Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.

One of the obstacles to AIDS vaccine development is the variability of HIV-1 within individuals and within infected populations, enabling viral escape from highly specific vaccine induced immune responses. An understanding of the different immune mechanisms capable of inhibiting HIV infection may be of benefit in the eventual design of vaccines effective against HIV-1 variants. To study this we first compared the immune responses induced in Rhesus monkeys by using two different immunization strategies based on the same vaccine strain of HIV-1. We then utilized a chimeric simian͞HIV that expressed the envelope of a dual tropic HIV-1 escape variant isolated from a later time point from the same patient from which the vaccine strain was isolated. Upon challenge, one vaccine group was completely protected from infection, whereas all of the other vaccinees and controls became infected. Protected macaques developed highest titers of heterologous neutralizing antibodies, and consistently elevated HIV-1-specific T helper responses. Furthermore, only protected animals had markedly increased concentrations of RANTES, macrophage inf lammatory proteins 1␣ and 1␤ produced by circulating CD8 ؉ T cells. These results suggest that vaccine strategies that induce multiple effector mechanisms in concert with ␤-chemokines may be desired in the generation of protective immune responses by HIV-1 vaccines.

Hydroxyurea inhibits the activity of ribonucleotide reductase (ribonucleoside-diphosphate reductase; 2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) in' bacteria and mammalian cells. The reductase from Escherichia coli consists of two nonidentical subunits (Bi and B2) and hydroxyurea acts by specifically destroying a tyrosine free radical of B2 required. for enzyme activity. The mammalian enzyme also consists of two nonidentical subunits (MI and M2), only one of which (MI) has been obtained in pure form. By continuous culture at stepwise-increasing drug concentrations, we have now obtained a 3T6 mouse fibroblast cell line with a 100-fold increased resistance to hydroxyurea. Extracts from resistant cells showed a 3-to 15-fold increase in reductase activity. The amount of MI protein was not increased. The amount of M2 protein could not be measured directly, but the M2 activity in extracts from resistant cells was increased 27-fold. Furthermore, the packed resistant cells (but not normal-cells) showed an EPR spectrum very similar to that of the tyrosine radical of'the bacterial B2 subunit. We propose that, resistance to hydroxyurea is caused either by overproduction of the complete M2 subunit or by increased generation of the tyrosine radical within the M2 protein.It seems that either alternative mirrors a possible normal regulatory mechanism for the activity of the reductase. In bacteria as well as in mammalian cells hydroxyurea inhibits DNA synthesis by interaction with the enzyme ribonucleotide reductase (ribonucleoside-diphosphate reductase; 2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) (for review see ref. 1). In Escherichia coli this enzyme consists of two nonidentical subunits, called proteins Bi and B2. The B2 subunit contains iron and a tyrosine free radical characterized by a specific EPR signal and essential for enzyme activity. Incubation ofprotein B2 with hydroxyurea irreversibly destroys the radical and the enzyme activity.The best-characterized mammalian reductase is the enzyme from calf thymus (2). This enzyme also appears to consist of two subunits, proteins MI and M2 (3). Only the Ml subunit has so far been obtained in homogeneous form; it appears in many respects to be equivalent to the bacterial B1 subunit. The properties of M2 are poorly characterized because of limited purification. M2 appears to contain iron but no evidence for a free radical has as yet been obtained. Hydroxyurea inhibits the thymus enzyme in a reversible fashion.In this communication we report the results. from experiments with 3T6 mouse fibroblasts with an up to 100-fold increase in resistance to hydroxyurea. Resistant cell lines were obtained by growing the parent line over a period of 2 years in the presence of stepwise increasing concentrations of hydroxy- The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §17...

One of the functions of N-linked glycans of viral glycoproteins is protecting otherwise accessible neutralization epitopes of the viral envelope from neutralizing antibodies. The aim of the present study was to explore the possibility to obtain a more broadly neutralizing immune response by immunizing guinea pigs with gp160 depleted of three N-linked glycans in the CD4-binding domain by site-directed mutagenesis. Mutant and wild type gp160 were formulated into immunostimulating complexes and injected s.c. into guinea pigs. Both preparations induced high serum antibody response to native gp120 and V3 peptides. Both preparations also induced antibodies that bound equally well to the V3 loop or the CD4-binding region, as determined by a competitive enzyme-linked immunosorbent assay (ELISA). The sera from animals, immunized with mutated glycoprotein, did not neutralize nonrelated HIV strains better than did sera from animals, immunized with wild type glycoprotein. Instead, a pattern of preferred homologous neutralization was observed, i.e., sera from animals, immunized with mutant gp160, neutralized mutant virus better than wild type virus, and vice versa. These data indicated that elimination of the three N-linked glycans from gp160 resulted in an altered local antigenic conformation but did not uncover hidden neutralization epitopes, broadening the immune response.