Biochemical and immunologic comparison of virus-like particles for a rotavirus subunit vaccine.

A parenterally administered rotavirus vaccine composed of virus-like particles (VLPs) is being evaluated for human use. VLPs composed of bovine VP6 and simian VP7 (SA11, G3) proteins (6/7-VLPs) or of bovine VP2, bovine VP6, and simian VP7 (SA11, G3) proteins (2/6/7-VLPs) were synthesized and purified from Sf9 insect cells co-infected with recombinant baculoviruses. 6/7- and 2/6/7-VLP administered parenterally (i.m.) in mice had comparable immunogenicity, but the 2/6/7-VLPs were more homogeneous and stable. The inclusion of the VP2 capsid contributed to particle formation and stability. The adjuvant QS-21 significantly enhanced the immunogenicity of 2/6/7-VLPs over A10H or saline alone. Equivalent serum neutralizing antibody responses were induced over the range of 1-15 microg/dose of 2/6/7-VLPs administered with the range of 5-20 microg/dose of QS-21. The immunogenicity of 2/6/7-VLPs and inactivated SA11 virus were comparable. 2/6/7-VLPs are a promising candidate for a parenterally delivered rotavirus subunit vaccine.

Heterotypic protection from rotavirus infection in mice vaccinated with virus-like particles.

Virus-like particles (VLPs) composed of rotavirus VP2, VP6, and VP7 of G1 or G3 serotype specificity were produced in insect cells coinfected with recombinant baculoviruses expressing single rotavirus genes. The VLPs were purified and subsequently evaluated for immunogenicity and protection in the adult mouse model of rotavirus infection. Mice were vaccinated twice intramuscularly with G1 VLPs formulated with Quillaja saponaria (QS-21) or adsorbed to aluminium hydroxide (AlOH), or with G1 VLPs alone. G3 VLPs, G1 plus G3 VLPs, inactivated SA11 virions formulated with QS-21, or adjuvants were similarly inoculated as controls. Mice were examined for serum and fecal antibody responses by ELISA or microneutralization assays. Protective efficacy of the VLP vaccine formulations against oral challenge with the G3 murine ECwt rotavirus was assessed by comparing the antigen shed in stool of the VLP-vaccinated mice to that of the adjuvant-immunized mice. G1 VLPs in QS-21 induced significantly higher serum and intestinal antibody titers than G1 VLPs in AlOH or G1 VLPs alone. QS-21 also heightened serum and fecal antibody responses to G3 VLPs. These QS-21-augmented antibody responses were further characterized by equivalent IgG1 and IgG2a titers in sera, suggesting that G1 or G3 VLPs in QS-21 induced a balanced Th1/Th2 response. G1 VLPs in QS-21 induced partial protection (88%) against oral challenge with the heterotypic ECwt virus, whereas G3 VLPs in QS-21 induced complete protection (100%). In contrast, G1 VLPs when formulated with AlOH induced a predominant Th2 response and did not protect (1%) mice from virus challenge. Our results indicate that the type of adjuvant used clearly influences both antibody responses to rotavirus VLPs and the protective efficacy against rotavirus infections. These data have important implications for the development of parenteral vaccines to ameliorate rotavirus disease.

Synthesis of rotavirus-like particles in insect cells: comparative and quantitative analysis.

When the three major structural proteins, VP2, VP6, and VP7, of rotavirus are co-expressed in insect cells infected with recombinant baculoviruses, they self-assemble into triple-layered virus-like particles (VLPs) that are similar in morphology to native rotavirus. In order to establish the most favorable conditions for the synthesis of rotavirus VLPs, we have compared the kinetics of 2/6/7-VLP synthesis in two different insect cell lines: High Five cells propagated in Excell 405 medium and Spodoptera frugiperda 9 cells in Excell 400 medium. The majority of VLPs produced in both cell lines were released into the culture medium, and these released VLPs were predominantly triple-layered and were found to be stable for the period of six or seven days examined. The optimal synthesis of VLPs depended upon the cell line and the culture medium used as well as the time of harvesting infected cell cultures. The highest yield of VLPs was obtained from High Five cultures in the late phase of infection when the yield was at least 5-fold higher than that from S. frugiperda 9 cultures on a per cell basis. Our results demonstrate the usefulness of High Five cells for the production of VLPs as potential rotavirus subunit vaccines.

