Report of a consultation on the optimization of clinical challenge trials for evaluation of candidate blood stage malaria vaccines, 18-19 March 2009, Bethesda, MD, USA.

Development and optimization of first generation malaria vaccine candidates has been facilitated by the existence of a well-established Plasmodium falciparum clinical challenge model in which infectious sporozoites are administered to human subjects via mosquito bite. While ideal for testing pre-erythrocytic stage vaccines, some researchers believe that the sporozoite challenge model is less appropriate for testing blood stage vaccines. Here we report a consultation, co-sponsored by PATH MVI, USAID, EMVI and WHO, where scientists from all institutions globally that have conducted such clinical challenges in recent years and representatives from regulatory agencies and funding agencies met to discuss clinical malaria challenge models. Participants discussed strengthening and harmonizing the sporozoite challenge model and considered the pros and cons of further developing a blood stage challenge possibly better suited for evaluating the efficacy of blood stage vaccines. This report summarizes major findings and recommendations, including an update on the Plasmodium vivax clinical challenge model, the prospects for performing experimental challenge trials in malaria endemic countries and an update on clinical safety data. While the focus of the meeting was on the optimization of clinical challenge models for evaluation of blood stage candidate malaria vaccines, many of the considerations are relevant for the application of challenge trials to other purposes.

Synthetic malaria peptide vaccine elicits high levels of antibodies in vaccinees of defined HLA genotypes.

A multiple antigen peptide (MAP) malaria vaccine containing minimal Plasmodium falciparum circumsporozoite protein repeat epitopes was assessed for safety and immunogenicity in volunteers of known class II genotypes. The MAP/alum/QS-21 vaccine formulation elicited high levels of parasite-specific antibodies in 10 of 12 volunteers expressing DQB1*0603, DRB1*0401, or DRB1*1101 class II molecules. In contrast, volunteers of other HLA genotypes were low responders or nonresponders. A second study of 7 volunteers confirmed the correlation of class II genotype and high responder phenotype. This is the first demonstration in humans that a peptide vaccine containing minimal T and B cell epitopes composed of only 5 amino acids (N, A, V, D, and P) can elicit antibody titers comparable to multiple exposures to irradiated P. falciparum-infected mosquitoes. Moreover, the high-responder phenotypes were predicted by analysis of peptide/HLA interactions in vitro, thus facilitating the rational design of epitope-based peptide vaccines for malaria, as well as for other pathogens.

Immunogenicity and efficacy in aotus monkeys of four recombinant Plasmodium falciparum vaccines in multiple adjuvant formulations based on the 19-kilodalton C terminus of merozoite surface protein 1.

The immunogenicity and protective efficacy of four versions of recombinant C-terminal 19-kDa epidermal growth factor-like region of the major surface protein 1 (rMSP1(19)) of Plasmodium falciparum was studied in Aotus monkeys. Vaccination with each of the four rMSP1(19) constructs elicited high levels of antibodies to MSP1(19) but only one construct, the 19-kDa fragment expressed as a secreted fusion protein from Saccharomyces cerevisiae (yP30P2MSP1(19)), induced a high degree of protective immunity in Aotus nancymai against lethal P. falciparum challenge. Protective formulation required Freund's adjuvant; vaccination with yP30P2MSP1(19) in six other adjuvants that are suitable for human use induced lower levels of antibody response and no protection. These results emphasize the need to continue the search for an adjuvant that is comparable to Freund's adjuvant in potency and is safe for use in humans.

Efficacy of vaccines containing rhoptry-associated proteins RAP1 and RAP2 of Plasmodium falciparum in Saimiri boliviensis monkeys.

