INFECTION AND IMMUNITY, Sept. 1983, p. 1234-1244
0019-9567/83/091234-11$02.00/0
Copyright © 1983, American Society for Microbiology

Vol. 41, No. 3

Precipitating Cross-Reactions Among Pneumococcal Types

MICHAEL HEIDELBERGER
Department of Pathology, New York University Medical Center, New York, New York 10016

Received 20 April 1983/Accepted 7 June 1983

Data accumulated over many years are brought together on cross-reactions of
46 among the more than 80 pneumococcal serological types, with the idea of
correlating cross-reactions with the structures of the relevant type-specific
capsular polysaccharides, insofar as these have been determined. The precipitin
reaction was carried out with the polysaccharides and antibodies raised in horses,
rabbits, and a mule. Quantitative values (micrograms of antibody nitrogen per
milliliter of antiserum at 0 to 1°C) are given in many instances and discussed,
together with arbitrary qualitative data, in terms of the known structures of the
polysaccharides. Some precise relationships are uncovered, and an attempt is
made to determine why some of the cross-reactions are reciprocal and why others

are only unilateral.

 

Even before the structures of any of the
capsular type-specific polysaccharides of Strep-
tococcus pneumoniae (pneumococci) were
known, unilateral and reciprocal cross-reactions
between the many serological types were re-
corded (46, 58) as a result of attempts to raise
type-specific diagnostic antisera in rabbits.

The development and use of purified microbi-
al polysaccharides as vaccines are presently
expanding. There is also anticipation that dem-
onstrated cross-reactivities might afford some
measure of cross-protection. These consider-
ations and the rapidly increasing knowledge of
the structures of the relevant polysaccharides
prompted this collection of published and hither-
to unpublished data accumulated over the years.
Cross-reactions among the pneumococcal (Pn)
types, as demonstrated by the precipitin reac-
tion, are shown in the tables. Data, mainly
quantitative, from earlier papers have been add-
ed to make the tables more complete and be-
cause recently identified chemical structures of
the Pn capsular polysaccharides have made
some of the previously described cross-reac-
tions more explicable. Quantitative values of
antibody nitrogen precipitated are given for
most of the more marked reactions. Data are
given for cross-reactions of 46 serological types
of S. pneumoniae with anti-Pn sera of 36 types.
Cross-reactivity was measured by precipitation
of antisera by type-specific capsular polysaccha-
rides. Although the precipitin reaction is rela-
tively insensitive, this was considered an advan-
tage since positive results might be all the more
significant in relating chemical structure to im-
munological specificity, the main objective of
the study. Predictions of structural features of
Pn polysaccharides of unknown structure could

often be made from cross-reactivity in antisera
to types for which the structure of the capsular
polysaccharides was known. In most instances
these predictions were verified by subsequent
studies.

The cross-precipitations are assembled in Ta-
ble 1, and the reciprocal ones are brought to-
gether in Table 2 and treated separately. Insofar
as possible, the results are discussed in relation
to the chemical structures of the type-specific
capsular polysaccharides involved as dominant
immunogens in the raising of antisera and as
antigens in the cross-reactions.

MATERIALS AND METHODS

All tests were carried out at or near 0°C to obtain
maximal values. My co-workers and I have repeatedly
called attention to the usually larger differential shown
by cross-reactions between 0 and 37°C than is ob-
served with homologous precipitation. This is because
cross-reacting antibodies usually combine with only a
portion of the repeating unit of a polysaccharide,
sometimes a single sugar. It therefore is not surprising
that cross-precipitation at low temperatures does not
always parallel immunogenicity in humans or other
animals at 37°C or higher (cf. reference 41). An ex-
treme example of the difference in precipitation be-
tween 0 and 23°C is given in reference 19.

Methods used and sources of most of the polysac-
charides and antisera have been described in the
papers cited. Antisera containing appreciable amounts
of antibodies to the group-specific C-polysaccharide
were absorbed with this polysaccharide before use;
this is denoted by the letter C after the number of the
serum. Qualitative tests were allowed to stand in a
cold room at 3 to 5°C for 6 days or more and were read
ona scale of — to ++++; quantitative determinations
were placed in a cold bath at 0 to 1°C for 2 days to 2
weeks or longer, depending on the rapidity of precipi-
tation. Qualitative data are given within parentheses

1234
VOL. 41, 1983

when carried out in antisera which were negative or
weakly positive in other tests. Quantitative figures
within parentheses refer to single analyses instead of
the usual mean of duplicates. The following abbrevia-
tions are used: S, specific capsular polysaccharide;
Gal, galactose; Glc, glucose; Rham, rhamnose; Man,
mannose; GalA, galacturonic acid; GlcA, glucuronic
acid; GlcNAc, N-acetylglucosamine; dp, depyruvylat-
ed. Except as indicated, the antisera used had been
raised in horses. All sugars are in the pyranose form
unless indicated as furanoses by f, as in Galf.

