Nirbhay Kumar - US grants

Malaria remains one of the major health problems in the world. Malaria vaccines are directed against three stages of the parasite: the sporozoites, the merozoites and the sexual forms (gametocytes/gametes). Studies proposed here are related to malaria immunity against the gametes/gametocytes (transmission blocking immunity). In this form of immunity, various mechanisms operate in the midgut of the misquitoes and suppress the infectivity of the parasites. By preventing spread of the diesase via the mosquito vector, immunity against the gametes could reduce the risk of malarial infection to the human population and stop the spread of parasites which may become resistant to other vaccines. Earlier studies have suggested several gamete surface antigens as potential candidates for a transmission blocking vaccine. Various biochemical studies proposed are aimed at characterization of the antigenic nature of the epitopes in these proteins. Their evaluation as vaccine candidate would depend upon successful gene cloning, sequence analysis and expression in an appropriate system.

Malaria remains a major cause of human suffering with hundreds of million infections and many million deaths in the world annually. A major goal of research on human malaria is the development of vaccines targeting different stages of the parasite's life cycle in the human host and in the mosquito vector. The overall long term goal of our research is to develop a vaccine that would reduce or stop malaria transmission. This form of immunity, known as malaria transmission- blocking immunity, is largely antibody-mediated and operates in the mosquito midgut to prevent sexual reproduction of the parasites. A 27 kDa protein of Plasmodium falciparum (human malaria parasite) gametocytes has been identified as a target antigen for the development of transmission-blocking vaccine. Immunization in mice with various recombinant fragments of the 27 kDa protein elicited antibodies capable of suppressing malaria transmission. The linear B cell epitope recognized by transmission-blocking monoclonal antibodies was mapped using recombinant overlapping fragments of the 27 kDa protein expressed in E. coli and synthetic peptides. To facilitate rational vaccine development, we have also mapped, in addition to the B cell epitope, several helper T cell epitopes recognized by the 27 kDa protein- specific murine and human T cell clones. It now appears feasible to combine the mapped B and T cell epitopes to develop a subunit vaccine construct. A major goal of the studies proposed in this application is to investigate the biological mechanism and molecular basis of malaria transmission-blocking mediated by monoclonal antibodies or antibodies induced by the recombinant 27 kDa protein. Studies are also proposed to clone the 27 kDa protein homologue in an evolutionarily-related parasite P. gallinaceum which will offer an opportunity for vaccine trials using an animal malaria model system. Finally, studies are also proposed to construct a hybrid of two target antigens of transmission- blocking immunity: the 27 kDa gametocyte protein and the 25 kDa zygote/ookinete protein by recombinant cloning in Pichia pastoris and in DNA vaccine vectors. Evaluation of these two antigens either combined individually or as a hybrid molecule would identify an effective vaccine construct. Overall, the proposed studies would lead to an improved biological understanding of the sexual stages of human malaria parasite and suggest ways to produce an effective malaria transmission-blocking vaccine.

The protozoan parasites of the genus Plasmodium have an obligate sexual phase in their life cycle. Sexual stages play a pivotal role in the transmission of parasites from a vertebrate host to an insect vector. Infections in humans by the asexual stages of P. falciparu on the other hand, are responsible for hundreds of millions of clinical cases and millions of death annually. Male and female gametes must fuse in the mosquito midgut for parasite transmission to occur. It is also during the sexual reproduction in the mosquitoes that novel genotypes are produced by self and random fertilization between male and female gametes. Infectivity of parasites in the mosquitoes, therefore, requires initial differentiation of parasites in the vertebrate host to form male and female gametocytes. The molecular basis for this sexual differentiation is not known. a number of proteins expressed in the sexual stages of P. falciparum have been identified and their genes cloned but their functional role in the biology of differentiation and develop of P. falciparum remains to be elucidated. Studies are proposed to investigate genes which are differentially expressed between the asexual and sexual stages. An approach based on subtraction hybridization will be employed using stage-specific cDNA libraries. Cloned genes will be characterized with respect to encoded protein products, subcellular localization and stage-specific expression of RNA transcripts in various developmental stages of gametocytes. Finally, studies based on genetic transformation of parasites will evaluate the functional role of these genes and their encoded products in the sexual differentiation and development of P. falciparum. a molecular and biochemical understanding of the process of the sexual differentiation will suggest novel mechanisms and is likely to identify new strategies for interfering with the sexual development of P. falciparum.

