Journal of General Virology (1991), 72, 1855-1861. Printed in Great Britain

1855

Analysis of the neutralization epitopes on human rotavirus VP7 recognized by monotype-specific monoclonal antibodies Nobumichi Kobayashi,* Koki Taniguchi, Tomoko Urasawa and Shozo Urasawa Department o f Hygiene, Sapporo Medical College, South-l, West-17, Chuo-ku, Sapporo 060, Japan

Three anti-VP7 monoclonal antibodies (MAbs) which neutralized only two strains (K8 and S12) of five serotype 1 human rotaviruses (HRVs) were obtained, and neutralization epitopes recognized by these 'monotype-specific' MAbs were analysed by epitope mapping and sequencing of the VP7 genes. Neutralizationresistant mutants of K8 and S 12 were selected by the monotype-specific MAbs and serotype 1-specific MAbs prepared previously. Cross-neutralization tests between MAbs and neutralization-resistant mutants of K8 and S12 indicated that epitopes of monotypespecific MAbs operationally overlap with those of serotype 1-specific and cross-reactive MAbs recogniz-

ing the S 1 region. Sequence analyses of the VP7 genes indicated that VP7s of strains K8 and S12, which belong to a monotype of serotype 1 viruses, possessed amino acids at positions 42 and 87 different from other serotype 1 HRVs. Furthermore, amino acid substitution sites of representative mutants of K8, selected by the monotype-specific MAbs, were identified at positions 96, 97 and 100. These results imply that amino acids in variable region B (amino acids 87 to 101) are involved in the monotype-specific neutralization epitope as well as serotype-specific neutralization

Introduction

by MAbs have been suggested to arise by antigenic variation in the neutralization epitopes (Green et al., 1988; Nishikawa et al., 1989). However, the identification of amino acids associated with monotype specificity and the functional relationships between epitopes recognized by serotype-specific and monotype-specific MAbs have not been studied. In this study, we isolated hybridoma clones secreting monotype-specific anti-VP7 MAbs which neutralized only a subset of serotype 1 HRV strains. By using these monotype-specific MAbs and serotype 1-specific MAbs prepared previously (Kobayashi et al., 1991 a, Taniguchi et al., 1985) for selecting neutralization-resistant mutants, the monotype-specific neutralization epitopes on VP7 were characterized.

Group A rotaviruses are recognized world-wide as major viral pathogens in infantile gastroenteritis (Kapikian & Chanock, 1990). Two rotavirus outer capsid proteins, VP4 and VP7, are independently involved in virus neutralization (Hoshino et al., 1985; Offit & Blavat, 1986), and antigenic analyses of these proteins are essential for the development of rotavirus vaccines. VP7, which is encoded by R N A segment 7, 8 or 9 depending on the strain (Greenberg et al., 1983 ; Kalica et al., 1981 ; Ward et al., 1988), defines serotype specificity determined by neutralization tests (Hoshino et al., 1984). So far, 12 serotypes have been identified in group A rotaviruses (Estes & Cohen, 1989; Taniguchi et al., 1990; Urasawa et al., 1990), and seven of these (serotypes 1, 2, 3, 4, 8, 9 and 12) have been detected in humans. Although most of the monoclonal antibodies (MAbs) directed at VP7 show a serotype-specific reaction (Taniguchi et al., 1987; Urasawa et al., 1988), it has been reported that a serotype can be classified into minor antigenic variants distinguishable by MAbs. Such antigenic variants within a serotype were termed 'monotype' by Coulson (1987), and have been observed in human rotavirus (HRV) strains of serotypes 1 to 4 (Taniguchi et al., 1985; Coulson et al., 1985; Coulson, 1987; Gerna et al., 1988; Kobayashi et al., 1991 a). The monotypes differentiated 0001-0141 © 1991 SGM

epitopes.

