Organ-specific testosterone-insensitive response of miRNA expression of C57BL/6 mice to Plasmodium chabaudi malaria

Increasing evidence critically implicates miRNAs in the pathogenesis of diseases, but little is known in context with infectious diseases. This study investigates as to whether the testosterone-induced persistent susceptibility to blood-stage malaria of Plasmodium chabaudi coincides with changes in miRNA expression of the anti-malaria effectors sites spleen and liver. Female C57BL/6 mice were treated with vehicle or testosterone (T) for 3?weeks. Then, T treatment was discontinued for 12?weeks before challenge with 10(6) P. chabaudi-parasitized erythrocytes. The miRNA expression was examined after 12?weeks of T withdrawal and during infections at peak parasitemia on day?8 p.i. using miRXplore? microarray technology. P. chabaudi infections induce an organ-specific response of miRNA expression. We can identify 25 miRNA species to be downregulated by more than 2-fold in the spleen and 169 miRNA species in the liver. Among these 194 miRNA species, there are 12 common miRNA species that are downregulated by 0.48-0.14-fold in both spleen and liver, which are miR-194, miR-192, miR-193A-3P, miR-145, miR-16, miR-99A, miR-99B, miR-15A, miR-152, let-7G, let-7B, and miR-455-3P. Only in the liver, there is an upregulation of the miR-142-5p by 2.5-fold and miR-342-3p by 5.1-fold. After 12?weeks of T withdrawal, the spleen exhibits only the miR-200A that is upregulated by 2.7-fold. In the liver, miR-376B, miR-493*, and miR-188-3P are upregulated by 2.4-fold, 2.2-fold, and 2.1-fold, respectively, and miR-347, miR-200A, and miR-200B are downregulated by approximately 0.4-fold. Upon infection, however, these changes are not sustained, i.e., the miRNA expressions of both spleen and liver of T-pretreated mice exhibit the same response to P. chabaudi malaria as that of vehicle-treated control mice. Our data suggest (1) that the P. chabaudi-induced downregulation of miRNA expression in spleen and liver is required to allow the upregulation of their numerous target genes in response to infection, and (2) that the T-induced persistent susceptibility to P. chabaudi does not affect the responsiveness of miRNA expression in spleen and liver to blood-stage malaria.     

CD28 costimulation is required for the expression of T-cell-dependent cell-mediated immunity against blood-stage Plasmodium chabaudi malaria parasites

Mice suppress the parasitemia of acute blood-stage Plasmodium chabaudi malaria by an antibody- or T-cell-dependent cell-mediated mechanism of immunity (AMI and CMI, respectively) or by both mechanisms. To determine whether CD28 costimulation is required for expression of these polar immune responses, we first compared the time courses of P. chabaudi malaria in CD28-deficient (CD28(-/-)) and CD28-intact (CD28(+/+)) mice. Acute infections in both knockout (KO) and control mice followed similar time courses, with the period of descending parasitemia being prolonged approximately 2 weeks in KO mice followed by intermittent low-grade chronic parasitemia. Infected CD28(-/-) mice produced primarily the immunoglobulin M antibody, which upon passive transfer provided partial protection against P. chabaudi challenge, suggesting that the elimination of blood-stage parasites by CD28(-/-) mice was achieved by AMI. To determine whether CD28(-/-) costimulation is required for the expression of CMI against the parasite, we compared the time courses of parasitemia in B-cell-deficient double-KO (J(H)(-/-) x CD28(-/-)) mice and control (J(H)(-/-) x CD28(+/+)) mice. Whereas control mice suppressed parasitemia to subpatent levels within approximately 2 weeks postinoculation, double-KO mice developed high levels of parasitemia of long-lasting duration. Although not required for the suppression of acute P. chabaudi parasitemia by AMI, CD28 costimulation is essential for the elimination of blood-stage parasites by CMI.     

