Infectious simian/human immunodeficiency virus with human immunodeficiency virus type 1 subtype C from an African isolate: rhesus macaque model.

Human immunodeficiency virus type 1 (HIV-1) subtype C is responsible for more than 56% of all infections in the HIV and AIDS pandemic. It is the predominant subtype in the rapidly expanding epidemic in southern Africa. To develop a relevant model that would facilitate studies of transmission, pathogenesis, and vaccine development for this subtype, we generated SHIV(MJ4), a simian/human immunodeficiency virus (SHIV) chimera based on HIV-1 subtype C. SHIV(MJ4) contains the majority of env, the entire second exon of tat, and a partial sequence of the second exon of rev, all derived from a CCR5-tropic, primary isolate envelope clone from southern Africa. SHIV(MJ4) replicated efficiently in human, rhesus, and pig-tailed macaque peripheral blood mononuclear cells (PBMCs) in vitro but not in CEMx174 cells. To assess in vivo infectivity, SHIV(MJ4) was intravenously inoculated into four rhesus macaques (Macaca mulatta). All four animals became infected as determined through virus isolation, PCR analysis, and viral loads of 10(7) to 10(8) copies of viral RNA per ml of plasma during the primary infection phase. We have established a CCR5-tropic SHIV(MJ4)/rhesus macaque model that may be useful in the studies of HIV-1 subtype C immunology and biology and may also facilitate the evaluation of vaccines to control the spread of HIV-1 subtype C in southern Africa and elsewhere.

Extraction of human Langerhans cells: a method for isolation of epidermis-resident dendritic cells.

Langerhans cells (LCs) are immature dendritic cells in the epidermis that play a central role in T-lymphocyte mediated skin immunity. Upon activation with antigenic stimuli, they differentiate drastically into mature dendritic cells while migrating from the epidermis to regional lymph nodes. Thus, in order to study biological details of immature LCs, it is crucial to isolate epidermis-resident, immature LCs without dermal dendritic cell contamination. Methods for extracting LCs from human skin as well as in vitro derivation of LC-like cells from hematopoietic progenitor cells have been described previously, but the cell preparations can potentially contain a significant number of dendritic cells that are not identical to epidermal LCs. Here, we describe a technique by which purely epidermis-resident LCs are extracted from human skin. Following digestion of human skin with dispase, the epidermis was separated mechanically without any attached dermal component. The trypsinized epidermal cells were then fractionated by centrifugation with a discontinuous density gradient composed of bovine albumin and sodium metrizoate. The LC-enriched preparation thus obtained contained 80% to >90% CD1a+, E-cadherin+ cells that expressed Birbeck granules and the Lag protein. Consistent with their being at an immature stage, the freshly isolated LCs lacked the expression of CD83, a marker for mature dendritic cells. The purified LCs were able to activate allogeneic T cells, indicating that the cells retained T-cell stimulation ability even after extraction. Thus, the present work offers an opportunity for precise in vitro studies of epidermal LCs.

Host and viral factors affecting the decreased immunogenicity of Sabin type 3 vaccine after administration of trivalent oral polio vaccine to rural Mayan children.

Factors affecting immunogenicity of the first 2 doses of oral poliovirus vaccine (OPV) among unimmunized Mayan infants were prospectively evaluated. The relative impact of multiple variables, including mass or routine vaccination, concurrent enteric bacterial (salmonella, shigella, and campylobacter) and viral (adenovirus 40/41, astrovirus, nonpolio enteroviruses, and rotavirus) infections, interference among Sabin vaccine viruses, and preexisting poliovirus antibodies were studied. Sera were available from 181 infants after 2 OPV doses. Seroresponses were 86% to Sabin type 1, 97% to Sabin type 2, and 61% to Sabin type 3 vaccines. Mass versus routine vaccination and preexisting poliovirus antibodies did not affect immunogenicity. By multiple logistic regression analysis, fecal shedding of homologous Sabin strains was associated with increased seroresponses to all Sabin types, especially to Sabin type 3. Decreased OPV immunogenicity was primarily attributable to interference of Sabin type 3 by Sabin type 2. OPV formulations with higher doses of Sabin type 3 could improve immunogenicity among infants in developing countries.

