Brent House

A Field Trial to Assess a Blood-Stage Malaria Vaccine

BACKGROUND Blood-stage malaria vaccines are intended to prevent clinical disease. The malaria vaccine FMP2.1/AS02(A), a recombinant protein based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, has previously been shown to have immunogenicity and acceptable safety in Malian adults and children. METHODS In a double-blind, randomized trial, we immunized 400 Malian children with either the malaria vaccine or a control (rabies) vaccine and followed them for 6 months. The primary end point was clinical malaria, defined as fever and at least 2500 parasites per cubic millimeter of blood. A secondary end point was clinical malaria caused by parasites with the AMA1 DNA sequence found in the vaccine strain. RESULTS The cumulative incidence of the primary end point was 48.4% in the malaria-vaccine group and 54.4% in the control group; efficacy against the primary end point was 17.4% (hazard ratio for the primary end point, 0.83; 95% confidence interval [CI], 0.63 to 1.09; P=0.18). Efficacy against the first and subsequent episodes of clinical malaria, as defined on the basis of various parasite-density thresholds, was approximately 20%. Efficacy against clinical malaria caused by parasites with AMA1 corresponding to that of the vaccine strain was 64.3% (hazard ratio, 0.36; 95% CI, 0.08 to 0.86; P=0.03). Local reactions and fever after vaccination were more frequent with the malaria vaccine. CONCLUSIONS On the basis of the primary end point, the malaria vaccine did not provide significant protection against clinical malaria, but on the basis of secondary results, it may have strain-specific efficacy. If this finding is confirmed, AMA1 might be useful in a multicomponent malaria vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00460525.).

Adenovirus-5-Vectored P. falciparum Vaccine Expressing CSP and AMA1. Part B: Safety, Immunogenicity and Protective Efficacy of the CSP Component

Background A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge. Methodology/Principal Findings NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected. Significance The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection. Trial Registration ClinicalTrials.gov NCT00392015

Adenovirus 5-Vectored P. falciparum Vaccine Expressing CSP and AMA1. Part A: Safety and Immunogenicity in Seronegative Adults

Background Models of immunity to malaria indicate the importance of CD8+ T cell responses for targeting intrahepatic stages and antibodies for targeting sporozoite and blood stages. We designed a multistage adenovirus 5 (Ad5)-vectored Plasmodium falciparum malaria vaccine, aiming to induce both types of responses in humans, that was tested for safety and immunogenicity in a Phase 1 dose escalation trial in Ad5-seronegative volunteers. Methodology/Principal Findings The NMRC-M3V-Ad-PfCA vaccine combines two adenovectors encoding circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). Group 1 (n = 6) healthy volunteers received one intramuscular injection of 2×10∧10 particle units (1×10∧10 each construct) and Group 2 (n = 6) a five-fold higher dose. Transient, mild to moderate adverse events were more pronounced with the higher dose. ELISpot responses to CSP and AMA1 peaked at 1 month, were higher in the low dose (geomean CSP = 422, AMA1 = 862 spot forming cells/million) than in the high dose (CSP = 154, p = 0.049, AMA1 = 423, p = 0.045) group and were still positive at 12 months in a number of volunteers. ELISpot depletion assays identified dependence on CD4+ or on both CD4+ and CD8+ T cells, with few responses dependent only on CD8+ T cells. Intracellular cytokine staining detected stronger CD8+ than CD4+ T cell IFN-γ responses (CSP p = 0.0001, AMA1 p = 0.003), but similar frequencies of multifunctional CD4+ and CD8+ T cells secreting two or more of IFN-γ, TNF-α or IL-2. Median fluorescence intensities were 7–10 fold higher in triple than single secreting cells. Antibody responses were low but trended higher in the high dose group and did not inhibit growth of cultured P. falciparum blood stage parasites. Significance As found in other trials, adenovectored vaccines appeared safe and well-tolerated at doses up to 1×10∧11 particle units. This is the first demonstration in humans of a malaria vaccine eliciting strong CD8+ T cell IFN-γ responses. Trial Registration ClinicalTrials.gov NCT00392015

