Molecular and immunological tools for the evaluation of the cellular immune response in the neotropical monkey Saimiri sciureus, a non-human primate model for malaria research.

BACKGROUND: The neotropical, non-human primates (NHP) of the genus Saimiri and Aotus are recommended by the World Health Organization as experimental models for the study of human malaria because these animals can be infected with the same Plasmodium that cause malaria in humans. However, one limitation is the lack of immunological tools to assess the immune response in these models. The present study focuses on the development and comparative use of molecular and immunological methods to evaluate the cellular immune response in Saimiri sciureus. METHODS: Blood samples were obtained from nineteen uninfected Saimiri. Peripheral blood mononuclear cells (PBMC) from these animals and splenocytes from one splenectomized animal were cultured for 6, 12, 18, 24, 48, 72 and 96 hrs in the presence of phorbol-12-myristate-13-acetate and ionomycin. The cytokine levels in the supernatant were detected using human and NHP cytometric bead array Th1/Th2 cytokine kits, the Bio-Plex Pro Human Cytokine Th1/Th2 Assay, enzyme-linked immunosorbent assay, enzyme-linked immunospot assays and intracellular cytokine secretion assays. Cytokine gene expression was examined through TaqMan® Gene Expression Real-Time PCR using predesigned human gene-specific primers and probes or primers and probes designed based on published S. sciureus cytokine sequences. RESULTS: The use of five assays based on monoclonal antibodies specific for human cytokines facilitated the detection of IL-2, IL-4 and/or IFN-γ. TaqMan array plates facilitated the detection of 12 of the 28 cytokines assayed. However, only seven cytokines (IL-1A, IL-2, IL-10, IL-12B, IL-17, IFN-ß, and TNF) presented relative expression levels of at least 70% of the gene expression observed in human PBMC. The use of primers and probes specific for S. sciureus cytokines facilitated the detection of transcripts that showed relative expression below the threshold of 70%. The most efficient evaluation of cytokine gene expression, in PBMC and splenocytes, was observed after 6-12 hrs of culture, except for LTA in PBMC, whose expression was best analysed after 24 hrs of culture. CONCLUSIONS: Real-time PCR facilitates the analysis of a large number of cytokines altered during malaria infection, and this technique is considered the best tool for the evaluation of the cellular immune response in S. sciureus.

Reactivity of HLADR antibody manifests expression of surface MHC II molecules on peripheral blood T lymphocytes in new world monkeys.

BACKGROUND: Major histocompatibility complex (MHC) class II molecules expressed on B cells, monocytes and dendritic cells present processed peptides to CD4+ T cells as one of the mechanisms to combat infection and inflammation. AIM: To study MHC II expression in a variety of nonhuman primate species, including New World (NWM) squirrel monkeys (Saimiri boliviensis boliviensis), owl monkeys (Aotus nancymae), common marmosets (Callithrix spp.), and Old World (OWM) rhesus (Macaca mulatta), baboons (Papio anubis). METHODS: Two clones of cross-reactive mouse anti-human HLADR monoclonal antibodies (mAb) binding were analyzed by flow cytometry to evaluate MHC II expression on NHP immune cells, including T lymphocytes in whole blood (WB) and peripheral blood mononuclear cells (PBMC). RESULTS: MHC class II antibody reactivity is seen with CD20+ B cells, CD14+ monocytes and CD3+ T lymphocytes. Specific reactivity with both clones was demonstrated in T lymphocytes: this reactivity was not inhibited by purified CD16 antibody but was completely inhibited when pre-blocked with purified unconjugated MHC II antibody. Freshly prepared PBMC also showed reactivity with T lymphocytes without any stimulation. Interestingly, peripheral blood from rhesus macaques and olive baboons (OWM) showed no such T lymphocyte associated MHCII antibody reactivity. DISCUSSION & CONCLUSION: Our results from antibody (MHC II) reactivity clearly show the potential existence of constitutively expressed (with no stimulation) MHC II molecules on T lymphocytes in new world monkeys. These results suggest that additional study is warranted to evaluate the functional and evolutionary significance of these finding and to better understand MHC II expression on T lymphocytes in new world monkeys.

Species-specific inhibition of foamy viruses from South American monkeys by New World Monkey TRIM5{alpha} proteins.

