Dose-Dependent Infectivity of Aseptic, Purified, Cryopreserved Plasmodium falciparum 7G8 Sporozoites in Malaria-Naive Adults.

Direct venous inoculation of 3.2 × 103 aseptic, purified, cryopreserved, vialed Plasmodium falciparum (Pf) strain NF54 sporozoites, PfSPZ Challenge (NF54), has been used for controlled human malaria infection (CHMI) in the United States, 4 European countries, and 6 African countries. In nonimmune adults, this results in 100% infection rates. We conducted a double-blind, randomized, dose-escalation study to assess the infectivity of the 7G8 clone of Pf (PfSPZ Challenge [7G8]). Results showed dose-dependent infectivity from 43% for 8 × 102 PfSPZ to 100% for 4.8 × 103 PfSPZ. PfSPZ Challenge (7G8) will allow for more complete assessment by CHMI of antimalarial vaccines and drugs.

Feasibility of direct venous inoculation of the radiation-attenuated Plasmodium falciparum whole sporozoite vaccine in children and infants in Siaya, western Kenya.

PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites, is administered by direct venous inoculation (DVI) for maximal efficacy against malaria. A critical issue for advancing vaccines that are administered intravenously is the ability to efficiently administer them across multiple age groups. As part of a pediatric safety, immunogenicity, and efficacy trial in western Kenya, we evaluated the feasibility and tolerability of DVI, including ease of venous access, injection time, and crying during the procedure across age groups. Part 1 was an age de-escalation, dose escalation trial in children aged 13 months-5 years and infants aged 5-12 months; part 2 was a vaccine efficacy trial including only infants, using the most skilled injectors from part 1. Injectors could use a vein viewer, if needed. A total of 1222 injections (target 0.5 mL) were initiated by DVI in 511 participants (36 were 5-9-year-olds, 65 were 13-59-month-olds, and 410 infants). The complete volume was injected in 1185/1222 (97.0%) vaccinations, 1083/1185 (91.4%) achieved with the first DVI. 474/511 (92.8%) participants received only complete injections, 27/511 (5.3%) received at least one partial injection (<0.5 mL), and in 10/511 (2.0%) venous access was not obtained. The rate of complete injections by single DVI for infants improved from 77.1% in part 1 to 92.8% in part 2. No crying occurred in 51/59 (86.4%) vaccinations in 5-9-year-olds, 25/86 (29.1%) vaccinations in 13-59-month-olds and 172/1067 (16.1%) vaccinations in infants. Mean administration time ranged from 2.6 to 4.6 minutes and was longer for younger age groups. These data show that vaccination by DVI was feasible and well tolerated in infants and children in this rural hospital in western Kenya, when performed by skilled injectors. We also report that shipping and storage in liquid nitrogen vapor phase was simple and efficient. (Clinicaltrials.gov NCT02687373).

Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion.

The Plasmodium falciparum gene encoding erythrocyte binding antigen-175 (EBA-175), a putative receptor for red cell invasion (Camus, D., and T. J. Hadley. 1985. Science (Wash. DC). 230:553-556.), has been isolated and characterized. DNA sequencing demonstrated a single open reading frame encoding a translation product of 1,435 amino acid residues. Peptides corresponding to regions on the deduced amino acid sequence predicted to be B cell epitopes were assessed for immunogenicity. Immunization of mice and rabbits with EBA-peptide 4, a synthetic peptide encompassing amino acid residues 1,062-1,103, produced antibodies that recognized P. falciparum merozoites in an indirect fluorescent antibody assay. When compared to sera from rabbits immunized with the same adjuvant and carrier protein, sera from rabbits immunized with EBA-peptide 4 inhibited merozoite invasion of erythrocytes in vitro by 80% at a 1:5 dilution. Furthermore, these sera inhibited the binding of purified, authentic EBA-175 to erythrocytes, suggesting that their activity in inhibiting merozoite invasion of erythrocytes is mediated by blocking the binding of EBA-175 to erythrocytes. Since the nucleotide sequence of EBA-peptide 4 is conserved among seven strains of P. falciparum from throughout the world (Sim, B. K. L. 1990. Mol. Biochem. Parasitol. 41:293-296.), these data identify a region of the protein that should be a focus of vaccine development efforts.

Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum.

A 175-kilodalton erythrocyte binding protein, EBA-175, of the parasite Plasmodium falciparum mediates the invasion of erythrocytes. The erythrocyte receptor for EBA-175 is dependent on sialic acid. The domain of EBA-175 that binds erythrocytes was identified as region II with the use of truncated portions of EBA-175 expressed on COS cells. Region II, which contains a cysteine-rich motif, and native EBA-175 bind specifically to glycophorin A, but not to glycophorin B, on the erythrocyte membrane. Erythrocyte recognition of EBA-175 requires both sialic acid and the peptide backbone of glycophorin A. The identification of both the receptor and ligand domains may suggest rational designs for receptor blockade and vaccines.