DESCRIPTION (Adapted from the Applicant's Abstract): The goal of this application is to develop a transmission blocking vaccine to Plasmodium falciparum. This vaccine is meant to induce antibodies which bind to antigens present on the surface of male and female gameites and parasites developing in the mosquito midgut. The main candidate antigen is Pfs25, a surface protein that is expressed at the onset of gametogenesis. The vaccine will be based on DNA vaccines given either alone or in a prime-boast regimen with protein vaccines.

The Malaria Research and Training Program in Zimbabwe (MRTPZ) will link the research and educational opportunities of the Molecular Microbiology and Immunology (MMI) Department of the Johns Hopkins School of Public Health with the internationally known Blair Research Institute (BRI) and the Biomedical Research and Training Institute (BRTI) for the purpose of invigorating the existing malaria prevention and control infrastructure of Zimbabwe. The long-term objective will be to fortify and sustain a center of excellence in an African malaria endemic setting. The MRTPZ will knit comprehensive research training of two pre-doctoral and two masters level students at MMI with home country education training in immunologic aspects of malaria transmission control, characterization of drug resistance patterns, vector control strategies, community involvement in malaria control and research methods training. The BRI is the lead government agency charged with management of scientific research and training on Zimbabwe health problems. Malaria control is one of the three priority areas in the national health strategy. The Department of MMI conducts research and trains scientists in the basic mechanisms of infectious diseases and host responses with the underlying School of Public Health philosophy that employs state-of-the-art scientific approaches to public health problems of global significance. The malaria research projects, initiated at MMI but conducted in Zimbabwe will provide a structure for the application of theoretical knowledge gained from comprehensive courses in molecular biology, microbiology, immunology, ecology and-population genetics to the issues related to malaria epidemiology, vector biology, immunology and vaccines, anti malaria drugs, pathogenesis and health systems and operational methods. An essential aspect of the training program, includes a requirement to participate in seminars, research forums, journal clubs, national and international scientific meetings and to participate in the various training courses to be offered in Zimbabwe jointly by the faculty from Hopkins, BRI and BRTI. The training program will be structured to meet the program goals and closely supervised by the MRTPZ advisory committee.

Malaria parasites are responsible for 300-500 million infections and 2-3 million deaths annually. Transmission of malaria between vertebrate hosts involves an obligate sexual developmental cycle in the anopheline mosquito vector. The sexual stages are absolutely essential for malaria transmission. The molecular mechanisms underlying sexual differentiation and development in Plasmodium falciparum remain largely unknown. After the initial commitment to the sexual cycle, Plasmodium gametocytes undergo what appears to be a multi-step growth and development process, possibly involving several gene products. The goal of this proposal is to investigate the functional involvement of two proteins (Pfg27 and Pfs16), abundantly expressed early during gametocytogenesis. We have recently disrupted the gene for Pfg27 by homologous recombination, resulting in the loss of sexual phenotype in the transformed parasites. These studies for the first time have shown that Pfg27, a protein expressed in stage I and II gametocytes is critical for gametocyte development. Studies on Pfs16 disruption will further elucidate sexual differentiation process in P. falciparum. Investigations on stably transformed parasites will also offer an opportunity to evaluate transcriptional control mechanisms, sexual stage- specificity of promoters and complementation of disrupted genes. A combination of immunochemical and molecular (yeast-two-hybrid) approaches will be employed to investigate protein-protein interactions involving Pfg27 and Pfs16 and other cellular proteins. These studies could possibly identify novel proteins as interacting functional partners, thus advancing our understanding of the biological roles of Pfg27 and Pfs16 in the sexual differentiation and development of transmission competent parasites. The proposed studies will thus offer a novel molecular genetic approach to dissect mechanisms underlying sexual development in P. falciparum. Such information could lead to rational development of immunological and/or chemotherapeutic arsenal to prevent transmission of P. falciparum malaria.

