Stephen L. Hoffman

Protection of Humans against Malaria by Immunization with Radiation-Attenuated Plasmodium falciparum Sporozoites

During 1989-1999, 11 volunteers were immunized by the bites of 1001-2927 irradiated mosquitoes harboring infectious sporozoites of Plasmodium falciparum (Pf) strain NF54 or clone 3D7/NF54. Ten volunteers were first challenged by the bites of Pf-infected mosquitoes 2-9 weeks after the last immunization, and all were protected. A volunteer challenged 10 weeks after the last immunization was not protected. Five previously protected volunteers were rechallenged 23-42 weeks after a secondary immunization, and 4 were protected. Two volunteers were protected when rechallenged with a heterologous Pf strain (7G8). In total, there was protection in 24 of 26 challenges. These results expand published findings demonstrating that immunization by exposure to thousands of mosquitoes carrying radiation-attenuated Pf sporozoites is safe and well tolerated and elicits strain-transcendent protective immunity that persists for at least 42 weeks.

Primaquine Therapy for Malaria

Primaquine is the only available drug for preventing relapse of malaria, and confusion surrounds its use. This review examines the wide range of clinical applications of primaquine described in the medical literature between 1946 and 2004. The risk of relapse of Plasmodium vivax malaria without primaquine therapy ranged from 5% to 80% or more, depending largely upon geographic location. Supervision of therapy profoundly impacts the risk of relapse, and almost all reports of malaria resistant to primaquine are associated with lack of such supervision. We nonetheless suspect that there is widespread resistance to the standard course of primaquine therapy, which is 15 mg primaquine base daily for 14 days. Clinical evidence confirms that a course of 15 mg daily for just 5 days, a regimen widely used in areas where malaria is endemic, has no discernible efficacy. This review supports a recommendation for a regimen of 0.5 mg/kg primaquine daily for 14 days, on the basis of superior efficacy and good tolerability and safety in nonpregnant persons without glucose-6-phosphate dehydrogenase deficiency.

Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine

SUMMARY Annually, malaria causes >300 million clinical cases and 1 million deaths, is responsible for the loss of >1% of gross domestic product (GDP) in Africa and is a serious concern for travelers. An effective vaccine could have a dramatic impact on the disease. For 20 years, scientists have tried to develop modern, recombinant `subunit malaria vaccines. This has been difficult. In fact, there is only one recombinant protein vaccine on the market for any disease, and no vaccines based on synthetic peptides, recombinant viruses, recombinant bacteria or DNA plasmids. Most vaccines are based on attenuated or inactivated whole pathogens or material derived directly from the infectious agent. It is in that context that our recent report summarizing the protection of humans with attenuated Plasmodium falciparum (Pf) sporozoites produced at four different sites over 25 years is important. In studies utilizing live mosquitoes as the vaccine delivery mechanism, there was complete protection against malaria in 93% of volunteers (13/14) and 94% of challenges (33/35). Sanarias goal is to develop and commercialize a non-replicating, metabolically active Pf sporozoite vaccine. Three practical questions must be addressed before manufacturing for clinical trials: (1) can one administer the vaccine by a route that is clinically practical; (2) can one produce adequate quantities of sporozoites; and (3) can sporozoites be produced with the physical characteristics that meet the regulatory, potency and safety requirements of regulatory authorities? Once these questions have been answered, Sanaria will demonstrate that the vaccine protects >90% of human recipients against experimental challenge with Pf sporozoites, can be produced with an efficiency that makes it economically feasible, and protects >90% of African infants and children from infection, and thus from severe morbidity and mortality. By producing a vaccine for travelers, Sanaria will provide the infrastructure, regulatory foundation and funds necessary to speed licensure, manufacturing and deployment of the vaccine for the infants and children who need it most.

Meta-Analysis: Convalescent Blood Products for Spanish Influenza Pneumonia: A Future H5N1 Treatment?

