The prevalence of antibodies to simian T-cell leukaemia/lymphotropic virus (STLV-I) in non-human primate colonies in Kenya.

Retroviruses closely related to the human T-cell leukaemia/lymphotrophic virus type I (HTLV-I) have been detected in several, non-human, primate species. These retroviruses are called simian T-lymphotrophic virus type I (STLV-I). Infection with STLV-I has been associated with lymphoma and leukaemia in macaques, baboons, African green monkeys and gorillas. However, no STLV-I infection has been detected in New World primates, although STLV-II has been detected in spider monkeys. When sera from 10 species of non-human primates maintained at the Institute of Primate Research were screened for STLV-I infection, anti-STLV-I antibodies were detected in 12%, 12%, 23% and 38% of the olive baboons, yellow baboons, African green monkeys and lowland Sykes' monkeys, respectively. Western-blot studies confirmed these results. To date, no clinical disease has been linked with STLV-I infection in these colonies. The relatively high prevalence of anti-STLV-I antibodies in these non-human primates offers an opportunity for studies on the transmission, phylogenetic relationships and natural history of STLV-I in primate colonies.

Characterization of antigens expressed in normal baboon trophoblast and cross-reactive with HIV/SIV antibodies.

Electron microscopic studies have revealed the presence of endogenous retroviral (ERV) particles in normal primate placental tissues. These particles have ultrastructural similarities to type C retroviral particles and are mainly associated with the trophoblast. In normal human placental tissues, they have antigenic similarity with exogenous retroviruses, such as the human immunodeficiency virus (HIV), and may have a role to play in the regulation of cellular gene expression, syncytiotrophoblast formation or pregnancy-related immunosuppression. In this study, a panel of antibodies (polyclonal and monoclonal antibodies) against viral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) proteins were assessed by immunohistochemistry and immunoblotting, for their cross-reactivity with ERV particles isolated from normal baboon placental tissues. The antibodies (anti-HERV-K RT, anti-ERV3 env, anti-HIV-1 p17, anti-HIV-2 gp120) reacted positively with the syncytiotrophoblast and each antibody recognized one or two proteins of molecular weights (MW) 38, 58 or 64 kDa present in the baboon placental villous tissues and SIV-infected molt-4 Cl8 cells, but not in uninfected cells. The results of this study confirm the specific expression of retroviral cross-reactive antigens in normal baboon placental tissues and suggest placental cellular proteins may have antigenic similarity with those recognized by anti-HIV/SIV antibodies. The role of these retroviral-related proteins expressed at the maternal-fetal interface remain unclear.

Ancistrobertsonine A and related naphthylisoquinoline alkaloids from Ancistrocladus robertsoniorum.

From the as yet unexplored East African Liana, Ancistrocladus robertsoniorum, several naphthylisoquinoline alkaloids have been isolated, based mainly on High Speed Countercurrent Chromatography (HSCCC). The structure of the new compound, ancistrobertsonine A, was elucidated by chemical and spectroscopic methods. Furthermore, the known alkaloids ancistrocladine, its atropo-diastereomer, hamatine, and its regioisomer, ancistrobrevine B, were isolated.

Retarded development of exoerythrocytic stages of the rodent malaria parasite Plasmodium berghei in human hepatoma cells by extracts from Dioncophyllaceae and Ancistrocladaceae species.

Retarded development of exoerythrocytic stages of the rodent malaria parasite Plasmodium berghei in human hepatoma cells by extracts from Dioncophyllaceae and Ancistrocladaceae species. International Journal for Parasitology 27: 29-32. Naphthylisoquinoline alkaloid-containing extracts (10 micrograms ml-1) of species belonging to the Dioncophyllaceae and the Ancistrocladaceae, 2 small tropical plant families, display pronounced in vitro activities against exoerythrocytic stages of Plasmodium berghei (Anka), developing in human hepatoma cells (Hep G2). The highest activities were obtained with CH2Cl2 root and bark extracts, and a CH2Cl2/NH3 leaf extract from Triphyophyllum peltatum, a CH2Cl2/NH3 root extract from Ancistrocladus abbreviatus, and a CH2Cl2 leaf extract from A. tectorius. The degrees of growth inhibition ranged within 27.7-70.0%. The commercially available drug primaquine diphosphate (25 micrograms ml-1) caused a comparable effect (62.1%) in the same test system.

Prevalence of antibodies against simian immunodeficiency virus (SIV) and simian T-lymphotropic virus (STLV) in a colony of non-human primates in Kenya, East Africa.

