Monkey Peripheral Blood Mononuclear Cells Research Articles

Genetic variation of the SIVagm transmembrane glycoprotein in naturally and experimentally infected primates

An in-frame stop codon prematurely truncating the transmembrane glycoprotein (TMP) is a common feature of many simian immunodeficiency virus, African green monkey strain (SIVagm) molecular clones. The purpose of this study was to investigate the native form of the SIVagm TMP in a naturally infected African green monkey (AGM) and to study the fate of the stop codon following the passage of SIVagm in primates. Polymerase chain reaction was used to clone the entire intracellular portion of the TMP from: (1) peripheral blood mononuclear cells (PBMC) of the naturally infected AGM 155; (2) an isolate of SIVagm155 in rhesus PBMC and (3) PBMC from pig-tailed macaques and AGM experimentally infected with an SIVagm molecular clone encoding a truncated TMP. PBMC of the naturally infected AGM contained a 'swarm' of related virus genotypes that encoded a full-length TMP, whereas tissue-culture passage in rhesus PBMC resulted in a prematurely truncated form of the TMP. This premature stop codon persisted in PBMC of monkeys experimentally infected with an SIVagm molecular clone. Both macaques and AGM of same subspecies as AGM 155 (Cercopithecus pygerythrus) and other subspecies (C. aethiops and C. sabaeus) became infected with SIVagm155. Genetic drift of this region of env, as assessed by calculation of the nucleotide substitution/site/year rate, was similar to that of other retroviruses. The native form of the SIVagm TMP is a full-length gp40, similar to the SIV macaque (SIVmac) strain and HIV-1. However, passage of SIVagm in tissue culture can result in point mutations that introduce a premature stop codon. This stop codon persists during subsequent in vivo passage of SIVagm in primates. This contrasts with similar studies in macaques infected with SIVmac, in which reversion of the TMP stop codon was observed.

Evidence for the Cooperation of gp120 Amino Acids 322 and 448 in SIVmac Entry

The substitution of glycine for valine at amino acid 448 in the C4 domain of SIVmac239 envelope glycoprotein gp120 resulted in little or no viral infectivity for CEMX174 cells and rhesus monkey peripheral blood mononuclear cells. The block to viral replication was located at an early stage including virus entry into cells since the mutant virus was severely impaired in its ability to form newly synthesized viral DNA during the initial 14 hr after exposure to cells. Envelope glycoprotein with the 448V → G mutation bound to soluble CD4 in a manner similar to wild type in an in vitro assay and was also translated, processed, and cleaved in a manner similar to wild-type envelope protein. When CEMx174 cells were transfected with 448V → G mutant proviral DNA and held for longer than 40 days, a variant appeared in one culture that was able to replicate with near wild-type kinetics. Sequencing of viral DNA from cultures infected with this variant revealed that the original 448V → G mutation was retained in eight of eight clones analyzed but that six of eight clones analyzed had an additional mutation of V → I at amino acid 322 of gp120. Amino acid 322 in SIVmac gp120 is located in a region which corresponds to V3 of HIV. Analysis of site-specific mutants demonstrated that the mutation of V → I at position 322 can indeed compensate for the 448V → G mutation, resulting in efficient virus entry and replication by the double mutant. These results identify single amino acids in gp120 that are critical for early events in SIVmac replication and suggest that the "V3" and C4 domains of SIVmac gp120 cooperate to effect virus entry.

Protection of monkeys by a split vaccine against SIVmac depends upon biological properties of the challenge virus

To investigate the role of the anti-cellular immune response in the protection of rhesus macaques against infection with the simian immunodeficiency virus SIVmac. To determine the biological differences between SIV challenge stocks grown either on human T-cell lines or on monkey peripheral blood mononuclear cells (MPBMC). A protective SIVmac split vaccine was administered to rhesus macaques and their anti-, B- and T-cell response monitored. Vaccinees and controls were challenged with SIVmac grown either on human or on monkey cells. The in vivo replication rate of, and the immune response to, the two viruses was compared. Five rhesus macaques were immunized with a total of 2 mg each of purified SIVmac251/32H grown on the human C8166 T-cell line. The antibody and proliferative T-cell responses were evaluated by enzyme-linked immunosorbent assay and T-cell proliferation assay, respectively. Four protected animals and four controls were reboosted and challenged with MPBMC-grown SIVmac251 (SIVmac251/MPBMC). Cell-free virus load was determined by titration of plasma for SIV infectivity on C8166 cells and antigen with a core antigen capture assay. Protection from virus challenge with C8166-grown SIVmac251/32H or SIVmac251/MPBMC did not correlate with anti-cellular antibodies or proliferative T-cell reactivities. Control animals infected with SIVmac251/MPBMC showed high persistent antigenaemia and high plasma virus titres. Both were absent in controls infected with complement C8166-grown SIVmac251/32H. Whereas the latter always seroconverted against the full panel of viral polypeptides, SIVmac251/MPBMC-infected animals showed a drastically decreased antibody response. Neither the antibody nor the proliferative T-cell response to SIVmac correlates with protection from virus challenge. In contrast to SIVmac251/32H grown on C8166 cells, the MPBMC-grown challenge virus SIVmac251 appears to belong to the 'rapid-high' phenotype, possibly explaining the lack of protection against this SIV.

