Passive immunization against murine malaria with an IgG3 monoclonal antibody

Spleen cells of BALB/c mice that were immune to the 17X strain of P. yoelii were fused with P3X63Ag8 myeloma cells. Two hundred fifty-three of 1053 hybrid cells produced antibodies reactive with disrupted 17X parasites in a solid phase radioimmunoassay. One of these antibodies, McAb 302, reacted with the merozoites of the 17X (nonlethal) and 17XL (lethal) variants of P. yoelii. Of greater significance, McAb 302 passively protected mice against challenge infection with the lethal variant. Mice treated with this antibody before infection developed low-grade parasitemia (less than 0.3%) of short duration when challenged with P. yoelii 17XL . In contrast, control mice that had been untreated or injected with ascites fluid lacking McAb 302 uniformly died with fulminating malaria upon challenge with the same parasite. In other experiments, McAb 302 was shown capable of controlling blood parasite levels when administered to mice with patent P. yoelii 17XL infections. Although all control mice died, mice protected with a single dose of McAb 302 ultimately cleared their infections. Regardless of how passive immunization was performed, mice given McAb 302 were resistant to subsequent challenge with P. yoelii 17XL , indicating they had developed significant immunity during their initial controlled infections. McAb 302 also showed pronounced passive protective activity against the nonlethal 17X strain of P. yoelii, which is a parasite of reticulocytes. The protection afforded by McAb 302 was specific, because mice passively immunized with this antibody died when challenged with the unrelated P. vinckei. McAb 302 was shown to possess the IgG3 isotype and precipitated a 230-kd protein plus several smaller polypeptides from metabolically labeled parasite antigen preparation derived from both variants of P. yoelii. It did not react with similar preparations of other murine plasmodial species.     

Comparative histopathology of mice infected with the 17XL and 17XNL strains of Plasmodium yoelii

Plasmodium yoelii 17XL was used to investigate the mechanism of Plasmodium falciparum-caused cerebral malaria, although its histological effect on other mouse organs is still unclear. Here, histological examination was performed on mice infected with P. yoelii 17XL; the effect of P. yoelii 17XL infection on anemia and body weight loss, as well as its lesions in the brain, liver, kidney, lung, and spleen, also was investigated. Plasmodium yoelii 17XL-infected red blood cells were sequestered in the microcirculation of the brain and in the kidney. Compared with the nonlethal P. yoelii 17XNL strain, infection by P. yoelii 17XL caused substantial pulmonary edema, severe anemia, and significant body weight loss. Although P. yoelii 17XNL and 17XL produced a similar focal necrosis in the mouse liver, infection of P. yoelii 17XL induced coalescing of red and white pulp. Mortality caused by P. yoelii 17XL may be due to cerebral malaria, as well as respiratory distress syndrome and severe anemia. Plasmodium yoelii 17XL-infected rodent malaria seems to be a useful model for investigating severe malaria caused by P. falciparum.     

Attenuated Plasmodium yoelii lacking purine nucleoside phosphorylase confer protective immunity

Malaria continues to devastate sub-Saharan Africa owing to the emergence of drug resistance to established antimalarials and to the lack of an efficacious vaccine. Plasmodium species have a unique streamlined purine pathway in which the dual specificity enzyme purine nucleoside phosphorylase (PNP) functions in both purine recycling and purine salvage. To evaluate the importance of PNP in an in vivo model of malaria, we disrupted PyPNP, the gene encoding PNP in the lethal Plasmodium yoelii YM strain. P. yoelii parasites lacking PNP were attenuated and cleared in mice. Although able to form gametocytes, PNP-deficient parasites did not form oocysts in mosquito midguts and were not transmitted from mosquitoes to mice. Mice given PNP-deficient parasites were immune to subsequent challenge to a lethal inoculum of P. yoelii YM and to challenge from P. yoelii 17XNL, another strain. These in vivo studies with PNP-deficient parasites support purine salvage as a target for antimalarials. They also suggest a strategy for the development of attenuated nontransmissible metabolic mutants as blood-stage malaria vaccine strains.     

