Antigen of group B streptococci

The subject invention concerns a novel in vitro process for identifying and quantifying native antigens on potentially pathogenic group B streptococci bacteria present in a clinical specimen. The invention process is made possible by the discovery of novel bacterial markers denoted .gamma. and .delta. epitopes which are expressed by a variety of group B streptococcal strains.

BACKGROUND OF THE INVENTION 
Group B streptococci (GBS) are being increasingly recognized as important 
human pathogens. In addition to causing meningitis, bacteremia, 
endocarditis, bronchopneumonia, arthritis, peritonitis, wound infections, 
abscesses, and urinary tract infections in adults, as many as 80% of group 
B infections occur in neonates (Jelinkova, J. [1977]Current Topics in 
Microbiology and Immunology 76:127-165). Approximately 30% of pregnant 
women have been reported to be colonized by GBS. Despite this high 
carriage rate, neonatal infection occurs with an incidence of only 0.5% 
(Lim, D. V., Morales, W. J., Walsh, A. F., and Kazanis, D. [1986]J. Clin. 
Micro. 23:489-492). Predisposing factors to development of disease are 
premature birth, prolonged rupture of membranes, overt maternal infection, 
and deficiency of type specific antibody (Boyer, K. M. and Gotoff, S. P. 
[1986]New England J. Med. 314:1665-1669). To date no one has identified a 
bacterial marker which would predict which streptococcal strains are more 
likely to cause infection. 
The ability to subtype group B streptococci more comprehensively may be of 
value in predicting which organisms could cause sepsis, in particular 
neonatal sepsis. Recently, a receptor for the Fc region of human IgA was 
reported to be expressed on the surfaces of some strains of GBS 
(Russell-Jones, G. J., Gotschlich, E. C., and Blake, M. S. [1984] J. Exp. 
Med. 160:1467-1475). Western blot analysis of proteins extracted from 
these strains by treatment with detergent indicated that it may in fact be 
the .beta. antigen component of the c protein marker complex which has the 
ability to bind to IgA (Russell-Jones, G. J. and Gotschlich, E. C. [1984] 
J. Exp. Med. 160:1476-1484). Since IgA is the primary line of defense at 
mucosal surfaces, such as the vaginal epithelium, the ability of bacteria 
to bind this class of immunoglobulin molecules in a non-immune fashion 
might interfere with effective clearance of these microorganisms. 
GBS are typed based on the presence of type specific carbohydrate antigens 
expressed on their surfaces, i.e., Ia, Ib, II, III, IV, and V. In 
addition, a protein marker called the c protein has been used as a typing 
marker. Subsets of serotype Ia and II and virtually all serotype Ib have 
been reported to express components of the c protein. The c protein had 
been reported to consist of two acid extractable antigens called .alpha. 
and .beta.. A further level of sub-typing of group B streptococci, with 
respect to the c protein marker complex, would be desirable to identify 
potential pathogens. 
In addition to the need to identify and sub-type GBS, there is also an 
urgent need to identify means of preventing GBS infections. GBS infections 
now account for over 40% of all neonatal sepsis in the United States 
resulting in over 12,000 cases and 2,500 infant deaths annually 
(Strickland, D. M., E. R. Yeomans, G. D. V. Hankins 1990] Am. J. Obstet. 
Gynecol. 163:4-8). In addition, pregnancy related morbidity occurs in 
nearly 50,000 women annually (Baker, C. J. [1989]J. I. D. 161:917-921). 
The National Academy of Sciences estimated that the cost of GBS early 
onset sepsis and obstetric disease exceeded $500 million in 1985 and 
listed GBS as the fourth most important cause of preventable infectious 
mortality in the United States (Strickland et al., supra). As discussed 
above, as many as 30% of pregnant women have been reported to be colonized 
with GBS. Despite this high degree of colonization, only a small 
percentage of women actually give birth to septic infants. However, those 
infants who do become infected have a very high rate of morbidity 
(particularly permanent neurologic damage) and mortality. No GBS vaccine 
is currently available. Early onset sepsis occurs within hours to days of 
birth, is associated with vertical transmission from the mother, and is 
associated with a mortality rate as high as 50-60%. The causative GBS are 
not clearly delineated within a given serotype. Until recently, no 
bacterial determinants have been identified which are predictive of early 
onset sepsis. Late onset GBS sepsis occurs days to weeks after birth, is 
not necessarily associated with vertical transmission, and is associated 
primarily with strains expressing the type III carbohydrate. Serotype III 
GBS are often associated with meningitis. The mortality rate from late 
onset sepsis is approximately 20%, but neurologic sequelae occur in 
approximately 50% of patients with meningitis. 
Therefore, there is a great need to identify compositions and methods to 
reduce the susceptibility to GBS infection. 