Changing patterns of the IL-2/IL-2R pathway of lymphocyte activation following exposure to Plasmodium falciparum products: a study with squirrel monkey peripheral blood mononuclear cells

Changing patterns of the IL-2/IL-2R pathway of lymphocyte activation following exposure to Plasmodium falciparum products: a study with squirrel monkey peripheral blood mononuclear cells

Changing patterns of the IL-2/IL-2R pathway of lymphocyte activation following exposure toPlasmodium falciparumproducts: a study with squirrel monkey peripheral blood mononuclear cells

Squirrel monkeys are experimental hosts useful for studies on human malaria. In the present work, in vitro lymphocyte reactivity was measured by proliferation in the presence of Plasmodium falciparum-derived products, and found to depend on the previous malarial status of the animals and upon the source and/or the nature of the P. falciparum-derived material. Special attention was given to the IL-2/IL-2R pathway of lymphocyte activation. Culture supernatant from P. falciparum-parasitized erythrocytes exerted an inhibitory effect towards T lymphocytes obtained from P. falciparum-non-immune squirrel monkeys, when activated, for instance, by PHA. These lymphocytes did not incorporate tritiated thymidine (neither they did proliferate) although they expressed IL-2 alpha and beta binding chains and secreted IL-2 (or at least TCGF). This inhibitory effect could not be rescued by the addition of rhIL-2, although the assayed lymphocytes could retain the ability to continue their cell cycle progression and divide after removal of the P. falciparum-derived inhibitory product(s). The incidence of anti-mitogenic molecules which impair the IL-2/IL-2R pathway of lymphocyte activation in malaria related processes is discussed.

Reactivity of some monoclonal antibodies specific for human lymphocytes with vervet monkey peripheral blood mononuclear cells.

Reactivities of some monoclonal antibodies to human lymphocyte surface antigens were tested on vervet monkey peripheral blood mononuclear cells (PBMC), using flow cytometry and immunoperoxidase techniques. A number of antibodies were identified which reacted with similar populations of lymphocytes from vervet monkeys. These included antibodies that define T cells, suppressor/cytotoxic cell subset, pan B cells, monocytes and MHC class I and II. A number of anti-CD4 markers examined were unsuitable for use in the vervet system.

Experimental allergic encephalomyelitis in cynomolgus monkeys. Quantitation of T cell responses in peripheral blood.

Chronic relapsing-remitting experimental allergic encephalomyelitis (EAE) was induced in cynomolgus monkeys by a single immunization with a homogenate of human brain white matter (BH) in adjuvant. Proliferative T lymphocyte responses to BH, to myelin basic protein (MBP), but not to proteolipid protein, were detected in peripheral blood mononuclear cells (PBMC) of all animals and persisted until their death or, in surviving animals, for greater than 10 mo postimmunization. Responses of higher magnitude tended to be associated with fatal, compared with nonfatal, episodes of clinical EAE. The frequency of MBP-reactive T cells in PBMC of animals with acute EAE was quantitated with a soft agar colony system; the ratio of T cells that proliferated specifically to MBP was estimated at between 5 and 20 per 10(6) PBMC. A similar frequency of peptide-specific T cells was estimated from PBMC of monkeys immunized with a synthetic 14-mer peptide corresponding to a region near the carboxy terminus of MBP. Thus, autoantigen-reactive T cells can be detected in the circulation throughout the course of chronic EAE, are predictive of disease severity, and occur at a frequency similar to that estimated to be present in humans with multiple sclerosis.

Prevention of HIV-2 and SIV infections in cynomolgus macaques by prophylactic treatment with 3'-fluorothymidine.

The aim of this study was to determine the usefulness of human immunodeficiency virus type 2 (HIV-2) for in vivo evaluation of antiviral drugs in monkeys and to study if prophylactic treatment with 3'-fluorothymidine (FLT) could prevent infection against a low challenge dose of HIV-2 or simian immunodeficiency virus (SIV). Protection against infection was assessed by virus isolation and polymerase chain reaction (PCR) on monkey peripheral blood mononuclear cells (PBMC) as well as by antibody and viral antigen assays. Prophylactic treatment with FLT 3 x 5 mg/kg/day, starting 8 h prior to virus inoculation, prevented HIV-2 infection in 3 of 8 monkeys. In another experiment 2 of 4 monkeys resisted 2-10 monkey infectious doses (MID50) of SIV with the same prophylactic treatment. All control animals (HIV-2 n = 8, SIV n = 4) became infected. Thus, FLT treatment prevented HIV-2 and SIV infection in 5 of 12 animals.