Foamy virus evolution closely parallels that of the host species, indicating virus-host coadaptation. We studied simian foamy viruses (SFVs) from common marmosets, spider monkeys, and squirrel monkeys, New World monkey (NWM) species that share geographic ranges. The TRIM5alpha protein from each of these NWM species inhibited the replication of at least one of the SFVs associated with the other two species but did not affect the replication of its own SFV. Thus, TRIM5alpha has potentially shaped the evolution of SFVs in NWM hosts. Conversely, SFVs may have influenced the evolution of TRIM5 variants in New World primates.

Effects of relocation on immunological and physiological measures in female squirrel monkeys (Saimiri boliviensis boliviensis).

In the present study, we have quantified the effects of transport, relocation and acclimate/adapt to their new surroundings on female squirrel monkey. These responses are measured in blood samples obtained from squirrel monkeys, at different time points relative to their relocation from their old home to their new home. A group of squirrel monkeys we transported, by truck, for approximately 10 hours. Peripheral blood mononuclear cells (PBMCs) were assayed in order to evaluate the phenotype of lymphocyte subsets by flow, mitogen-specific immune responses of PBMCs in vitro, and levels of cytokines at various time points including immediately before transport, immediately upon arrival, and after approximately 150 days of acclimation. We observed significant changes in T cells and subsets, NK and B cells (CD4+, CD8+, CD4+/CD8+, CD16+, and CD20+). Mitogen specific (e.g. PHA, PWM and LPS) proliferation responses, IFN-γ by ELISPOT assay, and cytokines (IL-2, IL-4 and VEGF) significant changes were observed. Changes seen in the serum chemistry measurements mostly complement those seen in the hematology data. The specific goal was to empirically assess the effects of relocation stress in squirrel monkeys in terms of changes in the numbers and functions of various leukocyte subsets in the blood and the amount of time required for acclimating to their new environment. Such data will help to determine when newly arrived animals become available for use in research studies.

Immune pressure selects for Plasmodium falciparum parasites presenting distinct red blood cell surface antigens and inducing strain-specific protection in Saimiri sciureus monkeys.

The passive transfer of specific antibodies to a naive splenectomized Saimiri sciureus monkey infected with the Palo Alto FUP/SP strain of Plasmodium falciparum resulted in the emergence of parasites resistant to the transferred antibodies. Molecular typing indicated that the original and resistant parasites were isogenic. Saimiri monkeys primed with original parasites were fully susceptible to a challenge by the resistant ones, and vice versa. This absence of crossprotection indicates that strain-specific determinants would be the major targets of protective immunity developed in these monkeys. Phenotypic analysis showed that the surface of the infected red blood cells differed in both lines. Original parasites formed rosettes, autoagglutinated, presented characteristic knobs at the surface of the infected red blood cell, and did not agglutinate in the presence of a pool of human immune sera. In contrast, the resistant parasites did not form rosettes, did not spontaneously autoagglutinate, presented abnormal flattened knobs, and formed large aggregates in the presence of a pool of human immune sera. The presence of strain-specific determinants at the surface of the resistant parasites was confirmed by surface immunofluorescence and agglutination using homologous Saimiri serum. Neither the original nor the resistant parasites cytoadhered to an amelanotic melanoma cell line, suggesting that cytoadherence and agglutination can be dissociated. These results indicate that parasites that differ by the antigens exposed at the surface of the red blood cell induce strain-specific immunity. Furthermore they show that rosetting and nonrosetting parasites differ in their antigenic properties and do not crossprotect.

An outbreak of Toxoplasmosis amongst squirrel monkeys in an Israeli monkey colony.

An outbreak of Toxoplasmosis in a colony of squirrel monkeys (Saimiri sciureus) in Israel is described. Serological, pathological, and molecular findings of monkeys, as well as rodents and pigeons from the vicinity are summarized. Seventy-nine percent (19/24) of monkeys were T. gondii seropositive at titer 1:16 whilst 4% (1/24) were also seropositive at titer 1:64 using the Modified Agglutination Test (MAT). Eighty four percent (21/25) of rats were positive at titer 1:16 and 8% (2/25) of rats were positive at titer 1:32. DNA amplification of a 529bp repeated sequence of T. gondii was detected in the liver and lungs of all monkeys tested, 6/7 in myocardial extractions and 5/6 in brain extractions. Sequence analysis of the SAG2 locus disclosed that T. gondii detected was of Type III genotype. The source of disease was thought to be contamination of feed with infective feline oocysts. As a result of this study, the implementation of a program to capture and remove resident feral cats, to discontinue the feeding of stray cats, and to control rodent populations in the park was introduced.

