Elizabeth H. Duncan

Protection Induced by Plasmodium falciparum MSP142 Is Strain-Specific, Antigen and Adjuvant Dependent, and Correlates with Antibody Responses

Vaccination with Plasmodium falciparum MSP142/complete Freunds adjuvant (FA) followed by MSP142/incomplete FA is the only known regimen that protects Aotus nancymaae monkeys against infection by erythrocytic stage malaria parasites. The role of adjuvant is not defined; however complete FA cannot be used in humans. In rodent models, immunity is strain-specific. We vaccinated Aotus monkeys with the FVO or 3D7 alleles of MSP142 expressed in Escherichia coli or with the FVO allele expressed in baculovirus (bv) combined with complete and incomplete FA, Montanide ISA-720 (ISA-720) or AS02A. Challenge with FVO strain P. falciparum showed that suppression of cumulative day 11 parasitemia was strain-specific and could be induced by E. coli expressed MSP142 in combination with FA or ISA-720 but not with AS02A. The coli42-FVO antigen induced a stronger protective effect than the bv42-FVO antigen, and FA induced a stronger protective effect than ISA-720. ELISA antibody (Ab) responses at day of challenge (DOC) were strain-specific and correlated inversely with c-day 11 parasitemia (r = −0.843). ELISA Ab levels at DOC meeting a titer of at least 115,000 ELISA Ab units identified the vaccinees not requiring treatment (noTx) with a true positive rate of 83.3% and false positive rate of 14.3 %. Correlation between functional growth inhibitory Ab levels (GIA) and cumulative day 11 parasitemia was weaker (r = −0.511), and was not as predictive for a response of noTx. The lowest false positive rate for GIA was 30% when requiring a true positive rate of 83.3%. These inhibition results along with those showing that antigen/FA combinations induced a stronger protective immunity than antigen/ISA-720 or antigen/AS02 combinations are consistent with protection as ascribed to MSP1-specific cytophilic antibodies. Development of an effective MSP142 vaccine against erythrocytic stage P. falciparum infection will depend not only on antigen quality, but also upon the selection of an optimal adjuvant component.

Histidine affinity tags affect MSP142 structural stability and immunodominance in mice

Inclusion of affinity tags has greatly facilitated process development for protein antigens, primarily for their recovery from complex mixtures. Although generally viewed as supportive of product development, affinity tags may have unintended consequences on protein solubility, susceptibility to aggregation, and immunogenicity. Merozoite surface protein 1 (MSP1), an erythrocytic stage protein of Plasmodium falciparum and a candidate malaria vaccine, was used to evaluate the impact of a metal ion affinity‐tag on both protein structure and the induction of immunity. To this end, codon harmonized gene sequences from the P. falciparum MSP142 of FVO and 3D7 parasites were cloned and purified with and without a histidine (His) tag. We report on the influence of His‐affinity tags on protein expression levels, solubility, secondary structure, thermal denaturation, aggregation and the impact on humoral and cellular immune responses in mice. While the overall immunogenicity induced by His‐tagged MSP142 proteins is greater, the fine specificity of the humoral and cellular immune responses is altered relative to anti‐parasitic antibody activity and the breadth of T‐cell responses. Thus, the usefulness of protein tags may be outweighed by their potential impact on structure and function, stressing the need for caution in their use.

Evaluation of immunoglobulin purification methods and their impact on quality and yield of antigen-specific antibodies

BackgroundAntibodies are the main effectors against malaria blood-stage parasites. Evaluation of functional activities in immune sera from Phase 2a/b vaccine trials may provide invaluable information in the search for immune correlates of protection. However, the presence of anti-malarial-drugs, improper collection/storage conditions or concomitant immune responses against other pathogens can contribute to non-specific anti-parasite activities when the sera/plasma are tested in vitro. Purification of immunoglobulin is a standard approach for reducing such non-specific background activities, but the purification method itself can alter the quality and yield of recovered Ag-specific antibodies.MethodsTo address this concern, various immunoglobulin (Ig) purification methods (protein G Sepharose, protein A/G Sepharose, polyethylene glycol and caprylic acid-ammonium sulphate precipitation) were evaluated for their impact on the quality, quantity and functional activity of purified rabbit and human Igs. The recovered Igs were analysed for yield and purity by SDS-PAGE, for quality by Ag-specific ELISAs (determining changes in titer, avidity and isotype distribution) and for functional activity by in vitro parasite growth inhibition assay (GIA).ResultsThis comparison demonstrated that overall polyethylene glycol purification of human serum/plasma samples and protein G Sepharose purification of rabbit sera are optimal for recovering functional Ag-specific antibodies.ConclusionConsequently, critical consideration of the purification method is required to avoid selecting non-representative populations of recovered Ig, which could influence interpretations of vaccine efficacy, or affect the search for immune correlates of protection.

