Depleting regulatory T cells with arginine-rich, cell-penetrating, peptide-conjugated morpholino oligomer targeting FOXP3 inhibits regulatory T-cell function.

CD4+CD25+regulatory T cells (T(reg)) impair anti-tumor and anti-viral immunity. As there are higher T(reg) levels in cancer patients compared with healthy individuals, there is considerable interest in eliminating them or altering their function as part of cancer or viral immunotherapy strategies. The scurfin transcriptional regulator encoded by the member of the forkhead winged helix protein family (FOXP3) is critical for maintaining the functions of T(reg). We hypothesized that targeting FOXP3 expression with a novel arginine-rich, cell-penetrating, peptide-conjugated phosphorodiamidate morpholino (PPMO) based antisense would eliminate T(reg) and enhance the induction of effector T-cell responses. We observed that the PPMO was taken up by activated T cells in vitro and could downregulate FOXP3 expression, which otherwise increases during antigen-specific T-cell activation. Generation of antigen-specific T cells in response to peptide stimulation was enhanced by pre-treatment of peripheral blood mononuclear cells with the FOXP3-targeted PPMO. In summary, modulation of T(reg) levels using the FOXP3 PPMO antisense-based genomic strategy has the potential to optimize immunotherapy strategies in cancer and viral immunotherapy.

Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing.

Phosphorodiamidate morpholino oligomers (PMO) are synthetic antisense molecules that interfere with translation, pre-mRNA splicing and RNA synthesis. Like other gene-silencing technologies, PMO are poorly taken up by primary leukocytes without the use of physical or chemical delivery techniques. We sought an alternative delivery mechanism of PMO into immune cells that eliminates the need for such manipulations. Here we demonstrate the first use of arginine-rich cell-penetrating peptides (CPPs) to deliver PMO (P-PMO) directly into primary murine leukocytes for inhibition of gene expression and promotion of altered pre-mRNA splicing. We compared the P-PMO delivery efficacy of four arginine-rich CPPs including HIV Tat and penetratin, and one histidine rich CPP, and found that the (RXR)(4) peptide was the most efficacious for PMO delivery and targeted antisense effect. The delivery and antisense effects of P-PMO are time- and dose-dependent and influenced by the activation and maturation states of T cells and dendritic cells, respectively. Targeted expression of several genes using P-PMO is shown including surface signaling proteins (CD45 and OX-40), a cytokine (interleukin-2), and a nuclear transcription factor (Foxp3). Considering the abundance of naturally occurring alternatively spliced gene products involved in immune regulation, P-PMO offer an effective method for modulating gene activity for immunological research and applications beyond traditional antisense approaches.

Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers.

Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.

Lack of differences in nef alleles among HIV-infected asymptomatic long-term survivors and those who progressed to disease.

Sequences of the human immunodeficiency virus (HIV) nef gene in virus isolates from 12 long-term survivors (LTSs) and 7 progressors were compared to determine if any association existed between the sequences and the corresponding clinical status. The sequences of at least five clones were determined for each subject. Conceptual translations of the open reading frames (ORFs) were examined with respect to a consensus with a prototypic nef sequence (HIV-1SF2) and for conservation of functionally described motifs. Premature stops were observed at equivalent, yet low, frequencies among the different clinical groups: 2 of 60 (3.33%) and 1 of 45 (2.22%) respectively. No remarkable differences in protein motifs implicated in several activities ascribed to Nef were noted. An association between nef sequence characteristics and the clinical state was not found.

Differential effects of human immunodeficiency virus isolates on beta-chemokine and gamma interferon production and on cell proliferation.

All human immunodeficiency virus (HIV) isolates can grow readily in primary CD4(+) T cells, but they can be distinguished by their ability to replicate in macrophages and established T-cell lines. The macrophage-tropic viruses are generally non-syncytium inducing (NSI), whereas the T-cell-line-tropic viruses are syncytium inducing (SI) in cultured cells. We now demonstrate that infection of CD4(+) T cells by NSI and SI viruses shows a differential effect on production of beta-chemokines and gamma interferon. Infection by NSI viruses increased production of MIP-1alpha, MIP-1beta, and gamma interferon, whereas infection by SI viruses had no effect or decreased production of these cytokines. Production of RANTES was slightly increased during infection by both virus phenotypes. This differential effect of NSI and SI viruses was observed at the level of beta-chemokine mRNA as well as at the level of protein expression. Infection by NSI viruses also increased CD4(+) cell proliferation. These results may have relevance for a differential role of HIV strains in AIDS pathogenesis.

Human herpesvirus 8 detection in nasal secretions and saliva.

