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1.  Evans Blue Staining Reveals Vascular Leakage Associated with Focal Areas of Host-Parasite Interaction in Brains of Pigs Infected with Taenia solium 
PLoS ONE  2014;9(6):e97321.
Cysticidal drug treatment of viable Taenia solium brain parenchymal cysts leads to an acute pericystic host inflammatory response and blood brain barrier breakdown (BBB), commonly resulting in seizures. Naturally infected pigs, untreated or treated one time with praziquantel were sacrificed at 48 hr and 120 hr following the injection of Evans blue (EB) to assess the effect of treatment on larval parasites and surrounding tissue. Examination of harvested non encapsulated muscle cysts unexpectedly revealed one or more small, focal round region(s) of Evans blue dye infiltration (REBI) on the surface of otherwise non dye-stained muscle cysts. Histopathological analysis of REBI revealed focal areas of eosinophil-rich inflammatory infiltrates that migrated from the capsule into the tegument and internal structures of the parasite. In addition some encapsulated brain cysts, in which the presence of REBI could not be directly assessed, showed histopathology identical to that of the REBI. Muscle cysts with REBI were more frequent in pigs that had received praziquantel (6.6% of 3736 cysts; n = 6 pigs) than in those that were untreated (0.2% of 3172 cysts; n = 2 pigs). Similar results were found in the brain, where 20.7% of 29 cysts showed histopathology identical to muscle REBI cysts in praziquantel-treated pigs compared to the 4.3% of 47 cysts in untreated pigs. Closer examination of REBI infiltrates showed that EB was taken up only by eosinophils, a major component of the cellular infiltrates, which likely explains persistence of EB in the REBI. REBI likely represent early damaging host responses to T. solium cysts and highlight the focal nature of this initial host response and the importance of eosinophils at sites of host-parasite interaction. These findings suggest new avenues for immunomodulation to reduce inflammatory side effects of anthelmintic therapy.
doi:10.1371/journal.pone.0097321
PMCID: PMC4051593  PMID: 24915533
2.  PET Reveals Inflammation around Calcified Taenia solium Granulomas with Perilesional Edema 
PLoS ONE  2013;8(9):e74052.
Objective
Neurocysticercosis, an infection with the larval form of the tapeworm, Taeniasolium, is the cause of 29% of epilepsy in endemic regions. Epilepsy in this population is mostly associated with calcified granulomas; at the time of seizure recurrence 50% of those with calcifications demonstrate transient surrounding perilesional edema. Whether edema is consequence of the seizure, or a result of host inflammation directed against parasite antigens or other processes is unknown. To investigate whether perilesional edema is due to inflammation, we imaged a marker of neuroinflammation, translocater protein (TSPO), using positron emission tomography (PET) and the selective ligand 11C-PBR28.
Methods
In nine patients with perilesional edema, degenerating cyst or both, PET findings were compared to the corresponding magnetic resonance images. Degenerating cysts were also studied because unlike perilesional edema, degenerating cysts are known to have inflammation. In three of the nine patients, changes in 11C-PBR28 binding were also studied over time. 11C-PBR28 binding was compared to the contralateral un-affected region.
Results
11C-PBR28 binding increased by a mean of 13% in perilesional edema or degenerating cysts (P = 0·0005, n = 13 in nine patients). Among these 13 lesions, perilesional edema (n=10) showed a slightly smaller increase of 10% compared to the contralateral side (P = 0·005) than the three degenerating cysts. In five lesions with perilesional edema in which repeated measurements of 11C-PBR28 binding were done, increased binding lasted for 2-9 months.
Conclusions
Increased TSPO in perilesional edema indicates an inflammatory etiology. The long duration of increased TSPO binding after resolution of the original perilesional edema and the pattern of periodic episodes is consistent with intermittent exacerbation from a continued baseline presence of low level inflammation. Novel anti-inflammatory measures may be useful in the prevention or treatment of seizures in this population.
doi:10.1371/journal.pone.0074052
PMCID: PMC3773048  PMID: 24058514
3.  Quantitative Screening for Anticestode Drugs Based on Changes in Baseline Enzyme Secretion by Taenia crassiceps 
Neurocysticercosis (NCC), an infection of the brain with the larval stage of the Taenia solium tapeworm, is responsible for an estimated one-third of adult-onset epilepsy cases in regions of the world where it is endemic. Currently, anthelmintic drugs used for treatment of NCC are only partially effective, and there is, therefore, a pressing need for new therapeutic agents. Discovery of new anthelmintics with activity against T. solium has been limited by the lack of suitable sensitive assays that allow high-throughput screening. Using an in vitro culture system with Taenia crassiceps metacestodes, we demonstrate that changes in secretion of parasite-associated alkaline phosphatase (AP) and phosphoglucose isomerase (PGI) can be used to detect and quantify anthelmintic effects of praziquantel (PZQ), a drug with activity against T. solium. We applied two enzyme release assays to screen for anti-T. crassiceps activity in nonconventional antiparasitic drugs and demonstrate that nitazoxanide and artesunate induced release of both AP and PGI in differing time- and dose-related patterns. Furthermore, imatinib, a tyrosine kinase inhibitor previously reported to have parasiticidal activity against Schistosoma mansoni, also induced release of both AP and PGI in a dose-dependent manner, similar in pattern to that observed with the other anthelmintics. We also evaluated release of ATP into cyst supernatants as an indicator of drug effects but did not see any differences between treated and untreated cysts. These data provide the basis for rapid and quantitative screening assays for testing for anthelmintic activity in candidate anticestode agents.
