To identify the major antigenic determinant of native Salmonella flagella of antigenic type d, we constructed a series of mutated flicM genes with deletions and amino acid alterations in hypervariable region IV and in regions of putative epitopes as suggested by epitope mapping with synthetic octameric peptides (T. M. Joys and F. Schodel, Infect. Immun. 59:3330-3332, 1991). The expressed product of most of the mutant genes, with deletions of up to 92 amino acids in region IV, assembled into functional flagella and conferred motility on flagellin-deficient hosts. Serological analysis of these flagella with different anti-d antibodies revealed that the peptide sequence centered at amino acids 229 to 230 of flagellin was a dominant B-cell epitope at the surface of d flagella, because replacement of these two amino acids alone or together with their flanking sequence by a tripeptide specified by a linker sequence eliminated most reactivity with antisera against wild-type d flagella as tested by enzyme-linked immunosorbent assay or by Western immunoblot. Functional analysis of the mutated flagellin genes with or without an insert suggested that amino acids 180 to 214 in the 5' part of hypervariable region IV (residues 181 to 307 of the total of 505) is important to the function of flagella. The hybrid proteins formed by insertion of peptide sequence pre-Sl 12-47 of hepatitis B virus surface antigen into the deleted flagellins assembled into functional flagella, and antibody to the pre-Sl sequence was detected after immunization of mice with the hybrid protein. This suggests that such mutant flagellins containing heterologous epitopes have potential as vaccines.The bacterial flagellar filament consists of a single protein named flagellin (14). The flagellin monomer, after synthesis inside the cell, is believed to travel by a central channel through the rod, hook, and filament and to be added to the filament at its tip. The rigid helical structure of flagellar filaments enables them to impart translational movement to the cell when they are driven by rotary motors. This unique property suggests that the primary structure of flagellin may be subject to rigorous constraints to ensure its proper transportation and correct assembly into a functional structure. The Salmonella flagella carry the H antigen, one of the major antigens that elicit immune responses in the infected host (11). The H antigen is highly variable, with various serotypes (5). The antigenic properties of flagella have been studied by selection and genetic analysis of spontaneous, serum-selected flagellar antigen mutants (18)(19)(20), with results suggesting that only a small part of the flagellin molecule carries the flagellar antigen determinants. The cloning and sequencing of several Salmonella flagellin genes (13,36,37) showed highly conserved amino acid sequences at the ends and hypervariable regions at the middle of flagellin, a protein of 505 amino acids (aa). This result suggests that the structural and functional features common to different flag...
There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Adl310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigenspecific CTL response. The response induced by Adl310 was stronger than that by Ad1312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Adl310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.Activation of naive T cells requires not only recognition of an antigen-major histocompatibility complex on the surface of antigen-presenting cells (APC) by the T-cell receptor but also costimulatory signals, which can be provided by spleen cells of immunized mice (1). One such signal has been shown to result from interaction of costimulatory factors on the surface of APCs with specific molecules on the surface of T cells. Costimulatory factors on professional APCs include the B7-1 protein (CD80), which specifically interacts with the CD28 and CTLA-4 proteins on the surface of T cells resulting in a costimulatory signal for T-cell activation (2-9). B7-1 appears to be expressed only by professional APC (macrophages, dendritic cells, and activated B cells) and not other cells in vivo (3,10,11).The experiments described here were done to determine whether the cytotoxic T lymphocyte (CTL) response and antibody response are enhanced when cells expressing a foreign antigen in vivo are made to also express B7-1. This was done by delivering both genes for a viral antigen (hepatitis B surface antigen or HBsAg) and B7-1 in a recombinant viral vector to cells of BALB/c mice and comparing the immune response...
Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered major risk factors in the development of hepatocellular carcinoma (HCC) in humans and in animals. A high rate of mutations in the p53 tumor suppressor gene in hepatocellular carcinomas of predominantly hepatitis B virus (HBV) carrier patients has been recently related to dietary aflatoxin. Another member of the hepadnavirus family, the woodchuck hepatitis virus (WHV), infects woodchucks in a manner similar to that of HBV in humans. Therefore, it was of particular interest to determine whether the p53 gene in woodchuck HCCs associated with hepadnavirus infection and with exposure to AFB1 is affected in the same manner as in human HCCs. By direct PCR-sequencing, we analyzed exons 4-9 of the p53 gene in 13 HCCs from 12 woodchucks (two uninfected, ten WHV carriers). Six WHV carrier and two uninfected woodchucks were treated with AFB1. None of the analyzed HCC samples exhibited mutations, either in p53 gene exons 4-9, or in splicing donor-acceptor sites. The present data are consistent with our previous study that indicated a low rate of p53 mutations in HCCs of AFB1-treated ground squirrels, either infected or not infected with ground squirrel hepatitis virus, and in WHV carrier woodchucks not exposed to AFB1. Overall, our findings indicate that in woodchucks and in ground squirrels exposure to aflatoxin may affect the development of p53 mutations less than in humans.
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