Protective attenuated lentivirus immunization induces SIV-specific T cells in the genital tract of rhesus monkeys.

Live attenuated lentivirus immunization is the only vaccine strategy that elicits consistent protection against intravaginal challenge with pathogenic simian immunodeficiency virus (SIV). To determine the mechanism of protection in rhesus monkeys infected with attenuated simian-human immunodeficiency virus (SHIV)89.6, a detailed analysis of SIV Gag-specific T-cell responses in several tissues including the genital tract was performed. Six months after SHIV infection, antiviral T-cell responses were rare in the cervix; however, polyfunctional, cytokine-secreting, and degranulating SIV Gag-specific CD4(+) T cells were consistently found in the vagina of the immunized macaques. SIV-specific CD8(+) T cells were also detected in the vagina, blood, and genital lymph nodes of most of the animals. Thus, an attenuated SHIV vaccine induces persistent antiviral T cells in tissues, including the vagina, where these effector T-cell responses may mediate the consistent protection from vaginal SIV challenge observed in this model.

Anatomic site and immune function correlate with relative cytokine mRNA expression levels in lymphoid tissues of normal rhesus macaques.

Reverse transcriptase real-time polymerase chain reaction was used to determine pro-inflammatory, anti-viral and immunoregulatory cytokine mRNA expression levels in peripheral blood mononuclear cells (PBMC) of healthy juvenile, adolescent and adult rhesus macaques. Few age-related changes in cytokine mRNA expression levels were observed. Expression of interleukin 2 and Mx, a type I interferon-inducible gene, decreased with age, whereas interleukin 4 and macrophage inflammatory protein 1 (MIP-1) alpha and beta mRNA levels increased in older monkeys. Independent of age, the pro-inflammatory cytokines [tumour necrosis factor alpha (TNF-alpha) and chemokines] were expressed at higher mRNA levels in PBMC than the immunoregulatory cytokines (interleukins 2, 4, 12). Pro-inflammatory cytokine mRNA expression levels were highest in lymphoid tissues draining mucosal surfaces. Thus, a correlation exists between cytokine mRNA levels in lymphoid tissues and the anatomical site.

Antigen-dependent cytokine mRNA expression by individual rhesus macaque T helper cells by flow cytometry.

Specific patterns of cytokine secretion by CD4(+) T helper (Th) cells determine the nature of immune effector responses. Using a multiparameter, flow cytometric fluorescent in situ hybridization (FISH) assay that detected cytoplasmic mRNA within intact cells, we assessed antigen-specific cytokine expression in rhesus macaque Th cells. In the peripheral lymphocytes of immunized rhesus macaques, FISH detected antigen-induced cytokine gene expression in single Th cells. Analysis of simultaneous cytokine expression by single cells demonstrated that the recall immune response consisted of Th cells expressing either a Th1 (IL-2(+)/IFN-gamma(+)) or a Th2 (IL-4(+)/IL-6(+)) cytokine pattern. In addition to the classic Th subsets, Th cells expressing only one of two Th1 or Th2 defining cytokines were common following antigen restimulation. The data gathered with the FISH assay suggest that, in primates, the immune response to recall antigens consists of nonclassic Th cells, as well as a mixture of polarized Th1 and Th2 T cells.

Nasal immunization of nonhuman primates with simian immunodeficiency virus p55gag and cholera toxin adjuvant induces Th1/Th2 help for virus-specific immune responses in reproductive tissues.

Female rhesus macaques were nasally immunized with p55gag (p55) of SIV and cholera toxin as a mucosal adjuvant. Nasal immunization induced Ag-specific IgA and IgG Abs in mucosal secretions (e.g., cervicovaginal secretions, rectal washes, and saliva) and serum. Furthermore, high numbers of p55-specific IgA and IgG Ab-forming cells were induced in mucosal effector sites, i.e., uterine cervix, intestinal lamina propria, and nasal passage. p55-specific CD4+ T cells in both systemic and mucosal compartments expressed IFN-gamma and IL-2 (Th1-type)- as well as IL-5, IL-6, and IL-10 (Th2-type)-specific mRNA. Moreover, p55-specific CTL activity was demonstrated in lymphocytes from blood, tonsils, and other lymphoid tissues. These results show that nasal immunization with SIV p55 with cholera toxin elicits both Th1- and selective Th2-type cytokine responses associated with the induction of SIV-specific mucosal and serum Abs, and CTL activity. These results offer a promise for the development of protective mucosal immunity to SIV.

Mucosal phenotype of antiviral cytotoxic T lymphocytes in the vaginal mucosa of SIV-infected rhesus macaques.

CD8+ T lymphocytes are present in the vaginal epithelium and submucosa of women and female rhesus macaques. Antiviral cytotoxic T lymphocyte precursors were detected in the vaginal intraepithelial lymphocyte (IEL) population of SIV-infected monkeys. Monoclonal antibodies to adhesion molecules distinguish lymphocytes that recirculate through peripheral lymphoid tissues (e.g., L-selectin) from mucosal lymphocytes that traffic through peripheral blood to the gut (e.g., the integrins alpha4beta7 and alphaEbeta7). Cytolytic CD8+ T cell lines from either peripheral blood or the vaginal epithelium of SIV-infected monkeys were stained with antibodies against these molecules. Three of three vaginal epithelial cell lines had the phenotype: alpha4beta7+/alphaEbeta7+/L-selectin-. Two of three peripheral blood cell lines had this phenotype and the other was positive for all three molecules. These results suggest that cytolytic vaginal IELs have the same mucosal phenotype as has been described for human and murine gut IELs, and that their precursors are destined to traffic through peripheral blood and return to the vaginal mucosa.

Experimental measles. I. Pathogenesis in the normal and the immunized host.

An animal model to study measles pathogenesis and the correlates of protective immunity was established using rhesus monkeys. A measles isolate, obtained during an epidemic of measles in the primate colony at the University of California, Davis, was passaged through rhesus monkeys and amplified in rhesus mononuclear cells to create a pathogenic virus stock. Sequence analysis of the nucleoprotein and hemagglutinin genes of this isolate revealed strong homology with the Chicago 89 strain of measles virus. Conjunctival/intranasal inoculation of juvenile rhesus monkeys with this virus resulted in skin rash, pneumonia, and systemic infection with dissemination to other mucosal sites and to the lymphoid tissues. Inflammation and necrosis occurred in the lungs and lymphoid tissues and many cell types were infected with measles virus on Day 7 postinoculation (p.i.). The most commonly infected cell type was the B lymphocyte in lymphoid follicles. Measles antigen was found in follicular dendritic cells on Day 14 p.i. In contrast to naive monkeys infected with measles virus, animals vaccinated with the attenuated Moraten strain did not develop clinical or pathologic signs of measles after challenge. However, moderate to marked hyperplasia occurred in the lymph nodes and spleen of a vaccinated animal on Day 7 after pathogenic virus challenge, suggesting that an effective measles vaccine limits but does not prevent infection with wild-type measles virus.

Antiviral cytotoxic T lymphocytes in vaginal mucosa of simian immunodeficiency virus-infected rhesus macaques.

The mucosal immune system of the female reproductive tract is of central importance for protection against sexually transmitted diseases, including HIV; however, this arm of the immune system remains poorly understood. Antiviral CTL responses never have been documented in the genital tract and the role of CTL in this anatomic site is unknown. In this study, CD8+ intraepithelial lymphocytes (IEL) in the vaginas of six simian immunodeficiency virus (SIV)-infected female rhesus macaques were identified by immunohistochemistry to be CD2+ and TCR beta-chain+. In addition, the majority of CD8+ IEL contained TIA-1+ cytoplasmic granules that are associated with CTL activity. CD8+ T cells were isolated from the vaginal epithelium and submucosa and amplified by limiting dilution in the presence of feeder cells. SIV p55gag and/or gp160env-specific lysis was detected in cultures of vaginal epithelial but not submucosal CD8+ T lymphocytes. Estimated SIV-specific precursor CTL frequencies were higher in the vaginal CD8+ IEL population of chronically infected monkeys than in the same cells from acutely infected monkeys or a naive control monkey. These results provide the first demonstration that antiviral CTL are present in the vaginal epithelium, and suggest that a vaccine may be able to generate anti-HIV CTL in the genital mucosa.

Effects of viral virulence on intrauterine growth in SIV-infected fetal rhesus macaques (Macaca mulatta).