Differential induction of TGF-beta regulates proinflammatory cytokine production and determines the outcome of lethal and nonlethal Plasmodium yoelii infections

Transforming growth factor-beta is an essential moderator of malaria-induced inflammation in mice. In this study, we show that the virulence of malaria infections is dependent upon the cellular source of TGF-beta and the timing of its production. C57BL/6 mice infected with a nonlethal (Py17X) strain of Plasmodium yoelii produce TGF-beta from 5 days postinfection; this correlates with resolution of parasitemia, down-regulation of TNF-alpha, and full recovery. In contrast, infection with the lethal strain Py17XL induces high levels of circulating TGF-beta within 24 h; this is associated with delayed and blunted IFN-gamma and TNF-alpha responses, failure to clear parasites, and 100% mortality. Neutralization of early TGF-beta in Py17XL infection leads to a compensatory increase in IL-10 production, while simultaneous neutralization of TGF-beta and IL-10R signaling leads to up-regulation of TNF-alpha and IFN-gamma, prolonged survival in all, and ultimate resolution of infection in 40% of Py17XL-infected animals. TGF-beta production can be induced in an Ag-specific manner from splenocytes of infected mice, and by cross-linking surface CTLA-4. CD25(+) and CD8(+) cells are the primary source of TGF-beta following Py17X stimulation of splenocytes, whereas Py17XL induces significant production of TGF-beta from adherent cells. In mice immunized against Py17XL, the early TGF-beta response is inhibited and is accompanied by significant up-regulation of IFN-gamma and TNF-alpha and rapid resolution of challenge infections.     

Immunity to Plasmodium yoelii: kinetics of the generation of T and B lymphocytes that passively transfer protective immunity against virulent challenge

Adoptive immunization of A/Tru mice with splenic B cells or T cells from syngeneic donors with a primary, nonvirulent, Plasmodium yoelii (17X) infection confers on these recipients the capacity to resist a challenge infection with a virulent strain (YM) of P. yoelii. Unfractionated spleen cells as well as spleen cells enriched for T or B cells capable of transferring protective immunity were detected as early as Day 7 of the primary nonvirulent infection, and reached peak levels on Day 14. Spleen cells that were harvested from donor animals after resolution of the immunizing infection [on Days 21 or 28] were incapable of transferring protective immunity. The capacity of 7-day immune spleen cells to transfer immunity could be abolished by pretreatment with mitomycin C. In addition, it was found that immunocompetent recipient mice were required for successful adoptive immunization, since thymectomized, irradiated, bone marrow reconstituted mice infused with immune spleen cells failed to survive lethal challenge infections.     

Plasmodium yoelii: influence of antimalarial treatment on acquisition of immunity in BALB/c and DBA/2 mice

The effect of antimalarial drugs on immune responses to the malaria infection is evaluated in vivo using two experimental self-cured rodent models. BALB/c and DBA/2 mice were infected by Plasmodium yoelii 17XNL and 17XL strains, respectively, and then treated with different doses of antimalarial drugs: chloroquine (228mg/kg or 114mg/kg of the body weight) or artesunate (78mg/kg or 39mg/kg). The effect of antimalarial drugs on host immune responses was evaluated by parasitemia, splenocyte IFN-gamma production level, and parasite-specific IgG level in the serum, however, no significant differences were observed between drug-treated and untreated groups. Moreover, most of the infected mice of all groups showed the ability to resist homologous reinfection (challenged on day 60 post-infection), only a few mice experienced transient, low parasitemia. The rechallenged mice were accompanied by high level of parasite-specific IgG. Therefore, this research implicated that, for BALB/c and DBA/2 mice, chloroquine or artesunate treatment of blood-stage P. yoelii infections does not compromise acquired immunity to malaria in either primary infection or upon rechallenge.     

