Expression and immunogenicity of V3 loop epitopes of HIV-1, isolates SC and WMJ2, inserted in Salmonella flagellin.

Synthetic oligonucleotides corresponding to specific V3 loop portions of two HIV-1 isolates, SC and WMJ2, were expressed in the flagella of a Salmonella live-vaccine strain. Expression of the inserted epitopes in flagellin and their exposure at the surface of flagellar filaments were shown by immunoblotting and immunogold labeling with anti-flagellin (Salmonella d) and anti-HIV-1(IIIB) V3 loop peptide sera. Live recombinant Salmonella strains expressing either one of the two V3 loop inserts were administered intraperitoneally to BALB/c mice. All these animals developed antibodies specific for the heterologous glycoprotein 120 (gp120) of HIV-1 MN strain, as detected by enzyme-linked immunosorbent assays (ELISA), two of the sera had neutralizing activity against the heterologous HIV-1 MN strain. Moreover, oral administration of the live Salmonella recombinant strains to mice evoked specific IgA directed against gp120.

Enhancement of HSV-DNA infectivity, in Vero and RS cells, by a modified calcium-phosphate transfection technique.

Infectivity of herpes simplex virus (HSV) DNA was assessed by employing the calcium-phosphate transfection technique described by Chen and Okayama, originally applied to increase the efficiency of plasmid transfection by N, N-bis (2-hydroxyethyl)-2-aminoethane sulfonic acid (BES). The experimental conditions and efficiency of this transfection procedure were evaluated comparing the viral progeny titers obtained by the Chen and Okayama transfection method using DNA from wild-type strains of HSV-1 and HSV-2, as well as from mutant strains, with the viral progeny obtained by the most widely used transfection technique introduced by Graham and van der Eb. Furthermore, recombinant virus production was evaluated in marker transfer and marker rescue experiments, comparing both transfection techniques, using DNA fragments cotransfected with whole viral DNA into African green monkey (Vero) or rabbit skin (RS) cells. The viral production obtained from HSV-DNA transfected cells was enhanced approximately 1000-fold when the Chen and Okayama procedure was applied.

Immunogenicity of Nef protein of SIVSMM-PBj14 expressed in a live vaccine strain of Salmonella species.

The nef gene of an infectious molecular clone of SIVSMM isolate PBj14 was fused to the glutathione S-transferase gene of Schistosoma japonicum to generate plasmid pEMC100. The recombinant plasmid was placed in an aroA live vaccine Salmonella dublin strain, and the production of GST-Nef protein was induced by exposure to IPTG. The fusion protein was purified and administered as vaccine to BALB/c mice by i.p. injection. Several doses of the purified fusion protein produced an earlier anti-GST-Nef response, without an anti-GST response, than did IPTG-induced Salmonella live vaccine containing an equal amount (0.1 microgram) of fusion protein, apparently because of the transient immunosuppressive effect of live vaccine given by injection. The highest anti-GST-Nef titers were obtained by a third immunization schedule in which mice were treated with a priming inoculum of induced live vaccine followed, after the predicted immunosuppressed interval, by two i.p. doses of 1 microgram of purified GST-Nef protein with Ribi adjuvant. The data presented here demonstrate that SL5928 aroA, an attenuated S. dublin strain, can be used as a live vaccine carrier to express Nef protein of SIVSMM-PBj14, one of the most acutely pathogenic primate lentiviruses so far described.

c-fos proto-oncogene transient transcription is negatively affected in the ELa4-2 transformed rat cell line.

To study the effects of the regulatory phosphoprotein ICP4 of the Herpes simplex virus, (HSV), a DNA tumor virus, on the induction of gene expression by the epidermal growth factor (EGF), we have constructed a cell line, ELa4-2, which constitutively expresses the a-4 gene product. The ELa4-2 cells are derived from the rat fibroblast EL2, in which EGF induces a marked c-fos and c-myc proto-oncogene transcription. Here we report that in ELa4-2 cells, the gene expression induced by EGF was negatively affected in respect to that obtained stimulating the parental EL2 cells. In particular, we studied the c-fos and c-myc proto-oncogene transcription induced by EGF. We found that in ELa4-2 cells the c-fos induction was dramatically reduced in comparison with the c-fos induction obtained in the parental EL2 cells. On the contrary, the c-myc induction by EGF was not affected by the presence of ICP4. Finally, we compared the HSV infectivity in ELa4-2 versus the EL2 cells. We showed that the virus growth capability was reduced, in the cells expressing ICP4.

Fine mapping and characterization of the Syn 6 locus in the herpes simplex virus type 1 genome.

The syncytial mutant of herpes simplex virus type 1 (HSV-1), HSV-1(13) S11, which carries three distinct syncytial mutations, Syn 1, Syn 5 and Syn 6, was described previously. Syn 1 maps to the BamHI L fragment, map units (m.u.) 0.707 to 0.745; Syn 5 is located within the BamHI Q fragment, m.u. 0.296 to 0.317; Syn 6 lies in the junction fragment BamHI SP, m.u. 0.81 to 0.85. Although Syn 1 of HSV-1(13) S11 seems to be homologous to that of HSV-1(MP) and other syncytial mutants, and Syn 5 has been recently characterized, Syn 6 represents a novel syncytial locus which has yet to be characterized. In this paper we report the fine mapping of the Syn 6 locus. This mutation has been mapped, by marker rescue and marker transfer experiments, to the long repeat regions (RL) at both ends of the L component of the HSV genome in a restriction endonuclease fragment of approximately 1.6 kb designated BamHI-SacI C (approximate m.u. 0.01 to 0.02 and 0.81 to 0.82). In the internal copy of RL the sequences containing the Syn 6 mutation were bounded to the left by the 5' end of the alpha gene specifying ICP0 and to the right by the gamma 1 gene encoding ICP34.5.