Anita Kumar

Multistage Multiantigen Heterologous Prime Boost Vaccine for Plasmodium knowlesi Malaria Provides Partial Protection in Rhesus Macaques

ABSTRACT A nonhuman primate model for malaria vaccine development allowing reliable, stringent sporozoite challenge and evaluation of both cellular and antibody responses is needed. We therefore constructed a multicomponent, multistage DNA vaccine for the simian malaria species Plasmodium knowlesi including two preerythrocytic-stage antigens, the circumsporozoite protein (PkCSP) and sporozoite surface protein 2 (PkSSP2), and two blood stage antigens, apical merozoite antigen 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42), as well as recombinant canarypox viruses encoding the four antigens (ALVAC-4). The DNA vaccine plasmids expressed the corresponding antigens in vitro and induced antiparasite antibodies in mice. Groups of four rhesus monkeys received three doses of a mixture of the four DNA vaccine plasmids and a plasmid encoding rhesus granulocyte-monocyte colony-stimulating factor, followed by boosting with a single dose of ALVAC-4. Three groups received the priming DNA doses by different routes, either by intramuscular needle injection, by intramuscular injection with a needleless injection device, the Biojector, or by a combination of intramuscular and intradermal routes by Biojector. Animals immunized by any route developed antibody responses against sporozoites and infected erythrocytes and against a recombinant PkCSP protein, as well as gamma interferon-secreting T-cell responses against peptides from PkCSP. Following challenge with 100 P. knowlesi sporozoites, 1 of 12 experimental monkeys was completely protected and the mean parasitemia in the remaining monkeys was significantly lower than that in 4 control monkeys. This model will be important in preclinical vaccine development.

Protection of rhesus macaques against lethal Plasmodium knowlesi malaria by a heterologous DNA priming and poxvirus boosting immunization regimen

ABSTRACT We tested a cytokine-enhanced, multiantigen, DNA priming and poxvirus boosting vaccine regimen for prevention of malaria in the Plasmodium knowlesi-rhesus macaque model system. Animals were primed with a mixture of DNA plasmids encoding two preerythrocytic-stage proteins and two erythrocytic-stage proteins from P. knowlesi and combinations of the cytokines granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor alpha and were boosted with a mixture of four recombinant, attenuated vaccinia virus strains encoding the four P. knowlesi antigens. Two weeks after boosting, the geometric mean immunofluorescence titers in the immunized groups against sporozoites and infected erythrocytes ranged from 160 to 8,096 and from 1,810 to 5,120, respectively. The geometric mean anti-P. knowlesi circumsporozoite protein (PkCSP) titers ranged from 1,761 to 24,242. Peripheral blood mononuclear cells (PBMC) from the immunized monkeys produced gamma interferon (IFN-γ) in response to incubation with pooled peptides from the PkCSP at frequencies of 10 to 571 spot-forming cells/106 PBMC. Following challenge with 100 infectious P. knowlesi sporozoites, 2 of 11 immunized monkeys were sterilely protected, and 7 of the 9 infected monkeys resolved their parasitemias spontaneously. In contrast, all four controls became infected and required treatment for overwhelming parasitemia. Early protection was strongly associated with IFN-γ responses against a pool of peptides from the preerythrocytic-stage antigen, PkCSP. These findings demonstrate that a multistage, multiantigen, DNA priming and poxvirus boosting vaccine regimen can protect nonhuman primates from an otherwise lethal malaria sporozoite challenge.

ELISPOT assay for detection of peptide specific interferon-γ secreting cells in rhesus macaques

Abstract A reliable procedure to measure antigen specific T cell responses in rhesus macaques is required to determine the efficacy of vaccines and immunotherapies. The currently available T cell assays are poorly quantifiable or technically difficult to perform. Classical 51 Cr-release cytotoxic T cell (CTL) assays are cumbersome and difficult to quantitate reproducibly. Detection of specific T-cell using MHC-peptide tetrameric complexes is highly sensitive, but requires knowledge of MHC type and prior identification of T cell epitopes. We therefore developed a rhesus interferon-γ (IFN-γ) ELISPOT assay capable of detecting IFN-γ secretion in response to stimulation with pooled 20-mer peptides. Peripheral blood mononuclear cells (PBMCs) from rhesus monkeys immunized with a DNA vaccine and recombinant canary pox encoding the Plasmodium knowlesi circumsporozoite protein (PkCSP) were incubated with pools of peptides from PkCSP. Positive responses to peptide pools and individual peptides ranging from 100 to 450 spot forming cells (SFC)/10 6 PBMC were detected in four of four immunized monkeys and in zero of two control monkeys. In two monkeys studied in detail, the IFN-γ response was focussed on a single 20-mer peptide, QGDGANAGQPQAQGDGANAG, and was dependent on CD4 + , but not CD8 + , T cells. Background responses in control monkeys and preimmunization PBMCs ranged from 10 to 50 SFC/10 6 PBMC. The average within assay and between assay coefficients of variation (CV) for this peptide ELISPOT were 21.9 and 24.7%, respectively. This peptide IFN-γ assay will be a useful tool for evaluation of T cell responses in rhesus macaques.

Toward clinical trials of DNA vaccines against malaria.