BRIEF SUMMARY OF THE INVENTION 
This invention concerns novel bacterial markers which can be used to 
identify organisms that are potentially pathogenic to neonates. More 
specifically, the subject invention concerns novel bacterial markers 
denoted gamma and delta (.gamma. and .delta.) epitopes which can be used 
to classify further group B streptococci. These novel epitopes are 
expressed by a variety of group B streptococcal strains. Many of the group 
B streptococcal strains also express the known .alpha. and .beta. antigens 
within the c protein marker complex. The group B streptococcal strain 
denoted A909, which is the international typing strain available from the 
ATCC repository at 12301 Parklawn Drive, Rockville, Md., as ATCC 27591, 
can be used to express the known antigens .alpha. and .beta., and the 
novel antigens of the subject invention, .gamma. and .delta.. 
The discovery of the novel epitopes of the invention was enabled by a rapid 
two-stage radioimmunoassay (RIA) which identifies type specific antigens 
on the surfaces of group B streptococci. This improved rapid technique 
detects native unmodffied type specific antigens on bacterial surfaces 
objectively and economically with results obtained within three hours. The 
assay utilizes intact bacteria and does not require hot acid extraction. 
Consequently, this method detects acid stable as well as acid-labile 
antigens in their native unmodified form. The RIA is semi-quantitative and 
uses the same type specific antisera as precipitin testing. The classical 
precipitin test is dependent on optimal concentrations of both antigens 
and antibodies for precipitation to occur resulting in the potential for 
false negative results. In addition, hot acid extraction procedures have 
the potential to change the native configuration of the bacterial 
antigenic determinants. These problems are not encountered with the more 
sensitive RIA procedure. 
The subject invention further relates to the use of the .gamma. and .delta. 
antigens as immunogenic compositions to raise an immune response to GBS. 
Thus, these novel antigens can be used to reduce the susceptibility of a 
human or animal to GBS infection.

DETAILED DESCRIPTION OF THE INVENTION MATERIALS AND METHODS 
Using a radioimmunoassay (RIA) technique rather than conventional 
precipitin testing of bacterial hot acid extracts, we have identified two 
novel antigens, designated .gamma. and .delta., which are also reactive 
with c protein typing antiserum. The .gamma. antigen was detected on 
subsets of serotype Ia, Ib, and II GBS and could be identified in hot acid 
extracts by immunoelectrophoresis as well as by RIA. In contrast, the 
.delta. antigen was associated almost exclusively with serotype III 
strains and was not detected by conventional precipitin testing. 
To determine whether a correlation exists between expression of the 
individual c protein associated antigens and a GBS strain's associated 
with symptomatic infection in newborn infants, an epidemiologic study was 
undertaken (Chun, C. S. Y., L. J. Brady, M. D. P. Boyle, H. C. Dillon, E. 
M. Ayoub [1991]J. I. D. 163:786-791). Two hundred and fifty-five GBS 
isolates recovered from septic neonates, healthy colonized newborns, and 
colonized pregnant women were typed by RIA. Univariate analysis was used 
to evaluate the frequency of expression of the .alpha., .beta., .gamma., 
and .delta. antigens and stepwise logistic regression was used in the 
multivariate analysis to evaluate the association of combinations of these 
and carbohydrate serotype antigens with occurrence of disease. Both the 
.alpha. and .gamma. were expressed more frequently by GBS strains 
associated with early onset disease than late onset disease (p&lt;0.001 and 
p&lt;0.003, respectively). The association of the .gamma. antigen with early 
onset sepsis was found to be independent of serotype (p&lt;0.036) suggesting 
a potential role as a virulence factor in early onset disease. The .delta. 
antigen was significantly associated with strains from septic neonates 
(early and late) as compared with those from colonized healthy infants or 
pregnant females (p&lt;0.003) suggesting that it too may act as a potential 
virulence factor in neonatal GBS sepsis. 
Many in vitro studies have shown that expression of the c protein may 
interfere with effective clearance of GBS. Also, it has been found that 
antibodies to c protein constituents can provide protection against GBS in 
animal models (Payne, N. R., Y. Kim, P. Ferrieri [1987]Infect. and Immun. 
55:1243-1251; Madoff, L. C., J. L. Michel, D. L. Kasper [1991] Infect. and 
Immun. 59:204-210), and immunization by extracts containing c proteins 
have been reported to yield protective antibodies (Valtonen, M. V., D. L. 
Kasper, N. J. Levy [1986] Microbial Pathogen. 1:191-204). These factors, 
as well as the epidemiologic data described above, provide additional 
support for the use of our novel antigens in an immunogenic composition. 
Bacterial strains, media and growth conditions. Laboratory strains of group 
B streptococci were used. All strains were confirmed as group B 
streptococci by screening with a slide coagglutination test for 
identifying Groups A, B, C, and G streptococci, such as the PHADEBACT 
STREPTOCOCCUS TEST (Pharmacia Diagnostics, Piscataway, N.J.). Bacteria 
were grown as stationary cultures in Todd-Hewitt broth (Difco, Detroit, 
Mich.) for 18-24 hr at 37.degree. C. The bacteria were harvested by 
centrifugation and washed in 0.15M phosphate buffered saline (PBS), pH 
7.2. Stock cultures were stored in glycerol at -70.degree. C. Sodium azide 
was added to a final concentration of 0.02% to stored culture 
supernatants. 