Sera (165 samples in 1988 and 66, follow-up samples in 1989) were collected from olive baboons, African green monkeys, Syke's monkeys and grey mangabeys kept in a semi-free, breeding colony at the Institute of Primate Research (IPR) in Nairobi, Kenya. The levels of antibodies to simian T-lymphotropic virus (STLV) or simian immunodeficiency virus (SIV), and the reactivity patterns of positive sera to various lentivirus subgroup antigens, were then determined. The results of tests using enzyme-immunoassay kits were confirmed by western blots. The prevalence of antibodies which reacted with the Kenyan SIVagm(KEN) isolate was 28% in the African green monkeys tested and 34% in the Syke's monkeys. STLV seroprevalence was 25% in the African greens and 20% in the Syke's. No antibodies to either SIV or STLV were detected in the olive baboons or grey mangabeys. More SIV-positive samples were detected in western blots when SIVagm(KEN) was used as antigen than when SIVagm(CAR014), a geographically distinct isolate from the Central African Republic, was used. However, SIVagm(KEN)-positive sera were more reactive against SIVagm(CAR014) than SIVsmm and SIVmac subgroup antigens, indicating that the two isolates from the African green monkey, CAR014 and KEN, remain antigenetically close even though they were recovered in two geographically distinct regions. To date, no clinical disease has been linked with SIV and STLV infection in the African green or Syke's monkeys in the colony. However, the relatively high prevalence of anti-SIV and anti-STLV antibodies in these monkeys offers an opportunity for prospective studies on the transmission and natural history of both viruses in a single colony.

Reversible contraception in female baboons immunized with a synthetic epitope of sperm-specific lactate dehydrogenase.

In previous experiments, the sperm-specific isozyme of lactate dehydrogenase (LDH-C) had been purified from mouse testes and shown to suppress the fertility of female baboons by 70% compared to controls. Although these results demonstrated the feasibility of this approach for contraceptive vaccine development, it is not practical to purify enough of the protein from natural sources for human use. Therefore, a need exists to develop a contraceptive vaccine based on synthetic peptides. In the current study, baboon LDH-C cDNA was amplified by the reverse transcriptase-polymerase chain reaction technique. The amino acid sequences of human and baboon LDH-C were 99.3% identical, indicating that the human LDH-C would be an effective antigen in nonhuman primates. The immunodominant epitope of human LDH-C was identified, synthesized, and conjugated to diphtheria toxoid (DT). This construct was used to immunize 15 female baboons; 15 control animals were immunized with DT alone. The fertility of the experimental group was reduced by 75% as compared to the controls (p < 0.02). One year after the last immunization, the contraceptive effect was completely eliminated (no statistical difference between the groups). These results show that a synthetic peptide based on the sequence of human LDH-C is effective in preventing pregnancy in nonhuman primates. The effect is completely reversed 1 yr after the last immunization. The contraceptive effect is not related to serum antibody titers, and human LDH-C is only slightly more effective than mouse LDH-C in female baboons.

A distinct African lentivirus from Sykes' monkeys.

Asymptomatic infection with simian immunodeficiency virus (SIV) has been demonstrated in African Sykes' monkeys (Cercopithecus mitis albogularis), and virus isolation confirmed infection with a novel SIV from Sykes' monkeys (SIVsyk). Macaques inoculated with SIVsyk became persistently infected but remained clinically healthy. We utilized polymerase chain reaction amplification to generate a full-length, infectious molecular clone of SIVsyk. The genome organization of SIVsyk is similar to that of the other primate lentiviruses, consisting of gag, pol, vif, vpr, tat, rev, env, and nef. A unique feature is the absence of the highly conserved NF-kappa B binding site in the long terminal repeat. SIVsyk is genetically equidistant from other primate lentiviruses. Thus, SIVsyk represents a new group that is distinct from the four previously recognized primate lentivirus groups: human immunodeficiency virus type 1 (HIV-1), SIV from sooty mangabeys (SIVsmm) and HIV-2, SIV from African green monkeys (SIVagm), and SIV from mandrills (SIVmnd). The genetic differences between SIVsyk and SIVagm, isolates derived from monkeys of the same genus, underscore the potential for other distinct SIVs which have yet to be isolated and characterized.

Evolution of spontaneous endometriosis in the baboon (Papio anubis, Papio cynocephalus) over a 12-month period.

