In vitro susceptibility of Thai seronegative donor CD8+ T lymphocytes to human immunodeficiency virus-1 (HIV-1) infection

To determine whether CD8+ T lymphocytes from Thai donor cells are susceptible to HIV-1 infection, undepleted peripheral blood mononuclear cells (PBMC) and CD8-enriched PBMC were infected with HIV-1 Thai subtype B and CRF01_AE (E) primary isolates. Virus kinetics in HIV-1 infection of CD4+ and CD8+ T lymphocytes peaked at day 7 or 10 post infection (pi); the TCID50 used for cell infection was proportional to the level of p24 production in the cultures. We also found that the level of p24 antigen in the supernatants of infected undepleted PBMC was significantly higher than that of infected CD8-enriched PBMC. Interestingly, both single positive T lymphocytes (CD4+ and CD8+ T lymphocytes) as well as double positive CD4+/CD8+ T lymphocytes were infected with HIV-1. The double positive T lymphocytes in PBMC were found only in the presence of both CD4+ and CD8+ T lymphocytes. The majority of p24+/CD4-/CD8- T lymphocytes were HIV-1 infected CD4 down-modulated PBMC. This report provides direct evidence that single positive CD8+ T lymphocytes and double positive CD4+/ CD8+ T lymphocytes from Thai donors can be infected with HIV-1 subtypes B and E in vitro.     

HIV-1 IgA specific serum antibodies and disease progression during HIV-1 infection

OBJECTIVE: To evaluate the role of serum human immunodeficiency virus type 1 immunoglobulin A (HIV-1 IgA) antibodies in the progression of HIV-1 infection in relation to viral load and CD4 cell counts. METHODS: Sequential serum specimens were obtained from 218 homosexual men: 123 HIV-1 seropositives, 24 HIV-1 seroconverters, and 71 HIV-1 seronegatives. HIV-1 IgA antibodies were tested blindly by enzyme-linked immunosorbent assay and Western blot. T-lymphocyte subsets were measured by flow cytometry. Viral plasma load was determined by a sensitive branched DNA assay. RESULTS: HIV-1 IgA antibodies with a titer greater than or equal to 50 were detected among 50% of the seroconverters, 27% of the HIV-1-seropositive asymptomatic subjects, 25% of lymphadenopathy, and 23% of HIV-1-related symptomatic subjects. Among patients with the acquired immune deficiency syndrome, the prevalence of virus-specific IgA antibodies (55%) was significantly higher (p < 0.03) as compared with the HIV-1-seropositive asymptomatic subjects, lymphadenopathy and HIV-1-related symptomatic patients, but not versus the seroconverters (p = 0.8). IgA antibodies to HIV-1 gP160 were the most prevalent among all subjects tested. A significant decrease in CD4 cell counts was observed after HIV-1 seroconversion. Viral load was slightly higher among the seroconverters who demonstrated higher (> or =50) HIV-1 IgA levels. CONCLUSIONS: HIV-1 IgA serum antibodies did not predict the progression of the disease. Correlation between HIV-1 IgA antibodies titer, viral load, and CD4 cell counts was not detected.     

Analysis of the spontaneous in vitro anti-HIV-1 antibody secretion by peripheral blood mononuclear cells in HIV-1 infection.

We studied the spontaneous in vitro secretion of anti-HIV-1 antibodies by peripheral blood mononuclear cells (PBMC) from HIV-1-infected patients. Specific antibody production was detected in supernatants of PBMC cultures using an ELISA; HIV-1 specificity was confirmed by antigen adsorption and Western blotting. This antibody secretion was found to be an active phenomenon and was not due to a release of plasma antibodies passively adsorbed onto the cell membranes. In all positive supernatants, anti-HIV-1-secreted antibodies were directed against env-encoded antigens and many supernatants also contained antibodies to pol- and gag-encoded antigens. PBMC from all HIV-1-infected patients tested (140 adults and 18 infants) secreted anti-HIV-1 antibodies. This production was found during all the clinical stages of HIV-1 infection. Our results suggest that this spontaneous HIV-1-specific antibody secretion represents a marker of HIV-1 infection. Detection of these antibodies could be a valuable tool for early confirmation of HIV-1 infection in neonates born to HIV-1-seropositive mothers.     

