Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: Implications for glaucoma

Our group recently demonstrated that autoimmune T cells directed against central nervous system-associated myelin antigens protect neurons from secondary degeneration. We further showed that the synthetic peptide copolymer 1 (Cop-1), known to suppress experimental autoimmune encephalomyelitis, can be safely substituted for the natural myelin antigen in both passive and active immunization for neuroprotection of the injured optic nerve. Here we attempted to determine whether similar immunizations are protective from retinal ganglion cell loss resulting from a direct biochemical insult caused, for example, by glutamate (a major mediator of degeneration in acute and chronic optic nerve insults) and in a rat model of ocular hypertension. Passive immunization with T cells reactive to myelin basic protein or active immunization with myelin oligodendrocyte glycoprotein-derived peptide, although neuroprotective after optic nerve injury, was ineffective against glutamate toxicity in mice and rats. In contrast, the number of surviving retinal ganglion cells per square millimeter in glutamate-injected retinas was significantly larger in mice immunized 10 days previously with Cop-1 emulsified in complete Freund's adjuvant than in mice injected with PBS in the same adjuvant (2,133 ± 270 and 1,329 ± 121, respectively, mean ± SEM; P < 0.02). A similar pattern was observed when mice were immunized on the day of glutamate injection (1,777 ± 101 compared with 1,414 ± 36; P < 0.05), but not when they were immunized 48 h later. These findings suggest that protection from glutamate toxicity requires reinforcement of the immune system by antigens that are different from those associated with myelin. The use of Cop-1 apparently circumvents this antigen specificity barrier. In the rat ocular hypertension model, which simulates glaucoma, immunization with Cop-1 significantly reduced the retinal ganglion cell loss from 27.8% ± 6.8% to 4.3% ± 1.6%, without affecting the intraocular pressure. This study may point the way to a therapy for glaucoma, a neurodegenerative disease of the optic nerve often associated with increased intraocular pressure, as well as for acute and chronic degenerative disorders in which glutamate is a prominent participant.

The immunoprotective MHC II epitope of a chemically induced tumor harbors a unique mutation in a ribosomal protein

CD4+ T lymphocyte clones, generated from mice immunized with the methylcholanthrene-induced fibrosarcoma Meth A (H-2d), are restricted by I-Ed and recognize a unique antigen on Meth A. The antigen has been purified and characterized as the ribosomal protein L11. The antigenic epitope is contained within the sequence EYELRKHNFSDTG and is generated by substitution of Asn by His (italic) caused by a single point mutation. The tumor contains the wild-type and the mutated alleles. Immunization of BALB/cJ mice with the mutated epitope but not with the wild-type epitope protects mice against a subsequent challenge with the Meth A sarcoma. Adoptive transfer of CD4+ clones into BALB/c mice renders the mice specifically resistant to Meth A sarcoma. The mutated L11 epitope is thus shown to be an immunoprotective epitope in vivo by several criteria.

Immunity to retroviral infection: The Friend virus model

Friend virus infection of adult immunocompetent mice is a well established model for studying genetic resistance to infection by an immunosuppressive retrovirus. This paper reviews both the genetics of immune resistance and the types of immune responses required for recovery from infection. Specific major histocompatibility complex (MHC) class I and II alleles are necessary for recovery, as is a non-MHC gene, Rfv-3, which controls virus-specific antibody responses. In concordance with these genetic requirements are immunological requirements for cytotoxic T lymphocyte, T helper, and antibody responses, each of which provides essential nonoverlapping functions. The complexity of responses necessary for recovery from Friend virus infection has implications for both immunotherapies and vaccines. For example, it is shown that successful passive antibody therapy is dependent on MHC type because of the requirement for T cell responses. For vaccines, successful immunization requires priming of both T cell and B cell responses. In vivo depletion experiments demonstrate different requirements for CD8+ T cells depending on the vaccine used. The implications of these studies for human retroviral diseases are discussed.

Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy

Most tumor-associated antigens represent self-proteins and as a result are poorly immunogenic due to immune tolerance. Here we show that tolerance to carcinoembryonic antigen (CEA), which is overexpressed by the majority of lethal malignancies, can be reversed by immunization with a CEA-derived peptide. This peptide was altered to make it a more potent T cell antigen and loaded onto dendritic cells (DCs) for delivery as a cellular vaccine. Although DCs are rare in the blood, we found that treatment of advanced cancer patients with Flt3 ligand, a hematopoietic growth factor, expanded DCs 20-fold in vivo. Immunization with these antigen-loaded DCs induced CD8 cytotoxic T lymphocytes that recognized tumor cells expressing endogenous CEA. Staining with peptide-MHC tetramers demonstrated the expansion of CD8 T cells that recognize both the native and altered epitopes and possess an effector cytotoxic T lymphocyte phenotype (CD45RA+CD27−CCR7−). After vaccination, two of 12 patients experienced dramatic tumor regression, one patient had a mixed response, and two had stable disease. Clinical response correlated with the expansion of CD8 tetramer+ T cells, confirming the role of CD8 T cells in this treatment strategy.

Peripheral anti-Aβ antibody alters CNS and plasma Aβ clearance and decreases brain Aβ burden in a mouse model of Alzheimer's disease

Active immunization with the amyloid β (Aβ) peptide has been shown to decrease brain Aβ deposition in transgenic mouse models of Alzheimer's disease and certain peripherally administered anti-Aβ antibodies were shown to mimic this effect. In exploring factors that alter Aβ metabolism and clearance, we found that a monoclonal antibody (m266) directed against the central domain of Aβ was able to bind and completely sequester plasma Aβ. Peripheral administration of m266 to PDAPP transgenic mice, in which Aβ is generated specifically within the central nervous system (CNS), results in a rapid 1,000-fold increase in plasma Aβ, due, in part, to a change in Aβ equilibrium between the CNS and plasma. Although peripheral administration of m266 to PDAPP mice markedly reduces Aβ deposition, m266 did not bind to Aβ deposits in the brain. Thus, m266 appears to reduce brain Aβ burden by altering CNS and plasma Aβ clearance.

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety.

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

Vaccination with syngeneic, lymphoma-derived immunoglobulin idiotype combined with granulocyte/macrophage colony-stimulating factor primes mice for a protective T-cell response.

The idiotype of the Ig expressed by a B-cell malignancy (Id) can serve as a unique tumor-specific antigen and as a model for cancer vaccine development. In murine models of Id vaccination, formulation of syngeneic Id with carrier proteins or adjuvants induces an anti-idiotypic antibody response. However, inducing a potent cell-mediated response to this weak antigen instead would be highly desirable. In the 38C13 lymphoma model, we observed that low doses of free granulocyte/macrophage colony-stimulating factor (GM-CSF) 10,000 units i.p. or locally s.c. daily for 4 days significantly enhanced protective antitumor immunity induced by s.c. Id-keyhole limpet hemocyanin (KLH) immunization. This effect was critically dependent upon effector CD4+ and CD8+ T cells and was not associated with any increased anti-idiotypic antibody production. Lymphocytes from spleens and draining lymph nodes of mice primed with Id-KLH plus GM-CSF, but not with Id-KLH alone, demonstrated significant proliferation to Id in vitro without any biased production of interferon gamma or interleukin 4 protein or mRNA. As a further demonstration of potency, 50% of mice immunized with Id-KLH plus GM-CSF on the same day as challenge with a large s.c. tumor inoculum remained tumor-free at day 80, compared with 17% for Id-KLH alone, when immunization was combined with cyclophosphamide. Taken together, these results demonstrate that GM-CSF can significantly enhance the immunogenicity of a defined self-antigen and that this effect is mediated exclusively by activating the T-cell arm of the immune response.

