Characterization of a synthetic peptide mimicking trypsin-cleavage site of rotavirus VP4.

A synthetic peptide corresponding to the trypsin cleavage site on the 84 k protein of bovine rotavirus was synthesized (VP4-peptide). This synthetic peptide could be cleaved by trypsin and therefore possessed the enzyme binding site present on the authentic protein. Further proof that this peptide mimicks the authentic trypsin cleavage site was the specific reaction of anti-peptide serum with the 84 k protein. The reaction of anti-peptide serum with infectious virus neutralized infectivity thereby supporting the biological importance of this site. Another interesting characteristic of this peptide was its ability to bind to the nucleocapsid protein resulting in a laddering effect on the nucleocapsid monomer (45 k), dimer (90 k) and trimer (135 k) [Gorzilia et al., J. Gen. Virol. 66, 1889-1900 (1985); Sabara et al., J. Virol. 53, 58-66 (1985); Sabara et al., J. Gen. Virol. 67, 201-212 (1986)]. Definitive proof of binding was provided by the fact that the increments in the ladder corresponded to the molecular weight of the synthetic peptide and that anti-peptide serum specifically reacted with the ladder formations. The laddering of the nucleocapsid could be eliminated by incubation with trypsin thus further supporting the formation of a synthetic peptide-nucleocapsid complex. Due to the ability of the peptide to bind to trypsin and to the nucleocapsid protein its biological activity was investigated. It appeared that increasing concentrations of the peptide reduced the rate of virus plaque formation, thereby suggesting that virus replication was inhibited. These results illustrate two features of this synthetic peptide which warrant further investigation; (1) its capacity to mimic an enzyme cleavage site and, (2) its ability to complex tightly to another protein. In protection-challenge experiments performed using a murine model, animals immunized with VP4-peptide provided protection passively, to neonates suckling on the immune dams, against a virulent rotavirus. The potential applications of this peptide in rotavirus diagnosis, therapy and synthetic peptides based vaccine is discussed.

Formulation of the purified fusion protein of respiratory syncytial virus with the saponin QS-21 induces protective immune responses in Balb/c mice that are similar to those generated by experimental infection.

The feasibility of employing a vaccine composed of the purified fraction 21 of Quillaja saponaria (QS-21) and the fusion (F) protein of respiratory syncytial virus (RSV) to induce protective immune responses in the lower respiratory tract of Balb/c mice was examined. Our goal was to compare local and systemic immune responses with those induced following immunization with the protein adsorbed to aluminium hydroxide (F/ALOH) adjuvant or by experimental infection. Sera from mice vaccinated with the QS-21 formulation (F/QS-21) contained elevated anti-F protein IgG antibody titres that were dependent on the dose of QS-21 employed. Similar to the immune responses generated by experimental infection, the sera from mice vaccinated with F/QS-21 possessed greater capacity to neutralize virus infectivity that was associated with the generation of heightened complement-fixing IgG2a antibody titres. In contrast, vaccination with F/ALOH elicited systemic immune responses that were characterized by a predominance of protein-specific antibodies of the IgG1 subclass and lower neutralizing antibody titres. The capacity of F/QS-21 to facilitate local pulmonary immune responses was also examined and found to be similar to those induced by experimental infection. After virus challenge, a 90-fold increase in the number of F protein-specific antibody-secreting cells was observed and associated with the clearance of virus from the infected lungs. Moreover, elevated levels of antigen-dependent killer cell activity were detected and appeared to be mediated by class I major histocompatibility complex restricted CD8+ T cells. Additional characterization of the pulmonary immune response was performed on the cellular infiltrates obtained after bronchoalveolar lavage and on formalin-fixed lung tissue. The local protective immune responses induced after challenge of the groups immunized with F/QS-21 or infectious virus were significantly different from those elicited in naive control mice injected with adjuvant alone, or in mice immunized with F/ALOH. The cellularity of the lavage fluids from the former groups was characterized by a significantly greater percentage of lymphocytes and less neutrophils. In similar fashion histological evaluation of the lungs from mice immunized with F/QS-21 or infectious virus revealed significantly elevated local immune responses after challenge. In conclusion, the results suggest that formulation with F/QS-21 alters the qualitative and quantitative nature of the immune response to the F glycoprotein when compared with the traditional aluminium-based adjuvants.

Molecular determinants of rotavirus virulence: localization of a potential virulence site in a murine rotavirus VP4.

