Daniel E. Mccallus

Facilitated DNA inoculation induces anti-HIV-1 immunity in vivo

Vaccine design against HIV-1 is complicated both by the latent aspects of lentiviral infection and the diversity of the virus. The type of vaccine approach used is therefore likely to be critically important. In general, vaccination strategies have relied on the use of live attenuated material or inactivated/subunit preparations as specific immunogens. Each of these methodologies has advantages and disadvantages in terms of the elicitation of broad cellular and humoral immune responses. Although most success has been achieved with live attenuated vaccines, there is a conceptual safety concern associated with the use of these vaccines for the prevention of human infections. In contrast, subunit or killed vaccine preparations enjoy advantages in preparation and conceptual safety; however, their ability to elicit broad immunity is more limited. In theory, inoculation of a plasmid DNA that supports in vivo expression of proteins, and therefore presentation of the processed protein antigen to the immune system, could be used to combine the features of a subunit vaccine and a live attenuated vaccine. We have designed a strategy for intramuscular DNA inoculation to elicit humoral and cellular immune responses against expressed HIV antigens. Uptake and expression are significantly enhanced if DNA is administered in conjunction with the facilitating agent bupivacaine-HCl. Using this technique we have demonstrated functional cellular and humoral immune responses against the majority of HIV-1 encoded antigens in both rodents and non-human primates.

DNA inoculation induces cross clade anti-HIV-1 responses.

Nucleic acid or DNA immunization represents a novel approach to vaccine and immune therapeutic development. The direct injection of expression cassettes into a living host results in in vivo gene expression and immune activation. In the case of HIV-1 it has been shown by our laboratory that facilitated injection mimicks aspects of live attenuated vaccines and that both humoral and cellular responses can be induced upon injection of a nucleic acid sequence directly into a host target tissue. Antisera from HIV-1 plasmid expression cassette-immunized animals contain anti-HIV envelope glycoprotein immune responses. The antiserum neutralizes HIV-1 infection and inhibits cell to cell infection in vitro. Cellular immune responses have also been evaluated. We observed both T cell proliferation and isotype switching consistent with the production of relevant T helper immune responses in immunized animals. Furthermore it was demonstrated that CTL lysis of relevant env-expressing targets was similarly induced. These studies further define the importance of evaluating this new technology for vaccine and immune therapeutic development for HIV-1 as well as for other human viral pathogens.

Isolated VH4 Heavy Chain Variable Regions Bind DNA Characterization of a Recombinant Antibody Heavy Chain Library Derived from Patient(s) with Active SLE

In many autoimmune diseases autoantibodies are intimately involved in disease manifestations. Molecular characterization of these autoantibodies should provide insights into the pathogenesis of these diseases, as well as suggest novel avenues for development of therapeutics. While some prior studies suggest that DNA binding may be a characteristic of individual heavy chain variable regions, the ability of these V regions to bind DNA in isolation has not been investigated. We have utilized a bacterial vector for cloning and expressing isolated antibody heavy chain variable regions. RNA was extracted from peripheral blood mononuclear cells of patients with active SLE, cDNA synthesized and heavy chain V regions amplified with VH specific oligonucleotide primers. The V H fragments were cloned into a bacterial expression plasmid including the pelB leader peptide to direct appropriate expression. Recombinant antibodies were screened for binding to32P-labeled double-stranded plasmid DNA and later also characterized for binding to single-stranded DNA. Binding was confirmed by standard ELISA methodology. Sequence analysis of seven DNA binding VH fragments revealed that they utilized the VH gene family previously described to be associated with autoimmune responses, with a JH6 segment. On VH sequence analysis only one residue substitution in the consensus sequence is needed to form a V H4 germline gene. Potential contact residues with DNA were delineated by three-dimensional structure analysis. We concluded that the DNA binding characteristics of VH regions can be examined in the absence of light chain. DNA binding specificity appears to be a property of the germline VH4 gene. Analysis of such V regions can aid in the identification of hypervariable region contact residues important for DNA binding.

Ocular tissue involvement in HIV infection: Immunological and pathological aspects

The CNS afflictions in AIDS are myriad and suggest a tropism of HIV to neural tissue. Ocular involvement is a frequent manifestation of the HIV infection, resulting in a high incidence of blindness within this patient population. Ocular lesions include cotton wool spots, presumably from HIV-induced microvasculopathy, retinal hemorrhage in cytomegalovirus retinitis and conjunctival Kaposis sarcoma. These manifestations have been noted in up to 71% of AIDS patients. In fact, ocular disease is often the presenting symptom in an HIV-infected individual. Despite the high incidence of ocular involvement in AIDS patients, the etiology and pathogenesis of these manifestations are not well understood. The immunosuppressive action of HIV is the most likely primary cause for the development of ocular complications in AIDS. Here we review some of the important immunological and pathological features of AIDS affliction in the eye.

Utilization of Anti-Idiotypic Antibodies as Molecular Probes of Virus—Receptor Interaction

Knowledge of the molecular basis of ligand receptor interactions may lead to strategies for the design of substances that can prevent viral-mediated pathology. We will describe our work in two different viral systems. First we review our studies of the reovirus type 3 system. We have utilized the reovirus type 3—cellular receptor interaction as a model system to develop such a strategy. We demonstrate the utility of monoclonal anti-idiotypic (anti-Id) antibodies as starting points for the rational design of peptides that mimic the specificities of the receptor-binding structures of viruses. In this model system we were able to specifically determine the receptor interaction sites by sequence homology with the viral hemagglutinin. Based on these sequences we produced functionally active mimics that interfere with virus binding and maintain, when suitably constructed, many properties of the hemagglutinin.