Immunoregulation in murine malaria. Susceptibility of inbred mice to infection with Plasmodium yoelii depends on the dynamic interplay of host and parasite genes

Inbred and H-2 congenic mouse strains were tested for their ability to resist infections with the non-lethal 17X or with the lethal YM isolates of Plasmodium yoelii. DBA/2 and B10.D2 mice, which best resisted infections with non-lethal P. yoelii, were exquisitely susceptible to infection with lethal isolates of this malaria species. In contrast, B6 and B10 mice, which were susceptible to infection with non-lethal P. yoelii, were resistant to infection with the lethal isolates. This reversal of host response phenotype was influenced by H-2 genes, as evidenced by the divergent responses of the H-2 congenic strains B10 and B10.D2. However, a survey of mouse strains sharing common H-2 genes, but expressing different genetic backgrounds, demonstrated that genes outside the H-2 complex also influence the outcome of P. yoelii infections. By enumerating the numbers of P. yoelii-specific antibody-secreting cells in the spleens of infected mice, it was demonstrated that B6 mice, although susceptible to infection with non-lethal P. yoelii, nonetheless made a far stronger anti-parasite response after infection than did resistant DBA/2 mice. Using FACS analysis it was shown that infected B6 mice also produced large amounts of antibodies which bound to the surface of uninfected RBC. Thus, in B6 mice infected with non-lethal P. yoelii, a strong parasite-induced immune response was associated with susceptibility rather than resistance to infection. When T cell-deficient nude mice and their normal littermates were infected with the different isolates of P. yoelii, the nude mice had lower levels of parasitemia and higher RBC counts during the early stages of these infections, and lived longer than did normal littermates after infection with the lethal isolate. These data and the data from studies of B6 and DBA/2 mice support the idea that a strong immune response may be associated with susceptibility rather than resistance to P. yoelii, at least during the early stages of the infection. The finding that a single strain of mouse may present as resistant to infection with one P. yoelii isolate yet be exquisitely susceptible to infection with another suggests that the outcome of these murine malaria infections is dependent on a dynamic interplay between host and parasite genes. Thus, when genetic variability exists in both the host and the parasite populations, as would occur in nature, there may be little directed evolutionary change toward one phenotype or another.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunization with extracellular vesicles excreted by Toxoplasma gondii confers protection in murine infection,activating cellular and humoral responses

The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EVs), purified from Toxoplasma gondii are able to stimulate the protective immunity of experimental mice when administered, as challenge, a highly virulent strain. EVs excreted from T. gondii tachyzoites (RH strain) were purified by chromatography and used for immunization assays in inbred mouse groups (EV-IM). Chronic infected (CHR) and naive (NI) mice were used as control groups, since the immune response is well known. After immunizations, experimental groups were challenged with 100 tachyzoites. Next, parasitemias were determined by real-time PCR (qPCR), and survival levels were evaluated daily. The humoral response was analyzed by detection of IgM, IgG, IgG1 and IgG2a, and opsonization experiments. The cellular response was evaluated in situ by immunohistochemistry on IFN-γ, IL-10, TNF-α and IL-17 expression in cells of five organs (brain, heart, liver, spleen and skeletal muscles). EV immunization reduced parasitemia and increased the survival index in two mouse lineages (A/Sn and BALB/c) infected with a lethal T. gondii strain. EV-IM mice had higher IgG1 levels than IgM or IgG2a. IgGs purified from sera of EV-IM mice were able to opsonize tachyzoites (RH strain), and mice that received these parasites had lower parasitemias, and mortality was delayed 48 h, compared with the same results from those receiving parasites opsonized with IgG purified from NI mice. Brain and spleen cells from EV-IM mice more highly expressed IFN-γ, IL-10 and TNF-α. In conclusion, EV-immunization was capable of inducing immune protection, eliciting high production of IgG1, IFN-γ, IL-10 and TNF-α.     

Suppression of lethal Plasmodium yoelii malaria following protective immunization requires antibody-, IL-4-, and IFN-gamma-dependent responses induced by vaccination and/or challenge infection