HIV-1 Langerhans' cell tropism associated with heterosexual transmission of HIV.

Heterosexual transmission by vaginal intercourse accounts for most transmission of human immunodeficiency virus-type 1 (HIV-1) in Africa and Asia but is less important in the HIV-1 epidemics of the United States and Western Europe. Epithelial Langerhans' cells (LCs) represent a possible source of initial cell contact for vaginal infection. Fifteen primary isolates of HIV-1 from U.S. homosexuals and 18 HIV-1 isolates from Thailand heterosexuals were evaluated for growth in LCs of U.S. origin. All the viruses from the Thai heterosexuals, which were subtype E, grew more efficiently in the LCs than any of the viruses from the U.S. homosexuals, which are subtype B. These results suggest that LC tropism is associated with the efficiency of heterosexual transmission of HIV.

Altered histopathology in protein-deprived mice during Sendai virus pneumonia: evidence for delayed inflammatory response and recovery.

The morphology of the lungs and airway during the course of respiratory infection caused by Sendai virus was examined in normal (20% protein diet) and malnourished (2% protein diet) BALB/c mice. Mortality in normal Sendai-infected mice was 0 compared with 71% in the infected malnourished group. Virus was isolated until day 6 in normally fed mice and until day 9 in the malnourished group. Pulmonary inflammation was largely mononuclear and began in the normally nourished animals on day 3, peaked at day 6, and reverted almost to normal by 30 days. In the malnourished group, inflammation was delayed by about 1 day and fell further behind during the first week. It peaked 10-13 days after infection and was still present with little resolution by day 30. These findings may have relevance to the high mortality of acute respiratory diseases in children of the developing world.

Protective activity of a human respiratory syncytial virus immune globulin prepared from donors screened by microneutralization assay.

To explore the feasibility of preparing a human immune globulin specific for respiratory syncytial virus (RSV) by screening plasma donors, the ability of seven RSV antibody assays to identify plasma-yielding IgG with high virus-neutralizing and animal-protective activities was compared. IgG prepared from plasma units selected by microneutralization assay had significantly higher activity in protecting mice from respiratory RSV challenge than did IgGs prepared from plasmas selected by three direct ELISAs using purified F protein, G protein, or RSV-infected cell lysate, by two competitive ELISAs with RSV neutralizing monoclonal antibodies directed to the F2 or F3 epitopes of the F protein, or by plaque reduction neutralization. Relative to IgG made from unselected plasma, microneutralization-screened IgG was enriched fivefold by plaque-reduction neutralization assays done with or without complement. The microneutralization assay identified RSV antibodies with highest animal protective activity. This assay will be useful for identifying plasma donors for the preparation of a human immune globulin with high protective activity against RSV and deserves further evaluation for prediction of protective antibody concentrations in children.

Sendai virus infection of normal and protein malnourished mice: response of airway leukocytes to infection.

We examined the cells recovered by bronchoalveolar lavage (BAL) during the course of respiratory infection caused by Sendai virus in normal (20% protein diet) and malnourished (2% protein diet) mice. As in our previous experiments, mortality in normal mice was 32% in comparison with 87% in the malnourished group. Virus was isolated until the 5th day in normally fed mice and until the 9th day in the malnourished group. BAL fluids contained 97% macrophages before infection in both groups. During infection there was a progressive lymphocyte response, reaching a peak of 60-70% on day 5 in the normal mice and on days 7-9 in the malnourished group. Subtyping of BAL cells by flow cytometry indicated that in uninfected animals lymphocytes were largely CD4-bearing. On days 3 and 5 post-infection most mononuclear cells were Thy 1.2-positive, but lacked both CD4 and CD8 markers and were therefore probably natural killer cells. Beginning on day 5, in both diet groups CD8-positive cells rose to become the predominant subset. In the 20% protein diet group, CD8-positive cells reached a maximum of 60% on day 7, whereas in the 2% protein diet group this level was not reached until day 9. These results were consistent in three separate experiments. In malnourished mice the delayed appearance of CD8-bearing cells in the airway may contribute to the higher mortality and delayed virus clearance during Sendai virus infection.