doi:10.1128/AAC.01022-12
PMCID: PMC3553704  PMID: 23229489
4.  In Vitro Analysis of Albendazole Sulfoxide Enantiomers Shows that (+)-(R)-Albendazole Sulfoxide Is the Active Enantiomer against Taenia solium 
Albendazole is an anthelmintic drug widely used in the treatment of neurocysticercosis (NCC), an infection of the brain with Taenia solium cysts. However, drug levels of its active metabolite, albendazole sulfoxide (ABZSO), are erratic, likely resulting in decreased efficacy and suboptimal cure rates in NCC. Racemic albendazole sulfoxide is composed of ABZSO (+)-(R)- and (−)-(S) enantiomers that have been shown to differ in pharmacokinetics and activity against other helminths. The antiparasitic activities of racemic ABZSO and its (+)-(R)- and (−)-(S) enantiomers against T. solium cysts were evaluated in vitro. Parasites were collected from naturally infected pigs, cultured, and exposed to the racemic mixture or to each enantiomer (range, 10 to 500 ng/ml) or to praziquantel as a reference drug. The activity of each compound against cysts was assayed by measuring the ability to evaginate and inhibition of alkaline phosphatase (AP) and parasite antigen release. (+)-(R)-ABZSO was significantly more active than (−)-(S)-ABZSO in suppressing the release of AP and antigen into the supernatant in a dose- and time-dependent manner, indicating that most of the activity of ABZSO resides in the (+)-(R) enantiomer. Use of this enantiomer alone may lead to increased efficacy and/or less toxicity compared to albendazole.
doi:10.1128/AAC.01465-12
PMCID: PMC3553705  PMID: 23229490
5.  Corticosteroid use in neurocysticercosis 
Expert review of neurotherapeutics  2011;11(8):1175-1183.
The cystic larvae of Taenia solium commonly infect the human nervous system, resulting in neurocysticercosis, a major contributor to seizure disorders in most of the world. Inflammation around the parasites is a hallmark of neurocysticercosis pathophysiology. Although mechanisms regulating this inflammation are poorly understood, anti-inflammatory drugs, particularly corticosteroids, have been long used alone or with anthelmintics to manage disease and limit neurological complications and perhaps damage to neural tissues. Only scarce controlled data exist to determine when and what type of corticosteroids and the treatment regime to use. This article revisits the mechanisms of action, rationale, evidence of benefit, safety and problems of corticosteroids in the context of neurocysticercosis, as well as alternative anti-inflammatory strategies to limit the damage caused by inflammation in the CNS.
doi:10.1586/ern.11.86
PMCID: PMC3721198  PMID: 21797658
central nervous system; corticosteroids; cysticercosis; neurocysticercosis; seizures; Taenia solium
6.  Neurocysticercosis—More Than a Neglected Disease 
doi:10.1371/journal.pntd.0001964
PMCID: PMC3636045  PMID: 23638190
7.  Disseminated Strongyloides stercoralis Infection in HTLV-1-Associated Adult T-Cell Leukemia/Lymphoma 
Acta Haematologica  2011;126(2):63-67.
A 55-year-old woman with human T-cell lymphotropic virus type-1 (HTLV-1)-associated adult T-cell leukemia (ATL) and a history of previously treated Strongyloides stercoralis infection received anti-CD52 monoclonal antibody therapy with alemtuzumab on a clinical trial. After an initial response, she developed ocular involvement by ATL. Alemtuzumab was stopped and high-dose corticosteroid therapy was started to palliate her ocular symptoms. Ten days later, the patient developed diarrhea, vomiting, fever, cough, skin rash, and a deteriorating mental status. She was diagnosed with disseminated S. stercoralis. Corticosteroids were discontinued and the patient received anthelmintic therapy with ivermectin and albendazole with complete clinical recovery.