Context Studies of Spanish influenza that evaluated effects of transfusion with influenza-convalescent blood products might offer insights regarding potential treatments for H5N1 influenza. Contribution This review of 8 controlled studies published in English-language medical literature between 1918 to 1925 found that transfusion with influenza-convalescent human blood products may have reduced risk for death in hospitalized patients with Spanish influenza complicated by pneumonia. Transfusions caused some chill reactions. Cautions Studies had many methodologic limitations. Implications Studies from the Spanish influenza era support the idea that convalescent human H5N1 plasma could be an effective, accessible treatment that should be studied in clinical trials. The Editors The world is bracing for a potential H5N1 influenza pandemic. During the Spanish influenza pandemic, an estimated 30% of the worlds population became ill and 50 million people died (1). An H5N1 influenza pandemic could be equally or more severe. Unfortunately, effective vaccines will be difficult to produce before a novel human pandemic strain emerges and will take substantial time to manufacture and distribute in quantity. It is sobering that the worlds annual production capacity for influenza vaccine is 300 million doses (2)enough for 4.5% of the worlds population. These facts have caused some governments to develop response plans to pandemic influenza that involve creating antiviral stockpiles and increasing the capacity to handle surges in the need for medical care. Patients with H5N1 influenza often develop a fatal case of acute respiratory distress syndrome or multiple organ dysfunction syndrome that is similar to the syndromes reported in patients with Spanish influenza who developed pneumonia-like complications (35). To treat patients with H5N1 influenza, the World Health Organization recommends hospitalization with early use of oseltamivir and supportive care (3). Despite these treatments, 30% to 80% of hospitalized patients with H5N1 influenza have died, and an oseltamivir-resistant virus has developed in some patients (3, 4). A case series report of Vietnamese patients with H5N1 influenza suggested that supportive care may be the only option available (4). Even if more effective standard pharmaceutical treatments are produced, it is unlikely that sufficient quantities will be rapidly or widely available because of financial, logistical, and health care delivery limitations. Passively delivered anti-influenza antibodies in convalescent human plasma obtained from H5N1 survivors may offer a novel treatment approach and possible solution to these problems. Passive antibodies have been used to prevent or treat such diseases as rabies, measles, hepatitis B, cytomegalovirus, and respiratory syncytial virus (6), and convalescent human plasma may have efficacy in the treatment of severe acute respiratory syndrome (7, 8). The modern plasmapheresis systems in many hospitals and blood collection centers currently produce large volumes of plasma for treating coagulopathies and other conditions (9, 10). The same infrastructure, personnel, and regulatory framework could produce convalescent plasma for the treatment of H5N1 influenza. To help assess the potential treatment efficacy of convalescent plasma in reducing mortality in current patients with H5N1 influenza, we conducted a review of studies from the Spanish influenza era that used influenza-convalescent human blood products to treat patients with Spanish influenza complicated by pneumonia (influenza pneumonia). Methods Data Sources and Searches We developed and followed a protocol for the literature review and also followed standard reporting guidelines (11). The medical literature during the 1920s was not centrally indexed in an electronic or text database. Two authors first conducted a preliminary survey and study of the original medical literature published about Spanish influenza. This was done to gain an understanding of the scientific concepts, research methods, medical practices, and vocabulary used during that era to aid in the development of our review and search strategy. Subsequently, 1 author conducted a manual review of the indexes of the following medical journals from 1918 to 1925: Journal of the American Medical Association, Boston Medical and Surgical Journal (now New England Journal of Medicine), British Medical Journal, Canadian Medical Association Journal, Lancet, Archives of Internal Medicine, The Military Surgeon (United States), and Naval Medical Bulletin (United States). We searched 3 terms in the journal indexes: influenza, serotherapy, and pneumonia. We then searched subindexes or article titles that were listed under the 3 categories for any of the following terms: influenza, serotherapy, pneumonia, serum, plasma, blood, bronchopneumonia, convalescent, intravenous, and transfusion. Potentially relevant articles were obtained and reviewed. We also reviewed references of relevant articles. Of note, many of the source journals provided an indexed abstract section of articles drawn from other English-language and nonEnglish-language journals. Original articles on our topic were often published as an abstract by other journals, and the articles often cross-referenced each other. For practical reasons, including feasibility and resource constraints, we limited our searches to years in which relevant studies were likely to be published. Study Selection Two authors selected studies published in an English-language medical journal that met inclusion criteria defined a priori (Figure 1). Studies had to have used convalescent whole blood, plasma, or serum obtained from humans who had recovered from Spanish influenza as the treatment product and had to indicate the type, route, and volume of the product that was used. The treatment and control groups had to have included hospitalized patients with a diagnosis of influenza complicated by pneumonia, and investigators had to report mortality rates. The treatment group had to include at least 10 patients. The control group had to receive standard care and could not be assigned to receive, as a group, an alternative experimental therapy, such as an equine-derived antipneumococcus serum. Studies had to be conducted in a hospital setting during the Spanish influenza pandemic of 1918 to 1920. We excluded studies if they were reported only as an editorial, commentary, or abstract or as a translated synopsis of a nonEnglish-language study. Figure 1. Flow diagram of trial identification and selection. *Most of these studies were excluded on the basis of multiple criteria. Our rationale for the detailed inclusion and exclusion criteria was as follows. Hospitalized patients were likely to have had very severe illness and a more reliable diagnosis of influenza pneumonia than were patients whose illness was diagnosed and treated by general practitioners in the home. Although strains of Spanish influenza probably circulated before 1918 and certainly did so after 1920, the accuracy of a diagnosis of Spanish influenza pneumonia was likely to be reasonably good during years when herd immunity was low, the virus was virulent, and large epidemics occurred. Because scientific concepts, research methods, medical practices, and vocabulary have changed markedly since 1920, we restricted our analysis to articles that we could carefully scrutinize and for which we could reasonably reliably determine the primary clinical condition of patients, the treatment that was given, and characteristics of the treatment and control groups. Data Extraction and Quality Assessment Two authors independently extracted data about study characteristics, outcomes, adverse events, and quality. Disagreements were resolved by consensus. The quality of each study was assessed by using a 27-item checklist that was developed to assess the methodologic quality of randomized and nonrandomized studies of health care interventions (12). The quality scores could range from 0 to 27, with higher scores indicating better quality. Data Synthesis and Analysis We used as the principal measure of effect the range of absolute risk differences in death between the treatment and control groups. We conducted a planned subgroup analysis of mortality among patients who received early treatment (after <4 days of illness) compared with those who received late treatment (after 4 days). We also calculated overall crude case-fatality rates and pooled absolute risk differences in death by using the random-effects model of DerSimonian and Laird (13). Heterogeneity was assessed visually by using Galbraith plots (14) and statistically by using the I 2 statistic (15). To exclude the possibility that any one study was excessively influencing the results, we conducted a sensitivity analysis by excluding each study one at a time. We used the method of Egger and colleagues (16) to assess for statistical evidence of possible publication bias. All analyses were performed by using Stata software, version 9.1 (Stata Corp., College Station, Texas). Role of the Funding Source No funding was received for this review. Results Study Selection and Evaluation We searched hundreds of titles in the topic indexes and retrieved 72 manuscripts for screening (Figure 1). Many of these studies focused on the isolation and identification of the influenza bacillus or known bacterial pathogens or used various animal-derived antipneumococcus serums or other preparations for treatment. In 27 reports, influenza-convalescent human blood products were used to treat patients with Spanish influenza, with or without pneumonia complications. Of these, 8 studies described in 10 reports met all of our inclusion criteria (1726). No included study was identified solely from the citation review. We excluded 17 articles that were small case reports, were incomplete or noninterpretable, were written in a non-English language, or involved o