Increased number of single-LTR HIV-1 DNA junctions correlates with HIV-1 antigen expression and CD4+ cell decline in vivo

Human immunodeficiency type 1 (HIV-1) DNA in peripheral blood cells of HIV-1 infected individuals may be present as integrated and/or unintegrated DNA. Several reports have indicated that a major proportion of HIV-1 DNA in the asymptomatic phase is linear, full-length, and unintegrated and in the symptomatic phase either circular unintegrated or integrated in the host genome. We developed a quantitative polymerase chain reaction (PCR) technique to detect single-LTR HIV-1 DNA junctions, reflecting the presence of unintegrated single-LTR circles. In vitro infection of a CD4+ T-cell line resulted first in the increase of single-LTR junctions followed by syncytium formation and a rise of p24 antigen production. The number of single-LTR HIV-1 DNA junctions was further studied in two acutely infected individuals and in 21 long-term infected individuals. The number of single-LTR junctions was significantly correlated with CD4+ cell decline, p24 antigen expression, and total HIV-1 DNA content of peripheral blood mononuclear cells (PBMC). Single-LTR HIV-1 DNA junctions were absent from PBMC containing other forms of HIV-1 DNA in four of nine non/slow progressors relative to 2 of 12 rapid progressors/AIDS patients. We conclude from our data that quantitative detection of single-LTR HIV-1 DNA junctions can be used as an early DNA marker of the transition from clinical latency to active replication in the peripheral blood. © 1995 Wiley-Liss, inc.     

Differential maturation of avidity of IgG antibodies to gp41, p24 and p17 following infection with HIV-1

We have evaluated solid-phase ELISA IgG antibody avidity studies as a means of identifying cases of recent HIV-1 infection. Although separate studies on the avidity of anti-gp41 and anti-p24 antibodies in seroconvertors have been reported, a comparison of the ability of patients to simultaneously mature their immune response to more than one HIV antigen immediately following seroconversion appears to be lacking. We have demonstrated a maturation in anti-gp41 avidity which reflects the time since seroconversion in all cases. In contrast, however, only some patients produced high-avidity anti-p24 or anti-p17 antibodies during the same time span. While the avidity of anti-gp41 antibodies remained high in cases of non-recent HIV infection, even in the face of advanced disease, we have confirmed the findings of others that the avidity of anti-p24 falls before the onset of ARC or AIDS. Therefore, whilst the avidity of anti-gp41 antibodies could reliably be of value in identifying cases of recent HIV infection, the avidity of anti-p24 or anti-p17 antibodies could not, but may be of prognostic value, even at an early stage. The time taken to reach maximum anti-p17, anti-p24 and anti-gp41 titres was variable, but anti-gp41 titres, like anti-gp41 avidity, remained high. In contrast, anti-p24 titres fell, even during the early follow-up period in some seroconvertors. Anti-p24 antibody avidity, however, appeared to be a better predictor of disease progression in ‘remote’ cases than anti-p24 titre. The avidity and titres of these antibodies are presented in relation to the clinical details, p24 antigen status, CD4 and CD8 counts where these are known.     

T lymphocyte interferon-gamma production induced by Plasmodium falciparum antigen is high in recently infected non-immune and low in immune subjects.

Interferon (IFN) alpha and gamma were measured by radio-immunoassays in supernatants from cultures of peripheral blood mononuclear cells (PBMC) or purified T cell subsets incubated with either Plasmodium falciparum schizont-enriched malaria antigen (mAg), uninfected red blood cells (RBC) or pokeweed mitogen (PWM). Cell donors were 24 clinically immune, healthy African adult native residents of a P. falciparum-endemic region, Haut-Ogooué, Gabon, and seven non-immune, European temporary residents with a history of a single to a few malaria infections during the previous 1 to 9 months. When PBMC were cultured in medium alone or with RBC antigen no or low titres of IFN-gamma were detected. PBMC proliferation and IFN-gamma production observed in the presence of mAg were dose dependent and significantly correlated. When cultured with mAg, PBMC from non-immune Europeans produced significantly higher levels of IFN-gamma than did PBMC from clinically immune Africans. No such difference was found when PBMC were cultured with PWM. The mAg-induced IFN-gamma production was due mainly to CD4+ T cells and was not enhanced by CD8+ T cell depletion. No IFN-alpha was detected in culture supernatants. Thus, P. falciparum antigens are able to induce in vitro production of IFN-gamma by CD4+ T cells; however, in this sample, individuals considered to be clinically resistant to malaria were low producers of IFN-gamma.     