Source of group B streptococci typing antiserum. Rabbit anti-type Ia, Ib, 
II, and III carbohydrate antigens, as well as rabbit anti-c protein 
antigen, were kindly provided by Dr. R. Facklam, Center for Disease 
Control, Atlanta, Ga. 
Adsorption of anti-c protein typing antiserum. Bacterial strains expressing 
the reactivities to be depleted from the anti-c antiserum were chosen as 
appropriate adsorbents after characterization in the two-stage RIA 
described below. The bacteria from 5 ml of a Todd-Hewitt broth overnight 
culture were pelleted by centrifugation and washed once with 2 ml of PBS, 
pH 7.2. A 100 .mu.l sample of anti-c protein antiserum was added to the 
washed bacterial pellet and rotated at 4.degree. C for 1 hour. The 
adsorption was repeated at least twice or until all reactivity against the 
adsorbing strain was eliminated as detected by the two-stage RIA. 
Source of protein A. The type I bacterial Fc receptor, staphylococcal 
Protein A, was purchased from Pharmacia Fine Chemicals, Piscataway, N.J. 
Iodination of protein A. Protein A was radioiodinated by the mild 
lactoperoxidase method using ENZYMOBEADS a reagent used to facilitate the 
iodination of proteins (Bio-Rad, Richmond, CA) (Reis, K. J., Ayoub, E. M., 
and Boyle, M. D. P. [1983] J. Immunol. Meth. 59:83-94). The labeled 
protein was separated from free iodine by passage over a gel filtration 
matrix used for desalting, such as G25 column (PL 10, Pharmacia) and 
collected in veronal buffered saline, pH 7.4 containing 0.001M Mg.sup.++, 
0.00015M Ca.sup.++, and 0.1% gelatin (VBS gel, Reis et al., supra). 
Proteins labeled by this method routinely have a specific activity of 
approximately 0.3 mCi/mg (Reis et al. supra). 
Hot acid extracts of group B streptococci. Laboratory strains to be hot 
acid extracted were inoculated into 10 ml Todd-Hewitt broth starter 
culture tubes. After 6-8 hours of growth the cultures were transferred to 
vessels containing 150 ml of sterile Todd-Hewitt broth and grown to late 
log phase at 37.degree. C (18-24 hours). The bacteria were harvested by 
centrifugation and extracted following the method of Lancefield 
(Wilkinson, H. W., Facklam, R. R., Wortham, E. C. [1973] Infect. Immun. 
8:228). Essentially, the bacterial pellet was resuspended in approximately 
1 ml 0.2N HCl in 0.85% NaCl. These suspensions were boiled for 10 minutes 
then adjusted to neutral pH by the addition of 0.2N NaOH. Suspensions were 
centrifuged at 10,000.times.g for 30 minutes to remove bacterial debris 
and the supernatants were decanted. 
Precipitin assay for..typing .group B streptococci. Hot acid extracts were 
reacted with antisera of appropriate specificity and reactivity determined 
by precipitation in capillary tubes or by radial immunodiffusion in 
agarose gel (Jensen, N. E. [1979] Acta Path. Microbiol. Scand. 87:77-83). 
Immunoelectrophoresis. Hot acid extracts of GBS strains were concentrated 
10-fold using a MINICON MACROSOLUTE CONCENTRATOR (Amicon, Danvers, Mass.). 
The concentrated hot acid extracts were applied to the wells of an agarose 
plate (HYLAND DIAGNOSTICS AGAROSE GEL IEP SYSTEM, Malvern, Pa.) which was 
then subjected to electrophoresis at 30 mAmps for 40 minutes. The agarose 
from the pre-cut troughs was removed and anti-c protein marker typing 
serum was applied. The plates were incubated overnight at 4.degree. C in a 
moist chamber. After being soaked in PBS, pH 7.2, for 48 hours and in 
deionized water for 12 hours, the plates were stained with 0.7% amido 
black in 7% acetic acid for 5 minutes. Destaining was done with 7% acetic 
acid until all background color was removed. 
Radioimmunoassay for determination of subtypes of group B streptococcal 
strains. A two-stage radioimmunoassay (RIA) was employed to detect type 
specific antigens on the bacterial cell surface. Laboratory strains were 
inoculated into 10 ml Todd-Hewitt broth starter culture tubes and grown to 
late log phase at 37.degree. C (18 to 24 hours). The bacteria were 
pelleted by centrifugation for 8 minutes at 1,000.times.g, and the 
bacterial pellets were resuspended in 2 ml PBS, pH 7.4. Tubes containing 
100 .mu.l of bacterial suspension were incubated for 1 hour at 37.degree. 
