neutralizing monoclonal antibodies and a corresponding set of escape mutants (6, 25, 26, 37, 38). In VP4, eight neutralization

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1 JOURNAL OF VROLOGY, Mar. 1991, p X/91/ $02.00/0 Copyright 1991, American Society for Microbiology Vol. 65, No. 3 The Amino-Terminal Half of Rotavirus SA114fM VP4 Protein Contains a Hemagglutination Domain and Primes for Neutralizing Antibodies to the Virus MARCELA LZANO,t SUSANA LOPEZ, AND CARLOS F. ARAS* Departamento de Biologia Molecular, Centro de nvestigacion sobre ngenieria Genetica y Biotecnologia, Universidad Nacional autonoma de Mexico, Apartado postal 510-3, Colonia Miraval, Cuernavaca, Morelos 62271, Mexico Received 2 August 1990/Accepted 28 November 1990 We have previously reported the synthesis in Escherichia coli of polypeptide MS2-VP8', which contains the amino-terminal half of the SA114fM VP4 protein fused to MS2 bacteriophage polymerase sequences (C. F. Arias, M. Lizano, and S. L6pez, J. Gen. Virol. 68: , 1987). n this work we have synthesized the carboxy-terminal half of the VP4 protein also fused to the MS2 polymerase.' This protein, designated MS2-VP5', was recognized by sera to the complete virion and was able to induce antibodies to the virus when administered to mice; however, these antibodies had no neutralizing activity. The two chimeric polypeptides were tested for their ability to agglutinate erythrocytes and to prime the immune system of mice. Bacterial lysates enriched for the MS2-VP8' hybrid polypeptide, but not those enriched for the MS2-VP5' protein or those containing proteins from the host E. coli strain, had hemagglutinating activity. This hemagglutination was inhibited by sera to SA114fM rotavirus. n addition, a single dose of the MS2-VP8' polypeptide was able to prime the immune system of mice for an augmented neutralizing antibody response when the animals were subsequently immunized with purified SA114fM virus. Rotaviruses are the single most important etiologic agents of severe dehydrating infantile gastroenteritis in developed and developing countries, and the recognition of the high morbidity and mortality rates caused by these viruses has focused rotavirus research efforts on prevention of the disease and its control (17). Rotaviruses are composed of an RNA genome contained in a double-layered capsid. The genome is made up of 11 segments of double-stranded RNA, with sizes ranging from about 650 to 3,500 bp (10). The capsid consists of at least six protein classes, four of which (VP1, VP2, VP3, and VP6) make up the inner layer, whereas the other two (VP4 and VP7) are in the surface layer of the virus (9, 11, 21). VP7, a 37-kDa glycoprotein, induces neutralizing antibodies that specify the viral serotype (4, 13). VP4, which also induces neutralizing antibodies to the virus (13, 39), is an 86.5-kDa protein encoded by the fourth genomic RNA segment (23, 28), and gene reassortment studies have demonstrated that the viral phenotypes for hemagglutination (HA) and trypsin-enhanced plaque formation segregate with this gene (15). n vitro treatment of virions with trypsin results in the specific cleavage of VP4 to polypeptides VP5 and VP8, of approximately 60 and 26.5 kda, respectively, with a concomitant enhancement of viral infectivity (9, 11). The activation of the virus by trypsin has been associated with the entry of the virus by direct cell membrane penetration (12, 16). n addition, VP4 has been shown to be a determinant of virulence in mice orally infected with heterologous host rotaviruses (33). Recently, the sites involved in rotavirus neutralization have been located on VP7 and VP4 proteins with the use of * Corresponding author. t Present address: Departamento de nvestigaci6n Basica, nstituto Nacional de Cancerologia, San Fernando 22, Tlalpan 14,000, Mexico City, Mexico neutralizing monoclonal antibodies and a corresponding set of escape mutants (6, 25, 26, 37, 38). n VP4, eight neutralization domains were identified; three of them were located to VP5, while the remaining five were spread broadly over VP8. Most antigenic determinants on VP8 were serotype specific, while the epitopes on VP5 were mostly heterotypic. Antibodies directed to both VP5 and VP8 neutralize the virus, inhibit hemagglutination, and passively protect mice against rotavirus challenge in vivo (26, 30, 34, 38). On the basis of these data, we chose to construct recombinant expression plasmids to produce in bacteria the aminoand carboxy-terminal halves of the VP4 protein, representing primarily polypeptides VP8 and VP5, respectively, with the aim of further localizing the HA function of the virus, and to test the potential of these two regions to'be used as subunit vaccines. Previously, we had synthesized in Escherichia coli the 45% amino-terminal coding sequence of the SA114fM rotavirus VP4 protein as a fusion polypeptide with the first 98 amino acids of the