Reduced protective efficacy of a blood-stage malaria vaccine by concurrent nematode infection

Helminth infections, which are prevalent in areas where malaria is endemic, have been shown to modulate immune responses to unrelated pathogens and have been implicated in poor efficacy of malaria vaccines in humans. We established a murine coinfection model involving blood-stage Plasmodium chabaudi AS malaria and a gastrointestinal nematode, Heligmosomoides polygyrus, to investigate the impact of nematode infection on the protective efficacy of a malaria vaccine. C57BL/6 mice immunized with crude blood-stage P. chabaudi AS antigen in TiterMax adjuvant developed strong protection against malaria challenge. The same immunization protocol failed to induce strong protection in H. polygyrus-infected mice. Immunized nematode-infected mice produced significantly lower levels of malaria-specific antibody than nematode-free mice produced. In response to nematode and malarial antigens, spleen cells from immunized nematode-infected mice produced significantly lower levels of gamma interferon but more interleukin-4 (IL-4), IL-13, and IL-10 in vitro than spleen cells from immunized nematode-free mice produced. Furthermore, H. polygyrus infection also induced a strong transforming growth factor beta1 response in vivo and in vitro. Deworming treatment of H. polygyrus-infected mice before antimalarial immunization, but not deworming treatment after antimalarial immunization, restored the protective immunity to malaria challenge. These results demonstrate that concurrent nematode infection strongly modulates immune responses induced by an experimental malaria vaccine and consequently suppresses the protective efficacy of the vaccine against malaria challenge.     

Cellular changes and apoptosis in the spleens and peripheral blood of mice infected with blood-stage Plasmodium chabaudi chabaudi AS

Infection with blood-stage Plasmodium chabaudi chabaudi AS results in splenomegaly, peripheral leukocytosis, and a major activation of the immune system. The frequencies and absolute numbers of T-cell, B-cell, and macrophage populations in spleen and peripheral blood from P. chabaudi-infected BALB/c mice were compared and found to be significantly altered during acute infection. The kinetics of the redistribution of the different cell types in spleen and peripheral blood were different, with T and B cells appearing in the blood when their frequencies and absolute numbers in the spleen were low. The frequency and absolute number of apoptotic cells in the spleen were increased during acute P. chabaudi infection and involved both T cells, B cells, and macrophages. Both Fas and Fas-ligand expression were increased in the spleen. Taken together, our data provide new information on the complex cellular interactions that take place in the immune system during blood-stage malaria infection in a mouse model.     

Vaccination with novel immunostimulatory adjuvants against blood-stage malaria in mice

An important aspect of malaria vaccine development is the identification of an appropriate adjuvant which is both capable of stimulating a protective immune response and safe for use by humans. Here, we investigated the feasibility of using novel immunostimulatory molecules as adjuvants combined with a crude antigen preparation and coadsorbed to aluminum hydroxide (alum) as a vaccine against blood-stage Plasmodium chabaudi AS malaria. Prior to challenge infection, immunization of genetically susceptible A/J mice with the combination of malaria antigen plus recombinant interleukin-12 (IL-12) in alum induced a Th1 immune response with production of high levels of gamma interferon (IFN-gamma) and diminished IL-4 levels by spleen cells stimulated in vitro with parasite antigen compared to mice immunized with antigen alone, antigen in alum, or antigen plus IL-12. Mice immunized with malaria antigen plus recombinant IL-12 in alum had high levels of total malaria-specific antibody and immunoglobulin G2a. Compared to unimmunized mice, immunization with antigen plus IL-12 in alum induced the highest level of protective immunity against challenge infection with P. chabaudi AS, which was evident as a significantly decreased peak parasitemia level and 100% survival. Protective immunity was dependent on CD4(+) T cells, IFN-gamma, and B cells and was long-lasting. Replacement of IL-12 as an adjuvant by synthetic oligodeoxynucleotides (ODN) containing CpG motifs induced a similar level of vaccine-induced protection against challenge infection with P. chabaudi AS. These results illustrate that it is possible to enhance the potency of a crude malaria antigen preparation delivered in alum by inclusion of immunostimulatory molecules, such as IL-12 or CpG-ODN.     

