Pending problem of "silent" human immunodeficiency virus infection.

The problem of "silent" HIV infection is reviewed. Overall, the number of proven "silent" infection in several at-risk populations, including HIV exposed health-care workers, homosexuals, IV drug addicts and children born to HIV-infected mothers, has been very low. Contrary to these observations, we describe a very high prevalence of HIV specific immunity and positive HIV specific PCR signals in an Ethiopian immigrant population recently arrived in Israel. The interpretation of these findings is not entirely clear but we suggest that host immunity and probably different handling of the infection may account for the longer persistence of viral components in the body. Further studies are required to determine the amount and nature of these viral elements and, most importantly, whether they are still infectious.

Evidence for simian immunodeficiency virus-specific IgM and IgG response in peripheral blood mononuclear cells of serum enzyme-linked immunosorbent assay-negative nonhuman primates.

In vitro polyclonal activation of peripheral blood mononuclear cells (PBMCs) from 70% of the simian immunodeficiency virus (SIV) serum enzyme-linked, immunosorbent assay (ELISA)-negative sooty mangabeys leads to synthesis and release of low but significant and reproducible levels of SIV-reactive antibodies, as determined by ELISA and Western blot analysis. The predominant isotype of SIV-reactive antibodies in the pokeweed mitogen (PWM) supernatant fluids from serum ELISA-negative mangabeys is IgM, whereas the predominant isotype of SIV-reactive antibodies in seropositive mangabeys is IgG. Depletion of CD8+ cells led to a marked increase in the levels of SIV-reactive antibodies detected in supernatant fluids from PWM-induced cultures from the serum ELISA-negative mangabeys. No evidence for such SIV-reactive antibodies has been found, to date, in similar unfractionated or CD8+ T-cell-depleted PWM-induced PBMC cultures from uninfected macaques. Supernatant fluids from PWM cultures of PBMCs from a select group of serum ELISA-negative mangabeys, when concentrated five times, were shown to give a Western blot profile against SIV, similar to the profile seen with plasma from seropositive infected macaques and mangabeys. Evidence is presented to show that these serum ELISA-negative mangabeys are most likely latently infected with SIV. This evidence, which was obtained in samples from such ELISA-negative mangabeys, includes the detection of reverse transcriptase activity and the presence of SIV p27 in supernatant fluids of phytohemagglutinin-stimulated PBMCs in vitro. In addition, the data show the presence of CD8+ T cells that regulate SIV-specific Ig synthesis and show the detection of gag sequences by the polymerase chain reaction. Thus, the PWM assay described herein may provide a valuable additional tool for detection of lentivirus infection before or in the absence of seroconversion.

A new look at HIV transmission from seropositive mothers to their infants: the facts beyond serology.

Once the curtain of maternal antibodies is removed (12-18 months) only a fraction of the infants are seropositive. Some babies from whom virus has been isolated or detected in their cells subsequently become seronegative. What does the negative serology of these children really tell us about exposure to HIV? It is suggested that seroconverting is only one of the ways to respond to an HIV exposure from an infected mother; it is not the only or the best way. Some form of tolerance to HIV, emerging after in utero exposure of the fetus, could theoretically lead to a seronegative state despite infection. Based on monkey studies with simian immunodeficiency virus (SIV), this tolerance could offer protection against pathological outcome of the infection. Seronegative yet infected/exposed children of HIV-positive mothers exist, though their number remains unknown. They might hold the key to a protective immunity to HIV.

Early and complete detection of HIV exposure.

Currently, HIV diagnosis relies on serology. Yet in groups at high risk for HIV serology is not sufficient because of the window period between infection and seroconversion. There is a growing body of reports on HIV-infected yet seronegative individuals. Some tests have been developed to identify exposure to HIV by its effect on the cells of the immune system that would differentiate following exposure to the foreign antigens. Detection, in vitro, of HIV-specific B and T cells in seronegative, at risk individuals has been reported. In only some of these individuals was an HIV infection confirmed by other methods. These new assays to detect HIV immunity enable us to identify two new groups among seronegative, at risk individuals; namely those with immunity to HIV and a detectable HIV infection (silent carriers), and those with immunity and no proof of infection. Both groups have been exposed to HIV yet are not being detected by serology. Both might hold information on other forms of HIV immunity, possibly a protective one. Thus there could be an important role for other immunological assays in early detection of HIV exposure.

Inhibition of cellular activation of retroviral replication by CD8+ T cells derived from non-human primates.

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 (FEc1) 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 FEc1 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 FEc1 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.

'Silent' HIV infection among wives of seropositive HIV carriers in the Ethiopian community in Israel.