RESULTS AND DISCUSSION

Cross-precipitation of other Pn type-specific
polysaccharides in anti-Pni. S3 gave small pre-
cipitates in three of the four equine anti-Pni sera
tested: 27 yg of antibody nitrogen per ml at 0°C
was the largest amount, vith 884C. S33 and
S10A (34 U.S.) precipitated 10 and 27 yg of N,
respectively, from 1057C. Although the signifi-
cance of these small reactions is not clear, a D-
Galf residue of S10A (11, 50) might fit into
antibody spaces designed for S1, the structure of
which is given (44) as:

-/3)-2-acetamino-4-amino-2,4,6-trideoxy-D-Gal
-a-(1 — 3)-p-GalA-a-(1 — 4)-p-GalA-a-(i-},

S2, S5, S6, S6B, and anti-Pn sera (references
17, 18, and 28). The structure of S2 is (37):

CROSS-REACTIONS OF PNEUMOCOCCAL TYPES

1235

appreciable (18), S5 failed to react in the anti-
Pn2 sera at hand.

S6B differs from S6 only in its 1,4-linkage
between L-Rham and ribitol (36); in S6 the con-
nection is 1,3- (52). If the specificities are deter-
mined almost entirely by the sugars, this would
explain why S6 and S6B precipitate practically
the same amount of antibody from anti-Pn6.
Otherwise S6B cross-reacts very weakly (+)
only in anti-Pn28 and therefore is omitted from
Table 1. Reciprocal cross-reactions of type 2 in
anti-Pn19, anti-Pn20, and anti-Pn23 are given in
Table 2 and discussed in a separate section, as
are the other reciprocal reactions encountered.

S4 and dp S4. Removal of pyruvic acid from
its acetal linkage on positions 2 and 3 of p-Gal
(43) in S4 occurs without marked degradation,
but dpS4 precipitates far less antibody (21) from
anti-Pn4, showing that the 2,3-pyruvyl-D-Gal is
immunodominant. However, dpS4 is more
cross-reactive than S4 (Table 1), owing to the
liberation of two hydroxyl groups of the Gal.
Moreover, dpS4 precipitates C-reactive protein
and antibodies to Pn C-polysaccharide almost
quantitatively (33). N-Acetyl-p-galactosamine is
the sugar common to PnC and dpS4, but the
extent of the reaction is unusual if only a single
sugar in the repeating units is actually the cause.
The 1,4-linked p-Gal (32) in dpS4 presumably is

 

3)-L-Rham-a-(1 — 3)-L-Rham-a-(1 > 3)-L-Rham-B-(1 > pecan

Teac ~ 6)-D-Gle-c-(1'?

Accordingly, there is no 1,4,6-linked Glc, as
previously thought, nor is there GlcA in the
main chain, so the discussion of the Pn2-Pn20
cross-reaction in reference 30 requires revision.
The immunodominant group of S2 is the isomal-
touronic side chain (29), making it possible that
the phospho-«-p-glucosyl residue of S20 (52a),

 

responsible for the acquisition of cross-precipi-
tation in anti-Pn8: intact $4 does not precipitate.
Reactivity of both forms in anti-Pn14 probably
occurs because the 1,3-linked N-acetyl-p-galac-
tosamine of S4 (34) fits partially into antibody
sites designed for 1,3-linked p-Gal.

S7 and S7B. Although the structure of S7 is

 

—Porancied — 6)-B-D-Glc-(1 — 3)-B-p-Galf-(1 > 3)-B-p-Gle-(1 — 3)-a-D-GleNAc-(1 > ofo +
OH J*

(-OAc, partial)

with or without the adjacent a-1,6-linked D-Glc,
might fit partially into combining sites on anti-S2
molecules designed for insomaltouronic acid. As
$18, like S2, has Glc in 1,4- and 1,6-linkages and
1,3-linked L-Rham in addition (16), it is not
surprising that S2 precipitates anti-Pn18. The
reverse reaction does not occur with the anti-
Pn2 sera available.

A tentative structure for S5 retains the 1,2-
linked p-glcA (B. Lindberg et al., unpublished
data). Although precipitation of anti-Pn5 by S2 is

B-D-Galf-(1t*

 

not yet fully known, nonreducing lateral end
groups of p-Gal in the repeating unit have been
identified (10), as surmised in reference 30 and
predicted in reference 56 because of the cross-
reaction of S7 in anti-Pn14. Precipitation in anti-
Pn19 is probably due to the 1,4-linked D-Glc, as
well as to 1,2-linked L-Rham in S19 (47) and S7.
The long-known reaction of S7B in rabbit anti-
Pn24 (7) was confirmed. Several other weak
cross-reactions were observed (Table 1). The
structure is not known.
1236 HEIDELBERGER

INFECT. IMMUN.

TABLE 1. Cross-precipitation of Pn capsular polysaccharides in anti-Pn sera

Cross-precipitation® in anti-Pn:

Polysaccharide*

 

 

