Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to surface proteins vp3 and vp7.

Monoclonal antibodies directed against two rotavirus surface proteins (vp3 and vp7) as well as a rotavirus inner capsid protein (vp6) were tested for their ability to protect suckling mice against virulent rotavirus challenge. Monoclonal antibodies to two distinct epitopes of vp7 of simian rotavirus strain RRV neutralized RRV in vitro and passively protected suckling mice against RRV challenge. A monoclonal antibody directed against vp3 of porcine rotavirus strain OSU neutralized three distinct serotypes in vitro (OSU, RRV, and UK) and passively protected suckling mice against OSU, RRV, and UK virus-induced diarrhea. The role of vp3 in eliciting protection against heterotypic rotavirus challenge should be considered when developing a vaccine with cloned rotavirus genes. Alternatively, immunization with a reassortant rotavirus containing vp3 and vp7 from two antigenically distinct rotavirus parents might protect against diarrhea induced by two or more rotavirus serotypes.     

Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge.

A baculovirus-expressed VP4 protein derived from the simian rhesus rotavirus (RRV) was used to parenterally immunize murine dams. VP4-immunized dams developed high levels of neutralizing antibodies against RRV and low levels of cross-reactive neutralizing antibodies against human strains Wa, ST3, and S2 and animal strains SA-11, NCDV, and Eb. Newborn mice suckled on VP4-immunized dams were protected against a virulent challenge dose of the simian strain RRV and against murine rotavirus Eb. The cross-reactive nature of the serum-neutralizing response generated by VP4 immunization and the protective efficacy of the immunization suggest that recombinant-expressed VP4 proteins should be considered as viable vaccine candidates.     

Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles

The recognition that rotaviruses are the major cause of life-threatening diarrheal disease and significant morbidity in young children has focused efforts on disease prevention and control of these viruses. Although the correlates of protection in children remain unclear, some studies indicate that serotype-specific antibody is important. Based on this premise, current live attenuated reassortant rotavirus vaccines include the four predominant serotypes of virus. We are evaluating subunit rotavirus vaccines, 2/6/7-VLPs and 2/4/6/7-VLPs, that contain only a single VP7 of serotype G1 or G3. In mice immunized parenterally twice, G3 virus-like particles (VLPs) induced a homotypic, whereas G1 VLPs induced a homotypic and heterotypic (G3) serum neutralizing immune response. Administration of three doses of G1 or G3 VLPs induced serum antibodies that neutralized five of seven different serotype test viruses. The inclusion of VP4 in the VLPs was not essential for the induction of heterotypic neutralizing antibody in mice. To confirm these results in another species, rabbits were immunized parenterally with two doses of 2/4/6/7-VLPs containing a G3 or G1 VP7, sequentially with G3 VLPs followed by G1 (G3/G1) VLPs, or with live or psoralen-inactivated SA11. High-titer homotypic serum neutralizing antibody was induced in all rabbits, and low-level heterotypic neutralizing antibody was induced in a subset of rabbits. The rabbits immunized with the G1 or G3/G1 VLPs in QS-21 were challenged orally with live G3 ALA rotavirus. Protection levels were similar in rabbits immunized with homotypic G3 2/4/6/7-VLPs, heterotypic G1 2/4/6/7-VLPs, or G3/G1 2/4/6/7-VLPs. Therefore, G1 2/4/6/7-VLPs can induce protective immunity against a live heterotypic rotavirus challenge in an adjuvant with potential use in humans. Following challenge, broad serum heterotypic neutralizing antibody responses were detected in rabbits parenterally immunized with G1, G3/G1, or G3 VLPs but not with SA11. Immunization with VLPs may provide sufficient priming of the immune system to induce protective anamnestic heterotypic neutralizing antibody responses upon subsequent rotavirus infection. Therefore, a limited number of serotypes of VLPs may be sufficient to provide a broadly protective subunit vaccine.     

Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains.

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.     

Immunization with baculovirus-expressed recombinant rotavirus proteins VP1, VP4, VP6, and VP7 induces CD8+ T lymphocytes that mediate clearance of chronic rotavirus infection in SCID mice.

Clearance of chronic murine rotavirus infection in SCID mice can be demonstrated by adoptive transfer of immune CD8+ T lymphocytes from histocompatible donor mice immunized with a murine homotypic rotavirus (T. Dharakul, L. Rott, and H.B. Greenberg, J. Virol 64:4375-4382, 1990). The present study focuses on the protein specificity and heterotypic nature of cell-mediated clearance of chronic murine rotavirus infection in SCID mice. Heterotypic cell-mediated clearance was demonstrated in SCID mice infected with EDIM (murine) rotavirus after adoptive transfer of CD8+ T lymphocytes from BALB/c mice that were immunized with a variety of heterologous (nonmurine) rotaviruses including Wa (human, serotype 1), SA11 and RRV (simian, serotype 3), and NCDV and RF (bovine, serotype 6). This finding indicates the serotypic independence of T-cell-mediated rotavirus clearance. To further identify the rotavirus proteins that are capable of generating CD8+ T cells that mediate virus clearance, donor mice were immunized with SF-9 cells infected with a baculovirus recombinant expressing one of the following rotavirus proteins: VP1, VP2, NS53 (from RF), VP4, VP7, NS35 (from RRV), VP6, and NS28 (from SA11). SCID mice stopped shedding rotavirus after receiving CD8+ T cells from mice immunized with VP1, VP4, VP6, and VP7 but not with VP2, NS53, NS35, NS28, or wild-type baculovirus. These results suggest that heterotypic cell-mediated clearance of rotavirus in SCID mice is mediated by three of the major rotavirus structural proteins and by a putative polymerase protein.     

Comparative analysis of the VP3 gene of divergent strains of the rotaviruses simian SA11 and bovine Nebraska calf diarrhea virus.

The gene encoding outer capsid protein VP3 of subpopulations of two animal rotaviruses, simian SA11 and Nebraska calf diarrhea virus (NCDV), was analyzed. Two laboratory strains of simian SA11 rotavirus (SA11-SEM and SA11-FEM) differed with respect to VP3. This dimorphism was indicated by a difference in electrophoretic mobility and a difference in reactivity with anti-VP3 monoclonal antibodies. The overall VP3 amino acid homology between the two SA11 VP3 proteins was 82.7%, whereas the VP3 protein of SA11-FEM was 98.5% homologous in amino acid sequence to NCDV VP3, suggesting that SA11-FEM VP3 was derived by gene reassortment in the laboratory during contamination with a bovine rotavirus. A comparison of the deduced amino acid sequence of the VP3 of two virulent NCDV strains and an attenuated NCDV strain (RIT 4237), revealed only five amino acid differences which were scattered throughout the protein but did not involve the trypsin cleavage sites. Of interest, the VP3 of the standard strain of NCDV which is virulent for cows differed in only one amino acid (position 23, Gln to Lys) from the VP3 of an NCDV mutant which was attenuated both for cows and for children.     

Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope

The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcalpha3-Galbeta) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcalpha3Galbeta is preferred to NeuAcalpha3Galbeta. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.     

Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen.