A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer.

Endogenous murine angiostatin, identified as an internal fragment of plasminogen, blocks neovascularization and growth of experimental primary and metastatic tumors in vivo. A recombinant protein comprising kringles 1-4 of human plasminogen (amino acids 93-470) expressed in Pichia pastoris had physical properties (molecular size, binding to lysine, reactivity with antibody to kringles 1-3) that mimicked native angiostatin. This recombinant Angiostatin protein inhibited the proliferation of bovine capillary endothelial cells in vitro. Systemic administration of recombinant Angiostatin protein at doses of 1.5 mg/kg suppressed the growth of Lewis lung carcinoma-low metastatic phenotype metastases in C57BL/6 mice by greater than 90%; administration of the recombinant protein at doses of 100 mg/kg also suppressed the growth of primary Lewis lung carcinoma-low metastatic phenotype tumors. These findings demonstrate unambiguously that the antiangiogenic and antitumor activity of endogenous angiostatin resides within kringles 1-4 of plasminogen.

Continuous administration of endostatin by intraperitoneally implanted osmotic pump improves the efficacy and potency of therapy in a mouse xenograft tumor model.

In the first Phase I clinical trials of endostatin as an antiangiogenic therapy for cancer, the protein was administered as an i.v. bolus for approximately 20-30 min each day. This protocol was based on experimental studies in which animals were treated by s.c. bolus once a day. However, it was not clear in the previous studies whether this schedule could be maximized further. Therefore, we developed experimental models involving continuous administration of endostatin to determine the potency and efficacy of this approach. Endostatin was administered to tumor-bearing mice either s.c. or i.p. in single bolus doses. The efficacy of these regimens was compared with endostatin administered continuously via an i.p. implanted mini-osmotic pump. Our results show that endostatin remains stable and active in mini-osmotic pumps for at least 7 days. We show that endostatin injected i.p. is rapidly cleared within 2 h, whereas endostatin administered continuously via mini-osmotic pump maintains systemic concentrations of 200-300 ng/ml for the duration of administration. Furthermore, continuous i.p. administration of endostatin results in more effective tumor suppression at significantly reduced doses (5-fold), compared with bolus administration. Additional experiments using a human pancreatic cancer model in severe combined immunodeficient mice showed that there was a significant decrease in the microvessel density between the treatment groups and the control group. These data show that continuous administration of human endostatin results in sustained systemic concentrations of the protein leading to: (a) increased efficacy manifested as increased tumor regression; and (b) an 8-10-fold decrease in the dose required to achieve the same antitumor effect as the single daily bolus administration of endostatin. On the basis of this approach, an additional clinical trial has been designed and initiated and is under way in two countries.

Cell invasion by the vertebrate stages of Plasmodium.

Protozoans of the genus Plasmodium are the causative agents of malaria; they have a complex life cycle involving vertebrate and arthropod hosts and have three distinct invasive stages. Although the invasive stages probably invade cells using similar mechanisms, each stage has a different host cell specificity and utilizes different receptors to enter cells.

Delineation of functional regions on Plasmodium falciparum EBA-175 by antibodies eluted from immune complexes.

After several infections and drug cure, Aotus spp. monkeys are protected against homologous Plasmodium falciparum challenge. Sera from these monkeys passively transfers protection (Diggs CL, Hines F, Wellde BT. Exp Parasitol 1995;80:291-6). Antibodies in these sera agglutinate merozoites emerging from ruptured schizonts forming immune clusters and thereby presumably prevent merozoite invasion of erythroyctes (Lyon JA, Haynes JD, Diggs CL, Chulay JD, Pratt-Rossiter JM. J Immunol 1986;136:2252-8). It is reasoned that antibodies eluted from the immune clusters of merozoites must recognize exposed epitopes with functional relevance to the survival of the live, invasive merozoites. The erythrocyte binding protein, EBA-175 was used as a model to study this hypothesis. We expressed and purified nine overlapping fragments of EBA-175 as recombinant proteins and probed them in immunoblots with antibodies eluted from the immune clusters. Only the two regions of EBA-175 previously shown to be functionally relevant were recognized by the eluted antibodies. One was region II which includes the erythrocyte binding domain of this parasite ligand which binds to its receptor glycophorin A. The other was the region containing EBA-peptide 4, antibodies to which block binding of EBA-175 to erythrocytes and inhibit invasion of merozoites into erythrocytes. Immunization of mice with the region II recombinant protein induced antibodies that recognize merozoites in immunofluorescence assays, identified a 175 kDa band on unreduced immunoblots, blocked binding of EBA-175 to erythrocytes and inhibited merozoite invasion of erythrocytes. These findings corroborate the importance of EBA-175 region II and EBA-peptide 4, and suggest that antibodies eluted from immune clusters can be used to identify protective epitopes exposed on invasive merozoites from other P.falciparum proteins.

