Comparison of ELISA with electro-chemiluminescence technology for the qualitative and quantitative assessment of serological responses to vaccination.

BACKGROUND: Profiling immune responses induced by either infection or vaccination can provide insight into identification of correlates of protection. Furthermore, profiling of serological responses can be used to identify biomarkers indicative of exposure to pathogens. Conducting such immune surveillance requires readout methods that are high-throughput, robust, and require small sample volumes. While the enzyme-linked immunosorbent assay (ELISA) is the classical readout method for assessing serological responses, the advent of multiplex assays has significantly increased the throughput and capacity for immunoprofiling. This report describes the development and assay performance (sensitivity, linearity of detection, requirement for multiple dilutions for each sample, intra- and inter-assay variability) of an electro-chemiluminescence (ECLIA)-based multiplex assay. METHODS: The current study describes the development of a multiplex ECLIA-based assay and characterizes the sensitivity, linear range, and inter- and intra-assay variability of the ECLIA platform and its agreement with the traditional ELISA. Special emphasis was placed on potential antigenic competition when testing closely related antigens in the multiplex format. RESULTS: Multiplexing of antigens in ECLIA provides significant practical benefits in terms of reducing sample volume requirements and experimental time. Beyond the practical advantages of multiplexing, the ECLIA provides superior assay performance when compared to the ELISA. Not only does ECLIA show good agreement with the ELISA assay, but the linear range of ECLIA is also sufficiently wide to permit single-dilution measurements of concentration without the need to do serial dilutions. The lack of antigenic competition allows the simultaneous testing of closely related antigens, such as plate antigens representing different alleles of the same protein, which can inform about cross-reactivities-or lack thereof-of serological responses. CONCLUSION: The advantages of the newly developed tool for assessing the antigen profiles of serological responses may ultimately lead to the identification of biomarkers associated with various disease stages and or protection against disease.

Impact of a Rapid Decline in Malaria Transmission on Antimalarial IgG Subclasses and Avidity.

Understanding how immunity to malaria is affected by declining transmission is important to aid vaccine design and understand disease resurgence. Both IgG subclasses and avidity of antigen-specific responses are important components of an effective immune response. Using a multiplex bead array assay, we measured the total IgG, IgG subclasses, and avidity profiles of responses to 18 P. falciparum blood stage antigens in samples from 160 Ugandans collected at two time points during high malaria transmission and two time points following a dramatic reduction in transmission. Results demonstrated that, for the antigens tested, (i) the rate of decay of total IgG following infection declined with age and was driven consistently by the decrease in IgG3 and occasionally the decrease in IgG1; (ii) the proportion of IgG3 relative to IgG1 in the absence of infection increased with age; (iii) the increase in avidity index (the strength of association between the antibody and antigen) following infection was largely due to a rapid loss of non-avid compared to avid total IgG; and (iv) both avid and non-avid total IgG in the absence of infection increased with age. Further studies are required to understand the functional differences between IgG1 and IgG3 in order to determine their contribution to the longevity of protective immunity to malaria. Measuring changes in antibody avidity may be a better approach of detecting affinity maturation compared to avidity index due to the differential expansion and contraction of high and low avidity total IgG.

High-Density Peptide Arrays for Malaria Vaccine Development.

The development of an efficacious and practicable vaccine conferring sterile immunity towards a Plasmodium infection represents a not yet achieved goal. A crucial factor for the impact of a given anti-plasmodial subunit vaccine is the identification of the most potent parasitic components required to induce protection from both infection and disease. Here, we present a method based on a novel high-density peptide array technology that allows for a flexible readout of malaria antibodies. Peptide arrays applied as a screening method can be used to identify novel immunogenic antibody epitopes under a large number of potential antigens/peptides. Ultimately, discovered antigen candidates and/or epitope sequences can be translated into vaccine prototype design. The technology can be further utilized to unravel antibody-mediated immune responses (e.g., involved in the establishment of semi-immunity) and moreover to confirm vaccine potency during the process of clinical development by verifying the induced antibody responses following vaccination.

Artesunate versus chloroquine infection-treatment-vaccination defines stage-specific immune responses associated with prolonged sterile protection against both pre-erythrocytic and erythrocytic Plasmodium yoelii infection.