Methods Cells and viruses. MA-104cells, a continuous cell line derived from foetal rhesus monkeykidney, were used for propagationof rotaviruses (Urasawa et al., 1981). The followinghuman and animal rotaviruses were employed:KU, Wa, SI2, K8 and M37 (serotype1); $2, HN126, AK26, DS-1 and 1076(serotype2); YO, $3, P, P2, AK35, MEN-13, SA11 and RRV (serotype3); Hochi, Hosokawaand ST-3(serotype4); NCDV (serotype6); 69M (serotype 8) and WI61 (serotype9). Hybridoma production and characterization of MAbs. Purified HRV strain K8, serotype 1(Urasawaet al., 1984;Taniguchi et al., 1989)was

1856

N. Kobayashi and others

injected into an adult female BALB/c mouse as described previously (Taniguchi et al., 1985). The spleen cells were then fused with myeloma PAl cells (Stocker et al., 1982) obtained from the Japanese Cancer Research Resource Bank. A fluorescent-focusreduction neutralization (FFN) test was performed to detect hybridomas producing neutralizing MAbs (N-MAbs) as described previously (Urasawa et al., 1988). The protein specificityof the MAbs was determined by an FFN test using two reassortant strains derived from two different HRVs (K8 and P): PxK8-A2 with VP4 derived from strain P (serotype 3) and VP7 derived from strain K8 (serotype 1) and PxK8-BI withVP4 derived from strain K8 and VP7 derived from strain P (Kobayashi et al., 1991b). An immunoprecipitation test was also carried out as described previously (Taniguchi et al., 1985). The reactivities of MAbs to rotaviruses were examined by FFN tests and ELISA as described (Urasawa et al., 1988, 1989). Neutralization-resistant mutant. Neutralization-resistant mutants of serotype 1 HRV strains K8 and S12 (Urasawa et al., 1984) were prepared by two successive selections with the N-MAbs obtained in this study and serotype 1-specificand cross-reactiveN-MAbs prepared previously (Kobayashi et al., 1991a; Taniguchi et al.. 1985). Epitope regions on VP7 recognized by the previously produced MAbs have been identified as S1 (MAbs KU-3A, KU-6A11, KU-4, KU-6BG and YO-4C2) and $2 (MAbs KS-3H16 and KS-6D6) (Kobayashi et al., 1991a). RNA sequencing analysis. The nucleotide sequences of genes encoding VP7 of some neutralization-resistant mutants and serotype 1 HRV strains were determined by the primer extension method using five oligonucleotide primers as described previously (Gorziglia et al., 1986; Green et aL, 1988).

Results Preparation o f monotype-specific N - M A b s In neutralization tests using 21 H R V strains with different serotype specificity, we detected three M A b s (K8-12G2, K 8 - 2 D l l and K8-10C8) which neutralized only K8 and S12 strains out of five serotype 1 strains examined (Table 1), although one of them (MAb K810C8) reacted with all serotype 1 strains except M37 in ELISA. The protein specificity of the three N - M A b s was determined as VP7, since all three N - M A b s neutralized the reassortant PxK8-A2 (VP4 : P, VP7 : K8), whereas no neutralization activity was shown against the other reassortant PxK8-BI ( V P 4 : K 8 , V P 7 : P ) (Table 2). However, no viral protein was immunoprecipitated by any of these MAbs. The three anti-VP7 M A b s prepared in this study were designated monotype-specific and were distinguishable from other serotype 1-specific M A b s (KU-3A, K U - 6 A l l , KU-4, K U - 6 B G , K8-3H16 and K8-6D6) prepared previously and employed in this study. Antigenic analysis o f neutralization epitopes recognized by monotype-specific M A b s To analyse the neutralization epitopes recognized by the monotype-specific N-MAbs, antigenic mutants resistant