Immunophenotypic characterization of owl monkey peripheral blood mononuclear cells.

A two-phase study was initiated to delineate the peripheral blood lymphocyte populations present in owl monkeys and to correlate those populations with immune response and parasitism during malaria infection. The goal of phase I of the study was to elucidate a monoclonal antibody panel that could be used to characterize peripheral blood mononuclear cell (PBMC) populations with flow cytometric techniques. Forty-two monoclonal antibodies (reported to be reactive with human and macaque lymphocyte antigens) were screened for activity to owl monkey PBMC. Eleven monoclonals were found to react: anti-H42A (MHC Class II DP-like); anti-TH14B (MHC Class II DR-like); and anti-TH81A5 (MHC Class II DQ-like); anti-H58A (MHC Class I); anti-DH59B (granulocyte and monocyte); anti-B1 (B cell); anti-T4 (CD4); anti-Leu3a (CD4); anti-Leu11a (CD16); anti-60.3 (CD18); and anti-OKM1 (NK and monocyte). In a preliminary retrospective study correlating antibody titers, parasitemia values, and MHC Class I and Class II marker profiles on PBMC to test antigens used in malaria vaccine trials, a significant negative association was observed between cells bearing MHC Class II molecules and the other elements of the comparison. In summary, an appropriate panel of monoclonal antibodies has been identified for characterizing PBMC in owl monkeys, and preliminary studies indicate a possible association between clinical outcome and expressed phenotypic PBMC markers.

SIV/HIV recombinants and their use in studying biological properties.

A series of chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency viruses (SIV) were constructed. Viability of the recombinant viruses was dependent on the position of recombination. Infectious chimeric viruses between HIV-1 and SIVAGM (isolated from an African green monkey) and those between HIV-1 and SIVMAC (isolated from a rhesus monkey) were examined for host cell tropism. Viral determinants that restrict the replication of SIVAGM in human MT-4 cells and that of HIV-1 in macaque monkey peripheral blood mononuclear cells (PBMC) mapped to the 5' half of the virus genome. One HIV-1/SIVMAC chimera which contained the HIV-1 env gene was shown to replicate in macaque PBMC in vitro and to infect macaque monkeys in vivo. This HIV-1/SIVMAC chimera will be useful for a variety of AIDS pathogenesis and vaccine studies.

Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells

We constructed five chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVMAC) and four SIVMAC mutants by recombinant DNA techniques. Three chimeric clones and all mutants with an alteration in either the vif, vpx, vpr, or nef gene were infectious to human CD4-positive cell lines. The susceptibility of macaque monkey peripheral blood mononuclear cells (PBMC) to infection by these mutants and chimeras was examined in vitro. Macaque PBMC supported the replication of wild-type and vpx, vpr, and nef mutant SIVMAC strains. A chimera carrying the long terminal repeats (LTRs), gag, pol, vif, and vpx of SIVMAC and tat, rev, vpu, and env of HIV-1 was also replication competent in PBMC. In contrast, HIV-1, the vif mutant of SIVMAC, a chimera containing rev and env of SIVMAC, and a chimera containing vpx, vpr, tat, rev, and env of SIVMAC did not grow in PBMC. Western immunoblotting analysis of the replicating chimera in PBMC confirmed the hybrid nature of the virus. These data strongly suggested that the sequence important for macaque cell tropism lies within the LTR, gag, pol, and/or vif sequences of the SIVMAC genome.

Peripheral blood mononuclear cells in the squirrel monkey Saimiri sciureus: Characterization and functional aspects of T lymphocytes

Characterization and functional aspects of squirrel monkey peripheral blood mononuclear cells (PBMC), and mainly T cells, are described in the present paper; this should enable the study of cellular immune responses in an experimental model for malaria. PBMC were obtained from Ficoll-Hypaque gradient separation and fractionated into T cells and non-T cells by means of E-rosetting techniques and adherence to plastic dishes. PBMC subset phenotypes were characterized by means of monoclonal antibodies (mAb) directed against human leukocyte differentiation antigens (Ag), fluoresceinated lectins, anti-surface Ig (squirrel-monkey-specific) antibodies (Ab) and latex bead ingestion assays. PBMC functions were assayed through lymphoblastic transformation tests (LTT) in the presence of either numerous mitogenic, co-mitogenic and anti-mitogenic lectins or anti-human leukocyte differentiation Ag mAb. We sought to standardize reference values for lymphocyte phenotypes and functions in normal squirrel monkeys (prior to experimental infection). We also present evidence that splenectomy (generally rendered necessary for experimental human malaria infection) performed six months prior to the present investigation did not modify PBMC numbers and functions in the tested animals.