Virus-like particles as a rotavirus subunit vaccine.

Rotavirus subunit vaccines are being evaluated for use in humans. The virus-like particles (VLPs) for these vaccines are produced in insect cells coinfected with combinations of baculovirus recombinants expressing bovine RIF VP2 and simian SA11, VP4, VP6, or VP7 rotavirus proteins. VLPs were administered parenterally to mice and rabbits, and the immunogenicity and protective efficacy of the vaccines were evaluated. Rabbits vaccinated with VP2/4/6/7 or VP2/6/7 VLP combinations developed high levels of rotavirus-specific serum antibody and fecal IgG but not fecal IgA. The induction of fecal IgG was associated with total or partial protection from oral challenge with ALA rotavirus. Heterotypic serum and fecal neutralizing antibody was induced in mice vaccinated parenterally with G1 VP2/6/7 or VP2/4/6n VLPs. VLPs were highly immunogenic when administered in QS21 adjuvant, inducing serum neutralizing antibody titers comparable to those induced by SA11 virus. VLPs are effective immunogens when administered parenterally and may be an effective subunit vaccine.

Sequence diversity of small, round-structured viruses in the Norwalk virus group.

We have determined the nucleotide sequences of a highly conserved region of the RNA-dependent RNA polymerase of the prototype Snow Mountain agent (SMA) and of four other small, round-structured viruses (antigenically Norwalk virus [NV]-like or SMA-like) following reverse transcription-PCR amplification of viral RNA obtained from human stools. The stool samples were either from volunteers administered SMA or from sporadic cases and outbreaks of gastroenteritis that occurred in Japan and the United Kingdom between 1984 and 1992. The GLPSG and YGDD RNA polymerase motifs were in the proper locations in the sequences of the five SRSVs, but each sequence was distinct from the 8FIIa prototype NV sequence and from each other. Analysis of the sequences and reactivities in a new NV antigen enzyme-linked immunosorbent assay showed that the five viruses could be divided into two groups (serogroups) with NV and SMA, respectively, being the prototypes. The sequences of the capsid region and a nonstructural region (2C) were determined from one strain from each group. One virus (SRSV-KY-89/89/J), isolated in Japan and antigenically similar to the prototype NV (isolated 21 years earlier in Ohio), showed a remarkable level of sequence similarity to NV. KY-89 and the 8FIIa NV showed 87.2% nucleotide similarity over 2,516 continuous nucleotides amounting to 96 to 98.9% amino acid similarity in three distinct domains in two open reading frames. Between the prototype SMA and NV, the polymerase region showed 63% nucleotide and 59% amino acid similarity, respectively. Two other antigenically SMA-like isolates (SRSV-925/92/UK and SRSV-OTH-25/89/J), from the United Kingdom and Japan, showed 80% nucleotide and 88 to 92% amino acid similarity in the polymerase region to the prototype SMA isolated 16 and 13 years earlier in the United States. The capsid region of the antigenically SMA-like OTH-25 virus showed 53% nucleotide and 65% amino acid similarity to the prototype NV capsid region. Domains of sequence diversity and conversation were identified within the capsid protein of these two distinct prototype serotypes of virus. These results indicate that NV-like and SMA-like agents are still circulating, and sequence comparisons will be useful to identify and classify distinct viruses in the NV group.

Norwalk virus infection of volunteers: new insights based on improved assays.

Norwalk virus infection is a common cause of gastroenteritis in humans. The clinical features and virologic and immunologic responses following oral administration of Norwalk virus to 50 volunteers were monitored. New ELISAs using recombinant virus particles as the antigen source were used to assess the pattern of virus shedding and the specific immune responses. Forty-one subjects (82%) became infected; 68% were symptomatic and 32% were asymptomatic. The proportion of subjects infected was similar for those with and without preexisting antibody (82% vs. 60%; P > .2). The magnitude of seroconversion was highest in subjects who had vomiting. The peak of viral shedding was between 25 and 72 h, and virus first appeared in stool at 15 h. Specimens collected 7 days after inoculation remained positive. These results show a higher infection rate, more subclinical infections, and longer virus excretion following Norwalk virus inoculation than previously recognized.

Subclass-specific serum antibody responses to recombinant Norwalk virus capsid antigen (rNV) in adults infected with Norwalk, Snow Mountain, or Hawaii virus.

Subclass-specific antibody responses to the Norwalk virus capsid protein in adults challenged with Norwalk, Snow Mountain, or Hawaii virus were evaluated by solid-phase enzyme immunoassay using recombinant Norwalk virus capsid antigen (rNV). Fourfold or greater serum immunoglobulin G (IgG) antibody responses to rNV were detected in 15 of 20 volunteers challenged with Norwalk virus, and serum IgA and IgM antibody responses to rNV were seen in almost all subjects who had rNV IgG responses. Serum rNV IgG antibody responses also were detected in 6 of 15 volunteers challenged with Snow Mountain virus and 2 of 12 volunteers challenged with the Hawaii virus. However, the magnitude of antibody response and the geometric mean postchallenge rNV IgG antibody titers were lower in subjects challenged with Snow Mountain or Hawaii virus, and serum IgA and IgM responses generally did not occur.

Field trial of rhesus rotavirus or human-rhesus rotavirus reassortant vaccine of VP7 serotype 3 or 1 specificity in infants. The Elmwood, Panorama, and Westfall Pediatric Groups.

Orally administered live rhesus monkey rotavirus vaccine (RRV, VP7 serotype 3) and human-rhesus reassortant rotavirus vaccine (DxRRV, VP7 serotype 1) were evaluated in a placebo-controlled field trial of 223 infants 2-4 months old. Both vaccines were mildly reactogenic but were generally well tolerated in the 10 days after vaccination. RRV and DxRRV were immunogenic, inducing serum antibody responses in 78% and 71% of the vaccines, respectively. Efficacy of RRV vaccine was 66% (P = .01) and of DxRRV vaccine 77% (P = .002) against rotavirus-associated illness in the first season after vaccination. Efficacy of RRV vaccine against rotavirus-associated illness over three rotavirus seasons was 51.2% (P = .045) and of DxRRV vaccine was 67.3% (P = .006). RRV vaccine provided heterotypic protection of 58.5% (P = .041) and DxRRV vaccine provided homotypic protection of 72.8% (P = .005) over three seasons against the predominant serotype 1 rotavirus.

Antigenic relatedness among the Norwalk-like agents by serum antibody rises.

The Norwalk, Snow Mountain (SMA), and Hawaii agents are etiologically associated with separate outbreaks of acute viral gastroenteritis. Previous cross-challenge of volunteers, immune electron microscopy, and/or enzyme-immunoassay analysis suggested that these agents are antigenically distinct. We examined paired sera from human volunteers challenged with these agents for the presence of homologous and heterologous serum antibody titer rises to the agents. Two-way cross-reactions occurred between Hawaii agent and SMA. A one-way cross-reaction occurred between Norwalk agent and SMA, as volunteers challenged with Norwalk agent had heterologous serum antibody titer rises to SMA, but the reverse did not occur. The Norwalk and Hawaii agents had minimal cross-reaction, with only one volunteer challenged with Hawaii agents having a heterologous rise to Norwalk agent. These observations indicate varying degrees of antigenic relatedness among these agents.

Comparison of three enzyme immunoassays to tissue culture for the diagnosis of rotavirus gastroenteritis in infants and young children.

Three enzyme immunoassays (EIAs), Rotazyme II, IDL, and Pathfinder, were evaluated for rotavirus detection in stool and rectal swab specimens from children with symptomatic gastroenteritis and compared with virus isolation in primary African green monkey kidney cells. Of 125 specimens tested, 49 were rotavirus positive by tissue culture isolation; of these 49, 40 were positive by Rotazyme II, 43 were positive by IDL, and 46 were positive by Pathfinder EIAs. As compared with tissue culture isolation, the Rotazyme II, IDL, and Pathfinder EIAs had sensitivities of 82, 88, and 94%, specificities of 90, 99, and 95%, and overall agreements of 86, 94, and 94%, respectively.

Foodborne Snow Mountain agent gastroenteritis with secondary person-to-person spread in a retirement community.

A variety of small round-structured viruses are being recognized with increasing frequency as a cause of gastroenteritis in the community, but have rarely been reported to cause outbreaks in hospitals or extended-care facilities. From March 20 through April 15, 1988, an outbreak of gastroenteritis occurred in a retirement facility in the San Francisco Bay area. Illness was characterized by diarrhea, nausea, and vomiting; two residents died. Attack rates were 46% (155 of 336) in residents and 37% (28 of 75) in employees. During the initial outbreak period, illness among residents was associated with two shrimp meals served in the facility dining hall (odds ratio = 6.7). Person-to-person transmission probably occurred: The risk of becoming ill one or two days after a roommate became ill was significantly greater than that of becoming ill at other times during the outbreak (risk ratio = 6.5). Microbiologic examinations for bacterial and parasitic enteric pathogens were negative; however, 27-nm viral particles were detected by immune electron microscopy and by blocking enzyme immunoassay to Snow Mountain agent in stools obtained at the onset of illness from one of six ill residents. Seroconversion (greater than fourfold antibody rise) to Snow Mountain agent was detected in acute- and convalescent-phase serum specimens from five of six ill residents as measured by enzyme immunoassay, but not for Norwalk agent as measured by radioimmunoassay. This report of an outbreak of Snow Mountain agent gastroenteritis in an extended-care facility documents that these difficult-to-identify 27-nm viruses can cause outbreaks in inpatient settings.

Viral gastroenteritis aboard a cruise ship.

A 32-nm small round structured virus (SRSV), possibly related to the Snow Mountain agent (SMA), was implicated as the cause of recurrent outbreaks of gastroenteritis on a cruise ship. There was no identifiable relation to food or water consumption, but the risk of gastroenteritis among passengers who had shared toilet facilities was twice that of those who had a private bathroom and the rate of illness was related to the number of passengers sharing a communal restroom (ie, with one or more toilets): contaminated bathrooms may be an important vehicle for person-to-person spread of this enteric agent. In each cabin, index patients who had vomited in their cabins were more likely to have had cabinmates who subsequently became ill than were index patients who had not vomited. These epidemiological findings implicate vomitus in the transmission of viral gastroenteritis and they are consistent with the transmission of viral agents by airborne droplets or person-to-person contact. New strategies for prevention of viral gastroenteritis should include protection against environmental contamination by viruses in airborne droplets or vomitus.

Development of an enzyme immunoassay for the Hawaii agent of viral gastroenteritis.

The Hawaii agent is a Norwalk-like virus of acute gastroenteritis in humans which is antigenically distinct from the prototype Norwalk agent. We established a solid phase sandwich type microtiter enzyme immunoassay (EIA) for Hawaii antigen employing sera and stools from experimentally challenged volunteers as reagents. This assay detected the Hawaii agent in stools from 3 of 8 volunteers who were ill after oral challenge with the Hawaii agent, including one specimen which was positive to a dilution of 1/320. Virus shedding occurred on days 3 to 7 after challenge. The Hawaii-positive stools did not react in the EIAs for Norwalk and Snow Mountain agent (SMA), nor did Norwalk or SMA-positive stools react in the Hawaii EIA. Human rotavirus, enteric adenovirus, feline calicivirus, and several enteroviruses also did not react in the Hawaii EIA. A blocking EIA to detect serum antibody to the Hawaii agent was also developed employing a diarrheal stool containing Hawaii as a source of antigen. Serum antibody rises were detected in 15 of 16 individuals with experimentally induced illness after challenge and in 3 of 5 individuals who remained well after challenge. The EIA for the Hawaii agent should permit epidemiologic studies of the Hawaii agent to be carried out as well as allow further characterization of the Hawaii virion.

Field trial of rhesus rotavirus vaccine in infants.

Orally administered rhesus rotavirus vaccine (RRV) was evaluated in a placebo-controlled study in 176 infants (ages 2 to 4 months). Eighty-eight infants received a dose of 10(4) plaque-forming units of the vaccine, and 88 received the placebo. RRV was well-tolerated but mildly reactogenic in the 10 days after vaccination. There were mild febrile reactions (greater than or equal to 38 degrees C rectally) in 40% of the vaccinees and in 16% of the placebo recipients (P = 0.001). More of the vaccinees had loose stools than did the placebo recipients (P less than 0.05). RRV was immunogenic and induced a 4-fold or greater rise in serum neutralizing antibody responses in 67% of the vaccinees; however, breast-fed infants were less likely to develop a seroresponse than infants who were not breast-fed. Despite the good immunogenicity of RRV the overall incidence of rotavirus-associated illnesses was similar between the vaccine and placebo recipients. The failure of RRV in Rochester may be related to the fact that the circulating rotaviruses were predominantly serotype 1 and RRV is a serotype 3 rotavirus. Because the serotypes of rotavirus that predominate may vary from year to year, a polyvalent preparation may be necessary to provide effective vaccination against rotaviruses.

Production of a monoclonal antibody against the Snow Mountain agent of gastroenteritis by in vitro immunization of murine spleen cells.

The Snow Mountain agent (SMA) is a 27- to 32-nm noncultivatable virus that causes acute gastroenteritis in humans. SMA is morphologically similar to but immunologically distinct from the Norwalk agent. SMA has been partially purified from the stool of experimentally infected volunteers and contains a single structural protein of Mr 62,000 as well as one or more non-virion-associated soluble proteins. Further characterization of this important human pathogen and other Norwalk-like viruses has been hindered by the lack of reagents with which to study them. To further characterize SMA, we developed a monoclonal antibody to SMA using in vitro immunization--a technique that permitted use of small quantities of antigen for immunization. The monoclonal antibody, SM-4, was specific for SMA and did not react with the Norwalk or Hawaii agents. In addition, SM-4 reacted with purified virion but not with the soluble protein. SM-4 also blocked the ability of labeled postinfection human IgG to bind to purified virion. Finally, both SM-4 and human postinfection sera specifically recognized the Mr 62,000 virion-associated protein. Thus, SM-4 is directed against an epitope present on the SMA structural protein that is not shared by the Norwalk or Hawaii agents and that is not present on the soluble protein. The availability of a monoclonal antibody against SMA should facilitate further purification and characterization of this agent. The techniques utilized in these studies provide a method for the production of additional monoclonal antibodies to this group of viruses and also should be useful for the study of other occult viral agents.

Snow Mountain agent gastroenteritis from clams.

A 1983 investigation of two clambake-related gastroenteritis outbreaks in Rochester, New York, showed that 84 (43%) of 196 persons interviewed had an acute illness characterized by watery diarrhea, vomiting, and abdominal cramps. None of the ill persons were hospitalized or had complications. Illness was associated with eating raw (p = 0.002) or baked (p less than 0.01) hard-shell clams, with the risk of illness increasing with the total number of clams consumed (p less than 0.01). The median incubation period and duration of illness were 36 and 44 hours, respectively. Stool samples obtained 2-4 days after onset of illness were negative for commonly recognized bacterial and viral pathogens. However, of 31 persons whose stools were tested, the stool of only one ill person was positive by enzyme-linked immunosorbent assay for the Snow Mountain agent, one of the Norwalk-like viruses. Paired serum specimens from six (67%) of nine ill and two (29%) of seven well persons showed a fourfold or greater rise in antibody titer to Snow Mountain agent. Persons who ate clams were more likely to seroconvert to Snow Mountain agent (eight of 12) than were those who did not eat clams (zero of four) (p = 0.04). The clams were harvested off the coast of southern Massachusetts in late October, when harvest waters were documented to be contaminated by untreated municipal sewage. This report describes the first documented outbreak of shellfish-associated gastroenteritis attributed to Snow Mountain agent of which we are aware.

Foodborne Snow Mountain agent gastroenteritis in a school cafeteria.

In 1984, an outbreak of gastroenteritis occurred at a school with 1,860 students in Brooklyn, NY. In a single-stage cluster sample of 375 students, 129 (34%) had illnesses that met our case definition of vomiting or diarrhea. The mean incubation period was 26 hours, and the mean illness duration was 24 hours. All case students had eaten in the cafeteria on at least one day between Nov 13 and 16, compared with 174/214 (81%) noncase students (P = 10(-8), Fisher exact test). Foods implicated were french fries (relative risk 1.7, 95% confidence limits 1.4, 2.0) and hamburgers (relative risk 1.6, 95%, confidence limits 1.2, 2.1). Two cafeteria employees had served those foods while affected by diarrhea. By a recently developed blocking enzyme-linked immunosorbent assay, six of 11 (55%) case students showed fourfold antibody increases between acute- and convalescent-phase serum samples for Snow Mountain agent, a Norwalk-like virus, compared with one of ten (10%) noncase students (P = .04, Fisher exact test). We strongly suspect, but cannot document conclusively, that the Snow Mountain agent was spread to students on a vector of hot foods contaminated by ill food handlers. Implicated foods conferred low relative risks and could only have accounted for 74% of cases of illness. The strong association between cafeteria exposure and illness, therefore, suggests that additional modes of spread occurred.

Novel agents of viral enteritis in humans.

Considerable information has recently emerged regarding certain "groups" of novel viruses associated with gastroenteritis in humans. The viruses reviewed here are 20-35 nm in diameter and can be detected in the stools of acutely ill individuals with gastroenteritis. These viruses can be conveniently divided into four "groups": Norwalk-like agents, caliciviruses, astroviruses, and other small round viruses. The evidence for the etiologic association of these agents with gastroenteritis varies from agent to agent but is most extensive for the Norwalk-like agents, of which the Norwalk serotype is a major cause of epidemic gastroenteritis. Human caliciviruses appear to be relatively common causes of gastroenteritis in children, particularly in Japan and the United Kingdom. Astroviruses have been reported as occasional causes of gastroenteritis in children and adults in various parts of the world. The epidemiological significance of the other small round viruses is unknown. Because of the difficulty of cultivating these agents in vitro, biochemical and antigenic characterization of these agents is incomplete. An understanding of the pathogenesis of disease and of the roles of immune responses to infection is similarly at a primitive stage. The recent development of sensitive and efficient assays for detection of several of these agents and the reports that certain strains of human caliciviruses and astroviruses can be cultivated in vitro should facilitate characterization and epidemiological studies of these agents. Systematic, prospective epidemiological studies of these agents in well-defined populations of various age groups are sorely needed for definition of the relative importance of each agent in human disease. Such information is essential for the consideration of appropriate control measures.