163 research outputs found
Immune-mediated competition in rodent malaria is most likely caused by induced changes in innate immune clearance of merozoites
Malarial infections are often genetically diverse, leading to competitive interactions between parasites. A quantitative understanding of the competition between strains is essential to understand a wide range of issues, including the evolution of virulence and drug resistance. In this study, we use dynamical-model based Bayesian inference to investigate the cause of competitive suppression of an avirulent clone of Plasmodium chabaudi (AS) by a virulent clone (AJ) in immuno-deficient and competent mice. We test whether competitive suppression is caused by clone-specific differences in one or more of the following processes: adaptive immune clearance of merozoites and parasitised red blood cells (RBCs), background loss of merozoites and parasitised RBCs, RBC age preference, RBC infection rate, burst size, and within-RBC interference. These processes were parameterised in dynamical mathematical models and fitted to experimental data. We found that just one parameter μ, the ratio of background loss rate of merozoites to invasion rate of mature RBCs, needed to be clone-specific to predict the data. Interestingly, μ was found to be the same for both clones in single-clone infections, but different between the clones in mixed infections. The size of this difference was largest in immuno-competent mice and smallest in immuno-deficient mice. This explains why competitive suppression was alleviated in immuno-deficient mice. We found that competitive suppression acts early in infection, even before the day of peak parasitaemia. These results lead us to argue that the innate immune response clearing merozoites is the most likely, but not necessarily the only, mediator of competitive interactions between virulent and avirulent clones. Moreover, in mixed infections we predict there to be an interaction between the clones and the innate immune response which induces changes in the strength of its clearance of merozoites. What this interaction is unknown, but future refinement of the model, challenged with other datasets, may lead to its discovery
Biological properties of the digestive vacuole of Plasmodium falciparum: Activation of complement and coagulation
Plasmodium falciparum is an intracellular protozoan parasite that has been associated with humans since the dawn of time and causes severe forms of malaria. It is a major health problem around the globe and causes highest toll of death among children less than five years of age in developing countries. An infected female Anopheles mosquito injects malaria sporozoites into the skin while taking a blood meal. The sporozoites, which are released into the blood stream, reaches the liver where they undergo exoerythrocytic schizogony. After exoerythrocytic schizogony, millions of merozoites are released into the blood stream and infect new red blood cells, where they undergo erythrocytic schizogony in a cyclic manner. The erythrocytic schizogony stage of Plasmodium life cycle is where all clinical manifestations of malaria as a disease become apparent. The clinical symptoms like fever, headache, jaundice, vomiting have been associated with hyperparasitemia and these clinical symptoms coincide with the cyclical release of malaria parasites during schizonts rupture. A severe form of malaria develops as a consequence of capillaric sequestration of parasitized red blood cells (pRBC) and rosetting of pRBC with uninfected red blood cells which obstruct the blood flow to the brain. Activation of complement and coagulation, and increase in vascular permeability further aggravates severity of the disease which can lead to microcirculatory disturbances with comatous death as the ultimate outcome.
Rupture of each Plasmodium falciparum infected red blood cell releases 8-32 infective merozoites along with a single digestive vacuole into the blood stream. The released digestive vacuole is an organelle in which hemozoin is surrounded by an intact membrane. We have discovered that the digestive vacuoles have the capacity to dually activate the complement and coagulation systems. Activation of complement and coagulation requires an intact DV membrane. The complement and coagulation activating properties of the DV are inhibited by low molecular weight dextran sulfate. In non-immune serum, DVs are opsonised with complement C3b and rapidly phagocytosed by polymorphonuclear granulocytes (PMN). Upon rupture, DVs lost its functional activities and the extracted malaria pigment from the DV organelle is not engulfed by the PMN. Liberated merozoites are not opsonized in non-immune serum and escape phagocytosis. High titered anti-malarial antibodies from immune patients mediate some uptake of the merozoites, but to an extent that is not sufficient to markedly reduce re-invasion rates. Engulfment of DVs by PMN induces a respiratory burst, but the generated reactive oxygen species (ROS) are unable to suppress the infective capacity of invading merozoites. Finally, ingested DVs drive the PMN into a state of functional exhaustion. Upon challenging of PMN with bacteria after DV ingestion, the ability to phagocytose bacteria prevails, but their capacity to mount a respiratory burst is reduced and microbicidal activity is compromised. We propose that these events might be linked to the development of septicemic episodes in patients with severe malaria in sub-Saharan African countries
Studies evaluating the possible evolution of malaria parasites in response to blood-stage vaccination
Drug resistance is one of the most medically relevant forms of pathogen evolution.