doi:10.1159/000324799
PMCID: PMC3080579  PMID: 21474923
Adult T-cell leukemia; Alemtuzumab; Corticosteroid; Disseminated Strongyloides; HTLV-1; Human T-cell lymphotropic virus type-1
8.  Filarial Infection Suppresses Malaria-Specific Multifunctional Th1 and Th17 Responses in Malaria and Filarial Coinfections 
The mechanisms underlying the modulation of both the malaria-specific immune response and the course of clinical malaria in the context of concomitant helminth infection are poorly understood. We used multiparameter flow cytometry to characterize the quality and the magnitude of malaria-specific T cell responses in filaria-infected and -uninfected individuals with concomitant asymptomatic Plasmodium falciparum malaria in Mali. In comparison with filarial-uninfected subjects, filarial infection was associated with higher ex vivo frequencies of CD4+ cells producing IL-4, IL-10, and IL-17A (p = 0.01, p = 0.001, and p = 0.03, respectively). In response to malaria Ag stimulation, however, filarial infection was associated with lower frequencies of CD4+ T cells producing IFN-γ, TNF-α, and IL-17A (p < 0.001, p = 0.04, and p = 0.04, respectively) and with higher frequencies of CD4+IL10+T cells (p = 0.0005). Importantly, filarial infection was associated with markedly lower frequencies of malaria Ag-specific Th1 (p < 0.0001), Th17 (p = 0.012), and “TNF-α” (p = 0.0008) cells, and a complete absence of malaria-specific multifunctional Th1 cells. Filarial infection was also associated with a marked increase in the frequency of malaria-specific adaptive regulatory T/Tr1 cells (p = 0.024), and the addition of neutralizing anti–IL-10 Ab augmented the amount of Th1-associated cytokine produced per cell. Thus, among malaria-infected individuals, concomitant filarial infection diminishes dramatically the frequencies of malaria-specific Th1 and Th17 T cells, and alters the quality and magnitude of malaria-specific T cell responses.
doi:10.4049/jimmunol.1003778
PMCID: PMC3407819  PMID: 21411732
9.  At Homeostasis Filarial Infections Have Expanded Adaptive T Regulatory but Not Classical Th2 Cells 
Despite the well-documented immune suppression associated with human helminth infections, studies characterizing the immune response at the single-cell level are scanty. We used multiparameter flow cytometry to characterize the type of effector (Th1, Th2, and Th17) and regulatory (natural T regulatory cells [nTregs] and adaptive Treg cells [aTreg/type 1 regulatory cells (Tr1s)]) CD4+ and CD8+ T cells in filaria-infected (Fil+) and -uninfected (Fil−) individuals at homeostasis (in the absence of stimulation). Frequencies of CD4+ lymphocytes spontaneously producing IL-4, IL-10, and IL-17A were significantly higher in Fil+, as were those of IL-10+/IL-4+ double-producing CD4+ cells. Interestingly, frequencies of Th17 and aTreg/Tr1s but not classical Th1 or Th2 cells were significantly increased in Fil+ compared to Fil− individuals. Although the frequency of nTreg was increased in Fil+, IL-10 was overwhelmingly produced by CD4+CD25− cells. Moreover, the concentration of IL-10 produced spontaneously in vitro strongly correlated with the integrated geometric mean fluorescence intensity of IL-10–producing aTreg/Tr1s in Fil+. Together, these data show that at steady state, IL-10–producing aTreg/Tr1 as well as nTreg and effector Th17 CD4+ cells are expanded in vivo in human filarial infections. Moreover, we have established baseline ex vivo frequencies of effector and Tregs at homeostasis at a population level.
doi:10.4049/jimmunol.0904067
PMCID: PMC3407820  PMID: 20357251
10.  Expanded Numbers of Circulating Myeloid Dendritic Cells in Patent Human Filarial Infection Reflect Lower CCR1 Expression 
APC dysfunction has been postulated to mediate some of the parasite-specific T cell unresponsiveness seen in patent filarial infection. We have shown that live microfilariae of Brugia malayi induce caspase-dependent apoptosis in human monocyte-derived dendritic cells (DCs) in vitro. This study addresses whether apoptosis observed in vitro extends to patent filarial infections in humans and is reflected in the number of circulating myeloid DCs (mDCs; CD11c−CD123lo) in peripheral blood of infected microfilaremic individuals. Utilizing flow cytometry to identify DC subpopulations (mDCs and plasmacytoid DCs [pDCs]) based on expression of CD11c and CD123, we found a significant increase in numbers of circulating mDCs (CD11c+CD123lo) in filaria-infected individuals compared with uninfected controls from the same filaria-endemic region of Mali. Total numbers of pDCs, monocytes, and lymphocytes did not differ between the two groups. To investigate potential causes of differences in mDC numbers between the two groups, we assessed chemokine receptor expression on mDCs. Our data indicate that filaria-infected individuals had a lower percentage of circulating CCR1+ mDCs and a higher percentage of circulating CCR5+ mDCs and pDCs. Finally, live microfilariae of B. malayi were able to downregulate cell-surface expression of CCR1 on monocyte-derived DCs and diminish their calcium flux in response to stimulation by a CCR1 ligand. These findings suggest that microfilaria are capable of altering mDC migration through downregulation of expression of some chemokine receptors and their signaling functions. These observations have major implications for regulation of immune responses to these long-lived parasites.
