Identification of a neutralizing epitope within minor repeat region of Plasmodium falciparum CS protein

Malaria remains a major cause of morbidity and mortality worldwide with 219 million infections and 435,000 deaths predominantly in Africa. The infective Plasmodium sporozoite is the target of a potent humoral immune response that can protect murine, simian and human hosts against challenge by malaria-infected mosquitoes. Early murine studies demonstrated that sporozoites or subunit vaccines based on the sporozoite major surface antigen, the circumsporozoite (CS) protein, elicit antibodies that primarily target the central repeat region of the CS protein. In the current murine studies, using monoclonal antibodies and polyclonal sera obtained following immunization with P. falciparum sporozoites or synthetic repeat peptides, we demonstrate differences in the ability of these antibodies to recognize the major and minor repeats contained in the central repeat region. The biological relevance of these differences in fine specificity was explored using a transgenic P. berghei rodent parasite expressing the P. falciparum CS repeat region. In these in vitro and in vivo studies, we demonstrate that the minor repeat region, comprised of three copies of alternating NANP and NVDP tetramer repeats, contains an epitope recognized by sporozoite-neutralizing antibodies. In contrast, murine monoclonal antibodies specific for the major CS repeats (NANP)n could be isolated from peptide-immunized mice that had limited or no sporozoite-neutralizing activity. These studies highlight the importance of assessing the fine specificity and functions of antirepeat antibodies elicited by P. falciparum CS-based vaccines and suggest that the design of immunogens to increase antibody responses to minor CS repeats may enhance vaccine efficacy

Phase I Trial of an Alhydrogel Adjuvanted Hepatitis B Core Virus-Like Particle Containing Epitopes of Plasmodium falciparum Circumsporozoite Protein

The objectives of this non-randomized, non-blinded, dose-escalating Phase I clinical trial were to assess the safety, reactogenicity and immunogenicity of ICC-1132 formulated with Alhydrogel (aluminum hydroxide) in 51 healthy, malaria-naive adults aged 18 to 45 years. ICC-1132 (Malariavax) is a recombinant, virus-like particle malaria vaccine comprised of hepatitis core antigen engineered to express the central repeat regions from Plasmodium falciparum circumsporozoite protein containing an immunodominant B [(NANP)3] epitope, an HLA-restricted CD4 (NANPNVDPNANP) epitope and a universal T cell epitope (T*) (amino acids 326—345, NF54 isolate). We assessed an Alhydrogel (aluminum hydroxide)-adjuvanted vaccine formulation at three ICC-1132 dose levels, each injected intramuscularly (1.0 mL) on study days 0, 56 and 168. A saline vaccine formulation was found to be unstable after prolonged storage and this formulation was subsequently removed from the study. Thirty-two volunteers were followed for one year. Local and systemic adverse clinical events were measured and immune responses to P. falciparum and hepatitis B virus core antigens were determined utilizing the following assays: IgG and IgM ELISA, indirect immunofluorescence against P. falciparum sporozoites, circumsporozoite precipitin (CSP) and transgenic sporozoite neutralization assays. Cellular responses were measured by proliferation and IL-2 assays. Local and systemic reactions were similarly mild and well tolerated between dose cohorts. Depending on the ICC-1132 vaccine concentration, 95 to 100% of volunteers developed antibody responses to the ICC-1132 immunogen and HBc after two injections; however, only 29—75% and 29—63% of volunteers, respectively, developed malaria-specific responses measured by the malaria repeat synthetic peptide ELISA and IFA; 2 of 8 volunteers had positive reactions in the CSP assay. Maximal transgenic sporozoite neutralization assay inhibition was 54%. Forty-seven to seventy-five percent demonstrated T cell proliferation in response to ICC-1132 or to recombinant circumsporozoite protein (rCS) NF-54 isolate. This candidate malaria vaccine was well tolerated, but the vaccine formulation was poorly immunogenic. The vaccine may benefit from a more powerful adjuvant to improve immunogenicity

"Before we used to get sick all the time": perceptions of malaria and use of long-lasting insecticide-treated bed nets (LLINs) in a rural Kenyan community

<p>Abstract</p> <p>Background</p> <p>Malaria is a leading global cause of preventable morbidity and mortality, especially in sub-Saharan Africa, despite recent advances in treatment and prevention technologies. Scale-up and wide distribution of long-lasting insecticide-treated nets (LLINs) could rapidly decrease malarial disease in endemic areas, if used properly and continuously. Studies have shown that effective use of LLINs depends, in part, upon understanding causal factors associated with malaria. This study examined malaria beliefs, attitudes, and practices toward LLINs assessed during a large-scale integrated prevention campaign (IPC) in rural Kenya.</p> <p>Methods</p> <p>Qualitative interviews were conducted with 34 IPC participants who received LLINs as part of a comprehensive prevention package of goods and services. One month after distribution, interviewers asked these individuals about their attitudes and beliefs regarding malaria, and about their use of LLINs.</p> <p>Results</p> <p>Virtually all participants noted that mosquitoes were involved in causing malaria, though a substantial proportion of participants (47 percent) also mentioned an incorrect cause in addition to mosquitoes. For example, participants commonly noted that the weather (rain, cold) or consumption of bad food and water caused malaria. Regardless, most participants used the LLINs they were given and most mentioned positive benefits from their use, namely reductions in malarial illness and in the costs associated with its diagnosis and treatment.</p> <p>Conclusions</p> <p>Attitudes toward LLINs were positive in this rural community in Western Kenya, and respondents noted benefits with LLIN use. With improved understanding and clarification of the direct (mosquitoes) and indirect (e.g., standing water) causes of malaria, it is likely that LLIN use can be sustained, offering effective household-level protection against malaria.</p