Improved detection of HIV p24 antigen in serum after acid pretreatment

HIV-1 p24 antigen was detected in 554 sera (509 from HIV-1 seropositive individuals and 45 sera from seronegative controls) using a conventional method with acid pretreatment of the sample in order to separate the p24 antigen/anti-p24 antibody immune complexes. In asymptomatic individuals there was a substantial increase in antigen detection (48.2 % vs 8.4 %). Similar results were also observed in ARC (59.1 % vs 12.2 %) and AIDS patients (85.7 % vs 37.1 %). It can be concluded that the acid treatment improves the sensitivity of conventional techniques to detect HIV-1 p24 antigen.     

Specific Immune Response to Human Immunodeficiency Virus (HIV)-1 in Patients Assessed Through the Production of Interferon-γ and Interleukin-4 in HIV-1 p24-Activated Whole Blood Cultures: Relationship with the Viral Load in Plasma

We studied the in vitro HIV-1 antigen-stimulated production of IFN-gamma and IL-4 in HIV-1-infected patients and its relationship with viral replication as assessed through the plasma level of HIV-1 RNA. The levels of IFN-gamma and IL-4 were higher in supernatants of stimulated whole blood cultures than in stimulated peripheral blood mononuclear cell cultures, therefore whole blood cultures were used in the rest of the study. Specific IFN-gamma and IL-4 responses to HIV-1 p24 antigen were observed in HIV-1-infected patients but not in healthy controls (n = 23). A lower proportion of individuals with a positive IFN-gamma response to HIV-1 p24 was observed in patients at a declining clinical stage: 62% in asymptomatic patients (CDC group A, n = 16) versus 19% in symptomatic patients (CDC groups B and C, n = 21; P = 0.007, chi2 testing), whereas the proportion of individuals with a positive IL-4 response to HIV-1 p24 was almost similar in both groups of patients (25% versus 23.8%). Increased IL-4 production by HIV-1 p24-activated immunocompetent cells of patients and a predominant IL-4 response to HIV-1 p24 (with IL-4/IFN-gamma > 1) were positively correlated with an increased viral load. In contrast, there was no correlation between the mitogen-stimulated production of IL-4 and IFN-gamma and the viral load in plasma. The CD8 T cells from whole blood of patients, but not from controls played a significant role in the HIV-1 p24-activated production of IFN-gamma and IL-4. In conclusion, HIV-1-antigen-stimulated whole blood appears to be a valuable tool to study the production of IL-4 in HIV-1-infected patients. The cytokine profile pattern in response to epitopes of HIV-1 gag p24 may play an important role in the host immune response to HIV-1.     

A longitudinal study of the IgG antibody response to HIV-1 p17 gag protein in HIV-1+ patients with haemophilia: titre and avidity.

The IgG response to HIV-1 p17 gag protein was studied for up to 6 years in 12 HIV-1-infected patients with haemophilia, who had seroconverted between 1982 and 1985. To assess any prognostic value, p17 IgG titres were compared with p24 IgG titres, CD4 cell counts and p24 antigenaemia. p17 IgG avidity index was also examined. A strong similarity was found between the IgG titre to HIV-1 p17 and that to p24. In patients who developed AIDS the decline in p17 IgG titres could precede by several years the drop in CD4 cells to under 200 cells/microliters; whereas some long-term asymptomatic patients (CDCII) had increasing p17 IgG titres and stable CD4 cell counts. Declining p17 and p24 IgG titres were not always associated with an increase in p24 antigenaemia. IgG titres were found to be better predictors of disease progression than CD4 cell counts or p24 antigenaemia. Patients who developed AIDS during the study were also characterized by a lower p17 IgG avidity than patients who remained asymptomatic. This result suggests that IgG avidity could have prognostic relevance and be of importance for host resistance to AIDS onset.     