doi:10.4049/jimmunol.1001605
PMCID: PMC3403815  PMID: 20956349
12.  A Species-Specific Approach to the Use of Non-Antimony Treatments for Cutaneous Leishmaniasis 
We used a species-specific approach to treat 10 patients with cutaneous leishmaniasis diagnosed using polymerase chain reaction. Non-antimony treatments (oral miltefosine, ketoconazole, and liposomal amphotericin B) were chosen as an alternative to pentavalent antimony drugs based on likely or proven drug efficacy against the infecting species. Leishmania Viannia panamensis was diagnosed in three patients and treated successfully with oral ketoconazole. Miltefosine treatment cured two patients with L. infantum chagasi. A wide variety of Leishmania responded to liposomal amphotericin B administered for 5–7 days. Three patients with L. V. braziliensis, one patient with L. tropica, and two patients with L. infantum chagasi were treated successfully. One person with L. V. braziliensis healed slowly because of a resistant bacterial superinfection, and a second patient with L. infantum chagasi relapsed and was retreated with miltefosine. These drugs were reasonably well-tolerated. In this limited case series, alternative non-antimony–based regimens were convenient, safe, and effective.
doi:10.4269/ajtmh.2011.10-0437
PMCID: PMC3005496  PMID: 21212212
13.  Plasmodium falciparum apical membrane antigen 1 vaccine elicits multifunctional CD4 cytokine-producing and memory T cells 
Vaccine  2009;27(38):5239-5246.
The Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading vaccine candidate and was tested for safety and immunogenicity in human Phase I Clinical Trials. PBMC from vaccine recipients were analyzed by flow cytometric methods to determine the nature of T cell responses and AMA1-reactive memory T cells. Both CD4 and CD8 T cells produced a number of cytokines following AMA1 re-stimulation, with IL-5-producing cells at the highest frequency, consistent with a Th2 bias. The relative frequency of multifunctional cells synthesizing Th1 cytokines IFN-γ, IL-2 and TNF-α changed after each vaccination. Interestingly, median fluorescence intensity measurements revealed that cells producing more than one cytokine contributed greater quantities of each cytokine than cell populations that produced each of the cytokines alone. AMA1 vaccination also elicited the development of memory cell populations, and both central and effector memory T cells were identified concurrently after the AMA1 vaccination. The detailed profile of multifunctional T cell responses to AMA1 presented here will advance our ability to assess the immunogenicity of human malarial vaccines.
doi:10.1016/j.vaccine.2009.06.066
PMCID: PMC2744949  PMID: 19591795
Plasmodium falciparum apical membrane antigen 1 (PfAMA1); Multifunctional T cell responses; Central and effector memory T cells
14.  Patent Filarial Infection Modulates Malaria-Specific Type 1 Cytokine Responses in an IL-10-Dependent Manner in a Filaria/Malaria Coinfected Population 1 
The effect of filarial infections on malaria-specific immune responses was investigated in Malian villages co-endemic for filariasis and malaria. Cytokines were measured from plasma and Ag-stimulated whole blood from individuals with Wuchereria bancrofti (Wb) and/or Mansonella perstans (Mp) infections (Fil+; n = 19) and those without evidence of filarial infection (Fil−; n = 19). Plasma levels of IL-10 (geometric mean [GM] 22.8 vs. 10.4) were higher in Fil+ compared with Fil−, whereas levels of IP-10 were lower in Fil+ (GM = 66.3 vs. 110.0). Fil+ had higher levels of spontaneously secreted IL-10 (59.3 vs. 6.8 pg/ml) and lower levels of IL-2 (1.0 vs. 1.2 pg/ml) than did Fil−. Although there were no differences in levels of Staphylococcus aureus enterotoxin B-induced cytokines between the two groups, Fil+ mounted a lower IL-12p70 (1.11 vs. 3.83 pg/ml; p = 0.007), IFN-γ (5.44 vs. 23.41 pg/ml; p = 0.009), and IP-10 (29.43 vs. 281.7 pg/ml; p = 0.007) response following malaria Ag (MalAg) stimulation compared with Fil−. In contrast, Fil+ had a higher MalAg-specific IL-10 response (7318 pg/ml vs. 3029 pg/ml; p = 0.006) compared with those without filarial infection. Neutralizing Ab to IL-10 (but not to TGFβ) reversed the downregulated MalAg-specific IFN-γ and IP-10 (p < 0.001) responses in Fil+. Together these data demonstrate that filarial infections modulate the Plasmodium falciparum-specific IL-12p70/IFN-γ secretion pathways known to play a key role in resistance to malaria and thata they do so in an IL-10-dependent manner.
doi:10.4049/jimmunol.0900257
PMCID: PMC2789677  PMID: 19561105
Helminth; Protozoan; Cytokines
15.  Phase 1 Trial of the Plasmodium falciparum Blood Stage Vaccine MSP142-C1/Alhydrogel with and without CPG 7909 in Malaria Naïve Adults 
PLoS ONE  2010;5(1):e8787.