C with 100 .mu.l of a 1:400 dilution of each type specific antiserum; 
anti-Ia, anti-Ib, anti-II, anti-III or anti-c antibody following selective 
adsorption. Normal rabbit serum was included in each assay as a control 
for specificity. Following incubation, the tubes were centrffuged for 8 
minutes at 1,000.times.g and the bacterial pellet washed with 2 ml PBS, pH 
7.2, to remove unbound antibodies. The pellets were resuspended after the 
wash by vortexing, and bacterial-bound antibody was quantitated by 
addition of 100 .mu.l of .sup.125 I labeled Protein A containing 
approximately 30,000 cpm. This Fc receptor has a high affinity for rabbit 
immunoglobulin and, therefore, binds to any bacterial-associated 
antibodies. The tubes were incubated for 1 hr at 37.degree. C. and the 
bacterial pellets washed twice with 2 ml of metal-free VBS containing 
0.01M ethylenediaminetetraacetic acid (EDTA) and 0.1% gelatin (EDTA-gel) 
to remove any labeled tracer not associated with a bacterial 
antigen-antibody complex. The bacterial-associated radioactivity was 
quantitated in a BECKMAN 5500 auto-gamma counter. The background level of 
radioactivity was determined to control tubes containing bacteria and 
radiolabeled Protein A only. Since the expression of a bacterial receptor 
that would bind antibody molecules non-specifically would complicate our 
assay, we have included a normal rabbit serum control in all of our 
assays. 
Following are examples which illustrate procedures, including the best 
mode, for practicing the invention. These examples should not be construed 
as limiting. All percentages are by weight and all solvent mixture 
proportions are by volume unless otherwise noted. 
EXAMPLE 1 
Testing of GBS Strains 
Fifty-three group B streptococcal (GBS) strains were tested for the 
presence of type specific carbohydrate antigens and protein antigens by 
the two-stage radioimmunoassay (RIA) described in the Methods. These 
strains were also typed by the conventional precipitin test of hot acid 
extracts, with reactivity detected by radial immunodiffusion as described 
in the Methods. There was an absolute correlation between the carbohydrate 
antigens detected by each assay (see Table 1). Strain HG782 was 
particularly interesting because it demonstrated two carbohydrate 
antigens. This was originally believed to be a mixture of two strains. 
Consequently, the RIA was repeated on cultures of fifteen individual 
colonies from this isolate and identical results were obtained. FIG. 1A-1D 
show analysis of type specific carbohydrate antigen on the surface of 
group B streptococci using the two-stage radioimmunoassay outlined in the 
Methods. The height of each line depicts the amount of .sup.125 I Fc 
receptor associated with each strain minus the background radioactivity, 
thus giving a measure of the quantity of each specific antiserum 
associated with the bacterial pellet. A comparison of the results obtained 
using this assay and the classical precipitin assays are presented in 
Table 1. The results presented in FIG. 1A-A3 demonstrate that variation in 
the quantities of type specific carbohydrate antigens expressed on various 
strains was detected by the two-stage RIA. The results obtained by the RIA 
were reproducible from day to day with each strain being typed on at least 
two occasions with identical results. In contrast to the semi-quantitative 
results obtained by RIA, precipitin tests of hot acid extracts indicated 
only the presence of a given carbohydrate. 
EXAMPLE 2 
Characterizing Antigens Within the c Protein Marker Complex 
When the two approaches, described in Example 1, were applied to 
characterizing antigens within the c protein marker complex, a much more 
complicated pattern emerged. Reactivity with antisera against the c 
protein complex was detected by RIA in 36/53 strains and by precipitin 
test in 20/53 strains (see Table 2). In order to analyze the reasons for 
these differences, the two-stage RIA was repeated with a series of anti-c 
protein antisera which had been selectively adsorbed with bacteria showing 
different c protein reactivity profiles (see FIG. 2A-2J). FIG. 2A-2E show 
analysis of c protein antigens on the surface of group B streptococci 
using the two-stage radioimmunoassay. The antiserum used in the assays is 
either the unabsorbed anti-c antiserum (bottom panel) or the same 
antiserum that has been adsorbed with selective bacteria demonstrating 
unique reactivities as detailed in Table 3. The height of each line 
depicts the amount of .sup.125 I labeled bacterial Fc receptor associated 
with each strain minus the background radioactivity, thus giving a measure 
of the quantity of each specific antiserum associated with the bacterial 
pellet. FIG. 2F-2J show further analysis of unique antigens within the c 
protein marker complex using antisera selective for .alpha., .beta., 
.gamma., and .delta. antigens of the c protein marker complex. These 
specific antisera were prepared by selective adsorption of the CDC anti-c 
typing serum as described in Table 3. Using this approach a variety of 
distinct binding patterns were observed which could be accounted for by a 
minimum of four separate epitopes. The bacteria were also tested with 
anti-c antisera which had been adsorbed with the group A strain R-28 
demonstrating that none of the reactivities detected were due to the R 
protein antigen (see FIG. 2F-2J). 
EXAMPLE 3 
Expression of Antigens by Strain A909-ATCC 27591 
The A909 strain expressed all four reactivities as detected by the RIA. 
