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A collaborative study was organized to identify monoclonal antibodies (MAbs) that may be broadly and potently neutralizing for a panel of human immunodeficiency virus type 1 (HIV-1) low-passaged adult and pediatric primary isolates in peripheral blood mononuclear cells. Five laboratories evaluated a coded panel of seven human MAbs to HIV-1 subtype B envelope V3, CD4 binding region, gp41, and other conformationally sensitive determinants. Each laboratory measured neutralizing activity of the MAbs against the laboratory isolate HIV(MN) and a panel of 9 subtype B primary isolates. Antibodies were classified as suitable candidates for future clinical studies if they could neutralize at least half of the 9 primary isolates at a concentration of < or = 25 microg/mL for 90% viral inhibition. The study identified three MAbs that met stated performance criteria: IgG1b12, 2G12, and 2F5. These results may provide a rationale for examining the clinical efficacy, either singly or in combination, of the three MAbs.

Objective: To evaluate human monoclonal antibodies (MAb) for neutralizing activity against primary HIV-1 isolates in peripheral blood mononuclear cells., Design: Neutralization activity data were obtained from 11 laboratories on a coded panel consisting of six human MAb to HIV envelope V3, CD4-binding region or gp41. Hyperimmune globulin against HIV-1 and normal human immunoglobulin G were supplied as controls. Each laboratory received pre-titered virus for use in the studies., Methods: Each laboratory measured neutralization of the MAb against laboratory strain HIVMN, genomic clone HIVJR-CSF, two subtype B and one subtype D primary isolates., Results: The titers of the centrally supplied virus stocks as determined by re-titration or back-titration varied among laboratories and were generally 10-100-fold less than provided. The neutralizing activity of each MAb varied by as much as a 1000-fold among laboratories. These differences may result from varying sensitivity in neutralization assay protocols and the differing susceptibility of primary cells to infection with HIV-1., Conclusions: To consolidate the data from multiple laboratories, the neutralization titers were compared by classifying antibodies as neutralizing if the antibody concentration for 50% virus inhibition was < or = 10 micrograms/ml. By this criterion, the CD4-binding region and gp41 MAb neutralized all four subtype B viruses and the subtype D isolate in a few of the laboratories. The V3 MAb neutralized only HIVMN and the closely related HIVJR-CSF viruses.

As part of the WHO Network for HIV Isolation and Characterization, we PCR amplified, cloned, and sequenced gp120 and gp160 genes from 12 HIV-1 isolates collected in four WHO-sponsored vaccine evaluation sites (Brazil, Rwanda, Thailand, Uganda). Envelope clones were derived from PBMC-grown isolates obtained from asymptomatic individuals within 2 years of seroconversion. Analysis of their deduced amino acid sequences identified all but one to contain an uninterrupted open reading frame. Transient expression and biological characterization of selected gp160 constructs identified six clones to encode full length and functional envelope glycoproteins. Phylogenetic analysis of their nucleotide sequences revealed that they represent HIV-1 subtypes A, B, C, and E. Since current knowledge of HIV-1 envelope immunobiology is almost exclusively derived from subtype B viruses, these reagents should facilitate future envelope structure, function and antigenicity studies on a broader spectrum of viruses. This should assist in the design and evaluation of effective vaccines against HIV-1.

