Biomedical subjects
Publications and source records attributed to R Ahmed.
Despite extensive documentation of prolonged antibody responses following vaccination or acute viral infection, the mechanisms behind long-term antibody production are not fully understood. We propose the hypothesis that long-lived plasma cells are an important, yet largely overlooked, component of long-term humoral immunity.
CD40 ligand is expressed on activated T cells and interacts with CD40 on B cells and monocytes. It is not known what role CD40 ligand plays in the generation of immune responses to viral infection. To address this issue, we examined virus-specific T- and B-cell responses in CD40 ligand-deficient (CD40L-/-) mice following infection with lymphocytic choriomeningitis virus (LCMV). We found that primary anti-LCMV specific antibody responses were severely impaired in CD40L-/- mice, with the defect being most striking for antibody of the immunoglobulin G1 (IgG1) isotype. Interestingly, low levels of LCMV-specific antibodies of the IgG2a, IgG2b, and IgG3 isotypes were made in the CD40L-/- mice, showing that IgG1 responses are totally dependent on CD40L but that at least some IgG2a, IgG2b, and IgG3 responses can be CD40L independent. However, unlike CD40L+/+ mice, CD40L-/- mice were unable to sustain virus-specific antibody responses and showed a gradual decline in serum antibody levels over time. The CD40L-/- mice were also deficient in the generation of memory B cells. In contrast to the severely impaired humoral responses, CD40L-/- mice generated potent virus-specific CD8+ cytotoxic T-lymphocyte responses after LCMV infection and were able to clear the virus. These results show that CD40L does not play a role in generating primary virus-specific CD8+ cytotoxic T-lymphocyte responses but does affect the primary antibody response and the generation of memory B cells.
Listeria monocytogenes is a facultative intracellular bacterium that is able to escape phagocytic vesicles and replicate in the cytoplasm of infected cells. As with viral vectors, this intracytoplasmic life cycle provides a means for introducing foreign proteins into the major histocompatibility complex class I pathway of antigen presentation. Using recombinant L. monocytogenes (rLM) strains expressing the full-length nucleoprotein (NP) or a single cytotoxic T-lymphocyte (CTL) epitope from lymphocytic choriomeningitis virus (LCMV), we analyzed antiviral CTL responses induced by rLM vaccination. After vaccination, rLM was cleared from the host within 7 days while inducing an LCMV-specific ex vivo CD8+ effector CTL response. Virus-specific CTL memory was maintained for 6 months postvaccination, as demonstrated by vigorous secondary CTL responses after in vitro stimulation. A single immunization with rLM that expressed either the full-length NP gene or the CTL epitope alone resulted in LCMV NP-specific CTL precursor frequencies of approximately 1/10(4) CD8+ T cells. A second rLM vaccination resulted in enhanced virus-specific CTL activity and in vitro proliferation. rLM-vaccinated mice were protected against chronic viral infection by an accelerated virus-specific memory CTL response. These mice cleared infectious virus as well as viral antigen, suggesting that sterilizing immunity was achieved. In contrast to mice that received wild-type LM, rLM-vaccinated mice were protected from virally induced immunosuppression and splenic atrophy associated with chronic LCMV infection. The ability to elicit long-term cell-mediated immunity is fundamental in designing vaccines against intracellular pathogens, and these results demonstrate the efficacy of recombinant LM vaccination for inducing protective antiviral CTL memory.
Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2Ld-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8+ T cells.
Cottontail rabbit papillomavirus is the major papillomavirus animal model with which to study host-virus interactions. As with human papillomaviruses, papillomas may spontaneously regress, persist, or progress to carcinoma. Here we show that the majority (88%) of regressor rabbits had antibody to the nonstructural protein E2 compared to 29% in animals with persisting papilloma. The antibody response to other nonstructural viral proteins was the same for rabbits with regressing and persisting papilloma. The cellular immune response was measured by an in vitro proliferation assay. The responses to E6 and E7 were infrequent and similar in papilloma-bearing and in regressor rabbits and no rabbits responded to E1. In contrast, the response to E2 was more frequent in regressor rabbits. These data suggest that E2-specific immune responses may play a role in tumor regression.
Intracranial inoculation of neonatal mice of certain inbred strains with Pichinde virus has been found to be fatal, but Balb/c neonates survive such infection. Survival of Balb/c mice after neonatal inoculation was not linked to the major histocompatability complex. Virus was gradually cleared in surviving Balb/c mice but could be detected in the brain and kidneys for up to 9 months after infection. These animals were not immunologically tolerant but exhibited high antibody titers to viral antigens. MHC restricted cytotoxic T cell activity was also demonstrable in persistently infected mice following challenge with high titered virus. Pathological changes consistent with glomerulonephritis were observed in the kidneys and surviving mice were runted compared to normals. This model differs from the widely studied persistent infection of mice with lymphocytic choriomeningitis virus (LCMV) and provides a unique model for the study of the genetics of resistance to viral infection, mechanisms of persistence and pathological processes in chronic viral infections.