to each antibody were selected from strain K8 or S12 parental viruses. As shown in Table 3, the reactivities of these mutants were examined by cross-neutralization tests using the three monotype-specific MAbs, six serotype 1-specific M A b s and one cross-reactive M A b prepared previously (Taniguchi et al., 1985, 1988; Kobayashi et al., 1991a). A mutant was regarded as being resistant to a M A b when the neutralization titre of the mutant was reduced to less than one-sixteenth that of the parental K8 or S12 strain. All the clones of EV-K812G2 and EV-K8-10C8 and all the mutants of S12 were resistant to the three monotype-specific MAbs. The mutants selected by M A b K8-2D11 were still neutralized by M A b K8-12G2. These results indicate that the epitopes recognized by the three monotype-specific M A b s operationally overlap with each other to form a single antigenic region. Furthermore, of the K8 mutants selected by the monotype-specific MAbs, resistance to neutralization was observed against only the two serotype 1-specific MAbs, K U - 3 A and KU-6A11, which recognized the S1 region of VP7 identified previously (Kobayashi et al., 1991 a). None of the S12 mutants was resistant to any serotype 1-specific or cross-reactive MAbs. To confirm the operational overlapping of the epitopes recognized by monotype-specific and serotype-specific MAbs in another way, resistant mutants of K8 were also prepared by selection with serotype 1-specific MAbs. In cross-neutralization tests (Table 4), mutants of K8 selected by M A b s recognizing the S1 region (KU-3A, KU-6A11, KU-4, K U - 6 B G and YO-4C2) were resistant to all of the monotype-specific MAbs. However, mutants selected by K8-3H 16 and K8-6D6, which recognized the $2 region, were not resistant to any monotype-specific MAbs. These results indicate that the epitopes of all the monotype-specific MAbs have an operational overlap with those of serotype 1-specific M A b s recognizing the S1 region. Analysis o f the VP7 gene by R N A sequencing The VP7 genes of strains K8 and S12 representing a monotype of serotype 1 viruses were sequenced by primer extension, and their deduced amino acid sequences were compared with those of reference serotype 1 strain K U . Only two and three VP7 amino acids of strains K8 and S12, respectively, were different from those of strain K U : at position 42, isoleucine in K8 and S12 replaced valine in K U ; at position 65, alanine in S12 replaced threonine in K U ; at position 87, asparagine in K8 and S12 replaced threonine in K U (Table 5). The two positions, 42 and 87, where amino acid differences were observed between K U and two monotype strains are located in the two separate variable regions, A (amino

Monotype-specific epitopes on rotavirus VP7

1857

Table 1. Reactivity of the monotype-specific MAbs with rotavirus strains ELISA test with*

Neutralizing titre* of MAb Rotavirus strain (serotype) K U (1) Wa (1) K8 (1) S12 (1) M37 (1) HRV strains (2, 3, 4, 8 and 9):~ Animal rotaviruses (3 and 6)§

K8-12G2

K8-2D11

K8-10C8

K8-12G2

K8-2DI 1

K8-10C8

< 100 < 100 6400 3200 < 100

< 100 < 100 6400 3200 < 100

< 100 < 100 12800 6400 < 100

+ + -

+ + -

+ + + + -

< 100

< 100

< 100

-

-

-

< 100

< 100

< 100

-

-

-

* Neutralizing titres are expressed as the reciprocal of the highest dilution of ascitic fluid that reduced the fluorescent focus count by more than 60%. t Reactivity in ELISA is expressed as + or - . + , A positive reaction in which the sum of A492 × 1000 for the two test wells minus the sum of A492 × 1000 for the two control wells exceeds 300. 3~The following HRV strains were tested: $2, AK26, HN126, DS-1 and 1076 (serotype 2); YO, $3, P2, P, AK35 and MeN-13 (serotype 3); Hochi, Hosokawa and ST-3 (serotype 4); 69M (serotype 8); WI61 (serotype 9). § Serotype 3 simian rotaviruses SA11 and RRV, and serotype 6 bovine rotavirus N C D V were tested.

Determination of protein specificity of monotype-specific MAbs by neutralization test with rotavirus reassortants

T a b l e 2.