1 2 3 4 5 6 7 8 9 10 u
Homologous 1,024 4,000 600 2,390 4,060 724 893 1,288 1,655 864 792
Sl ++ + ++ - = - z - + -
2 (-) + - 637 21° + + + ¥ +
3 +++ + + + + 205 + + -
4 0 - ~- - - + - + + -
dp4 26 ++ 20 765 + 10 + 70 ++ ++ +
5 + of ++ ++ - - 58 - ++ -
6 (-) or (~) (-) (-) (+) (-) (-) (+) (+)
7 (-) (-) (-) (+) (-) (-) 62 + (-) +
7B (48 U.S.) + + + + - - + - - - -
8 (-) (-) 13 (+) {~) (-) (-) (-) (-) (-)
9 - - + + - - - 9 - -
9A (33 U.S.) (-) (+++) (-) (+) (-) (-) (-) (-) (++++) (~)
10 [ (-)to(+) } (+++) [ (-) to (+) J -
10A G4 U.S.) +++ + (-) (+) (+) (-) + +++ (-) $1 (+)
11 (+) (-) (-) (-) (-) ++ (+) (+) (-) (-)
11A (43 U.S.) +H ~- (-) +44 (-) (-) (-) (-) (-) 125
12 (+ (-) (+) + (+) + + (-) (-) - -
12A (83 U.S.) - + + - ~ +++ - - +++ - -
13 - - - - - ++ - - ~ 25 -
14 + + - + + 17° 39 - - 19 -
15 i i - (78) ~ + - + 62 (++)
15A (30 U.S.) - - - (77) - - - - - 28 -
15B (54 U.S.) - - + +42 - - - - ~ - (~)
1SC (77 U.S.) ++ + + +++ + + ++ ++ - ++ +
16 (-) (-) (-) (+) (-) (-) ++ (-) (-) (-) (=)
18 - + - - - 10 v 4y - + -
18A (44 U.S.) - - - - - - - - + + (++)
18C (56 U.S.) - - ++ + - - - ~ -
19 (++) 20° (++) (-) (~) (-) 19 105 (-) (-) -
19A (57 U.S.) + - ++ ++ + ++ - ++ ~ ++
20 + 56 - + _ + - + + 16% -
22 ++ +44 - - - - - ++ ~ +++ -
23 (-) 7 { (-) to (+) ] -
24 - - + 47" +44 - - 68 ~ + -
24A (65 U.S.) - - - - - - - - - -
25 + - ~ +++ ++ - + +++ i 43 -
27 - - ~ 140" - - - - 11° (-)
28 - +++ - - RA - - - + (-)
29 - + - + - ++ - - ++ 61 (-)
32 (-) (~) (-) (++) (-) (=) (+) (++) (+) (-) (+)
32A (67 U.S.) - + ++ + _ + ++ + + + -
37 [ All (—) or (+) ] +
72 ( All — or + through 15

 

S9 (Danish 9N). A tentative structure, possibly
overly complex, has been proposed for S9 (14).
Simpler repeating units were shown to occur in
other members of the group i.e., in 9A (type 33
U.S.) (6) and 9V (type 68) (49). Cross-reactions
within the group are discussed in reference 55:
p-GlcA was immunodominant in 9A, 9V, and 9L,
since reduction of -COOH to -CH,OH greatly
diminished serological activity. In the present
study, only small cross-reactions of 9N were

found with anti-Pn’s 3, 8, 12, 14, 18, 19, 22, and
25. S9 and the capsular polysaccharides of all of
these types except possibly $25, the structure of
which is not known, contain 1,4-linked D-Glc in
their repeating units. This may be the cause of
these minor precipitations.

S10 and S10A (34 U.S.) The structure assigned
for S10 (M. B. Perry, V. Daoust, and R. Lowe,
Abstr. W.H.O. 3rd Int. Conf. on Immunity,
Immunization, and Cerebrospinal Meningitis,
VoL, 41, 1983 CROSS-REACTIONS OF PNEUMOCOCCAL TYPES = 1237

TABLE 1—Centinued

 

 

 

12 14 15 16 18 19 20 22 23 25 27 28 29
1,240 1,010 70 872 «2,200 «2,250 355 878 420 620 277 78S 389
1 ~ + + + + - - - 3° + - ++
2 + + - + - F 4 +4 ++ ++ ~ ~ ++
3 £ + ~ - + - - - - + ~ ~ +
4 5 65 - - - - - ++2 + 16° 18” - ++
4 t 537 - 25 - ++ 29 ++ ++ x" 25% + ++
3 ++ + + - ++ a i + + - a -
6 (~) 15° (-) (-) ++ + + = - (+) ff) (© +
7 +e 3 (-) (-) 91 Yo +e (-) 220 (+++) (+) ++ (+)
7B * + = - ++ + + - ++ + ~
8 - (-) - - is 12% (-)— (#) (-) + ~ ©) ©)
9 + 11! - ~ 2 (-) = 10 x ++ - -
9A
10 ~ fo +t +++ - + 39 + ~ axe 86 + 57
WA = (#) ++ (-) @ © © @© @) © IB 5 - 2%
u (-) 448 + - + - + - + + ~ ~
A (-) (682) ++4++ 278 -) () - - +++ - ~ ++
12 45 - ~ (-) (4) (€) (-) (-) (+) (-) () -
WA +4+4+ + + ~ + + - - - ~ - ~
13 - +t - ~ ~ ~ ++ - ~ ++ ~ ~ ~
14 (~-) (21) (-) ~ + — +++ - ++ - - ++
15 (-) 47 (-) S} (-) ++ - - te +44 ~ ++
15A - ttt 301 - - + - + 7 ++ 95 ~ ~
15B (-) +444 293 (+) +44 (-) (+) - ++ - + - -
15C ++ +b +44 - - + - -~ +++ +42 - ++
16 (+) ~) (-) (-) (4) (4) - ~ ~ +2 +
18 - - ++ +++ + + +++ + - + ~
18A (+) (2) G) Geet) FH) - ® © &® ©} -
18C - - - 150 1,685 - = - ++ - - ~ -
19 + + - + ~ + - - - - +
194
20 + z ~ +++ + ~ ++ ++ - -
22 oa - ~ - - + + ++ (- or +)
23 + x - + ~ + ns (+) (++) (+) (++) (-}
24 - ++ - + - ~- = - + ++ + + ++
24A - ++ - ~ - + - ~- 109 ++ ~ 189 -~
25 = - ~ + ~ + - (+) -) () © (-)
27 (~) (2) (-) (+) + + + 12¥ + +
28 (~) (+) (~) (+) (-) (4) = - - +t ++z
23 ++ {-) (+) (-) (-) + - ++ + -
32 G+) ) ¢) (-) (-) 28 (+) (+) (+++) (~) (++2) (+) (*)
32A - ++ - +e +++ - + ~ 13400 +44 38 ++ 00 +H
37 34 (++) (+) G) (-} (+)
nR ] 290 (~) -) ©) (~) (-) (-) i) () (~)