MS2 phage polymerase (2). This bacterial product, designated MS2-VP8', was able to induce hemagglutination-inhibiting (HA) as well as neutralizing antibodies to SA114fM rotavirus. Here, we report the-expression in E. coli of the carboxy-terminal half of the SA114fM VP4 protein (designated MS2-VP5') and the comparative analysis of both fusion polypeptides with regard to their antigenicity, immunogenicity, and HA activity. MATERALS AND METHODS Virus and cells. Simian rotavirus SAil was obtained from M. K. Estes, Baylor College of Medicine, Houston, Tex. Human rotavirus strain Wa was obtained from Y. Hoshino, National nstitutes of Health, Bethesda, Md. The NCDV strain of bovine rotavirus was provided by R. S. Spendlove, University of Utah, Logan. The origin of the simian rotavi-

2 1384 LZANO ET AL. (a) (b) nt '. gene4 psr Hho Fnudl.BomH f VP4 gene (nt ) Hindl Klenow FG. 1. Construction of a vector expressing the 3' half of rotavirus SA114fM gene 4. (a) Relative position of the viral cdna insert in plasmid psr4-4 with respect to the full-length gene 4. This cdna contains nucleotides (nt) 1057 to 2252 of the coding strand of the gene. (b) Steps involved in the construction of expression plasmid puma451ml. rus strain SA114fM, used for the gene expression studies we described, is detailed in Results. All rotavirus strains were grown in MA104 cells, and strain SA114fM was purified as described previously (9). Bacteria and plasmids. E. coli RR1 (X)- [F-, hsds20 (rbj mb,-) ara-14 proa2 lacyl galk2 rspl20 (Smr) xyl-5 mtl-l supe44 X] was obtained from A. Covarrubias, National University of Mexico, Cuernavaca, Morelos. E. coli M5219 (36) is the designation for strain M72 lacz(am) trp(am) Smr (Xbio252 c857 AH1), which carries a temperature-sensitive repressor gene (c857) for the A PL promoter. Plasmids pplc24 (36), psr4-4 (23), and puma481ml (2) have been reported earlier. Construction of a plasmid for expression of the 3'-terminal half of SAHl4OM gene 4. Plasmid DNA manipulations were carried out essentially as described before (27). The viral cdna in plasmid psr4-4 (23), used for the construction of the recombinant expression plasmid, represents nucleotides 1057 to 2252 of SAl14fM gene 4 (Fig. la). The polypeptide product it encodes was designated VP5' since it makes up 75% of the sequence of VP5. The strategy used for the construction of the expression plasmid is depicted in Fig. lb. Plasmid psr4-4 was digested with Hha and Fnud, and the resulting 1,085-bp DNA fragment was ligated into the Hind site of pplc24, located immediately downstream of the first 98 amino acids of the bacteriophage MS2 polymer- J. VROL. ase. To make the ends of these two DNA fragments suitable for ligation, they were made flush. The Hha-generated 3'-protruding ends of the insert were made blunt by using the exonucleolytic activity of the Klenow enzyme and the 3'-recessive ends of the vector were filled in by the polymerase activity of the same enzyme. n the resulting plasmid, designated puma450ml, the rotavirus information was not in frame with the MS2 sequences. To correct the frame of the hybrid gene, plasmid puma450ml was digested with BamH; its ends were filled in with the Klenow fragment; and the plasmid was religated. This treatment caused a four-nucleotide insertion at the junction region, which left the two coding DNA fragments in a continuous reading frame. This plasmid was designated puma451ml (Fig. lb). The constructed chimeric plasmid was transformed into E. coli RR1 (A) and, after being characterized with restriction endonucleases, was transferred to E. coli M5219, allowing temperature-dependent controlled expression of the A PL promoter. Electrophoretic and immunoblot analysis of the recombinant viral proteins. Bacteria were grown and induced as described previously (2). Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE; 10:0.26% acrylamide-bisacrylamide) following the method of Laemmli (19), and the percentage of the hybrid protein in a sample was estimated by staining the gel with Coomassie brilliant blue and scanning with a densitometer. For immunoblot analysis, the chimeric proteins were enriched about threefold by low-speed centrifugation of a bacterial lysate produced as described previously (2) and were subsequently separated by electrophoresis and transferred to nitrocellulose paper essentially by the method of Tsang et al. (40). The identification of the viral epitopes on the bacterial proteins was achieved by incubation with an anti-rotavirus SA114fM hyperimmune serum and subsequent incubation with 125-labeled protein A, as described by Arias et al. (2). Filters were dried and autoradiographed. Animals and immunization schedule. Male BALB/c mice 7 to 9 weeks of age were screened on day 0 for neutralizing antibodies to SAl1 rotavirus and for hemagglutination-inhibiting antibodies, as described below. All preimmune sera had hemagglutination-inhibiting antibody