Derivation of neutralization protein

Neutralizing titre* of MAb

Rotavirus strains

VP4

VP7

K8-12G2

K8-2D11

K8-10C8

K8 P PxK8-A2 PxK8-BI

K8 P P K8

K8 P K8 P

6400 < 100 12800 < 100

6400 < 100 3200 < 100

12 800 < 100 6400 < 100

VP7

VP7

VP7

Ascribed protein specificity of MAb * See footnote for Table 1.

acids 39 to 50) and B (87 to 101), where amino acid sequences diverge between different serotypes (Glass et al., 1985; Green et al., 1987). Furthermore, VP7 genes of the four representative mutants of K8 selected by monotype-specific MAbs were sequenced. As shown in Table 6, when compared with the sequence of parental strain K8, each mutant was found to have sustained a single amino acid substitution at position 96, 97 or 100, which are situated in variable region B.

Discussion In this study we found that the epitopes of the monotypespecific MAbs have an operational overlap with those of

serotype 1-specific MAbs recognizing the S1 region, one of the two serotype-specific epitope regions identified previously (Kobayashi et al., 1991a). However, only some mutants of K8 selected by monotype-specific MAbs were resistant to the two serotype l-specific MAbs, and none of the S12 mutants selected by monotype-specific MAbs was resistant to serotypespecific MAbs. In contrast, all the K8 mutants selected by serotype-specific MAbs recognizing the S1 region were resistant to at least one of the monotype-specific MAbs examined. These findings imply that the antigenic variations after selection with the monotype-specific MAbs may not have a significant influence on the neutralizing activity of the serotype-specific MAbs, whereas antigenic changes in serotype-specific neutral-

6400 12800

6400 I>25600

-

-

-

-

-

-

6400 1600 6400

-[1

K8-2D11

12 800 12800

-

K8-10C8

6400 3200

1600 3200 6400 1600 6400 6400 6400 6400 3200

KU-3A (S1)§

/> 25 600 >125600

6400 12800 12 800

6400

6400

-

KU-6A11 (S1)

6400 />25600

12 800 3200 6400 6400 /> 25 600 />25600 6400 >/25 600 i> 25 600 6400 12 800 ~>25 600

KU-4 (S1)

6400 6400

12800 6400 3200 3200 12800 6400 3200 3200 3200 3200 3200 3200

KU-6BG (S1)

Serotype 1-specific M A b s t

1600 1600

6400 3200 1600 3200 3200 1600 3200 1600 800 1600 1600 1600

K8-3HI6 ($2)

6400 6400

/> 25 600 >/25 600 >~25 600 6400 >/25 600 ~>25600 6400 6400 12800 1600 3200 3200

K8-6D6 ($2)

6400 6400

/> 25 600 3200 6400 6400 12 800 12800 6400 /> 25 600 >/25 600 3200 3200 3200

YO-4C2 (S1)

Cross-reactive MAbt

* See footnote for T a b l e 1. t T h e s e M A b s h a v e been c h a r a c t e r i z e d p r e v i o u s l y ( T a n i g u c h i et al., 1985, 1988; K o b a y a s h i et al., 1991 a). A n t i - V P 7 M A b Y O - 4 C 2 s h o w s c r o s s - r e a c t i v i t y in n e u t r a l i z a t i o n tests a g a i n s t serotypes 1, 3, 4 a n d 9 r o t a v i r u s strains. ;~ M u t a n t s of K8 or S 12 were d e s i g n a t e d ' E V ' or 'SV' followed by the n a m e of the M A b s used for t h e i r selection, respectively. T h r e e i n d e p e n d e n t m u t a n t clones o f K8 selected w i t h M A b s K8-12G2, K8-2D11 a n d K8-10C8 w e r e d i s t i n g u i s h e d b y 'I', 'II', or ' I I I ' . § S1 a n d $2 in p a r e n t h e s e s i n d i c a t e t w o f u n c t i o n a l l y i n d e p e n d e n t e p i t o p e regions on VP7 identified p r e v i o u s l y ( K o b a y a s h i et al., 1991 a). [1-, N e u t r a l i z i n g titre less t h a n o n e - s i x t e e n t h the titre a g a i n s t the p a r e n t a l strains K 8 or S12.