Characterization of the spleen B-cell compartment at the early and late blood-stage Plasmodium chabaudi malaria

Polyclonal B-cell activation is a feature of the early spleen cell response to blood-stage Plasmodium chabaudi malaria. Immunity to blood-stage malaria is guaranteed by the generation of B cells able to produce parasite-specific antibodies mainly from the immunoglobulin (Ig)G2a isotype. In the present study, we characterized the spleen B-cell compartment during blood-stage P. chabaudi infection. The numbers of B220(+) and B220(LOW) CD138(+) (plasma) cells increased sharply between days 4 and 7 post-infection (p.i.). At this time B220(+) cells expressed surface (s)IgM, but nearly all B220(LOW) CD138(+) cells showed concomitantly intracellular (i)IgM and IgG2a. Both follicular and marginal zone B cells were activated expressing high amounts of CD69. At day 40 p.i., B220(LOW) CD138(+) cell population was still increased but, differently from acute infection, 61.1% of these cells were positive for iIgG2a while only 14.2% expressed iIgM. Moreover, at days 20 and 40 p.i., 29.2% and 13.0% of B220(+) cells expressed sIgG2a, respectively. According to cell size and expression of CD80, CD86, CD11b, CD44 and CD38, B220(+) sIgG2a(+) cells had a phenotype characteristic of activated/memory B cells. Furthermore, 14.1% of B220(+) sIgG2a(+) cells at day 30 p.i. expressed a marginal zone B-cell phenotype. Importantly, B cells from 40-day-infected mice were very efficient in presenting parasite antigens leading to proliferation of both CD4(+) and CD8(+) cells. Our results contribute for understanding the dynamics of B cells during P. chabaudi infection, underlying the mechanisms of antigen presentation and antibody production, which are essential for the acquisition of protective immunity against malaria.     

Central role of endogenous gamma interferon in protective immunity against blood-stage Plasmodium chabaudi AS infection

The role of endogenous gamma interferon (IFN-gamma) in protective immunity against blood-stage Plasmodium chabaudi AS malaria was studied using IFN-gamma gene knockout (GKO) and wild-type (WT) C57BL/6 mice. Following infection with 10(6) parasitized erythrocytes, GKO mice developed significantly higher parasitemia during acute infection than WT mice and had severe mortality. In infected GKO mice, production of interleukin 12 (IL-12) p70 and tumor necrosis factor alpha in vivo and IL-12 p70 in vitro by splenic macrophages was significantly reduced compared to that in WT mice and the enhanced nitric oxide (NO) production observed in infected WT mice was completely absent. WT and GKO mice had comparable numbers of total nucleated spleen cells and B220(+) and Mac-1(+) spleen cells both before and after infection. Infected WT mice, however, had significantly more F4/80(+), NK1.1(+), and F4/80(+)Ia(+) spleen cells than infected GKO mice; male WT had more CD3(+) cells than male GKO mice. In comparison with those from WT mice, splenocytes from infected GKO mice had significantly higher proliferation in vitro in response to parasite antigen or concanavalin A stimulation and produced significantly higher levels of IL-10 in response to parasite antigen. Infected WT mice produced more parasite-specific immunoglobulin M (IgM), IgG2a, and IgG3 and less IgG1 than GKO mice. Significant gender differences in both GKO and WT mice in peak parasitemia levels, mortality, phenotypes of spleen cells, and proliferation of and cytokine production by splenocytes in vitro were apparent during infection. These results thus provide unequivocal evidence for the central role of endogenous IFN-gamma in the development of protective immunity against blood-stage P. chabaudi AS.     

Changes in oxidative burst capacity during murine malaria and the effect of vaccination.

