Fluorometric enzyme immunoassay for measurement of infectious feline leukemia virus and its neutralization.

A fluorometric enzyme immunoassay using an alkaline phosphatase-conjugated monoclonal antibody was developed to quantitate feline leukemia virus (FeLV) infection. Monoclonal antibodies, directed against the FeLV structural protein p27, were conjugated with alkaline phosphatase using a modified maleimide method. The enzyme immunoassay requires only 4 days to reproducibly measure FeLV production instead of the 12 days required for the commonly used transformation assay using C81 cells. A linear correlation was found between the virion associated reverse transcriptase activity and the amount of intracellular p27 as determined by the fluorometric enzyme immunoassay. An immunocytochemical assay using the same conjugated monoclonal with a different substrate gave visible plaques in infected cell monolayers and was therefore used to titrate FeLV in plaque-forming units. The results obtained by all the procedures followed single hit kinetics for FeLV infection. The fluorometric enzyme immunoassay was adapted to measure FeLV neutralizing antibodies, allowing a sensitive and accurate determination of neutralizing titers.

In vitro measurement of antigen-specific cell-mediated immune responses using recombinant HIV-1 proteins adsorbed to latex microspheres.

Recombinant proteins representing full-length and truncated forms of the human immunodeficiency virus type 1 envelope protein gp160 were produced in E. coli and sf9 insect cells. These proteins were denatured and reduced as a function of purification. We adsorbed these proteins onto latex microspheres and used the protein-coated particles as a vehicle to present the antigen in vitro to splenic mononuclear cells from immune mice. Recombinant proteins presented on the latex particles induced antigen-specific proliferative responses that were dependent on the antigen concentration. The proliferative responses were similar to those produced against an identical protein used in soluble form and equivalent protein concentrations. Latex microspheres coated with recombinant proteins could also induce precursor cytotoxic T lymphocytes to mature to functional effector cells in vitro. The use of the latex microspheres to present recombinant proteins as antigens allowed for the use of denatured proteins in our assay that were not soluble in aqueous solutions, such as cell culture media. This system of delivering recombinant proteins in vitro should greatly facilitate the use of recombinant proteins in assays involving live cells.

Methylation and a polymorphic restriction site adjacent to human beta-interferon gene.

High-molecular-weight DNA from cultured human fibroblast cells and tissues was analyzed with the restriction endonucleases Msp I and Hpa II for the presence of methylated sites in the sequences flanking the beta-interferon gene. The majority of the DNAs analyzed were methylated in the restriction site (CCGG) for these two enzymes and thus were sensitive to cleavage by Msp I, but resistant to Hpa II. A polymorphic Msp I restriction site was identified approximately 1000 bp upstream from the beta-interferon gene. The results show an association of the beta-interferon gene with Msp I fragments of either 2.7 kb or 4.2 kb, which are inherited as Mendelian alleles. An unusual Msp I site upstream from the beta-interferon gene was present in 22% of the DNA from peripheral leukocytes of healthy individuals, and in 36% of the DNA from leukocytes of individuals with different forms of leukemia. Induction of beta-interferon with poly(rl X rC) did not alter the methylation pattern in the sequences flanking the beta-interferon gene, and the levels of beta-interferon induced in cells by poly(rl X rC) could not be directly related to the presence or absence of a polymorphic Msp I restriction site in the 5'-flanking region.

Effect of interferon on retrovirus replication.

Interferon block of retrovirus replication can be explained as a consequence of interferon induced changes in physiology (e.g. fluidity) of cellular plasma membranes. Thus, both the membrane associated virus uptake and assembly of progency virus are altered in interferon treated cells (Fig. 1). The relative unsensitivity of retroviral mRNA to the enzymatic systems alternating efficiency of viral mRNA translation in interferon treated cells may be only a relative phenomenon, since the inhibition of viral protein synthesis is observed under conditions of limited viral expression.

Physical and chemical properties of an oncornavirus associated with a murine adrenal carcinoma cell line.

A type C oncornavirus has been isolated from a continuous cell line of murine adrenal carcinoma in culture. The particles have a buoyant density of 1.165 g/cm(3), exhibit typical type C morphology by electron microscopy, possess an RNA-dependent DNA polymerase, and have a high molecular weight RNA (6.1 x 10(6)) which can be denatured to a homogeneous lower molecular weight species (3.2 x 10(6)) when extracted from rapidly harvested "immature" virions. The virus is related antigenically to other mammalian oncornaviruses and exhibits a similar, although much more complex, sodium dodecyl sulfate gel electrophoretic profile of virion proteins when compared to the profiles of other type C RNA tumor viruses.

Physical properties of moloney murine leukemia virus high-molecular-weight RNA: a two subunit structure.

The high-molecular-weight RNA of Moloney murine leukemia virus (MuLV) was analyzed by sedimentation equilibrium ultracentrifugation. Molecular weights of 7.2 x 10(6) and 3.4 x 10(6) were found for the native and subunit forms, respectively, indicating that the native structure is a dimer. S20,w and frictional coefficients were determined for MuLV RNA by analytical velocity centrifugation as a function of ionic strength. The apparent S20,w of native MuLV RNA was 47.3, 57.4, and 66.5 in 0.01, 0.1, and 0.20 M Na+, respectively; the corresponding frictional coefficients were 5.44, 4.48, and 3.87. Native RNA was estimated by circular dichroism to be 85% helical, whereas denatured RNA was 54% helical. Thermal denaturation profiles were obtained from uv absorbance scans. Melting temperatures of 57 and 68 C were obtained for high-molecular-weight RNA in 0.01 M Na+ and 0.122 M Na+, 1mM Mg2+, respectively. van't Hoff plots of the thermal denaturation data gave enthalpies for the helix-coil transition of 21,600 cal (ca. 90,500 J) per mol of cooperatively melting unit in high salt and 19,600 cal (ca. 82,100 J) per mol in low salt, consistent with both base stacking and pairing. The melting of Mu LV RNA occurred over a broad temprange and van't Hoff plots were linear over most of the melting range, indicating a noncooperative process of helix stabilization.

