Identification of human immunodeficiency virus type 1 (HIV-1)-infected individuals is of paramount importance for the control of the spread of AIDS worldwide. Currently, the vast majority of screening centers throughout the world rely on serological techniques. As such, clinically asymptomatic but HIV-infected, seronegative individuals are rarely identified. In this report we show that 18% (30/165) of seronegative individuals who were considered to be a unique cohort of patients at high risk for HIV infection had circulating B cells that, upon in vitro polyclonal activation with pokeweed mitogen, produced antibodies reactive with HIV. Furthermore, polymerase chain reaction analysis of DNA obtained from aliquots of the peripheral blood mononuclear cells from these seronegative but pokeweed mitogen assay-positive individuals tested revealed the presence of HIV-specific sequences in a significant number of samples. In addition, depletion of CD8' T cells from peripheral blood mononuclear cells of HIV-1-seronegative individuals prior to in vitro culture with pokeweed mitogen resulted in increased sensitivity for detecting HlIV-reactive antibodies. This assay has obvious epidemiological implications, especially in the case of high-risk groups, and also provides a simple technique to enhance detection of HIV-infected individuals. Of further interest is the determination of the mechanisms related to the lack of HIV-specific antibodies in the serum of these infected individuals.

SUMMARY To test the hypothesis that CD8+ T cells inhibit viral replication at the level of cellular activation, an Epstein‐Barr virus (EBV)‐transformed cell line (FEcl) from a simian immunodeficiency virus (SIV)‐seropositive sooty mangabey monkey was transfected with a human CD4 gene and shown to be replication‐competent for HIV‐1, HIV‐2 and SIV. Utilizing a dual‐chamber culture system, it was found that inhibition of viral replication can be mediated by a soluble factor. The FEcl cell line was transiently transfected with an LTR‐driven CAT reporter gene. It was found that autologous CD8+ T cells markedly inhibited CAT activity. Furthermore, co‐transfection of the FEcl cell line with an LTR‐driven tat plasmid and LTR‐CAT was able to quantitatively mitigate the suppressive effect. Thus, this inhibition appears to be directed at cellular mechanisms of viral transcription. Control transfections with an LTR‐driven CAT plasmid with a mutation at the NFkB binding site yielded no CAT activity, suggesting that most viral replication as measured by CAT activity is dependent, to a large extent, upon cellularly derived NFkB binding proteins.

Background: Undetectable HIV infection in blood banks poses a serious threat to public health. Thus, donations from high school students are preferred over adult samples in Kenyan blood banks, due to lower HIV infection prevalence within this population, as detected by conventional serology testing. However, the number of recently infected individuals remains difficult to identify, as HIV-induced immunological window periods can span months. This study focuses on the potential contribution of a novel mode of diagnostic testing in revealing early, seronegative HIV carriers. Methods and Findings: Stimmunology, an in vitro lymphocyte stimulation technique, was used to detect early HIV infection among random samples of adult and adolescent blood donors. The Stimmunology protocol unveiled a significant number of early, pre-seroconversion HIV carriers both among adult and teenage Kenyan populations, undetected by typical serological diagnostic kits. Both populations demonstrated a significant increase in HIV-specific antibody formation following activation using the Stimmunology assay. The younger population exhibited a higher proportion of early HIV infection (0.45) than the adult (0.27) population. Conclusions: While blood samples of young donors are preferred over adult donations, these data demonstrate a worrisome ratio of early, seronegative HIV carriers within this population. This simple, cost-effective, and reliable HIV-boosting antibody assay can be used in a resource-poor setting to increase blood supply safety and quality. Incorporation of Stimmunology into basic blood bank testing and into diagnostic protocols can also decrease undesirable disease transmission.

Polymerase chain reaction techniques were used to identify simian immunodeficiency virus (SIV) SIVsmm gag sequences in genomic DNA isolated from peripheral blood mononuclear cells from naturally infected MATERIALS AND METHODS Animals. The rhesus macaques and sooty mangabey monkeys used in this study were housed in colonies at the Yerkes Regional Primate Research Center. All animals were maintained according to the guidelines of the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, and the Health and Human Services guidelines for the care and use of laboratory animals. Media. RPMI 1640 (GIBCO, Grand Island, N.Y.) was used throughout this study supplemented with 50 ,ug of gentamicin sulfate (Roche, Nutley, N.J.) per ml, 2 mM L-glutamine (GIBCO), and 10% fetal bovine serum (Hyclone, Fort Logan, Utah). PBMC isolation. PBMCs were separated from fresh heparinized venous blood onto 54% Percoll (

The window period between infection and seroconversion varies based on viral genetics, dose and route of transmission, host genetics, and environmental factors. The in vitro Stimmunology blood pretreatment assay was utilized to promote the synthesis of HIV-specific antibodies in efforts to define the window period between infection and seroconversion. Ethiopians seeking immigration to Israel while in refugee camps provided a unique cohort to perform a comparative analysis of the window period between HIV-1 infection and diagnosis utilizing conventional Ab-ELISA and our laboratory established an in vitro Stimmunolog assay. This population was considered unique due to its exposure to an environment with a high seroprevalence rate for a defined period of time. Unlinked blood samples were tested and validated before and after Stimmunology. Diagnostic screening was conducted in Israel. A total of 285 and 537 random samples were tested from the 1992 and 1998 immigrant population, respectively. Analysis of HIV prevalence, incidence, and window period length among the immigrants was measured by comparing results obtained on samples prior to and following analysis by Stimmunology as compared with standard ELISA-based assay. This novel assay that promotes the in vitro stimulation of antibody synthesis led to the diagnosis of 2.7% and 0.36% of the 1992 and 1998 immigrant groups, respectively, as HIV infected individuals during the window period. Using mathematical modeling, the length of the window period in the surveyed population was estimated to range from 2 to 5 months. Stimmunology-aided detection of early seronegative HIV-infected individuals provided a reliable and consistent mode of identifying infection in seronegative HIV-1-infected individuals. Applying mathematical modeling to the data obtained enabled us to define the window period length, which was found to extend beyond previous estimates. These results suggest a need for the reevaluation of the techniques that are employed to assess the true incidence and prevalence of HIV-1 infection, especially in populations within sub-Saharan Africa.