Solution Structure And Protective Efficacy Of A Multi-stage Recombinant Malaria Vaccine

Malaria remains one of the most serious life-threatening parasitic disease in the world. Because of the rapid spread of drug-resistant parasites and insecticide-resistant mosquitoes, traditional anti-malaria measures are faced with rigorous challenges. New tools for control malaria are urgently needed. Developing a safe, effective malaria vaccine is one of the most possible solutions. Malaria vaccine is feasibile supported by the data of epidemiological study and results in lab. Immunity against malaria can be induced in the residents living in malaria endemic areas naturally. People over 10 ages rarely damaged by severe malaria in highly malaria endemic areas. Passive transfer of IgG reduced the plasmodium density of 99% in children with severe malaria, which was purified from West African adult's sera who living in malaria-endemic areas. In addition, repeated inoculation of irradiated attenuated plasmodium sporozoite can induce completely protective immunity to malaria. These facts support the development of malaria vaccine. Malaria vaccine research has lasted for nearly thirty years, and a group of malaria vaccine antigens contains currently each phase of life cycle have been identified. These antigens can produce high level of specificity antibodies in animal experiments, and show some protection effect in vitro or animal models. Some of which has entered the clinical research, and shows substantial immune protection effect, such as: RTS, S based on PfCSP has enterd phase III trials, and the phase II trial of RTS, S/AS01E in African children showed certain protective effect (39.2%~ 45.8%), lasting 15 months at least. However, there are many difficulties in malaria vaccine development, including: (1) The lack of understanding the mechanisms about the naturally protective immunity to malaria, and existing vaccine antigens showed weak immunogenicity and cannot produce effective immune protection when used alone; (2) The lack of suitable animal models to evaluate the prorective effect of vaccine in vivo; (3) Antigenic variation of Plasmodium falciparum and multiple mechanism of immune evasion; (4) The limitation of human vaccine adjuvants with strong effect.It is a trend to develop a multi-stage combination malaria vaccine composed of antigens expressed at different stage of life cycle. Because the life cycle is complex, it is widely believed that an effective vaccine must be compsed of antigens taget to different stage of growth and development, enhancing the effect of the vaccine and overcoming problems such as antigen variability. The combination malaria vaccine can induced protective immunity to kill erythrocytic and exoerythrocytic plasmodium when the antigen contain rythrocytic and exoerythrocytic antigens. In addition, the selection of human vaccine adjuvants is also important to malaria vaccine development. Research has shown that extremely high immune response is needed to malaria protective immune. So it is necessary to select or develop a human vaccine adjuvant with strong effect, combinate with malaria vaccine antigens.A series of works have been done in our lab. PfCP-2.9 is an erythrocytic malaria vaccine candidate which was designed and developed in our lab, composed of PfMSP119 and PfAMA1-III. PfCP-2.9 shows good prospects for development in previous lab and clinical research: (1) The PfCP-2.9 was produced by Pichia pastoris at highly output, soluble and high stability; (2) It showed strong immunogenicity in mice, rabbit, monkey animal models, and induced high level antibodies of ten times more than single components in rabbits; (3) Rabbit and rhesus monkeys immune sera showed good growth parasite growth inhibition in vitro; (4) Two phase I clinical trials of this vaccine candidate formulated in Montanide ISA 720 were completed recently, demonstrating the safety, tolerability, and immunogenicity of the vaccine in humans. However, an important issues——the conformation of PfCP-2.9 remains unknown. Especially, PfCP-2.9 is compsed of two disulfide bond rich domains. It is widespread concern whether PfMSP119 and PfAMA1-III form natural conformation and whether there are interaction between two domains.NMR technology was used in this research to analysis the solution structure of PfCP-2.9. We prepared 15N-labled and 13C,15N-labled PfCP-2.9, 15N-labled PfMSP119 for analyzation of the solution structure, through compare the chemical shift of amine acid in chimeric protein and single domain. In addition, we carried out immunology and preclinical study of a multi-stage malaria vaccine based on PfCP-2.9 and PfCSP-2 antigen. This research has achieved main results are as follows:1. Solution structure analysis of chimeric protein vaccine candidate PfCP-2.9We prepared 15N-labled and 13C,15N-labled PfCP-2.9, 15N-labled PfMSP119 in Pichia pastoris expression system for NMR analysis. In the process of preparing samples, we met problems such as protein degradation and low expression amount. We get the final smples through using small cans of fermentation and strict control and optimization fermentation conditions.The three-dimensional (3D) HNCA, HNCACB, CBCA(CO)NH, HNCO, and HN(CA)CO experiments were carried out for the backbone assignments. HBHA(CO)NH, (H)CC(CO) NH, CC(CO)NH, and 15N-TOCSY-HSQC experiments were performed for side-chain assignments. Backbone assignments were obtained for 39 out of 116 residues of the PfAMA1-III domain, 12 out of 28 residues of the hinge region, and 71 out of 97 residues of the PfMSP119 domain. Side-chain chemical shifts were also partially obtained. For the PfAMA-1(III) domain, the assigned residues are spread over different regions of this domain, including both well-structured and disordered regions. In particular, many residues in the core structural region were assigned. For the PfMSP119 domain, the majority of the residues in this domain were assigned.3D 15N-edited