Evaluation of the safety and immunogenicity of Plasmodium falciparum apical membrane antigen 1, merozoite surface protein 1 or RTS,S vaccines with adjuvant system AS02A administered alone or concurrently in rhesus monkeys.

In an effort to broaden the immune response induced by the RTS,S/AS02(A),vaccine, we have evaluated the immunogenicity of the RTS,S antigen when combined with MSP1(42) and with AMA1, antigens derived from the asexual blood stage. The objectives of this study were (i) to determine whether MSP1(42) and AMA1 vaccines formulated with the AS02(A) Adjuvant System were safe and immunogenic in the rhesus monkey model; (ii) to investigate whether MSP1(42) or AMA1 induced immune interference to each other, or to RTS,S, when added singly or in combinations at a single injection site; (iii) in the event of immune interference, to determine if this could be reduced when antigens were administered at separate sites. We found that MSP1(42) and AMA1 were safe and immunogenic, eliciting antibodies, and Th1 and Th2 responses using IFN-gamma and IL-5 as markers. When malaria antigens were delivered together in one formulation, MSP1(42) and RTS,S reduced AMA1-specific antibody responses as measured by ELISA however, only MSP1(42) lowered parasite growth inhibitory activity of anti-AMA1 antibodies as measured by in vitro growth inhibition assay. Unlike RTS,S, MSP1(42) significantly reduced AMA1 IFN-gamma and IL-5 responses. MSP1(42) suppression of AMA1 IFN-gamma responses was not seen in animals receiving RTS,S+AMA1+MSP1(42) suggesting that RTS,S restored IFN-gamma responses. Conversely, AMA1 had no effect on MSP1(42) antibody and IFN-gamma and IL-5 responses. Neither AMA1 alone or combined with MSP1(42) affected RTS,S antibody or IFN-gamma and IL-5 responses. Immune interference by MSP1(42) on AMA1 antibody responses was also evident when AMA1, MSP1(42) and RTS,S were administered concurrently at separate sites. These results suggest that immune interference may be complex and should be considered for the design of multi-antigen, multi-stage vaccines against malaria.

Preclinical evaluation of the safety and immunogenicity of a vaccine consisting of Plasmodium falciparum liver-stage antigen 1 with adjuvant AS01B administered alone or concurrently with the RTS,S/AS01B vaccine in rhesus primates.

Several lines of evidence suggest that targeting pre-erythrocytic-stage parasites for malaria vaccine development can provide sterile immunity. The objectives of this study were (i) to evaluate preclinically the safety and immunogenicity of a new recombinant pre-erythrocytic-stage antigen, liver-stage antigen 1 (LSA1), in nonhuman primates; and (ii) to investigate the potential for immune interference between LSA1 and the leading malaria vaccine candidate, RTS,S, by comparing the immune responses after single-antigen vaccination to responses after simultaneous administration of both antigens at separate sites. Using a rhesus monkey model, we found that LSA1 formulated with the GlaxoSmithKline proprietary adjuvant system AS01B (LSA1/AS01B) was safe and immunogenic, inducing high titers of antigen-specific antibody and CD4+ T-cell responses, as monitored by the production of interleukin-2 and gamma interferon, using intracellular cytokine staining. RTS,S/AS01B vaccination was well tolerated and demonstrated robust antibody and moderate CD4+ T-cell responses to circumsporozoite protein (CSP) and HBsAg. Positive CD8+ T-cell responses to HBsAg were detected, whereas the responses to CSP and LSA1 were negligible. For both LSA1/AS01B and RTS,S/AS01B, no statistically significant differences were observed between individual and concurrent administration in the magnitude or duration of antibody and T-cell responses. Our results revealed that both pre-erythrocytic-stage antigens were safe and immunogenic, administered either separately or simultaneously to rhesus monkeys, and that no significant immune cross interference occurred with concurrent separate-site administration. The comparison of the profiles of immune responses induced by separate-site and single-site vaccinations with LSA1 and RTS,S warrants further investigation.

Effects of hydroxypyridinone iron chelators in combination with antimalarial drugs on the in vitro growth of Plasmodium falciparum.