This is the Lancefield prototype la/c strain which is used by CDC to 
prepare the c protein typing antiserum used in this study. In an attempt 
to identify the nature of the reactive antigens, a 10-fold concentrated 
hot acid extract of this strain was analyzed by immunoelectrophoresis 
(IEP) (see FIG. 3 ). FIG. 3 shows immunoelectrophoresis of a ten-fold 
concentrate of a hot acid extract of prototype Ia/c strain A909, developed 
against polyclonal rabbit anti-c protein marker typing serum. Two of the 
resulting precipitin arcs correspond to the previously reported .alpha. 
and .beta. antigens observed by Bevanger (Bevanger, L. and Iversen, 0.-J. 
[1981] Acta Path. Microbiol. Scand. 89:205-209). In addition, a 
precipitation line corresponding to a third reactivity was observed (see 
FIG. 3). This antigen corresponded to the .gamma. reactivity demonstrated 
by RIA. The .gamma. epitope has not been detected by IEP in the past and 
can only be seen after concentration of the hot acid extracts. The fourth 
reactivity, .delta., could not be detected by IEP even when hot acid 
extracts were concentrated 50-fold. The inability of hot acid extracts of 
.delta.-bearing strains to competitively inhibit the .delta.-reactivity in 
the two-stage RIA indicates that either .delta. is not acid-extractable or 
it is acid labile. In contrast, solubilized antigens present in hot acid 
extracts of .alpha., .beta., and .gamma. bearing strains were able to 
inhibit those corresponding reactivities in the two-stage RIA. 
These results suggest that the c protein marker complex represents a more 
complicated antigenic structure than has been recognized previously. 
Furthermore, since the precipitin test is a qualitative one and is 
dependent on optimal concentrations of antigen and type specific antisera, 
low levels of one or more components of the c protein complex may lead to 
false negative results. However, low levels of any surface antigen would 
be detectable in the more sensitive RIA. 
EXAMPLE 4 
Preparation of Monospecific Antisera 
Monospecific antisera against .beta., .gamma., and .delta. epitopes have 
been prepared by selective absorption with combinations of strains bearing 
all other reactivities (see Table 3). Streptococcus agalactiae strains 
PF65BV, PF549AR-B, TC795, and Perihew (PEH) were deposited with the 
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 
20852 USA, on Jun. 10, 1991 and were assigned the accession numbers ATCC 
55191, ATCC 55192, ATCC 55193, and ATCC 55194, respectively. 
The subject cultures have been deposited under conditions that assure that 
access to the cultures will be available during the pendency of this 
patent application to one determined by the Commissioner of Patents and 
Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The 
deposits are available as required by foreign patent laws in countries 
wherein counterparts of the subject application, or its progeny, are 
filed. However, it should be understood that the availability of the 
deposits does not constitute a license to practice the subject invention 
in derogation of patent rights granted by governmental action. 
Further, the subject culture deposits will be stored and made available to 
the public in accord with the provisions of the Budapest Treaty for the 
Deposit of Microorganisms, i.e., they will be stored with all the care 
necessary to keep them viable and uncontaminated for a period of at least 
five years after the most recent request for the furnishing of a sample of 
a deposit, and in any case, for a period of at least thirty (30) years 
after the date of deposit or for the enforceable life of any patent which 
may issue disclosing the cultures. The depositor acknowledges the duty to 
replace the deposits should the depository be unable to furnish a sample 
when requested, due to the condition of a deposit. All restrictions on the 
availability to the public of the subject culture deposits will be 
irrevocably removed upon the granting of a patent disclosing them. 
Specific antisera against a could not be generated since we were unable to 
find a strain that expressed .gamma. that did not also express a. Strains 
expressing .alpha. alone could be identified by comparison of results 
using .gamma. specific, versus .alpha. plus .gamma. specific, antisera. 
Each of the 53 strains was characterized based on its reactivity with each 
of the individual carbohydrate typing antisera, as well as the polyclonal 
unabsorbed, and monospecific absorbed, anti-c protein complex typing sera 
(see Tables 1 and 2). 
EXAMPLE 5 
Summary of Incidence of Antigens 
Table 4 summarizes the incidence of each antigen detected by the two-stage 
RIA. There were 2/53 strains found to have no type specific carbohydrate. 
Type Ia and Ib antigens were each detected on 9/53 strains and type II and 
III antigens were each seen on 16/53 strains. As mentioned, one strain 
bears both type II and III antigens. At least one component of the c 
protein marker complex was seen on 68% or 36/53 of the group B 
streptococcal strains tested by RIA. The .alpha. antigen was the most 
widely detected epitope and was present on 27/53 strains. A subset of 
strains bearing the .alpha. antigen expressed the &gt;, reactivity. Seventeen 
of the 27 .alpha.-positive strains expressed the .gamma. epitope which was 
never detected on a strain that did not also have the .alpha. antigen. The 
.delta. reactivity was detected on 10 strains. With the exception of the 
two strains which bear all four RIA reactivities (both type Ia/c), this 
epitope was detected only on type III strains and was the only c protein 
component associated with these strains. The .beta. antigen was the least 
common c protein reactivity found on GBS and was detected on the surfaces 
of 8/53 strains screened. 