Use of chitinase to facilitate detection of protozoan, helminth and single copy genes in squashed whole mosquitoes.

The application of DNA probes to detect foreign DNA in whole arthropods has been limited by the inability of the probes to distinguish between small quantities of target DNA and the background signal generated by non-specific hybridization of mosquito material. We report that treatment of nitrocellulose filters upon which mosquitoes have been squashed with chitinase and proteinase K eliminates non-specific hybridization of DNA probes to mosquito components. Using this technique we have been able to detect a single larva of Brugia malayi, sporozoites of Plasmodium falciparum and Plasmodium berghei, and a single-copy gene in directly squashed vector mosquitoes. Use of this simple, rapid technique should facilitate the successful use of DNA probes in field studies.

A DNA probe cloned in Escherichia coli for the identification of Brugia malayi.

This report describes a specific and sensitive DNA probe for the identification of Brugia malayi. A genomic DNA library produced from subperiodic B. malayi microfilariae was screened to detect clones containing DNA sequences which are highly repeated within the parasite genome. Several clones were further analyzed to identify those which hybridize specifically with B. malayi DNA but not with DNA from B. pahangi and Dirofilaria immitis. From these, clone pBm15 was selected because it hybridized with high sensitivity to B. malayi DNA as detected by autoradiography. Clone pBm15 was sensitive enough to detect two infective larvae or five microfilariae or 300 pg of purified B. malayi microfilarial DNA. This study forms the basis for the development of a specific and sensitive DNA probe for the identification of B. malayi in field specimens.

Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum.

EBA-175 is a soluble 175-kDa Plasmodium falciparum antigen that is released into culture supernatants during rupture of schizont-infected erythrocytes. EBA-175 binds to erythrocytes and binding is sialic acid-dependent. A clone expressing the gene encoding EBA-175 was obtained previously by screening a genomic DNA expression library with antibodies that had been affinity-purified from EBA-175. Antibodies were raised against a 43-amino-acid peptide (EBA-peptide 4) synthesized according to the deduced amino acid sequence. Antibodies to peptide 4 and affinity-purified antibodies specific for EBA-175 were used to characterize further EBA-175 giving the following results: (1) EBA-175 differs biochemically and immunologically from other reported malarial antigens; (2) the EBA-175s from six geographical isolates of P. falciparum are antigenically conserved; (3) EBA-175 is expressed during schizogony as a 190-kDa protein which is larger than the culture supernatant form of the antigen. The 190-kDa form of the protein is recovered from the cell pellet in schizont-infected erythrocytes and partitions to the soluble fraction when extracted with detergent; (4) release of soluble EBA-175 into the culture supernatant coincides with schizont rupture; (5) there was no observable change in pI (pI = 6.86) by isoelectric focusing between the cellular and supernatant species of the protein; and (6) release of EBA-175 into the culture supernatant is inhibited by the addition of chymostatin and leupeptin. The continued research into the role of EBA-175 during erythrocyte invasion may aid in vaccine development for malaria.

Two alleles of the 175-kilodalton Plasmodium falciparum erythrocyte binding antigen.

EBA-175, erythrocyte binding antigen 175, is a 175-kDa antigen of Plasmodium falciparum which has been shown to be involved in the recognition of erythrocytes by merozoites and may be involved in the process of erythrocyte invasion. Invasion of erythrocytes by Camp strain merozoites is inhibited by pre-treatment of red blood cells by EBA-175 from the heterologous strain, FCR-3. The sequence of the Camp strain has been published and we report here the sequence of the FCR-3 strain. The sequences are nearly identical except for a 423-bp segment in the FCR-3 strain, F-segment, that is not found in the Camp strain and a 342-bp segment, C-segment, present in the Camp strain but not in the FCR-3 strain. The locations of these two segments are different in Camp and FCR-3 EBA-175 genes and there is little DNA or amino acid sequence homology between them. The essentially dimorphic alleles, F-segment and C-segment, are conserved in all isolates examined to date. Evidence of genetic cross-over between the FCR-3 and the Camp EBA-175 genes was not observed in the analysis of a limited number of wild isolates. The continued study of the biological relevance of these sequence divergences in EBA-175 may further elucidate the sequence of events resulting in merozoite invasion of erythrocytes.