Effect of respiratory syncytial virus infection on mice with protein malnutrition.

Respiratory syncytial virus (RSV) pulmonary infection was produced in BALB/c mice fed protein-deficient diets in an effort to understand the severity of viral pneumonia in infants in developing countries. As in previously published experiments with Sendai virus, animals on the deficient diet became clinically malnourished, and certain aspects of their cell-mediated immunity were altered. The course of RSV infection in protein-deprived mice was essentially identical to that in normally nourished animals. The titer of virus recovered from lung homogenates over time, as well as the histologic picture of bronchiolitis, were identical under all experimental conditions. This model, unlike that of Sendai virus infection, fails to demonstrate an effect of protein malnutrition on RSV infection.

A comparative study of virus isolation, polymerase chain reaction, and antigen detection in children of mothers infected with human immunodeficiency virus.

We report on an investigation designed to compare the polymerase chain reaction (PCR) with culture and p24 measurement for the diagnosis of human immunodeficiency virus (HIV) infection in infants and children. Forty-five children born of mothers with antibodies to HIV type 1 were studied; P24 antigen was measured in plasma, and HIV-1 proviral DNA was sought in peripheral blood mononuclear cells after amplification by PCR. In 26 cases, blood specimens were cultured for HIV; in all but two instances cultures were established at the same time that the PCR test was performed. Primer pairs in three regions of the proviral genome were used for the PCR test. There was good agreement between the results obtained from PCR tests and from cultures; of 24 children in whom both tests were done at the same time, 10 had positive results on both the culture and the PCR test, 1 had positive results on the PCR test but negative culture results, and 13 had negative results on both tests (concordance 96%). Measurement of p24 antigen in plasma was, in contrast, an insensitive marker of infection: 6 of 12 infants with positive cultures had positive p24 test results, and 8 of 18 infants had positive PCR test results. Sixteen children with subsequent seronegativity for HIV-1 had negative PCR results. This study provides further evidence that the PCR test is a valid alternative to viral culture for the diagnosis of pediatric HIV infection.

Sendai virus infection of mice with protein malnutrition.

Sendai virus pneumonia was produced in BALB/c mice fed protein-deficient diets in an effort to understand the severity of viral pneumonia in infants in developing countries. Animals on the deficient diet became clinically malnourished, and some aspects of cellular immunity were altered. In protein-deprived animals, the 50% lethal dose of intranasally administered Sendai virus was over 1,000-fold lower, pulmonary virus titers were higher, the infection was prolonged, and lung infection was established at a lower inoculum than in normal animals.

Rescue of a wild-type MDCK cell by a ouabain-resistant mutant.

When wild-type MDCK cells (W-MDCK) were cocultured in mixed monolayers with a ouabain-resistant mutant (R-MDCK), the wild-type cells were protected from the effect of ouabain up to concentrations as high as 100 microM. Rescue depended on the dose of ouabain and on the proportion of each cell type in the coculture. The survival of R-MDCK cells at 1 microM ouabain was not endangered by varying from 1:9 to 9:1 the proportion of W-MDCK cells to be rescued. Ouabain binding revealed two kinds of binding sites in R-MDCK cells, one with high and the other with low affinity. Only the high affinity site was present in W-MDCK cells. Electron probe analysis of individual cells revealed that rescued cells kept a high K and a low Na intracellular contents, similar to control cells. Histograms of intracellular K/Na in cocultured cells treated with ouabain were unimodal. Using microinjection of Lucifer yellow or electrophysiological techniques we estimated that at most 13% of the R-MDCK and W-MDCK cells may be connected at a given time through cell-to-cell junctions. Therefore permanent cell-to-cell communication did not seem to play a central role in the rescue. W-MDCK cells cocultured with R-MDCK cells and subsequently separated, were not rescued. Thus rescue did not seem to depend on the transfer from R-MDCK to W-MDCK cells of either ouabain-resistant Na-K pumps or of information to synthesize them. It is speculated that intercellular communications were sporadic events, so that all cells may become intermittently connected and rescued.