Alphonse J. Langlois

Polypeptides of mammalian oncornaviruses. IV. Structural components of murine leukemia virus released as soluble antigens in cell culture.

Abstract Supernatant fluids from mouse cultures productively infected with Friend leukemia virus contain, in addition to virus, large quantities of the major virion glycoprotein (gp71) in soluble form. The release of this antigen in cell culture is of practical value for its large-scale isolation in nondenatured form. It may also have bearing on how the immune system of the mouse deals with virus infection and leukemogenesis.

Converted-Cell Focus Formation in Culture by Strain MC29 Avian Leukosis Virus.

Summary Infection of chick embryo cell monolayers in high multiplicity with strain MC29 leukosis virus in conventional tissue culture induces rapid, massive morphological conversion within 4 to 7 days. Treatment of the cells in appropriately lower inoculation multiplicities results in the occurrence of infectious centers distinguished as foci of converted cells. When the infected cells are overlaid with agar, the number of foci occurring in 7 days is closely proportional to virus dose. This relationship is applicable to bioassay of strain MC29 leukosis virus with reproducibility and precision of results compatible with practical quantitative studies on the agent. The bioassay procedure and the rapid massive culture conversion make available a unique avian virus-tumor system for studies on cell-virus interrelationships in the induction of neoplasia.

Recombinant subunit vaccines as an approach to study correlates of protection against primate lentivirus infection

Using pathogenic simian immunodeficiency virus (SIV) infection of macaques as a model, we explored the limits of the protective immunity elicited by recombinant subunit vaccines and examined factors that affect their efficacy. Envelope gp 160 vaccines, when used in a live recombinant virus-priming and subunit-protein-boosting regimen, protected macaques against a low-dose, intravenous infection by a cloned homologous virus SIVmne E11S. The same regimen was also effective against intrarectal challenge by the same virus and against intravenous challenge by E11S grown on primary macaque peripheral blood mononuclear cells (PBMC). However, only limited protection was observed against uncloned SIVmne. Priming with live recombinant virus was more effective than immunization with subunit gp 160 alone, indicating a potential advantage of native antigen presentation and the possible role of cell-mediated immunity in protection. Whole gp 160 was more effective than the surface antigen (gp 130), even though both antigens elicited similar levels of neutralizing antibodies. Animals immunized with the core (gag-pol) antigens failed to generate any neutralizing antibody and were all infected following challenge. However, their proviral load was 10-100-fold lower than that of the control animals, indicating that immune mechanisms such as cytotoxic T lymphocytes (CTL) may play a role. Finally, animals immunized with both the core and the envelope antigens generated significant protective immunity, even with relatively low neutralizing antibodies. Taken together, these results indicate that multiple mechanisms may contribute to protection. It may therefore be advantageous to incorporate multiple antigens in the design of recombinant subunit vaccines against acquired immunodeficiency syndrome (AIDS).

Normal chicken cells (chf−) express a surface antigen which cross-reacts with determinants of the major envelope glycoprotein (gp85) of avian myeloblastosis virus☆

Abstract Using rabbit antisera raised against the purified major envelope glycoprotein (gp85) of avian myeloblastosis virus (AMV), we were unable to differentiate between normal chicken cells carrying the chicken-helper factor (chf+) from those lacking this endogenous virus information (chf−) in several serological assays, including competition radioimmunoassay and membrane immunofluorescence. Radioimmunoprecipitation analysis of the material recognized in uninfected chf(+) and chf(-) chicken cells by the rabbit anti-AMV gp85 antisera revealed the presence in immune precipitates of both cell types of a high molecular weight glycoprotein (∼125,000), while low amounts of gp85 were precipitated only from the chf(+) cells. In contrast, antisera raised against the gp85s of three other avian oncornaviruses did not recognize the 125,000-dalton molecule in either cell type, although gp85 was again precipitated from the chf(+) chicken cells. The high molecular weight glycoprotein (denoted NCG) appears to represent a normal chicken cell surface glycoprotein which is antigenically cross-reactive with unique determinants of AMV gp85, presumably dependent on the carbohydrate portion of the molecules. The significance of this cross-reactivity is discussed, as well as the interpretation of previous results obtained through the use of these rabbit anti-AMV gp85 antisera.