A single-gene substitution reassortant 11-1 was generated from two porcine rotaviruses, OSU (serotype 5) and Gottfried (serotype 4). This reassortant derived 10 genes, including gene 4 encoding VP3, from the OSU strain and only gene 9, encoding a major neutralization glycoprotein (VP7), from the Gottfried strain and was thus designated VP3:5; VP7:4. Oral administration of this reassortant to colostrum-deprived gnotobiotic newborn pigs induced a high level of neutralizing antibodies not only to Gottfried VP7 but also to OSU VP3, thus demonstrating that VP3 is as potent an immunogen as VP7 in inducing neutralizing antibodies during experimental oral infection. Gnotobiotic piglets infected previously with the reassortant were completely resistant to oral challenge with the virulent Gottfried strain (VP3:4; VP7:4), as indicated by failure of symptoms to develop and lack of virus shedding. Similarly, prior infection with the reassortant induced almost complete protection against diarrhea and significant restriction of virus replication after oral challenge with the virulent OSU strain (VP3:5; VP7:5). Thus, it appears that (i) the immune system of the piglet responds equally well to two rotavirus outer capsid proteins, VP3 and VP7, during primary enteric rotavirus infection; (ii) antibody to VP3 and antibody to VP7 are each associated with resistance to diarrhea; and (iii) infection with a reassortant rotavirus bearing VP3 and VP7 neutralization antigens derived from two viruses of different serotype induces immunity to both parental viruses. The relevance of these findings to the development of effective reassortant rotavirus vaccines is discussed.     

Molecular and antigenic characterization of porcine rotavirus YM, a possible new rotavirus serotype.

In 1983, we isolated a porcine rotavirus (strain YM) that was prevalent in several regions of Mexico, as judged by the frequency of its characteristic electropherotype. By a focus reduction neutralization test, rotavirus YM was clearly distinguished from prototype rotavirus strains belonging to serotypes 1 (Wa), 2 (S2), 3 (SA11), 4 (ST3), 5 (OSU), and 6 (NCDV). Minor, one-way cross-neutralization (1 to 5%) was observed when antisera to the various rotavirus strains were incubated with rotavirus YM. In addition, the YM virus was not neutralized by neutralizing monoclonal antibodies with specificity to serotypes 1, 2, 3, and 5. The subgroup of the virus was determined to be I by enzyme-linked immunosorbent assay. To characterize the serotype-specific glycoprotein of the virus at the molecular level, we cloned and sequenced the gene coding for VP7. Comparison of the deduced amino acid sequence with reported homologous sequences from human and animal rotavirus strains belonging to six different serotypes further supported the distinct immunological identity of the YM VP7 protein.     

Comparison of mucosal and systemic humoral immune responses and subsequent protection in mice orally inoculated with a homologous or a heterologous rotavirus.

Rotaviruses are the single most important cause of severe diarrhea in young children worldwide, and vaccination is probably the most effective way to control the disease. Most current live virus vaccine candidates are based on the host range-restricted attenuation of heterologous animal rotaviruses in humans. The protective efficacy of these vaccine candidates has been variable. To better understand the nature of the heterologous rotavirus-induced active immune response, we compared the differences in the mucosal and systemic immune responses generated by heterologous (nonmurine) and homologous (murine) rotaviruses as well as the ability of these infections to produce subsequent protective immunity in a mouse model. Sucking mice were orally inoculated with a heterologous simian or bovine rotavirus (strain RRV or NCDV) or a homologous murine rotavirus (wild-type or tissue culture-adapted) strain EHP at various doses. Six weeks later, mice were challenged with a virulent murine rotavirus (wild-type strain ECW) and the shedding of viral antigen in feces was quantitated. Levels of rotavirus-specific serum immunoglobulin G (IgG) and fecal IgA prior to challenge were measured and correlated with subsequent viral shedding or protection. Heterologous rotavirus-induced active protection was highly dependent on the strain and dose of the virus tested. Mice inoculated with a high dose (10(7) PFU per mouse) of RRV were completely protected, while the protection was diminished in animals inoculated with NCDV or lower doses of RRV. The ability of a heterologous rotavirus to stimulate a detectable intestinal IgA response correlated with the ability of the virus to generate protective immunity. Serum IgG titer did not correlate with protection. Homologous rotavirus infection, on the other hand, was much more efficient at inducing both mucosal and systemic immune responses as well as protection regardless of the virulence of the virus strain or the size of the immunizing dose.     

