Characterization of anti-anti-idiotypic antibodies that bind antigen and an anti-idiotype

Two mouse monoclonal anti-anti-idiotopic antibodies (anti-anti-Id, Ab3), AF14 and AF52, were prepared by immunizing BALB/c mice with rabbit polyclonal anti-idiotypic antibodies (anti-Id, Ab2) raised against antibody D1.3 (Ab1) specific for the antigen hen egg lysozyme. AF14 and AF52 react with an “internal image” monoclonal mouse anti-Id antibody E5.2 (Ab2), previously raised against D1.3, with affinity constants (1.0 × 109 M−1 and 2.4 × 107 M−1, respectively) usually observed in secondary responses against protein antigens. They also react with the antigen but with lower affinity (1.8 × 106 M−1 and 3.8 × 106 M−1). This pattern of affinities for the anti-Id and for the antigen also was displayed by the sera of the immunized mice. The amino acid sequences of AF14 and AF52 are very close to that of D1.3. In particular, the amino acid side chains that contribute to contacts with both antigen and anti-Id are largely conserved in AF14 and AF52 compared with D1.3. Therapeutic immunizations against different pathogenic antigens using anti-Id antibodies have been proposed. Our experiments show that a response to an anti-Id immunogen elicits anti-anti-Id antibodies that are optimized for binding the anti-Id antibodies rather than the antigen.

DNA immunization: Induction of higher avidity antibody and effect of route on T cell cytotoxicity

Immunizations of mice with plasmid DNAs encoding ovalbumin (OVA), human Ig, and hen egg lysozyme were compared with doses of soluble protein (without adjuvant) that induced similar IgG responses. The route of immunization influenced the magnitude of the antibody (Ab) response in that intradermal (i.d.) injection elicited higher IgG Ab levels than i.m. injection in both DNA- and protein-immunized mice. Although total IgG levels were similar to soluble protein controls, the avidity of the anti-OVA Abs generated by DNA immunization were 100- and 1,000-fold higher via the i.m. or i.d. route, respectively. However, despite the generation of high-avidity Ab in DNA-immunized mice, germinal centers could not be detected in either DNA- or protein-immunized mice. Examination of the IgG subclass response showed that IgG2a was induced by i.m. DNA immunization, coinciding with elevated interferon γ production, whereas a dominant and elevated IgG1 response, coinciding with detectable interleukin 4 production, was generated after i.d. immunization with DNA or soluble OVA and hen egg lysozyme but not human Ig protein. As expected, cytotoxic T cell (CTL) responses could be detected only after DNA immunization. I.d. immunization produced the strongest CTL responses early (2 weeks) but was similar to i.m. later. Therefore, DNA immunization can differ from protein immunization by its ability to induce rapid CTL responses and higher avidity Ab, both of which are advantageous for vaccination.

Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines

We have reported previously that murine bone marrow-derived dendritic cells (DC) pulsed with whole tumor lysates can mediate potent antitumor immune responses both in vitro and in vivo. Because successful therapy was dependent on host immune T cells, we have now evaluated whether the systemic administration of the T cell stimulatory/growth promoting cytokine interleukin-2 (IL-2) could enhance tumor lysate-pulsed DC-based immunizations to further promote protective immunity toward, and therapeutic rejection of, syngeneic murine tumors. In three separate approaches using a weakly immunogenic sarcoma (MCA-207), the systemic administration of nontoxic doses of recombinant IL-2 (20,000 and 40,000 IU/dose) was capable of mediating significant increases in the potency of DC-based immunizations. IL-2 could augment the efficacy of tumor lysate-pulsed DC to induce protective immunity to lethal tumor challenge as well as enhance splenic cytotoxic T lymphocyte activity and interferon-γ production in these treated mice. Moreover, treatment with the combination of tumor lysate-pulsed DC and IL-2 could also mediate regressions of established pulmonary 3-day micrometastases and 7-day macrometastases as well as established 14- and 28-day s.c. tumors, leading to either significant cure rates or prolongation in overall survival. Collectively, these findings show that nontoxic doses of recombinant IL-2 can potentiate the antitumor effects of tumor lysate-pulsed DC in vivo and provide preclinical rationale for the use of IL-2 in DC-based vaccine strategies in patients with advanced cancer.

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.

Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen.

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.

Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy.

Like human gliomas, the rat 9L gliosarcoma secretes the immunosuppressive transforming growth factor beta (TGF-beta). Using the 9L model, we tested our hypothesis that genetic modification of glioma cells to block TGF-beta expression may enhance their immunogenicity and make them more suitable for active tumor immunotherapy. Subcutaneous immunizations of tumor-bearing animals with 9L cells genetically modified to inhibit TGF-beta expression with an antisense plasmid vector resulted in a significantly higher number of animals surviving for 12 weeks (11/11, 100%) compared to immunizations with control vector-modified 9L cells (2/15, 13%) or 9L cells transduced with an interleukin 2 retroviral vector (3/10, 30%) (P < 0.001 for both comparisons). Histologic evaluation of implantation sites 12 weeks after treatment revealed no evidence of residual tumor. In vitro tumor cytotoxicity assays with lymph node effector cells revealed a 3- to 4-fold increase in lytic activity for the animals immunized with TGF-beta antisense-modified tumor cells compared to immunizations with control vector or interleukin 2 gene-modified tumor cells. These results indicate that inhibition of TGF-beta expression significantly enhances tumor-cell immunogenicity and supports future clinical evaluation of TGF-beta antisense gene therapy for TGF-beta-expressing tumors.

In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render DNA immunogenic. We report that in vivo expression of a single DNA-binding protein, the polyoma virus T antigen, is sufficient to initiate production of anti-double-stranded DNA and anti-histone antibodies but not a panel of other autoantigens. Expression of a mutant, non-DNA-binding T antigen did result in strong production of antibodies to the T antigen, but only borderline levels of antibodies to DNA and no detectable antibodies to histones. Nonexpressing plasmid DNA containing the complete cDNA sequence for T antigen did not evoke such immune responses, indicating that DNA by itself is not immunogenic in vivo. The results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus.

Peptide mimicry of the meningococcal group C capsular polysaccharide.

Sequence analysis of the variable regions of the heavy and light chains of the anti-idiotypic antibody 6F9, which mimics the meningococcal group C capsular polysaccharide (MCP), was performed. The immunogenic site on 6F9 responsible for inducing an anti-MCP antibody response was determined by means of sequence and computer model analysis of these data. Complementarity-determining region 3 (CDR3) was found to be unique in that the sequence tract YRY was exposed on the surface. A synthetic peptide spanning the CDR3 domain was synthesized and complexed to proteosomes (meningococcal group B outer membrane protein). Immunizations of BALB/c mice with the peptide-proteosome complex resulted in a significant anti-MCP antibody response. Immunized mice were protected against infection with a lethal dose of Neisseria meningitidis serogroup C.