Background
Merozoite surface protein 142 (MSP142) is a leading blood stage malaria vaccine candidate. In order to induce immune responses that cover the major antigenic polymorphisms, FVO and 3D7 recombinant proteins of MSP142 were mixed (MSP142-C1). To improve the level of antibody response, MSP142-C1 was formulated with Alhydrogel plus the novel adjuvant CPG 7909.
Methods
A Phase 1 clinical trial was conducted in healthy malaria-naïve adults at the Center for Immunization Research in Washington, D.C., to evaluate the safety and immunogenicity of MSP142-C1/Alhydrogel +/− CPG 7909. Sixty volunteers were enrolled in dose escalating cohorts and randomized to receive three vaccinations of either 40 or 160 µg protein adsorbed to Alhydrogel +/− 560 µg CPG 7909 at 0, 1 and 2 months.
Results
Vaccinations were well tolerated, with only one related adverse event graded as severe (Grade 3 injection site erythema) and all other vaccine related adverse events graded as either mild or moderate. Local adverse events were more frequent and severe in the groups receiving CPG. The addition of CPG enhanced anti-MSP142 antibody responses following vaccination by up to 49-fold two weeks after second immunization and 8-fold two weeks after the third immunization when compared to MSP142-C1/Alhydrogel alone (p<0.0001). After the third immunization, functionality of the antibody was tested by an in vitro growth inhibition assay. Inhibition was a function of antibody titer, with an average of 3% (range −2 to 10%) in the non CPG groups versus 14% (3 to 32%) in the CPG groups.
Conclusion/Significance
The favorable safety profile and high antibody responses induced with MSP142-C1/Alhydrogel + CPG 7909 are encouraging. MSP142-C1/Alhydrogel is being combined with other blood stage antigens and will be taken forward in a formulation adjuvanted with CPG 7909.
Trial Registration
ClinicalTrials.gov Identifier: NCT00320658
doi:10.1371/journal.pone.0008787
PMCID: PMC2809736  PMID: 20107498
16.  Phase 1 Trial of Malaria Transmission Blocking Vaccine Candidates Pfs25 and Pvs25 Formulated with Montanide ISA 51 
PLoS ONE  2008;3(7):e2636.
Background
Pfs25 and Pvs25, surface proteins of mosquito stage of the malaria parasites P. falciparum and P. vivax, respectively, are leading candidates for vaccines preventing malaria transmission by mosquitoes. This single blinded, dose escalating, controlled Phase 1 study assessed the safety and immunogenicity of recombinant Pfs25 and Pvs25 formulated with Montanide ISA 51, a water-in-oil emulsion.
Methodology/Principal Findings
The trial was conducted at The Johns Hopkins Center for Immunization Research, Washington DC, USA, between May 16, 2005–April 30, 2007. The trial was designed to enroll 72 healthy male and non-pregnant female volunteers into 1 group to receive adjuvant control and 6 groups to receive escalating doses of the vaccines. Due to unexpected reactogenicity, the vaccination was halted and only 36 volunteers were enrolled into 4 groups: 3 groups of 10 volunteers each were immunized with 5 µg of Pfs25/ISA 51, 5 µg of Pvs25/ISA 51, or 20 µg of Pvs25/ISA 51, respectively. A fourth group of 6 volunteers received adjuvant control (PBS/ISA 51). Frequent local reactogenicity was observed. Systemic adverse events included two cases of erythema nodosum considered to be probably related to the combination of the antigen and the adjuvant. Significant antibody responses were detected in volunteers who completed the lowest scheduled doses of Pfs25/ISA 51. Serum anti-Pfs25 levels correlated with transmission blocking activity.
Conclusion/Significance
It is feasible to induce transmission blocking immunity in humans using the Pfs25/ISA 51 vaccine, but these vaccines are unexpectedly reactogenic for further development. This is the first report that the formulation is associated with systemic adverse events including erythema nodosum.
Trial Registration
ClinicalTrials.gov NCT00295581
doi:10.1371/journal.pone.0002636
PMCID: PMC2440546  PMID: 18612426
17.  Phase 1 Study of Two Merozoite Surface Protein 1 (MSP142) Vaccines for Plasmodium falciparum Malaria 
PLoS Clinical Trials  2007;2(4):e12.
