Sanjai Kumar

Expression and antigenicity of Plasmodium falciparum major merozoite surface protein (MSP119) variants secreted from Saccharomyces cerevisiae

Four antigenic variants of the 19-kDa carboxy terminal fragment of Plasmodium falciparum merozoite surface protein, MSP1 (MSP1(19)), were expressed in Saccharomyces cerevisiae as a histidine-tagged, secreted polypeptides (rMSP1(19)s). Structural analysis of the rMSP1(19)s indicated that a single amino acid change (E to Q) in the first EGF-like domain of the yeast-secreted rMSP1(19) proteins caused a significant change in their disulfide bond-dependent conformation. The antigenicity of the rMSP1(19)s were qualitatively and quantitatively analyzed by direct and competitive binding ELISAs. The data indicate that conserved and variant B cell determinants of MSP1(19), as well as epitopes that are known targets of protective antibodies, were recreated authentically in the rMSP1(19)s. Secretion of histidine-tagged rMSP1(19)s using the expression system described may be an efficient and effective means of producing a properly folded immunogen for a human vaccine against the blood stages of P. falciparum.

The real difficulties for malaria sporozoite vaccine development: nonresponsiveness and antigenic variation

Abstract Antibody against the circumsporozoite coat (CS) protein and cytolytic T lymphocytes (CTL), probably also specific for the CS protein, can block sporozoite-induced malaria parasite infection. These mechanisms of immunity define current strategies for sporozoite vaccine development. In this article, Michael Good and colleagues emphasize that these approaches demand that the CS protein is immunogenic in all members of the population. Unfortunately, the CS protein is not immunogenic for T cells in up to 40% of adults in a region of Africa where malaria is endemic, and studies in mice suggest that immunogenicity is under the control of the immune response (lr) genes. Furthermore, the regions of the CS protein that are immunogenic for T cells are variant. Both the variation and the poor immunogenicity may be the result of selection pressures acting against hepatocytes bearing immunogenic parasites, which will be recognized and killed by CTL.

Cellular mechanisms in immunity to blood stage infection.

We studied mechanisms of immunity to blood stage infection in the mouse malarias Plasmodium vinckei and Plasmodium yoelii 17X. Infection with P. vinckei was uniformly lethal, whereas P. yoelii 17X caused a self-limited, nonlethal infection. Transfer of immune CD4+ T cells conferred protection against P. yoelii in nude mice. Previous studies by others had suggested that immunity to P. yoelii may be related to MHC class I expression on reticulocytes and found that CD8+ T cells alone transferred protection in immunodeficient mice. However, in our experiments, immune CD8+ T cells failed to transfer protection. In the P. vinckei system, both B cell-deficient and immunologically intact mice developed immunity to P. vinckei after parasite infection and drug cure. In vivo depletion of CD4+ T cells abrogated immunity in these immune mice. Adoptive transfer of CD4+ T cells failed to protect nude or normal mice from P. vinckei infection, but the transfer of immune CD4+ T cells reconstituted immunity in CD4-depleted immune mice. Splenectomy of immune mice resulted in the complete loss of immunity. Despite the fact that immunity to P. vinckei could be achieved with live parasite infection and drug cure, immunization of mice with killed P. vinckei with various adjuvants failed to protect mice from live challenge. In contrast, immunization with killed P. vinckei antigens in combination with attenuated Salmonella typhimurium SL3235 induced a high degree of protective immunity. These results suggest that induction of immunity against virulent malarias requires both induction of CD4+ T cells and certain splenic alterations caused by parasite infection or S. typhimurium.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and immunogenicity of the C-terminus of a major blood-stage surface protein of Plasmodium vivax, Pv20019, secreted from Saccharomyces cerevisiae

The carboxy-terminus of the major merozoite surface protein of Plasmodium has been shown to be the target of protective immunity in a number of non-vivax malaria parasite species. In an effort to develop a protective vaccine for Plasmodium vivax, the most prevalent form of human malaria, we expressed in Saccharomyces cerevisiae the 19-kDa a carboxy-terminus of Pv200 as a His6-tagged, secreted polypeptide. Five of seven H-2 congenic mouse strains elicited antibodies that recognized yeast produced PV200(19) by ELISA. The vaccine appears to be immunogenic and widely recognized, and to contain one or more helper T cell epitopes that may allow boosting with subsequent natural infections.

Autologous lymphoblastoid cell lines stably transfected with Plasmodium falciparum circumsporozoite protein as targets in cytotoxic T-lymphocyte assays☆

To produce cell lines that can be used as a continuous source of antigen presenting cells for stimulating T-cell lines and clones and as targets in cytotoxic T-lymphocyte (CTL) assays, we used a retroviral vector with a simian virus (SV40) early promotor to transfer a Plasmodium falciparum circumporozoite (PfCSP) gene into human EBV transformed B-lymphoblastoid cell lines (B-LCL). We herein report successful, stable transfection and cell surface expression of this gene, as confirmed by PCR, Western blot analysis and immunoelectron microscopy. One of three successfully transfected autologous cell lines expressed PfCSP on the cell surface and was lysed by CD8+ T-cell dependent CTL from a donor volunteer who had been immunized with irradiated P. falciparum sporozoites. Such cell lines should provide excellent tools for characterizing human CD8+ T-cell responses against Plasmodium sp. proteins.

Immune Response Regulations in Parasitic Infections

The yearly death toll in the major infectious diseases plaguing primarily tropical and subtropical areas of the world is estimated to be appr. 18 million (WHO, Geneva, reproduced in Science, 1992, 256:1135). These figures do not account for the enormous background of morbidity caused by these diseases and their disastrous consequences both for those directly affected and for the socioeconomic development of the countries concerned. Seen in these perspectives, it may appear surprising that only a relatively minor fraction of worldwide research in immunology during the past decades has dealt with the immunology of infection. This situation seems, however, to be changing slowly partly because of the increasing need to fight infectious diseases by immunological manipulation, i.e. with vaccines. This has led to the recognition that construction of modern vaccines requires better insights in the basic mechanisms involved in immunity to infection. Much of the research in this area has dealt with the characterization and cloning of pathogen derived antigens expected to give rise to protective immunity in the infected host. However, more recently, attention has also been given to the various factors regulating immune protection. Some of these regulations will be exemplified and discussed in the following.