Ruth D. Ellis

A Randomized Controlled Phase 2 Trial of the Blood Stage AMA1-C1/Alhydrogel Malaria Vaccine in Children in Mali

A double blind, randomized, controlled Phase 2 clinical trial was conducted to assess the safety, immunogenicity, and biologic impact of the vaccine candidate Apical Membrane Antigen 1-Combination 1 (AMA1-C1), adjuvanted with Alhydrogel. Participants were healthy children 2-3 years old living in or near the village of Bancoumana, Mali. A total of 300 children received either the study vaccine or the comparator. No impact of vaccination was seen on the primary endpoint, the frequency of parasitemia measured as episodes >3000/microL/day at risk. There was a negative impact of vaccination on the hemoglobin level during clinical malaria, and mean incidence of hemoglobin <8.5 g/dL, in the direction of lower hemoglobin in the children who received AMA1-C1, although these differences were not significant after correction for multiple tests. These differences were not seen in the second year of transmission.

Phase 1 trial of AMA1-C1/Alhydrogel plus CPG 7909: an asexual blood-stage vaccine for Plasmodium falciparum malaria

Background Apical Membrane Antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. This is the first reported use in humans of an investigational vaccine, AMA1-C1/Alhydrogel, with the novel adjuvant CPG 7909. Methods A phase 1 trial was conducted at the University of Rochester with 75 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine. Participants were sequentially enrolled and randomized within dose escalating cohorts to receive three vaccinations on days 0, 28 and 56 of either 20 µg of AMA1-C1/Alhydrogel®+564 µg CPG 7909 (n = 15), 80 µg of AMA1-C1/Alhydrogel® (n = 30), or 80 µg of AMA1-C1/Alhydrogel+564 µg CPG 7909 (n = 30). Results Local and systemic adverse events were significantly more likely to be of higher severity with the addition of CPG 7909. Anti-AMA1 immunoglobulin G (IgG) were detected by enzyme-linked immunosorbent assay (ELISA), and the immune sera of volunteers that received 20 µg or 80 µg of AMA1-C1/Alhydrogel+CPG 7909 had up to 14 fold significant increases in anti-AMA1 antibody concentration compared to 80 µg of AMA1-C1/Alhydrogel alone. The addition of CPG 7909 to the AMA1-C1/Alhydrogel vaccine in humans also elicited AMA1 specific immune IgG that significantly and dramatically increased the in vitro growth inhibition of homologous parasites to levels as high as 96% inhibition. Conclusion/Significance The safety profile of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine is acceptable, given the significant increase in immunogenicity observed. Further clinical development is ongoing. Trial Registration ClinicalTrials.gov NCT00344539

The TLR9 Ligand CpG Promotes the Acquisition of Plasmodium falciparum-Specific Memory B Cells in Malaria-Naive Individuals

Despite the central role of memory B cells (MBC) in protective immune responses, little is understood about how they are acquired in naive individuals in response to Ag exposure, and how this process is influenced by concurrent activation of the innate immune system’s TLR. In this longitudinal study of malaria-naive individuals, we examined the MBC response to two candidate malaria vaccines administered with or without CpG, a TLR9 ligand. We show that the acquisition of MBC is a dynamic process in which the vaccine-specific MBC pool rapidly expands and then contracts, and that CpG enhances the kinetics, magnitude, and longevity of this response. We observed that the percentage of vaccine-specific MBC present at the time of reimmunization predicts vaccine-specific Ab levels 14 days later; and that at steady-state, there is a positive correlation between vaccine-specific MBC and Ab levels. An examination of the total circulating MBC and plasma cell pools also suggests that MBC differentiate into plasma cells through polyclonal activation, independent of Ag specificity. These results provide important insights into the human MBC response, which can inform the development of vaccines against malaria and other pathogens that disrupt immunological memory.

Phase 1 trial of the Plasmodium falciparum blood stage vaccine MSP142-C1/alhydrogel with and without CPG 7909 in malaria naïve adults.