K8 K8 K8 K8 K8 K8 K8 K8 K8 S12 S 12 S 12

EV-K8-12G2-I EV-K8-12G2-II EV-K8-12G2-III EV-K8-2D11-I E V - K 8 - 2 D 11-II EV-K8-2D11-III E V - K 8 - 1 0 C 8-1 EV-K8-10C8-II EV-K8-10C8-III SV-K8-12G2 S V - K 8 - 2 D 11 SV-K8-10C8 P a r e n t strains K8 S12

K8-12G2

Monotype-specific MAbs

N e u t r a l i z i n g titre* of M A b

Antigenic analysis o f the resistant mutants o f K8 and S12 selected with monotype-specific anti-VP7 M A b s

Parent strain

3.

Antigenic mutant strains:~

Table

r~

00

1859

Monotype-specific epitopes on rotavirus VP7

T a b l e 6. Nucleotide and amino acid changes detected in the antigenic mutants o f K8 selected with anti-VP7 monotypespecific MAbs

T a b l e 4. Antigenic analysis o f resistant mutant o f K8 selected with serotype 1-specific anti-VP7 MAbs Neutralizing titre* of monotype-specific M A b Antigenic m u t a n t straint EV-YO-4C2 EV-KU-3A-I EV-KU-3A-II EV-KU-3A-III EV-KU-6A11-I EV-KU-6A11-II E V - K U - 6 A 11-III EV-KU-4 EV-KU-6BG EV-K8-3H16 EV-K8-6D6

Selecting M A b (epitope region):~ K8-12G2 YO-4C2 (S1) K U - 3 A (S1) K U - 3 A (S1) K U - 3 A (SI) K U - 6 A I 1 (SI) KU-6A11 (S1) KU-6A11 (SI) K U - 4 (SI) K U - 6 B G (S1) K8-3H16 ($2) K8-6D6 ($2)

MAb-resistant mutant

K8-2Dll

K8-10C8

3200 -

-§

6400 12800 3200 3200 6400 6400 6400

1600 6400

3200 3200 3200 -

1600 1600 3200 3200

Nucleotide change

EV-K8-12G2-I EV-K8-2D1 l-I EV-K8-10C8-I EV-K8-10C8-1I

G--,A A-G G--*A G~T

(337)* (347) (337) (335)

Codon change GAC-*AAC GAC-~GGC GAC~AAC GGT~GTT

A m i n o acid change (97)$ (100) (97) (96)

Asp~Asn Asp~Gly Asp--,Asn Gly~Val

* The n u m b e r in parentheses shows the position of the nucleotide substitution. t T h e n u m b e r in parentheses shows the position of the amino acid substitution.

6400 6400

available. However, the reactivities of S 12 with anti-VP4 MAbs prepared previously strongly suggest that the VP4 antigenicity of S12 is similar to that of K U and Wa, but appears to be different from that of K8 (Kobayashi et al., 1990; Taniguchi et al., 1985). Three monotype-specific MAbs prepared in this study neutralized K8 of VP4 serotype 3 and S12 which seems to have VP4 serotype 1A, while they did not neutralize K U or Wa which also belong to VP4 serotype 1A. These findings indicate that the limited neutralization activity of the monotypespecific MAbs may not be caused by the presence of different VP4 antigens, although functional interactions between VP4 and VP7 have been suggested previously (Burns et al., 1989; Chen et al., 1989; Nagesha et al., 1989). It is interesting that the two strains of one monotype, K8 and S12, possessed the same amino acids at positions 42 (isoleucine) and 87 (asparagine), which were different from those of other serotype 1 HRVs. Although these sites are included in variable regions A (amino acids 39 to 50) and B (87 to 101), an asparagine residue at position

