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A statistical generalizability analysis gauges the degree to which a single assessment of a parameter successfully estimates that measure over repeated assessments for that individual. The generalizability of enumerative and functional immune parameters was estimated for two species of macaque monkeys assessed every 3 months between 18 and 42 months of age. Subjects were cross-balanced by species (bonnet, Macaca radiata, n=22; pigtail, Macaca nemestrina, n=21), sex (male, n=21; female, n=22), and brief early maternal separation with reunion (control, n=21; separated, n=22). Cell subset analysis showed the best generalizability (35-69%). Natural cytotoxicity also performed well (44-70%), but when computed on a lysis per cell basis, removing the effect of cell phenotype, it was less stable (15-48%). For most immune parameters, at least 5 assessments would be necessary to establish conventionally reliable (0.80) characterizations of long-term, stable individual differences in immunity, and three for minimally reliable (0.60) characterizations. More reactive parameters, as well as more behaviorally reactive species, yielded more generalizable results. Cell subsets that are typically most sensitive to acute stress (CD8, CD16) were more stable than other subsets (CD4, CD20). Behaviorally reactive species (pigtail) yielded more stable natural cytotoxicity results than the less reactive species (bonnet). Sex and rearing condition (early, brief maternal separation) did not substantially affect generalizability, although females tended to generate more stable results than did males.

Background: The human sperm-associated antigen 9 (hSPAG9) is of special interest attributing to the findings indicating that SPAG9 is an acrosomal molecule. SPAG9 is not only restricted to acrosomal compartment but also persists in equatorial segment post-acrosome reaction, which is a key location in sperm-egg interaction., Methods and Results: Immunogenicity studies in macaques were carried out with recombinant hSPAG9 (rhSPAG9) adsorbed on alum, which resulted in high titres of anti-rhSPAG9 antibodies as determined by enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis employing anti-rhSPAG9 antibodies generated in monkeys indicated that antibodies specifically reacted with native SPAG9 from macaque and human sperm and rhSPAG9 protein. Furthermore, indirect immunofluorescence experiments demonstrated SPAG9 localization in the acrosomal compartment of macaque and human sperm. In addition, monkey antibodies against rhSPAG9 significantly inhibited the human spermatozoa adherence or penetration in zona-free hamster oocytes., Conclusion: These results demonstrate that rhSPAG9 adsorbed on alum is highly immunogenic in subhuman primate model and therefore represents a suitable sperm-based vaccine immunogen for fertility trials in macaque.

Bonnet monkeys develop an enhanced disease after immunization with the formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine that is characterized by increased viral replication in perivascular sites of the lung. These sites contain many mononuclear cells, which are known to be permissive for RSV replication. To test the hypothesis that FI-RSV vaccine stimulates the production of enhancing antibodies that serve to increase the replication of RSV in macrophages, in vitro studies were done. Antibody-dependent enhancement was observed in animals immunized with FI-RSV but not in control animals with primary and tertiary infections or those immunized with FI-Vero cell culture. In the presence of serum samples from animals immunized with FI-RSV, an increased number of U937 cells was infected. The enhancement index correlated positively with the pathologic scores of the FI-RSV-vaccinated monkeys.

Sperm agglutination antigen-1 (SAGA-1) is a human male reproductive tract glycoform of CD52. Unique modification of CD52 N-linked oligosaccharide chains in the epididymis and vas deferens results in the appearance of a carbohydrate epitope that is localized over the entire surface of human spermatozoa. SAGA-1 was characterized by the sperm-inhibitory murine monoclonal antibody (mAb) S19, and it is the target antigen of a human mAb (H6-3C4) associated with antibody-mediated infertility. Collectively, sperm surface localization, antibody inhibition of sperm function, and potential reproductive-tissue specificity identify SAGA-1 as an attractive candidate contraceptive immunogen. To establish an animal model for the study of SAGA-1 in immunologic infertility and immunocontraceptive development, we investigated the appearance of the S19 carbohydrate epitope in nonhuman primates. The S19 mAb demonstrated little to no immunoreactivity by Western blot analysis with protein extracts of spermatozoa from the baboon, marmoset, bonnet, cynomolgus, and pigtailed macaques. Immunohistochemical analysis identified CD52 in the bonnet monkey epididymis; however, the N-linked carbohydrate moiety recognized by the S19 mAb, and unique to SAGA-1, was absent. In contrast, the S19 carbohydrate epitope was identified in chimpanzee sperm extracts by Western blot analysis and in chimpanzee epididymal tissue sections by immunohistochemical analysis, indicating that it is conserved in this close relative of the human. Chimpanzee testis, seminal vesicle, and prostate do not express the S19 epitope. Although anti-CD52 immunoreactivity was identified in the spleen, the carbohydrate moiety recognized by the S19 mAb was absent, corroborating data in the human that demonstrated tissue-specific glycosylation of sperm CD52. Immunofluorescent analysis indicated that the chimpanzee homologue of sperm CD52 was present over the entire spermatozoon. In addition, the S19 mAb agglutinated chimpanzee spermatozoa in a manner similar to the effect observed on human spermatozoa. These data indicate that the distinctive carbohydrate moiety of human sperm CD52 is present in the chimpanzee, and they identify the chimpanzee as the most appropriate primate model to study the potential of this unique CD52 glycoform as a contraceptive immunogen.