 

* $17 was + only in anti-Pn7 and anti-Pn9; S17A was ++ in anti-Pnil and + in anti-Pn's 12, 15, and 20. They are omitted from
the table.

* Data are expressed either qualitatively (see text) or quantitatively (as micrograms of antibody N per milliliter).

© From reference 1.

¢ From reference 18.

* From reference 28,

/ From reference 30.

* From reference 21.

* From reference 16.

‘ From reference 49a.

4 In anti-Pnl 1057C; ~ in anti-Pnl 884.

* 44+ in rabbit anti-Pnl1 59 (New York State).

' From reference 22.

™ Mean of 43 and 50.

* dpS27 gave 176 yg of N per mi.

° dpS27 precipitated 45.

” Precipitation by dpS27.
1238 HEIDELBERGER

Marburg, 1980) is:

INFECT. IMMUN.

Q
—parsonoto-.o — 6)-B-D-Galf-(1 — 3)-a-p-Gal-(1 — 4)-B-p-GalNAc-(1 > .0-Gghf

OH

and that for S10A (11, 50) is:

B-p-Gal-(1!¢

9
—pamiioi.0f.0--0carc —> 3)-p-Gal-(1 — 4)-p-GalNAc-(1 > sooanf

OH
(-OAc, partial)

In spite of structural similarities, cross-precip-
itation of S10 in rabbit anti-Pnl10A (R13 N.Y.
State) was only about 80 of 1,965 wg of N per ml
and 6% of the total in the reverse direction with
H627. There were marked differences in the
cross-reactivities of S10 and S10A in other anti-
sera (Table 1). Rabbit anti-10A was tested only
with S1, $8, $14, S25, S27, and S29, giving +,
+,2, ++, -, and ++, respectively.

S11 and S11A (43 U.S.). The structure for $11
(M. B. Perry, personal communication) is:

p-Galf-(116

 

respectively, but S11A gave +++ + and 278 pg
of N per ml (Table 1). As pointed out in refer-
ence 38, S11A and S18 have the probable se-
quence —3)-D-Gal-(1—>4)-a-p-Glc-(1-6)-p-Glc-
(1- and also glycerophosphate and O-acetyl
groups (16) in common, so that massive cross-
reactivity is to be expected. The structure of S16
is not known.

$12, S12A (83 U.S.), and S13. The only
marked cross-reaction of S12 was in anti-Pn14
635C and was predictable since both S12 (15, 42)

 

6)-a-D-GlcNAc-(1 — 4)-a-p-Gal-(1 — 3)-B-p-Gal-(1 > 4)-B-p-Glc-(* ] -

OT
Ribitol-P-O
OH
and that for S11A (38) is:
OAc

-OAc J

2 |
6)-a-D-Glc-(1 — 4)-a-D-Gal-(1 -> 3)-B-p-Gal-(1 — 4)-B-p-Glc-(1

4

|o-B-0.CH,-CH(OH)-CH,OH
OH

Removal of the acetyl groups from S11A abol-
ished the precipitin reaction in a rabbit anti-
Pni1A (38). The smaller-than-expected precipi-
tation of S11A in anti-Pn11 613 (16%) is perhaps
due to blocking of antibodies by the as yet
unlocated -OAc groups in S11 and S11A. Both
polysaccharides react heavily in anti-Pn15 528C,
probably because of the -OAc sugar in their
repeating units, since O-deacetylation of S15, as
with S11A, abolishes its precipitation in homolo-
gous rabbit serum. Precipitation of S4, S25, and
S29 in a potent rabbit anti-11A was ++, +, and
++, respectively. Precipitation by S11 in anti-
Pn16 594C and anti-Pn18 495 was only + and —,

-OAc

 

and S14 (4, 45) have nonreducing lateral end
groups of p-Gal in their repeating units. S12A
precipitated anti-Pn12 very heavily and anti-Pn6
and anti-Pn9 only moderately. S12A differs from
S12 only in the substitution of N-acetyl-p-gluco-
samine for N-acetyl-D-galactosamine in the main
chain. (K. Leontein, B. Lindberg, J. Lonngren,
and D. J. Carlo, personal communication).