Adherent spleen and liver cells from mice infected with Plasmodium yoelii 17X or P. chabaudi AS were tested for production of reactive oxygen intermediates to measure their state of activation. Phorbol myristate acetate (PMA) was used to trigger the respiratory burst and production of superoxide anions was measured by the reduction of nitroblue tetrazolium. Spleen cells from mice infected with P. chabaudi showed an early increase in oxidative activity on day 3, and when the oxidative capacity of the whole spleen was calculated, it was maximal on day 9, just as the mice began to recover. In mice infected with P. yoelii, spleen cells showed an early peak in activity on day 5, and then returned to normal, although the mice did not recover for a further 2-3 weeks. However the total oxidative capacity of the spleen remained high throughout the infection. Mice vaccinated against P. yoelii with a killed blood-stage vaccine showed increased activity on day 3 (spleen) and day 5 (liver), compared with infected control mice. Thus macrophages in these organs could, if given an appropriate trigger, release high levels of these potentially toxic molecules during infection.     

Activation of liver macrophages in murine malaria is enhanced by vaccination.

During blood-stage infection of mice with a lethal variant of Plasmodium yoelii, cells in both spleen and liver became activated to reach a peak at day 5. In mice protected by vaccination, activation was accelerated after infection. The most striking difference observed was in the 10-fold greater yield of infiltrating cells, including macrophages, obtained from the liver just before the mice recovered. Their capacity to give an oxidative burst and their cytotoxic activity against tumour cells was also more than 10 times normal. This suggests that the recruitment of inflammatory cells to the liver plays an important role in the protection of vaccinated mice against malaria.     

Role of interferon-gamma and tumor necrosis factor in host resistance to Plasmodium chabaudi AS

The contribution of the T cell- and macrophage-derived cytokines, interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF), respectively, in the cell-mediated mechanisms leading to acquired immunity to blood-stage Plasmodium chabaudi AS was investigated. To examine the contribution of IFN-gamma, resistant C57BL-derived mice were treated during infection with two different neutralizing, anti-murine IFN-gamma mAbs. Such treatment impaired the ability of the host to limit parasite multiplication just before and at the time of the peak parasitemia but did not abrogate the development of acquired immunity resulting in control and elimination of acute infection. The requirement of endogenous IFN-gamma around the time of the peak parasitemia was confirmed by quantification of IFN-gamma production in vitro by spleen cells from infected animals in response to malaria antigen. To investigate the role of TNF, resistant C57BL/6 and susceptible A/J mice were treated with rTNF during P. chabaudi AS infection. Treatment with 10(3) or 10(5) U rTNF resulted in increased resistance in A/J hosts (that is, increased survival and a less severe course of infection); there was no difference between control and treated C57BL/6 mice in the course of infection but there was increased mortality among the animals treated with rTNF. Splenic macrophages harvested from C57BL/6 mice during infection were found to produce high levels of TNF from day 3 to day 28 post-infection. In conclusion, both IFN-gamma and TNF appear to contribute to host resistance to blood-stage infection with P. chabaudi AS.     

Protection against Plasmodium chabaudi malaria induced by immunization with apical membrane antigen 1 and merozoite surface protein 1 in the absence of gamma interferon or interleukin-4

Strategies to optimize formulations of multisubunit malaria vaccines require a basic knowledge of underlying protective immune mechanisms induced by each vaccine component. In the present study, we evaluated the contribution of antibody-mediated and cell-mediated immune mechanisms to the protection induced by immunization with two blood-stage malaria vaccine candidate antigens, apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-1). Immunologically intact or selected immunologic knockout mice were immunized with purified recombinant Plasmodium chabaudi AMA-1 (PcAMA-1) and/or the 42-kDa C-terminal processing fragment of P. chabaudi MSP-1 (MSP-1(42)). The efficacy of immunization in each animal model was measured as protection against blood-stage P. chabaudi malaria. Immunization of B-cell-deficient JH(-/-) mice indicated that PcAMA-1 vaccine-induced immunity is largely antibody dependent. In contrast, JH(-/-) mice immunized with PcMSP-1(42) were partially protected against P. chabaudi malaria, indicating a role for protective antibody-dependent and antibody-independent mechanisms of immunity. The involvement of gammadelta T cells in vaccine-induced PcAMA-1 and/or PcMSP-1(42) protection was minor. Analysis of the isotypic profile of antigen-specific antibodies induced by immunization of immunologically intact mice revealed a dominant IgG1 response. However, neither interleukin-4 and the production of IgG1 antibodies nor gamma interferon and the production of IgG2a/c antibodies were essential for PcAMA-1 and/or PcMSP-1(42) vaccine-induced protection. Therefore, for protective antibody-mediated immunity, vaccine adjuvants and delivery systems for AMA-1- and MSP-1-based vaccines can be selected for their ability to maximize responses irrespective of IgG isotype or any Th1 versus Th2 bias in the CD4(+)-T-cell response.     