To document the spontaneous evolution of endometriosis, a repeat laparoscopy was performed in 11 baboons after 10 and/or 12 months. The mean number of endometriotic lesions had increased significantly after 10 months (P less than 0.02) because of a high proportion of new lesions (82%). These implants were mainly subtle (67%) and localized on the uterine peritoneum (58%). Progression of endometriosis did not go beyond revised AFS stage I. Additionally, repeat laparoscopy in 10 baboons with an initially normal pelvis showed an endometriosis incidence of 70% after 10 to 12 months. Remodeling of the lesions was apparent in both groups after 12 months. These results suggest that endometriosis is moderately progressive in the baboon. It is possible that multiple laparoscopies could favor the development of endometriosis.

Morphological characteristics of spontaneous endometriosis in the baboon (Papio anubis and Papio cynocephalus).

The histopathology of spontaneous endometriosis was studied on 20 pelvic implants biopsied at laparoscopy in 15 healthy baboons. Endometriosis was confirmed by histopathology in 10 of these animals (66%). Typical (n = 3) and subtle (n = 13) endometriotic lesions were confirmed by histopathology in 100 and 61%, respectively. Suspected disease-bearing lesions (n = 4) were confirmed in 50%. Implants could be classified as active (n = 5), inactive (n = 3), atrophic (n = 2) or stromal endometriosis (n = 3). The histological findings for typical and subtle implants were similar to those reported in humans.

Anti-sperm and anti-ovum vaccines: the selection of candidate antigens and the outcome of preclinical studies.

A clear conceptual basis for anti-sperm and other modes of immunological birth control exists in spite of the limited basic research done on surface antigens of sperm or egg. In the normal physiological condition, the immune system does not respond to sperm, egg or fetus. Compared with anti-egg or anti-fetus immunocontraception, an anti-sperm vaccine has two theoretical advantages. First it would work in both males and females; second, it would not raise problems of autoimmunity in the female if a sperm-specific protein is used. However, for the development of such contraceptive vaccines the following scientific questions need to be resolved: (1) what surface protein to use as an antigen; (2) what adjuvant to use in humans for an anti-fertility vaccine; (3) how to maintain a high titre; (4) how the contraceptive effect will be terminated; and (5) what are the potential side-effects.

PIP: Certain sperm antigens are auto- or isoimmunogenic, and naturally occurring sperm antibodies have been implicated in 10-15% of unexplained human infertility. Thus, efforts to develop an immunocontraceptive for human application have targeted sperm antigens. The World Health Organization Task Force on Contraceptive Vaccines designated the human sperm SP-10 as a primary vaccine candidate. The presence of SP-10 mRNA was demonstrated in baboons and macaques, and it is an ideal primate model for evaluating human efficacy. At a workshop on worldwide research on all monoclonal antibodies against sperm, HS-11 and HS-63 were selected for further evaluation because of their high specificity and significant anti-fertility effects. Isoenzyme LDH-C4 is a primary sperm antigen: it provokes antibodies that are absolutely cell specific and do not cross-react with somatic LDH isozymes. Immunization of both males and females with purified LDH-C4 results in an immune response and antibodies suppress fertility in female mice, rabbits and baboons. The PH-20 protein from guinea pigs has a required function in sperm-zona binding, and a 100% effective contraception is obtained when either male or female guinea pigs are immunized with purified PH-20. Regarding zona pellucida antigens (ZP), the porcine zona pellucida contains the major glycoprotein families identified as ZP1, ZP2 and ZP3. In a human in vitro fertilization system, only antibodies against ZP3 completely suppressed sperm-egg interaction. Passive administration of anti-zona pellucida antibodies results in long-term reversible contraception. The development of antigens capable of evoking an immune response, thereby preventing fertility without ovarian histopathology, is under way. A zona pellucida contraceptive vaccine containing 8-cell, but not T-cell, zona pellucida epitopes may prevent ovarian pathology.

Electron-microscopic localization of baboon acrosomal antigens using antisperm monoclonal antibody.

A sperm antigen corresponding to baboon sperm monoclonal antibody 1A9 was localized in the testis and ejaculated sperm in this animal, using the immunofluorescence technique and immunogold labelling. Immunohistochemical studies of the baboon testis showed that the antigenic determinant was localized in the late spermatid cells and spermatozoa close to the seminiferous tubules. Immunofluorescence studies indicate that the protein was localized on the acrosome region of ejaculated baboon sperm. At the electron-microscopic level, gold particles indicative of the presence of this determinant recognized by 1A9 monoclonal antibody were detected on the inner acrosomal region of ejaculated baboon sperm.