MSP-1p42-specific antibodies affect growth and development of intra-erythrocytic parasites of Plasmodium falciparum

BackgroundAntibodies are the main effector molecules in the defense against blood stages of the malaria parasite Plasmodium falciparum. Understanding the mechanisms by which vaccine-induced anti-blood stage antibodies work in protecting against malaria is essential for vaccine design and testing.MethodsThe effects of MSP-1p42-specific antibodies on the development of blood stage parasites were studied using microscopy, flow cytometry and the pLDH assay. To determine allele-specific effects, if present, allele-specific antibodies and the various parasite clones representative of these alleles of MSP-1 were employed.ResultsThe mode of action of anti-MSP-1p42 antibodies differs among the parasite clones tested: anti-MSP-1p42 sera act mainly through invasion-inhibitory mechanisms against FVO parasites, by either preventing schizonts from rupturing or agglutinating merozoites upon their release. The same antibodies do not prevent the rupture of 3D7 schizonts; instead they agglutinate merozoites and arrest the development of young parasites at the early trophozoite stage, thus acting through both invasion- and growth inhibitory mechanisms. The second key finding is that antibodies have access to the intra-erythrocytic parasite, as evidenced by the labeling of developing merozoites with fluorochrome-conjugated anti-MSP-1p42 antibodies. Access to the parasite through this route likely allows antibodies to exert their inhibitory activities on the maturing schizonts leading to their inability to rupture and be released as infectious merozoites.ConclusionThe identification of various modes of action by which anti-MSP-1 antibodies function against the parasite during erythrocytic development emphasizes the importance of functional assays for evaluating malaria vaccines and may also open new avenues for immunotherapy and vaccine development.

Molecular adjuvants for malaria DNA vaccines based on the modulation of host-cell apoptosis.

Malaria represents a major global health problem but despite extensive efforts, no effective vaccine is available. Various vaccine candidates have been developed that provide protection in animal models, such as a gene gun-delivered DNA vaccine encoding the circumsporozoite protein (CSP) of Plasmodium berghei. A common shortcoming of most malaria vaccines is the requirement for multiple immunizations leaving room for improvement even for established vaccine candidates such as the CSP-DNA vaccine. In this study, we explored whether regulating apoptosis in DNA vaccine transfected host cells could accelerate the onset of protective immunity and provide significant protection after a single immunization. A pro-apoptotic gene (Bax) was used as a molecular adjuvant in an attempt to mimic the immunostimulatory apoptosis triggered by viral or virus-derived vaccines, while anti-apoptotic genes such as Bcl-XL may increase the life span of transfected cells thus prolonging antigen production. Surprisingly, co-delivery of either Bax or Bcl-XL greatly reduced CSP-DNA vaccine efficacy after a single immunization. Co-delivery of Bax for three immunizations still had a detrimental effect on protective immunity, while repeated co-delivery of Bcl-XL had no negative impact. The fine characterization of humoral and cellular immune response modulated by these two molecular adjuvants revealed a previously unknown effect, i.e., a shift in the Th-profile. These results demonstrate that pro- or anti-apoptotic molecules should not be used as molecular adjuvants without careful evaluation of the resulting immune response. This finding represents yet another example that strategies to enhance vaccine efficacy developed for other model systems such as viral diseases cannot easily be applied to any vaccine.

Nijmegen-Bethesda Assay to Measure Factor VIII Inhibitors

Hemophilia A is an inherited bleeding disorder caused by a deficiency of factor VIII coagulant activity (FVIII:C). Patients are treated with infusions of either plasma-derived or recombinant factor VIII. However, some patients develop inhibitory antibodies (inhibitors) to infused factor VIII which render it ineffective. The original Bethesda method was developed to standardize measurement of inhibitors in a factor VIII neutralization assay. One Bethesda unit is defined as that amount of inhibitor that results in 50% residual FVIII:C activity of a defined test mixture. In the Nijmegen modification of the original Bethesda method, the pH and the protein concentration of the test mixture is further standardized. As a result, the FVIII:C in the test mixture is less prone to artifactual deterioration and the test has improved specificity. Even with a standardized procedure a number of factors can affect the performance of the test and it is important for laboratory staff to be aware of their impact on the result outcome.

Identification and targeting of tumor escape mechanisms: a new hope for cancer therapy?

Conventional cancer therapy is administered in the form of surgery, radiotherapy or chemotherapy. Immunotherapy is the latest asset to the panel of anti-cancer treatments. This approach appears favorable over the other more conventional methods for various reasons: (1) it is highly specific for cancer cells and, therefore, low toxicity should be expected; (2) it recognizes and eliminates cancer cells regardless of their phase in the cell cycle; (3) tumors that developed drug resistances would still be a suitable target for immunotherapy. (4) Immunotherapy offers the possibility of preventive immunization of high-risk patients. Due to the diverse mechanisms that result in the transformation of cells and subsequent tumor development, not all cancers respond similarly to treatment. Significant effort is currently invested in the characterization of the underlying regulatory network in individual cancers responsible for tumorigenesis. Understanding tumors better allows on one hand the identification of essential pathways that can be intercepted to kill the transformed cells more specifically. On the other hand, these insights also allow us to exclude therapeutic strategies with little chance of success when dealing with tumor escape mutants thus saving valuable time and resources. Any tumor therapy puts selective pressure on tumors thus favoring the outgrowth of therapy-resistant variants. This review summarizes current knowledge on tumor escape mechanisms and some of the efforts to overcome these mechanisms.