Observations on the sporozoite transmission of Plasmodium vivax to monkeys.

Saimiri boliviensis monkeys were infected via sporozoites with the Salvador I strain of Plasmodium vivax that had been stored frozen for periods ranging from 12 to 5,312 days. Prepatent periods ranged from 16 to 53 days.

Blood typing in Saimiri sciureus monkeys: influence of anti-red blood cell alloantibodies on Plasmodium falciparum parasitaemia in vivo.

The Saimiri sciureus monkey is a well-established host for experimental studies with human malaria parasites. During the course of iterative inoculations with Plasmodium falciparum parasitised red blood cells (RBC), anti-RBC alloantibodies were detected in the sera of two of eight Saimiri monkeys. These anti-RBC antibodies were further used to investigate RBC phenotypes in 35 colony-reared Saimiri monkeys by flow cytometry. Three RBC phenotypes (named I-III) were observed. Their distribution was I (86%), II (11%) and III (3%). Using the Palo Alto FUP-2 strain, a variant P. falciparum line insensitive to hyperimmune serum and the passive transfer of anti-RBC alloantibodies, a dramatic drop in parasite growth was documented in an incompatible monkey.

Flow cytometry identification and characterization of mononuclear cell subsets in the neotropical primate Saimiri sciureus (squirrel monkey).

BACKGROUND: The neotropical primate squirrel monkey is used in many areas of biomedical research including neuroendocrinology, immunology and infectious diseases. However, research has been hampered by the lack of immunological tools for this primate. METHODS: A series of 67 commercially available monoclonal antibodies to human CD antigens or cytokines were tested on Saimiri mononuclear cells and the specificity was assessed by double staining using flow cytometry. RESULTS: Monoclonal antibodies defining the main mononuclear cells subsets (monocytes, B, T, including CD4 and CD8 T cells) as well as activation markers have been identified. The conditions to specifically identify the various cell subsets using two color flow cytometry and establish their relative proportions have been set-up. We also have established normal values of the main circulating mononuclear cell subsets for adult Saimiri sciureus monkeys from the breeding unit of Institut Pasteur in French Guiana. The distribution between spleen, blood and lymph nodes has been compared. CONCLUSIONS: These tools allow documenting the phenotype of most Saimiri mononuclear cell subsets and assessing their activation level. This opens new perspectives for vaccinology and immunopathology research in this experimental non-human primate host, in particular for malaria research.

A two-site sandwich immunoradiometric assay of squirrel monkey (Saimiri sciureus) IgM using monoclonal antibodies.

Nine hybrid clones secreting antibodies to squirrel monkey (Saimiri sciureus) IgM were produced and two of these (1F1G5 and 5H11B3) were selected for further studies. These non-precipitating monoclonal antibodies reacted with two distinct repetitive antigenic determinants, probably of the conformational type, only present on the native or SDS-denatured IgM molecule. Reduction of the pentamer with 2-mercaptoethanol led to complete destruction of the corresponding epitopes. 1F1G5 antibodies from ascitic fluids were used in the purification of monkey IgM by affinity chromatography. The characteristics of 1F1G5 and 5H11B3 MAbs permitted the development of a solid-phase two-site immunoradiometric assay for the measurement of IgM levels in serum specimens taken from healthy animal donors of both sexes.

Measurement of squirrel monkey serum IgG levels by a two-site sandwich radioimmunoassay with monoclonal antibodies.