Objectives:
To assess the safety and immunogenicity of two vaccines, MSP142-FVO/Alhydrogel and MSP142-3D7/Alhydrogel, targeting blood-stage Plasmodium falciparum parasites.
Design:
A Phase 1 open-label, dose-escalating study.
Setting:
Quintiles Phase 1 Services, Lenexa, Kansas between July 2004 and November 2005.
Participants:
Sixty healthy malaria-naïve volunteers 18–48 y of age.
Interventions:
The C-terminal 42-kDa region of merozoite surface protein 1 (MSP142) corresponding to the two allelic forms present in FVO and 3D7 P. falciparum lines were expressed in Escherichia coli, refolded, purified, and formulated on Alhydrogel (aluminum hydroxide). For each vaccine, volunteers in each of three dose cohorts (5, 20, and 80 μg) were vaccinated at 0, 28, and 180 d. Volunteers were followed for 1 y.
Outcome Measures:
The safety of MSP142-FVO/Alhydrogel and MSP142-3D7/Alhydrogel was assessed. The antibody response to each vaccine was measured by reactivity to homologous and heterologous MSP142, MSP119, and MSP133 recombinant proteins and recognition of FVO and 3D7 parasites.
Results:
Anti-MSP142 antibodies were detected by ELISA in 20/27 (74%) and 22/27 (81%) volunteers receiving three vaccinations of MSP142-FVO/Alhydrogel or MSP142-3D7/Alhydrogel, respectively. Regardless of the vaccine, the antibodies were cross-reactive to both MSP142-FVO and MSP142-3D7 proteins. The majority of the antibody response targeted the C-terminal 19-kDa domain of MSP142, although low-level antibodies to the N-terminal 33-kDa domain of MSP142 were also detected. Immunofluorescence microscopy of sera from the volunteers demonstrated reactivity with both FVO and 3D7 P. falciparum schizonts and free merozoites. Minimal in vitro growth inhibition of FVO or 3D7 parasites by purified IgG from the sera of the vaccinees was observed.
Conclusions:
The MSP142/Alhydrogel vaccines were safe and well tolerated but not sufficiently immunogenic to generate a biologic effect in vitro. Addition of immunostimulants to the Alhydrogel formulation to elicit higher vaccine-induced responses in humans may be required for an effective vaccine.
Editorial Commentary
Background: Generally, adults living in parts of the world where malaria is common develop protective immunity against the parasite. This means they may get infected but not become ill as a result. However, there are individuals, such as pregnant women and children under the age of five, who are more likely to develop symptoms of malaria due to no (or reduced) natural immunity. A successful malaria vaccine would stimulate an individual's immune system to respond to the malaria parasite and prevent serious clinical disease. Many different groups are currently developing potential vaccines. Several candidates are based on a protein called MSP1 (merozoite surface protein 1) which is found on the surface of the blood-stage form of the malaria parasite. However, in nature parasites carry different versions of the MSP1 protein, and ideally a successful vaccine would bring about immune responses against these different versions. The researchers carrying out this trial wanted to compare the safety and immune responses against candidate vaccines representing two different MSP1 proteins, which covered many different parasite lines. As a phase 1 trial, the study was carried out in healthy adult volunteers. Sixty individuals were assigned to receive an injection of the vaccines, either containing a recombinant protein analogous to the FVO parasite line (termed MSP142-FVO) or the 3D7 parasite line (termed MSP142-3D7) at three different dose levels. The trial's primary objective was to assess safety, which was done by collecting data on any abnormal signs or symptoms up to 14 d after each of three vaccinations. These outcomes were graded and then defined as related to the vaccine or not. The researchers also looked at antibody levels in participants' blood against different variants of the MSP1 protein, as well as using in vitro tests to see whether antibodies from vaccinated individuals could prevent malaria parasites from growing in lab culture.
What the trial shows: The safety outcomes of the trial showed that the most common type of side effect experienced by the volunteers was pain at the injection site. The vast majority of such events were graded as mild, although there was one single case of a severe event (high levels of pain experienced by one volunteer at the injection site). There was no significant association between the chance of side effects and the vaccine dosage that an individual received. Following vaccination, antibody levels against the protein on which the vaccine was based were detected, although these levels dropped over time. The researchers did not see a strong association between the vaccine dosage that individuals received and the level of antibody response. However, the two vaccines when compared seemed to be equally good at raising an immune response and both caused antibodies to be raised corresponding to different variants of the MSP1 protein. However, the antibodies raised did not seem to be particularly effective at preventing malaria parasites from growing in lab culture.