EXAMPLE 6 
Testing a Clinical Specimen 
A clinical specimen, for example, body fluids, swabs of tissue and tissue 
itself, and the like can be tested, for example, in accord with the 
following procedures: 
1. Vaginal cultures can be obtained using a dual swab transport device for 
anaerobic or aerobic bacteria that contains modified Stuarts' transport 
media, such as CULTURETTE II dual swabs (Marion Scientific Corp., Kansas 
City, Mo.); 
2. Inoculate each swab into 2 ml LIM GROUP B STREP BROTH (Gibco 
Laboratories, Madison, Wis.) and incubate for at least 6 hours at 
37.degree. C.; 
3. Test broth cultures for GBS by slide coagglutination (PHADEBACT STREP B 
test, Pharmacia Diagnostics, Piscataway, N.J.); 
4. Isolate GBS by streaking onto Blood Agar Plates (Difco); 
5. Inoculate isolated GBS colonies into 10 ml Todd-Hewitt broth tubes and 
incubate for 6 to 24 hours at 37.degree. C.; and 
6. Conduct two stage RIA as described in the Materials and Methods section, 
supra. 
Other examples of clinical specimens are cerebral spinal fluid, amniotic 
fluid, cervical swabs, blood, lung lavage, urethral swabs, rectal swabs, 
throat swabs, umbilical swabs, saliva, and the like. 
The subject invention can be used to identify and quantify native antigens 
on potentially pathogenic group B streptococci bacteria present in 
clinical specimens of mammals, for example, humans and cows. Particularly 
important are pregnant mammals. 
In addition to the previously-described uses, the subject invention also is 
useful for epidemiological studies. For example, the invention can be used 
to trace zoonotic sources of group B streptococcal outbreaks. Further, the 
invention can be used to trace nosocomial sources of group B streptococcal 
outbreaks in hospitals, in particular, newborn nurseries. Further, there 
may be as yet undiscovered biological properties of the novel .gamma. and 
.delta. antigens described herein, analogous to the ability of the .beta. 
antigen to bind IgA, which would impact on their capacity to cause 
disease. In addition, the association of these antigens with the 
pathogenicity of GBS makes these antigens particularly advantageous for 
use in immunogenic compositions such as vaccines. Such immunogenic 
compositions are described in greater detail below. 
The novel antisera of the subject invention can be employed in a variety of 
immunotechnology procedures. The antisera can be immobilized and tagged 
with an appropriate label, such as a radioisotope, an optical label, e.g., 
a fluorescent tag, an enzyme, an electron dense ligand, and the like. The 
label may be detected by a gamma counter if the label is a radioactive 
gamma emitter, or by a fluorimeter, if the label is a fluorescent 
material. In the case of an enzyme, label detection may be done 
colorimetrically employing a substrate for the enzyme. All of these 
procedures are well known in the art. See "Applications of Bacterial Fc 
Receptors in Immunotechnology," Boyle, Michael D. P., BioTechniques 
Nov./Dec. 1984, pp. 334-339. This publication and the publications recited 
therein are incorporated herein by reference thereto. 
Immobilization supports or substrates which can be used in the subject 
invention processes are any inert material generally used in 
immunochemical assays. Examples of such materials are beads formed of 
glass, polystyrene, polypropylene, dextran or other material. Other 
suitable solid phases include tubes or plates formed from or coated with 
these materials. Commercially available supports are agarose beads, 
IMMUNOBEADS (a solid phase affinity support reagent available in an 
activated form for the immobilization of proteins to the support) and AFI 
GEL 15 (a solid phase affinity support reagent for the immobilization of 
ligands that possess a primary amino group) activated beads. These are all 
trademarks of Bio-Rad, Richmond, Ca. 
The antisera of the invention can be utilized in any immunoassay method 
which involves the ability of an antibody to recognize or react with an 
antigen or antigenic determinant (epitope) and the detection or assay of 
the resulting antigenantibody complex. 
As disclosed previously, the components to be assayed may be coupled or 
bonded to any assayable ligand such as a radioisotope, fluorescent tag, 
bio-assayable enzyme, electron dense tag, and the like. Those skilled in 
the art, having been exposed to the principles of the present invention, 
will be cognizant of the types of assayable ligands and methods for 
coupling them to the components of the methods of the present invention 
without the exercise of undue experimentation or inventive faculties. 
For convenience and standardization, reagents for the performance of the 
assay can be assembled in assay kits. Two types of kits can be used. 
One kit would provide for the rapid detection of unique antigens in which 
immobilized antiserum to native components within the c protein marker 
complex, for example, .alpha., .beta., .gamma., and .delta., are mixed 
with group B streptococcal bacteria and agglutination is measured. A 
positive agglutination reaction indicates the presence of the 
corresponding antigen to the immobilized antibody. Thus, this type of kit 
would contain 
(a) immobilized antiserum; .alpha., .beta., .gamma., .delta.; 
(b) a culture of intact group B streptococcal bacteria; 
(c) a negative control using immobilized normal serum; and 
(d) a positive control using bacteria known to express the desired antigen. 