Prevalence and laparoscopic appearance of spontaneous endometriosis in the baboon (Papio anubis, Papio cynocephalus).

The prevalence of spontaneous endometriosis was investigated by laparoscopy in 52 baboons (Papio anubis and Papio cynocephalus) of proven fertility. Clinical endometriosis was diagnosed in 9 (17%) and 4 (8%) baboons with or without a previous hysterotomy, respectively. Endometriosis was confirmed by histology in 75% of these animals. The 37 endometriotic lesions were classified as typical (13%), subtle (57%), or suspicious (30%); and the percentage of histological confirmation was 100%, 61%, and 50%, respectively. Lesions were found on the uterosacral ligaments and in Douglas' pouch (46%), on the uterine peritoneum and the uterovesical fold (38%), and on uterine-omental adhesions (11%). Only 5% of the lesions were localized on the ovarian ligament, whereas ovarian endometriosis was not found. This study for the first time demonstrates that spontaneous endometriosis occurs in healthy baboons with proven fertility. It also shows that the laparoscopic appearances, the histological aspect, and the localization of the pelvic lesions are comparable to those found in women. We therefore conclude that the baboon is a good animal model for the study of endometriosis.

Isolation from African Sykes' monkeys (Cercopithecus mitis) of a lentivirus related to human and simian immunodeficiency viruses.

Analysis of serum samples from 100 wild-caught or colony-born Sykes' monkeys (Cercopithecus mitis) in Kenya revealed that 59 animals had antibodies cross-reactive to human immunodeficiency virus type 2 (HIV-2) and to simian immunodeficiency viruses (SIVs). A lentivirus, designated SIVsyk, was isolated from five of six seropositive asymptomatic Sykes' monkeys, but in four cases isolation was possible only after depletion of CD8+ lymphocytes and cocultivation of the CD4(+)-enriched cell population with peripheral blood mononuclear cells from seronegative Sykes' monkeys. SIVsyk resembled other SIVs and HIVs morphologically, had an Mg2(+)-dependent reverse transcriptase enzyme, and replicated in and was cytopathic for CEMx174 and Sup-T1 cells. SIVsyk differred substantially from other SIVs, however, in that it failed to replicate in normal human, mangabey, and macaque peripheral blood mononuclear cells and serum from seropositive Sykes' monkeys immunoprecipitated env antigens from HIV-1 as well as from HIV-2, SIVsmm, and SIVagm. These data demonstrate a high prevalence of natural infection in Sykes' monkeys in Kenya with a lentivirus that appears to be unique with respect to its host range and antigenic cross-reactivity.

Stage-specific expression of a protein of the acrosome of baboon sperm during spermiogenesis detected with a monoclonal antibody.

A single monoclonal antibody, BSA4, raised against baboon epididymal sperm was used to study the ontogeny of the baboon sperm acrosome region during testicular spermiogenesis. This antibody is not species-specific but is restricted to the acrosome region in all other sperm examined (human, rat, and mouse). In the baboon, treatment of epididymal sperm with 0.05% Triton-X results in complete loss of anterior acrosome staining. Such treated sperm display a distinct equatorial staining. Antibody BSA4 reacts with a determinant (molecular weight, 43,000 d) that first appears in postmeiotic round spermatids during spermiogenesis. When tested for an effect on the fertilization process in vitro, the antibody BSA4 displayed significant inhibition, indicating a possible functional role for the determinant on mouse sperm. Using the avidin-biotin immunoperoxidase method, several stages of acrosome development were recognized: ie, cap, acrosome, and maturation stages of spermiogenesis. The antibody staining was restricted to the developing acrosome at all stages, indicating that the equatorial region is part of the acrosome and is expressed with temporal specificity during spermatogenesis in the baboon.

Monoclonal antibodies to baboon syncytiotrophoblast and cytotrophoblast.

Three monoclonal antibodies (BM2, BM3 and BM4) were obtained from two hybridomas produced after murine immunization with total placental cell dispersion obtained from animals at day 33 of gestation. Immunohistochemical studies showed that antigens recognised by these three antibodies were located on villous trophoblast of fixed placental sections at day 33 of pregnancy. Other fixed normal baboon tissues tested were non-reactive with these antibodies. Monoclonal antibody BM2 was found to stain villous cytotrophoblast; BM3 and BM4 reacted with syncytiotrophoblast but with different staining characteristics. Immunoblot studies showed that BM2, BM3 and BM4 antibodies recognized antigens corresponding to 30.9, 31.3 and 42.3 kDa, respectively.