Monoclonal antibodies against the squirrel monkey Saimiri sciureus IgG have been produced for a more specific analysis of the antibody-related immunological aspects in experimental human or monkey malaria. Two monoclonal antibodies, 3D8/D5 and 3F11/G10, out of 64 reacted with distinct epitopes on the IgG present throughout the complete population without interfering with each other. The 2 monoclonal antibodies were used to develop a highly specific, reliable and sensitive two-site sandwich radioimmunoassay for the measurement of the serum IgG levels in 83 animals. The antibodies also allowed us to produce by a simple immunoabsorbent technique a highly purified IgG standard easy to calibrate and store. The assay permits the detection of IgG levels as low as 0.48 ng/ml. The standard curve is linear between 3.9 and 125 ng protein/ml and allows by a simple mathematical equation an accurate measurement of the serum IgG levels.

Ascites production in the squirrel monkey (Saimiri sciureus).

A method is described for inducing the production of large amounts of ascitic fluid (AF) in the squirrel monkey Saimiri sciureus. The total amount of protein in the induced AF is close to 60% of that in the serum. Electrophoretic analysis of serum and AF samples from the same monkey revealed similar protein patterns, including gamma globulins. Antibody titers against Plasmodium falciparum in infected monkeys, measured by indirect immunofluorescence, were also comparable in serum and AF.

Manipulating blood T cells and B cells from squirrel monkeys: some technical considerations.

The squirrel monkey Saimiri sciureus is an experimental host for a range of human pathogens, and for the assessment of vaccine candidate antigens and vaccine strategies. This experimental host is thus particularly suitable for the follow-up of humoral responses. To understand some of the mechanisms that underlie the defense against experimental pathogens, there is a need of basic knowledge on cellular immune effectors also. The authors report here their experience in characterizing squirrel monkey blood T and B lymphocytes, and in studying in vitro induced activation and proliferation of T and B cells. Particular emphasis is given to the in vitro differentiation of squirrel monkey B cells into immunoglobulin secreting cells, with respect to Plasmodium falciparum antigens.

Antigens linked to synthetic microspheres induce immune responses in primates in the absence of adjuvant.

Although most strategies of vaccination require immunopotentiation to induce efficient immune responses, the development of new adjuvants for human vaccines is highly limited by safety problems. In order to overcome this problem, we developed a new vaccine formulation based on the covalent linkage of protein or peptide to synthetic microspheres. In previous experiments performed in mice, we demonstrated that these particulate antigens induce strong antigen-specific CD4+ T cell proliferative responses in the absence of adjuvant. In the present study, we analyzed the immunogenicity in primate Saimiri sciureus monkeys of two different proteins linked to synthetic microspheres. Immune responses induced by these particulate proteins administered without adjuvant were compared to those stimulated by the soluble antigens injected with alum. We currently demonstrated that, in monkeys, particulate antigens administered without adjuvant, induced good PBMC proliferative response and antibody production. Furthermore, the analysis of antibody responses using mAbs specific for different Saimiri sciureus immunoglobulins showed that the antibody response profiles were different in monkeys immunized with soluble versus particulate form of antigens. Results of this study demonstrate that particulate form of antigen may stimulate qualitatively different immune responses as compared to alum and therefore suggest that this new antigen formulation could be an attractive candidate for the development of vaccines.

Cytokine secretion in squirrel monkeys.

The squirrel monkey, a non-human New World primate, has several endocrine peculiarities, including a 10-fold higher plasma cortisol concentration than Old World primates, such as man. Glucocorticoids are known to have immunomodulatory properties. We therefore measured cytokine levels in supernatants of in vitro cultures of mononuclear cells from the peripheral blood of squirrel monkeys and humans. We stimulated monocytes and lymphocytes with lipopolysaccharide (LPS) and phytohemagglutinin (PHA) in the presence or absence of hydrocortisone. Squirrel monkey monocytes secreted a more than 100-fold lower level of interleukin-1 beta (IL-1 beta) but a four-fold higher level of transforming growth factor beta (TGF-beta) than human monocytes, whereas the secretion of other cytokines, such as tumor necrosis factor alpha (TNF-alpha), TNF-beta and interleukin 2 (IL-2), did not differ between squirrel monkeys and humans. However, in squirrel monkey lymphocytes, the PHA-stimulated secretion of TNF-alpha was much greater than that of TNF-beta. Our results support the view that in squirrel monkeys there are subtle adaptations in some immune functions, particularly linked to the hypothalamic-pituitary-adrenal (HPA) system rather than a global suppression of the immune system.