DESCRIPTION (provided by applicant): The long-term objective of the Malaria Training and Research Capacity Building in Southern Africa is to create a center of excellence for malaria research in Southern Africa. The proposed program builds upon our previous success with the Malaria Research and Training Program in Zimbabwe. We propose to continue to strengthen our current trainees (two PhD) while extending malaria research and training to Zambia and neighboring Southern African countries. In addition to maintaining strong research ties with our former trainees, we propose to create specific training to satisfy immediate needs of the local research community. This would occur in the form of short-term training in state-of-the-art research methods and techniques offered by Johns Hopkins faculty. In addition, four different training courses will be offered in Zambia: (1) Plasmodium Biology and Treatment, (2) Malaria Control: Monitoring and Evaluation, (3) Basic Immunology and Microbial Immunity and (4) Entomology. The courses will be offered in partnership between Johns Hopkins University faculty and scientists from various centers in Zambia, especially the School of Medicine University Teaching Hospital, University of Zambia, Malaria Institute at Macha and Tropical Disease Research Center at Ndola. These courses will also involve our former training grant colleagues from Zimbabwe. The various malaria research and training activities conducted in Zambia will provide a structure for the application of knowledge gained from training courses and specialized targeted research training in molecular biology, microbiology, immunology, ecology, molecular epidemiology, genomics and population genetics to local issues resulting in the creation of a comprehensive research community of more than a dozen scientists in the region.