Sterile protection against malaria infection can be achieved through vaccination of mice and humans with whole Plasmodium spp. parasites. One such method, known as infection-treatment-vaccination (ITV), involves immunization with wild type sporozoites (spz) under drug coverage. In this work, we used the different effects of antimalarial drugs chloroquine (CQ) and artesunate (AS) on blood stage (BS) parasites to dissect the stage-specific immune responses in mice immunized with Plasmodium yoelii spz under either drug, as well as their ability to protect mice against challenge with spz or infected RBCs (iRBCs). Whereas CQ-ITV induced sterile protection against challenge with both spz and iRBCs, AS-ITV only induced sterile protection against spz challenge. Importantly, AS-ITV delayed the onset of BS infection, indicating that both regimens induced cross-stage immunity. Moreover, both CQ- and AS-ITV induced CD8(+) T cells in the liver that eliminated malaria-infected hepatocytes in vitro, as well as Abs that recognized pre-erythrocytic parasites. Sera from both groups of mice inhibited spz invasion of hepatocytes in vitro, but only CQ-ITV induced high levels of anti-BS Abs. Finally, passive transfer of sera from CQ-ITV-treated mice delayed the onset of erythrocytic infection in the majority of mice challenged with P. yoelii iRBCs. Besides constituting the first characterization, to our knowledge, of AS-ITV as a vaccination strategy, our data show that ITV strategies that lead to subtle differences in the persistence of parasites in the blood enable the characterization of the resulting immune responses, which will contribute to future research in vaccine design and malaria interventions.

Protective immunity against malaria after vaccination.

A good understanding of the immunological correlates of protective immunity is an important requirement for the development of effective vaccines against malaria. However, this concern has received little attention even in the face of two decades of intensive vaccine research. Here, we review the immune response to blood-stage malaria, with a particular focus on the type of vaccine most likely to induce the kind of response required to give strong protection against infection.

An essential role for C5aR signaling in the optimal induction of a malaria-specific CD4+ T cell response by a whole-killed blood-stage vaccine.

The protective immunity induced by the whole-killed parasite vaccine against malarial blood-stage infection is dependent on the CD4(+) T cell response. However, the mechanism underlying this robust CD4(+) T cell response elicited by the whole-killed parasite vaccine is still largely unknown. In this study, we observe that immunization with Plasmodium yoelii-parasitized RBC lysate activates complement C5 and generates C5a. However, the protective efficacy against P. yoelii 17XL challenge is considerably reduced, and the malaria-specific CD4(+) T cell activation and memory T cell differentiation are largely suppressed in the C5aR-deficient (C5aR(-/-)) mice. An adoptive transfer assay demonstrates that the reduced protection of C5aR(-/-) mice is closely associated with the severely impaired CD4(+) T cell response. This is further confirmed by the fact that administration of C5aR antagonist significantly reduces the protective efficacy of the immunized B cell-deficient mice. Further study indicates that the defective CD4(+) T cell response in C5aR(-/-) mice is unlikely involved in the expansion of CD4(+)CD25(+)Foxp3(+) T cells, but strongly linked to a defect in dendritic cell (DC) maturation and the ability to allostimulate CD4(+) T cells. These results demonstrate that C5aR signaling is essential for the optimal induction of the malaria-specific CD4(+) T cell response by the whole-killed parasite vaccine through modulation of DCs function, which provides us with new clues to design an effective blood-stage subunit vaccine and helps us to understand the mechanism by which the T cell response is regulated by the complement system.

Comparison of modeling methods to determine liver-to-blood inocula and parasite multiplication rates during controlled human malaria infection.

Controlled human malaria infection is used to measure efficacy of candidate malaria vaccines before field studies are undertaken. Mathematical modeling using data from quantitative polymerase chain reaction (qPCR) parasitemia monitoring can discriminate between vaccine effects on the parasite's liver and blood stages. Uncertainty regarding the most appropriate modeling method hinders interpretation of such trials. We used qPCR data from 267 Plasmodium falciparum infections to compare linear, sine-wave, and normal-cumulative-density-function models. We find that the parameters estimated by these models are closely correlated, and their predictive accuracy for omitted data points was similar. We propose that future studies include the linear model.

Plasmodium yoelii blood-stage antigens newly identified by immunoaffinity using purified IgG antibodies from malaria-resistant mice.