A vaccine trial was conducted with rhoptry-associated proteins 1 and 2 (RAP1 and RAP2) of Plasmodium falciparum in Saimiri boliviensis monkeys to compare the ability of parasite-derived (PfRAP1 and 2) and recombinant proteins (rRAP1 and 2) to induce protective immune responses and to find adjuvants suitable for use in humans. Eight groups of 6 monkeys each were immunized with parasite-derived or recombinant RAP1 and 2 with Freund's complete adjuvant (FCA) followed by Freund's incomplete adjuvant (FIA), Montanide ISA720 adjuvant, or CRL1005 adjuvant. Recombinant RAP1 and RAP2 were also administered separately, with Montanide ISA720. After 3 immunizations, monkeys were challenged by iv inoculation of 50,000 parasites of the Uganda Palo Alto strain of P. falciparum. Of the animals vaccinated using FCA/FIA, 1 of 6 control monkeys, 3 of 6 immunized with PfRAP1 and 2, and 2 of 6 with rRAP1 and 2 did not require drug treatment. Of the monkeys vaccinated with Montanide ISA720 adjuvant, 0 of the 6 control monkeys, 2 of 6 immunized with RAP1 and 2, 1 of 6 immunized with rRAP1, and 4 of 6 immunized with RAP2 did not require drug treatment. Two of 6 monkeys immunized with PfRAP1 and 2 with CRL1005 did not require treatment. All groups receiving RAP1, RAP2, or both had a significant decrease in initial parasite multiplication rates and there was a significant negative correlation between anti-RAP2 antibody and multiplication rates. Animals were rechallenged with the homologous parasite 126 days after the first challenge. Of the monkeys that did not require drug treatment after the first challenge, none developed detectable parasitemia following rechallenge.

Phase I trial of two recombinant vaccines containing the 19kd carboxy terminal fragment of Plasmodium falciparum merozoite surface protein 1 (msp-1(19)) and T helper epitopes of tetanus toxoid.

The safety and immunogenicity of 2 yeast-derived, blood-stage malaria vaccines were evaluated in a phase l trial. Healthy adults were given 2 or 3 doses of alum-adsorbed vaccine containing the 19 kDa carboxy-terminal fragment of the merozoite surface protein-1 (MSP-1(19)) derived from the 3D7 or the FVO strain of Plasmodium falciparum fused to tetanus toxoid T-helper epitopes P30 and P2. The first 2 doses of MSP-1(19) were well tolerated. Hypersensitivity reactions occurred in 3 subjects after the third dose of MSP-1(19), including bilateral injection site reactions in 2 (one with generalized skin rash), and probable histamine-associated hypotension in 1. Serum antibody responses to MSP-1(19) occurred in 5/16, 9/16 and 0/8 subjects given 20 microg of MSP-1(19), 200 microg of MSP-1(19), and control vaccines (hepatitis B or Td), respectively. Both MSP-1(19) vaccines were immunogenic in humans, but changes in formulation will be necessary to improve safety and immunogenicity profiles.

High dose chemoradiotherapy followed by esophagectomy for adenocarcinoma of the esophagus and gastroesophageal junction: results of a phase II study of the Eastern Cooperative Oncology Group.

