A DNA sequence coding for mature human leukocyte interferon D (LeIF-D) was linked with DNA fragments of the 5'-flanking sequences of the Saccharomyces cerevisiae (yeast) alcohol dehydrogenase I gene in a plasmid capable of autonomous replication and selection in both yeast and Escherichia coli. Yeast cells transformed by these plasmids synthesize up to 1 x 10(6) molecules of biologically active LeIF-D per cell.

Four hybrid human leukocyte interferon (LeIF or IFN-alpha) genes have been constructed by in vitro recombination of LeIF-A (IFN-alpha 2) and LeIF-D (IFN-alpha 1) genes at common restriction endonuclease sites located within their coding regions. These hybrid genes have been expressed in E. coli under trp promoter control. The interferons produced [LeIF-AD (BglII), -AD (PvuII), -DA (BglII), -DA (PvuII)] have antiviral properties distinct from the parental molecules LeIF-A and -D, varying considerably in their abilities to inhibit plaque formation by different viruses in a range of mammalian cells. All six of the cloned LeIFs exhibit the heat stability, pH 2 stability and antigenic specificity of natural leukocyte interferons.

Five human interferon-alpha (leukocyte) subtypes derived from genes cloned in Escherichia coli have been compared for their ability to induce antiviral activity against vesicular stomatitis virus infection of various mammalian cell cultures. These interferons, designated LeIF-A (IFN-alpha 2), -B, -C, -D (IFN-alpha 1) and LeIF-F, show different relative activities when assayed on human, bovine, hamster, mouse, rabbit and monkey cell lines. As with a natural human buffy-coat interferon-alpha preparation, three subtypes (LeIF-B, -C and -D) showed considerable activity on RK-13 rabbit cells, but two (LeIF-D and -F) also showed some activity on mouse L-929 cells. Of the five interferon subtypes examined, LeIF-F demonstrated the highest degree of species specificity.

Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4(+) and CD8(+) T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania infantum proteins, cysteine peptidase A (CPA), histone H1, KMP-11, and Leishmania eukaryotic initiation factor (LeIF) were analyzed for the prediction of binding epitopes to H2(d) MHC class I and II molecules, using online available algorithms. Based on in silico analysis, eight peptides including highly scored MHC class I- and II-restricted epitopes were synthesized. Peptide immunogenicity was validated in MHC compatible BALB/c mice immunized with each synthetic peptide emulsified in complete Freund's adjuvant/incomplete Freund's adjuvant. CPA_p2, CPA_p3, H1_p1, and LeIF_p6 induced strong spleen cell proliferation upon in vitro peptide re-stimulation. In addition, the majority of the peptides, except of LeIF_p1 and KMP-11_p1, induced IFN-γ secretion, while KMP-11_p1 indicated a suppressive effect on IL-10 production. CPA_p2, CPA_p3, LeIF_p3, and LeIF_p6 induced IFN-γ-producing CD4(+) T cells indicating a TH1-type response. In addition, CPA_p2, CPA_p3, and H1_p1 induced also the induction of CD8(+) T cells. The induction of peptide-specific IgG in immunized mice designated also the existence of B cell epitopes in peptide sequences. Combining immunoinformatic tools and experimental validation, we demonstrated that CPA_p2, CPA_p3, H1_p1, H1_p3, CPA_p2, LeIF_p3, and LeIF_p6 are likely to include potential epitopes for the induction of protective cytotoxic and/or TH1-type immune responses supporting the feasibility of peptide-based vaccine development for leishmaniasis.

One of several human leukocyte interferon subtypes A (LeIF-A), obtained in purified form from a gene cloned in Escherichia coli, stimulated human peripheral blood natural killer cell activity, whereas another human leukocyte interferon subtype D (LeIF-D) had no effect with the use of K562 as target cells. With Daudi as target cells, both LeIF-A and LeIF-D stimulated natural killer cell activity. A hybrid human leukocyte interferon, NH2-terminal 61 amino acids and COOH-terminal 104 residues of LeIF-A and LeIF-D, respectively (LeIF-AD) showed greater stimulation than did LeIF-A, but the stimulation did not exceed that of natural buffy coat interferon. A mixture of equal antiviral units of LeIF-A and LeIF-D was no more effective than was LeIF-A alone. The cloned interferon subtypes showed differential effects on the proliferation of three human leukemic cell lines: Daudi (B-cell lymphoblastoid leukemia); BALL 1 (B-cell acute lymphoblastic leukemia); CCRF-HSB-2 (T-cell acute lymphoblastoid leukemia). Growth of Daudi cells was generally most sensitive to all the interferons tested, LeIF-A, -D, -AD, and a buffy coat preparation; no viable cells remained after 120-hr exposure to 1000-unit/ml doses of the interferons. BALL 1 was relatively resistant to the interferon subtypes tested including LeIF-AD, but this cell lines was very sensitive to a preparation of natural buffy coat interferon. CCRF-HSB-2 showed some sensitivity to all the interferons with greatest sensitivity to LeIF-A (10% of the viable cells were detected after 1000 units/ml exposure for 120 hr). In contrast to the leukemic cell lines tested, human amnion cells (WISH) and the human erythroid leukemia, K562, were resistant to the antiproliferative activity of the interferons.

A pronounced genetic polymorphism of the interferon type I gene family has been assumed on the basis of RFLP analysis of the genomic region as well as the large number of sequences published compared to the number of loci. However, IFNA2 is the only locus that has been carefully analyzed concerning gene frequency, and only naturally occurring rare alleles have been found. We have extended the studies on a variation of expressed sequences by studying the IFNA1, IFNA2, IFNA10, IFNA13, IFNA14, and IFNA17 genes. Genomic white-blood-cell DNA from a population sample of blood donors and from a family material were screened by single-nucleotide primer extension (allele-specific primer extension) of PCR fragments. Because of sequence similarities, in some cases "nested" PCR was used, and, when applicable, restriction analysis or control sequencing was performed. All individuals carried the interferon-alpha 1 and interferon-alpha 13 variants but not the LeIF D variant. At the IFNA2 and IFNA14 loci only one sequence variant was found, while in the IFNA10 and IFNA17 groups two alleles were detected in each group. The IFNA10 and IFNA17 alleles segregated in families and showed a close fit to the Hardy-Weinberg equilibrium. There was a significant linkage disequilibrium between IFNA10 and IFNA17 alleles. The fact that the extent of genetic polymorphism was lower than expected suggests that a majority of the previously described gene sequences represent nonpolymorphic rare mutants that may have arisen in tumor cell lines.

Highly purified preparations of two recombinant-DNA derived human leukocyte interferon subtypes (LeIF-A and LeIF-D) and a similarly derived fibroblast interferon were compared for efficacy against herpes simplex virus, type 1, infection of the rabbit cornea. LeIF-D appeared to be more effective than LeIF-A especially when compared on the basis of interferon units. The fibroblast interferon showed no significant effects. The greater activity of LeIF-D compared with LeIF-A could be due to greater direct antiviral effects, as observed in rabbit cell cultures.