Immunization with Plasmodium yoelii merozoite surface protein (PyMSP)-8 protects mice from lethal malaria but does not prevent infection. Using this merozoite surface protein-based vaccine model, we investigated vaccine- and infection-induced immune responses that contribute to protection. Analysis of prechallenge sera from rPyMSP-8-immunized C57BL/6 and BALB/c mice revealed high and comparable levels of Ag-specific IgG, but differences in isotype profile and specificity for conformational epitopes were noted. As both strains of mice were similarly protected against P. yoelii, we could not correlate vaccine-induced responses with protection. However, passive immunization studies suggested that protection resulted from differing immune responses. Studies with cytokine-deficient mice showed that protection was induced by immunization of C57BL/6 mice only when IL-4 and IFN-gamma were both present. In BALB/c mice, the absence of either IL-4 or IFN-gamma led to predictable shifts in the IgG isotype profile but did not reduce the magnitude of the Ab response induced by rPyMSP-8 immunization. Immunized IL-4-/- BALB/c mice were solidly protected against P. yoelii. To our surprise, immunized IFN-gamma-/- BALB/c mice initially controlled parasite growth but eventually succumbed to infection. Analysis of cytokine production revealed that P. yoelii infection induced two distinct peaks of IFN-gamma that correlated with periods of controlled parasite growth in intact, rPyMSP-8-immunized BALB/c mice. Maximal parasite growth occurred during a period of sustained TGF-beta production. Combined, the data indicate that induction of protective responses by merozoite surface protein-based vaccines depends on IL-4 and IFN-gamma-dependent pathways and that vaccine efficacy is significantly influenced by host responses elicited upon infection.     

IL-17 is a critical component of vaccine-induced protection against lung infection by lipopolysaccharide-heterologous strains of Pseudomonas aeruginosa

In a murine model of acute fatal pneumonia, we previously showed that nasal immunization with a live-attenuated aroA deletant of Pseudomonas aeruginosa strain PAO1 elicited LPS serogroup-specific protection, indicating that opsonic Ab to the LPS O Ag was the most important immune effector. Because P. aeruginosa strain PA14 possesses additional virulence factors, we hypothesized that a live-attenuated vaccine based on PA14 might elicit a broader array of immune effectors. Thus, an aroA deletant of PA14, denoted PA14DeltaaroA, was constructed. PA14DeltaaroA-immunized mice were protected against lethal pneumonia caused not only by the parental strain but also by cytotoxic variants of the O Ag-heterologous P. aeruginosa strains PAO1 and PAO6a,d. Remarkably, serum from PA14DeltaaroA-immunized mice had very low levels of opsonic activity against strain PAO1 and could not passively transfer protection, suggesting that an antibody-independent mechanism was needed for the observed cross-serogroup protection. Compared with control mice, PA14DeltaaroA-immunized mice had more rapid recruitment of neutrophils to the airways early after challenge. T cells isolated from P. aeruginosa DeltaaroA-immunized mice proliferated and produced IL-17 in high quantities after coculture with gentamicin-killed P. aeruginosa. Six hours following challenge, PA14DeltaaroA-immunized mice had significantly higher levels of IL-17 in bronchoalveolar lavage fluid compared with unimmunized, Escherichia coli-immunized, or PAO1DeltaaroA-immunized mice. Antibody-mediated depletion of IL-17 before challenge or absence of the IL-17 receptor abrogated the PA14DeltaaroA vaccine's protection against lethal pneumonia. These data show that IL-17 plays a critical role in antibody-independent vaccine-induced protection against LPS-heterologous strains of P. aeruginosa in the lung.     

Delineation of epitopes on the Py235 rhoptry antigen of Plasmodium yoelii YM

The 235-kDa antigenic rhoptry protein Py235 of Plasmodium yoelii is encoded by a large, highly polymorphic gene family. Monoclonal antibodies to some of these antigens have been shown to attenuate the virulence of the lethal YM strain of the parasite, converting a potentially fatal YM infection to a fulminating one typical of the nonlethal 17X strain, by inducing a switch in target cell preference from mature red blood cells to reticulocytes. The reason for this is not known but would suggest that antigenic determinants of Py235 may be useful in or as subunit vaccines. To identify such determinants, we constructed an epitope expression library of one Py235 variant and screened the library with the antibodies. Thus, we mapped 5- and 12-amino acid epitopes to the C-terminus of the antigen. Both epitopes were more reactive with protective than with nonprotective monoclonal antibodies. This may explain the differential protection conferred by these antibodies upon their passive transfer into mice.     