The following synthetic peptides were made as immunogens for development of a zona-based contraceptive vaccine: P1, KQPFWLLQGGASRAETSVQPVLVE [amino acids (aa) 23-45 with an additional K at the N-terminus]; P2, FSEEKLVFSLRLMEENC (aa 164-179 with an additional C at the C-terminus and T170 replaced by V); and P3, CSFSKSSNSWFPVEGPADICQCC (aa 300-322). The aa are numbered on the basis of bonnet monkey ZP3 precursor protein. Antibodies against an additional peptide P4, KGDCGTPSHSRRQPHVVSQWSRSA (aa 324-347), significantly inhibits human sperm-oocyte binding. In addition, antibodies against cocktail of peptide-diphtheria toxoid conjugates also significantly inhibit the binding of spermatozoa to zona pellucida in a hemizona assay. These results will further help in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to ZP3.

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for immunocontraception. Studies on this potential use can be facilitated by the availability of recombinant proteins. A cDNA lambda gt11 library was constructed using poly(A)+ mRNA isolated from bonnet monkey (Macaca radiata) ovaries and was screened for bonnet monkey ZP1 using a 404-basepair (bp) human ZP1 fragment (nucleotides 818-1221) as probe. Bonnet monkey ZP1 cDNA comprises 1617 nucleotides and encodes a polypeptide of 539 amino acid residues that share 92.0% identity with human ZP1. The major difference between bonnet monkey ZP1 and human ZP1 is the deletion of a 28-amino acid domain (amino acid residues 100-127 corresponding to human ZP1). An internal fragment (1317 bp) of bonnet monkey ZP1, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by polymerase chain reaction. The amplified Sac I and Kpn I restricted fragment was cloned in a frame downstream of the T5 promoter under the lac operator control for expression in the pQE-30 vector. Recombinant ZP1 (r-ZP1) was expressed as a polyhistidine fusion protein in Escherichia coli strains SG13009[pREP4] and ompT and Ion protease-deficient BL21 (plysS). SDS-PAGE analysis and immunoblotting with a murine monoclonal antibody, MA-410 (raised against porcine ZP3alpha--a homologue of bonnet monkey ZP1--and cross-reactive with bonnet monkey zona pellucida), revealed major bands of 51 and 40 kDa besides truncated fragments. Optimum expression of r-ZP1 was observed at 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG). Immunization of male rabbits with r-ZP1 purified on nickel-nitrilotriacetic acid (NTA) resin under denaturing conditions and of female rabbits with r-ZP1 conjugated with diphtheria toxoid-generated antibodies reactive with r-ZP1 in ELISA. Moreover, immune sera, when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The information on the sequence of bonnet monkey ZP1 and the availability of the recombinant protein will help toward better understanding and evaluation of the contraceptive potential of homologous immunization in a nonhuman primate model.

Individual differences in the response to maternal separation in nonhuman primate infants have been attributed to (among other variables) presence or absence of processes that may model social support in humans. Alternative attachments to other members of the social group buffer the infant against a depressive response to maternal separation. This hypothesis was tested in a group of bonnet macaques by manipulating the presence or absence of alternative juvenile attachment figures (friends) during separation. Infants who retained such attachments showed fewer behavioral evidences of depression when separated from their mothers. These infants without friends also showed changes in lymphocyte activation by mitogens or natural cytotoxicity that were not evident in the infants with juvenile friends. Across all separated infants, natural cytotoxicity was positively correlated with juvenile affiliative behavior directed toward the infants during the separation. These results support the hypothesis that social support, available from alternative attachments, can modulate the response to loss, and can account for some of the individual differences seen in these responses.

