The biophysical properties of the tobacco mosaic tobamovirus (TMV) coat protein (CP) make it possible to display foreign peptides on the surface of TMV. The immunogenic epitopes G5-24 from the rabies virus (RV) glycoprotein, and 5B19 from murine hepatitis virus (MHV) S-glycoprotein were successfully displayed on the surface of TMV, and viruses accumulated to high levels in infected leaves of Nicotiana tabacum Xanthi-nn. The peptide RB19, which contains an arginine residue plus the 5B19 epitope fused to the CP (TMV-RB19), resulted in the induction of necrotic local lesions on inoculated leaves of N. tabacum Xanthi-nn and cell death of infected BY2 protoplasts. RNA dot blot assays confirmed that expression of the acidic and basic pathogenesis-related PR2 genes were induced in infected Xanthi-nn leaf tissue. TMV that carried epitope 31D from the RV nucleoprotein did not accumulate in inoculated tobacco leaves. Analysis of hybrid CPs predicted that the isoelectric points (pI):charge value was 5.31:-2 for wild-type CP, 5.64:-1 for CP-RB19, and 9.14:+2 for CP-31D. When acidic amino acids were inserted in CP-RB19 and CP-31D to bring their pI:charge to near that of wild-type CP, the resulting viruses TMV-RB19E and TMV-4D:31D infected N. tabacum Xanthi-nn plants and BY2 protoplasts without causing cell death. These data show the importance of the pI of the epitope and its effects on the hybrid CP pI:charge value for successful epitope display as well as the lack of tolerance to positively charged epitopes on the surface of TMV.
Hybrids of tobacco mosaic virus (TMV) were constructed with the use of fusion to the coat protein peptides of 10 or 15 amino acids, containing the 5B19 epitope from the spike protein of murine hepatitis virus (MHV) and giving rise to TMV-5B19 and TMV-5B19L, respectively. The TMV hybrids were propagated in tobacco plants, and the virus particles were purified. Immunogold labeling, with the use of the monoclonal MAb5B19 antibody, showed specific decoration of hybrid TMV particles, confirming the expression and display of the MHV epitope on the surface of the TMV. Mice were immunized with purified hybrid viruses after several regimens of immunization. Mice that received TMV-5B19L intranasally developed serum IgG and IgA specific for the 5B19 epitope and for the TMV coat protein. Hybrid TMV-5B19, administered by subcutaneous injections, elicited high titers of serum IgG that was specific for the 5B19 epitope and for coat protein, but IgA that was specific against 5B19 was not observed. Mice that were immunized with hybrid virus by subcutaneous or intranasal routes of administration survived challenge with a lethal dose (10 ؋ LD 50 ) of MHV strain JHM, whereas mice administered wild-type TMV died 10 d post challenge. Furthermore, there was a positive correlation between the dose of administered immunogen and protection against MHV infection. These studies show that TMV can be an effective vaccine delivery vehicle for parenteral and mucosal immunization and for protection from challenge with viral infection.Several plant viruses have been developed for use as vectors for the expression and delivery of foreign peptides (1-7). These examples include immunogenic epitopes that can be used in vaccines to confer protective immunity against human and animal diseases. We have demonstrated that a hybrid tobacco mosaic virus (TMV), containing the 13 amino acid sequence of the murine zona pellucida ZP3 epitope fused to a region near the C terminus of the coat protein (CP) successfully elicited the production of anti-ZP3 antibody in parenterally immunized animals. Anti-ZP3 antibody accumulated around the ova of immunized animals; however, it did not prevent fertilization (5).Murine hepatitis virus (MHV), a member of the Coronaviridae family, is responsible for a variety of acute and chronic diseases in its natural host. The strain JHM of MHV induces demyelinating encephalomyelitis with high mortality, and surviving mice exhibit chronic demyelination. MHV contains at least three dominant structural proteins: the membrane glycoprotein (M protein), the nucleocapsid protein (N protein), and the spike glycoprotein (S protein) (8). The S protein is posttranslationally processed to yield the S1 and the S2 proteins (9). The S protein has been the focus of many studies because of its important role in viral attachment to target cellular receptors (10), its membrane penetration, and its ability to induce cell fusion (11). The S protein is a critical determinant of viral pathogenicity and has been shown to contain major immunodomina...
Excessive collagen deposition plays a critical role in the development of fibrosis, and early or active fibrosis may be more susceptible to therapeutic intervention than later stages of scarring. However, at present there is no simple method for assessing the collagen-synthesizing and secreting activity of fibroblasts in human tissues. Type I procollagen carboxyterminal domains are proteolytically removed during collagen secretion. Thus, antibodies to these domains should stain fibroblasts synthesizing type I collagen but not extracellular collagen fibrils which could mask the signal from the cells. We developed and characterized a monoclonal antibody (Anti-pC) specific for the carboxyterminal propeptide of type I procollagen.
Daily treatment of rats with the thymothyroid hormone leucogenenol after their immunosuppression with anti-lymphocyte globulins causes the rats to regain their immunocompetency after 7 days of treatment, in contrast to well over a month, which is required for untreated controls. Immunocompetency was mesured by the ability of the rat to respond to the injection of sheep erythrocytes with the formation of normal concentrations of antibody-forming cells in their lymphoid tissues and normal titers of hemolysins in their serum. These results indicate that leucogenenol increases the rate of development of T cells, as well as B cells, from committed precursors.
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