Background Merozoite surface protein 142 (MSP142) is a leading blood stage malaria vaccine candidate. In order to induce immune responses that cover the major antigenic polymorphisms, FVO and 3D7 recombinant proteins of MSP142 were mixed (MSP142-C1). To improve the level of antibody response, MSP142-C1 was formulated with Alhydrogel plus the novel adjuvant CPG 7909. Methods A Phase 1 clinical trial was conducted in healthy malaria-naïve adults at the Center for Immunization Research in Washington, D.C., to evaluate the safety and immunogenicity of MSP142-C1/Alhydrogel +/− CPG 7909. Sixty volunteers were enrolled in dose escalating cohorts and randomized to receive three vaccinations of either 40 or 160 µg protein adsorbed to Alhydrogel +/− 560 µg CPG 7909 at 0, 1 and 2 months. Results Vaccinations were well tolerated, with only one related adverse event graded as severe (Grade 3 injection site erythema) and all other vaccine related adverse events graded as either mild or moderate. Local adverse events were more frequent and severe in the groups receiving CPG. The addition of CPG enhanced anti-MSP142 antibody responses following vaccination by up to 49-fold two weeks after second immunization and 8-fold two weeks after the third immunization when compared to MSP142-C1/Alhydrogel alone (p<0.0001). After the third immunization, functionality of the antibody was tested by an in vitro growth inhibition assay. Inhibition was a function of antibody titer, with an average of 3% (range −2 to 10%) in the non CPG groups versus 14% (3 to 32%) in the CPG groups. Conclusion/Significance The favorable safety profile and high antibody responses induced with MSP142-C1/Alhydrogel + CPG 7909 are encouraging. MSP142-C1/Alhydrogel is being combined with other blood stage antigens and will be taken forward in a formulation adjuvanted with CPG 7909. Trial Registration ClinicalTrials.gov Identifier: NCT00320658

Impact on Malaria Parasite Multiplication Rates in Infected Volunteers of the Protein-in-Adjuvant Vaccine AMA1-C1/Alhydrogel+CPG 7909

Background Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria. Methods In this phase I/IIa open-label clinical trial five healthy malaria-naive volunteers were immunised with AMA1/C1-Alhydrogel+CPG 7909, and together with three unvaccinated controls were challenged by intravenous inoculation of P. falciparum infected erythrocytes. Results A significant correlation was observed between parasite multiplication rate in 48 hours (PMR) and both vaccine-induced growth-inhibitory activity (Pearson r = −0.93 [95% CI: −1.0, −0.27] P = 0.02) and AMA1 antibody titres in the vaccine group (Pearson r = −0.93 [95% CI: −0.99, −0.25] P = 0.02). However immunisation failed to reduce overall mean PMR in the vaccine group in comparison to the controls (vaccinee 16 fold [95% CI: 12, 22], control 17 fold [CI: 0, 65] P = 0.70). Therefore no impact on pre-patent period was observed (vaccine group median 8.5 days [range 7.5–9], control group median 9 days [range 7–9]). Conclusions Despite the first observation in human experimental malaria infection of a significant association between vaccine-induced in vitro growth inhibitory activity and in vivo parasite multiplication rate, this did not translate into any observable clinically relevant vaccine effect in this small group of volunteers. Trial Registration ClinicalTrials.gov [NCT00984763]

A randomized and controlled Phase 1 study of the safety and immunogenicity of the AMA1-C1/Alhydrogel + CPG 7909 vaccine for Plasmodium falciparum malaria in semi-immune Malian adults.