* See footnote for Table 1. t M u t a n t s were designated ' E V ' followed by the n a m e of the M A b s used for their selection. Three independent m u t a n t clones selected with M A b s K U - 3 A a n d KU-6A11 were distinguished as I, II or III. ~/See footnote § of Table 3. § See footnote IIof Table 3. Neutralizing titres of M A b s to the parent strain K8 are shown in Table 3.

ization epitopes may have a considerable influence on the function of monotype-specific epitopes. Although the five serotype 1 HRV strains were examined for their reactivities with the monotypespecific MAbs, serological groups of VP4 (VP4 serotypes, P types) (Estes & Cohen, 1989: Gorziglia et al., 1990) of these strains were different. According to the classification by Gorziglia et al. (1990), strains K U and Wa belong to VP4 serotype IA, whereas strains M37 and K8 belong to VP4 serotypes 2 and 3, respectively. The VP4 serotype of strain S12 is undetermined because not enough serological and genetic data on this strain are

Table 5. Differences in amino acid sequences o f VP7 among serotype 1 H R Vs A m i n o acid position* on the VP7 sequence Serotype 1 HRVs KU Wa K8 S12 M37 Variable region§ of the amino acid position

16

37

42

65

I -.~

F

V

T V T Q E A . . . . . . . . . . . A I A R G

I I V

S A

66

68

72

74

75

87

V .

T .

I

94

97 123 147 170 208 218 291

N

D

S N

E

-

S . . N

B

C

D

. N N -

. . S

B

B

. .

. .

. .

I V . .

Q -

V I

K -

R

-

R

E

E

. . -

. .

A m i n o acid identity to the V P 7 o f K U ( ~ ) t 100.0 98-8 99.4 99-1 96.3

* A m i n o acid positions where different a m i n o acids were found a m o n g the five H R V strains are shown. VP7 a m i n o acid sequences of K U , W a and M37 were published previously (Green et al., 1987; Richardson et al., 1984; Taniguchi et al., 1988). t A m i n o acid identity is expressed as the ratio ( ~ ) of identical a m i n o acids as compared with the whole VP7 a m i n o acid sequence of strain KU. -, Identical a m i n o acid to K U at the given position. § The designation of the variable regions described by Glass et al. (1985) was used.

1860

N. Kobayashi and others

87 of the strains K8 and S12 is considered to be associated with a monotype-specific neutralization epitope because it has been shown that the preceding 50 amino acids, including region A, are cleaved from VP7 before the generation of mature virus particles (Stirzaker et al., 1987). The amino acid substitutions of K8 mutants selected by the monotype-specific MAbs were detected at positions 96, 97 or 100, which are close or identical to the positions (94, 96, 97 and 99) mapped by serotype 1specific and cross-reactive MAbs in other rotavirus mutants (Dyall-Smith et al., 1986; Mackow et al., 1988; Taniguchi et al., 1988). These findings clearly indicate that variable region B (amino acids 87 to 101) is associated with neutralization of both serotype-specific and monotype-specific MAbs. Our conclusion that amino acids in variable region B are related to the monotypes may be supported by the previous observation on the serotype-specific neutralization epitope described by Green et al. (1988). They concluded that amino acid substitution at position 94 in VP7 of serotype 1 strain M37 might be associated with escape from neutralization by an anti-VP7 MAb to serotype 1 HRV. Although monotypic epitopes were identified only in variable region B in the present study, it is possible that monotype-specific MAbs also map in other variable regions, because variation with single or double amino acid changes within strains of the same serotype have also been found in variable regions C, D, E and F (Green et al., 1987, 1988). This study was supported in part by grant no. 02770238 from the Ministry of Education, Science, and Culture of Japan.

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(Received 14 January 1991 ; Accepted 12 April 1991)