Validation of an enzyme-linked immunosorbent assay for the quantification of human IgG directed against the repeat region of the circumsporozoite protein of the parasite Plasmodium falciparum.

BACKGROUND: Several pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development. Vaccine immunogenicity is commonly evaluated by the determination of anti-CSP antibody levels using IgG-based assays, but no standard assay is available to allow comparison of the different vaccines. METHODS: The validation of an anti-CSP repeat region enzyme-linked immunosorbent assay (ELISA) is described. This assay is based on the binding of serum antibodies to R32LR, a recombinant protein composed of the repeat region of P. falciparum CSP. In addition to the original recombinant R32LR, an easy to purify recombinant His-tagged R32LR protein has been constructed to be used as solid phase antigen in the assay. Also, hybridoma cell lines have been generated producing human anti-R32LR monoclonal antibodies to be used as a potential inexhaustible source of anti-CSP repeats standard, instead of a reference serum. RESULTS: The anti-CSP repeats ELISA was shown to be robust, specific and linear within the analytical range, and adequately fulfilled all validation criteria as defined in the ICH guidelines. Furthermore, the coefficient of variation for repeatability and intermediate precision did not exceed 23%. Non-interference was demonstrated for R32LR-binding sera, and the assay was shown to be stable over time. CONCLUSIONS: This ELISA, specific for antibodies directed against the CSP repeat region, can be used as a standard assay for the determination of humoral immunogenicity in the development of any CSP-based P. falciparum malaria vaccine

A combined analysis of immunogenicity, antibody kinetics and vaccine efficacy from phase 2 trials of the RTS,S malaria vaccine.

BACKGROUND: The RTS,S malaria vaccine is currently undergoing phase 3 trials. High vaccine-induced antibody titres to the circumsporozoite protein (CSP) antigen have been associated with protection from infection and episodes of clinical malaria. METHODS: Using data from 5,144 participants in nine phase 2 trials, we explore predictors of vaccine immunogenicity (anti-CSP antibody titres), decay in antibody titres, and the association between antibody titres and clinical outcomes. We use empirically-observed relationships between these factors to predict vaccine efficacy in a range of scenarios. RESULTS: Vaccine-induced anti-CSP antibody titres were significantly associated with age (P = 0.04), adjuvant (P <0.001), pre-vaccination anti-hepatitis B surface antigen titres (P = 0.005) and pre-vaccination anti-CSP titres (P <0.001). Co-administration with other vaccines reduced anti-CSP antibody titres although not significantly (P = 0.095). Antibody titres showed a bi-phasic decay over time with an initial rapid decay in the first three months and a second slower decay over the next three to four years. Antibody titres were significantly associated with protection, with a titre of 51 (95% Credible Interval (CrI): 29 to 85) ELISA units/ml (EU/mL) predicted to prevent 50% of infections in children. Vaccine efficacy was predicted to decline to zero over four years in a setting with entomological inoculation rate (EIR) = 20 infectious bites per year (ibpy). Over a five-year follow-up period at an EIR = 20 ibpy, we predict RTS,S will avert 1,782 cases per 1,000 vaccinated children, 1,452 cases per 1,000 vaccinated infants, and 887 cases per 1,000 infants when co-administered with expanded programme on immunisation (EPI) vaccines. Our main study limitations include an absence of vaccine-induced cellular immune responses and short duration of follow-up in some individuals. CONCLUSIONS: Vaccine-induced anti-CSP antibody titres and transmission intensity can explain variations in observed vaccine efficacy

Production of a Transgenic Mosquito Expressing Circumsporozoite Protein, a Malarial Protein, in the Salivary Gland of Anopheles stephensi (Diptera:Culicidae)

We are producing a transgenic mosquito, a flying syringe, to deliver a vaccine protein to human beings via the saliva the mosquito deposits in the skin while biting. The mosquito produces a vaccine protein in the salivary gland (SG) and deposits the protein into the host's skin when it takes the host's blood. We chose circumsporozoite protein (CSP), currently the most promising malaria vaccine candidate, to be expressed in the SG of Anopheles stephensi. To transform the mosquitoes, plasmid containing the CSP gene under the promoter of female SG-specific gene, as well as the green fluorescent protein (GFP) gene under the promoter of 3xP3 as a selection marker in the eyes, was injected into more than 400 eggs. As a result, five strains of GFP-expressing mosquitoes were established, and successful CSP expression in the SG was confirmed in one strain. The estimated amount of CSP in the SG of the strain was 40ng per mosquito. We allowed the CSP-expressing mosquitoes to feed on mice to induce the production of anti-CSP antibody. However, the mice did not develop anti-CSP antibody even after transgenic mosquitoes had bitten them several times. We consider that CSP in the SG was not secreted properly into the saliva. Further techniques and trials are required in order to realize vaccine-delivering mosquitoes