$13 (57) gave only small cross-precipitations
(Table 1). In anti-Pn6, reactivity might be due to
a small amount of anti-ribitolphosphate in the
antiserum; in anti-Pn14 and anti-Pn20, it might
be due to 1,3-linked D-Gal.

S14. New methods have established a simpler
VoL. 41, 1983

structure for S14 (45):

CROSS-REACTIONS OF PNEUMOCOCCAL TYPES

1239

anti-Pn12, and anti-Pn27, but the structure of

 

4)-B-D-Glc-(1 > 6)-B-D-GlcNAc-(1 —> 3)-B-D-Gal-(1
B-p-Gal-1!4 :

Anti-Pn14 635C has been a useful reagent for
detecting and predicting the presence of im-
munodominant lateral p-Gal in many other mi-
crobial and plant polysaccharides. Cross-precip-
itation of S14 in anti-Pn22 is apparently caused
by the 1,4-linked p-Glc residues in the repeating
units of S14 and S22 (9).

S15, S15A, S15B, and S15C. The structure of
S15 (48) is:

 

S16 is not known.

S17 and S17A (78 U.S.). S17 and S17A are
omitted from Table 1 because the strongest
reaction was of S17A, ++ in anti-Pnl1 613.
However, S17 (for structure, see M. B. Perry,
D. R. Bundle, V. Daost, and D. J. Carlo, Can. J.
Biochem. Cell Biol., in press) precipitated mas-
sively in a rabbit antistreptococcal group F, type
4 serum (31), as did the group F type 4 polysac-

 

eee 3)-B-D-Gal-(1 — 4)-B-p-Glc-(1 — 3)-a-p-Gal-(1 > 2)-D-Gal-1
-OAc

-OAc

$15 and S15A precipitated anti-Pn4 609C to the
same extent. Possibly the 1,3-linked D-Gal resi-
dues of S15 fitted partially into combining sites
of anti-Pn4 designed for the 1,3-linked p-Gal-
NAc residues of S4. The structures of S15A,
S15B, and S15C have not yet been determined,
but S15C has the same main chain as S15B but
lacks the -O-acetyl group of the latter (P. E.
Jansson, unpublished data). In this group, S15
reacted most heavily in anti-Pn10 627C, S15A
and S15C precipitated less, and S15B did not
react at all. The 1,3-linked residues of D-Gal in
S15 and S29 are apparently the mediators of the
reaction of S15 in anti-Pn29, whereas the 1,4-

 

t
(CH) NCH BOF O a

 

charide in rabbit anti-Pn17 (N.Y. State no. 29).
S17A has several structural differences from that
of S17 (P. E. Jansson et al., in press).

S18, S18A (44 U.S.) (22), and S18C (56 U.S.).
Two alternative probable structures have been
proposed for S18 (16). 1,4-Linked pD-glucosyl
residues in $18 and S23 are undoubtedly respon-
sible for the cross-reaction of $18 in anti-Pn23.
As the structures of S16, S18A, and S18C are
not known, reasons for the heavy cross-precipi-
tation of the $18 group in anti-Pn16 cannot be
given.

$19 and S19A (57 U.S.). The structure of S19
(47) is:

O
—forp-osanniacd — 4)-a-D-Glc-(1 — 2)-a-L-Rham-(1 — ofof
OH J"

 

linked residues of p-Glc are undoubtedly the
reason for precipitation of S15 in anti-Pn20 616.
S15A and S15B precipitate anti-Pn15 to the same
extent but differ sharply in anti-Pn14, anti-Pn18,
and anti-Pn27. Because of the strong reaction of
S15B in anti-Pn14, S15B may be expected to
have a lateral nonreducing end group of p-Gal in
its repeating unit; S15SC gave only +. Although
SISA precipitated anti-Pn4 609 quite strongly
(Table 1), the reciprocal reaction in a potent
rabbit anti-Pn15A was only ++. Precipitation of
S4 in rabbjt anti-Pn15B was only (+). $20,
however, gave ++, but the reciprocal reaction
was only (+).

S16. Small cross-reactions occur in anti-Pn7,

In anti-Pn2 and anti-Pn7, S19 gave definite pre-
cipitates, but S19A (40, 41) did not (Table 1).
Precipitation of anti-Pn8 1008 by S19 was much
stronger than that by S19A, probably because of
inhibition by the side chains of S19A on the 1,4-
linked Glc. S19A reacted weakly in anti-Pn4,
anti-Pn5, anti-Pn6, and anti-Pn10, in which S19
was negative. Except for 1,3-linked p-Gal in $10
and S19A, the reasons for the other reactions are
obscure.

S20 (p. 1237) and S21. The strong precipitation
of $20 in anti-Pn18 appears to be due to the 1,6-
linked p-glucosyl and 1,3-linked D-galactosy!
residues in the repeating units of both $18 and
S20. The latter residues and those of 1,4-linked
1240 HEIDELBERGER

p-Gle probably account for the weaker reaction
in anti-Pn27. S21, — to + in all antisera tested, is
omitted from Table 1.

$22. As in other instances, the stronger reac-
tions (Table 1) of S22 (9) could be due to the 1,4-
linked p-linked D-Glc of the polysaccharides of
the types involved.