To date, vaccines have not failed with the same depressing regularity as drugs. Does
that then make vaccines evolution-proof? In the face of vaccination, pathogens are
thought to evolve in two ways: by evolving epitope changes at the antigenic target of
vaccination (epitope evolution); or by evolving changes at other antigenic loci, some
of which may involve virulence (virulence evolution). The fundamental difference
between these two forms of evolution is that virulence evolution could lead to
disease outcomes in unvaccinated people that are more severe than would have been
seen prior to evolution. One of the theoretical assumptions of virulence evolution is
that more virulent parasites will have a selective advantage over less virulent
parasites in an immunized host, and are thus more likely to be transmitted. The
assumption is that more virulent parasites may be competitively more superior in
mixed infections, or may be better able to evade/modulate the host immune response.
Thus, the aim of this thesis was to experimentally test whether more virulent
parasites have a within-host selective advantage in an immunized host or whether
vaccine efficacy is more likely to depend on genetic differences at the targeted sites
of vaccination.
I used clones (genotypes) of the rodent malaria Plasmodium chabaudi originally
derived from wild-caught Thicket (Thamnomys rutilans) rats to infect laboratory
mice and a rodent analogue of the candidate blood-stage malaria vaccine apical
membrane antigen 1 (AMA-1). I found that within-host selection did not depend on
parasite virulence, and that protective efficacy depended on genotype-specific
differences at the vaccine target. Vaccine-induced protection was not enhanced by
including a number of allelic variants. However, such genotype-specific responses
were only observed when the vaccine was tested against genetically distinct P.
chabaudi parasites. When one P. chabaudi genotype was serially passaged through
naïve mice the derived line was more virulent and was subsequently less well
controlled by vaccine-induced immunity. In other experiments I found within host
competition not to be immune-mediated. Thus my results suggest that vaccination
has the potential to select for more virulent parasites but that the selective advantage
is likely to be independent of competition. The selective advantage may be
attributable to the enhanced immune evasion of more virulent parasites. However,
without genetic markers of virulence, the mechanisms that mediate this selection
remain unknown.
My thesis contributes towards a growing body of evidence that vaccines have the
potential to differently alter the within-host parasite dynamics of particular pathogen
genotypes and that the selection imposed is likely to be system specific, depending
on the fine specificity of the vaccine-induced responses and the identity of infecting
parasites. Although vaccine potency may not be enhanced by including more than
one allelic variant of an antigen, multi-valent vaccines may be one of the best ways
to avoid the inadvertent selection for more virulent malaria parasites
Plasmodium chabaudi limits early Nippostrongylus brasiliensis-induced pulmonary immune activation and Th2 polarization in co-infected mice
<p>Abstract</p> <p>Background</p> <p>Larvae of several common species of parasitic nematodes obligately migrate through, and often damage, host lungs. The larvae induce strong pulmonary Type 2 immune responses, including T-helper (Th)2 cells as well as alternatively activated macrophages (AAMφ) and associated chitinase and Fizz/resistin family members (ChaFFs), which are thought to promote tissue repair processes. Given the prevalence of systemic or lung-resident Type 1-inducing pathogens in geographical areas in which nematodes are endemic, we wished to investigate the impact of concurrent Type 1 responses on the development of these Type 2 responses to nematode larval migration. We therefore infected BALB/c mice with the nematode <it>Nippostrongylus brasiliensis</it>, in the presence or absence of <it>Plasmodium chabaudi chabaudi </it>malaria parasites. Co-infected animals received both infections on the same day, and disease was assessed daily before immunological measurements were taken at 3, 5, 7 or 20 days post-infection.</p> <p>Results</p> <p>We observed that the nematodes themselves caused transient loss of body mass and red blood cell density, but co-infection then slightly ameliorated the severity of malarial anaemia. We also tracked the development of immune responses in the lung and thoracic lymph node. By the time of onset of the adaptive immune response around 7 days post-infection, malaria co-infection had reduced pulmonary expression of ChaFFs. Assessment of the T cell response demonstrated that the Th2 response to the nematode was also significantly impaired by malaria co-infection.</p> <p>Conclusion</p> <p><it>P. c. chabaudi </it>co-infection altered both local and lymph node Type 2 immune activation due to migration of <it>N. brasiliensis </it>larvae. Given recent work from other laboratories showing that <it>N. brasiliensis</it>-induced ChaFFs correlate to the extent of long-term lung damage, our results raise the possibility that co-infection with malaria might alter pulmonary repair processes following nematode migration. Further experimentation in the co-infection model developed here will reveal the longer-term consequences of the presence of both malaria and helminths in the lung.</p
Characterizing the gut microbiota during plasmodium infection and antimalarial treatment.