Another type of kit uses immobilized antigens from the c protein marker 
complex to quantify levels of specific antibodies. This testing can be 
used to identify individuals having a potential risk for infection by 
group B streptococcal bacteria carrying the particular antigen. This type 
of kit would contain 
(a) immobilized antigen; 
(b) Fc reporter, e.g., a radioisotope, an optical label, an enzyme, an 
electron dense ligand, and the like; 
(c) normal serum control (no antibodies to any c protein marker); and 
(d) positive control containing specific antibody to the corresponding 
immobilized antigen. 
If the label is an enzyme, an additional element of the kit can be the 
substrate for the enzyme. 
Examples of enzymes which can be used as a label are lactoperoxidase, 
horseradish peroxidase, alkaline phosphatase, glucose oxidase or 
.beta.-glucuronidase. 
Examples of radioisotope labels which can be used are .sup.125 I, .sup.131 
I, .sup.3 H, .sup.14 C or .sup.35 S. 
Examples of electron dense ligand labels which can be used are ferritin, 
gold or horse-radish peroxidase. 
EXAMPLE 7 
Vaccines 
The novel .gamma. and .delta. antigens described herein can be used 
advantageously in an immunogenic composition such as a vaccine. Such a 
composition, when administered to a person or animal, raises antibodies or 
other immune response which reduces the susceptibility of that human or 
animal to GBS infection. 
Vaccines comprising one or both of the GBS antigens, disclosed herein, and 
variants thereof having antigenic properties, can be prepared by 
procedures well known in the art. For example, such vaccines can be 
prepared as injectables, e.g., liquid solutions or suspensions. Solid 
forms for solution in, or suspension in, a liquid prior to injection also 
can be prepared. Optionally, the preparation also can be emulsified. The 
active antigenic ingredient or ingredients can be mixed with excipients 
which are pharmaceutically acceptable and compatible with the active 
ingredient. Examples of suitable excipients are water, saline, denrose, 
glycerol, ethanol, or the like, and combinations thereof. In addition, if 
desired, the vaccine can contain minor amounts of auxiliary substances 
such as wetting or emulsifying agents, pH buffering agents, or adjuvants 
such as aluminum hydroxide or muramyl dipeptide or variations thereof. 
Also, cholera toxin subunit B or other agents which stimulate antibody 
production at mucosal sites can be used. In the case of peptides, coupling 
to larger molecules such as KLH or tetanus toxoid sometimes enhances 
immunogenicity. The vaccines are conventionally administered parenterally, 
by injection, for example, either subcutaneously or intramuscularly. 
Additional formulations which are suitable for other modes of 
administration include suppositories and, in some cases, oral 
formulations. For suppositories, traditional binders and carriers include, 
for example, polyalkalene glycols or triglycerides. Suppositories can be 
formed from mixtures containing the active ingredient in the range of 
about 0.5% to about 10%, preferably about 1 to about 2%. Oral formulations 
can include such normally employed excipients as, for example, 
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, 
sodium saccharine, cellulose, magnesium carbonate, and the like. These 
compositions can take the form of solutions, suspensions, tablets, pills, 
capsules, sustained release formulations or powders and contain from about 
10% to about 95% of active ingredient, preferably from about 25% to about 
70%. 
The compounds can be formulated into the vaccine as neutral or salt forms. 
Pharmaceutically acceptable salts include the acid addition salts (formed 
-with the free amino groups of the peptide) and which are formed with 
inorganic acids such as, for example, hydrochloric or phosphoric acids, or 
such organic acids as acetic, oxalic, tartaric, mandelic, and the like. 
Salts formed with the free carboxyl groups can also be derived from 
inorganic bases such as, for example, sodium, potassium, ammonium, 
calcium, or ferric hydroxides, and such organic bases as isopropylamine, 
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. 
The vaccines are administered in a manner compatible with the dosage 
formulation, and in such amount as will be therapeutically effective and 
immunogenic. The quantity to be administered can depend on the subject to 
be treated and the degree of protection desired. Advantageously, methods 
known to promote mucosal immunity can be combined with systemic immunity 
promoters to maximize protection against GBS. Also, the .gamma. and 
.delta. antigens of the subject invention may be combined with 
carbohydrate antigenic components to enhance the immunogenic response and 
provide a broader range of protection. The combination of these antigens 
may be, for example, through chemical coupling. Precise amounts of active 
ingredient required to be administered depend on the judgment of the 
practitioner and can be peculiar to each individual. However, suitable 
dosage ranges are of the order of about several hundred micrograms active 
ingredient per individual. Suitable regimes for initial administration and 
booster shots are also variable, but are typified by an initial 
administration followed in one or two week intervals by a subsequent 
injection or other administration. 
Advantageously, the vaccines of the subject invention can be formulated and 
administered in a manner designed specifically to induce mucosal immunity. 
It should be understood that the examples and embodiments described herein 
are for illustrative purposes only and that various modifications or 
changes in light thereof will be suggested to persons skilled in the art 
and are to be included within the spirit and purview of this application 
and the scope of the appended claims. 
