antiserum neutralisation (X and Y, Figure 2) were not detected.

Antiserum-neutralising effect of proteins of fraction lb by SDS-PAGE

In preparative experiments, the proteins demonstrated in fraction 1b by SDS-PAGE that were associated with biological activity were eluted from regions (about 2 cm) of the gels corres¬ ponding roughly to 60 and 20 kDal (regions marked X and Y on Figure 2) and, also, the principal outer membrane protein region around 35 kDal. The concentrated eluates were used to absorb the antiserum against BS4 (agar) together with an eluate from a control portion of the gel; the results of four experiments are summarised in Table 2. Clearly the antiserum-neutralising activity lies with the protein(s) of the 20 kDal region. TABLE 2

Viable gonococci in phagocyte deposits after 1 hour at 37 C

Antiserum Absorbed with + (% of total microscopic count) -V

(1) (2) (3) (4)

- - 12.5 25.1 7.7 17.7

+ - 1.4 0.5 1.0 1.3

+ fraction 1b 15.1 7.0 5.8 ND

+ 60 kDal region 1.6 1.4 1.1 2.7

+ 35 kDal region 3.6 0.1 2.3 2.4

+ 20 kDal region 19.1 12.9 13.9 14.0

+ control gel 1.0 1.1 2.6 2.0

* As for Table 1.

4 Absorption was done as described in the text; the concentration

12 -1 ooff aallll aabbssoorrbbeennttss was equivalent to 1 x 10 gonococci ml + As for Table 1.

EXAMPLE 2 This Example illustrates the use of the proteinaceous material of the invention in diagnosis.

First, the optimum amount of fraction 1b (0.5 μg ml protein) to use as antigen in a survey of patients serum was obtained by reacting logarithmic dilutions of the fraction (10 μg - 0.001 μg protein ml- ) against logarithmic dilutions (1/10 - 1/10 ) of hyper-immune rabbit serum against strain BS4 (agar) .

The fraction 1b was coated onto the plastic wells of a "Titertek" multiwell plate by a conventional method. 100 micro- litre volumes were used. Human patient serum and sheep anti-human IgG labelled with alkaline phosphatase were added to the wells. After incubation and washing the enzyme activity of the solid phase was measured to determine the extent of neutralisation. In two similar comparisons of sera from 19 gonococcal patients and from 11 persons with no history of gonorrhoea using 0.5 μg ml (protein) of fraction 1b as antigen, the two types of sera were easily distinguished at 1/10, 1/100 and 1/1000 dilutions. (A 1/10 dilution signi¬ fies 10 μl serum: 90 μl diluent.) Thus, with a threshold value (mean of negative serum group + 2 SD; positive: negative ratio of 1.00) determined for each serum dilution, only one normal serum was above the threshold value when 1/10 and 1/100 dilutions were examined; and omitting this sample, the mean absorbance in ELISA of the positive groups was 3-fold higher than the negative groups. At 1/1000 dilution of sera, all patients' sera were above the threshold value and all the control sera below. These results are highly encouraging. EXAMPLE 3

Tests were carried out to establish whether monoclonal antibody to the H8 antigen of Cannon et al. , loc. cit. neutralised resistance of gonococci (strain BS4) to killing by phagocytes. The BS4 gonococci were incubated in tissue culture medium containing the antibody (1 hour, 37 C) before mixing with human phagocytes in the test described in Example 1. In all of five such experiments, the resistance of strain BS4 (agar) to killing by phagocytes was not neutralised at all by the monoclonal antibody even when concen¬ trated 8-fold; in contrast antiserum against whole BS4 (agar) organisms greatly reduced the resistance. EXAMPLE 4

Fraction 1b prepared as described in Example 1 (8-10 ml: derived from 5 x 10 gonococci ml and containing about 1 mg ml protein) was re-fractionated on a column (1 m x 2.5 mm) of "Sephadex G25" with glycine/NaOH (0.1 M, ^"pH 9.59) containing sodium cholate 1% (w/v) as eluant. Appropriate fractions were taken from the region where components of 20 kDal would elute. This region was indicated by preliminary calibration of the column with the following marker materials: ribonuclease A, chymotrypsinogen A, ovalbumin, bovine serum albumin and blue dextran 13.7, 15.0, 43.0, 67.1 and 2000 kDal respectively) , under the same conditions as for the gonococcal fraction. The final material was freeze-dried and desalted on a column of "Sephadex G25" (1 m, 15 mm) using water as eluant. The eluate was freeze-dried, dissolved in phosphate-buffered saline pH 7.2 (volume 1/25 of that of the original solution of fraction 1b) and kept at -20 C. It contained between 1 and 1.5 g ml- of protein.