Impairment of protective immunity to blood-stage malaria by concurrent nematode infection

Helminthiases, which are highly prevalent in areas where malaria is endemic, have been shown to modulate or suppress the immune response to unrelated antigens or pathogens. In this study, we established a murine model of coinfection with a gastrointestinal nematode parasite, Heligmosomoides polygyrus, and the blood-stage malaria parasite Plasmodium chabaudi AS in order to investigate the modulation of antimalarial immunity by concurrent nematode infection. Chronic infection with the nematode for 2, 3, or 5 weeks before P. chabaudi AS infection severely impaired the ability of C57BL/6 mice to control malaria, as demonstrated by severe mortality and significantly increased malaria peak parasitemia levels. Coinfected mice produced significantly lower levels of gamma interferon (IFN-gamma) during P. chabaudi AS infection than mice infected with malaria alone. Concurrent nematode infection also suppressed production of type 1-associated, malaria-specific immunoglobulin G2a. Mice either infected with the nematode alone or coinfected with the nematode and malaria had high transforming growth factor beta1 (TGF-beta1) levels, and concurrent nematode and malaria infections resulted in high levels of interleukin-10 in vivo. Splenic CD11c(+) dendritic cells (DC) from mice infected with malaria alone and coinfected mice showed similarly increased expression of CD40, CD80, and CD86, but DC from coinfected mice were unable to induce CD4(+) T-cell proliferation and optimal IFN-gamma production in response to the malaria antigen in vitro. Importantly, treatment of nematode-infected mice with an anthelmintic drug prior to malaria infection fully restored protective antimalarial immunity and reduced TGF-beta1 levels. These results demonstrate that concurrent nematode infection strongly modulates multiple aspects of immunity to blood-stage malaria and consequently impairs the development of protective antimalarial immunity.     

Immunization of mice with phosphatidylcholine drastically reduces the parasitaemia of subsequent Plasmodium chabaudi chabaudi blood-stage infections.

It has been suggested that phospholipids and antibodies directed against phospholipids are important in the pathology of malaria. We have investigated the influence of immunizations with phospholipids on the course of subsequent blood-stage Plasmodium chabaudi chabaudi infections in ICR inbred mice. We observed a significant reduction in the parasitaemia following immunization with phosphatidylcholine (PC), but not with phosphatidylethanolamine (PE) immunization. At the peak of the infection, PC-immunized mice displayed a T-helper 2 (Th2)-type cytokine production pattern, whereas PE-immunized or non-treated controls displayed a cytokine production pattern of the T-helper 1 (Th1) type. Serum immunoglobulin transfer from PC-immunized mice protected naive mice in a similar fashion to PC-immunization, demonstrating that the observed reduction of parasitaemia was caused by the presence of PC-specific antibodies.     