Strain improvement of agriculturally important animals will require efficient techniques for gene delivery, the ability to regulate the expression of the newly introduced genes and, most important, the identification of genes whose appropriate expression could cause improvement of the animal. We have developed a series of avian retroviral vectors that can be used to introduce new genetic information into the germ line of chickens, for which transgenics cannot be created by direct microinjection of DNA into fertilized eggs. We have identified a 220-bp segment of the chicken skeletal muscle alpha-actin gene that can cause other genes to be expressed specifically in striated muscle. This chicken promoter shows correct tissue specificity in transgenic mice and presumably could be used in other mammalian species. The skeletal muscle alpha-actin promoter has been inserted into the avian retroviral vectors and the promoter is functional in cultured cells infected by these retroviral vectors. The tissue specificity of the expression of the skeletal muscle alpha-actin promoter carried by the retroviral vectors will soon be tested in vivo. We are studying two types of genes that might be useful in strain improvement; genes that could produce dominant resistance to infection by pathogenic viruses, and genes that could play critical roles in muscle development. Expression of the envelope glycoprotein of retroviruses can specifically block the cellular receptor that viruses use to infect a susceptible cell. Expression of the avian leukosis virus subgroup A envelope in transgenic chickens prevents infection by pathogenic viruses of the same subgroup. We are attempting to block reticuloendotheliosis virus infection by expressing the reticuloendotheliosis envelope glycoprotein. We have shown that we can block infection in cultured cells, and we are now creating retroviral vectors for experiments in vivo. We have also begun to study the cellular homologue of the ski oncogene, which has been shown to stimulate the differentiation of quail myoblasts in vitro. Biologically active cDNAs have been isolated; we have now begun to analyse the effects of expressing the c-ski proteins in the whole animal.

The molecular characterization of the proviral DNA genome of Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, is described. An analysis of unintegrated viral DNAs present in acutely infected cells revealed open and closed circular molecules and linear species. The size of the M-PMV linear proviral DNA is determined to be 8.1 kbp in length. A preliminary screening of restriction enzymes indicated that many of those commonly used for cloning (EcoRI, SalI, ClaI, XhoI) did not cut the provirus. Digestion of a mixture of linear and circular forms of unintegrated DNA with HindIII produced a set of restriction fragments 2.3-3 kbp in length. These subgenomic fragments where cloned into the bacterial plasmid pAT153, and two classes of M-PMV subgenomic clones isolated. The first of these contained fragments that spanned the ends of the linear genome and presumably were derived from circular proviruses. Six of the seven clones in this class contained a single long terminal repeat (LTR), represented by pMP6, while the seventh, pMP9, contains two LTRs. Digestion of the latter clone with an enzyme that cleaves once within the LTR allowed the length of the M-PMV LTR to be determined as 350 bp. Both the LTR containing clones and the second class of subgenomic clones have been used in developing a detailed restriction map of the M-PMV proviral DNA and in orienting it with regard to transcription of viral RNA. Thus, pMP6/pMP9 contain sequences from the LTR-gag region of the genome and the second class of subclones (represented by pMP1) span the env-coding region. No clones containing the pol-coding region have been isolated. In order to determine the nature of M-PMV-related endogenous sequences in the chromosomal DNA of Old World primates, EcoRI-digested primate DNA was hybridized at low stringency to the subgenomic clones and then washed under conditions of low, moderate, and high stringencies. Multiple sequences closely related to the LTR-gag region of the M-PMV genome, were detected. Sequences more distantly related to the env region were also found in Old World monkeys. Ape and human DNAs were shown to contain sequences related to the LTR-gag region of the M-PMV genome, but were only weakly detectable at low stringency.

Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, unlike most retroviruses, preassembles core structures in the cytoplasm of infected cells during morphogenesis. We have shown previously (J. Bradac and E. Hunter, 1984, Virology 138, 260-275) that M-PMV virions contain 5 gag-encoded polypeptides. In this report, the translational order of the proteins encoded on the gag and env genes of M-PMV has been determined by pactamycin mapping. The proposed order of the viral proteins on their respective precursors is env: NH2-gp85-gp20-COOH; gag: NH2-p10-pp24/pp16-p12-p27-p14-COOH. Myristic acid-labeled virions contained a single radioactive protein, p10, supporting the mapping of this molecule to the amino terminus. From these studies it is clear that M-PMV contains an unusual additional gag polypeptide, p12, for which no function has been assigned to date. A remarkable similarity exists in the size and organization of M-PMV and mouse mammary tumor virus (MMTV) gag polypeptides; suggesting a common ancestor to these two viruses.