TABLE 1 
______________________________________ 
Summary of Precipitin Assay and Two Stage Radioimmunoassay 
to Type Group B Streptococci for Carbohydrate Antigens 
Detected by Detected by 
Strain Precipitin Test 
RIA 
______________________________________ 
N86K No type No type 
NP1AR No type No type 
HG824 Ia Ia 
SS617 Ia Ia 
HG346 Ia Ia 
PF534AR Ia Ia 
J46 Ia Ia 
HG783 Ia Ia 
HG784 Ia Ia 
HG381 Ia Ia 
A909 Ia Ia 
HG812 Ib Ib 
HG806 Ib Ib 
2AR Ib Ib 
TC795 Ib Ib 
PF549AR-B Ib Ib 
HG805 Ib Ib 
HG769 Ib Ib 
TC137 Ib Ib 
SS618 Ib Ib 
HG811 II II 
PF58AV II II 
PF536AR II II 
PF549AR-NB II II 
PF541AR II II 
J44 II II 
PF65BV II II 
PF610AR II II 
NPF1AV II II 
HG818 II II 
HG819 II II 
9B200 II II 
HG768 II II 
HG804 II II 
HG774 II II 
HG782 II II, III 
HG820 III III 
VC75 III III 
HG780 III III 
HG757 III III 
HG814 III III 
HG754 III III 
HG802 III III 
HG738 III III 
J48 III III 
J52 III III 
PEH III III 
J51 III III 
HG786 III III 
HG828 III III 
HG770 III III 
HG771 III III 
______________________________________ 
TABLE 2 
______________________________________ 
Distribution of .alpha., .beta., .gamma., and .delta. Antigens on Strains 
Reactive with 
Anti-c Typing Antisera Detected by RIA 
c Protein c Protein 
Reactivity Marker Antigens 
Carbohydrate 
Detected by Detected 
Strain Subtype Precipitin Test 
by RIA 
______________________________________ 
HG346 Ia + .alpha. 
PF534AR Ia + .alpha. 
J46 Ia + .alpha. 
HG783 Ia - .alpha. 
HG784 Ia - .alpha. 
HG381 Ia + .alpha., .beta., .gamma., .delta. 
A909 Ia + .alpha., .beta., .gamma., .delta. 
HG812 Ib + .alpha., .gamma. 
HG806 Ib + .alpha., .gamma. 
2AR Ib + .alpha., .gamma. 
TF795 Ib + .beta. 
PF549AR-B 
Ib + .alpha., .beta., .gamma. 
HG805 Ib + .alpha., .beta., .gamma. 
HG769 Ib + .alpha., .beta., .gamma. 
TC137 Ib + .alpha., .beta., .gamma. 
SS618 Ib + .alpha., .beta., .gamma. 
J44 II + .alpha. 
PF65BV II + .alpha. 
PF25AR 11 + .alpha. 
PF610AR II + .alpha. 
NPF1AV II + .alpha. 
HG818 II - .alpha., .gamma. 
HG819 II - .alpha., .gamma. 
9B200 11 + .alpha., .gamma. 
HG768 II - .alpha., .gamma. 
HG804 II - .alpha., .gamma. 
HG774 11 - .alpha., .gamma. 
HG782 II, III - .alpha., .gamma. 
J48 III - .delta. 
J52 III - .delta. 
JB3 III - .delta. 
J51 III - .delta. 
HG786 III - .delta. 
HG828 III - .delta. 
HG770 III - .delta. 
HG771 III - .delta. 
______________________________________ 
TABLE 3 
______________________________________ 
Depletion of Component Reactivities from Anti-Ibc 
Typing Antiserum 
Strain(s) Used Reactivities 
Reactivities 
For Adsorption Depleted Remaining 
______________________________________ 
R-28 (Group A) R .alpha., .beta., .gamma., .delta. 
PF65BV .alpha. .beta., .gamma., .delta. 
TC795 .beta. .alpha., .gamma., .delta. 
J48 .delta. .alpha., .beta., .delta. 
TC795, PEH .beta., .delta. 
.alpha., .gamma. 
9B200, PEH .alpha., .gamma., .delta. 
.beta. 
PF65BV, TC795, J48 
.alpha., .beta., .delta. 
.gamma. 
PF549AR-B .alpha., .beta., .gamma. 
.delta. 
HG381 .alpha., .beta., .gamma., .delta. 
NONE 
______________________________________ 
TABLE 4 
______________________________________ 
Summary of Typing Results (53 Strains) 
Carbohydrate % of % of 
Antigens No. All Strains 
Ic Strains 
______________________________________ 
No Type 2 3.8 
Ia 9 16.0 
Ib 9 17.0 
II 16 30.2 
III 16 30.2 
II/III 1 1.9 
Ic protein marker 
36 67.9 100.0 
.alpha. 27 50.9 75.0 
.beta. 8 15.1 22.2 
.gamma. 17 32.1 47.2 
.delta. 10 18.9 27.8 
______________________________________