Using standard in vitro drug susceptibility methods, we assessed the antimalarial activity of 3 orally administered iron chelators (hydroxypyridinones) alone and in combination with conventional antimalarials drugs (quinine, mefloquine, artesunate, tetracycline, atovaquone) against a chloroquine-resistant Plasmodium falciparum isolate. When tested alone, all iron chelators and antimalarial compounds inhibited the growth of the parasites. IC50 values for iron chelators were 60-70 microM, whereas the IC50 values for antimalarial drugs were in nM ranges, with artesunate being the most potent. The derived isobolograms for the interaction of hydroxypyridinones and antimalarial drugs showed addition or mild antagonism, similar to desferroxamine (Sum of Fractional Inhibitory Concentration, sigma FIC < 0.5 or > 4.0). Despite the absence of synergy with conventional drugs, intrinsic antimalarial activity of hydroxypyridinones supports the continued assessment of these iron chelators as treatment adjuncts.

Isolation and characterization of rhesus blood dendritic cells using flow cytometry.

Recognition of dendritic cells (DCs) as initiators and modulators of immune responses and growing use of rhesus monkeys for the preclinical optimization of vaccine formulations prompted characterization of the phenotype and function of isolated rhesus peripheral blood DCs. We developed a flow cytometric method to directly identify and isolate DCs from rhesus peripheral blood whereby a T cell depleted population negative for CD3, CD14, CD16 and CD20 but positive for CD83 yielded a cell population with surface markers, morphology, and a cytokine profile similar to human myeloid DCs. Rhesus blood DCs were more effective than monocytes and B cells in mixed lymphocyte reactions and in the presentation of recombinant malaria blood stage antigen MSP-1((42)) to autologous T cells. The ability to isolate rhesus blood DC from peripheral blood should be a useful tool for immunological investigations.

Whole blood cultures to assess the immunostimulatory activities of CpG oligodeoxynucleotides.

Specially designed oligodeoxynucleotide (ODN) sequences known as 'CpG' ODNs elicit innate and acquired immune responses. In general, screening of new CpG ODNs has been conducted by conventional lymphoproliferative assays or expression of activation markers in peripheral blood mononuclear cell (PBMC) cultures. Here, we compared conventional in vitro human PBMC assays with whole blood assays for screening the immunostimulatory properties of CpG ODNs. Commercially available DNA preparations and mycobacterial-based adjuvants were used as comparators. Activation was assessed by flow cytometry and cytokine production. CpG ODNs, identified by four-letter codes, consisted of 2006 (strong human cell stimulant), 1826 (strong murine cell stimulant), 1840 (weak immunostimulant), and 2041, a non-CpG ODN. In both test systems, and in accordance with previous reports, 2006 was an effective up-regulator of CD40 on human dendritic cells (DC1, DC2), monocytes, and B cells, and of CD69 on NK cells. In contrast to murine cells exposed to CpG ODNs, IL-12 (p40) and IFN-gamma production in human immune cells was negligible, but greatly enhanced by adding GM-CSF. Like 2006, two comparator mycobacterial adjuvant formulations activated DC1, DC2, monocytes and natural killer (NK) cells, but only 2006 had a strong effect on B cells. The usefulness of the whole blood assay was further demonstrated by studies in small volumes of umbilical cord mononuclear cells, that like adult blood cells, showed up-regulation of CD40 expression on B cells, DC, and monocytes, and CD69 on NK cells. The whole blood assay, in conjunction with flow cytometry, is useful for assessing the immunological properties of CpG ODN sequences.

The immune modulation of B-cell responses by Porphyromonas ginginvalis and interleukin-10.

BACKGROUND: Polyclonal B-cell activation induced by periodontopathic bacteria has been cited as being important for elevated numbers of B cells, but the role of bacteria in the pathogenesis of periodontal disease remains unknown. In this study, we used an in vitro model to investigate the activation of immune cells by the periodontopathic bacterium Porphyromonas gingivalis in healthy subjects. METHODS: Peripheral blood mononuclear cells (PBMC) or purified subsets of lymphocytes were stimulated with sonicated extracts of P. gingivalis for 24 hours. Cells were harvested and monitored for expression of CD69 by flow cytometry. Cytokine production (IL-10, IL-12, and IL-15) in P. gingivalis-stimulated PBMC cultures was measured by ELISA. To identify IL-10 producer cells, a cell depletion experiment was used and confirmed by the ability of the purified cell population to produce IL-10. To evaluate the effect of P. gingivalis and IL-10, the proliferative response of purified B cells was assessed by [3H] thymidine uptake. RESULTS: PBMC cultured with P. gingivalis led to a large number of activated B and natural killer (NK) cells as monitored by CD69 expression. When positively sorted cells were used, the bacterium itself could directly activate only B cells but not NK cells, alphabeta, and gammadelta T cells. Measurement of B-cell regulatory cytokine production in P. gingivalis-stimulated PBMC cultures revealed a large amount of IL-10 but no detectable IL-12 or IL-15; the major producing cells were monocytes, not B cells or alphabeta T cells. When IL-10 was added to B cells in the presence of bacteria, significantly increased B-cell proliferative responses were observed. CONCLUSIONS: These results suggest that P. gingivalis, both directly and indirectly via macrophage IL-10, may play an important role in polyclonal B-cell activation associated with periodontal disease.