Dendritic cells induce immunity and long-lasting protection against blood-stage malaria despite an in vitro parasite-induced maturation defect

Dendritic cells (DC) suffer a maturation defect following interaction with erythrocytes infected with malaria parasites and become unable to induce protective malaria liver-stage immunity. Here we show that, by contrast, maturation-arrested DC in vitro are capable of the successful induction of antigen-specific gamma interferon (IFN-gamma) and interleukin 4 (IL-4) T-cell responses, antibody responses, and potent protection against lethal blood-stage malaria challenge in vivo. Similar results were found with DC pulsed with intact parasitized Plasmodium yoelii or Plasmodium chabaudi erythrocytes. Cross-strain protection was also induced. High levels of protection (80 to 100%) against lethal challenge were evident from 10 days after a single immunization and maintained up to 120 days. Interestingly, correlation studies versus blood-stage protection at different time points suggest that the immune effector mechanisms associated with protection could change over time. Antibody-independent, T-cell- and IL-12-associated protection was observed early after immunization, followed by antibody and IL-4-associated, IFN-gamma-independent protection in long-term studies. These results indicate that DC, even when clearly susceptible to parasite-induced maturation defect effects in vitro, can be central to the induction of protection against blood-stage malaria in vivo.     

A Th1-associated increase in tumor necrosis factor alpha expression in the spleen correlates with resistance to blood-stage malaria in mice.

We investigated the kinetics of tissue-specific mRNA expression and systemic production of tumor necrosis factor alpha (TNF-alpha) and the kinetics of splenic expression of mRNAs of gamma interferon (INF-gamma) and interleukin-4 (IL-4), cytokines that may regulate TNF-alpha production, during the early phase of blood-stage infection with Plasmodium chabaudi AS. Northern blot analysis revealed that resistant C57BL/6 mice, which clear the infection by 4 weeks, had higher levels of TNF-alpha mRNA in the spleen and liver early during infection that did susceptible A/J mice, which succumb to the disease 10 days after initiation of infection. Treatment of resistant mice with a polyclonal anti-TNF-alpha antibody confirmed the protective role of TNF-alpha early during the course of infection. Furthermore, resistant C57BL/6 mice also expressed high levels of mRNA of IFN-gamma (a Th1 marker) and low levels of mRNA of IL-4 (a Th2 marker) in the spleen, whereas susceptible A/J mice had low levels of IFN-gamma mRNA but high levels of TNF-alpha mRNA in the liver and had high levels of TNF-alpha protein in serum, as measured by enzyme-linked immunosorbent assay, later during infection just before death occurred. These results demonstrate that a Th1-associated increase in TNF-alpha mRNA expression in the spleen early during infection correlates with resistance to P. chabaudi AS, whereas increased TNF-alpha mRNA levels in the liver and excessive levels of the TNF-alpha protein in serum later during infection correlate with susceptibility. Thus, the role of the TNF-alpha during malaria appears to depend on the timing and site of its expression and the presence of cytokines regulating its production.     

Differential induction of helper T cell subsets during blood-stage Plasmodium chabaudi AS infection in resistant and susceptible mice.

The induction of T helper cell subsets during the course of non-lethal or lethal blood-stage Plasmodium chabaudi AS infection was investigated using inbred strains of mice which differ in the level of resistance to this intraerythrocytic parasite. Resistant C57Bl/6 mice experience a non-lethal course of infection characterized by moderate levels of both parasitaemia and anaemia and resolution of primary acute infection by 4 weeks, while susceptible A/J mice experience lethal infection with fulminant parasitaemia and severe anaemia. T helper subset function was assessed during infection by determining the kinetics of spleen cell production in vitro of the Th1-derived cytokine, interferon-gamma (IFN-gamma), and of the Th2-derived cytokine, IL-5, using sandwich ELISAs. Spleen cells from resistant C57Bl/6 mice were found to produce high levels of IFN-gamma within 1 week of infection in response to both the mitogen concanavalin A (Con A) and malaria antigen. Furthermore, CD4+ T cells were found to be the source of IFN-gamma while both CD4+ and CD8+ T cells were found to produce IL-5. Decreased IFN-gamma production after day 10 was concomitant with significant production of IL-5 between 2 and 3 weeks post infection. In contrast, spleen cells from susceptible A/J mice produced high levels of IL-5 within the first week of infection. In addition, these animals were found to have high serum levels of IL-5. These results, thus, confirm previous observations that resolution of primary blood-stage P. chabaudi infection occurs by sequential activation of Th1 CD4+ T cells followed by activation of the Th2 subset, and in addition, suggest that induction of a strong Th2 response early in infection may lead to a severe and lethal course of malaria.     