S23. The weak reaction in anti-Pn2 might be
caused by 1,4-linked pD-Glc even though (or
because) it is substituted by a phosphoryl group
on S23 (Perry et al., Abstr. W.H.O. 3rd Int.
Conf. in Immunity, Immunization, and Cerebro-
spinal Meningitis, 1980.) D-GicPO, might even
fit loosely into the combining sites of anti-Pn2
meant for p-GIcA.

S24 and 824A (65 U.S.) (composition and struc-
tures not known). $24 precipitated in anti-Pn4
and anti-Pn 8 (Table 1), but S24A did not; both
reacted equally weakly in anti-Pn14 and nearly
equally weakly in mule anti-Pn25. S24A was the
only Pn S tested which precipitated strongly in
anti-Pn28 510C, giving 189 of wg N per ml. It
also gave 109 wg of N in anti-Pn23 912C; recipro-
cal reactions were negligible in the rabbit anti-
Pn24A available.

S25. Precipitation was ++ in anti-PnS and
+++ in anti-Pn8. S25 contains p-GalA (13), but
the structure has not been determined. Cross-
reactions are shown in Tables 1 and 2.

S27. S27 (5) was the first Pn S in which
pyruvic acid was found (21); because of the
cross-reactivity of the extracellular polysaccha-
ride of pyruvyl-containing Klebsiella type K32
in both anti-Pn27 and anti-Pn4, pyruvic acid was
suspected in S4 as well and was identified (21).

$28. The structure of S28 is not known. Cross-
reactions are given in Table 1.

$29. S29 has two residues of 1,6-linked p-Galf
in its repeating unit (51), and these might func-
tion as somewhat hindered nonreducing end
groups. This would explain the reactivity of $29
in anti-Pn6 and anti-Pn14. Both S29 and S14
have 1,3-linked p-galactopyranose in their re-
peating units. This could reinforce the precipita-
tion in anti-Pni4 and account for that in anti-Pn9
as well, since S9 contains 1,3-linked p-GalNAc
which would be partly equivalent immunologi-
cally to similarly bound D-Gal.

S32 and S32A (67 U.S.) (structures unknown).
$32 contains Gal, Glc, Rham, and uronic acid (1)
and does not precipitate as strongly in anti-Pn23
(a long-known cross-reaction [46, 58]) as does
S32A (Table 1). The latter, at least, may there-
fore be assumed to have nonreducing lateral end
groups of L-Rham in its repeating units (P. Allen,
B. Prescott, and M. Heidelberger, unpublished
data); phosphorylcholine is also a constituent.
S32A reacts in anti-Pn’s4, 14, 16, 18, and 25,
whereas S32 does not, but gives 28 wg of N in
anti-Pn19 631C. Both cross-reacted roughly
equally in anti-Pn27.

INFECT. IMMUN.

S37. The only noteworthy cross-reaction of
the unusual glucan S37 (39) (not tested in all
antisera) was in anti-Pn12 296C, giving 34 pg of
N per ml. S37 is said to have the nonreducing
lateral end groups D-Glc-B-(1—2)-p-Glc-B-(1-, in
contrast to the corresponding a-linked kojibiosyl
of $12 (15).

$72. All tests of S72 were — to +, except for
heavy precipitation in anti-Pn16 594C (290 pg of
N per mi). As structural formulas have not been
proposed for S16 or S72 (12) and there are
several sugars in common, an explanation is
presently lacking.

RECIPROCAL CROSS-REACTIONS

‘Reciprocal, as well as unilateral, cross-reac-
tions were often encountered in early efforts to
obtain a reliable supply of strictly type-specific,
diagnostic rabbit anti-Pn sera (46, 58). In this
part of the present paper, an attempt is made to
find out why some cross-precipitations between
serological types are unilateral and others are
reciprocal. One factor that might be involved is
that different animals of a single species produce
antisera mainly reactive toward different por-
tions of the repeating units of bacterial polysac-
charides (see, for example, reference 28). There-
fore, if one could have a sufficiently large
collection of potent antisera, possibly all cross-
precipitations would be reciprocal. It is also
likely that a target sugar in the main chain of the
more highly branched or more highly O-acetylat-
ed of two polysaccharides would be relatively
inaccessible to cross-reactive antibodies. This
would tend to make a reaction unilateral. These
considerations apply also to bacterial agglutina-
tion, especially as this is a precipitation reaction
at the cellular surface (23).

Data on reciprocal cross-precipitations among
the Pn types are summarized in Table 2. Except
for certain intergroup reactions, those are omit-
ted which are recorded as less than + + in either
direction. Types 2,5 and 2,6 are included, even
though S5 and S6 failed to precipitate several
anti-Pn2 sera, since types 5 and 6, originally 2A
and 2B, were ‘‘weakly and incompletely aggluti-
nated’’ (3) in anti-Pn2.