In mid 1997 the first malaria DNA vaccine will enter clinical trials. This single gene DNA vaccine encoding the Plasmodium falciparum circumsporozoite protein (PfCSP) will be studied for safety and immunogenicity. If these criteria are met, a multi‐gene DNA vaccine designed to induce protective CDS8+ T cell responses against P. falciparum infected hepatocytes will be subsequently assessed for safety, immunogenicity and capacity to protect immunized volunteers against experimental challenge with P. falciparum sporozoites. Our perspectives on malaria vaccine development in general,1 and on a multi‐gene DNA vaccine in particular,2 have been recently reviewed. Herein, we review the rationale and experimental foundation for the anticipated P. falciparum DNA vaccine trials.

Protection of rhesus monkeys by a DNA prime/poxvirus boost malaria vaccine depends on optimal DNA priming and inclusion of blood stage antigens.

Background We have previously described a four antigen malaria vaccine consisting of DNA plasmids boosted by recombinant poxviruses which protects a high percentage of rhesus monkeys against Plasmodium knowlesi (Pk) malaria. This is a multi-stage vaccine that includes two pre-erythrocytic antigens, PkCSP and PkSSP2(TRAP), and two erythrocytic antigens, PkAMA-1 and PkMSP-1(42kD). The present study reports three further experiments where we investigate the effects of DNA dose, timing, and formulation. We also compare vaccines utilizing only the pre-erythrocytic antigens with the four antigen vaccine. Methodology In three experiments, rhesus monkeys were immunized with malaria vaccines using DNA plasmid injections followed by boosting with poxvirus vaccine. A variety of parameters were tested, including formulation of DNA on poly-lactic co-glycolide (PLG) particles, varying the number of DNA injections and the amount of DNA, varying the interval between the last DNA injection to the poxvirus boost from 7 to 21 weeks, and using vaccines with from one to four malaria antigens. Monkeys were challenged with Pk sporozoites given iv 2 to 4 weeks after the poxvirus injection, and parasitemia was measured by daily Giemsa stained blood films. Immune responses in venous blood samples taken after each vaccine injection were measured by ELIspot production of interferon-γ, and by ELISA. Conclusions 1) the number of DNA injections, the formulation of the DNA plasmids, and the interval between the last DNA injection and the poxvirus injection are critical to vaccine efficacy. However, the total dose used for DNA priming is not as important; 2) the blood stage antigens PkAMA-1 and PkMSP-1 were able to protect against high parasitemias as part of a genetic vaccine where antigen folding is not well defined; 3) immunization with PkSSP2 DNA inhibited immune responses to PkCSP DNA even when vaccinations were given into separate legs; and 4) in a counter-intuitive result, higher interferon-γ ELIspot responses to the PkCSP antigen correlated with earlier appearance of parasites in the blood, despite the fact that PkCSP vaccines had a protective effect.

The development of a multivalent DNA vaccine for malaria

According to the latest report from the World Health Organization (December 1996), malaria is still a public health problem in more than 90 countries, inhabited by a total of some 2.4 billion people or 40% of the world’s population. Worldwide incidence of the disease is estimated to be 300–500 million clinical cases each year. Mortality due to malaria is estimated to be in the range of 1.5–2.7 million deaths annually. The vast majority of deaths occur among young children in Africa, especially in remote rural areas with poor access to health services. Immunity to malaria seems to occur among residents of malarious areas with increasing age which prevents an even greater number of deaths. However, this type of protection is short-lived and requires frequent exposure to the malaria parasite.

HLA-A*01-Restricted Cytotoxic T-Lymphocyte Epitope from the Plasmodium falciparum Circumsporozoite Protein

ABSTRACT Here, we report the identification of a novel CD8+cytotoxic T-lymphocyte epitope on the Plasmodium falciparumcircumsporozoite protein (3D7; amino acids 310 to 319 [EPSDKHIKEY]) that is restricted by HLA-A*01 and is recognized by human volunteers immunized with irradiated P. falciparum sporozoites. HLA-A*01 is the second most common HLA allele among Caucasians.

Cytotoxic T lymphocyte (CTL) adherence assay (CAA): a non-radioactive assay for murine CTL recognition of peptide-MHC class I complexes

Cytotoxic T lymphocytes (CTL) form an important immune surveillance system against intracellular pathogens. Here we describe a simple, visual assay for identifying peptides specifically recognized by CTL, based on the discovery that CTL develop increased adhesive properties upon TCR triggering. Several CTL lines were shown to pellet to the bottom of a round bottom 96-well plate in the absence of peptide. In contrast, these same CTL lines incubated with their cognate peptide, allowing them to present peptide to each other, adhered to the sides of the well and were readily distinguished by macroscopic visual examination of the plate after 4-5 h or overnight incubation. This CTL adherence assay (CAA) demonstrated peptide specificity and MHC restriction, and was titratable with peptide concentration. With this technique, a minimal-sized, malaria CTL epitope was correctly identified from a panel of overlapping nonamers, although the adherence pattern of two mono-substituted, variant peptides was less predictive of lytic activity. Also, substitutions in an HIV-1 envelope CTL epitope that reduced lytic activity were correctly predicted. Inhibitors of RNA and protein synthesis, upon preincubation, abrogated the adherence, indicating, at minimum, a need for live cells. Wortmannin, a PI-3 kinase inhibitor, inhibited the peptide specific adherence, consistent with a role for TCR or integrin signal transduction in CAA. Other cytoskeletal and metabolic inhibitors had no effect. Adherence of the T cells may involve low affinity, nonspecific interactions since wells coated with FCS, BSA or milk powder all produced an effective CAA in the presence of peptide under serum free conditions. Consequently, CAA may represent a rapid, simple method for screening large numbers of peptides to find cytolytic epitopes for a given CTL line and may identify additional epitopes causing T cell activation and adherence but not cytolytic activity.

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.