We have previously described the phenomenon of 'silent HIV carriers', i.e. individuals with HIV specific immunity and a positive PCR for HIV-1, yet HIV seronegative. In the present study, we have looked for such 'silent' carriers among wives of individuals infected with HIV in Africa (Ethiopia). In addition to determining HIV serology, peripheral blood mononuclear cells (PBMC) were tested by PCR for HIV-1 and for their ability to generate specific antibodies to HIV upon polyclonal B-cell activation (P-BAT). Out of 16 wives so tested, three were HIV seropositive and among the 13 seronegatives, eight were P-BAT positive and five were both P-BAT and PCR positive. These findings suggest that (1) 'silent' HIV carriers may indeed be present in African populations; (2) interpretation of the 'silent' carrier phenomenon is not clear and will depend on clinical follow-up and the ability to culture virus from such carriers; and (3) results of HIV serology in this population and probably in other African populations should be viewed with caution.

Maternal transmission of SIVsmm in rhesus macaques.

Fifteen SIV-infected rhesus monkeys delivered 13 livebirths and two stillbirths; one livebirth died at three days of age. While all infants were culture-negative for SIV at birth, nine had maternal antibodies that disappeared by six months of age. Three infants subsequently seroconverted and became virus positive at 9-15 months. Milk samples from all mothers were virus-negative at parturition but samples from four animals were virus-positive at nine and 12 months. This study documents maternal transmission of SIV and suggests transmission by breast-feeding.

Transmission of retroviral infection by transfusion of seronegative blood in nonhuman primates.

Techniques such as polyclonal B cell activation with pokeweed mitogen (PWM) and polymerase chain reaction (PCR) analysis have documented the existence of simian immunodeficiency virus (SIV)-and human immunodeficiency virus type 1-seronegative but infected humans and nonhuman primates. To establish whether blood from such seronegative but PWM- and PCR-positive monkeys can transmit infection, naive macaques were transfused with whole blood (n = 2) or cultured cells and supernatant fluid (n = 2) from two seronegative but PWM- and PCR-positive sooty mangabeys. After transfusion, three of the four recipients seroconverted, and peripheral blood mononuclear cells from all four recipients secreted SIV-reactive antibodies upon polyclonal activation in vitro and were SIV-positive by PCR that used highly specific gag primer pairs and probe. In addition, CD8+ cells from all four recipients markedly inhibited replication of SIV in autologous cells in vitro. These data suggest caution in the sole use of serologic tests for the detection of retroviral infection and document the ability of such blood samples to transmit infection.

Detection of occult simian immunodeficiency virus SIVsmm infection in asymptomatic seronegative nonhuman primates and evidence for variation in SIV gag sequence between in vivo- and in vitro-propagated virus.

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 asymptomatic seropositive and seronegative sooty mangabeys (Cercocebus atys) and from experimentally infected but asymptomatic rhesus macaques (Macaca mulatta). The results indicate that most if not all SIV-seronegative mangabeys from the colony at the Yerkes Primate Center are in fact infected with SIVsmm despite their lack of humoral immune response, confirming previous immunological and virological observations made by our laboratory. Sequence analysis of these particular gag fragments from the mangabey revealed an average of 88% nucleotide sequence homology but 97% amino acid identity with the previously published sequence of the SIVsmmH4 clone. The significance of this finding relative to the asymptomatic state of SIV-infected mangabeys and disease-susceptible SIV-infected rhesus macaques is discussed.

Polyclonal B-cell activation reveals antibodies against human immunodeficiency virus type 1 (HIV-1) in HIV-1-seronegative individuals.

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 HIV-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.

Does stem cell self renewal and progenitor cell commitment operate through an effector-memory cell mechanism?

We propose a model for stem cell self renewal and transition into commitment towards a variety of cell lineages. In this model the production of both "effector cells" (as represented by the mature cells in the different cell lineages) and of progenitor "memory" lymphocytes, takes place concomitantly. The experimental evidence supporting this model is as follows: Pure lymphocytic suspensions (PLS) are established and persist in culture when nude mouse-spleen and lymph-node cells are maintained on X-irradiated fibroblast monolayers in the presence of the S-phase cytotoxic agent cytosine arabinoside (Ara-C). From these PLS the following colony types can be initiated by the corresponding inducing (stimulating) factors (CSF): histiocytes (tissue macrophages) - CSF-1; granulocytes-macrophages (GM) - CSF-GM; mast cells - MMSF; granular-NK mucus secreting cells - IL-2; and multilineage colonies - IL-3. Mitotically active blast cells (formed by transformation of lymphocytes), condense into motile small cells when the stimulatory factor is removed. These "memory" lymphocytes are committed as they carry the receptors for the specific CSF; they respond by retransformation into blast cells. A dramatic increase in mast-cell colony forming cells is found in bone marrow, spleen and lymph-nodes of mice infected with Schistosoma mansoni. By maintaining PLS with both Ara-C and each of the CSFs and then titrating the incidence of CFC in the residual PLS, we find that each one of the CSFs acts on an independent set of cells in the PLS to produce the corresponding colony type. Finally, the concept suggests that the various blast cells carrying the receptors, undergo condensation into memory lymphocytes when dissociated from the environment prevailed by the corresponding CSF. In this way pluripotential blast-cells condense into motile lymphocytes which are committed to pluripotentiality.