Lymphocyte activation after non-thermal trauma.

BACKGROUND: Severe injury causes immunological changes that may contribute to a poor outcome. Longitudinal characterization of lymphocyte response patterns may provide further insight into the basis of these immunological alterations. METHODS: Venous blood obtained seven times over 2 weeks from 61 patients with injury severity scores above 20 was assessed for lymphocyte phenotypic and activation markers together with serum levels of interleukin (IL) 2, IL-4, soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4), soluble CD8 (sCD8) and interferon gamma. RESULTS: Severe injury was associated with profound changes in the phenotypic and activation profile of circulating lymphocytes. Activation was indicated by increased numbers of T cells expressing CD25, CD69 and CD71, and raised serum levels of IL-2, sIL-2R, sCD4 and sCD8. Relatively higher levels of sIL-2R and sCD4 were found in patients with sepsis syndrome. CONCLUSION: Polytrauma is associated with dramatic alterations in the phenotypic and activation profile of circulating lymphocytes which are generally independent of clinical course. In contrast, several lymphocyte soluble factors, including sCD4 and sIL-2R, paralleled the clinical course. These data provide new insight into lymphocyte responses after injury and suggest that further assessment of soluble factors as clinical correlates, including those related to lymphocyte activation or generalized inflammation, may be warranted.

Impairment of Plasmodium falciparum growth in thalassemic red blood cells: further evidence by using biotin labeling and flow cytometry.

Certain red blood cell (RBC) disorders, including thalassemia, have been associated with an innate protection against malaria infection. However, many in vitro correlative studies have been inconclusive. To better understand the relationship between human RBCs with thalassemia hemoglobinopathies and susceptibility to in vitro infection, we used an in vitro coculture system that involved biotin labeling and flow cytometry to study the ability of normal and variant RBC populations in supporting the growth of Plasmodium falciparum malaria parasites. Results showed that both normal and thalassemic RBCs were susceptible to P falciparum invasion, but the parasite multiplication rates were significantly reduced in the thalassemic RBC populations. The growth inhibition was especially marked in RBCs from alpha-thalassemia patients (both alpha-thalassemia1/alpha-thalassemia2 and alpha-thalassemia1 heterozygote). Our observations support the contention that thalassemia confers protection against malaria and may explain why it is more prevalent in malaria endemic areas.

Regulation of leukocyte adhesion molecules CD11b/CD18 and leukocyte adhesion molecule-1 on phagocytic cells activated by malaria pigment.

There is increasing evidence that inappropriate immune activation induced by parasite products occurs in malaria disease. To further elucidate the role of Plasmodium falciparum-derived products on host immune activation, we studied the expression of leukocyte adhesion molecules (CD11b/CD18 and LAM-1) on neutrophils and monocytes in response to malaria pigment using flow cytometry. Exposure of leukocytes to isolated malaria pigment derived from ruptured schizonts resulted in significant up-regulation of CD11b/CD18 expression and down-regulation of LAM-1 on both neutrophils and monocytes. In contrast, culture supernatants (pigment free) from ruptured schizonts did not alter the expression of CD11b/CD18 and LAM-1. The increase of CD11b/CD18 and the loss of LAM-1 expression occurred simultaneously with the earliest response detected at 10 min and a plateau reached by 60 min. The effect of malaria pigment on leukocyte adhesion molecules was inhibited by EDTA in a dose-dependent manner. Phagocytosis of malaria pigment was also suppressed by EDTA. This observation suggests that phagocytosis of malaria pigment may be a prerequisite for the effect of malaria pigment on the regulation of CD11b/CD18 and LAM-1 expression. Regulation of leukocyte adhesion molecules through up-regulation of CD11b/CD18 and down-regulation of LAM-1 by malaria pigment could promote leukocyte adherence to endothelium in vivo. This increased adherence of malaria pigment-activated leukocytes might induce cytokine (tumor necrosis factor alpha and interleukin-1beta)-mediated increases in capillary permeability resulting in local tissue edema, and a cytokine-mediated increase in adhesion molecule expression causing vascular clogging by adherent red blood cells, and in severe disease by adherent leukocytes.