Studies with a simian immunodeficiency virus (SIV)-infected fetal monkey model were conducted with a focus on fetal growth and viral pathogenesis. Twenty-six fetuses were inoculated in utero via ultrasound guidance with an uncloned pathogenic strain of SIV or vehicle during the second or third trimesters [gestational day (GD) 65, 110, or 130], sonographically monitored weekly (biometrics, blood flow), then necropsied at incremental time points postinfection. Peripheral blood hematologic (complete blood counts, clinical chemistries), immunologic (immunophenotyping), and endocrine studies [insulin-like growth factor (IGF), IGF-binding proteins (IGFBP)] were conducted. Severe intrauterine growth restriction (IUGR), oligohydramnios, and altered lymphocyte counts were noted for fetuses infected on GD 65. Less severe effects were detected for fetuses inoculated at the later time points, with severity dependent upon the length of SIV infection in utero. IGF studies indicated significant reductions in IGF-I and elevated immunoreactive levels of IGFBP-3 in infected fetuses during the third trimester. Parallel studies conducted with four fetuses infected on GD 65 with a nonpathogenic, molecularly cloned virus (SIVmac1A11) resulted in normal fetal growth, with no effects on hematopoiesis or IGF/IGFBP levels, and no evidence of clinical disease. Taken together, these studies show that (1) infection of fetuses during the early second trimester with an uncloned pathogenic strain of SIV results in severe IUGR and a disruption in the molar ratio of IGF:IGFBP-3, and (2) outcome of fetal SIV infection is determined by the timing of infection and the virulence of the viral inoculum.

Gag-specific cytotoxic responses to HIV type 1 are associated with a decreased risk of progression to AIDS-related complex or AIDS.

The duration of human immunodeficiency virus (HIV-1) infection prior to the development of AIDS is variable, and for most patients the exact time of infection is not known. A group of 38 HIV-1-infected subjects was tested while asymptomatic for comparative cytotoxic lymphocyte responses to the Gag and envelope antigens of HIV-1. Twenty of the 38 patients had no detectable primary cytotoxic T lymphocyte (CTL) response to Gag, and this was associated with a relative risk of 1.89 for progression to ARC or AIDS during the subsequent 3 to 40 months of observation when compared with patients who had Gag-specific CTL activity at the beginning of the observation period. In contrast, no significant association was observed between envelope-specific cytotoxic activity and disease progression. Other patient characteristics, including CD4+ T lymphocyte counts and antibody levels to the p24gag protein, measured at the start of observation, did not correlate with disease progression during the observation period. This suggests that the anti-Gag CTL response may be protective during HIV-1 infection.

Cytotoxic mechanisms in vitro against Epstein-Barr virus infected lymphoblastoid cell lines in rheumatoid arthritis.

Impaired regulation of latent infection with Epstein-Barr virus (EBV) may contribute to the pathogenesis of rheumatoid arthritis (RA) by allowing uncontrolled polyclonal B cell activation. The control of EBV infection in vitro is dependent on several cytotoxic lymphoid cell populations. The present report examines the suppression of early lymphoblastoid outgrowth by natural killer (NK) like cells and the ability to form cytotoxic T lymphocytes (CTLs) specific for EBV in vitro. The latter was measured by a regression assay of EBV induced lymphoblastoid transformation. In this assay the regression of B cell outgrowth at four and six weeks is due to the generation of CTLs specific for EBV. Patients with RA were defective in this ability to generate CTLs. Eight out of nine patients with RA had a geometric mean at the 50% regression end point equal to or greater than 20 X 10(5) cells/ml. In contrast, the geometric mean for all control donors was less than 4 X 10(5) cell/ml. NK activity was measured by a conventional 51Cr release assay with K562 targets. Patients with RA did not have significantly different activity from that of controls (RA patients, n = 4, 45.6 +/- 19.7% (means +/- SD) at 50:1, effector:target; normals, n = 5, 56.6 +/- 5.7%). No spontaneous NK activity was detected against allogeneic or autologous EBV infected B cell targets. When peripheral mononuclear cells from patients were incubated for six days with interleukin-2, lysis of EBV infected targets was seen. No difference in this activity was seen between RA and control studies. Overall, these studies show that patients with RA are defective in their ability to generate CTLs specific for EBV in vitro.

Kinetics of tumor cell cytostasis by sea star factor-activated macrophages.

Macrophages obtained from Wistar/Furth (W/Fu) and Fischer (F344) rats receiving intraperitoneal injection of 0.2 mg of sea star factor (SSF) exert a profound suppressive effect on proliferation of tumor cells in vitro. The cytostatic effect of these activated macrophages is not immunologically specific, and is demonstrably effective against syngeneic, allogeneic, or xenogeneic targets (the latter of murine origin, P815-X2 of DBA/2 strain). Evidence suggests that cytostasis can be resolved into three discrete temporally progressive steps: cytoadhesion, macrophage-dependent, and macrophage-independent states. Interference with the first stage by the use of high molecular weight dextran blocks development of macrophage-dependent cytostasis. Suppression of this second stage by selective macrophage toxicity using silica blocks the final event of irreversible macrophage-independent cytostasis. Taken together, these data suggest that suppression of tumor cell division by SSF-activated macrophages requires direct contact for a relatively prolonged period between viable effector macrophages and tumor target cells to achieve maximal cytostasis.