Plasmodium, the parasitic cause of malaria, is a global pathogen, annually causing 216 million infections and 445,000 deaths. As drug resistance continues develop and no effective vaccine is available, it is critical to understand the factors underlying the severity of this disease. Plasmodium is an extra-gastrointestinal tract infection where the parasite infects red blood cells causing clinical malaria. However, recent publications have pointed to interactions between the gut microbiota and malaria. With this in mind, the role of the gut microbiota in malaria infection was studied. C57BL/6 mice from different vendors displayed differential resistance and susceptibility to severe malaria, and cecal contents transplanted from these mice to germ-free mice recapitulated the observed phenotypes. Similarly, resistant mice possessed a much more robust humoral immune response than susceptible mice, which is critical for Plasmodium clearance. When the cecal contents from resistant and susceptible mice were sequenced, Lactobacillus and Bifidobacterium genera were enriched in resistant mice. Moreover, treating susceptible mice with probiotics containing these bacterial genera after antibiotic administration led to a lower parasite burden. These observations point to a previously unknown role for the microbiota in modulating the severity of malaria. To further characterize the interactions between the host and gut microbiota in malaria, different components of gut homeostasis were investigated in both mild and severe disease. While intestinal permeability increased in both resistant and susceptible mice, there were no significant differences between the two groups. However, susceptible mice were shown to have greater numbers of lamina propria immune cells as well as greater abundances of cecal metabolites and bile acids during infection compared to resistant mice. Consistent with the decreased abundance of bile acids, histology showed much greater and prolonged damage and hemozoin deposition in the livers of susceptible mice compared to resistant mice. Despite these differences, the microbiota composition of resistant and susceptible mice became more similar during infection, although these changes were not associated with susceptibility or resistance when the altered cecal contents were transferred into germ-free mice. However, there were distinct differences in the functional capacity of the resistant and susceptible microbiota during infection. Susceptible mice showed significant increases in genes related to bacterial motility and flagellar assembly. Overall, there are profound differences in gut homeostasis during severe and mild Py infection. Finally, it was investigated whether antimalarial drugs, particularly clinically relevant artemisinin combination therapies (ACTs), could disrupt the gut microbiota. As previously shown, the composition of the gut microbiota alone can modulate the severity of Py infection; if ACTs change the microbiota composition, future infections could be more severe. To test this hypothesis, two common ACTs, artesunate plus amodiaquine and artemether plus lumefantrine, were used to orally treat mice while fecal pellets were collected to characterize the gut microbiota before and after treatment. After either ACT treatment, the overall species abundance in mice was similar to baseline. While alpha diversity remained unchanged by any treatment, there were minor, inconsistent changes in beta diversity that returned to baseline. With these findings, it does not appear that ACTs change the gut microbiota. This work has greatly increased the scientific knowledge concerning the three-fold interaction between host, gut microbiota, and Plasmodium. While much work still needs to be done, these findings can provide a contextual foundation on which future work can be built
Immunity against sexual stage Plasmodium falciparum and Plasmodium vivax parasites.
