Effects of whole blood lysis and fixation on the infectivity of human T-lymphotropic virus type 1 (HTLV-I).

Whole blood lysis and fixation methods for flow cytometric (FCM) analysis were tested for their ability to reduce the infectivity of human T-lymphotropic virus type 1 (HTLV-I). Our goals were to: (1) determine the effects of 1.0 and 2.0% paraformaldehyde (PF) fixation on HTLV-I infected cell lines and (2) assess the infectivity of blood samples containing HTLV-I-infected cells following processing with 5 commercially available products (Immuno-lyse, ImmunoPrep/Q-Prep, FACS lysis solution, GenTrak lyse and fix reagent, and Ortho-mune lysing reagent) compared to ammonium chloride lysis with either 0.1 or 1.0% PF fixation. Infectivity was determined by monitoring HTLV-I p24 antigen production in cocultures of treated leukocytes with uninfected peripheral blood mononuclear cells (PBMC). Each method effectively reduced the viability of treated leukocytes. Commercial lysis/fixation methods significantly reduced HTLV-I infectivity compared to prepared ammonium chloride/PF-based methods. For all preparations, increasing the time of fixation (e.g., 60 min) effectively reduced viral infectivity. Taken together, these data suggest that commercially available fixatives greatly reduce, but do not eliminate the risk of HTLV-I infection during processing of viral-infected cells for FCM analysis.

Detection of simian immunodeficiency virus and human immunodeficiency virus type 2 capsid antigens by a monoclonal antibody-based antigen capture assay.

We have tested the ability of a monoclonal antibody-based simian immunodeficiency virus (SIV) p27 capsid antigen assay to detect SIV antigen in supernatants from a variety of infected cell cultures. The antigen capture assay has a sensitivity of approximately 30 pg of SIV p27 capsid antigen/ml. The assay detected SIV p27 capsid antigen in cell culture supernatants from all six strains tested, detected the replication of SIV following the inoculation of the virus in peripheral blood mononuclear cell cultures earlier compared to reverse transcriptase assay, and was more sensitive in detection of the SIV antigen compared to human immunodeficiency virus type 1 (HIV-1) antigen capture assays. The SIV antigen capture assay was used to detect SIV antigen from serum samples and tissue cultures from eight of eight SIVB670-infected rhesus macaques (Macaca mulatta). Similar samples from four control rhesus macaques were negative when tested by the assay. The SIV antigen was detected in virus-infected monkeys during early time periods following inoculation (1 to 3 weeks) or during episodes of CD4+ lymphocytopenia and clinically evident disease. In addition, the SIV antigen capture assay positively identified each of three different HIV-2 strains in cell culture supernatants. The SIV antigen capture assay provides a sensitive and specific method to monitor SIV and HIV-2 capsid antigen in cell cultures and from infected animals. The assay will be an important tool in the utilization of SIV and HIV-2 primate models for HIV-induced acquired immunodeficiency disease syndrome.

Development and characterization of a monoclonal antibody to class II MHC antigens in rhesus macaques.

The characterization of a monoclonal antibody with specificity for a monomorphic determinant on rhesus macaque class II antigens is described. This antibody, designated 2D16, is an IgG2b immunoglobulin which also displayed useful cross-reactivity with lymphoreticular cells and cell lines of other species including man, bonnet and stumptail macaques, sheep, dog and horse. Limited polymorphism of the 2D16 epitope was observed in the dog.