BACKGROUND: To assess the toxicity, local response, and survival associated with multimodality therapy in a cooperative group setting, patients with biopsy-proven clinical Stage I or II adenocarcinoma of the esophagus (staged according to 1983 American Joint Committee on Cancer criteria) or gastroesophageal junction were treated with concomitant radiation and chemotherapy followed by esophagectomy. METHODS: Radiotherapy was administered in daily 2-gray (Gy) fractions 5 days a week until a total of 60 Gy was reached. 5-fluorouracil (5-FU) was infused continuously at a dose of 1000 mg/m2/day for 96 hours on Days 2-5 and 28-31. On Day 2, a 10 mg/m2 bolus of mitomycin was injected intravenously. Esophagectomy was performed 4-8 weeks following completion of the radiotherapy. RESULTS: During the 18-month study period (August 1991 through January 1993), 46 eligible patients were accrued from 21 institutions. Eight patients were Stage I and 38 Stage II. Eighty-seven percent of patients (40 of 46) received 6000 centigray (cGy), and all received >5000 cGy. Seventy-eight percent of patients (36 of 46) received >90% of the planned 5-FU dose. Follow-up ranged from 11 to 36 months (median, 22 months). There were eight treatment-related deaths; two were preoperative (from adult respiratory distress syndrome) and six were postoperative. Complete or partial response prior to esophagectomy was observed in 63% of cases, stable disease in 15%, and progression in 20%. Thirty-three patients underwent esophagectomy (transhiatal, n=14; Ivor Lewis, n=16; other, n=3). No tumor was found in the specimens resected from 8 of these 33 patients; this represented a pathologic complete response rate of 17% overall and 24% for those who underwent esophagectomy. Overall median survival was 16.6 months, 1-year survival 57%, and 2-year survival 27%. Survival was significantly worse for patients with circumferential cancers (median, 18.1 months vs. 8.3 months; P <0.05). CONCLUSION: High dose radiation therapy with concurrent 5-FU and mitomycin may be administered to patients with esophageal adenocarcinoma with acceptable morbidity. However, in a cooperative group setting, esophagogastrectomy following intensive chemoradiotherapy is associated with excessive morbidity and mortality. Circumferential tumor growth is a significant adverse prognostic factor.

Plasmodium falciparum: passive immunization of Aotus lemurinus griseimembra with immune serum.

Owl monkeys (Aotus lemurinus griseimembra) were immunized against Plasmodium falciparum by infection and drug cure. After challenge, 3 of 4 monkeys developed extended prepatent periods and low grade parasitemias followed by self cure. The fourth monkey did not develop a patent infection. Immune monkey serum passively transferred at the time of challenge conferred immunity to 20 naive monkeys. Immunity was characterized by extended prepatent periods in 19 monkeys, low levels of parasitemia (< or = 1%) followed by self cure in 12 animals, and lack of detectable infection in 3 recipient monkeys. Immune serum collected from monkeys undergoing repeated challenges afforded more protection than serum from singly infected monkeys. However, single doses of hyperimmune serum appeared to be as effective as multiple doses. Normal serum had no effect on the course of infection in 12 monkeys. These studies confirm that owl monkeys can be immunized by infection and cure and demonstrate that this immunity can, in large part, be transferred to nonimmune recipients with serum from immune donors.

Malaria vaccine research.

In our report "Activation of Raf as a result of recruitment to the plasma membrane" (3 June, p. 1463) (1), panels E and F of figure 1 on page 1464 were incorrect. The correct photographs appear below. In addition, the [See figure in the PDF file] second sentence of the legend to figure 1 should have read, "The Raf constructs were tagged at the COOH-terminus with a Glu-Glu epitope (MEYMPME) (24) for c-Raf, or at the NH(2)-terminus with both the Glu-Glu and the Myc (MEQKLISEEDL) (23) epitopes for RafCAAX"; the next-to-the-last sentence of the legend to figure 1 should have read, "The c-Raf constructs in (A through D) are Glu-Glu-tagged and were detected by using an anti Glu-Glu antibody, and the RafCAAX and Raf6QCAAX constructs used in E and F were detected by using the antibody to Raf COOH-terminal peptide"; and the third sentence of note 26 should have read, "After blocking with 5% milk in phosphate-buffered saline (M-PBS), cells were incubated with a mouse monoclonal antibody to Glu-Glu or a rabbit polyclonal antibody to a 20-amino acid COOH-terminal peptide of Raf-1 (Santa Cruz Biotechnology, Santa Cruz, California), washed, and incubated with donkey antibodies to mouse or rabbit IgG combined with Texas Red (Jackson) in M-PBS, washed, and mounted in FITC-Guard (Testog)."

The major merozoite surface protein as a malaria vaccine target.