Types 2,5; 2,6; 2,19; 2,20; and 2,22. Reciprocal
cross-precipitation of types 2,5 is undoubtedly
due to the lateral nonreducing end groups of pD-
GlcA in the repeating unit of S2 and 1,2-linked
b-GicA in S5. Like 1,2-linked D-Gal in S6 (52),
1,2-linked p-GlcA in S5 may react serologically
as a somewhat hindered lateral end group (18).
The cross-reaction may also be reinforced by the
1,4-linked p-Glc in the main chains of both S2
and SS, a feature presumably also involved in
the two-way 2,19 reaction. The rather small
precipitation in the 2,6 pair is referable to the
1,3-linked L-Rham in the repeating units of both.
It is difficult to account for the 2,20 pair unless
 

 

VOL. 41, 1983 CROSS-REACTIONS OF PNEUMOCOCCAL TYPES 1241
TABLE 2. Reciprocal cross-precipitation of pneumococcal types?
Polysaccharide Antiserum Polysaccharide Antiserum
2° V555 63, V606 79, VI681 21 5, 6, 19, 20, 22 11464 +, [1513 0, 20,
VI771 155, X1X631 8, 56, +++
XX616 44, XXIIS66 ++
3 VIII1008 205 8 111792 113
4 RXXIV ++, MXXV 16, 24, 25, 27 IV609 50, +++,
XXVII668 18° 1407
5 RXXIV +++ 24 V555 ++
6 XIV635 15, XVITI495 ++, 14, 18, 29 VI 681 17, 10, ++
XXIX641 ++
7 XIV 34, XVIII 91 14, 18 VII937 35, 7
8 XVIII 14, XIX 127, RXXIV 18, 19, 24 VIII 47, 105, 68
+++
10 RXA < 79, XIV 15, XV628 10A, 14, 15, 15A, X627 51, 79, 62, 28
++, RXVA, +++ XX 20, 25, 27, 29 163, 43, 11°, 61
35, MXXV 49, XXVII 86,
XXIX 57
10A XXIX 20 29 RXA ++
11 RXIA +++ A XI1613 125
11A XV (682), XVI594 ++++, 15, 16, 18 RXIA ++, ++++4,
XVIII 278 ++
14 XV 21, RXVA +++ 15, 15A XIV 47, +++
15 RXVA ++++, RXVB 15A, 15B, 18 XV 301, 293, ++
++++, XVIII 57
15A RXVB ++++, XXVII 95 15B, 27 RXVA 7%, +++
18 RXVIIA 321", RXXXIIA 18A, 32A/ XVII 357", +++
+++!

 

 

* Qualitative data are expressed ona scale of — to + +++; quantitative values are expressed as micrograms of
antibody nitrogen per milliliter of antiserum. M, Mule; R, rabbit (antisera without these letters were raised in
horses). Sera containing appreciable antibody to Pn group-specific C-polysaccharide were absorbed with C
before use. In this table serum types are given in Roman numerals to avoid confusion with serum numbers and

amounts of precipitated nitrogen.

» See text for reason why group pairs 2,6 and 6,2 are included.

© dpS4 gave 25 pg of N.

4 dpS27 gave 176 yg of N.
* dpS27 gave 45 ng of N.

F Single determination only.
® Test was not made.

* From reference 22.

‘P. Z. Allen, B. Prescott, and M. Heidelberger, unpublished data.

the glucose PO, of S20 could fit loosely into
combining sites of anti-Pn2 designed for GlcA.
As for types 2 and 22, the polysaccharides of
both contain sugars bound in the same ways, yet
the cross-precipitations are small.

Types 3,8. Since one-half of S8 consists of the
repeating unit of S3 (35), heavy mutual cross-
precipitation is to be expected. This long-known
reciprocal cross-reaction has been extensively
discussed (24, 25), as have also the inhibitory
properties of oligosaccharides formed on partial
hydrolysis of each S (8). The strong reciprocal
cross-precipitation between types 8 and 19 may
now be accounted for by the presence of a- and
B-1,4-linked p-Glc (35) in S8 and by a-1,4-linked
D-Gle and #-1,4-linked p-ManNAc (partially
equivalent immunologically to p-Glc) in S19.

Types 4,24; 4,25; and 4,27. S24 and S25, of
unknown structure, appear not to have been
tested for pyruvic acid. This acid, however, has

little to do with the reciprocal cross-reactivity of
4,27, as precipitation is greater with the dp
polysaccharides (Table 1). A possible reason for
the reaction is the partial serological equivalence
of 1,4-D-ManNAc in S4 and 1,4-linked p-Glc in
$27.

Types 5,24. There is no explanation for the
reciprocal reactions of 5,24 at present.

Types 6,14; 6,18; and 6,29. The rather weak
reciprocal cross-reactions of 6,14 are accounted
for by the evidence (28) that the 1,2-linked p-Gal
of S6 shows the serological behavior of a partial-
ly hindered lateral end group of D-Gal such as
exists unobstructed in S14 (see above). The still
weaker 6,18 reactions reflect the presence of
1,3-linked L-Rham in both polysaccharides. In
the 6,29 pair, the two 1,6-linked pD-Galf residues
of S29 might fit loosely into antibody sites
designed for the p-Gal of S6.

Types 7,14 and 7,18. Reciprocal cross-reactiv-
1242 HEIDELBERGER

ity of types 7 and 14 is due to the nonreducing
lateral end groups of p-Gal in the repeating units
of S7 and $14, with probable reinforcement by
the B-1,4-linked p-glucosy! residues present in
both. The 7,18 pair also has 1,4-linked p-Glc
and, in addition, 1,3-linked L-Rham.