Strengths and limitations: Strengths of this study include a comparison of three different dosage levels of the vaccines under study, as well as a comparison of two vaccines based on the same protein, representing different parasite lines. Limitations to the study include the small number of participants, which makes the trial underpowered to detect all but large differences in side effects between the groups being compared. A placebo arm was not included in the trial, so it is not possible to be sure that the numbers of side effects observed here can be attributed to the vaccines or not. Finally, the procedure for assigning individuals to the two different vaccines involved alternation, rather than true randomization, which could have minimized the risk of bias.
Contribution to the evidence: The trial reported here is an essential step in vaccine development. The results provide the first evidence relating to safety for these two vaccines, and do not raise any safety concerns at this stage. Although the vaccines raised an immune response, the antibodies raised did not seem to have much of an effect on malaria parasites in vitro. While these vaccines are safe, alternative MSP1 vaccine formulations anticipated to bring about a greater immune response will likely be studied before proceeding to field studies.
doi:10.1371/journal.pctr.0020012
PMCID: PMC1847697  PMID: 17415408
18.  Phase 1 Clinical Trial of Apical Membrane Antigen 1: an Asexual Blood-Stage Vaccine for Plasmodium falciparum Malaria  
Infection and Immunity  2005;73(6):3677-3685.
Apical membrane antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. A phase 1 trial was conducted with 30 malaria-naïve volunteers to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of recombinant proteins based on sequences from the FVO and 3D7 clones of Plasmodium falciparum. The proteins were expressed in Pichia pastoris and adsorbed on Alhydrogel. Ten volunteers in each of three dose groups (5 μg, 20 μg, and 80 μg) were vaccinated in an open-label study at 0, 28, and 180 days. The vaccine was well tolerated, with pain at the injection site being the most commonly observed reaction. Anti-AMA1 immunoglobulin G (IgG) was detected by enzyme-linked immunosorbent assay (ELISA) in 15/28 (54%) volunteers after the second immunization and in 23/25 (92%) after the third immunization, with equal reactivity to both AMA1-FVO and AMA1-3D7 vaccine components. A significant dose-response relationship between antigen dose and antibody response by ELISA was observed, and the antibodies were predominantly of the IgG1 isotype. Confocal microscopic evaluation of sera from vaccinated volunteers demonstrated reactivity with P. falciparum schizonts in a pattern similar to native parasite AMA1. Antigen-specific in vitro inhibition of both FVO and 3D7 parasites was achieved with IgG purified from sera of vaccinees, demonstrating biological activity of the antibodies. To our knowledge, this is the first AMA1 vaccine candidate to elicit functional immune responses in malaria-naïve humans, and our results support the further development of this vaccine.
doi:10.1128/IAI.73.6.3677-3685.2005
PMCID: PMC1111886  PMID: 15908397
19.  Analysis of Human Peripheral Blood Samples from Fatal and Nonfatal Cases of Ebola (Sudan) Hemorrhagic Fever: Cellular Responses, Virus Load, and Nitric Oxide Levels 
Journal of Virology  2004;78(19):10370-10377.
Peripheral blood samples obtained from patients during an outbreak of Ebola virus (Sudan species) disease in Uganda in 2000 were used to phenotype peripheral blood mononuclear cells (PBMC), quantitate gene expression, measure antigenemia, and determine nitric oxide levels. It was determined that as the severity of disease increased in infected patients, there was a corresponding increase in antigenemia and leukopenia. Blood smears revealed thrombocytopenia, a left shift in neutrophils (in some cases degenerating), and atypical lymphocytes. Infected patients who died had reduced numbers of T cells, CD8+ T cells, and activated (HLA-DR+) CD8+ T cells, while the opposite was noted for patients who survived the disease. Expression levels of cytokines, Fas antigen, and Fas ligand (TaqMan quantitation) in PBMC from infected patients were not significantly different from those in uninfected patients (treated in the same isolation wards), nor was there a significant increase in expression compared to healthy volunteers (United States). This unresponsive state of PBMC from infected patients despite high levels of circulating antigen and virus replication suggests that some form of immunosuppression had developed. Ebola virus RNA levels (virus load) in PBMC specimens were found to be much higher in infected patients who died than patients who survived the disease. Similarly, blood levels of nitric oxide were much higher in fatal cases (increasing with disease severity), and extremely elevated levels (≥150 μM) would have negatively affected vascular tone and contributed to virus-induced shock.
doi:10.1128/JVI.78.19.10370-10377.2004
PMCID: PMC516433  PMID: 15367603
20.  Persistent Infection with Ebola Virus under Conditions of Partial Immunity 
Journal of Virology  2004;78(2):958-967.