The initial nucleotide sequence of DNA transcribed from avian myeloblastosis virus 70 S RNA by RNA-dependent DNA polymerase

Abstract Denaturation of avian myeloblastosis virus 70 S RNA resulted in the release of one major and several minor species of 4 S RNA which were separated by electrophoresis in polyacrylamide gels. The major 4 S RNA component (one to two molecules per 70 S RNA) was shown to function in the 70 S RNA complex as the primer for the initiation of DNA synthesis in vitro by the viral RNA-dependent DNA polymerase. The covalent linkage between primer RNAs and nascent DNAs was found to involve an adenosine residue at the 3′-termini of the RNAs and a deoxyadenosine residue at the 5′-termini of the DNAs. By nearest neighbor analyses, a single unique nucleotide sequence, dApdApdTpdGpd-ApdApdGpdC OH , was determined to be present at the 5′-termini of the nascent DNA transcripts.

Strain MC29 avian leukosis virus release by chick embryo cells infected with the agent.

Summary Release of strain MC29 avian leukosis and Rous sarcoma (RSV) viruses by infected and/or morphologically altered chick embryo cells was measured by counts of physical particles and infectious units (FFU). Particles and FFU were liberated in parallel to a maximum rate at 3-4 days after low multiplicity culture inoculation. The ratios of particles/FFU, corrected for virus inactivation, were 878 and 232 for MC29 and RSV, respectively. Morphologically altered MC29 and RSV cells had a population doubling time of 24 and 42 hr, and the half-lives of the 2 agents were 6 and 10.5 hr, respectively, under simulated culture conditions.

BAI strain A avian (myeloblastosis) leukosis virus from myeloblast tissue culture.

Summary A combined culture-dialysis chamber was devised for producing BAI strain A virus in vitro in amounts greatly increasing the scope of physical and chemical studies of the agent. The yield of virus was approximately 15 mg dry wt/day/chamber. Infectivity of the culture virus was the same on a particle basis as that of virus from leukemic chicks. Properties of the culture virus were indicated by gradient centrifugation, gel electrophoresis and by the character of RNA extracted from the agent.

HIV-1 Neutralizing Antibody and Approaches to the Envelope Diversity Problem

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of AIDS in 1983–84, there has been considerable effort to design an efficacious vaccine. Principally for safety reasons, defined subunit polypeptides and proteins devoid of genomic material have been considered as alternatives to attenuated and/or killed virus. Much of the effort to develope such a component vaccine against HIV has been directed at the envelope gene products. These have included recombinant gp l60 (1,2,3), recombinant gp 120 (4,5,6), native viral gp 120 (7,8,9), bacterially expressed envelope fragments (10), and a large number of synthetic peptides (11–19). In these early studies each of the various reagents were tested for their ability to raise neutralizing antibodies in experimental animals. When successful, resultant sera were usually found to be active only against homologous virus, i.e. were isolate restricted. The results suggested that variable sites on the envelope represented the major target of neutralizing antibody raised by the various experimental immunogens.

Restricted Neutralization of Divergent HTLV-III/LAV Isolates by Antibodies to the Major Envelope Glycoprotein

By analogy to other retroviruses, the major envelope glycoprotein - gp120 - of HTLV-III/LAV is a probable target for neutralizing antibody. This antigen has been purified from H9 cells chronically infected with the HTLV-IIIB prototype strain. Several goats immunized with the gp120 produced antibodies that neutralized infection of H9 by the homologous virus isolate. These same sera failed to neutralize the divergent HTLV-IIIRF isolate. Individuals infected with HTLV-III/LAV commonly develop antibodies to gp120 which could be isolated using the gp120 antigen coupled to an immunoadsorbent resin. The antibody fraction that bound tightly to such a resin was found to neutralize the IIIB but not the RF isolate in a fashion similar to that of the goat anti-gp120 sera. However, the nonbinding fraction (effluent) from the resin also contained neutralizing activity which was able to block infection by both virus isolates with similar efficacy. Human antibodies to the other virus envelope gene product, the transmembrane gp41, were also affinity-purified utilizing the recombinant peptide 121, but they failed to influence infection by either virus isolate.