titers of 1:200, while no neutralizing antibodies were detected at a dilution of 1:50, the lowest dilution tested. To ensure the rotavirus-free status of the mice used, the preimmune sera were also tested for rotavirus-specific antibodies, at a dilution of 1:25, by enzyme-linked immunoassay (1). All sera tested showed only background reactivity by this assay, equivalent to that obtained with sera from VAF/Plus mice, recently acquired from Charles River Breeding Laboratories, nc., Portage, Mich. Two groups of eight seronegative mice each were inoculated with bacterial proteins from E. coli M5219 containing either plasmid puma481ml or puma451ml, which had been enriched for the hybrid polypeptide MS2- VP8' or MS2-VP5', respectively, as described above. Mice from the first group received 100,ug of protein per injection, while mice in the second group were injected with 260,g each time. n both cases, about 30 jig of the administered bacterial protein corresponded to VP4 sequences. A third group of four mice received per injection 100,ug of bacterial proteins derived from E. coli M5219 transformed with vector pplc24 and treated the same way as the recombinant plasmid-carrying bacteria. Finally, a fourth group, composed of two mice, received 50 pug of purified SA114fM virus per injection. Each of the four groups received five injections, on days 0, 14, 29, 44, and 59. Mice were bled on day

3 VOL. 65, 1991 ROTAVRUS SA114fM VP4 PROTEN 1385 (a) H2N [ VP Oa r6 361 aa 1 COOH --r-.m L-1 MS2 polymerase VP5' 2 (b) X L S W D R S D Q A L P V *--TTG TCA TGG GAT CGA 1CC GAC CAA GCT CTT CCA GTTooo... =-.V.=- - r = MS2 polymerase Linker VP5' S D P S W A V G A Q P X TCT GAC COG MC TTGGCTGM 1C CGC GTT. * * * * * GGG GGCAG CCA TGA VP5' Linker p PLc24 FG. 2. (a) Schematic representation of the expected MS2-VP5' polypeptide directed by puma451ml. Asterisk represents the cleavage sites associated with trypsin enhancement of rotavirus infectivity. (b) Nucleotide sequence and the encoded amino acid sequence of the junction regions, boxed in panel a, predicted on the basis of plasmid construction. Numbers above the amino acids represent the position of that amino acid in the original protein. X, Termination codon. 66. Each serum sample was heat inactivated at 56 C for 30 min and stored at -20 C. To study the ability of the recombinant proteins to prime the immune system of mice, three different groups of 10 mice each received one dose of either bacterial extracts enriched for the MS2-VP8' or MS2-VP5' hybrid polypeptides or control bacterial proteins. Two weeks later, all mice were injected with 50,ug of purified rotavirus SA114fM. Sera were collected at days 0 and 15 and weekly thereafter for 5 weeks. The sera were treated as mentioned above and kept at -20 C. The mice in all immunization schedules described above were injected subcutaneously with 200,l of an emulsion of the respective antigen and adjuvant. Freund complete adjuvant was used for the first immunization; for subsequent injections, incomplete adjuvant was used. Neutralizing antibody assay. Neutralization antibody titers were determined by an immunoperoxidase focus reduction assay as described by Arias et al. (2). The titer of neutralizing antibodies in a serum sample was defined as the highest serum dilution at which a reduction of at least 60% in the number of infected cells was observed as compared with controls in which phosphate-buffered saline (PBS) had been used instead of serum. HA and HA tests. Serial twofold dilutions in PBS of rotavirus SA114fM or bacterial lysates enriched for the recombinant proteins, prepared as described above, were made in 96-well (V-shaped bottom) microtiter plates. To 50,ul of these dilutions, 50,ul of 0.4% human type 0 erythrocytes in 0.2% bovine serum albumin in PBS was added, and the plates were kept at room temperature for 90 min before the titers were recorded. For the HA assay, the sera tested were serially diluted in 0.2% bovine serum albumin in PBS and then mixed with an equal volume of this diluent containing 4 HA units of SA114fM virus or 4 HA units of the MS2-VP8' bacterial protein. After incubation for 60 min at room temperature, 50-,l aliquots of these mixtures were mixed with human type 0 erythrocytes as described above, and the inhbition of HA was scored after incubation at room temperature for 90 min. RESULTS Synthesis in E. coli of the fusion protein MS2-VP5'. From the known nucleotide sequence of the various DNA components of plasmid puma451ml (Fig. 2), we expected it to direct the synthesis of the fusion product MS2-VP5', consisting of 517 amino acids: 98 of the MS2 polymerase, 6 coded by the vector at the MS2-VP5' junction region, 361 of VP5 (amino acids 389 to 749 of VP4), 2 linker encoded, and 50 of pbr322. Previously, we had described the construction of the expression plasmid puma481ml, which directs the synthesis in E. coli of the chimeric protein