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During investigation of a gastroenteritis outbreak in a chronic care institution, Norwalk virus was found in stool specimens from two individuals and bacterial isolates presumptively identified as Bacillus cereus were isolated from four individuals (including one with Norwalk virus) and spice. Phage typing confirmed all Bacillus clinical isolates were phage type 2. All clinical isolates were subsequently identified as B. thuringiensis when tested as a result of a related study (L. Leroux, personal communication). Eight of 10 spice isolates were phage type 4. All B. cereus and B. thuringiensis isolates showed cytotoxic effects characteristic of enterotoxin-producing B. cereus. An additional 20 isolates each of B. cereus and B. thuringiensis from other sources were tested for cytotoxicity. With the exception of one B. cereus, all showed characteristic cytotoxic patterns.
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Lymphocytic choriomeningitis virus clone 13 (LCMV clone 13), a variant isolated from the spleens of neonatally infected mice, causes persistent infections in mice infected as adults. Such persistently infected mice succumb to a normally sublethal dose of Histoplasma capsulatum, and their macrophages contain overwhelming numbers of yeast cells of the fungus. Both LCMV clone 13 and H. capsulatum yeast cells target and replicate in macrophages of the host. We sought to study the effects of LCMV clone 13 on the ability of macrophages to control growth of H. capsulatum in vitro. We show that the growth of H. capsulatum within macrophages was not directly affected by the presence of LCMV clone 13. However, macrophages containing LCMV clone 13 did not respond fully to gamma interferon (IFN-gamma) stimulation. Such unresponsiveness resulted in proliferation of the fungus within macrophages cultured in the presence of IFN-gamma. The addition of anti-IFN-alpha/beta antibodies to LCMV clone 13-infected macrophage cultures restored macrophage responsiveness to IFN-gamma. These results indicate that production of IFN-alpha/beta by LCMV clone 13-infected macrophages antagonizes their responsiveness to IFN-gamma. Such antagonism may be one of the mechanisms by means of which certain viruses cause immune suppression and susceptibility to opportunistic infections.
Bacillus cereus is responsible for an increasing number of food poisoning cases. By using 12 bacteriophages isolated from sewage, a typing scheme for B. cereus isolates from outbreaks or sporadic cases of food poisoning was developed. The phages belonged to three morphotypes. Ten phages with contractile tails and icosahedral heads were members of the Myoviridae family, and two phages with noncontractile tails belonged to the Siphoviridae family. Phage 11 represented a new species. It had an isometric head and a very long contractile tail with long wavy tail fibers and was one of the largest viruses known. The vast majority of 166 B. cereus strains (161, or 97%) isolated from food poisoning cases were typeable. Of 146 strains isolated from 18 outbreaks, 142 (97%) could be divided into 17 phage types. A good correlation, on the order of 80 to 100%, between phage types of strains isolated from suspected foods and those of strains isolated from stools of symptomatic patients was observed. Most Bacillus thuringiensis strains were also typeable, providing further evidence of the close relatedness of B. cereus and B. thuringiensis. This phage typing scheme can be a valuable epidemiological tool in tracing the origins of food poisoning caused by B. cereus.
Cottontail rabbit papillomavirus is the major animal model for cancer-associated papillomaviruses. Here we show that vaccination with the nonstructural proteins E1 and E2 induces the regression of virus-induced papillomas and that vaccination is equally effective when proteins are given with and without adjuvant. There was no correlation between antibody levels and regression, suggesting that tumor regression may be due to a cell-mediated response.
Antiviral antibody production is often sustained for long periods after resolution of an acute viral infection. Despite extensive documentation of this phenomenon, the mechanisms involved in maintaining long-term antibody production remain poorly defined. As a first step towards understanding the nature of long-term humoral immunity, we examined the anatomical location of antibody-producing cells during acute viral infection. Using the lymphocytic choriomeningitis virus (LCMV) model, we found that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appears in the bone marrow and constitutes the major source of long-term antibody production. Following infection of adult mice, LCMV-specific antibody-secreting cells (ASC) peaked in the spleen at 8 days postinfection but were undetectable in the bone marrow at that time. The infection was essentially cleared by 15 days, and the ASC numbers in the spleen rapidly declined while an increasing population of LCMV-specific ASC began to appear in the bone marrow. Compared with the peak response at 8 days postinfection, time points from 30 days to more than 1 year later demonstrated greater-than-10-fold reductions in splenic ASC. In contrast, LCMV-specific plasma cell numbers in the bone marrow remained high and correlated with the high levels of antiviral serum antibody. The presence of LCMV-specific plasma cells in the bone marrow was not due to persistent infection at this site, since the virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and PCR assays. The immunoglobulin G subclass profile of antibody-secreting cells derived from bone marrow and the spleen correlated with the immunoglobulin G subclass distribution of LCMV-specific antibody in the serum. Upon rechallenge with LCMV, the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. Bone marrow ASC populations and LCMV-specific antibody levels in the serum did not change during the early phase of the reinfection, but both increased about two-fold by 15 days postchallenge. After both primary and secondary viral infections, LCMV-specific plasma cells were maintained in the bone marrow, showing that the bone marrow is a major site of long-term antibody production after acute viral infection. These results documenting long-term persistence of plasma cells in the bone marrow suggest a reexamination of our current notions regarding the half-life of plasma cells.