The efficient spread of malaria from infected humans to mosquitoes is a major challenge for malaria elimination initiatives. Gametocytes are the only Plasmodium life stage infectious to mosquitoes. Here, we summarize evidence for naturally acquired anti-gametocyte immunity and the current state of transmission blocking vaccines (TBV). Although gametocytes are intra-erythrocytic when present in infected humans, developing Plasmodium falciparum gametocytes may express proteins on the surface of red blood cells that elicit immune responses in naturally exposed individuals. This immune response may reduce the burden of circulating gametocytes. For both P. falciparum and Plasmodium vivax, there is a solid evidence that antibodies against antigens present on the gametocyte surface, when co-ingested with gametocytes, can influence transmission to mosquitoes. Transmission reducing immunity, reducing the burden of infection in mosquitoes, is a well-acknowledged but poorly quantified phenomenon that forms the basis for the development of TBV. Transmission enhancing immunity, increasing the likelihood or intensity of transmission to mosquitoes, is more speculative in nature but is convincingly demonstrated for P. vivax. With the increased interest in malaria elimination, TBV and monoclonal antibodies have moved to the center stage of malaria vaccine development. Methodologies to prioritize and evaluate products are urgently needed
Loss and recovery of Humoral Immunity to Influenza Virus following Malaria Infection
The mechanisms of maintenance of humoral immunity to infectious pathogens, particularly the contributions of memory B cells and long-lived plasma cells in maintaining specific serum antibody titres, are not well understood. Furthermore, it is not clear whether sequential heterologous humoral immune responses and disease pathology can result in the dysregulation and loss of previously acquired antibody-mediated immune responses to unrelated antigens. Here, depletion of memory B cells using anti-hCD20 monoclonal antibodies in hCD20 transgenic mice was used to dissect the role of memory B cells and long-lived plasma cells in maintaining long-term serum antibodies after intranasal Influenza A infection. Next, an experimental model of sequential infections with Influenza A/PR/8/34 and Plasmodium chabaudi chabaudi (AS) was set up, with a 15-20 week interval between the infections, in order to investigate whether sequential infection with P. chabaudi would affect pre-established humoral immunity to Influenza A. This study demonstrates that memory B cells are essential for the maintenance of long-lived serum Ab titres to Influenza A, as depletion of memory B cells results in the eventual loss of long-lived plasma cells and serum antibodies. Sequential infection with P. chabaudi results in the loss of pre-established serum antibodies to Influenza A by inducing the loss of long-lived plasma cells in an FcγRI,II,III-dependent manner, and this renders mice susceptible to secondary infection with Influenza A. However, this loss of pre-established humoral immunity is temporary, as serum antibodies do eventually return to normal levels. These findings demonstrate a mechanism shared by memory B cells and long-lived plasma cells which ensures that serum antibodies are maintained for long periods of time in the face of continuous generation and incorporation of new specificities throughout the lifetime of the host. A more complete understanding of the parameters that affect the longevity of immunological memory and how heterologous infections influence this will be vital in our understanding of the effect of continuous exposure to infectious pathogens on the efficacy and longevity of previously established immune memory
The Ly-4+ T Lymphocyte Subset in the Host Immune Response to the Asexual Stages of Plasmodium chabaudi chabaudi
T cells play a major role in acquired immunity to the asexual erythrocytic stages of malaria parasites. In different host/parasite combinations there is evidence that human CD4+ lymphocytes or their murine equivalent, Ly-4+, can act as helper cells in the production of protective Ab and also mediate cellular protective functions. The details of how the effector mechanisms operate in vivo, however, are not understood clearly. There is indirect evidence supporting an important role for Ly-4-bearing lymphocytes in the protective immune response to Plasmodium chabaudi chabaudi AS strain, a good animal model of P. falciparum infection. Experiments were performed to examine the nature of the Ly-4+ response to this parasite both in vivo and in vitro in order to characterise the cells responsible for the mediation of protective activity. Initial studies showed that during the course of a primary infection of P. c. chabaudi AS, there was a marked transient lymphocytosis in the peripheral blood, which occurred at a time just after peak parasitaemia (d 12-13 p. i. ). The adoptive transfer to syngeneic NIH recipients of either peripheral blood or splenic lymphocytes taken from donors at this early stage of infection conferred protection against homologous challenge. This was manifested in both competent and sublethally irradiated recipients as a reduced level and quickened remission of primary parasitaemia, and as a more rapid clearance of pRBC from the blood stream, compared to