Activation of gammadelta T cells in malaria: interaction of cytokines and a schizont-associated Plasmodium falciparum antigen.

A soluble Plasmodium falciparum antigen that specifically stimulates gammadelta T cells has been found associated predominantly with schizonts rather than ring forms, trophozoites, or gametocytes. This schizont-associated antigen (SAA) is resistant to protease digestion, is anionic at pH 8.5, is heat- and pH-resistant, and contains a phosphate group(s) that is crucial for biologic activity. Partially purified SAA induced proliferative responses and interferon-gamma production by gammadelta T cells. These stimulatory effects were greatly enhanced by monocyte-derived cytokines, interleukin (IL)-10, IL-12, and IL-1beta, but not by tumor necrosis factor-alpha. Taken together, these results suggest that concurrent stimulation of gammadelta T cells by SAA and by cytokines released from activated monocytes (IL-10, IL-12, IL-1beta) may represent the major mechanism underlying the selective activation of gammadelta T cells that is consistently observed in clinical cases of P. falciparum infection.

Flow cytometric assessment of hydroxypyridinone iron chelators on in vitro growth of drug-resistant malaria.

The resurgence of drug-resistant malaria makes urgent the evaluation of new antimalarial agents. This study describes a flow cytometric method (FCM) for testing in vitro drug susceptibility of Plasmodium falciparum malaria to several orally active hydroxypyridinone (CP) iron chelators and to the parenteral iron chelator desferrioxamine (DF). After exposure of parasites to various concentrations of iron chelating agents, aliquots of cultures were fixed with glutaraldehyde. The fixed samples were washed and stained for parasite DNA with propidium iodide and analyzed by flow cytometry. The remaining cells were pulsed with 3H-hypoxanthine, using the microdilution radioisotope method. Both CP and DF showed dose-dependent inhibition of parasite growth. Of the compounds studied, DF exerted a stronger inhibitory effect. Fifty percent of inhibitory concentrations (IC50) of CP and DF determined by DNA fluorescence profiles in the flow cytometer were consistent with those obtained from the radioisotope method and by microscopic examination. Moreover, the minimum inhibitory concentrations (MIC) of drug required to inhibit parasite growth, as detected by the decreasing DNA fluorescence intensity of the schizont, correlated with observed abnormal microscopic morphology. The validity of the MIC, as indicated by decreased fluorescence intensity, was confirmed by subsequent parasite culture. Our FCM study demonstrated the sensitivity of both chloroquine- and pyrimethamine-resistant malaria parasites to iron chelators. Addition of equimolar concentrations of ferric ion completely abolished the inhibitory effect of iron chelators, indicating the importance of iron for parasite growth and the primary effect of the compounds as iron (III) chelating agents. These data demonstrate that FCM provides a simple and reliable means for antimalarial drug susceptibility testing, and suggest that iron chelators have potential for the treatment of drug-resistant malaria.

Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry.

A novel culture system using biotin/streptavidin and flow cytometry was developed to compare maturation and growth rates in Plasmodium falciparum malaria parasites in two distinct red blood cell (RBC) populations. Biotin was used to label a selected RBC population which was then mixed with another distinct unbiotinylated RBC population. P. falciparum-infected RBCs were used to initiate co-cultures followed over 2-3 schizogonic growth cycles. Co-cultures were harvested and stained with streptavidin-fluorescein isothiocyanate (FITC) followed by fixation and staining of parasite DNA. The combination of biotin/streptavidin-FITC and DNA fluorochrome enabled simultaneous flow cytometric analysis of the two different RBC populations and of the parasitemias in each RBC population. We then used this system to study the in vitro susceptibility of RBCs from individuals with hemoglobin H (Hb H) disease to infection and growth of P. falciparum. Significant reduction in parasite multiplication was found in Hb H RBCs as compared with that in normal RBCs. This novel malaria culture system offers two major innovations: a method to compare directly the relative ability of any two red blood cell populations to support malaria parasite invasion and development under identical conditions, and a critical reduction in the volume of blood and reagents needed to assess parasite growth. The application of biotin-labeled RBCs in the flow cytometric analysis of parasite development may offer new insights in studies of the relationship between RBC defects and susceptibility to malaria parasites.

