CD8+ cytotoxic T lymphocytes (CTLs) are critical for protection against intracellular pathogens but often have been difficult to induce by subunit vaccines in animals. DNA vaccines elicit protective CD8+ T cell responses. Malaria-naïve volunteers who were vaccinated with plasmid DNA encoding a malaria protein developed antigen-specific, genetically restricted, CD8+ T cell-dependent CTLs. Responses were directed against all 10 peptides tested and were restricted by six human lymphocyte antigen (HLA) class I alleles. This first demonstration in healthy naïve humans of the induction of CD8+ CTLs by DNA vaccines, including CTLs that were restricted by multiple HLA alleles in the same individual, provides a foundation for further human testing of this potentially revolutionary vaccine technology.

The acute avian leukaemia retroviruses AMV and E26 both induce myeloblastosis in vivo and transform myeloblasts in vitro. Both viruses contain the oncogene v-myb first described for AMV. Unlike AMV, E26 has the additional capacity to induce erythroblastosis in vivo and to transform erythroblasts. Previous analyses indicated that the genome of E26 also contained nucleotide sequences distinct from v-myb and unrelated to viral replicative genes. Using a molecularly cloned E26 provirus, we have now identified a novel nucleotide sequence designated v-ets (for E-twenty-six specific) of approximately 1.5 kilobase pairs (kbp) located next to v-myb. v-ets possesses all the structural characteristics of a putative new oncogene: it has a conserved cellular counterpart c-ets which is transcribed in some normal chicken cells as a major 7.5-kb polyadenylated RNA. Although our results now await elucidation of their biological significance, we propose that v-ets could be a new oncogene accounting for the additional transforming properties of E26, or potentiating the transforming properties of the v-myb oncogene.

Candidate malaria vaccines have failed to elicit consistently protective immune responses against challenge with Plasmodium falciparum. NYVAC-Pf7, a highly attenuated vaccinia virus with 7 P. falciparum genes inserted into its genome, was tested in a phase I/IIa safety, immunogenicity, and efficacy vaccine trial in human volunteers. Malaria genes inserted into the NYVAC genome encoded proteins from all stages of the parasite's life cycle. Volunteers received three immunizations of two different dosages of NYVAC-Pf7. The vaccine was safe and well tolerated but variably immunogenic. While antibody responses were generally poor, cellular immune responses were detected in ú90% of the volunteers. Of the 35 volunteers challenged with the bite of 5 P. falciparum -infected Anopheles mosquitoes, 1 was completely protected, and there was a significant delay in time to parasite patency in the groups of volunteers who received either the low or high dose of vaccine compared with control volunteers.

The preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. Preliminary studies indicated that an African green monkey kidney cell line (Vero) is a suitable system for the primary isolation and cultivation of influenza A viruses (E. A. Govorkova, N. V. Kaverin, L. V. Gubareva, B. Meignier, and R. G. Webster, J. Infect. Dis. 172:250-253, 1995). We now demonstrate for the first time that Vero cells are suitable for isolation and productive replication of influenza B viruses and determine the biological and genetic properties of both influenza A and B viruses in Vero cells; additionally, we characterize the receptors on Vero cells compared with those on Madin-Darby canine kidney (MDCK) cells. Sequence analysis indicated that the hemagglutinin of Vero cell-derived influenza B viruses was identical to that of MDCK-grown counterparts but differed from that of egg-grown viruses at amino acid positions 196 to 198. Fluorescence-activated cell sorting analysis showed that although Vero cells possess predominantly ␣2,3 galactose-linked sialic acid, they are fully susceptible to infection with either human influenza A or B viruses. Moreover, all virus-specific polypeptides were synthesized in the same proportions in Vero cells as in MDCK cells. Electron microscopic and immunofluorescence studies confirmed that infected Vero cells undergo the same morphological changes as do other polarized epithelial cells. Taken together, these results indicate that Vero cell lines could serve as an alternative host system for the cultivation of influenza A and B viruses, providing adequate quantities of either virus to meet the vaccine requirements imposed by an emerging pandemic.

E26 is an acute leukaemia avian retrovirus which induces myeloblastosis and erythroblastosis in vivo and transforms erythroblasts and myeloblasts in vitro. It contains the oncogene v-myb (ref. 4), first described for avian myeloblastosis virus (AMV), as well as a second specific nucleotide sequence, v-ets located 3' to v-myb (refs 5,6). We have reported that v-ets has a cellular counterpart (c-ets) in chicken and human DNA. Now, using two independent methods--hybridization with human c-ets probe of sorted chromosomes and in situ hybridization--we report the localization of the ets locus on human chromosome 11 at bands q23-q24. This finding may be important, as specific breakpoints around this position have been reported for human malignancies such as acute monocytic leukaemia and Ewing's sarcoma.

Mill‐Hill‐2 virus (MH2) proviral DNA was cloned from a transformed non‐producer cell culture (MH2QB2) through insertion of randomly cut high mol. wt. cellular DNA in the lambdoid vector L47.1. Restriction analysis of a suitable recombinant phage by Southern DNA blotting and hybridization with different probes allowed us to characterize the genetic organization of the provirus and to identify a novel MH2‐specific sequence of at least 1.1kbp. Such a sequence, for which we propose the name v‐mil, from MilI‐Hill‐2 virus, is not homologous to v‐myc, the previously described oncogene of MH2, nor to avian leukaemia virus‐related sequences. Evidence is presented here that v‐mil has a cellular counterpart (c‐mil) phylogenetically conserved in birds and mammals, including man, and expressed as a single RNA species at least in some tissues. MH2 virus might thus be regarded, like avian erythroblastosis virus or E26, as another example of retroviruses having recombined with more than one cellular gene.

The highly attenuated NYVAC vaccinia virus strain has been utilized to develop a multiantigen, multistage vaccine candidate for malaria, a disease that remains a serious global health problem and for which no highly effective vaccine exists. Genes encoding seven Plasmodium falciparum antigens derived from the sporozoite (circumsporozoite protein and sporozoite surface protein 2), liver (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), and sexual (25-kDa sexual-stage antigen) stages of the parasite life cycle were inserted into a single NYVAC genome to generate NYVAC-Pf7. Each of the seven antigens was expressed in NYVAC-Pf7-infected culture cells, and the genotypic and phenotypic stability of the recombinant virus was demonstrated. When inoculated into rhesus monkeys, NYVAC-Pf7 was safe and well tolerated. Antibodies that recognize sporozoites, liver, blood, and sexual stages of P. falciparum were elicited. Specific antibody responses against four of the P. falciparum antigens (circumsporozoite protein, sporozoite surface protein 2, merozoite surface protein 1, and 25-kDa sexual-stage antigen) were characterized. The results demonstrate that NYVAC-Pf7 is an appropriate candidate vaccine for further evaluation in human clinical trials.

1 Human isolated pulmonary vessels were treated with cholinesterase (ChE) inhibitors to determine the role of these enzymes in regulating vascular muscle tone. In addition, kinetic parameters were determined for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in human pulmonary vessel homogenates. 2 Carbachol (CCh) and acetylcholine (ACh) were equipotent contractile agonists in human pulmonary arteries (pD 2 values, 5.28+0.05 and 5.65+0.16; E max , 0.91+0.26 and 0.98+0.30 g wt. for CCh and ACh, respectively; n=7). In venous preparations, ACh was ineective and CCh induced small contractions (E max , 0.08+0.04 g wt.; n=13). 3 In human pulmonary arteries following pretreatment with tetraisopropylpyrophosphoramide (iso-OMPA, 100 mM), an increased sensitivity to the contractile agonist ACh was observed (pD 2 values, 5.80+0.13 and 6.37+0.19 for control and treated preparations, respectively; n=5). This pretreatment had no eect on the CCh concentration response curve. In contrast, human pulmonary veins pretreated with iso-OMPA failed to elicit a contractile response to ACh. 4 Neither Iso-OMPA nor neostigmine elicited concentration-dependent contractions in human isolated pulmonary arteries or veins. These results suggest the absence of sucient spontaneous release of ACh to modulate human pulmonary vessel basal tone. 5 CCh was less potent than ACh in relaxing precontracted human isolated pulmonary arteries (pD 2 value, CCh: 6.55+0.15 and ACh: 7.16+0.13, n=4) and veins (pD 2 value, CCh: 4.95+0.13; n=5 and ACh: 5.56+0.17; n=6). Pretreatment of vessels with either iso-OMPA or neostigmine did not modify ACh relaxant responses in either type of preparation. 6 In human pulmonary veins, the ChE activity was two fold greater than in arteries (n=6). V max for AChE was 1.73+0.24 and 3.36+0.26 miu mg 71 protein in arteries and veins, respectively, whereas V ss for BChE was 1.83+0.22 and 4.71+0.17 miu mg 71 protein, in these respectively. 7 In human pulmonary arteries, BChE activity may play a role in the smooth muscle contraction but not on the smooth muscle endothelium-dependent relaxation induced by ACh. A role for ChE activity in the control of venous tone is presently dicult to observe, even though this tissue contains a greater amount of enzyme than the artery.