Experts gathered for two days in the summer of 1992 at the National Institutes of Health and the Walter Reed Army Institute of Research to discuss the potential of a major merozoite surface protein (MSP-1) in malaria vaccine development. The participants came in an exemplary spirit of co-operation, sharing ideas and unpublished data toward the common goal of a malaria vaccine. Their conclusions are presented here by Carter Diggs, Ripley Bollou and Lou Miller.

Small cell carcinoma of the lung. Treatment in the community.

The results of nonprotocol treatment of 232 patients with small cell lung cancer seen by a group of community-based medical oncologists over a 13-year period were evaluated. Factors associated with improved survival also were assessed. The following patient characteristics significantly improved survival: limited stage of disease at diagnosis, treatment of extensive (but not limited) disease with regimens including etoposide and cisplatin, tumor resection, age younger than 70 years, radiation therapy to the chest, and female sex (extensive disease only). Comparison of the data from this study with published results of protocol studies showed similar outcomes.

Independent instances of "souvenir" Asian skulls from the Tampa Bay area.

In the summer of 1984, police in Pinellas County, Florida, confiscated six identically colored imported Asian skulls (in a shipping case) from a private citizen. In May 1988, in nearby Hillsborough County, police confiscated a very similar skull from another private citizen, who allegedly had found it in an abandoned house. Aside from slight color differences between the six found in Pinellas County and the one found in Hillsborough County, the skulls are virtually identical in their osteological characteristics and condition and in the vital statistics derived from each. Each skull is as clean and dry as those typically sold by commercial scientific supply outlets in the United States. Each is edentulous (primarily premortem), between approximately 20 and 60 years of age at death, and morphologically Asian. Five of the seven are morphologically male, one is morphologically female, and one is a mosaic with respect to gender-related features. Police, medical examiners, coroners, and forensic anthropologists should be aware of such "souvenir" specimens, in the event that they encounter similar skulls. Discriminant function analyses for race and sex yield considerably conflicting results, which underscores the need for using extreme caution when interpreting forensic science estimates based on such techniques.

Cancer of unknown primary site. Deciding how far to carry evaluation.

Management of most patients who present with metastatic cancer from an unknown primary site is challenging. These patients often are debilitated from the onset of their disease, have symptoms that are hard to control, and respond poorly to systemic therapy. The decision regarding the extent of a frequently unrewarding diagnostic evaluation, especially in these days of cost containment, is difficult. Knowing that few will benefit from aggressive therapy, the treating physician should make quality of life the most important goal in caring for these patients.

Characterization of gp195 processed products purified from Plasmodium falciparum culture supernates.

Schizonts of the malaria parasite Plasmodium falciparum synthesize a 195 kDa surface glycoprotein (gp195) that is processed into several smaller products including one of 83 kDa, which, in the case of the Camp strain, is sequentially processed into 73 and 67 kDa products. gp195 and its processing intermediates larger than 83 kDa were not precipitated from culture supernates, but the 83 and 73 kDa products were precipitated by three monoclonal antibodies (McAbs). The 83 and 73 kDa products were affinity purified from culture supernates by adsorbing to McAb 7B2 coupled to Affigel 10 and eluting either with 0.2 N acetic acid, pH 2.8, or with 3 M potassium isothiocyanate (KSCN). The epitope recognized by McAb 7B2 was denatured by acid elution but could be regenerated by treating with 8 M urea followed by dialysis. The implications of renaturing antigens to regenerate epitopes should be considered in studies on the purification, function and immunogenicity of malaria antigens.

Our experiences with breeding of nine-banded armadillos (Dasypus novemcinctus) in captivity.

The nine-banded armadillo is considered the best animal model in the study of leprosy. Armadillos have never been successfully bred in the laboratory and therefore animals required for the experiments are captured from the wild and are likely to carry many diseases including leprosy. An attempt was made to breed them in captivity. Our attempt to house them in various combinations in their natural environment in the farmlands of Louisiana was successful. Although a significant increase in conception and delivery was recorded, consistent breeding of the animals was not obtained.