MS2-VP8', containing amino acids 42 to 387 of the SA114fM VP4 protein (2). Since the trypsin cleavage sites on VP4 are at amino acid positions 241 and 247, the MS2-VP8' protein contains 205 amino acids (83%) of VP8 plus 140 amino acids (26%) of VP5. The expression of the hybrid gene MS2-VP5', from puma451ml, is under the regulation of the lambda PL promoter. Thus, in the bacterial host M5219, which carries a temperature-sensitive lambda repressor gene (c857), the transcription from the lambda promoter was turned on by shifting the growing culture from 28 to 42 C. After heat induction, the proteins synthesized by E. coli M5219 containing plasmid puma451ml were analyzed by SDS-PAGE and compared with the proteins produced by the same strain containing plasmid puma481ml (coding for the MS2-VP8' hybrid protein) or pplc24. n the samples collected at 12 h postinduction, one protein was evident in the M5219 (puma451ml) lysates (Fig. 3a, lane 3) that was not present in the strain with the pplc24 vector (Fig. 3a, lane 2) and which migrated slightly above the already characterized fusion product MS2-VP8' (Fig. 3a, lane 1 [2]). This protein, synthesized by the puma451ml-bearing bacteria, had an

4 1386 LZANO ET AL. J. VROL. (a) (b) g- 0 MS2-VP8 - A _.1 ---,o *Po '_ f., MS2-VP8' -40 t : 'A M *10 0 "" mow do 40% FG. 3. Synthesis of the MS2-VP5' protein by plasmid-bearing cells. Analysis in an 11% SDS-polyacrylamide gel of (a) total cell proteins from E. coli M5219 containing plasmid puma481ml (lane 1), pplc24 (lane 2), or puma451ml (lane 3), after 12 h of temperature induction or (b) proteins in the pellet after low-speed centrifugation of the heat-induced bacterial lysates (see Materials and Methods) containing puma451ml (lane 1), puma481ml (lane 2), or pplc24 (lane 3). The proteins were visualized by Coomassie brilliant blue staining. The Mr markers (103) are as follows: 97.4, phosphorylase b; 68, bovine serum albumin; 43, ovalbumin; 25.7, chymotrypsinogen. The 57-kDa and the MS2-VP8' proteins are indicated. FG. 4. mmunoblot analysis of the proteins synthesized by plasmid puma451ml-bearing bacteria. The proteins in the pellet after low-speed centrifugation of bacterial lysates from E. coli M5219 containing plasmid puma481ml (lanes 1 and 4), puma 451ML (lanes 2 and 5), or pplc24 (lanes 3 and 6) were separated in an 11% SDS-polyacrylamide gel and were either stained with Coomassie brilliant blue (lanes 1, 2, and 3) or transferred to nitrocellulose paper (lanes 4, 5, and 6). The transferred proteins were incubated with a 500-fold dilution of anti-rotavirus SA114fM serum, and the bound antibody was identified with iodinated staphylococcal protein A. Arrows indicate the MS2-VP8' and MS2- VP5' polypeptides in lanes 4 and 5, respectively. apparent molecular weight of 57,000 which is very close, based on the plasmid construction, to the expected value of 56,000 calculated for the hybrid MS2-VP5' polypeptide. After scanning the gel with a densitometer, the 57-kDa protein was calculated to constitute approximately 2% of the total cellular protein. As occurred with the chimeric polypeptide MS2-VP8' (Fig. 3b, lane 2 [2]), the 57-kDa protein was also found to be insoluble, since most of it was found in the pellet after low-speed centrifugation of the bacterial cell lysate (Fig. 3b, lane 1). The SDS-PAGE and densitometric analyses of the pelleted bacterial proteins showed that the 57-kDa protein had been enriched about fivefold, representing approximately 10% of the sedimented proteins. Antigenicity of the MS2-VP5' polypeptide. The identity of the 57-kDa polypeptide was determined by analyzing its ability to bind anti-rotavirus antibodies. Cultures of E. coli M5219 containing puma451ml, puma481ml, or pplc24 were heat induced, and the proteins in the pellet resulting from a low-speed centrifugation of the bacterial lysates (see Materials and Methods) were separated by SDS-PAGE (Fig. 4). The proteins in the gel were either stained with Coomassie brilliant blue (lanes 1 to 3) or transferred to nitrocellulose paper (lanes 4 to 6). The blotted proteins were incubated with anti-rotavirus SA114fM serum and then probed with 1251-labeled protein A. The autoradiograph of the immunoblot (Fig. 4, lanes 4 to 6) showed that the anti-rotavirus serum reacted with both the 57-kDa protein synthesized by the bacteria containing puma451ml (lane 5) and the 52-kDa polypeptide (MS2-VP8') synthesized by the bacteria containing puma481ml (lane 4 [2]). These results indicate that the 57-kDa polypeptide is the MS2-VP5' expected fusion product. n addition to the MS2-VP8' polypeptide and the 57-kDa protein, some bands with lower molecular weight also reacted with the antiserum (lanes 4 and 5). These bands may represent proteolytic degradation from the cloned gene protein product, the products of internal signals of translation initiation in the chimeric mrna, or translation of prematurely terminated transcripts. HA and HA studies. The surface protein VP4 has been identified as responsible for the ability of rotaviruses to interact with and agglutinate erythrocytes. Since the two chimeric proteins expressed in E. coli are present in the form of aggregates, we considered that, if the viral HA domain were intact, one or both proteins could function as a multivalent entity capable of agglutinating human erythrocytes, as has been previously reported with an isolated E. coli recombinant protein derived from the reovirus hemagglutinin (29). Therefore, we tried to determine whether one or both of the fusion polypeptides possessed a functional HA domain. Lysates of bacteria harboring plasmid puma451ml, puma481ml, or pplc24, enriched by low-speed centrifugation for the recombinant proteins, were assayed for their ability to agglutinate human type 0 erythrocytes. n contrast to the pplc24- and puma451ml-transformed cell lysates, which showed no HA activity, the enriched lysate from bacteria containing plasmid puma481ml exhibited HA titers of 1:32 to 1:64 (Fig. 5a), which corresponded to an equivalent of 20 to 40,ug of the MS2-VP8' polypeptide per ml (1 HA unit was achieved with approximately 1,ug of the recombinant protein). The HA seemed to be specifically promoted by the rotavirus VP4 sequences in the MS2-VP8' polypeptide since it was inhibited by an anti-sa114fm rotavirus serum (Fig. Sb). These results indicate that in rotavirus SA114fM the HA domain resides within amino acids 42 to

5 VOL. 65, 1991 ROTAVRUS SA114fM VP4 PROTEN 1387 (a) PBS crontro; ,'S2-VPB' '. 4e )t %% M 5219 Control tetoot.'.n t. AN.\~A.' A s->... > MS2-VP5 SA114fM (b) PBS control SA114fM SA114fM + jnti-sa114fm MS2-V P8' MS2-VP83 anti-sa114fml PBS control anti-sal 14fM dilutioo (i CO Clc.c C<.. - C(> QOC OO > 995( Q)O ec 9X0D )vct.co&oocxdcd J) >D FG. 5. (a) HA activity of the recombinant polypeptides. Serial twofold dilutions of rotavirus SA114fM or bacterial lysates (protein concentration, -3 mg/ml) enriched for either of the hybrid polypeptides MS2-VP8' or MS2-VP5', or containing bacterial proteins from host E. coli M5219 treated in the same way as those from the plasmid-carrying bacteria, were made in PBS in a microtiter plate. An equal volume of a 0.4% (vol/vol) suspension of human type 0 erythrocytes was added to each well, and the agglutination reaction was carried out at room temperature for 90 min. The negative control is PBS mixed with the erythrocyte suspension. Numbers on top designate dilution factors of rotavirus or the bacterial lysates used. (b) nhibition of HA promoted by the MS2-VP8' polypeptide. Serial twofold dilutions of a hyperimmune serum to SA114fM virus were made in PBS. Four HA units of SA114fM virus or the MS2-VP8' polypeptide were added to each well. After 1 h of incubation at room temperature, human type 0 erythrocytes were added and incubation was continued for 90 min. The HA-negative controls are the same as in panel a, and the HA-positive controls are the virus or the recombinant protein incubated with PBS, instead of serum, before addition of the erythrocytes. 387 of VP4 and that this site of interaction with human erythrocytes is preserved in the recombinant protein MS2- VP8'. mmunogenicity of the recombinant protein. We had determined previously that the recombinant polypeptide MS2- VP8' was able to elicit neutralizing and hemagglutinationinhibiting antibodies when administered to mice (2). n this work we tested the immunogenicity of the MS2-VP5' chimeric protein in mice. As controls we included groups of mice inoculated with MS2-VP8' protein, whole SA114fM virions, or bacterial proteins derived from the host E. coli M5219, treated in the same way as those from the plasmidcarrying bacteria. After five immunizations, the MS2-VP5' polypeptide induced HA antibody titers of 1:1,000 to 1:2,000, comparable to those induced by the MS2-VP8' protein and the complete virion (Table 1). On the other hand, of the two recombinant proteins, only MS2-VP8' was able to elicit SA114fM neutralizing antibodies, reaching a titer of 1:1,600, as had been found previously (2). The sera of the mice immunized with TABLE 1. nduction of neutralizing (Nt) and HA antibodies by hybrid polypeptides MS2-VP8' and MS2-VP5' Antibody titer of sera taken on day: mmunogen Nt' HAb MS2-VP8' <50c 1, ,500 MS2-VP5' < ,400 Controld < SA114fM virus <50 34, ,000 a Titers are expressed as the reciprocal of the highest dilution of the serum that neutralized at least 60% of the immunoperoxidase foci in the assay. b Titers are expressed as the reciprocal of the highest dilution that completely inhibited HA of human type 0 erythrocytes by SA114fM virus. c Values represent mean of 10 mice for the MS2-VP8' and MS2-VP5' polypeptides and of 4 and 2 mice for the control proteins and the virus, respectively. d Bacterial proteins from host E. coli M5219 treated in the same way as those from the plasmid-carrying bacteria.