Codon Optimization of Gene Fragments Encoding Plasmodium falciparum Merzoite Proteins Enhances DNA Vaccine Protein Expression and Immunogenicity in Mice

ABSTRACT In contrast to conventional vaccines, DNA and other subunit vaccines exclusively utilize host cell molecules for transcription and translation of proteins. The adenine plus thymine content of Plasmodium falciparum gene sequences (∼80%) is much greater than that of Homo sapiens(∼59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of P. falciparumgenes encoded by DNA vaccines from that used by the parasite to those resembling mammalian codon usage would lead to increased P. falciparum protein expression in vitro in mouse cells and increased antibody responses in DNA-vaccinated mice. We synthesized gene fragments encoding the receptor-binding domain of the 175-kDaP. falciparum erythrocyte-binding protein (EBA-175 region II) and the 42-kDa C-terminal processed fragment of the P. falciparum merozoite surface protein 1 (MSP-142) using the most frequently occurring codon in mammals to code for each amino acid, and inserted the synthetic genes in DNA vaccine plasmids. In in vitro transient-expression assays, plasmids containing codon-optimized synthetic gene fragments (pS plasmids) showed greater than fourfold increased protein expression in mouse cells compared to those containing native gene fragments (pN plasmids). In mice immunized with 0.5, 5.0, or 50 μg of the DNA plasmids, the dose of DNA required to induce equivalent antibody titers was 10- to 100-fold lower for pS than for pN plasmids. These data demonstrate that optimizing codon usage in DNA vaccines can improve protein expression and consequently the immunogenicity of gene fragments in DNA vaccines for organisms whose codon usage differs substantially from that of mammals.