Investigations of cellular immune mechanisms to malaria during pregnancy in a malaria holoendemic region of Western Kenya

Bibliography: leaves 132-155.Women during pregnancy in holoendemic regions of malaria are at an increased risk for peripheral malaria infections with potential for developing placental malaria. The immunological basis of protection and pathogenesis are incompletely understood. This thesis investigates both processes. Research on maternal placental immune responses necessitates the collection of reliable placental intervillous blood; an appropriate method for placental blood collection was therefore first determined. Five documented methods of collection (perfusion, incision, biopsy, tissue grinding and prick) were compared for foetal blood contamination and mononuclear cell profiles using flow cytometry. Placental blood collection by prick was established as the most appropriate method and was subsequently used for further immunological investigations

Enhanced Immunogenicity of Plasmodium falciparum Peptide Vaccines Using a Topical Adjuvant Containing a Potent Synthetic Toll-Like Receptor 7 Agonist, Imiquimod ▿

Plasmodium sporozoites injected into the skin by malaria-infected mosquitoes can be effectively targeted by antibodies that block parasite invasion of host hepatocytes and thus prevent the subsequent development of blood stage infections responsible for clinical disease. Malaria subunit vaccines require potent adjuvants, as they lack known pathogen-associated molecular patterns found in attenuated viral or bacterial vaccines that function as Toll-like receptor (TLR) agonists to stimulate dendritic cells and initiate strong adaptive immune responses. A synthetic TLR7 agonist, imiquimod, which is FDA approved for topical treatment of various skin conditions, can function as a potent adjuvant for eliciting T-cell responses to intracellular pathogens and model protein antigens. In the current studies, the topical application of imiquimod at the site of subcutaneously injected Plasmodium falciparum circumsporozoite (CS) peptides elicited strong parasite-specific humoral immunity that protected against challenge with transgenic rodent parasites that express P. falciparum CS repeats. In addition, injection of a simple linear peptide followed by topical imiquimod elicited strong Th1 CD4+ T-cell responses, as well as high antibody titers. The correlation of high anti-repeat antibody titers with resistance to sporozoite challenge in vivo and in vitro supports use of this topical TLR7 agonist adjuvant to elicit protective humoral immunity. The safety, simplicity, and economic advantages of a topical synthetic TLR7 agonist adjuvant also apply to other vaccines requiring high antibody titers, such as malaria asexual or sexual blood stage antigens to prevent red blood cell invasion and block transmission to the mosquito vector, and to vaccines to other extracellular pathogens

Evaluation of Various Methods of Maternal Placental Blood Collection for Immunology Studies

The collection of maternal placental intervillous blood (IVB), without contamination of fetal blood and with an accurate mononuclear cell profile, is essential for immunological studies of placental malaria and other infectious diseases of the placenta. We have compared five documented methods of IVB collection: perfusion, incision, biopsy, tissue grinding, and puncture (prick) for fetal blood contamination and mononuclear cell profiles using flow cytometry. Twenty-five placentas were obtained from Plasmodium falciparum and human immunodeficiency virus-negative primigravid and secundigravid women delivering at Nyanza Provincial Hospital in Kisumu, western Kenya. Each of the five methods was performed on the same placenta. Fetal red blood cell contamination was significantly lower for the prick and perfusion methods (4.1% and 8.3%, respectively) than for incision (59.5%), biopsy (42.6%), and tissue grinding (19.9%). Significant variation was noted among the five methods in the percentages of monocytes, total T cells, CD4(+) and CD8(+) T cells, B cells, and NK cells. Further, a pairwise comparison of prick and perfusion, the two methods with low fetal blood contamination, showed that they were not different for fetal red blood cell contamination levels; however, prick yielded significantly higher percentages of CD4 T cells and CD4 memory T cells than perfusion. Collection by prick was determined to be the best method of intervillous blood collection for immunology studies, and perfusion represented the next best method of choice due to high sample volume yield. Overall, in considering the advantages/disadvantages of the two methods with low fetal cell contamination, we conclude that a combination of prick and perfusion is most suitable for immunology studies

Class II-Restricted Protective Immunity Induced by Malaria Sporozoites▿

The irradiated-sporozoite vaccine elicits sterile immunity against Plasmodium parasites in experimental rodent hosts and human volunteers. Based on rodent malaria models, it has been proposed that CD8+ T cells are the key protective effector mechanism required in sporozoite-induced immunity. To investigate the role of class II-restricted immunity in protective immunity, we immunized β2-microglobulin knockout (β2M−/−) mice with irradiated Plasmodium yoelii or P. berghei sporozoites. Sterile immunity was obtained in the CD8+-T-cell-deficient mice immunized with either P. berghei or P. yoelii sporozoites. β2M−/− mice with the BALB/c (H-2d) genetic background as well as those with the C57BL (H-2b) genetic background were protected. Effector mechanisms included CD4+ T cells, mediated in part through the production of gamma interferon, and neutralizing antibodies that targeted the extracellular sporozoites. We conclude that in the absence of class I-restricted CD8+ T cells, sporozoite-induced protective immunity can be effectively mediated by class II-restricted immune effector mechanisms. These results support efforts to develop subunit vaccines that effectively elicit high levels of antibody and CD4+ T cells to target Plasmodium preerythrocytic stages