Development of a sensitive ELISA for HIV-1 p24 antigen using a fluorogenic substrate for monitoring HIV-1 replication in vitro.

A two-site sandwich fluorescent-ELISA was optimized for the detection of HIV-1 p24 antigen produced by lymphoid cells infected with HIV-1 in vitro. To improve the sensitivity of the ELISA, a combination of streptavidin-beta-galactosidase and a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was employed for the enzymatic detection stage. Using recombinant p24 as standard antigen, a two-step assay detected as little as 0.7 pg/ml (3.10(-14) M) with an upper limit of 10,000 pg/ml. This detection range (approx. 50-70-times greater than ELISAs using a chromogenic detection) permitted an accurate and straightforward quantitation of p24 in culture supernatants. Overall, the fluorescent-ELISA had increased detectability, sensitivity and efficiency over existing ELISAs for HIV-1 p24.     

The affinity of IgG antibodies to gag p24 and p17 in HIV-1-infected patients correlates with disease progression.

The affinity of anti-gag antibody was studied for up to 9 years (1984-1993) in sera from 15 HIV-1+ patients with haemophilia. On the basis of their 1993 clinical status patients were divided into two groups: (i) patients who remained asymptomatic (n = 9); and (ii) those who progressed to AIDS between late 1987 and 1993. The affinity constants of antibody for p24 and p17 were determined by a double isotope fluid-phase radioimmunoassay; and the relationships between antibody affinity and titre, patient clinical course, CD4 cell counts and p24 antigenaemia were analysed. The affinity of p24- and p17-specific antibody was up to 100 times greater in asymptomatic patients than in patients who progressed to AIDS. Patients who developed AIDS either lost or failed to develop high-affinity antibodies early in the infection. Asymptomatic patients maintained high-affinity antibodies for several years; however, in some of these patients the affinity of anti-p24 and p17 antibodies subsequently fell later in the study period. The presence of low-affinity antibody and progressive reduction in the titre of specific antibody were earlier predictors of disease onset than CD4 cell counts. The failure to either develop or maintain high affinity gag-specific antibody suggests an early impairment of T helper function in individuals who progressed to AIDS. The presence of antibody of high affinity could be essential in controlling virus replication and the onset of AIDS.     

Human immunodeficiency virus type 1 detected in all seropositive symptomatic and asymptomatic individuals.

Between February 1987 and October 1988, peripheral mononuclear blood cells (PBMC) from 409 adult individuals antibody positive by Western (immuno-)blot for human immunodeficiency virus type 1 (HIV-1) (56 acquired immunodeficiency syndrome [AIDS] patients, 88 patients with AIDS-related complex, and 265 asymptomatic individuals) were consecutively cultured for HIV-1 or tested for the presence of HIV-1 DNA sequences by a polymerase chain reaction assay (PCR). We isolated HIV-1 or detected HIV-1 DNA sequences from the PBMC of all 409 HIV-1 antibody-positive individuals. None of 131 healthy HIV-1 antibody-negative individuals were HIV-1 culture positive, nor were HIV-1 DNA sequences detected by PCR in the blood specimens of 43 seronegative individuals. In addition, HIV-1 PCR and HIV-1 culture were compared in testing the PBMC of 59 HIV-1 antibody-positive and 20 HIV-1 antibody-negative hemophiliacs. Both methods were found to have sensitivities and specificities of at least 97 and 100%, respectively. In contrast, the sensitivities of serum HIV-1 antigen testing in AIDS patients and asymptomatic seropositive patients were 42 and 17%, respectively. Our ability to directly demonstrate HIV-1 infection in all HIV-1 antibody-positive individuals provides definitive support that HIV-1 antibody positivity is associated with present HIV-1 infection. Moreover, the sensitivities and specificities of PCR and culture for the detection of HIV-1 appear to be equivalent, and both methods are superior to testing for HIV-1 antigen in serum for the direct detection of HIV-1.     