Models for measuring and predicting malaria vaccine efficacy

In the past decade several candidate malaria vaccines have undergone clinical trials in artificial challenge studies and studies of natural infection under field conditions. GlaxoSmithKline’s RTS,S vaccine against Plasmodium falciparum infection has taken the lead, with Phase III trials in African children demonstrating 55.8% (97.5% CI, 51.3% - 59.8%) efficacy against clinical malaria and 34.8% (95% CI, 16.2% – 49.2%) efficacy against severe malaria. Mathematical models can contribute to multiple stages of malaria vaccine development, from measuring efficacy in clinical trials, understanding the relationship between naturally acquired and vaccine-induced immunity, identifying correlates of protection, and predicting the likely impact of vaccination programs in the field. When measuring vaccine efficacy in field trials under natural exposure to malaria, there are many factors which can bias estimates of efficacy. We demonstrate how heterogeneity in exposure can cause efficacy to be underestimated and heterogeneity in vaccine response can cause efficacy to be overestimated. Most infection-blocking vaccines rely on boosting some element of the pre-erythrocytic immune response, however the relationship between the naturally acquired pre-erythrocytic responses and protection from infection remains poorly understood. By analysing studies from a systematic of the published literature, I demonstrate that although many studies report a statistically significant relationship between cellular pre-erythrocytic immune responses and protection from infection, many studies do not have sufficient statistical power to evaluate the effects of the pre-erythrocytic immune response. Mathematical models are developed for investigating the relationship between pre-erythrocytic antibodies and protection from infection, and fitted to data from a longitudinal study of malaria infection in Kenyan adults. The relationship between antibodies to the antigens circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) and protection from infection is characterised using dose-response curves. Using data from an artificial challenge trial of the RTS,S malaria vaccine, I demonstrate that vaccine-induced protection from infection depends on both anti-CSP antibodies and CSP-specific T cells. I estimate that RTS,S causes a 97.7% (95% CI, 96.3% – 98/7%) reduction in the number of parasites entering the blood from the liver. The immune effector mechanisms determining the duration of vaccine-induced protection from infection are likely to be similar to those involved in naturally acquired immunity. Models of antibody kinetics were fitted to data from longitudinal studies of the antibody response to P. falciparum infection in Ghanaian and Gambian children, and the parameters determining the duration of antibody response are estimated. Upon licensure, a successful malaria vaccine is likely to be administered to young African children. A model of malaria transmission, extensively fitted to clinical data, is used to investigate the impact of vaccination in different transmission settings; the interaction between vaccines and other interventions such as insecticide treated nets; and the interaction between vaccination and naturally acquired immunity. Finally, the potential cost-effectiveness of vaccination is explored

T Cell Responses to the RTS,S/AS01E and RTS,S/AS02D Malaria Candidate Vaccines Administered According to Different Schedules to Ghanaian Children

BACKGROUND: The Plasmodium falciparum pre-erythrocytic stage candidate vaccine RTS,S is being developed for protection of young children against malaria in sub-Saharan Africa. RTS,S formulated with the liposome based adjuvant AS01(E) or the oil-in-water based adjuvant AS02(D) induces P. falciparum circumsporozoite (CSP) antigen-specific antibody and T cell responses which have been associated with protection in the experimental malaria challenge model in adults. METHODS: This study was designed to evaluate the safety and immunogenicity induced over a 19 month period by three vaccination schedules (0,1-, 0,1,2- and 0,1,7-month) of RTS,S/AS01(E) and RTS,S/AS02(D) in children aged 5-17 months in two research centers in Ghana. Control Rabies vaccine using the 0,1,2-month schedule was used in one of two study sites. RESULTS: Whole blood antigen stimulation followed by intra-cellular cytokine staining showed RTS,S/AS01(E) induced CSP specific CD4 T cells producing IL-2, TNF-α, and IFN-γ. Higher T cell responses were induced by a 0,1,7-month immunization schedule as compared with a 0,1- or 0,1,2-month schedule. RTS,S/AS01(E) induced higher CD4 T cell responses as compared to RTS,S/AS02(D) when given on a 0,1,7-month schedule. CONCLUSIONS: These findings support further Phase III evaluation of RTS,S/AS01(E). The role of immune effectors and immunization schedules on vaccine protection are currently under evaluation. TRIAL REGISTRATION: ClinicalTrials.gov NCT00360230

Assessment of humoral and cellular immune responses of the RTS,S/AS02D malaria vaccine candidate administered to infants living in a malaria endemic area in Mozambique