不同毒力疟原虫感染早期根治性治疗对再感染体液免疫应答的影响

为探讨不同毒力疟原虫感染早期根治性治疗对再感染体液免疫应答的影响,用致死型和非致死型约氏疟原虫感染DBA/2小鼠,感染后3 d进行根治性治疗,并于初次感染后90 d进行再感染。计数红细胞感染率和检测再感染前（0 d）和再感染后（1、3、5 d）不同时间点脾细胞中活化性B细胞百分率以及血清中IgG、IgG1和IgG2 a水平,结果显示,再感染后2组小鼠均出现短暂的低水平虫体血症,再感染后第3天活化性B细胞、IgG、IgG1和IgG2 a均出现有意义的升高,但在每一相同检测时间点,2组小鼠的虫体血症、活化性B细胞百分率、IgG、IgG1和IgG2 a水平均没有显著差异。这些结果表明,不同毒力疟原虫感染早期的根治性治疗并不影响宿主在再感染时产生有效的体液免疫应答,强毒株原虫感染也能获得与弱毒株相同的可抵御再感染的能力。     

Erythrocyte membrane-associated immunoglobulins during malaria infection of mice.

Immunoglobulin (Ig) is associated with erythrocyte membranes during infection of A/J mice with Plasmodium berghei. It was detected by agglutination of the cells with rabbit antibodies to mouse IgG and by a radioimmunoassay with 125I-labelled rabbit antibodies to mouse IgG. As shown by the degree of agglutination and of binding of radiolabeled antibodies to the erythrocyte membranes, the amount of Ig increased with time after infection and paralleled parasitemia and reticulocytosis. The erythrocyte-associated immunoglobulins are mainly IgG but IgM was also present on the cells of some mice. A large proportion of the Ig could be eluted at 37 degrees C and was analyzed by immunoprecipitation and acrylamide gel electrophoresis. A radioautographic study with 125I-labeled anti-mouse IgG revealed that both parasitized and nonparasitized reticulocytes of infected mice had much larger amounts of membrane-bound immunoglobulin than did mature, nonparasitized erythrocytes. The nature of the bonds between the Ig and the surface membrane of the reticulocytes is not known. The Ig could be part of immune complexes nonspecifically bound to the cell surface. However, since we have not detected Fc or C3d receptors on reticulocytes, it is possible that the Ig constitutes autoantibodies against reticulocytes or antibodies against parasitic antigens present on the cell membrane.     

Protection against malaria by anti-erythropoietin antibody due to suppression of erythropoiesis in the liver and at other sites

We have previously reported that erythropoiesis commences in the liver and spleen after malarial infection, and that newly generated erythrocytes in the liver are essential for infection of malarial parasites as well as continuation of infection. At this time, erythropoietin (EPO) is elevated in the serum. In the present study, we administered EPO or anti-EPO antibody into C57BL/6 (B6) mice to modulate the serum level of EPO. When mice were infected with a non-lethal strain (17NXL) of Plasmodium yoelii (blood-stage infection of 10(4) parasitized erythrocytes per mouse), parasitemia continued for 1 month, showing a peak at day 17. Daily injection of EPO (200 IU/day per mouse) from day five to day 14 prolonged parasitemia, whereas injection of anti-EPO antibody (1.5 mg/day per mouse) every second day from day five to day 28 decreased it. Erythropoiesis was confirmed in the liver, spleen and bone marrow by the appearance of nucleated erythrocytes (TER119+). When anti-EPO antibody was injected by the same protocol into mice infected with a lethal strain (17XL) of P. yoelii, all mice showed decreased parasitemia and recovered from the infection. These results suggest that the use of anti-EPO antibody after malarial infection may be of therapeutic value in severe cases of malaria.     

Passive transfer of antibodies protects immunocompetent and imunodeficient mice against lethal Ebola virus infection without complete inhibition of viral replication

Ebola hemorrhagic fever is a severe, usually fatal illness caused by Ebola virus, a member of the filovirus family. The use of nonhomologous immune serum in animal studies and blood from survivors in two anecdotal reports of Ebola hemorrhagic fever in humans has shown promise, but the efficacy of these treatments has not been demonstrated definitively. We have evaluated the protective efficacy of polyclonal immune serum in a mouse model of Ebola virus infection. Our results demonstrate that mice infected subcutaneously with live Ebola virus survive infection and generate high levels of anti-Ebola virus immunoglobulin G (IgG). Passive transfer of immune serum from these mice before challenge protected upto 100% of naive mice against lethal Ebola virus infection. Protection correlated with the level of anti-Ebola virus IgG titers, and passive treatment with high-titer antiserum was associated with a delay in the peak of viral replication. Transfer of immune serum to SCID mice resulted in 100% survival after lethal challenge with Ebola virus, indicating that antibodies alone can protect from lethal disease. Thus antibodies suppress or delay viral growth, provide protection against lethal Ebola virus infection, and may not require participation of other immune components for protection.     