Persistent inapparent HIV-1 infection of human neuroblastoma cells

Summary We have studied human immunodeficiency virus type 1 (HIV-1) infection in three different human neuroblastoma cell lines; SK-N-MC, IMR-32 and SH-SY5Y. In all of these cell lines the infection became productive. However, the virus expression was different as determined by the p 24 antigen capture assays from culture supernatants and immunochemical (APAAP) staining of cells. The medium of SK-N-MC cells contained 300 pg p 24 antigen per 10⁶ cells, 0.1–1% of the cells were p 24 antigen-positive and characteristic genomic and subgenomic HIV mRNA species were seen in Northern blotting. In infected IMR-32 and SH-SY5Y cell cultures, the HIV-1 production was below the level of detection. However, infectious virus was found by inoculating cultures of the lymphoid cell C 8166 with the cell-free supernatant fluid from the neuroblastoma cultures. The lymphoid cells became positive within one week. Moreover, phytohemagglutinin-stimulated normal human lymphocytes produced virus, if cocultured with any of the three infected neuroblastoma cell lines. The infection was persistent and has been followed, using the above techniques, for 4 months in the case of SK-N-MC and IMR-32 cells and 6 months in the case of SH-SY5Y cells. During this period, no alterations in cell morphology, viability, or proliferative capacity were seen. All three neuroblastoma lines were negative for the CD 4 receptor mRNA according to Northern hybridization and RNase protection assays. We conclude that HIV-1 produces persistent and inapparent infection in human neuroblastoma cells, using a CD 4-independent mechanism of entry to the cells.     

Immunoregulation of the in vitro anti-HBs antibody synthesis in chronic HBsAg carriers and in recently boosted anti-hepatitis B vaccine recipients.

We report a study on immunoregulation of in vitro antibody to hepatitis B surface antigen (anti-HBs) synthesis induced by pokeweed mitogen (PWM) from peripheral blood mononuclear cells (PBMC) in chronic hepatitis B surface antigen (HBsAg) carriers and in 'high responders', (anti-HBs RIA ratio greater than or equal to 20 in serum), recently boosted with anti-hepatitis B vaccine. Anti-HBs was detected in 11 days PBMC supernatants (SN) from 24 out of 36 'high responders', but in none from 31 chronic HBsAg carriers, despite detectable amounts of polyclonal IgG and antibody to hepatitis B core antigen (anti-HBc) were produced. The lack of anti-HBs production by chronic HBsAg carriers did not seem to be determined by suppressor influences because T lymphocytes from the majority of chronic HBsAg carriers, co-cultured with 'high responders' PBMC did not suppress anti-HBs production. Co-cultures between HBsAg carriers T4 positive (helper/inducer) cells and allogenic 'high responder' non-T cells produced anti-HBs antibody, indicating that HBsAg carrier T cells are not deficient in this allogenic helper function under PWM stimulation. Allogenic cocultures between HBsAg carrier non-T cells and 'high responder' T4 positive cells failed in anti-HBs production: a specific B lymphocyte defect might be involved in the lacking anti-HBs synthesis in chronic HBV patients. Antigen-induced specific anti-HBs synthesis experiments indicate that B cells themselves seem to be the target for HBsAg-induced suppression of anti-HBs antibody response.     

Liver-infiltrating T helper cells in autoimmune chronic active hepatitis stimulate the production of autoantibodies against the human asialoglycoprotein receptor in vitro.

Autoantibodies against the human asialoglycoprotein receptor (ASGPR) occur in the sera of patients with autoimmune liver disorders. Liver-infiltrating T cell clones that specifically recognize the ASGPR have been described in patients with autoimmune chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). Recently, we have shown that peripheral blood mononuclear cells (PBMC) from patients with AI-CAH or PBC but not chronic viral hepatitis secreted anti-ASGPR antibodies in vitro. In this study we characterized the influence of liver-infiltrating T cells on the secretion of ASGPR-specific autoantibodies by autologous B cells in cell culture supernatants. T cell clones from liver biopsies of three patients with chronic autoimmune liver disorders (one with AI-CAH, two with PBC) were isolated and investigated for their proliferative response to soluble ASGPR and their helper function provided to autoantibody-secreting B lymphocytes. PBMC from these patients secreted autoantibodies spontaneously in their cell culture supernatants and showed a proliferative response to ASGPR. T cell-depleted PBMC, however, lacked spontaneous antibody secretion. Four CD4+CD8- liver-infiltrating T cell clones showed a proliferative response to ASGPR and also induced spontaneous anti-ASGPR antibody production in cell culture supernatants when added to autologous T cell depleted PBMC. Activated supernatants of these T cell clones failed to induce antibody production. None of seven CD4+CD8- and two CD4-CD8+ T cell clones non-responding to ASGPR provided this help for antibody secretion. Anti-ASGPR secretion in vitro could not be inhibited by the addition of MoAbs raised against monomorphic determinants on HLA class II molecules. The addition of purified ASGPR or polyclonal-activating pokeweed mitogen showed no influence on the production of autoantibodies in these cultures. These data show that B lymphocytes require T cell help for the production of ASGPR-specific antibodies. This help can be provided by ASGPR-responsive T helper cells via cellular interactions.     