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2009Background: RTS,S candidate malaria vaccine has been shown to be highly immunogenic in children and infants, but the protective immune mechanisms still remain to be clearly elucidated. It is believed that RTS,S elicits a strong neutralizing humoral immune response directed against surface-exposed sporozoite proteins and cell mediated immune (CMI) responses characterized by predominantly CD4+ Th1 cells. The objective of this study was to investigate humoral and cell-mediated immune responses to the RTS,S/AS02D malaria vaccine and its association with protection against infection and disease by P. falciparum. Methodology and Principal Findings: This secondary data analysis from data of a phase I/IIb randomized, double-blind, controlled trial, included 154 healthy infants living in rural Mozambique, previously immunized with RTS,S/AS02D candidate malaria vaccine or the control Engerix-B™ vaccine. Antibodies against circumsporozoite protein (CSP) and hepatitis-B surface antigen (HBsAg) were measured with a standard ELISA. Fresh blood intracellular staining assay was performed to evaluate the expression of IL-2 and IFN-γ by CD4+ and CD8+ cells in response to in vitro stimulation of specific peptides. Data was evaluated for association with the risk of malaria detected by both active and passive case detection of infection over a period of 6 months post dose 3. Anti-HBs antibody geometric mean titers declined from 10,082 mIU/mL one month post Dose 3 to 2,751 mIU/mL at 12 months post Dose 3 in the RTS,S/AS02D group; anti-HBs v geometric mean titers were 392.4 mIU/mL and 263.9 mIU/mL, respectively in the Engerix- BTM group. Anti-CSP antibody geometric mean titers declined from 199.9 EU/mL one month post Dose 3 to 7.3 EU/mL at 12 months post Dose 3 in the RTS,S/AS02D group. Median stimulation indices of HBs-specific IL-2 and IFN-γ producing CD8+ T cells was higher in the RTS,S/AS02D group than in control group (Wilcoxon rank sum p-values for IFN-γ = 0.015, for IL-2 = 0.030) at 10.5 weeks post immunization. Median stimulation indices of anti-CSP specific IFN-γ producing CD8+ T cells at the same time point was 1.13 (IQR: 0.79 - 1.67; p=0.029). For specific IL-2-producing CD4+ T cells, the median SI was 1.14 (IQR: 0.74 – 1.60, p=0.043) at 10.5 weeks post dose three. The reduction in hazards of malaria infection were 18.3 % (95% CI: -267.9 – 81.8, p=0.793) and -12.0 % (95% CI: -295 – 68.2, p=0.86) for specific IL-2 CD4+ stimulation indices; For specific CD8+ IFN-γ stimulation indices the hazards were -103.6% (95% CI: -690.9 – 47.6; p=0.305) and 48.8% (95% CI: -97.0 – 86.7; p=0.33) at four and 10.5 weeks post immunization respectively. Conclusion: The RTS,S/AS02D vaccine was immunogenic and has elicited detectable levels of CSP specific cell mediated responses. No evidence of association was found between the antibodies anti-CSP and specific cell mediated responses and the risk of malaria

A monoclonal antibody-based immunoassay to measure the antibody response against the repeat region of the circumsporozoite protein of Plasmodium falciparum

Background: The malaria vaccine candidate RTS, S/AS01 (GSK Vaccines) induces high IgG concentration against the circumsporozoite protein (CSP) of Plasmodium falciparum. In human vaccine recipients circulating anti-CSP antibody concentrations are associated with protection against infection but appear not to be the correlate of protection. However, in a humanized mouse model of malaria infection prophylactic administration of a human monoclonal antibody (MAL1C), derived from a RTS, S/AS01-immunized volunteer, directed against the CSP repeat region, conveyed full protection in a dose-dependent manner suggesting that antibodies alone are able to prevent P. falciparum infection when present in sufficiently high concentrations. A competition ELISA was developed to measure the presence of MAL1C-like antibodies in polyclonal sera from RTS, S/AS01 vaccine recipients and study their possible contribution to protection against infection. Results: MAL1C-like antibodies present in polyclonal vaccine-induced sera were evaluated for their ability to compete with biotinylated monoclonal antibody MAL1C for binding sites on the capture antigen consisting of the recombinant protein encompassing 32 NANP repeats of CSP (R32LR). Serum samples were taken at different time points from participants in two RTS, S/AS01 vaccine studies (NCT01366534 and NCT01857869). Vaccine-induced protection status of the study participants was determined based on the outcome of experimental challenge with infected mosquito bites after vaccination. Optimal conditions were established to reliably detect MAL1C-like antibodies in polyclonal sera. Polyclonal anti-CSP antibodies and MAL1C-like antibody content were measured in 276 serum samples from RTS, S/AS01 vaccine recipients using the standard ELISA and MAL-1C competition ELISA, respectively. A strong correlation was observed between the results from these assays. However, no correlation was found between the results of either assay and protection against infection. Conclusions: The competition ELISA to measure MAL1C-like antibodies in polyclonal sera from RTS, S/AS01 vaccine recipients was robust and reliable but did not reveal the elusive correlate of protection