Results from tandem Phase 1 studies evaluating the safety, reactogenicity and immunogenicity of the vaccine candidate antigen Plasmodium falciparum FVO merozoite surface protein-1 (MSP142) administered intramuscularly with adjuvant system AS01

BackgroundThe development of an asexual blood stage vaccine against Plasmodium falciparum malaria based on the major merozoite surface protein-1 (MSP1) antigen is founded on the protective efficacy observed in preclinical studies and induction of invasion and growth inhibitory antibody responses. The 42 kDa C-terminus of MSP1 has been developed as the recombinant protein vaccine antigen, and the 3D7 allotype, formulated with the Adjuvant System AS02A, has been evaluated extensively in human clinical trials. In preclinical rabbit studies, the FVO allele of MSP142 has been shown to have improved immunogenicity over the 3D7 allele, in terms of antibody titres as well as growth inhibitory activity of antibodies against both the heterologous 3D7 and homologous FVO parasites.MethodsTwo Phase 1 clinical studies were conducted to examine the safety, reactogenicity and immunogenicity of the FVO allele of MSP142 in the adjuvant system AS01 administered intramuscularly at 0-, 1-, and 2-months: one in the USA and, after evaluation of safety data results, one in Western Kenya. The US study was an open-label, dose escalation study of 10 and 50 μg doses of MSP142 in 26 adults, while the Kenya study, evaluating 30 volunteers, was a double-blind, randomized study of only the 50 μg dose with a rabies vaccine comparator.ResultsIn these studies it was demonstrated that this vaccine formulation has an acceptable safety profile and is immunogenic in malaria-naïve and malaria-experienced populations. High titres of anti-MSP1 antibodies were induced in both study populations, although there was a limited number of volunteers whose serum demonstrated significant inhibition of blood-stage parasites as measured by growth inhibition assay. In the US volunteers, the antibodies generated exhibited better cross-reactivity to heterologous MSP1 alleles than a MSP1-based vaccine (3D7 allele) previously tested at both study sites.ConclusionsGiven that the primary effector mechanism for blood stage vaccine targets is humoral, the antibody responses demonstrated to this vaccine candidate, both quantitative (total antibody titres) and qualitative (functional antibodies inhibiting parasite growth) warrant further consideration of its application in endemic settings.Trial registrationsClinical Trials NCT00666380

Extended Safety, Immunogenicity and Efficacy of a Blood-Stage Malaria Vaccine in Malian Children: 24-Month Follow-Up of a Randomized, Double-Blinded Phase 2 Trial

Background The FMP2.1/AS02A candidate malaria vaccine was tested in a Phase 2 study in Mali. Based on results from the first eight months of follow-up, the vaccine appeared well-tolerated and immunogenic. It had no significant efficacy based on the primary endpoint, clinical malaria, but marginal efficacy against clinical malaria in secondary analyses, and high allele-specific efficacy. Extended follow-up was conducted to evaluate extended safety, immunogenicity and efficacy. Methods A randomized, double-blinded trial of safety, immunogenicity and efficacy of the candidate Plasmodium falciparum apical membrane antigen 1 (AMA1) vaccine FMP2.1/AS02A was conducted in Bandiagara, Mali. Children aged 1–6 years were randomized in a 1∶1 ratio to receive FMP2.1/AS02A or control rabies vaccine on days 0, 30 and 60. Using active and passive surveillance, clinical malaria and adverse events as well as antibodies against P. falciparum AMA1 were monitored for 24 months after the first vaccination, spanning two malaria seasons. Findings 400 children were enrolled. Serious adverse events occurred in nine participants in the FMP2.1/AS02A group and three in the control group; none was considered related to study vaccination. After two years, anti-AMA1 immune responses remained significantly higher in the FMP2.1/AS02A group than in the control group. For the entire 24-month follow-up period, vaccine efficacy was 7.6% (p = 0.51) against first clinical malaria episodes and 9.9% (p = 0.19) against all malaria episodes. For the final 16-month follow-up period, vaccine efficacy was 0.9% (p = 0.98) against all malaria episodes. Allele-specific efficacy seen in the first malaria season did not extend into the second season of follow-up. Interpretation Allele-specific vaccine efficacy was not sustained in the second malaria season, despite continued high levels of anti-AMA1 antibodies. This study presents an opportunity to evaluate correlates of partial protection against clinical malaria that waned during the second malaria season. Trial Registration Clinicaltrials.gov NCT00460525 NCT00460525