Malaria parasites, worldwide are responsible for 300-500 million new infections and 1-2 million deaths each year. An effective vaccine, however, remains elusive, partly due to antigenic diversity and the immune evasion strategies of the parasite. Recombination mechanisms are intimately linked with antigenic variation, a phenomenon of utmost significance for vaccine development against protozoan parasites like the malaria parasite. The long term objective of the proposed studies is to investigate mechanism(s) of genetic rearrangements associated with phenomenon like antigenic variation. An underlying tenet of the work proposed is that understanding the recombination mechanisms in Plasmodium will provide improved opportunities for the development of therapies. In other eukaryotes, homologous recombination (HR) plays a major role in chromosomal rearrangements, and Rad51 and Dmc1 proteins, the eukaryotic counterparts of bacterial RecA recombinase, are central molecules involved in HR during mitosis and meiosis. In eukaryotes, additional proteins like RPA and Rad54 functionally cooperate with Rad51 and mediate HR and the repair of damaged chromosomes. The hypothesis underlying proposed studies is that the Rad51 and other interacting proteins, as mediators of HR, play critical role(s) during growth and development of the parasite and facilitate gene rearrangements in P. falciparum. Molecular identification of Rad51 and Dmc1 homologues in P. falciparum and recent characterization of enzymatic properties of recombinant PfRad51, such as DNA strand exchange and ATPase activities, have strongly indicated a conserved functional role for PfRad51 in these organisms. We will test our hypothesis using biochemical as well as genetic approaches. Studies in the revised specific aims 1 and 2 will lead to in vitro characterization of the proteins involved in HR and thus probe into the biochemical basis of recombination and gene rearrangements in the parasites. PfRad51 gene knockout studies in the revised specific aim 3 will directly evaluate importance of PfRad51 in the erythrocytic growth of the parasite and analysis of repertoire of var gene transcripts. Moreover, studies on Dmc1 disruption (revised specific aim 3) will evaluate the role of meiosis specific recombinase during malaria transmission. The results of this study should be important in unraveling the recombination machinery and molecular and genetic basis for recombination and genetic rearrangements in P. falciparum.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise;2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues;and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): In this R21 application we propose to assess cellular, molecular and immune correlates of efficacy and safety of nanoparticle formulations using a well characterized malaria vaccine candidate as a model immunogen. A vaccine based on Pfs25 protein targeting the sexual stages of the parasite provides a direct approach to reduce malaria transmission. In Plasmodium falciparum, Pfs230 and Pfs48/45 proteins produced within the intra-erythrocytic gametocyte stages and Pfs25, expressed during the mosquito stage development of zygote into ookinete, represent well established target antigens of transmission-blocking antibodies. Antibodies recognizing specific conformational epitopes in these proteins are potent blockers of infectivity of malaria parasites in the mosquito. We have recently succeeded in recombinant expression and purification of re-folded Pfs25, for the first time in near native conformation, in E. coli. The purified protein (rPfs25) in experimental adjuvants elicited strong immunogenicity in mice. Better and safer adjuvants and delivery methods need to be developed for eventual human applicability. Nanoparticles are fast gaining acceptability as safe and effective vaccine adjuvants. We propose to develop Pfs25 - nanoparticle formulations (Aim 1) and evaluate functional immune response in inbred and outbred mice (Aim 3). We also propose to study the immune response-related host gene expression changes at the site of vaccine injection to gain mechanistic insights of Pfs25-nanoparticle vaccine efficacy (Aim 2). Finally studies are also proposed to investigate relevant vaccine safety parameters (Aim 4). These studies will provide better understanding of cellular and molecular correlates of immunogenic efficacy and safety of nanoparticle formulations, and also provide the basis for more in depth studies on vaccine development, in general.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise; 2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues; and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): Vaccines have provided the most cost-effective tool to control infectious diseases caused by viral and bacterial pathogens. However, there are no vaccines against any human parasitic infections and the development of an effective malaria vaccine remains an unachieved goal. Malaria infections account for nearly a million deaths out of ~ 300 million clinical cases globally on an annual basis. Vaccines based on antigens targeting the sexual stages of the parasite provide a direct approach to reduce malaria transmission. Antibodies recognizing specific conformational epitopes in these proteins are potent blockers of infectivity of malaria parasites in the mosquito. In this R21 application we propose to assess cellular, molecular and immune correlates of efficacy and safety of modified DNA vaccines using a well characterized malaria vaccine candidate, Pfs25, as a model immunogen. DNA vaccines induce a good initial immune response; however, in larger mammals they require heterologous boosting, eg. adjuvanted proteins to sustain functional antibody titers. Poor immunogenicity of DNA vaccines could result from an immune phenomenon described as T cell exhaustion or dysfunction resulting from upregulation of the programmed death 1 (PD-1) receptor in activated T cells and PD-L1 on antigen presenting cells. We propose to test this hypothesis through the novel addition of an RNAi sequence (designed to knockdown PD-L1) to a DNA vaccine and expect enhanced immunogenicity of DNA vaccines reflected in higher titer and longer lasting antibody responses. We will test our hypothesis by pursuing the following specific aims. In specific aim 1 we will develop DNA vaccines capable of silencing PD-L1 and expressing vaccine antigen within the same cell. Studies in specific aim 2 will provide a proof-of-principle through in vivo evaluation of potency of modified DNA vaccines and associated TFH cell responses. These studies will provide a better understanding of the cellular and molecular correlates of immunogenic efficacy and safety of DNA vaccines, and also provide the basis for more in depth studies on vaccine development across a broad spectrum.