Protective vaccination alters gene expression of the liver of Balb/c mice in response to early prepatent blood-stage malaria of <Emphasis Type="Italic">Plasmodium chabaudi</Emphasis>

Current knowledge about liver responses to blood-stage malaria and their modulation by vaccination is still unclear. This study investigated effects of protective vaccination on liver gene and lincRNA expression of Balb/c mice at early prepatency of Plasmodium chabaudi blood-stage malaria. When a blood-stage vaccine was used to induce >?80% survival of otherwise lethal malaria, significant differences (p?<?0.01) were detectable in global liver gene expression between vaccination-protected (potentially surviving) and non-protected non-vaccinated mice on day 1 p.i.. In the livers of protected mice, gene expression microarrays identified 224 and 419 genes, whose expression was up- and downregulated by >?3-fold, respectively. There were 24 genes upregulated by >?10-fold, including 10 IFN-inducible genes encompassing GTPases Irgm1, 2, and 3, and guanylate-binding protein Gbp11, the IL-1 decoy receptors Il1f9 and Il1ra1, the Il6 gene, and the gene for facilitated glucose transportation. Moreover, the IL-18 decoy receptor gene Il18bp, Gzmb, the genes Lif and Osmr encoding proteins of the IL-6 family, and the taurine transporter gene Slc6a6 were expressed >?3-fold in vaccinated mice. The genes Gbp10, 6, 4 were expressed by >?50% in vaccination-protected than in non-vaccinated mice. In addition, 43 lincRNA species were up- and 36 downregulated. Our data suggested novel regulatory elements of potential anti-malaria activity activated by protective vaccination in the liver, evidenced in response to early prepatent infections in vaccination-protected mice of otherwise lethal blood-stage malaria of P. chabaudi.     

Role of endogenous gamma interferon in host response to infection with blood-stage Plasmodium chabaudi AS.

The role of gamma interferon (IFN-gamma), a pluripotent lymphokine capable of activating macrophages, in acquired immunity to blood-stage malaria was investigated. C57BL-derived, lipopolysaccharide-resistant C57BL/10ScN mice, which were found to be resistant to intraperitoneal (i.p.) infection with 10(6) Plasmodium chabaudi AS parasitized erythrocytes, were treated with monoclonal anti-IFN-gamma antibody (MAb). Two MAbs were used: R4-6A2, a rat anti-mouse, neutralizing immunoglobulin G1, which was prepared against natural murine IFN-gamma, and DB-1, a murine anti-rat immunoglobulin G1 prepared against recombinant rat IFN-gamma, which can neutralize the murine molecule as well as the rat molecule. C57BL/10ScNH mice were injected i.p. with 200 micrograms of R4-6A2 1 day before infection and every 3 days through day 21. Control mice were treated with normal rat serum. In separate experiments, DB-1 (1.0 mg per week for 4 weeks) was administered i.p. to C57BL/10ScNH mice beginning on the day of infection; control mice were untreated. Control and MAb-treated mice were infected i.p. with 10(6) P. chabaudi AS parasitized erythrocytes, and the course and outcome of infection were determined. Control mice exhibited a course of infection that was characterized by a peak parasitemia between 30 and 40% parasitized erythrocytes and elimination of the parasite by 4 weeks. MAb-treated mice exhibited a significantly greater parasitemia 1 to 2 days before the peak parasitemia as well as a significantly greater peak parasitemia but also completely cleared the infection by 4 weeks. Thus, these results suggest that treatment with anti-IFN-gamma MAb impairs but does not completely abrogate host resistance to P. chabaudi AS. We also examined the kinetics of IFN-gamma production by spleen cells cultured in vitro with malaria antigen or concanavalin A. Spleen cells were recovered from individual C57BL/6 mice at various times after i.p. infection with 10(6) P. chabaudi AS parasitized erythrocytes. The amount of IFN-gamma produced was quantitated by enzyme-linked immunosorbent assay. In each case, the peak of IFN-gamma production occurred just before the peak parasitemia, followed by a decrease to little or no IFN-gamma production through 42 days postinfection. There was thus a parallel between the kinetics of production of IFN-gamma in vitro by spleen cells from infected animals and the requirement in vivo for the endogenous molecule just before and at the time of peak parasitemia. In conclusion, these results suggest that IFN-gamma-dependent and -independent mechanisms contribute to host resistance to P. chabaudi AS.     