Rad51 expression and localization in B cells carrying out class switch recombination.

Rad51 is a highly conserved eukaryotic homolog of the prokaryotic recombination protein RecA, which has been shown to function in both recombinational repair of DNA damage and meiotic recombination in yeast. In primary murine B cells cultured with lipopolysaccharide (LPS) to stimulate heavy chain class switch recombination, Rad51 protein levels are dramatically induced. Immunofluorescent microscopy shows that anti-Rad51 antibodies stain foci that are localized within the nuclei of switching B cells. Immunohistochemical analysis of splenic sections shows that clusters of cells that stain brightly with anti-Rad51 antibodies are evident within several days after primary immunization and that Rad51 staining in vivo is confined to B cells that are switching from expression of IgM to IgG antibodies. Following switch recombination, B cells populate splenic germinal centers, where somatic hypermutation and clonal proliferation occur. Germinal center B cells are not stained by anti-Rad51 antibodies. Rad51 expression is therefore not coincident with somatic hypermutation, nor does Rad51 expression correlate simply with cell proliferation. These data suggest that Rad51, or a highly related member of the conserved RecA family, may function in class switch recombination.

On the role of antigen in maintaining cytotoxic T-cell memory.

This study evaluated whether T-cell memory reflects increased precursor frequencies of specific long-lived T cells and/or a low-level immune response against some form of persistent antigen. Antivirally protective CD8+ T-cell memory was analyzed mostly in the original vaccinated host to assess the role of antigen in its maintenance. T-cell mediated resistance against reinfection was measured in the spleen and in peripheral solid organs with protocols that excluded protection by antibodies. In vivo protection was compared with detectable cytotoxic T-lymphocyte precursor frequencies determined in vitro. In the spleen, in vitro detectable cytotoxic T-lymphocyte precursor frequencies remained stable independently of antigen, conferring resistance against viral replication in the spleen during reinfection. In contrast, T-cell mediated resistance against reinfection of peripheral solid organs faded away in an antigen-dependent fashion within a few days or weeks. We show that only memory T cells persistently or freshly activated with antigen efficiently extravasate into peripheral organs, where cytotoxic T lymphocytes must be able to exert effector function immediately; both the capacity to extravasate and to rapidly exert effector function critically depend on restimulation by antigen. Our experiments document that the duration of T-cell memory protective against peripheral reinfection depended on the antigen dose used for immunization, was prolonged when additional antigen was provided, and was abrogated after removal of antigen. We conclude that T-cell mediated protective immunity against the usual peripheral routes of reinfection is antigen-dependent.

The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL). Immunization of mice with a carcinogen-induced colorectal tumor, CT26, engineered to secrete granulocyte/macrophage colony-stimulating factor, routinely generated both short-term and long-term CTL lines that not only lysed the parental tumor in vitro, but also cured mice of established tumor following adoptive transfer in vivo. When either short-term or long-term CTL lines were used to screen peptides isolated from CT26, one reverse-phase high performance liquid chromatography peptide fraction consistently sensitized a surrogate target for specific lysis. The bioactivity remained localized within one fraction following multiple purification procedures, indicating that virtually all of the CT26-specific CTL recognized a single peptide. This result contrasts with other tumor systems, where multiple bioactive peptide fractions have been detected. The bioactive peptide was identified as a nonmutated nonamer derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus. Adoptive transfer with CTL lines specific for this antigen demonstrated that this epitope represents a potent tumor rejection antigen. The selective expression of this antigen in multiple non-viral-induced tumors provides evidence for a unique class of shared immunodominant tumor associated antigens as targets for antitumor immunity.

Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction.

