HTLV-I infection in squirrel monkeys (Saïmiri sciureus) using autologous, homologous, or heterologous HTLV-I-transformed cell lines.

Peripheral blood mononuclear cells (PBMC) from three adult male squirrel monkeys (Saïmiri sciureus) were transformed by human T-cell leukemia/lymphoma virus type I (HTLV-I) by cocultivation with lethally irradiated human MT-2 cells. Three permanent monkey T-cell lines producing HTLV-I were obtained and characterized. Six weeks after inoculation seroconversion was observed in three of three monkeys inoculated with autologous transformed T cells and in two of three monkeys receiving homologous cells. Proviral DNA was detected in their PBMC at various times after inoculation, with the highest proviral load and antibody titers being found in monkeys infected with homologous cells. Monkeys inoculated with heterologous MT-2 cells did not seroconvert, and HTLV-I provirus was detected only transiently in their PBMC. To determine whether in vitro and in vivo HTLV-I infection of squirrel monkey cells led to a selection of monkey-adapted viral mutants, comparative sequencing of the proviral gp21 env between ex vivo monkey HTLV-I-infected PBMC, the inoculum, and MT-2 cells was done and no significant differences were detected. The squirrel monkey, which is naturally free of simian T-cell leukemia/ lymphoma virus, thus appears to be a suitable model for evaluating HTLV-I candidate vaccines and for studying the pathogenesis of HTLV-I.

Immunogenicity and efficacy trials with Plasmodium falciparum recombinant antigens identified as targets of opsonizing antibodies in the naive squirrel monkey Saimiri sciureus.

We have previously shown that Plasmodium falciparum recombinant antigens PfEB200, R23, and Pfi72 consistently inhibit opsonization of infected red blood cells by protective hyperimmune Saimiri sera, indicating that they present target epitopes involved in the phagocytosis of infected red blood cells. We report here an analysis of the immune response elicited in naive squirrel monkeys injected with the individual recombinant antigens or with a mixture of the three antigens combined with a synthetic peptide. In the three administration protocols investigated, there was no evidence for the production of antibody contributing to the phagocytosis of infected red blood cells, contrasting with the increase of opsonizing antibodies elicited by these antigens in monkeys with a prior (> or = 500 days) experience with malaria infection. However, the recombinant antigens were highly immunogenic, inducing specific antibody responses to P. falciparum and to the recombinant antigens. When the monkeys immunized with the antigen combination were challenged with blood-stage parasites, there was substantial protection: three of seven immunized animals self-cured and two others experienced a delayed peak of parasitemia. Taken together with our previous findings, these results suggest that PfEB200, R23, and Pfi72 constitute interesting vaccine candidates, and show that the presence of antibodies promoting phagocytosis of infected red blood cells is not a prerequisite for protection after immunization with these antigens in the Saimiri model.

Immunogenicity of HIV-1LAI gp160 and env peptides in squirrel monkey Saimiri sciureus using alumine and experimental adjuvants.

Since the identification of the HIV virus, important advances have been achieved in the definition of potential subunit vaccines. We investigated the immunogenicity of a recombinant gp160 antigen and of two gp41 peptides from HIV-1LAI associated with seven different adjuvant formulations in squirrel monkeys. All animals were immunized twice with gp160 and then with a gp41 peptide using the same formulation. All adjuvants used led to a subsequent antibody response against gp160, and 55% of the animals immunized developed anti-gp160 antibodies that could neutralize the virus in vitro. Specific anti-gp41 antibody response was also observed. Results obtained underlined the key role of the adjuvant formulation in the antibody response against a given part of the immunogen, and indicate that such immunogenicity-related investigation can be carried out conveniently in the squirrel monkey Saimiri sciureus.

Plasmodium falciparum: genetic diversity of several strains infectious for the squirrel monkey (Saimiri sciureus).

The squirrel monkey, Saimiri sciureus, is a useful experimental host for the human malaria parasite Plasmodium falciparum. Twelve strains of P. falciparum, including monkey-adapted strains, culture-derived strains, and one human isolate were injected into naive, splenectomized Saimiri monkeys of karyotype 14-7. Several parameters were recorded following inoculation such as parasitemia, body temperature, standard hematological parameters, gametocytemia, rosette formation, autoagglutination, as well as HRPI and PfEMP3 expression. Each strain was injected into two to four monkeys and induced a reproducible course of infection. Four distinct patterns of parasite development were observed. For each strain, a multilocus genotype was established by PCR using several polymorphic (Pf60, RESA, RESA2, MSA1, MSA2, Pf332, TRAP, GLURP, CSP, and HRPI) or conserved (EBA175, GARP, MDRI, and RNA POL III) markers. RFLP analysis was conducted for the Pf11.1 locus. This genotyping approach showed that 3 strains presented strictly similar patterns, typical of FUP/SP parasites. A group of 7 other strains presented a highly similar FUP/CP (FCR3-like) genetic background, while 4 other strains showed unique patterns. Infectiousness did not depend on a RESA deletion, as several strains developed successfully while present ng a wild-type RESA gene. Conversely, an interesting correlation was found between allelic diversity at the HRPI locus and the course of blood stage infection. The data presented here provide the first precise genotyping of several monkey-adapted strains, allowing a more rational approach in the study of the role of parasite diversity on host/parasite interactions.