The molecular basis of pathogenesis in vivo for a virulent mouse rotavirus (MRV) and a less virulent bovine rotavirus (BRV) were compared under in vitro and in vivo conditions. Obvious differences in the mobility of several genomic RNA segments were observed in one-dimensional gels. Under in vitro conditions, partial proteolytic peptide mapping identified differences between the two outer capsid proteins of these virus and no difference in inner capsid protein was observed. Since it has been observed by us and others that the gene coding for VP4 protein plays a significant role in determining virulence, the variability observed in the present study between the 84 k proteins (VP4) provided a basis for further investigations in order to locate a potential virulence determinant. A comparison of the carboxypeptidase digests of the MRV- and BRV-VP4 revealed an area of variability between amino acids 307 and 407, which may represent a site of virulence determinant. Under in vivo conditions the virulence of both parenteral BRV and MRV isolates and their corresponding reassortants (with replaced gene 4) were studied in murine and bovine hosts. Like their parents, BRV and MRV isolates, reassortants obtained by replacement of gene 4 in BRV with MRV gene 4 indicated that the dose of the virus isolate used and the clinical outcome in vivo was determined by gene segment 4. The implications of these findings to elucidate the molecular basis of pathogenesis of rotaviruses are discussed.

Characterization of two rotaviruses differing in their in vitro and in vivo virulence.

The proteins, genomic RNA and disassembly conditions and pathogenesis in vivo for a virulent mouse rotavirus (MRV) and a less virulent bovine rotavirus (BRV) were compared. An obvious difference in the mobility of several genomic RNA segments were observed in one-dimensional gels. Reassortants obtained by replacement of gene 4 in BRV with MRV gene 4 indicated that the dose of the virus used and the clinical outcome in vivo was determined by gene segment 4. Under in vitro conditions, a comparison of the inner capsid proteins by partial proteolytic peptide mapping did not reveal any difference between corresponding proteins. However, this technique did identify differences between the two corresponding outer capsid proteins of these viruses. These differences, in turn, may account for the increased stability of MRV, as compared to BRV, when subjected to calcium-chelating and chaotropic agents and may be one of the mechanisms involved in conferring virulence on the virus. The observed variability between the 84K proteins (VP4) provided a basis for further investigations in order to locate a potential virulence determinant, since it has been observed by us and others that the gene coding for this protein plays a role in determining virulence. A comparison of the carboxypeptidase digests of the MRV and BRV VP4 revealed an area of variability between amino acids 307 and 407, which may represent the site of a virulence determinant.

Biochemical and immunological characterization of a novel peptide carrier system using rotavirus VP6 particles.

A system which allows for the efficient attachment of synthetic peptides to spherical virus-like particles assembled from the VP6 rotavirus nucleocapsid protein is described. This attachment was shown to be mediated by peptide-protein interactions and did not require additional chemicals for conjugation. The resulting large macromolecular structure was highly immunogenic for both the VP6 protein and the coupled peptides. The antibody response to peptides bound to VP6 particles was of higher titre and longer duration than that induced by other carriers. In addition, the response to VP6-coupled peptides was not affected by prior exposure to rotavirus and exhibited a range of immunoglobulin subclasses in the absence of an adjuvant. These data demonstrate that assembled VP6 spherical particles are useful carriers for low doses of synthetic peptides.

Rotavirus particles function as immunological carriers for the delivery of peptides from infectious agents and endogenous proteins.

A major problem in the development of useful animal subunit vaccines has been the generation of immune responses to weakly immunogenic molecules. For this purpose a new and effective delivery system has been devised. This system is based upon the inner capsid of bovine rotavirus. Under the appropriate conditions, the inner capsid protein, designated BP6, can be made to self-assemble in vitro and form spherical particles. These particles possess an inherent capacity to target to cells of the immune system. Exploitation of these properties has led to the development of technology to couple antigens to the VP6 particles such that the sphere acts as a novel immunological carrier. This is based on a "binding peptide" derived from another rotavirus peptide, VP4, as well as on more traditional techniques of chemical coupling. We have coupled peptides or proteins to this carrier via the binding peptide and have shown that every epitope tested to date gave excellent immune responses. Furthermore, using this carrier, immunity has been developed without the use of adjuvants. This has far-reaching implications for animal and human immunization.

Effect of different routes of immunization with bovine rotavirus on lactogenic antibody response in mice.

The effect of different routes of immunization with either live or killed bovine rotavirus (BRV) on the production of lactogenic antibody response in mice was evaluated. The routes of immunization were intramuscular (IM), oral (O) or intradermal in the mammary region (IMam). Following immunization, serum antibody responses were monitored by an enzyme linked immunosorbent assay (ELISA). Following whelping, the mice were allowed to stay with their mother until sacrificed on alternate days post-parturition from day 1-11. Milk from their stomach was collected for antibody titration by ELISA and virus neutralization test. Regardless of the routes of immunization, a rapid increase in serum anti-rotavirus antibody titers was observed for the first 5 wk after immunization followed by a gradual decline. After parturition, the mean antibody titer of lacteal secretions, as determined by ELISA, increased gradually for 7 days with the greatest increase on day 9, followed by a decrease in anti-rotavirus antibody. These titers also correlated with antibody titers in milk as measured by virus neutralization test. The best lactogenic antibody response was observed when IMam X IM X 2 route of immunization was used with live BRV as the antigen. Interestingly, immunization via the oral route with killed BRV also resulted in good antibody responses. In contrast, in the group where killed BRV was used, animals receiving 3X orally had the highest antibody titer. The distribution of different antibody subtypes in milk samples revealed IgG to be the predominant antibody followed by IgM and IgA. Irrespective of the route of administration, there was an increase in IgA on day 9 as compared to day 1 in most of the groups. The significant role played by mucosal immunity in passive protection and the possible ways to modulate subtype specific lactogenic immune response are discussed.

Assessment of intestinal damage in rotavirus infected neonatal mice by a D-xylose absorption test.

A D-xylose absorption test has been standardized for use in newborn mice. It was used to measure small intestine dysfunction in neonate mice following infection with various isolates of rotaviruses. A xylose dose of 1 mg/g body weight was required to produce a maximum level of 100 micrograms/100 microliters of plasma 2 h after administration of D-xylose. The mice inoculated with rotavirus absorbed significantly less D-xylose compared with uninoculated control mice. A micromethod is described which proved to be suitable to quantitate D-xylose for determination of absorptive function of the small intestine in small animals such as newborn mice.

Biochemical evidence for the oligomeric arrangement of bovine rotavirus nucleocapsid protein and its possible significance in the immunogenicity of this protein.

The nucleocapsid protein of bovine rotavirus was shown to exist in trimeric units in both the virus particle and in infected cells, with the subunits linked by non-covalent interactions. These trimeric units complex further by disulphide bridges into larger units which may represent the hexameric structures observed by electron microscopy. Visualization of various nucleocapsid protein complexes was also achieved on polyacrylamide gels by treating virus preparations with urea at 37 degrees C or boiling in the presence and absence of 2-mercaptoethanol. Since virus particles devoid of nucleic acid were also broken down into trimeric subunits by such treatments, assembly of virus particles appears not to require an RNA-protein interaction. Four nucleocapsid-specific monoclonal antibodies with low neutralizing ability reacted with the monomeric (45,000 mol. wt., 45K), dimeric (90K), trimeric (135K) and trimeric pair (270K) subunits, indicating that a site responsible for neutralization is probably exposed after assembly of these subunits. Analysis of radiolabelled virus revealed that a high proportion (80%) of infectious particles could be immunoprecipitated by these monoclonal antibodies, suggesting that the virus particles are either partially double-shelled or have the nucleocapsid exposed on the surface. The monoclonal antibodies also cross-reacted with the nucleocapsid proteins of simian (SA11), pig (OSU), bovine (NCDV and UK) and human (Wa and ST4) rotaviruses in an immunoblot ELISA reaction. Since these six viruses belong to two different subgroups, it is likely that the antibodies did not recognize the subgroup-specific site, but a shared exposed antigenic determinant. Due to the hexameric configuration of the nucleocapsid in virus particles the neutralizing epitope may be repeatedly presented and, therefore, may contribute to the immunogenicity of this protein.

Inhibition of phagosome-lysosome fusion in macrophages by soluble extracts of virulent Brucella abortus.

Water extracts of virulent Brucella abortus were able to inhibit phagosome-lysosome fusion in unelicited murine peritoneal macrophages following ingestion of yeast. Extracts from an avirulent strain were unable to produce a similar effect. Lipopolysaccharide from B abortus did not appear to be involved with the ability of the extracts to inhibit fusion.

Synthetic hepatitis B surface antigen peptide vaccine.

A synthetic hepatitis B surface antigen (HBsAg) peptide was prepared containing amino acid residues 122-137 of the major HBsAg polypeptide. This peptide was cyclized by the introduction of an intrachain disulfide bond between cysteine residues at positions 124 and 137 because previous studies had shown that intact disulfide bonds are critical for maintenance of HBsAg activity. An anti-HBs response was produced in mice by free peptide entrapped in liposomes. However, the immunogenicity was enhanced by aggregation into micelles, and by coupling to tetanus toxoid. Analysis of the peptide with a panel of monoclonal antibodies showed that peptide 122-137 contained a conformation (discontinuous) group a epitope and a sequential (continuous) subgroup y epitope. In addition, the cyclic peptide inhibited a human anti-HBs idiotype-anti-idiotype reaction with specificity for group a determinant(s). The potential for synthetic peptides for hepatitis B virus vaccine development is discussed.