6 1388 LZANO ET AL. TABLE 2. Specificity of the neutralizing (Nt) antibody induced by the MS2-VP8' hybrid polypeptide Nt antibody titer' of sera from animals inoculated with the indicated immunogen Rotavirus MS2-VP8b NCDV SA114fM 4A8c 4B2 4B3 5B3 virus virus SA114fM ,600 1,600 3,200 6,400 SAl <100 <100 <100 < ,400 NCDV ,600 1,600 > 12,800 1,600 Wa <50 <50 <50 <50 NTd NT ST NT NT a See footnote a, Table 1. b The anti-ms2-vp8' serum was prepared in mice by inoculation of bacterial proteins from plasmid puma481ml-carrying bacteria, enriched for the hybrid polypeptide as described in Materials and Methods. c dentification number of mice. d NT, Not tested. MS2-VP5' did not show a neutralizing activity above the basal level (Table 1). The mice inoculated with purified SA114fM virions developed titers of neutralizing antibodies that were about 20-fold higher than those elicited by the recombinant protein MS2-VP8'. Altogether, these results suggest that both recombinant proteins were good immunogens but the neutralization epitopes they carry were in disadvantage (especially those on MS2-VP5'), compared with the same epitopes in the virion, to induction of neutralizing antibodies. Specificity of the MS2-VP8' neutralizing antibodies. The SAl strain of simian rotavirus used in this work, from which the rotavirus sequences of the MS2-VP8' gene were derived, was obtained originally from H. H. Malherbe, Gull Laboratories, Salt Lake City, Utah, in This strain was later found to have a faster-migrating gene 4 than the corresponding gene from the SAl strains handled in the laboratories of M. K. Estes of Baylor College of Medicine, Houston, Tex., and. H. Holmes, University of Melbourne, Melbourne, Australia (23), which were also obtained originally from H. Malherbe. Therefore, our SAl strain was renamed SA114fM (23). After the predicted amino acid sequence of the simian rotavirus SA114fM VP4 protein was published (22, 23), the sequences from the homologous proteins of bovine rotavirus strains C486 and NCDV (both of which belong to serotype 6) and the simian strain SAl (serotype 3) appeared (31, 32, 35). Unexpectedly, the SA114fM VP4 protein was almost identical (98% homology) to the bovine strains, while it only had an 82% homology to simian rotavirus SAl1. n light of these observations, we tested the specificity of the MS2- VP8' antibodies by a focus reduction neutralization assay. As expected from the sequence data, in addition to the SA114fM strain, the MS2-VP8' antibodies neutralized NCDV, but did not affect the infectivity of SAl rotavirus (Table 2). Accordingly, a hyperimmune antiserum to SA114fM neutralized both the NCDV and the SAl rotavirus strains, while an anti-ncdv serum neutralized SA114fM but not SAil. Since VP4 contains heterotypic neutralizing antibodies (26, 38), we tested the capacity of the MS2-VP8' antibodies to neutralize, in addition to the above-mentioned strains, rotavirus strains Wa (serotype 1) and ST3 (serotype 4). We found no neutralizing activity to rotavirus Wa; however, ST3 was neutralized to a titer fourfold lower than SA114fM (Table 2). a) L- 0.0 c 0' Cċ a) z 7500r 5000K O MS2-VP8 * MS2-VP5 o Control /J SA114fM Time after primary inoculation (days) J. VROL. FG. 6. Analysis of the secondary neutralizing antibody response to SA114fM virus of mice primed with the recombinant polypeptides. Titers shown represent the geometric mean of 10 mice. The animals received a single dose of the corresponding recombinant polypeptide, or the bacterial control proteins, on day 0 and were boosted ( ) 14 days later with SA114fM virus. Priming for a neutralizing antibody response by the fusion protein MS2-VP8'. Recently, several reports have appeared in which proteins or synthetic peptides of viral origin can specifically prime the immune system of animals for a virus-neutralizing antibody response after a single inoculation of intact virus (1, 8). This priming has even been achieved with immunogens that do not induce a detectable humoral response (7, 20). To determine whether the MS2- VP8' and MS2-VP5' fusion proteins were capable of priming for a neutralizing antibody response to SA114fM virus, groups of 10 mice each were inoculated with a single dose of either control bacterial proteins or bacterial proteins enriched for the MS2-VP8' or MS2-VP5' recombinant polypeptide. Two weeks after this initial immunization, all mice were inoculated with 50,ug of purified SA114fM particles, and the animals were bled weekly thereafter. At the moment of virus