Splenic gammadelta T cells regulated by CD4+ T cells are required to control chronic Plasmodium chabaudi malaria in the B-cell-deficient mouse

Little is known about the function and regulation of splenic gammadelta T cells during chronic Plasmodium chabaudi malaria. The splenic gammadelta T-cell population continues to expand, reaching levels equal to 4 times the number of splenocytes in an uninfected mouse. Splenic gammadelta T cells from J(H)-/- mice with chronic malaria expressed Vgamma1+ or Vdelta4+ in the same ratio as uninfected controls with Vgamma1 cells dominating, but the Vgamma2 ratio declined about twofold. Gammadelta T cells from G8 mice specific for the TL antigen increased only 2-fold in number, compared with 10-fold in BALB/c controls, but G8 gammadelta T cells failed to express the B220 activation marker. Elimination of the parasite by drug treatment caused a slow depletion in the number of splenic gammadelta, CD4+, and CD8+ T cells. Following challenge, drug-cured J(H)-/- mice exhibited nearly identical parasitemia time courses as na?ve controls. Depletion of either CD4+ T cells or gammadelta T cells from chronically infected J(H)-/- mice by monoclonal antibody treatment resulted in an immediate and significant (P < 0.05) exacerbation of parasitemia coupled with a marked decrease in splenic gammadelta T-cell numbers. The number of CD4+ T cells, in contrast, did not decrease in mice after anti-T-cell receptor gammadelta treatment. The results indicate that cell-mediated immunity against blood-stage malarial parasites during chronic malaria (i) requires the continued presence of blood-stage parasites to remain functional, (ii) is dependent upon both gammadelta T cells and CD4+ T cells, and (iii) lacks immunological memory.     

Mixed strain infections and strain-specific protective immunity in the rodent malaria parasite Plasmodium chabaudi chabaudi in mice

Important to malaria vaccine design is the phenomenon of "strain-specific" immunity. Using an accurate and sensitive assay of parasite genotype, real-time quantitative PCR, we have investigated protective immunity against mixed infections of genetically distinct cloned "strains" of the rodent malaria parasite Plasmodium chabaudi chabaudi in mice. Four strains of P. c. chabaudi, AS, AJ, AQ, and CB, were studied. One round of blood infection and drug cure with a single strain resulted in a partial reduction in parasitemia, compared with levels for na?ve mice, in challenge infections with mixed inocula of the immunizing (homologous) strain and a heterologous strain. In all cases, the numbers of blood-stage parasites of each genotype were reduced to similar degrees. After a second, homologous round of infection and drug cure followed by challenge with homologous and heterologous strains, the parasitemias were reduced even further. In these circumstances, moreover, the homologous strain was reduced much faster than the heterologous strain in all of the combinations tested. That the immunity induced by a single infection did not show "strain specificity," while the immunity following a second, homologous infection did, suggests that the "strain-specific" component of protective immunity in malaria may be dependent upon immune memory. The results show that strong, protective immunity induced by and effective against malaria parasites from a single parasite species has a significant "strain-specific" component and that this immunity operates differentially against genetically distinct parasites within the same infection.