Thomas C. Luke

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.

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

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

BACKGROUND Challenge 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. METHODS We 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. RESULTS At 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. CONCLUSIONS In 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.

Boosting of DNA Vaccine-Elicited Gamma Interferon Responses in Humans by Exposure to Malaria Parasites

ABSTRACT A mixture of DNA plasmids expressing five Plasmodium falciparum pre-erythrocyte-stage antigens was administered with or without a DNA plasmid encoding human granulocyte-macrophage colony-stimulating factor (hGM-CSF) as an immune enhancer. After DNA immunization, antigen-specific gamma interferon (IFN-γ) responses were detected by ELISPOT in 15/31 volunteers to multiple class I- and/or class II-restricted T-cell epitopes derived from all five antigens. Responses to multiple epitopes (≤7) were detected simultaneously in some volunteers. By 4 weeks after challenge with P. falciparum parasites, 23/31 volunteers had positive IFN-γ responses and the magnitude of responses was increased from 2- to 143-fold. Nonetheless, none was protected against malaria. Volunteers who received hGM-CSF had a reduced frequency of IFN-γ responses to class I peptides compared to those who only received plasmids expressing P. falciparum proteins before challenge (3/23 versus 3/8; P = 0.15) or after parasite challenge (4/23 versus 5/8; P = 0.015) but not to class II peptides before or after challenge. The responses to one antigen (P. falciparum circumsporozoite protein [PfCSP]) were similar among volunteers who received the five-gene mixture compared to volunteers who only received the PfCSP DNA plasmid in a previous study. In summary, DNA-primed IFN-γ responses were boosted in humans by exposure to native antigen on parasites, coadministration of a plasmid expressing hGM-CSF had a negative effect on boosting of class I-restricted IFN-γ responses, and there was no evidence that immunization with PfCSP DNA in the mixture reduced T-cell responses to PfCSP compared to when it was administered alone.

Hark back: Passive immunotherapy for influenza and other serious infections

The world is experiencing a pandemic of swine-origin influenza virus H1N1. A vaccine to prevent disease is now available, and millions have or will become ill before they can be vaccinated. The ability to use swine-origin influenza virus vaccines as a public health tool has been described as a “race against time.” Oseltamivir and related drugs are being used in an effort to reduce morbidity and mortality, but their efficacy for treating severe influenza is suboptimal, and possible wide-spread emergence of oseltamivir-resistant mutants is a concern. Another approach for prevention and treatment of serious influenza is infusion of hyperimmune plasma. The United States has thousands of licensed blood product collection centers that produce millions of liters of plasma licensed by the Food and Drug Administration on an annual basis for the treatment of serious conditions. Immunotherapy using infusion of convalescent plasma (or hyperimmune intravenous immunoglobulin) has been reported to be an effective treatment for severe influenza and other virulent pathogens in animal models and humans. Plasma obtained from those that have recovered or were early recipients of vaccine offers a resource for production of an immediately available and potentially effective therapy at the local, state, and national level. Past, current, and future uses of immunotherapy and current advisory body recommendations for this approach are presented.

Middle East Respiratory Syndrome

The Middle East respiratory syndrome is caused by a coronavirus that was first identified in Saudi Arabia in 2012. Periodic outbreaks continue to occur in the Middle East and elsewhere. This report provides the latest information on MERS.

Clinical trial in healthy malaria-naïve adults to evaluate the safety, tolerability, immunogenicity and efficacy of MuStDO5, a five-gene, sporozoite/hepatic stage Plasmodium falciparum DNA vaccine combined with escalating dose human GM-CSF DNA

When introduced in the 1990s, immunization with DNA plasmids was considered potentially revolutionary for vaccine development, particularly for vaccines intended to induce protective CD8 T cell responses against multiple antigens. We conducted, in 1997−1998, the first clinical trial in healthy humans of a DNA vaccine, a single plasmid encoding Plasmodium falciparum circumsporozoite protein (PfCSP), as an initial step toward developing a multi-antigen malaria vaccine targeting the liver stages of the parasite. As the next step, we conducted in 2000–2001 a clinical trial of a five-plasmid mixture called MuStDO5 encoding pre-erythrocytic antigens PfCSP, PfSSP2/TRAP, PfEXP1, PfLSA1 and PfLSA3. Thirty-two, malaria-naïve, adult volunteers were enrolled sequentially into four cohorts receiving a mixture of 500 μg of each plasmid plus escalating doses (0, 20, 100 or 500 μg) of a sixth plasmid encoding human granulocyte macrophage-colony stimulating factor (hGM-CSF). Three doses of each formulation were administered intramuscularly by needle-less jet injection at 0, 4 and 8 weeks, and each cohort had controlled human malaria infection administered by five mosquito bites 18 d later. The vaccine was safe and well-tolerated, inducing moderate antigen-specific, MHC-restricted T cell interferon-γ responses but no antibodies. Although no volunteers were protected, T cell responses were boosted post malaria challenge. This trial demonstrated the MuStDO5 DNA and hGM-CSF plasmids to be safe and modestly immunogenic for T cell responses. It also laid the foundation for priming with DNA plasmids and boosting with recombinant viruses, an approach known for nearly 15 y to enhance the immunogenicity and protective efficacy of DNA vaccines.

Feasibility of Using Convalescent Plasma Immunotherapy for MERS-CoV Infection, Saudi Arabia.

Efficacy testing will be challenging because of the small pool of donors with sufficiently high antibody titers.

A dengue DNA vaccine formulated with Vaxfectin® is well tolerated, and elicits strong neutralizing antibody responses to all four dengue serotypes in New Zealand white rabbits

A tetravalent DNA vaccine formulated with Vaxfectin adjuvant was shown to elicit high levels of neutralizing antibody against all four dengue virus serotypes (Porter et al.,16), warranting further testing in humans. In preparation for a phase 1 clinical testing, the vaccine and the adjuvant were manufactured under current good manufacturing practice guidelines. The formulated vaccine and the adjuvant were tested for safety and/or immunogenicity in New Zealand white rabbits using a repeat dose toxicology study. The formulated vaccine and the adjuvant were found to be well tolerated by the animals. Animals injected with formulated vaccine produced strong neutralizing antibody response to all four dengue serotypes.

Production of Potent Fully Human Polyclonal Antibodies against Ebola Zaire Virus in Transchromosomal Cattle

Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities.