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Novel ELISA method as exploratory tool to assess immunity induced by radiated attenuated sporozoites to decipher protective immunity
© The Author(s) 2017
Received: 11 September 2017
Accepted: 18 November 2017
Published: 29 November 2017
Whole parasite vaccines provide a unique opportunity for dissecting immune mechanisms and identify antigens that are targeted by immune responses which have the potential to mediate sterile protection against malaria infections. The radiation attenuated sporozoite (PfSPZ) vaccine has been considered the gold standard for malaria vaccines because of its unparalleled efficacy. The immunogenicity of this and other vaccines continues to be evaluated by using recombinant proteins or peptides of known sporozoite antigens. This approach, however, has significant limitations by relying solely on a limited number of known pathogen-associated immune epitopes. Using the full range of antigens expressed by the sporozoite will enable the comprehensive immune-profiling of humoral immune responses induced by whole parasite vaccines. To address this challenge, a novel ELISA based on sporozoites was developed.
The SPZ-ELISA method described in this report can be performed with either freshly dissected sporozoites or with cryopreserved sporozoite lysates. The use of a fixative for reproducible coating is not required. The SPZ-ELISA was first validated using monoclonal antibodies specific for CSP and TRAP and then used for the characterization of immune sera from radiation attenuated sporozoite vaccinees.
Applying this simple and highly reproducible approach to assess immune responses induced by malaria vaccines, both recombinant and whole parasite vaccines, (1) will help in the evaluation of immune responses induced by antigenically complex malaria vaccines such as the irradiated SPZ-vaccine, (2) will facilitate and accelerate the identification of immune correlates of protection, and (3) can also be a valuable assessment tool for antigen discovery as well as down-selection of vaccine formulations and, thereby, guide vaccine design.
To date, only whole parasite vaccines have been able to reproducibly induce high vaccine efficacy against malaria infection in humans [immunization under chemoprophylaxis (CPS), immunization with radiation attenuated sporozoites (PfSPZ), immunization with genetically attenuated sporozoites (GAS)] (reviewed in ). The question which SPZ-derived antigens mediate this protection has been the focus of intensive investigations, and the analysis of immune responses of vaccinees, who received whole parasite vaccines, has revealed novel antigens, such as the CelTOS protein, as well as some other antigens that are currently being evaluated for their ability to induce protective immunity when used in recombinant vaccines.
Major efforts have been undertaken to make the radiation attenuated sporozoite vaccine (PfSPZ) a viable alternative to recombinant vaccines against malaria. The PfSPZ-vaccine is based on the observation that large numbers of infectious bites from irradiated mosquitoes mediate sterile protection. The original approach required the immunization to take place close to a large insectary capable of supplying sufficient numbers of radiation attenuated, infected mosquitoes [2, 3]. More recently, radiation attenuated sporozoites have been cryopreserved and are eventually delivered by needle and syringe . Initial studies with this vaccine demonstrated that needle-based immunizations targeting the skin or muscle only had limited vaccine efficacy . This raises two questions: (1) what is the difference between vaccination with irradiated mosquitoes and injection of purified irradiated sporozoites in regards to immunomodulatory saliva components; (2) what is the impact of the delivery route and/or sporozoites dose of the needle-based vaccination (reviewed in ). Several clinical trials are underway or planned to address these questions and to establish a vaccine regimen that will provide complete, sterile and long-lasting protection. While the only reliable readout for vaccine efficacy currently available is a controlled human malaria infection (CHMI), the ability to identify a correlate or a surrogate marker of protection would greatly reduce the need for CHMIs thereby reducing the costs associated with vaccine design and accelerate the time line for an efficacious vaccine.
Apart from the obvious desire to develop a protective malaria vaccine, whole parasite vaccines also offer the opportunity to study immune mechanisms that lead to sterile protection. These findings can then be used to develop recombinant vaccines that emulate a sporozoite-based vaccine. Early studies with the radiation attenuated sporozoite (RAS) vaccine in preclinical models identified CD8+ T cells, IFN-γ, and antibodies as being essential for providing protection . While the role of CD8+ T cells in mediating protection in human vaccinees is less supported by data , they have been shown to be crucial in preclinical models . This could, in part, be due to the fact, that these cells may not be present in sufficiently large numbers in human peripheral blood, thus eluding detection. In contrast, the important role of antibodies in mediating protection induced by a variety of vaccine platforms has been well demonstrated [7–9].
Antibodies induced by whole parasite vaccines are mostly measured using conventional ELISA methods that employ either the full-length circumsporozoite protein (CSP) or the central repeat region of the CSP as plate antigen. While CSP is the major surface protein, using only a single antigen to assess the immunogenicity and magnitude of the vaccine induced humoral immune response provides quite limited insight into the vaccine-induced humoral immune response and may result in flawed conclusions and misdirect the search for immune correlates of protection.
This report describes a novel, simple, and highly reproducible ELISA protocol (SPZ-ELISA) based on employing Plasmodium falciparum sporozoites as plate antigen. To date, anti-sporozoite serological responses are captured by performing immunofluorescence assays with sporozoites [10–12]. Evaluating immune responses by microscopy is labour intense and, unless imaging systems are available, quantitation may be limited and standardization between laboratories is challenging. The high-throughput and ease of ELISA based assays offer an opportunity to evaluate serological responses recognizing sporozoites. There have been two reports on genetically attenuated Plasmodium yoelii sporozoite vaccines where sporozoite lysates were used in an ELISA format [13, 14]. To date, there are no reports or protocols describing the use of P. falciparum sporozoites as plate antigen in an ELISA. In reports where researchers utilize ELISA assays to detect or measure the number of sporozoites, monoclonal antibodies specific for CSP are used to capture sporozoites in a sandwich ELISA format  and such an assay has not been validated for the assessment of vaccine specific responses. The assay was validated using well-established monoclonal antibodies to CSP and Thrombospondin related adhesive protein (TRAP), and applied to pooled sera to establish the usefulness of the SPZ-ELISA as a novel tool for comprehensively evaluating ab responses to antigenically complex malaria antigens. It is proposed that this assay format will be able to serve as an additional tool for the ongoing search for immune correlates of protection against malaria.
Sporozoites were prepared by dissecting mosquitoes 16–20 days post blood feed using the Ozaki method . Sporozoites were either immediately coated onto ELISA plates or frozen as pellets for use as lysates.
For the detection of sporozoite antigens, the following antibodies were used: 2A10 (anti-CSP, BEI resources NIAID), clone SAI171C-5E2 (anti-TRAP, BEI resources), clones TH1 and TH3 (kind gift of Dr. Ted Hall, WRAIR), de-identified serum pools from radiation attenuated sporozoite-immunized (RAS) vaccinees . The “protected” pool consisted of six subjects and the “non-protected” pool consisted of five subjects. Mouse monoclonal 1D9 (ATCC, Manassas, VA) and pre-immune serum pool from all RAS vaccinees were used to determine background reactivity against sporozoites. Secondary antibodies (goat-anti mouse IgG-AP, goat-anti-human IgG-AP) were purchased from Southern Biotech (Birmingham, AL).
Freshly dissected sporozoites or thawed sporozoite pellets were suspended to the desired concentration with PBS (pH 7.4) and plated at 30 μl/well in Immulon 2HB plates (Thermo Scientific Waltham, MA). Plates were incubated for 2 h at room temperature (RT). The liquid was gently removed from wells and plates were either allow to air dry or fixed by adding 50 μl/well fixative (1% paraformaldehyde, 3% glutaraldehyde, or methanol). Plates were then blocked with PBS + 1% BSA (50 μl/well) for 1 h at RT. Primary antibodies were diluted with PBS + 1% BSA, added to the respective wells (50 μl/well), and plates were incubated for 2 h at RT. Unbound antibodies were removed by washing the plates three times with PBS. Secondary antibodies were diluted with PBS + 1% BSA (1:200) and added to each well (50 μl/well) for 1 h at RT. Plates were then washed with PBS and substrate (BluePhos, SeraCare, Gaithersburg, MD) was added. Plates were read on a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA) at 630 nm absorption.
Statistically significant differences between the various assay conditions were determined by using two-sided T tests (Minitab 17, State College, PA).
Results and discussion
Immune recognition of intact versus lysates by sporozoite specific antibodies
Air dried sporozoite material has higher immune reactivity with RAS immune antibodies
RAS-immune sera reacted significantly better with air dried than with methanol fixed lysates (p < 0.05, paired T test) regardless of the protective status of the vaccinees. When comparing the protected vs. non-protected groups, serum from protected vaccinees generated a significantly stronger signal when using air dried lysates (p < 0.01, paired T test). The reactivity of the protected vs. non-protected serum pools was not as significant when using methanol fixed sporozoite lysates and when coating with the higher antigen concentration; only at the lower SPZ concentration the difference was significant (p = 0.04, paired T test). These results suggest that the sporozoite ELISA may be a valuable tool for the analysis of whole sporozoite vaccines.
Reactivity of sporozoite-specific monoclonal antibodies in the SPZ-ELISA
The present study describes the development of an experimental approach to assess immune responses induced by malaria vaccines, in particular whole parasite vaccines. The use of reagents that can easily be made available to any laboratory was a major consideration and, therefore, the use of sporozoite lysates was tested regarding its impact on the resulting immunoreactivity in the ELISA method. The method described here represents an asset for the evaluation of immune responses induced by antigenically complex malaria vaccines such as the irradiated SPZ-vaccine and will, therefore, facilitate and accelerate the identification of immune correlates of protection.
TAK and EHD performed the experiments and contributed significantly to scientific discussions, TS provided crucial reagents for the assay and edited the manuscript, ESB-L designed the experiments and compiled the manuscript. All authors read and approved the final manuscript.
TAK is a participant of the Science and Engineering Apprentice Program (SEAP/CQL) and is enrolled at the Old Dominion University, Norfolk VA.
The authors would like to thank Drs. Wolfgang Leitner and Viseth Ngauy for reviewing the manuscript and scientific discussions.
The authors declare that they have no competing interests.
Availability of data and materials
The data and detailed protocol can be made available upon request from the corresponding author.
Consent for publication
Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors, and are not to be construed as official, or as reflecting the views of the Department of the Army or the Department of Defense. This paper has been approved for public release with unlimited distribution. The investigators have adhered to the policies for protection of human subjects as prescribed in AR 70-25.
Ethics approval and consent to participate
This work was supported by the US Military Infectious Disease Research Project and the US Army Medical Research and Materiel Command.
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