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.

Induction of opsonizing antibodies after injection of recombinant Plasmodium falciparum vaccine candidate antigens in preimmune Saimiri sciureus monkeys.

We have previously shown that Plasmodium falciparum recombinant antigens PfEB200, R23, and Pfi72 inhibit opsonization of infected erythrocytes by hyperimmune Saimiri sera, indicating that they contain target epitopes involved in the phagocytosis of infected erythrocytes. We have investigated in this study the immune response of Saimiri monkeys with previous experience of malaria infections (preimmune monkeys) after injection of these recombinant antigens, administered alone or simultaneously. The humoral response to the recombinant antigens was monitored by radioimmunoassay, and the response to P. falciparum blood stages was assayed by immunofluorescence. The relative proportion of protective versus nonprotective immunoglobulin subtypes was investigated by using 3A2/G6 and 3E4/H8 monoclonal antibodies, and the capacity of the antisera to promote in vitro phagocytosis of infected erythrocytes was evaluated. The antigens evoked in most cases a secondary-type antibody response, resulting in important increases in antigen-specific antibody titers and concomitantly in anti-P. falciparum titers. The ratio of 3A2/G6 to 3E4/H8 immunoglobulin subtypes varied with the immunogen used. Opsonizing antibodies were boosted in several animals, the most promising combination being the mixture of PfEB200 and R23 that induced long-lasting production in five of five animals. The detectable opsonizing activity appearing after immunization of the animals was antigen specific, as it was lost after adsorption of the recombinant antigens. The challenge of the animals with blood stage parasites confirmed previous findings showing a correlation between the presence of detectable opsonizing antibodies in serum and protection.

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.

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.

Assays for adjuvanticity of new formulations and of carrier proteins for inducing antibody responses to selected immunogens in the squirrel monkey Saimiri sciureus.

Different ways to improve antibody (Ab) responses following immunizations with selected antigens (TT and HSVgD) were investigated, and thus new adjuvant formulations and carrier molecules in a non-human primate experimental host, the squirrel monkey Saimiri sciureus, were assayed. Both quantitative and qualitative humoral responses were determined by means of radio-immunoassays using monoclonal Ab directed at Saimiri IgG. First, the adjuvanticity of the Syntex (SAF-1) adjuvant and of five new adjuvant formulations were assessed towards the selected Ag. This indicated that all the adjuvants induced similar antigen-specific Ab responses, although the adjuvants could modify to some extent the pattern of the qualitative Ab response. Second, we evaluated an adjuvant-free vaccine approach using a synthetic Ag from the circumsporozoite protein of Plasmodium falciparum as immunogen, this Ag being coupled to purified protein derivative (PPD) or to a recombinant heat shock protein from Mycobacterium tuberculosis. These constructs led to good antibody responses as well as an excellent memory effect. Bacille Calmette-Guérin (BCG) priming was required in conjunction with PPD as a carrier molecule to allow homogeneous Ab responses, whereas the heat shock protein construct gave a less homogeneous Ab response regardless of whether a BCG priming was done. We, in addition, discuss the relevance of Saimiri monkeys as experimental models for studies directed at evaluating the immunogenicity of further vaccine candidates.

Squirrel monkey (Saimiri sciureus) B lymphocytes: secretion of IgG directed to Plasmodium falciparum antigens, by primed blood B lymphocytes restimulated in vitro with parasitized red blood cells.

Blood B lymphocytes obtained from Plasmodium falciparum-immune Saimiri monkeys were assayed for their in vitro differentiation in immunoglobulin-secreting cells upon restimulation with P. falciparum-parasitized Saimiri red blood cells. Selected culture conditions enabled appropriately stimulated blood B cells to secrete 3F11/G10+ IgG, detected in the supernatants by means of a dot immunobinding assay. Primed blood B lymphocytes from P. falciparum-immune Saimiri monkeys were thus able to secrete IgG when restimulated by parasitized red blood cells in the presence of T cell- and monocyte-derived cytokines (recombinant human cytokines). These primed blood B cells, which were able to differentiate, were shown to secrete antibodies reactive with P. falciparum-infected red blood cells, as detected by means of an indirect immunofluorescence assay, and reactive with P. falciparum-infected red blood cell extracts, as detected by means of Western blot analysis. Furthermore, due to the possibility of discriminating between IgG subtypes in the squirrel monkey (3F11/G10+::3A2/G6+ IgG [associated with protection against the blood stages of P. falciparum] vs. 3F11/G10+::3E4/H8+ IgG [usually not functionally associated with protection]), we have attempted to estimate the respective proportions of each IgG subtype. In defined culture conditions, Saimiri monkey blood B cells preferentially secrete 3F11/G10+::3E4/H8+ IgG in response to parasitized red blood cells. We therefore discuss the conditions that would render this assay suitable for the selection, among P. falciparum blood stage antigens, of those that have major B-cell epitopes.