Impact of pre-existing MSP1 42 -allele specific immunity on potency of an erythrocytic Plasmodium falciparum vaccine

BackgroundMSP1 is the major surface protein on merozoites and a prime candidate for a blood stage malaria vaccine. Preclinical and seroepidemiological studies have implicated antibodies to MSP1 in protection against blood stage parasitaemia and/or reduced parasite densities, respectively. Malaria endemic areas have multiple strains of Plasmodium falciparum circulating at any given time, giving rise to complex immune responses, an issue which is generally not addressed in clinical trials conducted in non-endemic areas. A lack of understanding of the effect of pre-existing immunity to heterologous parasite strains may significantly contribute to vaccine failure in the field. The purpose of this study was to model the effect of pre-existing immunity to MSP142 on the immunogenicity of blood-stage malaria vaccines based on alternative MSP1 alleles.MethodsInbred and outbred mice were immunized with various recombinant P. falciparum MSP142 proteins that represent the two major alleles of MSP142, MAD20 (3D7) and Wellcome (K1, FVO). Humoral immune responses were analysed by ELISA and LuminexTM, and functional activity of induced MSP142-specific antibodies was assessed by growth inhibition assays. T-cell responses were characterized using ex vivo ELISpot assays.ResultsAnalysis of the immune responses induced by various immunization regimens demonstrated a strong allele-specific response at the T cell level in both inbred and outbred mice. The success of heterologous regimens depended on the degree of homology of the N-terminal p33 portion of the MSP142, likely due to the fact that most T cell epitopes reside in this part of the molecule. Analysis of humoral immune responses revealed a marked cross-reactivity between the alleles. Functional analyses showed that some of the heterologous regimens induced antibodies with improved growth inhibitory activities.ConclusionThe development of a more broadly efficacious MSP1 based vaccine may be hindered by clonally imprinted p33 responses mainly restricted at the T cell level. In this study, the homology of the p33 sequence between the clonally imprinted response and the vaccine allele determines the magnitude of vaccine induced responses.

Miniaturized Growth Inhibition Assay to Assess the Anti-blood Stage Activity of Antibodies

While no immune correlate for blood-stage specific immunity against Plasmodium falciparum malaria has been identified, there is strong evidence that antibodies directed to various malarial antigens play a crucial role. In an effort to evaluate the role of antibodies in inhibiting growth and/or invasion of erythrocytic stages of the malaria parasite it will be necessary to test large sample sets from Phase 2a/b trials as well as epidemiological studies. The major constraints for such analyses are (1) availability of sufficient sample quantities (especially from infants and small children) and (2) the throughput of standard growth inhibition assays. The method described here assesses growth- and invasion inhibition by measuring the metabolic activity and viability of the parasite (by using a parasite lactate dehydrogenase-specific substrate) in a 384-microtiter plate format. This culture method can be extended beyond the described detection system to accommodate other techniques commonly used for growth/invasion-inhibition.

The Impact of Immune Responses on the Asexual Erythrocytic Stages of Plasmodium and the Implication for Vaccine Development

Natural immunity against malaria is acquired after repeated infections for an extended period of time resulting in a state of immunological non-responsiveness against the malaria parasite. This state ultimately prevents the onset of severe disease such as cerebral malaria thereby reducing the risk of death from malaria. Individuals with acquired natural immunity to malaria still harbor parasites (albeit in low densities) in the blood, and therefore natural immunity against malaria is not sterile. For this reason, natural immunity to malaria cannot be compared to immunity achieved against other diseases where the immune response neutralizes and eliminates the pathogen. The second hallmark of natural immunity to malaria is that protection wanes once a “protected” individual leaves the malaria-endemic area indicating that protection depends on continued antigen exposure. Immunity to malaria is stageand species-specific and distinct immune mechanisms confer protection against the different developmental stages of the parasite. In the case of blood stage infection, passive transfer experiments with purified human immunoglobulins derived from immune individuals living in malaria-endemic areas have demonstrated that antibodies can mediate protection (Cohen et al., 1961; Butcher et al., 1970; BouharounTayoun et al., 1990). Mechanistic studies revealed that the effect of blood stage-specific antibodies on the asexual erythrocytic parasite depends on their antigen-specificity; antibodies can bind to merozoites, opsonize and target them towards phagocytic cells of the host (Groux and Gysin, 1990), or prevent invasion of new erythrocytes (Perkins, 1991). Once infected, antibodies against asexual blood stage antigens such as Pf332 or MSP-1 inhibit the intra-erythrocytic development of Plasmodium falciparum (Ahlborg et al., 1996; Siddique et al., 1998; Bergmann-Leitner et al., 2009). Antibodies directed to antigens expressed by sexual erythrocytic stages (gametocytes) have been shown to prevent transmission of malaria by blocking either the infection of the mosquito or the development in the mosquito (Lavazec and Bourqouin, 2008). As will be outlined in this chapter, it becomes increasingly clear that a blood stage vaccine may never be able to induce sterile protection, but can prevent mortality. Why bother developing blood stage vaccines? Extensive studies characterizing