Effects of serum from treated patients on antibody-dependent cell adherence to the infective larvae of Brugia malayi.

Adherence assays were used to demonstrate the in vitro effect of serum-dependent cellular adherence of human buffy coat cells to infective larvae of Brugia malayi in filariasis patients treated with antifilarial drugs. In this study, microfilaraemic patients were treated with either diethylcarbamazine citrate (DEC), mebendazole or levamisole hydrochloride. It was found that DEC and mebendazole decreased the motility of infective larvae due to a direct action of the drugs. Sera of levamisole-treated patients caused increased adherence of human buffy coat cells to infective larvae, leading to a decrease in motility and cuticular damage as confirmed by scanning electron microscopic studies. However, serum of levamisole-treated patients alone could cause a similar lethal effect on infective larvae. Studies with the indirect fluorescent antibody test suggested that IgM was involved in this phenomenon. Complement did not appear to be important.

Anemia in parasite- and recombinant protein-immunized aotus monkeys infected with Plasmodium falciparum.

Plasmodium falciparum-induced anemia was characterized in Aotus monkeys repeatedly immunized by infection with P. falciparum (FVO strain) parasites, then cross-challenged with CAMP strain, or in monkeys receiving blood stage challenges as part of malaria vaccine trials. In 4 studies, 25 (30.5%) of 82 monkeys had at least a 50% reduction in hematocrit; mean day of maximum parasitemia was 12.5, whereas the mean day of minimum hematocrit was 18.8 (P < 0.0009). Decreased hematocrit levels were not associated with reticulocytosis until parasite densities decreased significantly from peak levels. Direct antibody tests to detect IgG and C3d on the surface of erythrocytes were negative. Nonantibody/noncomplement-mediated lysis of uninfected erythrocytes seems to be the principal cause of the anemia, and it also seems that bone marrow suppression and lysis of infected erythrocytes contributed to the anemia. Partial immunity-whether induced by repeated immunization with whole parasites or with vaccine-seems important to the development of anemia.

Identification of Brugia malayi in vectors with a species-specific DNA probe.

We evaluated the potential value of a cloned sequence of genomic DNA of Brugia malayi as a species-specific probe. Clone pBm 15 reacted with all stages of 8 different geographic isolates of B. malayi and cross-hybridized with microfilariae of B. timori. It did not hybridize with Wuchereria bancrofti or with B. pahangi, W. kalimantani, Dirofilaria repens, Breinlia booliati or Cardiofilaria species, animal filariids that can be sympatric with B. malayi. P32-labeled clone pBm 15 correctly identified mosquitoes infected even with 1 infective larva of B. malayi. This specific DNA probe should be an invaluable tool to monitor control programs of Brugian filariasis.

Immune responses in human Brugia malayi infections: serum dependent cell-mediated destruction of infective larvae in vitro.

Serum dependent cell-mediated cytotoxicity of subperiodic Brugia malayi infective larvae in vitro was investigated. In vitro cellular adherence of normal human buffy coat cells to infective larvae of B. malayi was promoted by sera from patients with elephantiasis, tropical pulmonary eosinophilia (TPE) and amicrofilaraemic symptomatic filariasis, as well as by sera from normal subjects from filariasis endemic areas. However, strongest adherence was observed with TPE sera. Scanning electron microscopic (SEM) studies confirmed that the cellular adherence resulted in gross surface damage to the infective larvae. Studies with the indirect fluorescent antibody test (IFAT) suggested that IgG and/or IgM might be involved in the process of adherence. Complement did not appear to be important.

Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity.

Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

Safety and immunogenicity of a recombinant nonglycosylated erythrocyte binding antigen 175 Region II malaria vaccine in healthy adults living in an area where malaria is not endemic.

Erythrocyte binding antigen region II (EBA-175) is a conserved antigen of Plasmodium falciparum that is involved in binding of the parasite to the host's erythrocytes. We evaluated the safety and immunogenicity of a recombinant EBA-175 vaccine with aluminum phosphate adjuvant in healthy young adults living in the United States. Eighteen subjects/group received ascending doses (5, 20, 80, or 160 µg) of the vaccine at 0, 1, and 6 months; 8 subjects received placebo. Most of the injection site and systemic reactions were mild to moderate in intensity. After 2 or 3 doses of the vaccine at any concentration, antibody levels measured by enzyme-linked immunosorbent assay were significantly higher than those for the placebo group. Sera from subjects who received 3 doses of the vaccine at any concentration inhibited the growth of erythrocyte-stage P. falciparum at low levels compared to sera from placebo recipients or preimmune sera. In conclusion, the EBA-175 vaccine with adjuvant was safe and immunogenic in malaria-naïve subjects.