inoculation, no neutralizing activity was detectable in any of the mice groups; however, from that point, the titers of neutralizing antibodies in the sera of mice from the group that had been primarily immunized with the MS2-VP8' polypeptide increased rapidly and steadily, reaching means of about 1:4,000 and 1:8,000 at weeks 2 and 4, respectively (Fig. 6). On the other hand, the increase of the neutralization titers in the sera from mice that had received the MS2-VP5' polypeptide was slower and of a lower magnitude than in the MS2-VP8' group, reaching values of 1:400 and 1:1,000 at weeks 2 and 4, respectively; these titers were comparable to those obtained in the mice control group, which received bacterial proteins from the E. coli host M5219. These results are consistent with the idea that the immune system of mice had been primed by the MS2-VP8' polypeptide when it was exposed to the SA114fM virions. To learn about the mechanism through which MS2-VP8' is able to prime for an augmented antibody response to the SA114fM virus, we tested three different serum samples from mice that had been primed with the MS2-VP8' protein and subsequently immunized with the SA114fM virus for ability to neutralize SA114fM, SAl1, and NCDV rotavi-

7 VOL. 65, 1991 TABLE 3. Specificity of the neutralizing (Nt) antibody induced by SA114fM virus in mice primed with the MS2-VP8' polypeptide Rotavirus Nt antibody titera of sera from mice boosted with rotavirus SA114fM primarily inoculated with the indicated immunogen Control proteinsb MS2-VP8' 437c SA114fM < ,200 3,200 3,200 SAl ,600 3,200 3,200 NCDV ,600 3, Wa <200 <200 <200 <200 <200 a See footnote a, Table 1. b Control protein are the bacterial proteins from host E. coli M5219 treated in the same way as those from plasmid puma481ml-carrying bacteria (see Materials and Methods). c dentification number of mice. ruses. These sera neutralized all three viruses to a similar high titer, while the sera from control mice (inoculated with the complete virions but not primed with the recombinant polypeptide) only marginally neutralized the viruses (Table 3). These results suggest that the MS2-VP8' protein might be able to prime for a response to VP7 as well as to VP4 antigenic determinants. As expected, Wa rotavirus, whose VP4 and VP7 neutralizing epitopes are not related to those of the SA114fM virus (14), was not neutralized by the sera tested (Table 3). DSCUSSON The ability of rotaviruses to interact with and agglutinate erythrocytes has been ascribed to the virus surface protein VP4 by genetic studies (15). n addition, a full-length cdna copy of the RRV VP4 gene has recently been expressed by a recombinant baculovirus, and the synthesized VP4 protein was shown to have hemagglutinating activity (24), confirming the previous observations. n this study, we were able to determine directly that the amino-terminal half of VP4 (amino acids 42 to 387) contains an HA domain and that the HA observed was specific, since it was inhibited by antibodies to the virus. The detected HA domain might represent the viral HA domain, since the carboxy-terminal half of the protein was not able to hemagglutinate. These findings agree with the report of Kitaoka et al. (18), who suggested, by using hemagglutination inhibitory monoclonal antibodies directed to the VP4 of KUN virus, that the human rotavirus hemagglutinin site resides on the smaller trypsin cleavage products of the protein. However, we cannot rule out the existence of a different HA domain in the carboxy half of the protein which could not be in the correct conformation to be functional or the existence of an HA domain formed by the interaction of amino acids present in the two halves of the polypeptide. When the recombinant proteins containing the amino or carboxy halves of VP4 were used to immunize mice, only MS2-VP8', the protein containing the amino-terminal half of VP4, was able to induce neutralizing antibodies to the virus. These results correlate with the location of the neutralization epitopes on the VP4 protein of simian RRV and human KU rotavirus strains. Six different neutralization domains were described for RRV VP4 (26), five of which were located to the VP8 portion of the protein, while the sixth was mapped to VP5, around amino acid 393. For human rotavirus strain KU, three neutralizing antigenic sites were reported (38), all ROTAVRUS SA114fM VP4 PROTEN 1389 located to VP5 at amino acids 305, 392, and 433. Therefore, if the reported neutralizing epitopes are conserved in the