Flow cytometric immunophenotyping of lymphocyte subsets in samples that contain a high proportion of non-lymphoid cells.

Flow cytometric (FCM) immunophenotyping of peripheral blood from thalassemia patients presents technical difficulties because of the high proportion of immature red cells. The combination of forward scatter (FSC) and side scatter (SSC) with fluorescence associated with human leukocyte antigen/monocyte antigen (CD45/CD14) was unable to identify the lymphocyte population in thalassemia patients; therefore, it was necessary to exclude immature red cells to analyze lymphocyte subsets in these patients. A simultaneous three-color FCM method was developed, with the basis that transferrin receptor (CD71) or glycophorin A (glyco A) is present on all immature red cells, but is not expressed on CD45 positive leukocytes. In this study, the lymphocyte population was identified by gating out unwanted cell populations using the FSC/CD71-fluorescein isothiocyanate (FITC), FSC/glyco A-FITC, or FSC/CD45-peridinin chlorophyll protein (PerCP) profiles. The CD71-FITC negative cells, glyco A-FITC negative cells, or CD45-PerCP positive cells were identified, then analyzed on the basis of FSC/SSC to allow any remaining non-lymphocyte cells in FSC/SSC gate to be excluded. The cells in FSC/SSC gate were then analyzed using other irrelevant two-color antibodies. Of the three gating strategies, CD45-PerCP and glyco A-FITC methods are better than the CD71-FITC gating method. Both methods markedly increase the purity of lymphocytes in the analysis gate. Either method is easy, straightforward, requires a six-tube set of reagent tubes, and provides a reliable method for immunophenotyping lymphocyte subsets in preparations containing a large percentage of non-lymphoid cells.

Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites.

Cloning and characterization of mefloquine-resistant Plasmodium falciparum from Thailand.

Resistance to mefloquine in Plasmodium falciparum has begun to occur along the border of Thailand and Kampuchea. As a means of assessing the natural occurrence of mefloquine resistance, the admission and post-treatment parasite isolates from a mefloquine treatment failure were cloned and characterized. Clones from the admission isolate were susceptible to mefloquine in vitro (ID50 of 3.4 [2-5], G [95% CI] ng/ml) and showed a mixture of isozyme types for glucose phosphate isomerase (GPI types I and II). The post-treatment clones were resistant to mefloquine in vitro (ID50 of 17.3 [13-23] ng/ml) with only one isozyme (GPI type I) detected. These observations suggest that under mefloquine pressure a resistant parasite population was selected in the patient, indicating that the potential for mefloquine resistance already exists in the indigenous P. falciparum gene pool. In addition, the mefloquine-resistant clones showed decreased susceptibility in vitro to halofantrine suggesting possible cross-resistance to this new antimalarial drug currently under development.

Antimalarial drug susceptibility testing of Plasmodium falciparum in Thailand using a microdilution radioisotope method.

Antimalarial activity of chloroquine, quinine, mefloquine and halofantrine against 33 strains of P. falciparum isolated from naturally acquired malaria infections in Thailand was determined using a radioisotope microdilution method. A microtitration procedure was used to test isolates of P. falciparum against the 4 drugs simultaneously. The mean ID50 for chloroquine and quinine reflected known resistance to those drugs in Thailand. The mean ID50 for mefloquine and halofantrine showed susceptibility to these drugs. Four isolates of P. falciparum however had markedly decreased susceptibility to mefloquine (ID50 greater than 15 ng/ml); one case of which was confirmed as the first case of RII resistance for mefloquine in Thailand. Several parasite isolates were also observed to have decreased susceptibility to the new drug, halofantrine. These studies strongly recommend that in vitro testing be done in conjunction with field evaluation of new antimalarial drugs.