Types 8,18; 8,19; 8,24. Each repeating unit of
38 and $18 has two 1,4-linked p-Glc residues:
these appear to suffice for a limited degree of
reciprocal cross-reactivity. In the much heavier
cross-reactions of the 8,19 pair, it is possible that
the phosphoryl-(1->4)-8-D-ManNAc residues of
519 might fit to some extent into anti-Pn8 com-
bining sites intended for the cellobiouronic acid
groups of S8, aided and abetted by the 1,4-p-
glucosyl residues of $19. For the opposite direc-
tion, the cellobiouronic acid residues of S8 might
fit into the anti-Pn19 combining sites for PO,-
ManNAc residues. The composition and struc-
ture of S24 are not known.

9A, OL, 9N (9 U.S.), and 9V. Cross-precipita-
tion among the members of this group has been
studied quantitatively (55). Reciprocal crossing
between 9A and 9N is massive, as would be
expected from their similar main chains. Mutual
cross-reactivity of the 9A,9L pair is almost
complete, but the structure of 9L is not known.
9V has O-acetyl groups on GicA and Glc; these
probably give it an individual specificity since
SN does not precipitate the rabbit anti-9V used
(35).

Types 10,10A; 10,14; 10,15; 10,15A; 10,20;
10,27; 10A,25; and 10A,29. S10, SI0A, and S15
(see above for structures) have two 1,3-linked p-
galactosyl residues, and $14 and S27 have one in
their repeating units; these sugars appear to be
principally responsible for reciprocal reactions
10,10A; 10,14; 10,15; and 10,27. In the 10,20
pair the nonreducing lateral end groups of B-p-
Galf are probably the main cause, whereas S10A
and $29 have the same furanose in the chain of
sugars and 1,3-linked p-Gal in addition. The
structures of S15A and S825 are not yet available.

Types 11,11A and 11A,15. The structures of
$11, S11A, and S15 are given above. As all three
polysaccharides contain the sequence 1,3-8-p-
Gal-1,4-p-Glc in their repeating units, this ac-
counts for the reciprocal precipitation in antisera
to both pairs. Why the reaction of S11 and S11A
in anti-Pn15 is so massive is not clear.

Types 14,15 and 14,15A. The reason for these
reciprocal reactions appears to be the same Gal-
Glic sequence as described above, since this also
occurs in S14,

Types 15,15A; 15,15B; 15A4,15B; 15,18; and
15A,27. S18 also has the 1,3-p-Gal-1,4-p-Glc
sequence, but the anomeric forms of the sugars
were not determined (16). A rabbit anti-Pn15A
gave +++ with S27; S15A precipitated anti-
Pn27 668C similarly (95 pg of N per ml).

INFECT. IMMUN.

Types 18,18A and 18,32A. S18A contains all
components of S18 except the O-acetyl group
and has GIcNAc in addition (22), but its struc-
ture is not known, nor is that of S32A.

Types 19,19A. This reciprocal cross-precipita-
tion was demonstrated in reference 40. S19A
differs from $19 in that it has two side chains
which are lacking in $19 (40).

In summary, in most of the reciprocal cross-
reactions between Pn types of which the struc-
tures of the capsular polysaccharides are
known, at least two sugars or their immunologi-
cally partially equivalent amino analogs are pre-
sent in common. This is not, however, a rigid
requirement. In four of the pairs listed, a single
immunodominant sugar, or a presumably equiv-
alent combination, suffices to make the cross-
reactions reciprocal. This seems reasonable. In
only two instances, 2,6 and 6,8, probably due to
unusually favorable steric factors, does recipro-
cal cross-reactivity appear to be due to a single
non-immunodominant sugar in common. Ac-
cordingly, reciprocal cross-reactivity, like the
nonreciprocal reactions, has an orderly structur-
al basis. Where the chemical structures of the
capsular polysaccharides are known, it should
now be possible to predict the likelihood, but not
the certainty, of reciprocal cross-reactivity.

The use of more sensitive methods than those
in the current exploratory studies might make
such prediction more certain. For example, as
early as 1939 use of capsular swelling and agglu-
tination (46) in rabbit anti-Pn sera revealed the
following reciprocally cross-reactive pairs: 2,20;
3,8; 8,19; 10,20; 10,29; 13,17; 13,29; 18,28;
20,31; 22,31; and 23,32. In reference 58, the
pairs 8,19; 10,20; 10,29; 16,28; 20,31; and 23,32
are recorded. No information on the complete
structure of any Pn polysaccharide was available
at that time.

ACKNOWLEDGMENT

Data in the tables from papers cited by reference number
are from work carried out under grants from the National
Science Foundation which terminated in 1976.

Since 1976, polysaccharides or antisera or both have been
received with gratitude from the New York City Department
of Health Laboratories, kindness of P. May and Y. C. Faur;
the New York State Department of Health Research Labora-
tories, kindness of K. Amiraian; and from R. Austrian, J.
Henrichsen, and G. Schiffman, | am also indebted to Charles
Serhan, Corey Mallett, and Andrew Zakarian for technical
assistance and to the secretariat of the Department of Patholo-
gy of the New York University Medical Center for typing the
manuscript.

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