Detection of new SHV-12, SHV-5 and SHV-2a variants of extended spectrum Beta-lactamase in Klebsiella pneumoniae in Egypt

BackgroundKlebsiella pneumoniae outbreaks possessing extended-spectrum β-lactamase- (ESBL) mediated resistance to third-generation cephalosporins have increased significantly in hospital and community settings worldwide. The study objective was to characterize prevalent genetic determinants of TEM, SHV and CTX-M types ESBL activity in K. pneumoniae isolates from Egypt.MethodsSixty five ESBL-producing K. pneumoniae strains, isolated from nosocomial and community-acquired infections from 10 Egyptian University hospitals (2000–2003), were confirmed with double disc-synergy method and E-test. bla TEM, bla SHV and bla CTX-m genes were identified by PCR and DNA sequencing. Pulsed-field gel electrophoresis (PFGE) was conducted for genotyping.ResultsAll isolates displayed ceftazidime and cefotaxime resistance. bla TEM and bla SHV genes were detected in 98% of the isolates’ genomes, while 11% carried bla CTX-m. DNA sequencing revealed plasmid-borne SHV-12,-5,-2a (17%), CTX-m-15 (11%), and TEM-1 (10%) prevalence. Among SHV-12 (n=8), one isolate displayed 100% blaSHV-12 amino acid identity, while others had various point mutations: T17G (Leu to Arg, position 6 of the enzyme: n=2); A8T and A10G (Tyr and Ile to Phe and Val, positions 3 and 4, respectively: n=4), and; A703G (Lys to Glu 235: n=1). SHV-5 and SHV-2a variants were identified in three isolates: T17G (n=1); A703G and G705A (Ser and Lys to Gly and Glu: n=1); multiple mutations at A8T, A10G, T17G, A703G and G705A (n=1). Remarkably, 57% of community-acquired isolates carried CTX-m-15. PFGE demonstrated four distinct genetic clusters, grouping strains of different genetic backgrounds.ConclusionsThis is the first study demonstrating the occurrence of SHV-12, SHV-5 and SHV-2a variants in Egypt, indicating the spread of class A ESBL in K. pneumoniae through different mechanisms.

Towards an optimized inhibition of liver stage development assay (ILSDA) for Plasmodium falciparum

BackgroundExperimental vaccines targeting Plasmodium falciparum have had some success in recent years. These vaccines use attenuated parasites, recombinant sporozoite proteins, or DNA and virus combinations to induce cell-mediated immune responses and/or antibodies targeting sporozoite surface proteins. To capitalize on the success of these vaccines and understand the mechanisms by which these vaccines function, it is important to develop assays that measure correlates of protection in volunteers. The inhibition of liver stage development assay (ILSDA) tests antibodies for the ability to block sporozoite development in hepatocytes. As such the ILSDA is an excellent candidate assay to identify correlates of humoral protection, particularly against the liver stage of malaria infection. In addition, the ILSDA can be used as a tool to evaluate novel sporozoite antigens for future vaccine development. Historically the ILSDA has suffered from low sporozoite infection rates, absence of standardized reagents, and the subjectivity associated with the traditional primary outcome measures, which depend on microscopy of stained hepatocyte cultures. This study worked to significantly improve sporozoite infection rates in hepatocytes, modify key steps in the assay protocol to reduce experimental variability, and demonstrate the utility of the ILSDA in testing antibodies targeting the circumsporozoite protein.MethodsCryopreserved primary human hepatocytes, Plasmodium falciparum sporozoites, and circumsporozoite antibodies were used to optimize the ILSDA.ResultsInoculation of cryopreserved primary human hepatocytes with Plasmodium falciparum sporozoites improved liver stage development in the ILSDA compared to HCO4 cells. In the ILSDA, circumsporozoite antibodies suppressed liver stage development in cryopreserved primary human hepatocytes in a concentration-dependent manner. Antibody-mediated suppression of parasite development in the ILSDA at a 96-hour endpoint was more robust than the 24-hour endpoint.ConclusionsILSDA performance is improved by the use of cryopreserved primary human hepatocytes, expediting interactions between sporozoites and hepatocytes, and extending the assay endpoint.