This study shows that DNA vaccination can confer protection against a persistent viral infection by priming CD8+ cytotoxic T lymphocytes (CTL). Adult BALB/c (H-2d) mice were injected intramuscularly with a plasmid expressing the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) under the control of the cytomegalovirus promoter. The LCMV NP contains the immunodominant CTL epitope (amino acids 118 to 126) recognized by mice of the H-2d haplotype. After three injections with 200 micrograms of NP DNA, the vaccinated mice were challenged with LCMV variants (clones 13 and 28b) that establish persistent infection in naive adult mice. Fifty percent of the DNA-vaccinated mice were protected, as evidenced by decreased levels of infectious virus in the blood and tissues, eventual clearance of viral antigen from all organs tested, the presence of an enhanced LCMV-specific CD8+ CTL response, and maintenance of memory CTL after clearance of virus infection. However, it should be noted that protection was seen in only half of the vaccinated mice, and we were unable to directly measure virus-specific immune responses in any of the DNA-vaccinated mice prior to LCMV challenge. Thus, at least in the system that we have used, gene immunization was a suboptimal method of inducing protective immunity and was several orders of magnitude less efficient than vaccination with live virus. In conclusion, our results show that DNA immunization works against a persistent viral infection but that efforts should be directed towards improving this novel method of vaccination.
We have used a perforin-less (PO) mouse to explore alternate CTL-mediated lytic pathways. PO mice are unable to overcome an infection with LCMV in vivo. Nevertheless, splenocytes from infected mice show vigorous, antigen-specific cytotoxicity that requires the presence of the Fas antigen on target cells. The Fas lytic pathway is virtually indistinguishable, in terms of kinetics and magnitude of cytotoxicity, from perforin/granzyme-mediated lysis. It is rapidly induced in CTL upon occupation of the TcR, and requires protein synthesis for full expression. Upon removal of the activating signal, the capacity for fas-mediated lysis rapidly disappears. PO mice infected with LCMV also undergo what appears to be a CD8-mediated immunopathology, and rarely live beyond one month. The precise basis of this pathology is unknown at present. Given the widespread distribution of Fas in mice, particularly on inflamed tissues, the complete failure to clear virus from any tissue or organ is surprising.
Initial phytochemical screening of the seeds of Punica granatum has revealed the presence of Ursolic acid and beta-Sitosterol alongwith a long straightchain hydrocarbon - nonacosene. Presence of estrogens and glycosides have also been detected.
Mice lacking the perforin gene were generated by using targeted gene disruption in embryonal stem cells. When infected with lymphocytic choriomeningitis virus (LCMV), perforin-less (-/-) mice showed clear signs of having mounted an immune response based on activation of CD8 T cells but were unable to clear the LCMV infection. This failure to eliminate virus was accompanied by a failure to generate spleen cells capable of lysing LCMV-infected fibroblasts in vitro. Spleen cells from LCMV-infected -/- mice were able to lyse hematopoietic target cells after exposure to phorbol 12-myristate 13-acetate and ionomycin, provided the target cells expressed the Fas antigen. Spleen cells from -/- mice also responded to alloantigen in mixed leukocyte culture by blastogenesis and proliferation. The resulting cells were able to lyse hematopoietic target cells, although not as well as spleen cells from +/+ littermates sensitized in the same manner. However, lysis by -/- cells was again seen only if the target cells expressed Fas antigen. We conclude that perforin-less -/- mice retain and express the Fas lytic pathway as expressed in vitro but that this pathway is insufficient to clear an LCMV infection in vivo.
Memory is a hallmark of the immune system and ever since its recognition there has been considerable interest in understanding how immunity is maintained. The current model is that long-term memory is dependent on persistent antigenic stimulation. We report here results that challenge this view and provide evidence that antigen is not essential for the maintenance of CD8+ T-cell memory. We show that memory CD8+ cytotoxic T lymphocytes persist indefinitely in the absence of priming antigen, retain the memory phenotype (CD44hi), and provide protection against virus challenge. These findings suggest a re-evaluation of our current thinking on mechanisms involved in maintaining immunity and have implications towards designing effective vaccination strategies.