EXAMPLE 5

In experiments to examine the ability of the purified 20 kDal _1 protein (50-75 μg ml ) tissue culture medium) before mixing with phagocytes in the test described previously. Control organisms were those of the same strain but incubated (1 hour, 37 C) in tissue culture medium without the 20 kDal protein. In a typical experiment, treated gonococci showed a 2-fold enhancement of survival when compared with control organisms. EXAMPLE 6

This example describes preparation of a hybridoma cell line. Female Balb C mice were injected with the purified 20 kDal protein in physiological saline (approximately 1 mg protein/ml) . Emulsions were made with an equal volume of complete/incomplete

Freund's adjuvant (CFA/FA) and the mice were injected intraperito- neally according to the following schedule:

DAY 0 1st CFA, 50 μg protein

DAY 14 2nd FA, 75 μg protein

DAY 21 3rd FA, 75 μg protein

DAY 28 4th without adjuvant 100 μg protein

Finally, an intraveneous (tail vein boost) injection of 100 μg protein in saline (0.05 ml) was given 4 days before fusion, and the mice were test bled for antibody 1 day before fusion. A sandwich ELISA was carried out using immobilised 20 kDal protein, the mouse serum and enzyme labelled rabbit anti-mouse IgG. The test showed that antibodies to the protein had been made by the mouse. Hybridomas were then prepared by fusing spleen cells from the mice with mouse myeloma cells using the procedure described by BRL (UK) Ltd. in the instruction manual supplied with the "Hy BRL Prep. Kit" ("Reagents and Instructions for Hybridoma Production", 1982 BRL (UK) Ltd., Cambridge CB4 4BE, England), except that the myeloma cells used were NS-1 instead of NP-3 mentioned in the manual. From 5 microtitre plates of 96 wells per plate 85 wells contained hybridomas which showed by ELISA a reaction to fraction 1b. Of these 9 showed by ELISA a reaction to the 20 kDal protein.

Hybridomas obtained by this procedure can be screened against fraction 1b by Western blotting to detect cells which produce antibodies thereto, and the positive cells further tested against the 20 kDal protein itself.

103B

Claims

CLAIMS 1. A method of assay of human clinical gonorrhoea which comprises (1) incubating a sample, suspected of containing JN. gonorrhoea or antibodies to _N. gonorrhoea, from a body fluid of a human patient, (a) with a proteinaceous material of N. gonorrhoea strain BS4 (NCTC 11922), of relative molecular mass about 20 kDal and which neutralises antiserum to strain BS4 or (b) with antibodies to said proteinaceous material, respectively, and (2) detecting whether neutralisation between the proteinaceous material and the antibodies has taken place.

2. A method of assay of human clinical gonorrhoea which comprises (1) incubating a sample, suspected of containing N_. gonorrohoea or antibodies to II. gonorrhoea, from a body fluid of a human patient, (a) with a proteinaceous material obtainable from the strain of Neisseria gonorrhoeae BS4 (NCTC 11922), which strain is resistant to being killed by human phagocytes, said proteinaceous material being obtainable by (but not necessarily obtained by) extraction of the outer membrane vesicles (OMV) in 10 g/litre sodium cholate in an aqueous buffer at pH 9.5 and removing undissolved OMV, fractionating the extract and selecting a fraction comprising a proteinaceous material having a relative molecular mass of about 20 kDal and which neutralises antiserum to said phagocyte killing-resistant strain BS4 of Neisseria gonorrhoeae, or (b) with antibodies to said proteinaceous material, respectively, and (2) detecting whether neutralisation between the proteinaceous material and the antibodies has taken place.

3. A method according to Claim 2 wherein the proteinaceous material is substantially free of all proteins having other relative molecular masses.

4. A method according to Claim 2 wherein the proteinaceous material is obtainable by the defined method in which the fraction selected is that which gives a shoulder to the low molecular mass side of the UV absorption peak at 279 nm during continuous gel fractionation of the extract.

5. A method according to Claim 1, 2, 3 or 4 in the form of a sandwich assay wherein either the proteinaceous material or antibodies thereto are insolubilised and after incubation with the liquid sample, the product of the interaction is incubated with labelled anti-human immunoglobulin, unbound label is separated from the insoluble material and any labelled insoluble material is detected or measured.

6. A method according to Claim 1, 2, 3 or 4 in the form of a competitive or displacement assay wherein either (1) the protein- aceous material or (2) antibodies thereto are insolubilised and incubated (a) with the liquid sample and (b) (1) with an insufficiency of labelled antibody or (2) with an insufficiency of a labelled proteinaceous material, respectively, the unbound label is separated from the insoluble material and the label is detected or measured.

7. A kit -for use in a method of assay of human clinical gonorrhoea comprising a proteinaceous material as defined in Claim 1, 2, 3 or 4 and labelled anti-human immunoglobulin antibodies or labelled antibodies to the proteinaceous material.

8. A kit for use in a method of diagnosis of human clinical gonorrhoea, comprising antibodies to a proteinaceous material as defined in Claim 1, 2, 3 or 4 and labelled anti-human immuno¬ globulin antibodies or labelled proteinaceous material.

9. Antibodies to a proteinaceous material defined in Claim 1, 2, 3 or 4.

10. A hybridoma cell line secreting antibodies to a proteinaceous material defined in Claim 1, 2, 3 or 4.

11. Use as a component of a vaccine of a proteinaceous material defined in Claim 1, 2, 3 or 4.

103B