As the search for an effective human malaria vaccine continues, understanding immune responses to Plasmodium in rodent models is perhaps the key to unlocking new vaccine strategies. The recruitment of parasite-specific antibodies is an important component of natural immunity against infection in blood-stage malaria. Here, we describe the use of sera from naturally surviving ICR mice after infection with lethal doses of Plasmodium yoelii yoelii 17XL to identify highly immunogenic blood-stage antigens. Immobilized protein A/G was used for the affinity-chromatography purification of the IgGs present in pooled sera from surviving mice. These protective IgGs, covalently immobilized on agarose columns, were then used to isolate reactive antigens from whole P. yoelii yoelii 17XL protein extracts obtained from the blood-stage malaria infection. Through proteomics analysis of the recovered parasite antigens, we were able to identify two endoplasmic reticulum lumen proteins: protein disulfide isomerase and a member of the heat shock protein 70 family. Also identified were the digestive protease plasmepsin and the 39 kDa-subunit of eukaryotic translation initiation factor 3, a ribosome associated protein. Of these four proteins, three have not been previously identified as antigenic during blood-stage malaria infection. This procedure of isolating and identifying parasite antigens using serum IgGs from malaria-protected individuals could be a novel strategy for the development of multi-antigen-based vaccine therapies.

Epitope mapping of PfCP-2.9, an asexual blood-stage vaccine candidate of Plasmodium falciparum.

BACKGROUND: Apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP1) of Plasmodium falciparum are two leading blood-stage malaria vaccine candidates. A P. falciparum chimeric protein 2.9 (PfCP-2.9) has been constructed as a vaccine candidate, by fusing AMA-1 domain III (AMA-1 (III)) with a C-terminal 19 kDa fragment of MSP1 (MSP1-19) via a 28-mer peptide hinge. PfCP-2.9 was highly immunogenic in animal studies, and antibodies elicited by the PfCP-2.9 highly inhibited parasite growth in vitro. This study focused on locating the distribution of epitopes on PfCP-2.9. METHODS: A panel of anti-PfCP-2.9 monoclonal antibodies (mAbs) were produced and their properties were examined by Western blot as well as in vitro growth inhibition assay (GIA). In addition, a series of PfCP-2.9 mutants containing single amino acid substitution were produced in Pichia pastoris. Interaction of the mAbs with the PfCP-2.9 mutants was measured by both Western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: Twelve mAbs recognizing PfCP-2.9 chimeric protein were produced. Of them, eight mAbs recognized conformational epitopes and six mAbs showed various levels of inhibitory activities on parasite growth in vitro. In addition, seventeen PfCP-2.9 mutants with single amino acid substitution were produced in Pichia pastoris for interaction with mAbs. Reduced binding of an inhibitory mAb (mAb7G), was observed in three mutants including M62 (Phe491-->Ala), M82 (Glu511-->Gln) and M84 (Arg513-->Lys), suggesting that these amino acid substitutions are critical to the epitope corresponding to mAb7G. The binding of two non-inhibitory mAbs (mAbG11.12 and mAbW9.10) was also reduced in the mutants of either M62 or M82. The substitution of Leu31 to Arg resulted in completely abolishing the binding of mAb1E1 (a blocking antibody) to M176 mutant, suggesting that the Leu residue at this position plays a crucial role in the formation of the epitope. In addition, the Asn15 residue may also play an important role in the global folding of PfCP-2.9, as its substitution by Arg lead to reduced binding of most mAbs and abolishing the binding of mAb6G and mAbP5-W12. CONCLUSIONS: This study provided valuable information on epitopes of PfCP-2.9 vaccine candidate through generation of a panel of mAbs and a series of PfCP-2.9 mutants. The information may prove to be useful for designing more effective malaria vaccines against blood-stage parasites.

Enhanced CD8+ T cell immune responses and protection elicited against Plasmodium berghei malaria by prime boost immunization regimens using a novel attenuated fowlpox virus.

Sterile immunity can be provided against the pre-erythrocytic stages of malaria by IFN-gamma-secreting CD8(+) T cells that recognize parasite-infected hepatocytes. In this study, we have investigated the use of attenuated fowlpox virus (FPV) strains as recombinant vaccine vectors for eliciting CD8(+) T cells against Plasmodium berghei. The gene encoding the P. berghei circumsporozoite (PbCS) protein was inserted into an FPV vaccine strain licensed for use in chickens, Webster's FPV, and the novel FPV vaccine strain FP9 by homologous recombination. The novel FP9 strain proved more potent as a vaccine for eliciting CD8(+) T cell responses against the PbCS Ag. Sequential immunization with rFP9 and recombinant modified vaccinia virus Anakara (MVA) encoding the PbCS protein, administered by clinically acceptable routes, elicited potent CD8(+) T cell responses against the PbCS protein. This immunization regimen elicited substantial protection against a stringent liver-stage challenge with P. berghei and was more immunogenic and protective than DNA/MVA prime/boost immunization. However, further improvement was not achieved by sequential (triple) immunization with a DNA vaccine, FP9, and MVA.