SA114fM strain, our recombinant protein MS2-VP8' should encompass the amino acid sequence of at least six neutralization epitopes, while the MS2-VP5' protein would carry the sequence of only two of the mapped epitopes (around positions 392 and 433); however, the epitope in the 393 area may be truncated, since amino acid 393, which is critical for the corresponding neutralizing antigenic site, is located only 4 amino acids away from the junction region (Fig. 2). t is important to emphasize that the functions of HA, induction of neutralizing antibodies, and the priming effect reported in this work have not been assigned to VP8, but to the amino-terminal half of VP4 (represented in MS2-VP8') which contains, in addition to VP8 sequences, 140 amino acids of VP5. Despite that the MS2-VP5' protein was not able to induce neutralizing antibodies, it was shown to be immunogenic and to contain epitopes present in the VP4 protein as they are in the viral capsid, since it bound antibodies elicited by the complete viral particle, and it was able to induce antibodies that inhibited the hemagglutinating activity of the virus to a titer similar to that induced by the virus or the MS2-VP8' polypeptide. The antibodies induced by the MS2-VP8' protein had no neutralizing activity against SAl1 rotavirus, whereas they did neutralize NCDV and SA114fM. n addition, a hyperimmune antiserum to SA114fM neutralized both the NCDV and SAl1 rotavirus strains, while an NCDV antiserum neutralized SA114fM but not SAl1. These results indicate that the SA114fM and SAl1 strains share a common VP7, as known from their amino acid sequences (3, 5), while the SA114fM VP4 protein is antigenically more similar to NCDV VP4 than to SAl1 VP4. These results agree with the observations made by Nishikawa et al. (32) studying the rotavirus SAll-FEM strain, which also has a fast-migrating gene 4. The origin of the SA114fM strain is difficult to establish, but it may have arisen by reassortment in the laboratory between the SAl1 and NCDV rotavirus strains or by selection from the original Malherbe SAl1 stock, which might have consisted of a mixed population of viruses. The latter possibility is more likely since an SA114fM-like rotavirus was independently selected in Brazil at H. Pereira's laboratory after passing in vitro the Malherbe original SAl1 virus stock at a time when no bovine rotavirus existed in their laboratory (personal communication). The capacity of the MS2-VP8' antibodies to neutralize heterotypically was further tested with rotavirus strains Wa and ST3. While no neutralizing activity was detected against Wa, rotavirus ST3 was neutralized at a low but significant titer. The basis of this heterotypic neutralization is not clear, since most neutralizing epitopes mapped to VP8 seem to be homotypic (26). n addition, the sequence around amino acid 305 (present in the VP5 portion of the MS2-VP8' polypeptide), which has been shown to be a cross-reactive epitope among human rotavirus strains (38), is different in SA114fM and ST3 rotaviruses. However, a synthetic peptide representing amino acids 220 to 233 of the SA114fM VP4 protein has been shown recently to prime for neutralizing antibodies to rotavirus ST3 (1). The MS2-VP8' recombinant protein was able to prime the immune system of mice for an enhanced neutralizing antibody response to the SA114fM virus. That the titers of neutralizing antibodies obtained in response to the inoculation of primed animals with the complete SA114fM virus were similar for strains SAl1 and NCDV (as well as for the

8 1390 LZANO ET AL. SA114fM virus) suggests that the MS2-VP8' polypeptide is able to prime not only for an antibody response to VP4 epitopes but also for antigenic determinants present on VP7. Alternatively, since VP4 contains heterotypic epitopes, the priming with the MS2-VP8' polypeptide could have broadened the response to VP4 to encompass SAl1. Therefore, further experiments have to be done to conclusively prove whether the enhanced neutralizing activity is directed to VP7 as well as to VP4. On the other hand, the priming sera did not neutralize the Wa rotavirus strain, as would be expected since SA114fM and Wa viruses do not seem to share neutralizing antigenic determinants on either VP4 or VP7 (14). n this work, we have shown that the amino-terminal half of VP4, synthesized in E. coli, is able to hemagglutinate and to prime the immune system of mice for an augmented neutralizing antibody response. 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