? DESCRIPTION: Almost a million people are ill from malaria each day while severe malaria disease claims the lives of 600,000 people each year. A rugged, portable sensitive and accurate method to diagnose malaria without blood sampling and using the reagents would be ideal and addresses the critical problem of malaria diagnosis in field or in asymptomatic cases. The MalariSense technology is able to make a diagnosis through the skin within seconds without using reagents or a needle to obtain blood. The device works by shining a short safe laser pulse that penetrates the skin to small blood vessels. The malaria parasite has a crystal inside of itsel that is able to absorb the laser energy to make a small vapor nanobubble that expands and collapses. The collapsing vapor nanobubble generates a pressure wave that is able to be detected by an ultrasound detector. Uninfected red blood cells are not affected by the laser and do not make a vapor bubble. The proof of MalariSense concept was demonstrated in animals and humans by shining a laser on the ear and detecting the sound pings. In this project, the discovered mechanism will be translated into a field, easy-to-use diagnostic device to non- invasively detect both symptomatic and asymptomatic malaria infections in seconds, and to screen > 200,000 people per year with a single device with a diagnosis cost below 0.1$. This will be achieved through several connected activities: (1) Determine the methodology and specifications for the MalariSense technology; (2) Develop and prototype the MalariSense technology for field use; (3) Evaluate the MalariSense technology in endemic area, determine its performance and applications, and develop the platform for a global screening of malaria. The applications for analyzing mosquitoes, blood samples and small animals will also be developed and tested the ability to make the sensitive diagnosis of malaria through the skin opens up a whole new dimension to malaria diagnosis. The impact and applications are many. The ability to diagnose and treat asymptomatic people living in malaria areas will help greatly the strategies to eliminate malaria from regions. Another dimension of the device is the ability to enable the immediate global access and analysis of the raw data for the remote monitoring of malaria infection. The developed software, protocol and device will act as a platform for the future implementation of the global malaria diagnostic system to radically improve the malaria control and elimination in multiple settings. Just 1000 units will annually screen 200 million people to cover all current disease cases.

Abstract Malaria vaccine development has focused on antigens expressed during various stages of the life cycle of the parasite. Malaria transmission depends upon the development of intraerythrocytic sexual stages, ingestion by female anopheline mosquitoes and subsequent sexual development in mosquitoes leading to formation of sporozoites. An infected Anopheles mosquito initiates malaria infection cycle by injecting sporozoites which invade hepatocytes. Hence immune interventions aimed at blocking development of both the liver stage (pre-erythrocytic phase - PE) and sexual stage are expected to provide more effective strategy to protect against malaria. A transmission blocking vaccine (TBV) approach targeting antigens in the sexual stages (i.e. male and female gametocytes and gametes) and the mosquito stages of the parasite (i.e. zygote and ookinete) is believed to be of central importance in malaria elimination efforts. In Plasmodium falciparum, TBV target antigens include Pfs25, Pfs48/45 and Pfs230, with known orthologs in P. vivax. While a PE stage vaccine will prevent or reduce the development of blood stage parasites including gametocytes in an infected person, a TBV will block sexual reproduction of the gametocytes in the mosquito. A combination of vaccines targeting both PE and sexual/midgut stages, is expected to provide effective ways for interruption of malaria transmission, critical for elimination goal. Using knowledge gained from our published studies on Pfs25, Pvs28 and Pfs48/45, it is now possible to systematically evaluate a combination of these antigens along with PfCSP, an already well-established PE stage vaccine antigen. We propose to rationally develop and evaluate multi-stage (PE and sexual), multi-antigen (Pfs25, Pvs25, Pfs48/45, Pvs48/45 and PfCSP) and multi-species (P. falciparum and P. vivax) vaccine combinations to interrupt malaria transmission, a long-term and ultimate goal of our research. Using recombinant proteins, and DNA vaccines delivered by in vivo electroporation (EP), we will determine the potency of vaccine combinations comprised of target antigens from PE and sexual stages of P. falciparum (aim 1). In aim 2, we will evaluate and compare combination of vaccines targeting transmission of the two major Plasmodium spp. (P. falciparum and P. vivax). The goal of studies in aim 3 is to determine outcome and immune potency of DNA vaccines by enhancing delivery of DNA plasmids and uptake of antigen by antigen presenting cells in vivo. The proposed studies are expected to identify most potent vaccine combination(s) and provide a rational approach for advancing effective combination(s) to interrupt transmission of malaria, an important goal of malaria elimination strategies.