Natural killer cells in rhesus monkeys: Properties of effector cells which lyse Raji targets

Spontaneously occurring natural killer cell activity of rhesus monkey peripheral blood mononuclear cells was assayed against five human cell lines, three of which were Epstein-Barr virus (EBV) positive, including the human B cell line Raji. The lytic activity to Raji cells was high, significantly higher than to any other cell line tested. Raji cells are normally insensitive to spontaneous lysis by human NK cells, and the contrasting vigor of the rhesus monkey cytolytic activity to Raji prompted us to investigate the properties of this effector cell. We found the effector cell-mediating lysis of Raji to be nonadherent and phagocytic with lytic activity slightly enhanced in the E-rosette-forming cell (ERFC +) fraction and decreased in the ERFC − fraction. Further isolation of FcIgG receptor-positive and FcIgG receptor-negative subsets by rosetting resulted in significant enrichment of NK activity to Raji in the positive fraction and a loss of activity in the negative fraction. Depletion studies with various monoclonal antibodies (mAb's) confirmed that nearly all lytic activity was contained in the CD16 + (Leu 11b +) population, while subsets of effector cells expressed CD2 (9.6) and CD8 (OKT8). Depletion of CD4 (OKT4)-, HLADR (OKIa)-, or LFA1 (MAC-1)-positive populations failed to reduce NK activity. We compared the phenotypic properties of alloimmune effector cells exhibiting specificity for allogeneic donor targets with those exhibiting lysis of Raji targets. Results indicated that allospecific cytotoxic T lymphocytes expressed a CD16 −, CD2 + phenotype, a pattern distinct from that of the effector cell population recognizing Raji targets. The presence of CD2 mAb's in the culture had no effect on NK lytic activity. In contrast, mAbs CD8 and Leu 11b were inhibitory. This would suggest a functional role for CD8 and FcIgG molecules in the lysis of Raji cells by rhesus effectors. In summary, these studies describe a distinct population of effector cells in the blood of rhesus monkeys which exhibit spontaneous lytic activity to Raji cells and exhibit the properties of NK cells.

Effect of long-term feeding of corn oil or coconut oil diets on immune response and prostaglandin E 2 synthesis of squirrel and cebus monkeys

The in vitro and in vivo immune response was investigated in adult squirrel and cebus monkeys fed corn oil (CORN) or coconut oil (COCO) diets (31% calories as fat) since birth. The in vitro blastogenic response of lymphocytes cultured in calf serum or in autologous serum to T- and B-cell mitogens was compared between the two species fed the two types of fat. For in vivo measurements of immune competence, all monkeys received measles vaccine and their serum antibody titers were measured by hemagglutination inhibition assay along with measurement of IgG, IgM and IgA. Prostaglandin E 2 synthesis by peripheral blood mononuclear cells (PBMC) was measured following stimulation with calcium ionophore. Cells cultured in either calf or autologous serum responded in the same relative fashion to mitogens. However, culturing cells in autologous serum exaggerated the dietary differences found. Cebus monkey lymphocytes were more responsive than squirrel monkeys, but squirrel monkeys fed corn oil had the lowest blastogenic responses to T-cell and B-cell mitogens and lower antibody titers to measles vaccine. Cebus monkey PBMC also generated less PGE 2 than those from squirrel monkeys. The data suggest that genetic variation in phospholipid metabolism, possibly including PGE 2 synthesis, may underlie differences in the immune response to dietary fat unsaturation by these two species of monkeys.

Transformation of owl monkey T cells in vitro with Herpesvirus saimiri.

Owl monkey peripheral blood mononuclear cells were treated with phytohemagglutinin and expanded in interleukin 2 (IL-2)-containing medium. The cells were then exposed to Herpesvirus saimiri (HVS strain S295C)-infected owl monkey kidney monolayer cells. Four to 6 weeks later, the lymphocytes showed increased clumping and cell growth and the ability to grow in the absence of IL-2. Control lymphocyte cultures not exposed to HVS eventually died out at approximately 6-8 weeks, even in the presence of IL-2. Although infected lymphocytes grew continuously in the absence of IL-2, their growth was enhanced by addition of IL-2 to the cultures. Natural killer cell-like cytotoxicity and gamma-interferon release were also enhanced by IL-2. All cultures were positive for HVS antigens, infectious centers, or DNA. The reactivity of monoclonal antibodies to cell surface markers suggested that the resultant cell lines were comprised of activated T cells. The properties of the in vitro-transformed cells were similar to those of cells established from HVS-induced owl monkey tumors. Our results suggest that infection of T lymphocytes with HVS results in decreased dependence of T cells upon exogenous IL-2 for growth.