A double blind, randomized and controlled Phase 1 clinical trial was conducted to assess the safety and immunogenicity in malaria-exposed adults of the Plasmodium falciparum blood stage vaccine candidate Apical Membrane Antigen 1-Combination 1 (AMA1-C1)/Alhydrogel with and without the novel adjuvant CPG 7909. Participants were healthy adults 18-45 years old living in the village of Donéguébougou, Mali. A total of 24 participants received 2 doses one month apart of either 80 microg AMA1-C1/Alhydrogel or 80 microg AMA1-C1/Alhydrogel + 564 microg CPG 7909. The study started in October 2007 and completed follow up in May 2008. Both vaccines were well tolerated, with only mild local adverse events and no systemic adverse events judged related to vaccination. The difference in antibody responses were over 2-fold higher in the group receiving CPG 7909 for all time points after second vaccination and the differences are statistically significant (all p<0.05). This is the first use of the novel adjuvant CPG 7909 in a malaria-exposed population.

Anti-Apical-Membrane-Antigen-1 Antibody Is More Effective than Anti-42-Kilodalton-Merozoite-Surface-Protein-1 Antibody in Inhibiting Plasmodium falciparum Growth, as Determined by the In Vitro Growth Inhibition Assay

ABSTRACT Apical membrane antigen 1 (AMA1) and the 42-kDa merozoite surface protein 1 (MSP142) are leading malaria vaccine candidates. Several preclinical and clinical trials have been conducted, and an in vitro parasite growth inhibition assay has been used to evaluate the biological activities of the resulting antibodies. In a U.S. phase 1 trial with AMA1-C1/Alhydrogel plus CPG 7909, the vaccination elicited anti-AMA1 immunoglobulin G (IgG) which showed up to 96% inhibition. However, antibodies induced by MSP142-C1/Alhydrogel plus CPG 7909 vaccine showed less than 32% inhibition in vitro. To determine whether anti-MSP142 IgG had less growth-inhibitory activity than anti-AMA1 IgG in vitro, the amounts of IgG that produced 50% inhibition of parasite growth (Ab50) were compared for rabbit and human antibodies. The Ab50s of rabbit and human anti-MSP142 IgGs were significantly higher (0.21 and 0.62 mg/ml, respectively) than those of anti-AMA1 IgGs (0.07 and 0.10 mg/ml, respectively) against 3D7 parasites. Ab50 data against FVO parasites also demonstrated significant differences. We further investigated the Ab50s of mouse and monkey anti-AMA1 IgGs and showed that there were significant differences between the species (mouse, 0.28 mg/ml, and monkey, 0.14 mg/ml, against 3D7 parasites). Although it is unknown whether growth-inhibitory activity in vitro reflects protective immunity in vivo, this study showed that the Ab50 varies with both antigen and species. Our data provide a benchmark for antibody levels for future AMA1- or MSP142-based vaccine development efforts in preclinical and clinical trials.

A Phase 1 study of the blood-stage malaria vaccine candidate AMA1-C1/Alhydrogel® with CPG 7909, using two different formulations and dosing intervals

A Phase 1 study was conducted in 24 malaria naïve adults to assess the safety and immunogenicity of the recombinant protein vaccine apical membrane antigen 1-Combination 1 (AMA1-C1)/Alhydrogel with CPG 7909 in two different formulations (phosphate buffer and saline), and given at two different dosing schedules, 0 and 1 month or 0 and 2 months. Both formulations were well tolerated and frequency of local reactions and solicited adverse events was similar among the groups. Peak antibody levels in the groups receiving CPG 7909 in saline were not significantly different than those receiving CPG 7909 in phosphate. Peak antibody levels in the groups vaccinated at a 0,2 month interval were 2.52-fold higher than those vaccinated at a 0,1 month interval (p=0.037, 95% CI 1.03, 4.28). In vitro growth inhibition followed the antibody level: median inhibition was 51% (0,1 month interval) versus 85% (0,2 month interval) in antibody from samples taken 2 weeks post-second vaccination (p=0.056).

Phase 1 study of a combination AMA1 blood stage malaria vaccine in Malian children.