Detection of antibodies to human immunodeficiency virus by latex agglutination with recombinant antigen.

Recombinant human immunodeficiency virus (HIV) env antigen was attached to polystyrene particles, and these complexes were used to develop the first latex agglutination assay for antibodies to HIV. A total of 95 positive and 116 negative human serum samples were assayed for antibodies to HIV by latex agglutination, and results were compared with those of a commercial enzyme immunoassay. Latex agglutination was also compared with, and found to be completely concordant with, Western blot (immunoblot) analysis with virion antigens.

Comparison of six serological assays for human immunodeficiency virus antibody detection in developing countries.

Three commercially available assays for the detection of human immunodeficiency virus (HIV) antibodies-Vironostika enzyme immunoassay (EIA), Wellcozyme competitive EIA, and JLC Allaman indirect immunofluorescence assay--were tested on 300 serum samples from African subjects with and without HIV-related conditions. Two experimental assays both rapid and simple to perform (Biotech dip stick and Cambridge Bioscience latex agglutination) were also evaluated on the same serum samples. The results were compared with those of a commercial Western blot (WB) (immunoblot) assay from Biotech, used as the reference technique. All assays were tested in the laboratory of the AIDS Project in Kigali, Rwanda. Calculated specificity ranged from 90.8% (dip stick) to 98.6% (Vironostika EIA, Wellcozyme competitive EIA, and Cambridge Bioscience latex agglutination). Sensitivity ranged from 95.2% (Cambridge Bioscience latex agglutination) to 98.0% (Vironstika EIA) and JLC indirect immunofluorescence assay). However, the sensitivity of the latex agglutination test improved to 98.6% after the prozone effect was controlled for by serial twofold dilution of latex agglutination-negative, WB-positive samples. In situations with a high prevalence of HIV infection, any one of these tests can be regarded as an alternative to the more expensive, time-consuming, and difficult WB assay.

Generation of neutralizing anti-B19 parvovirus human monoclonal antibodies from patients infected with human immunodeficiency virus.

The prevalence of IgG antibodies to human B19 parvovirus (anti-B19) is elevated in individuals infected with human immunodeficiency virus (HIV), especially during the later stages of HIV infection. In subjects with high titers of IgG anti-B19, 86% (19 of 22) had circulating B cells producing anti-B19. Immortalization of these cells with Epstein-Barr virus and generation of heterohybridomas by fusion with a mouse X human heteromyeloma resulted in the production of two cell lines producing IgG1 kappa monoclonal antibodies (MAbs). Both of these MAbs were specific for conformational epitopes on the VP2 capsid protein of B19 parvovirus and both were capable of neutralizing 50% of the viral infectivity in a human erythroid colony-forming unit assay at < or = 1 micrograms of MAb/mL. These human MAbs are potentially useful in the treatment of acute B19 parvovirus infection.

Rapid latex agglutination assay using recombinant envelope polypeptide for the detection of antibody to the HIV.

Unscreened blood transfusions continue to be one of the major modes of transmission of the human immunodeficiency virus (HIV) in developing countries, such as in Central Africa, where 5% to 18% of blood donors are HIV seropositive. We evaluated a rapid latex agglutination assay using a novel recombinant envelope polypeptide of HIV for the detection of HIV antibodies among 2820 blood donors and clinical patients from diverse geographic regions, including on-site testing in Central Africa of 1600 blood donors. Overall, 29.2% of the serum samples were positive by Western blot assay. On a single determination, the latex agglutination slide test was found to be highly sensitive and specific compared with Western blot results in these populations with a relatively high prevalence of infection. Use of this assay will allow the immediate implementation of serologic screening for HIV in developing areas of the world, where standard screening procedures are impractical or not available, and in many other clinical settings, such as sexually transmitted diseases clinics and hospitals, where testing and counseling could be promptly implemented.

Candidate recombinant vaccine for human B19 parvovirus.

Recombinant baculoviruses were used to produce human B19 parvovirus empty capsids composed of only VP2 and VP2 capsids containing 4%, 25%, 35%, or 41% VP1 protein. Immunogenicity of the purified capsids, formulated with or without adjuvant, was evaluated in mice, guinea pigs, and rabbits. Sera were analyzed for total anti-B19 parvovirus antibodies, antibodies specific to the region unique to the VP1 capsid protein, and virus neutralizing antibodies. A relationship was observed between the development of antibodies specific to sequences unique to the VP1 protein and virus neutralization. The polypeptide composition of the empty capsid immunogens appeared to be important for elicitation of potent virus neutralizing activity. VP2 capsid immunogens devoid of VP1 protein, or consisting of only 4% VP1, the composition of naturally occurring virions, were generally poor at eliciting high levels of virus neutralizing activity. Capsids consisting of > or = 25% VP1 protein efficiently and consistently provoked vigorous B19 virus neutralizing responses. Recombinant empty capsids enriched for the VP1 protein should serve as the basis for a human B19 parvovirus vaccine.