The presence of human herpesvirus 8 (HHV-8) was determined by polymerase chain reaction (PCR) in nasal secretions and saliva from 14 HHV-8-seropositive persons, including 8 Kaposi's sarcoma patients: 7 were human immunodeficiency virus type 1-infected, 6 of whom were asymptomatic. HHV-8 was detected in one or both body fluids in 8 (57%) of 14 subjects. Parallel PCR testing revealed the concomitant presence of cytomegalovirus, Epstein-Barr virus, and HHV-6 in various combinations in these body fluids. These data indicate frequent shedding of multiple herpesviruses in nasal secretions and saliva, particularly in Kaposi's sarcoma patients. Both body fluids are therefore potential sources HHV-8 by nonsexual transmission.

Fish vaccine antigens produced or delivered by recombinant DNA technologies.

Current efforts to develop vaccines, particularly for aquacultured species, have turned largely to biotechnology because it provides the means to inexpensively produce sufficient quantities of the immunoprotective antigen. These efforts have resulted in several prototype vaccines for fish and the publication of a large number of articles on the subject. However, there are only a few recombinant DNA-based vaccines for aquaculture in the licensing pipeline. Continued funding of research on recombinant DNA vaccines comes from the recognition by industry and government funding agencies that this research can lead to an increased understanding of the mechanisms in protective immunity. This is especially important for fish and shellfish species since our knowledge of the immune mechanisms in these animals is pitifully meagre. This presentation discusses the relative merits of the different recombinant DNA technologies that have been used to produce viral vaccines for fish and the promising approaches that are under consideration to increase the efficacy of these vaccines. There are many approaches to antigen production by recombinant DNA techniques including: (i) the preparation of purified antigenic proteins produced from the cloned viral genes in a variety of vector/host expression systems, (ii) chemical synthesis or the use of fusion vectors to produce peptides corresponding to known epitopes, (iii) defined attenuations, i.e. specific genetic alterations, of live virus vaccines, (iv) the use of live bacterial or viral vectors to deliver resistance genes or viral antigens, (v) anti-idiotype antibodies, and (vi) DNA vaccines where purified plasmid DNA expressing the pathogen gene under a eucaryotic promoter is injected. All of these technologies have been used more or less successfully in the development of vaccines for aquacultured species. However, the requirements for safety, effectiveness, ease of application and low cost/dose restrict their commercial development for aquaculture. The ideal viral vaccine for aquaculture must be effective in preventing death, be inexpensive to produce and license, provide immunity of long duration, and be easily administered. In addition, these vaccines must not only provide protection against the lethal effects of virus infection but prevent the formation of virus persistence. This is especially true for infectious haematopoietic necrosis virus (IHNV) which has been shown to persist in survivors in the presence of high antibody levels. Since resolution of virus persistence is thought to be correlate with cell-mediated immunity, vaccines designed to augment the cell-mediated immunity must be developed for fish. Approaches that are being considered include the use of cytokines in combination with subunit vaccines and the use of specific MHC-I inducer adjuvants with the vaccine. The "tailoring" of vaccine immunogenicity using different combinations of antigen and adjuvant will be presented.

Gene expression in rainbow trout (Oncorhynchus mykiss) following intramuscular injection of DNA.

Expression of the firefly luciferase gene under the control of viral or fish promoters was observed in fish tissue after direct DNA injection of plasmid DNA. Plasmid DNA containing the firefly luciferase gene was injected into the skeletal muscle of rainbow trout (Oncorhynchus mykiss), and levels of luciferase activity were found to be dependent on the controlling promoter and the amount of injected DNA. Plasmids using the cytomegalovirus immediate early promoter (CMV-IEP) consistently produced the highest levels of luciferase activity. Maximal activity was observed five to seven days postinjection with 50 micrograms of DNA. This activity persisted in the tissues for as long as 115 days postinjection. When the DNA was examined up to two months postinjection, the predominant form was unreplicated, unintegrated DNA in linear and relaxed circular conformation. Expression of injected DNA was found predominantly within muscle cells along the injection path and in scattered muscle cells anterior to the injection site.

Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus.

Plasmid vectors encoding the infectious hematopoietic necrosis virus (IHNV) nucleoprotein or glycoprotein gene under the control of a cytomegalovirus promoter were used to immunize rainbow trout (Oncorhynchus mykiss) against IHNV. The plasmid DNA was injected into the skeletal muscle of rainbow trout fry, and immunization was determined by the detection of virus-neutralizing and enzyme-linked immunosorbent assay antibody activity, and by protection against live virus challenge. Fish injected with the glycoprotein-encoding plasmid pCMV4-G, either alone or in combination with the nucleoprotein-encoding plasmid pCMV4-N, generated glycoprotein-specific and virus-neutralizing antibody responses. The vaccinated fish were also protected from subsequent IHNV challenge. Fish receiving pCMV4-N alone did not produce measurable virus-specific antibody and were killed by IHNV infection. These studies show that DNA vaccination will protect rainbow trout against the lethal effects of IHNV infection.