Background Apical Membrane Antigen-1 (AMA1) is one of the leading blood stage malaria vaccine candidates. AMA1-C1/Alhydrogel® consists of an equal mixture of recombinant AMA1 from FVO and 3D7 clones of P. falciparum, adsorbed onto Alhydrogel®. A Phase 1 study in semi-immune adults in Mali showed that the vaccine was safe and immunogenic, with higher antibody responses in those who received the 80 µg dose. The aim of this study was to assess the safety and immunogenicity of this vaccine in young children in a malaria endemic area. Design This was a Phase 1 dose escalating study in 36 healthy children aged 2–3 years started in March 2006 in Donéguébougou, Mali. Eighteen children in the first cohort were randomized 2∶1 to receive either 20 µg AMA1-C1/Alhydrogel® or Haemophilus influenzae type b Hiberix® vaccine. Two weeks later 18 children in the second cohort were randomized 2∶1 to receive either 80 µg AMA1-C1/Alhydrogel® or Haemophilus influenzae type b Hiberix® vaccine. Vaccinations were administered on Days 0 and 28 and participants were examined on Days 1, 2, 3, 7, and 14 after vaccination and then about every two months. Results to Day 154 are reported in this manuscript. Results Of 36 volunteers enrolled, 33 received both vaccinations. There were 9 adverse events related to the vaccination in subjects who received AMA1-C1 vaccine and 7 in those who received Hiberix®. All were mild to moderate. No vaccine-related serious or grade 3 adverse events were observed. There was no increase in adverse events with increasing dose of vaccine or number of immunizations. In subjects who received the test vaccine, antibodies to AMA1 increased on Day 14 and peaked at Day 42, with changes from baseline significantly different from subjects who received control vaccine. Conclusion AMA-C1 vaccine is well tolerated and immunogenic in children in this endemic area although the antibody response was short lived. Trial Registration Clinicaltrials.gov NCT00341250

Lack of allele-specific efficacy of a bivalent AMA1 malaria vaccine

BackgroundExtensive genetic diversity in vaccine antigens may contribute to the lack of efficacy of blood stage malaria vaccines. Apical membrane antigen-1 (AMA1) is a leading blood stage malaria vaccine candidate with extreme diversity, potentially limiting its efficacy against infection and disease caused by Plasmodium falciparum parasites with diverse forms of AMA1.MethodsThree hundred Malian children participated in a Phase 2 clinical trial of a bivalent malaria vaccine that found no protective efficacy. The vaccine consists of recombinant AMA1 based on the 3D7 and FVO strains of P. falciparum adjuvanted with aluminum hydroxide (AMA1-C1). The gene encoding AMA1 was sequenced from P. falciparum infections experienced before and after immunization with the study vaccine or a control vaccine. Sequences of ama1 from infections in the malaria vaccine and control groups were compared with regard to similarity to the vaccine antigens using several measures of genetic diversity. Time to infection with parasites carrying AMA1 haplotypes similar to the vaccine strains with respect to immunologically important polymorphisms and the risk of infection with vaccine strain haplotypes were compared.ResultsBased on 62 polymorphic AMA1 residues, 186 unique ama1 haplotypes were identified among 315 ama1 sequences that were included in the analysis. Eight infections had ama1 sequences identical to 3D7 while none were identical to FVO. Several measures of genetic diversity showed that ama1 sequences in the malaria vaccine and control groups were comparable both at baseline and during follow up period. Pre- and post-immunization ama1 sequences in both groups all had a similar degree of genetic distance from FVO and 3D7 ama1. No differences were found in the time of first clinical episode or risk of infection with an AMA1 haplotype similar to 3D7 or FVO with respect to a limited set of immunologically important polymorphisms found in the cluster 1 loop of domain I of AMA1.ConclusionThis Phase 2 trial of a bivalent AMA1 malaria vaccine found no evidence of vaccine selection or strain-specific efficacy, suggesting that the extreme genetic diversity of AMA1 did not account for failure of the vaccine to provide protection.