control mice receiving unprimed lymphocytes. Although it was possible to transfer immunity with preparations enriched for either T or B cells, optimal protection was conferred by an unfractionated population containing both lymphocyte phenotypes, suggesting that there was a degree of synergistic activity between parasite-primed T and B cells in the control of malarial infection. This concept was supported further by examination of serum Ab titres for recipients of semi-immune T, B or T & B spleen cells. In each instance, the level of specific anti-P. c. chabaudi AS Ig reached a peak between d 31-33 p. i. , at or just prior to recrudescence, but the highest titres were recorded for recipients of a mixed splenic population. Since serum Ig levels were quite low during the first wave of patent parasitaemia, it suggested that resolution of acute infection was achieved largely through Ab-independent mechanisms of immunity. This correlated well with a significantly quicker remission of primary parasitaemia observed in sublethally irradiated recipients of semi-immune T cells, compared to similarly treated mice receiving the same inoculum size of either B or T & B cells. To dissect further the protective immune response in this model, splenic T lymphocytes were taken from P. c. chabaudi AS strain-infected NIH mice on d 16 and d 20 of primary infection and after resolution of secondary and tertiary infections, and each of these preparations established as cell lines in vitro using a lysed extract of pRBC as the source of antigenic stimulation. All four lines were maintained in long term culture and all proliferated specifically in response to P. c. chabaudi AS Ag processed and presented by syngeneic APC. It was shown that recognition of the APC/Ag complex by T cells was an MHC class ll-restricted phenomenon, each cell line requiring APC of compatible H-2 haplotype for an in vitro proliferative response. By using surface immunofluorescence and the complement-mediated cytotoxicity assay, each line was characterised phenotypically as Ly-4+, i. e. belonging to the helper/inducer T cell subset. In vivo, adoptive transfer of each Ly-4+ line was effective in conferring protective immunity to naive and to immunocompromised mice. This was demonstrable, compared to controls given naive T and/or B cells, as both a reduced level and shortened duration of primary parasitaemia, and as a quicker parasite elimination. Inoculation of the P. c. chabaudi AS-reactive lines into non-immune mice challenged with genotypic or phenotypic variant pRBC indicated that there was a strain-specific element of the immunity transferred. Although mice were able to control infection with heterologous parasites, the greatest protection was conferred against challenge with the homologous pRBC to which the lines had been raised. For the two Ly-4+ lines taken from reinfected mice, the protective activity against P. c. chabaudi AS challenge upon adoptive transfer into adult-thymectomised, irradiated and bone marrow-reconstituted mice was improved significantly by the cotransfer of additional naive B cells. This suggested that these Ly-4-bearing lymphocytes act by Ab-mediated mechanisms in vivo. (Abstract shortened by ProQuest.)
The leucine-rich repeat immune protein family in malaria vector mosquitoes
A novel mosquito-specific family of leucine-rich repeat immune proteins (LRIMs) was recently identified in Anopheles gambiae, the major vector of malaria in Africa. The founding family members, LRIM1 and APL1C, form a heterodimer circulating in the mosquito hemolymph and mediate killing of malaria parasites through their interaction with the complement C3-like effector, TEP1. This PhD thesis investigated the role of the LRIM family in mosquito immunity.
Transcriptional profiling demonstrated that different LRIMs show distinct responses to malarial, fungal, bacterial and viral infections as well as to blood feeding. Certain LRIMs are broadly induced whereas others respond specifically to particular immune challenges, suggesting that there is specificity within the LRIM family towards different infections. RNA interference-mediated gene silencing identified LRIM9 as a novel antagonist of the rodent malaria parasite, Plasmodium berghei, with a putative role in parasite melanisation. Silencing LRIM9 partially inhibits the activity of phenoloxidase, a key enzyme in the melanisation pathway, but does not affect tissue melanisation. Unlike the LRIM1/APL1C heterodimer, LRIM9 circulates as a monomer in the mosquito hemolymph and is not involved in antibacterial defence. As LRIM9 does not interact with TEP1 and is not involved in TEP1 activity against Plasmodium, its precise function in the mosquito immune system remains unclear. Importantly, LRIM9 is highly upregulated in female mosquitoes after blood feeding but does not function in mosquito reproduction.
The findings reported in this thesis indicate that the LRIM family has diversified to respond to infections with different microbes that mosquitoes encounter in their blood feeding lifestyle. LRIM9 is an important novel candidate for involvement in defence against malaria parasites. We hypothesise that LRIM9 is induced after blood feeding in anticipation of blood-borne infections, which is an original concept in mosquito immunity
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