Monoclonal antibody localization of sperm surface antigen secreted by the epididymis of the baboon (Papio cynocephalus).

A monoclonal antibody (BSA6) was generated against an antigenic determinant secreted by the epididymis of the baboon and present on the acrosomal surface of the spermatozoa. This determinant was first secreted by the principal cells of the proximal corpus region, as determined by fluorescent microscopy performed on Bouin-fixed epididymal tissue sections. The secretory product subsequently bound on the lateral acrosomal surfaces in the distal corpus region, but became uniformly distributed over the acrosomal region in the cauda epididymidis. The antigenic determinant had a molecular weight of 82,000 (western blot technique). The testis, caput and other somatic tissues were devoid of the antigen, indicating the restriction of the antigen to spermatozoa and epithelial cells of the corpus epididymidis. Examination of similar tissue from immature baboons indicated that the secretion of this antigen was age-dependent, secretion beginning at about 4 years of age.

Production and characterization of monoclonal antibodies specific for the transmembrane protein of simian immunodeficiency virus from the African green monkey.

Mouse monoclonal antibodies were produced against simian immunodeficiency virus (SIV) from the African green monkey (SIVAGM). The antibodies reacted with the transmembrane protein of all five SIVAGM isolates but not with those of SIVs from the rhesus macaque and mandrill or of human immunodeficiency virus type 1 or type 2, indicating that they recognize a species-specific epitope strongly conserved in SIVAGM. The transmembrane proteins of several SIVAGM isolates were found to vary in molecular size, even in the deglycosylated form after N-glycanase treatment, indicating heterogeneity of the SIVAGM isolates.

Characterization of baboon testicular antigens using monoclonal anti-sperm antibodies.

Sperm antigens that appear during spermatogenesis in the baboon were identified by using three monoclonal antibodies generated in culture from mice immunized with baboon caudal epididymal spermatozoa. Antibodies BSA1 and BSA2 recognize trypsin-sensitive 84,000 and 45,000 dalton determinants that are restricted to the tail and anterior acrosomal regions of the sperm, respectively, as determined by Western blot and immunofluorescence techniques. The tail antigen absent in 2- and 3-yr-old baboon testes first appears in spermatid cells at about 4 yr of age. In contrast, the acrosomal antigen recognized by BSA2 is present in 3-yr-old primitive testicular germ cells. In the mature testis, the 45,000 molecular weight determinant is predominantly localized in the nucleus of late pachytene spermatocytes and round spermatid cells as observed via the avidinbiotin immunoperoxidase method. Antibody BSA3 reacted only with sailidase-treated sections of adult testis. This trypsin-resistant determinant, not expressed on testicular sperm, is recognized by antibody BSA3 only on epididymal sperm, thus indicating a post-testicular sperm modification.

Serological survey and virus isolation of simian T-cell leukemia/T-lymphotropic virus type I (STLV-I) in non-human primates in their native countries.

Infection with a simian retrovirus (STLV-I) closely related to human T-lymphotropic virus type I (HTLV-I) was investigated in non-human primates living in their native countries in Africa and Asia. Serum antibodies cross-reacting with HTLV-I antigens were detected in 85 of 567 non-human primates of 30 species. Seropositive animals were found among African green monkeys, olive baboons, Sykes' monkeys, mandrills and patas monkeys in several countries in Africa, and cynomolgus monkeys, Celebes macaques and siamangs in Indonesia. The frequency of seropositivity was much higher in adult than in young African green monkeys, cynomolgus monkeys and Celebes macaques. STLV-Is were isolated by establishing II lines of virus-producing lymphoid cells in the presence of interleukin-2 from 5 species of seropositive non-human primates, i.e. the African green monkey, Sykes' monkey, Celebes macaque, cynomolgus monkey and siamang. All these cell lines had T-cell markers and Tac antigen, and the cell lines from the African green monkey and Sykes' monkeys were Leu2a+ while those from other species were Leu3a+. These cell lines expressed viral antigens reacting with human sera from adult T-cell leukemia (ATL) patients and monoclonal antibodies (MAbs) against p19 and p24 of HTLV-I core proteins, and produced virus particles having RNA-dependent DNA polymerase activity. Cellular DNAs from these cell lines contained provirus sequences homologous to HTLV-I, shown by Southern blot hybridization. The restriction patterns of these provirus genomes were different from those of HTLV-I and were also dissimilar in the different species.