Heteroimmunization of squirrel monkeys (Saimiri sciureus) with a purified porcine zona antigen (PPZA): immune response and biologic activity of antiserum.

The potential for utilization as a contraceptive vaccine of a 60,000 Mr glycoprotein component, purified porcine zona antigen (PPZA), isolated from porcine zonae, was investigated in the squirrel monkey. Immunization resulted in production and maintenance of high antibody titers for at least 1 year. Comparable immune profiles were obtained using either monkey or pig zonae in assay systems, but dose-dependent variations in immune response were not observed. In situ antibody binding to monkey zonae was detected, but significantly fewer ovulated eggs were obtained from immunized monkeys than from controls. Exposure of antibody-pretreated pig, monkey, and human zonae to homologous sperm resulted in total inhibition of sperm attachment for the respective species. Thus, the contraceptive potential of PPZA antibodies in these species is demonstrated.

Topographical study of the neurons containing hpGRF immunoreactivity in monkey hypothalamus.

Hypothalamic neurons producing growth hormone-releasing factor (GRF) have been characterized by immunohistochemistry in monkey hypothalamus, using an antiserum raised against hpGRF1-40, a peptide with GRF activity isolated from a human pancreatic tumor. Cell bodies with hpGRF immunoreactivity were found in arcuate and ventromedial nuclei. From these neurons, bundles of fibers innervate median eminence and appear to terminate in contact with portal vessels. In addition to median eminence, hpGRF immunoreactive fibers were found mostly in the anterior hypothalamus and the arcuate and ventromedial nuclei where they give perineuronal endings. These results correlate with earlier physiological data on hypothalamic control of growth hormone secretion and suggest that GRF is also involved in interneuronal relationships related or unrelated to neurohumoral control of pituitary secretions.

Variations in the immune response to Herpesvirus saimiri in squirrel and rhesus monkeys.

Humoral and cell-mediated immunity (CMI) to herpesvirus saimiri (HVS), an oncogenic lymphotropic herpesvirus, was studied in squirrel and rhesus monkeys. Natural antibody to HVS was found in five of six squirrel monkeys but there was no evidence of specific CMI directed against HVS. Rhesus monkeys did not show natural antibody or CMI against HVS antigens. Immunization with HVS, however, produced both antibody and specific CMI in the rhesus monkeys, but no CMI developed in the squirrel monkeys. These findings are important in the development of animal models for the treatment of tumors associated with lymphotropic herpesviruses.

Sequencing and analysis of genomic DNA and cDNA encoding TNF-alpha in the squirrel monkey (Saimiri sciureus).

If a number of cytokines and growth factors that have been characterized from human cells were investigated in non-human primates, results from such approaches would allow the development of assays to detect and quantitate cytokines in experimental models. Tumor necrosis factor-alpha (TNF-alpha) is an important pluripotent cytokine which plays a crucial role in host defense. As yet, no complete molecular data have been reported for the squirrel monkey TNF-alpha. Polymerase chain reaction (PCR) primers were used to trace introns, by comparing product sizes obtained using cDNA and genomic DNA as templates. The genomic DNA is composed of four exons and three introns with 1793 nucleotides. The corresponding cDNA is 702 nucleotides and phylogenetic analysis showed that the Saimiri sciureus was most closely related to that of the genus Aotus, a new-world primate, compared to old-world primates (genus Macaca and Papio). The deduced TNF-alpha protein consists of 233 amino acids with 82% identity to human, 95% to new-world monkeys and 79% to old-world monkeys. The cloned TNF-alpha cDNA will be useful to quantitate TNF-alpha at the mRNA level.

Presence of anti-ovalbumin IgE antibody in the sera of laboratory-reared squirrel monkeys (Saimiri sciureus) fed quail eggs.

In this study, we examined serum anti-ovalbumin (OVA) IgE and IgG antibodies in laboratory-reared squirrel monkeys (Saimiri sciureus), that were fed a boiled quail egg everyday. We found that 36 of 95 monkeys (38%) possessed specific IgE and 44% (42/95) had specific IgG against OVA. These antibody titers seemed to increase with age. There was, however, no apparent correlation between the anti-OVA IgE and IgG antibody titers.