IL-10 from CD4CD25Foxp3CD127 adaptive regulatory T cells modulates parasite clearance and pathology during malaria infection

The outcome of malaria infection is determined, in part, by the balance of pro-inflammatory and regulatory immune responses. Failure to develop an effective pro-inflammatory response can lead to unrestricted parasite replication, whilst failure to regulate this response leads to the development of severe immunopathology. IL-10 and TGF-beta are known to be important components of the regulatory response, but the cellular source of these cytokines is still unknown. Here we have examined the role of natural and adaptive regulatory T cells in the control of malaria infection and find that classical CD4+CD25(hi) (and Foxp3+) regulatory T cells do not significantly influence the outcome of infections with the lethal (17XL) strain of Plasmodium yoelii (PyL). In contrast, we find that adaptive IL-10-producing, CD4+ T cells (which are CD25-, Foxp3-, and CD127- and do not produce Th1, Th2, or Th17 associated cytokines) that are generated during both PyL and non-lethal P. yoelii 17X (PyNL) infections are able to down-regulate pro-inflammatory responses and impede parasite clearance. In summary, we have identified a population of induced Foxp3- regulatory (Tr1) T cells, characterised by production of IL-10 and down regulation of IL-7Ralpha, that modulates the inflammatory response to malaria.     

Allicin enhances host pro-inflammatory immune responses and protects against acute murine malaria infection

ABSTRACT: BACKGROUND: During malaria infection, multiple pro-inflammatory mediators including IFN-gamma, TNF and nitric oxide (NO) play a crucial role in the protection against the parasites. Modulation of host immunity is an important strategy to improve the outcome of malaria infection. Allicin is the major biologically active component of garlic and shows anti-microbial activity. Allicin is also active against protozoan parasites including Plasmodium, which is thought to be mediated by inhibiting cysteine proteases. In this study, the immunomodulatory activities of allicin were assessed during acute malaria infection using a rodent malaria model Plasmodium yoelii 17XL. METHODS: To determine whether allicin modulates host immune responses against malaria infection, mice were treated with allicin after infection with P. yoelii 17XL. Mortality was checked daily and parasitaemia was determined every other day. Pro-inflammatory mediators and IL-4 were quantified by ELISA, while NO level was determined by the Griess method. The populations of dendritic cells (DCs), macrophages, CD4+ T and regulatory T cells (Treg) were assessed by FACS. RESULTS: Allicin reduced parasitaemia and prolonged survival of the host in a dose-dependent manner. This effect is at least partially due to improved host immune responses. Results showed that allicin treatment enhanced the production of pro-inflammatory mediators such as IFN-gamma, TNF, IL-12 and NO. The absolute numbers of CD4+ T cells, DCs and macrophages were significantly higher in allicin-treated mice. In addition, allicin promoted the maturation of CD11c+ DCs, whereas it did not cause major changes in IL-4 and the level of anti-inflammatory cytokine IL-10. CONCLUSIONS: Allicin could partially protect host against P. yoelii 17XL through enhancement of the host innate and adaptive immune responses.     

约氏疟原虫Pys48核酸疫苗传播阻断效应的实验研究

探讨约氏疟原虫（Plasmodium yoelii17XL）Pys48核酸疫苗免疫BALB/c小鼠的特异性抗体产生特点及其效应。将Pys48核酸疫苗肌肉内注射免疫BALB/c小鼠,并以空质粒注射组作为对照,3次免疫后通过P.y17XL攻击小鼠;采用ELISA检测免疫后小鼠血清中特异性抗体水平;通过P.y17XL感染小鼠,取其感染后第3天含有配子体的血液进行体外培养,观察合子、动合子的形成数量。ELISA结果显示疫苗免疫组小鼠血清特异性抗体滴度明显高于对照组;而合子、动合子数量明显低于对照组。提示Pys48核酸疫苗具有良好的免疫原性,免疫小鼠后可以建立起有效的传播阻断效应。