Vaccination with synthetic peptides representing cytotoxic T lymphocyte (CTL) epitopes can lead to a protective CTL-mediated immunity against tumors or viruses. We now report that vaccination with a CTL epitope derived from the human adenovirus type 5 E1A-region (Ad5E1A234-243), which can serve as a target for tumor-eradicating CTL, enhances rather than inhibits the growth of Ad5E1A-expressing tumors. This adverse effect of peptide vaccination was rapidly evoked, required low doses of peptide (10 micrograms), and was achieved by a mode of peptide delivery that induces protective T-cell-mediated immunity in other models. Ad5E1A-specific CTL activity could no longer be isolated from mice after injection of Ad5E1A-peptide, indicating that tolerization of Ad5E1A-specific CTL activity causes the enhanced tumor outgrowth. In contrast to peptide vaccination, immunization with adenovirus, expressing Ad5E1A, induced Ad5E1A-specific immunity and prevented the outgrowth of Ad5E1A-expressing tumors. These results show that immunization with synthetic peptides can lead to the elimination of anti-tumor CTL responses. These findings are important for the design of safe peptide-based vaccines against tumors, allogeneic organ transplants, and T-cell-mediated autoimmune diseases.

DNA vaccine for hepatitis B: evidence for immunogenicity in chimpanzees and comparison with other vaccines.

Vaccination of two chimpanzees against hepatitis B virus (HBV) by intramuscular injection of plasmid DNA encoding the major and middle HBV envelope proteins induced group-, subtype- and preS2-specific antibodies. These were initially of IgM isotype, and then they were of IgG (predominantly IgGl) isotype. The chimpanzee injected with 2 mg of DNA attained >100 milli-international units/ml of anti-HBs antibody after one injection and 14,000 milli-international units/ml after four injections. A smaller dose (400 microg) induced lower and transient titers, but a strong anamnestic response occurred 1 year later. Comparison with responses in 23 chimpanzees receiving various antigen-based HBV vaccines suggests that the DNA approach is promising for prophylactic immunization against HBV.

Mass vaccination to control chickenpox: the influence of zoster.

The impact of transmission events from patients with shingles (zoster) on the epidemiology of varicella is examined before and after the introduction of mass immunization by using a stochastic mathematical model of transmission dynamics. Reactivation of the virus is shown to damp stochastic fluctuations and move the dynamics toward simple annual oscillations. The force of infection due to zoster cases is estimated by comparison of simulated and observed incidence time series. The presence of infectious zoster cases reduces the tendency for mass immunization to increase varicella incidence at older ages when disease severity is typically greater.

Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.

Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America.

The silver-haired bat variant of rabies virus (SHBRV) has been identified as the etiological agent of a number of recent human rabies cases in the United States that are unusual in not having been associated with any known history of conventional exposure. Comparison of the different biological and biochemical properties of isolates of this virus with those of a coyote street rabies virus (COSRV) revealed that there are unique features associated with SHBRV. In vitro studies showed that, while the susceptibility of neuroblastoma cells to infection by both viruses was similar, the infectivity of SHBRV was much higher than that of COSRV in fibroblasts (BHK-21) and epithelial cells (MA-104), particularly when these cells were kept at 34 degrees C. At this temperature, low pH-dependent fusion and cell-to-cell spread of virus is seen in BHK-21 cells infected with SHBRV but not with COSRV. It appears that SHBRV may possess an unique cellular tropism and the ability to replicate at lower temperature, allowing a more effective local replication in the dermis. This hypothesis is supported by in vivo results which showed that while SHBRV is less neurovirulent than COSRV when administered via the intramuscular or intranasal routes, both viruses are equally neuroinvasive if injected intracranially or intradermally. Consistent with the above findings, the amino acid sequences of the glycoproteins of SHBRV and COSRV were found to have substantial differences, particularly in the region that contains the putative toxic loop, which are reflected in marked differences in their antigenic composition. Nevertheless, an experimental rabies vaccine based on the Pittman Moore vaccine strain protected mice equally well from lethal doses of SHBRV and COSRV, suggesting that currently used vaccines should be effective in the postexposure prophylaxis of rabies due to SHBRV.