Protection of Aotus Monkeys by Plasmodium falciparum EBA-175 Region II DNA Prime—Protein Boost Immunization Regimen

Aotus monkeys received 4 doses of Plasmodium falciparum EBA-175 region II vaccine as plasmid DNA (Dv-Dv) or recombinant protein in adjuvant (Pv-Pv) or as 3 doses of DNA and 1 dose of protein (Dv-Pv). After 3 doses, antibody titers were approximately 10(4) in DNA-immunized monkeys and 10(6) in protein-immunized monkeys. A fourth dose did not significantly boost antibody responses in the Dv-Dv only or Pv-Pv only groups, but titers were boosted to approximately 10(6) in monkeys in the Dv-Pv group. Four weeks after the last immunization, the animals were challenged with 10(4) P. falciparum-parasitized erythrocytes. Peak levels of parasitemia were lower in the 16 monkeys that received region II-containing plasmids or proteins than in the 16 controls (geometric mean: 194,178 and 410,110 parasites/microL, respectively; P=.013, Students t test). Three of 4 monkeys in the Dv-Pv group did not require treatment. These data demonstrate that immunization with EBA-175 region II induces a significant antiparasite effect in vivo.

HLA-DR-Promiscuous T Cell Epitopes from Plasmodium falciparum Pre-Erythrocytic-Stage Antigens Restricted by Multiple HLA Class II Alleles

Previously, we identified and established the antigenicity of 17 CD8+ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN-γ responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8.7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.

Primaquine for Prevention of Malaria in Travelers

An expanding risk and range of endemic malaria threatens travelers. Primaquine is an old drug recently demonstrated to offer effective prophylaxis. Clinical trials conducted in Indonesia, Kenya, and Colombia showed that a primaquine base (30 mg per day) had protective efficacy against Plasmodium falciparum and Plasmodium vivax of 85%-93%. Among 339 children (age, >8 years) and adults taking this regimen for 12-52 weeks, there was no greater risk of adverse symptomatic events among primaquine users than among recipients of placebo in double-blind studies. Among 151 subjects evaluated after 20 or 52 weeks of daily primaquine therapy, methemoglobinemia was found to be mild (<13%; typically <6%) and transient (duration, <2 weeks). We consider primaquine base (0.5 mg/kg per day consumed with food) to be safe, well-tolerated, and effective prophylaxis against malaria for nonpregnant persons and those with normal glucose-6-phosphate dehydrogenase levels. Primaquines major advantage over most drugs for chemoprophylaxis is that it does not have to be taken before entering or beyond 3 days after leaving a malarious area.

Safety and Clinical Outcome of Experimental Challenge of Human Volunteers with Plasmodium falciparum-Infected Mosquitoes: An Update

BACKGROUNDnChallenge of volunteers by the bites of membrane-fed anopheline mosquitoes infected with Plasmodium falciparum was reported in 1986. In 1997, an analysis of experience with 118 volunteers indicated that mosquito inoculation of P. falciparum could be a safe, well-tolerated, reproducible, and efficient method of challenge.nnnMETHODSnWe reviewed the records of 47 volunteers challenged at our institution with the NF54 isolate of P. falciparum between 1998 and 2002. We also reviewed data from 17 published studies of experimental challenge conducted since 1996.nnnRESULTSnAt our institution, the time to onset of first symptoms (incubation period) was 8.9 days, and the time to first detectable parasitemia on blood smear (prepatent period) was 10.5 days. All volunteers became symptomatic. Most symptoms were mild to moderate, although 21% of volunteers had at least 1 severe symptom. None developed complicated or severe malaria, and all were cured. Laboratory assessments demonstrated modest, short-term abnormalities typical of malaria. Review of 17 published studies demonstrated that an additional 367 volunteers received experimental challenge safely with similar outcomes.nnnCONCLUSIONSnIn total, data from 532 volunteers demonstrate that experimental challenge is safe and results in predictable incubation and prepatent periods. Our findings support the continued use of this method for testing efficacy of vaccines and drugs against P. falciparum.

Enhancement of the immune response in rabbits to a malaria DNA vaccine by immunization with a needle-free jet device.

We compared the needle free jet device device Biojector with syringe/needle as a method to administer a DNA vaccine encoding the Plasmodium falciparum circumsporozoite protein (PfCSP) in albino rabbits. A group of three rabbits was injected by the intramuscular (IM) route using a syringe/needle combination, a second group IM with the Biojector device and a third group both IM and intradermal (ID) using the Biojector. When animals were immunized with the Biojector IM or IM/ID as compared to the syringe/needle IM, we observed 10- and 50-fold greater antibody titers, as measured by enzyme immunoassay (EIA) and indirect fluorescence antibody test (IFAT), respectively. We also observed that the Biojector conferred a greater ability to prime the immune system as compared with the needle. The subsequent boosting of all animals with a recombinant canary pox virus (ALVAC) expressing PfCSP induced significantly higher titers in both Biojector groups of rabbits as compared with the needle and naive animals. These results provided the foundation for a clinical trial using the same regime.