人类轮状病毒基因DNA免疫的初步研究

本文研究人类轮状病毒基因ＤＮＡ免疫及应用。通过构建重组质粒ｐＣＩ／ｖｐ７,ｐＣＩ／ｖｐ４及ｐＣＩ／ｖｐ６,以肌注法导入ＢＡＬＢ／ｃ小鼠肌肉组织,其中ｐＣＩ／ｖｐ７及ｐＣＩ／ｖｐ４能　有效引起机体免疫应答,而且对轮状病毒Ｗａ株有一定中和效应。从本次试验的结果来看　,人类轮状病毒ＤＮＡ疫苗能作为预防病毒性小儿腹泻的一种手段。     

Protection against rotavirus-induced gastroenteritis in a murine model by passively acquired gastrointestinal but not circulating antibodies.

Newborn mice suckled on dams immunized either orally or parenterally with primate rotavirus SA-11 were protected against diarrhea induced by SA-11 virus challenge. Experimental oral administration of milk from orally immunized dams protected suckling mice against challenge; protective activity was detected both in the anti-rotavirus immunoglobulin A (IgA) and IgG fractions, but IgA was more potent in vivo than IgG. Oral administration of milk from parentally immunized dams also protected suckling mice against challenge; in this case, protective activity was detected in the anti-rotavirus IgG fraction. In newborn mice foster-nursed by seronegative dams, circulating rotavirus-specific antibodies in high titer did not protect mice against oral SA-11 virus challenge. It appears that the most effective rotavirus vaccine will be that which induces an efficient production of antibodies active at the intestinal cell surface.     

Polypeptide specificity of antiviral serum antibodies in children naturally infected with human rotavirus.

Reassortants between serotype 3 SA11 and serotype 6 NCDV rotaviruses were used to determine the relative amounts of serum-neutralizing antibody to VP4 and VP7 of serotype 3 SA11 rotavirus in children after natural rotavirus exposure. Sera from Ecuadorian children of a population-based study and sera from children of a hospital-based study in Germany (excluding diarrhea patients) demonstrated high titers of VP7-specific but only low titers of VP4-specific antibodies. In contrast, paired sera from German children hospitalized with a symptomatic primary rotavirus gastroenteritis demonstrated a titer increase to VP4 more frequently than to VP7 protein by neutralization test and immunoblotting. For these rotavirus patients, we provided, previously, direct evidence for the development of cross-neutralizing antibodies.     

Comparative amino acid sequence analysis of VP4 for VP7 serotype 6 bovine rotavirus strains NCDV, B641, and UK.

In a previous study (S. Zheng, G. N. Woode, D. R. Melendy, and R. F. Ramig, J. Clin. Microbiol. 27:1939-1945, 1989), it was predicted that the VP7 serotype 6 bovine rotavirus strains NCDV and B641 do not share antigenically similar VP4s. In this study, gene 4 and the VP7 gene of B641 were sequenced, and the amino acid sequences were deduced and compared with those of NCDV and bovine rotavirus strain UK. Amino acid sequence homology in VP7 between the three strains was greater than 94%, confirming their relationship as VP7 serotype 6 viruses. VP4 of B641 showed amino acid homology to UK of 94% but only 73% homology to NCDV. Sequence comparison of a variable region of VP8 demonstrated amino acid homology of 53% between B641 and NCDV, whereas B641 and UK were 89% homologous in this region. These results confirm the earlier prediction that although the same serotype by VP7 reactivity, B641 and NCDV represent different VP4 serotypes. This difference in VP4 may have contributed to the lack of homotypic protection observed in calves, implicating VP4 as an important antigen in the active immune response to rotavirus infection in bovines.     

Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus.

Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neutralizing antibody to the VP4 and VP7 proteins of the vaccine virus. To do this, reassortant rotaviruses that contained the WC3 genome segment for only one of these two neutralization proteins were made. The segment for the other neutralization protein in these reassortants was from heterotypic rotaviruses that were serotypically distinct from WC3. Sera were examined from 31 infants who had no evidence of a previous rotavirus infection and the highest postvaccination WC3-neutralizing antibody titers (i.e., 160 to 600) of the 103 subjects administered the vaccine. A reassortant (3/17) that contained both neutralization proteins from the heterotypic rotaviruses, i.e., EDIM (EW strain of mouse rotavirus) VP7 and rhesus rotavirus VP4, was not neutralized by these sera (geometric mean titer [GMT], less than 20). A reassortant (E19) that contained EDIM VP7 and WC3 VP4 was also very poorly neutralized by these antisera (GMT = 20). In contrast, antibody titers to a reassortant (R20) that contained WC3 VP7 and rhesus rotavirus VP4 were higher than those against WC3 (GMTs of 458 and 313, respectively). Thus, VP7 appeared to be the dominant immunogen for production of neutralizing antibody after intestinal infection of previously uninfected infants vaccinated with WC3 bovine rotavirus.     

Passive immunity modulates genetic reassortment between rotaviruses in mixedly infected mice.

Genetic reassortment between simian rotavirus SA11 and rhesus rotavirus (RRV) occurs with high frequency following mixed infection of nonimmune suckling mice (J. L. Gombold and R. F. Ramig, J. Virol. 57:110-116, 1986). We examined the effects of passively acquired homotypic or heterotypic immunity on reassortment in vivo. Passively immune suckling mice obtained from dams immune to either serotype 3 simian rotavirus (SA11) or serotype 6 bovine rotavirus (NCDV) were infected orally with either SA11 or RRV or a mixture of SA11 and RRV (both serotype 3 viruses). At various times postinfection, signs of disease were noted and the intestines of individual mice were removed and homogenized for titration of infectious virus and isolation of progeny plaques. Electrophoresis of genomic RNA was used to identify reassortants among the viral progeny isolated from infected animals. No reassortants (less than 0.45%) were detected among 224 clones examined from mixedly infected, homotypically immune mice. Twenty-nine reassortants (10.66%) were identified among 272 progeny clones from mixedly infected, heterotypically immune mice. Thus, reassortment was reduced more than 50-fold by homotypic immunity and approximately threefold by heterotypic immunity compared with prior data obtained from mixed infections of nonimmune mice. In addition, reassortment between SA11 and RRV in nonimmune mice was shown to be dependent on the virus dose. Taken together, these results suggest that immune responses may modulate the frequency of reassortment by reducing the effective multiplicity of infection (by neutralization or other immune mechanisms), thereby preventing efficient mixed infection of enterocytes.     

Analysis of reassortment of genome segments in mice mixedly infected with rotaviruses SA11 and RRV.

Seven-day-old CD-1 mice born to seronegative dams were orally inoculated with a mixture of wild-type simian rotavirus SA11 and wild-type rhesus rotavirus RRV. At various times postinfection, progeny clones were randomly isolated from intestinal homogenates by limiting dilution. Analysis of genome RNAs by polyacrylamide gel electrophoresis was used to identify and genotype reassortant progeny. Reassortment of genome segments was observed in 252 of 662 (38%) clones analyzed from in vivo mixed infections. Kinetic studies indicated that reassortment was an early event in the in vivo infectious cycle; more than 25% of the progeny clones were reassortant by 12 h postinfection. The frequency of reassortant progeny increased to 80 to 100% by 72 to 96 h postinfection. A few reassortants with specific constellations of SA11 and RRV genome segments were repeatedly isolated from different litters or different animals within single litters, suggesting that these genotypes were independently and specifically selected in vivo. Analysis of segregation of individual genome segments among the 252 reassortant progeny revealed that, although most segments segregated randomly, segments 3 and 5 nonrandomly segregated from the SA11 parent. The possible selective pressures active during in vivo reassortment of rotavirus genome segments are discussed.