The prevalence of human T-lymphotropic virus (HTLV) type-1 antibodies was determined in the bonnet monkeys, living naturally, within about 30 km radius of Vellore (south India). Sera from 157 animals, collected between January 1982 and May 1993 were screened for the presence of HTLV-I infection by a particle agglutination test (PAT). When sera repeatedly reactive in PAT were subjected to indirect immunofluorescence and western blot tests, 63 (40%) were confirmed to be positive for HTLV-1 antibody. These findings are significant in the light of recent reports that HTLV infection is endemic to southern India.

A panel of immune parameters (lymphocyte activation by mitogens, natural cytotoxicity, and differential cell counts) was assessed in socially housed pigtail and bonnet macaques 1 and 2 weeks before, 48 h after, and 1 and 2 weeks after a competitive water test. Species differences were found in both baseline measures and the responses to the test: Immune measures observed during baseline periods were lower in pigtail macaques. Furthermore, only the pigtail macaques showed changes in mitogen activation and cytotoxicity at 48 h post-test. Dominance-related behaviors affected these responses both within and across social groups. The species differences may be accounted for by the differences in the behavioral responses of the two species to the test: Pigtail macaques consistently contested access to the water during the test, whereas bonnet macaques did not. These results suggest that the immune system can be modulated by psychosocial behavioral systems, particularly during times of stress.

Cross-protection between Japanese encephalitis (JE) and West Nile (WN) viruses was tested in bonnet macaques (Macaca radiata) immunized either with JE virus (JEV) or WN virus (WNV). JEV immunized monkeys were challenged by intranasal (i.n.) route with WNV and vice versa. Four control unimmunized monkeys were similarly infected either with WNV or JEV. Two of three control monkeys infected with WNV, developed paralysis followed by death. Virus was recovered from the central nervous system (CNS) of the both dead control monkeys and the histopathological examination of CNS revealed changes suggestive of viral encephalitis. The control monkey infected with JEV developed encephalitis and the virus was recovered from the blood and CNS. All the 3 JEV-immunized monkeys withstood WNV challenge, whereas only 2 of the 5 WNV immunized monkeys withstood the challenge with JEV. Out of 3 WNV-immunized monkeys surviving challenge with JEV, 2 revealed symptoms suggestive of mild encephalitis followed by complete recovery. The third monkey died on the 60th day post-infection (p.i.) without any symptoms and virus was recovered only from the olfactory lobe. These studies indicate that the immunization with JEV protects the bonnet macaques against WNV, whereas the WNV immunization only reduces the severity of the disease due to JEV.

An enzyme immunoassay (EIA) for the detection of rotaviral antibodies was developed, using a purified, cell culture-grown SA 11 viral antigen and alkaline phosphatase as an enzyme label. This technique was evaluated by comparative testing with tube neutralization and complement-fixation assays on a collection of simian sera. There was close correlation between positive and negative results obtained by EIA and by neutralization. The EIA was as easy to perform as complement fixation testing, but showed greater sensitivity and fewer nonspecific reactions. Thus, EIA was shown to be a very suitable test for routine detection of rotaviral antibodies in serum. Results of neutralization tests suggested that the monkeys (mostly rhesus macaques) in the present study were infected with viruses varying in their antigenic relatedness to SA 11 virus and to a British isolate of calf rotavirus.

In Southern India the prevalence of neutralising antibody to Berne virus was high in sera obtained from cattle (49%), horses (38%), and sheep (36%). Neutralising antibody was not detected in sera from humans and monkeys.

Five healthy female bonnet monkeys (Macaca radiata) of proven fertility and normal menstrual cyclicity have been actively immunised with the purified chicken egg white riboflavin carrier protein (cRCP). All the immunised animals exhibited specific antibodies to cRCP and the immunopotencies of their sera varied from 200 to 840 micrograms/ml at equivalent point. A definite fraction of antibodies in these sera specifically recognised the purified and 125I-labelled monkey RCP. Immunisation per se had no adverse effect on the animals' menstrual cyclicity, circulating levels of estrogen and progesterone and the riboflavin status as reflected by glutathione reductase activities and the total flavin contents of the erythrocytes. The fertility of these animals was monitored for a period extending up to 3 years after primary immunisation. Four out of the five animals exhibited termination of pregnancy once or more than once depending on their antibody titers. Towards the end of the study period, when the immune response was poor, all the animals delivered normal babies at term. Circulating anti-cRCP antibodies were monitored by 125I-labelled cRCP binding. The results show that pregnancy termination, owing to immuno-neutralisation of monkey RCP, occurred only in animals which had sufficiently high antibody titers. If the titers fell below a critical threshold level the pregnancies were carried to term.