Molecular Characterization of Multidrug Resistant Hospital Isolates Using the Antimicrobial Resistance Determinant Microarray

Molecular methods that enable the detection of antimicrobial resistance determinants are critical surveillance tools that are necessary to aid in curbing the spread of antibiotic resistance. In this study, we describe the use of the Antimicrobial Resistance Determinant Microarray (ARDM) that targets 239 unique genes that confer resistance to 12 classes of antimicrobial compounds, quaternary amines and streptothricin for the determination of multidrug resistance (MDR) gene profiles. Fourteen reference MDR strains, which either were genome, sequenced or possessed well characterized drug resistance profiles were used to optimize detection algorithms and threshold criteria to ensure the microarrays effectiveness for unbiased characterization of antimicrobial resistance determinants in MDR strains. The subsequent testing of Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae hospital isolates revealed the presence of several antibiotic resistance genes [e.g. belonging to TEM, SHV, OXA and CTX-M classes (and OXA and CTX-M subfamilies) of β-lactamases] and their assemblages which were confirmed by PCR and DNA sequence analysis. When combined with results from the reference strains, ∼25% of the ARDM content was confirmed as effective for representing allelic content from both Gram-positive and –negative species. Taken together, the ARDM identified MDR assemblages containing six to 18 unique resistance genes in each strain tested, demonstrating its utility as a powerful tool for molecular epidemiological investigations of antimicrobial resistance in clinically relevant bacterial pathogens.

Functional immunoassays using an in-vitro malaria liver-stage infection model: where do we go from here?

For more than 25 years, the ISI assay and ILSDA have been used to study the development of the malaria parasite in the liver, to discover and characterize sporozoite and liver-stage antigens, to support the development of malaria vaccine candidates, and to search for immunological correlates of protection in animals and in humans. Although both assays have been limited by low sporozoite invasion rates, significant biological variability, and the subjective nature of manually counting hepatocytes containing parasites as the read-out, they have nevertheless been useful tools for exploring parasite biology. This review describes the origin, application and current status of these assays, critically discusses the need for improvements, and explores the roles of these assays in supporting the development of an effective vaccine against Plasmodium falciparum malaria.

Etiologic Agents of Central Nervous System Infections among Febrile Hospitalized Patients in the Country of Georgia

Objectives There is a large spectrum of viral, bacterial, fungal, and prion pathogens that cause central nervous system (CNS) infections. As such, identification of the etiological agent requires multiple laboratory tests and accurate diagnosis requires clinical and epidemiological information. This hospital-based study aimed to determine the main causes of acute meningitis and encephalitis and enhance laboratory capacity for CNS infection diagnosis. Methods Children and adults patients clinically diagnosed with meningitis or encephalitis were enrolled at four reference health centers. Cerebrospinal fluid (CSF) was collected for bacterial culture, and in-house and multiplex RT-PCR testing was conducted for herpes simplex virus (HSV) types 1 and 2, mumps virus, enterovirus, varicella zoster virus (VZV), Streptococcus pneumoniae, HiB and Neisseria meningitidis. Results Out of 140 enrolled patients, the mean age was 23.9 years, and 58% were children. Bacterial or viral etiologies were determined in 51% of patients. Five Streptococcus pneumoniae cultures were isolated from CSF. Based on in-house PCR analysis, 25 patients were positive for S. pneumoniae, 6 for N. meningitidis, and 1 for H. influenzae. Viral multiplex PCR identified infections with enterovirus (n = 26), VZV (n = 4), and HSV-1 (n = 2). No patient was positive for mumps or HSV-2. Conclusions Study findings indicate that S. pneumoniae and enteroviruses are the main etiologies in this patient cohort. The utility of molecular diagnostics for pathogen identification combined with the knowledge provided by the investigation may improve health outcomes of CNS infection cases in Georgia.