Idiotypic mimicry of a cell surface DNA receptor: evidence for anti-DNA antibodies being a subset of anti-anti-DNA receptor antibodies.

Anti-idiotypic anti-DNA antibodies (anti-anti-DNA) have previously been described in both patients with systemic lupus erythematosus and healthy individuals. Jerne's hypothesis predicts that such antibodies would bear a paratope reactive with non-sequence specific DNA binding proteins. Here we have explored the notion of a molecular mimicry between anti-anti-DNA antibodies and antibodies to a previously described 28-29 kD cell surface DNA binding molecule. It was shown that affinity purified anti-anti-DNA antibodies inhibit the binding of DNA to cells and that MoAb to the 28-29 kD receptor react with anti-DNA antibodies. These findings indicate that a subset of anti-anti-DNA antibodies are idiotypically related to antibodies reactive with a cell surface DNA binding molecule. It is hypothesized that anti-DNA antibodies may arise when a convergence of genetic and environmental influences favours an unrestrained anti-idiotypic response to cell surface DNA binding molecule(s).

Bacterially expressed nucleoprotein of infectious hematopoietic necrosis virus augments protective immunity induced by the glycoprotein vaccine in fish.

The ribonucleoprotein gene of infectious hematopoietic necrosis virus (IHNV) has been expressed in Escherichia coli as a trpE fusion protein. This viral protein does not induce protective immunity to lethal IHNV infection in fish, and virus-neutralizing antibodies do not react with this viral protein. However, when it was administered with a bacterial lysate containing a region of the IHNV glycoprotein, there was enhanced resistance in immunized fish to lethal virus infection.

Epitope mapping and characterization of the infectious hematopoietic necrosis virus glycoprotein, using fusion proteins synthesized in Escherichia coli.

A characterization of the antigenic determinants (epitopes) of the glycoprotein (G) of infectious hematopoietic necrosis virus was made by expressing different regions of the G gene in Escherichia coli. A cDNA copy of the G gene was divided into four fragments by TaqI digestion, and the fragments were subcloned into pATH vectors, placing the expression of each G gene fragment under control of the trpE promoter. The resulting plasmids, pXL2, pXL3, and pXL7, encoded trpE-G fusion proteins subsequently detected with anti-infectious hematopoietic necrosis virus sera by Western immunoblots. A comparison of reactivities of the fusion proteins encoded by these plasmids was made by Western immunoblot and radioimmunoassay with a number of anti-G specific monoclonal antibodies (MAbs). The nonneutralizing MAb 136J reacted with the trpE-G fusion protein encoded by pXL3 and fusion proteins encoded by plasmids p52G and p618G, which were described in previous studies (R. D. Gilmore, Jr., H. M. Engelking, D. S. Manning, and J. C. Leong. Bio/Technology 6:295-300, 1988). Another nonneutralizing MAb, 2F, bound to the pXL3 fusion protein, and the neutralizing MAb RB/B5 recognized the pXL7 fusion protein. All fusion proteins were tested as vaccines in rainbow trout fry. Although significant protection was induced by all fusion proteins, the pXL3 fusion protein was most effective as a vaccine.

The production and characterization of murine monoclonal antibodies to a DNA receptor on human leukocytes.

Two murine mAb have been generated with a reactivity toward a 30,000 m.w. DNA binding protein found on the cell surface of human leukocytes; mAb 12A has an IgG1/k isotype, and mAb 24T has an IgG2b/k isotype. Both react with the DNA binding domain or adjacent region of the putative DNA receptor and inhibit the binding of [3H]DNA to PBMC at concentrations as low as 100 ng/ml. Stoichiometric studies indicate that both mAb react with monocytes and T cells with a kDa of 10(-7) M; about 0.5 x 10(6) molecules bind per cell at saturation. Flow cytometry indicated that 67% of lymphocytes and 98% of monocytes bore the DNA receptor. Dual labeling studies showed that 90% of B cells and 50% of T cells express the receptor; 50% of CD4+ T cells are receptor positive. Immunomatrices constructed with both mAb 12A and 24T allowed the receptor to be purified to a high degree of purity. A single protein of Mr 30,000 was readily observed after SDS-PAGE and silver staining of the gel; after electropheretic transfer of nitrocellulose this protein was shown to be a DNA binding molecule by use of a probe of biotin labeled DNA. These experiments provide further evidence to support the existence of a specific DNA receptor on human leukocytes; the availability of mAb to the receptor should be useful in its further characterization.