Immunohistochemical evidence for human immunodeficiency virus-1 infection of liver Kupffer cells

To investigate the possibility of human immunodeficiency virus-(HIV) 1 infection of liver cells, liver samples from 17 patients with either acquired immunodeficiency syndrome (AIDS, 13), AIDS-related complex (ARC, 3), or lymphadenopathy syndrome (LAS, 1) were studied. A monoclonal antibody directed against the p24 gag HIV-1 protein was used in an immunoperoxidase assay and yielded positive results in seven out of 17 samples. Staining by anti-p24 antibody was of three types: diffuse in Kupffer cells of most samples, inside granuloma in cells that were probably histiocytes, and in some sinusoidal cells whose origin was difficult to ascertain. Attempts to locate the CD4 membrane antigen showed that it was mainly present on endothelial sinusoidal cells. These results indicate that liver cells, including Kupffer cells, might be infected by HIV-1, and that these cells might be involved in certain liver lesions observed during HIV-1 infection, particularly sinusoidal abnormalities.     

T cell responses to peptides covering the gag p24 region of HIV-1 occur in HIV-1 seronegative individuals.

We demonstrate that peptides (16 amino acids long) covering the sequence of the HIV-1 core protein p24 induce significant proliferation in peripheral blood mononuclear cells (PBMC) of several (greater than 50%) healthy seronegative volunteers as well as seronegative homosexual men. The nature of this response was characterized and compared with those of HIV-infected patients. Several peptides induced responses; however, the most frequent responses in both seropositive and seronegative individuals were noted to the following peptides: 1 and 2 (aa 133-157); 6 and 7 (aa 183-207); 15 (aa 273-287); and 17 and 18 (aa 293-317). The response pattern was related to the disease stage of the patients; seronegative individuals as well as asymptomatic seropositive individuals (CDC II/III) responded to low concentrations of several peptides, but symptomatic patients (CDC IV) only responded to high concentrations of a few peptides. Cell separation studies of PBMC from healthy volunteers showed that the responding cells were CD4+ and expressed the CD45RO differentiation antigen. Furthermore, cord-blood mononuclear cells with less than 5% of CD45RO T cells did not proliferative to any of the peptides. Finally, CD4+ T cell lines specific for both peptides and p24 protein were successfully established from the PBMC of seronegative individuals confirming the data obtained with freshly isolated cells. These studies therefore suggest that the CD4+ cell response to p24 is not strictly disease related, instead, the response may be due to priming of the host with cross-reactive antigens.     

p24 antibody production in p24 antibody-negative HIV-infected subjects.

To determine if HIV-infected patients with no detectable serum antibodies to p24 are producing antibodies to p24 (anti-p24), blood was obtained from 49 HIV-infected patients at various stages of infection. Serum p24 antigen levels were measured and peripheral blood mononuclear cells were cultured for 1 week without mitogenic stimulation. The presence of anti-p24 in culture supernatants and sera was determined by radioimmunoprecipitation assays. Cells from 89% of the patients who had anti-p24 in their sera spontaneously synthesized anti-p24 in vitro. Similarly, cells from 83% of the HIV-infected patients who had no detectable anti-p24 in their sera spontaneously produced anti-p24 in vitro. Thus the absence of anti-p24 in serum did not reflect suppression in the ability of patients' cells to synthesize and secrete antibodies to p24. However, cells from patients whose sera contained anti-p24 spontaneously synthesized more anti-p24 than did cells from patients whose sera lacked anti-p24, suggesting that these two groups of patients may represent individuals with inherently high or low responses to p24 epitopes, respectively.