Ebola hemorrhagic fever in humans is associated with high mortality; however, some infected hosts clear the virus and recover. The mechanisms by which this occurs and the correlates of protective immunity are not well defined. Using a mouse model, we determined the role of the immune system in clearance of and protection against Ebola virus. All CD8 T-cell-deficient mice succumbed to subcutaneous infection and had high viral antigen titers in tissues, whereas mice deficient in B cells or CD4 T cells cleared infection and survived, suggesting that CD8 T cells, independent of CD4 T cells and antibodies, are critical to protection against subcutaneous Ebola virus infection. B-cell-deficient mice that survived the primary subcutaneous infection (vaccinated mice) transiently depleted or not depleted of CD4 T cells also survived lethal intraperitoneal rechallenge for ≥25 days. However, all vaccinated B-cell-deficient mice depleted of CD8 T cells had high viral antigen titers in tissues following intraperitoneal rechallenge and died within 6 days, suggesting that memory CD8 T cells by themselves can protect mice from early death. Surprisingly, vaccinated B-cell-deficient mice, after initially clearing the infection, were found to have viral antigens in tissues later (day 120 to 150 post-intraperitoneal infection). Furthermore, following intraperitoneal rechallenge, vaccinated B-cell-deficient mice that were transiently depleted of CD4 T cells had high levels of viral antigen in tissues earlier (days 50 to 70) than vaccinated undepleted mice. This demonstrates that under certain immunodeficiency conditions, Ebola virus can persist and that loss of primed CD4 T cells accelerates the course of persistent infections. These data show that CD8 T cells play an important role in protection against acute disease, while both CD4 T cells and antibodies are required for long-term protection, and they provide evidence of persistent infection by Ebola virus suggesting that under certain conditions of immunodeficiency a host can harbor virus for prolonged periods, potentially acting as a reservoir.
doi:10.1128/JVI.78.2.958-967.2004
PMCID: PMC368745  PMID: 14694127
21.  Enhanced Interleukin-12 and CD40 Ligand Activities but Reduced Staphylococcus aureus Cowan 1-Induced Responses Suggest a Generalized and Progressively Impaired Type 1 Cytokine Pattern for Human Schistosomiasis  
Infection and Immunity  2002;70(11):5903-5912.
Whole-blood-cell cultures from schistosomiasis patients were stimulated with a variety of T-cell-dependent and T-cell-independent stimuli to determine whether the defect in type 1 cytokine expression observed following helminth infection is associated with alterations in interleukin-12 (IL-12) or CD40 ligand (CD40L) responsiveness. Cultures from uninfected individuals produced abundant gamma interferon in response to Staphylococcus aureus Cowan 1 (SAC), while patients with intestinal and hepatosplenic disease displayed intermediate and weak responses, respectively. Importantly, the decrease in type 1 cytokine expression was not attributed to defects in IL-12- or CD40L-induced activity. Indeed, schistosomiasis patients displayed heightened responses and even produced more biologically active IL-12 when stimulated with SAC and CD40L than did uninfected controls. Finally, additional studies suggested only a partial role for IL-10, since intestinal patients were the only group that overproduced this downregulatory cytokine. Together, these studies demonstrate that the type 1 deficiency in chronic hepatosplenic schistosomiasis is not related to specific defects in IL-12, IL-10, or CD40L activity, although changes in the functional status of antigen-presenting cells appear to be involved.
doi:10.1128/IAI.70.11.5903-5912.2002
PMCID: PMC130279  PMID: 12379664
22.  Passive Transfer of Antibodies Protects Immunocompetent and Immunodeficient Mice against Lethal Ebola Virus Infection without Complete Inhibition of Viral Replication 
Journal of Virology  2001;75(10):4649-4654.
Ebola hemorrhagic fever is a severe, usually fatal illness caused by Ebola virus, a member of the filovirus family. The use of nonhomologous immune serum in animal studies and blood from survivors in two anecdotal reports of Ebola hemorrhagic fever in humans has shown promise, but the efficacy of these treatments has not been demonstrated definitively. We have evaluated the protective efficacy of polyclonal immune serum in a mouse model of Ebola virus infection. Our results demonstrate that mice infected subcutaneously with live Ebola virus survive infection and generate high levels of anti-Ebola virus immunoglobulin G (IgG). Passive transfer of immune serum from these mice before challenge protected upto 100% of naive mice against lethal Ebola virus infection. Protection correlated with the level of anti-Ebola virus IgG titers, and passive treatment with high-titer antiserum was associated with a delay in the peak of viral replication. Transfer of immune serum to SCID mice resulted in 100% survival after lethal challenge with Ebola virus, indicating that antibodies alone can protect from lethal disease. Thus antibodies suppress or delay viral growth, provide protection against lethal Ebola virus infection, and may not require participation of other immune components for protection.
doi:10.1128/JVI.75.10.4649-4654.2001
PMCID: PMC114218  PMID: 11312335

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