NOESY-HSQC spectrum (mixing time, 100 ms) was performed to confirm the chemical shift assignments.Nearly all of the assigned residues in PfCP-2.9 display chemical shifts highly similar to those reported for the individual domains PfAMA1-III and PfMSP119. Few atoms show bigger chemical shift differences, however this is considered by caused by differences of experimental conditions between cueernt study and previously published results. The overlay of the HSQC spectra of PfMSP119 and PfCP-2.9 demonstrated that the structure of the PfMSP119 domain in PfCP-2.9 is the same as PfMSP119 expressed alone. The chemical shift differences were within experimental errors for all peaks, with the exception of His150 and Ser238. His150 is preceded by the linker sequence in PfCP-2.9, whereas it is close to the N-terminus in PfMSP1-19. The residue Ser238 is close to the C-terminus of both proteins and could be easily affected by slight condition differences between samples. Moreover, no any signal disappearance was observed by comparing the two spectra. These data strongly demonstrated that the conformation of PfMSP1-19 domain in the fusion protein is identical to PfMSP1-19 domain alone.The transverse relaxation rates R2 of the two samples were measured to further investigate the possibility of line broadenings. The R2 values of these residues were compared between PfCP-2.9 and PfMSP119. The R2 values were overall higher for residues in PfCP-2.9 compared with those in PfMSP119, which is due to the higher molecular weight and slower molecular tumbling of PfCP-2.9. By overlaying the two datasets the trend of R2 values over residue number is essentially the same for the two samples. No significant increase of transverse relaxation rates were observed for any region. This result indicates that no residue chemical/conformational changes occurred in the PfMSP1-19 domain of PfCP-2.9, suggesting that the PfMSP1-19 domain is not likely to be involved in weak interactions with the AMA-1(III) domain or the linker region.2. Immunology and preclinical study of a multi-stage malaria vaccineExoerythrocytic antigen PfCSP-2 had been constructed in our lab to develop a multi -stage malaria vaccine. PfCSP-2 composed of the NANP repeat region and C-terminal flanking sequence of PfCSP of 3D7 strain. The antigen showed good prospects in previous study: high level expression in Pichia pastoris, highly stability and solubility, simple purification process, strong immunogenicity in mice and rabbits animal models. We carried out immunology study in different animal models, novel vaccine adjuvant and preclinical study of multi-stage malaria vaccine.(1) Selection of novel adjuvant: Vaccine is compoe of two important components——antigan and adjuvant. Adjuvant is as important as antigen in vaccine. We choose 5 adjuvants used in human, and evaluated the effect of these adjuvants in mouse and monkey models: MF59, GLA, MF59+GLA, Alum+GLA, Alum+CpG.64 BALB/c mice were randomized 8 groups, 8 mice in each group. BALB/c mice were immunized cervicodorsal subcutaneous 3 times with PfCP-2.9 formulated with MF59+GLA, MF59, GLA, ISA720, Freud's adjuvant, Alum and Alum+GLA, and take blood after every immune. Specific antibody levels of immune serum were detected by ELISA method. The results showed that Alum+GLA is the most effective adjuvant except two non-human adjuvants——Freud's adjuvant and ISA720, and the IgG purified from D56 sera of PfCP-2.9/Alum+GLA group showed parasite growth inhibition effect in vitro.15 rhesus monkeys were randomized 5 groups, 3 monkeys in each group. Rhesus monkeys were immunized intramscularly 3 times with PfCP-2.9+PfCSP-2 formulated with, ISA720, Alum+CpG, Alum+GLA, Alum and MF59+GLA, and take blood after every immune. Specific antibody levels of immune serum were detected by ELISA method. The results showed that the PfCP-2.9 specific antibody levels of Alum+GLA group is the highest, and the IgG purified from D56 sera of PfCP-2.9+PfCSP-2/Alum+GLA group showed parasite growth inhibition effect in vitro.We select the Alum+GLA for the adjuvant of multi-stage malaria vaccine after discussed with the experts of WHO and IDRI, comprehensive consideration of present effect and safty data in preclinical and clinical trials, and the actual effect of adjuvant will reflected in clinical trials.(2) Antigens dose ratio optimization: The two antigens of multi-stage malaria vaccine are faced with the problem of dose ratio. Therefore, we optimized the dose ratio in mouse models. 30 BALB/c mice were randomized 5 groups, 6 mice in each group. BALB/c mice were immunized cervicodorsal subcutaneous 3 times with PfCP-2.9: PfCSP-2 of ratio 1:1, 1:2, 2:1, 1:4, 4:1, and take blood after every immune. Specific antibody levels of immune serum were detected by ELISA method. Compare the PfCP-2.9 specific antibody levels, the ratio 1:1 group is higher than others, but there is no significant deviation between groups. Compare the PfCSP-2 specific antibody levels, the ratio 1:2 group is higher than others, but there is no significant deviation between ratio 1:1, 1:2, 2:1, 1:4 groups. Take together, we select the dose ratio 1:1 of PfCP-2.9: PfCSP-2 to the next phase experiment.(3) Mutual competitive inhibition analysis: In order to analyze mutual competitive inhibition between PfCP-2.9 and PfCSP-2 in multi-stage malaria vaccine, we perfomed experiments in mice and rabbit animal models. 24 BALB/c mice were randomized 4 groups, 6 mice in each group; 20 New Zealand rabbits were randomized 4 groups, 5 rabbits in each group. Animals were immunized cervicodorsal subcutaneous 3 times with PfCP-2.9, PfCSP-2, PfCP-2.9+PfCSP-2 and physiological saline formulated with Alum+ GLA. Specific antibody levels of immune serum were detected by ELISA method. Compare the specific antibody levels between antigen alone group and conbinant vaccine, there is no significant deviation. The results show there is no mutual competitive inhibition between PfCP-2.9 and PfCSP-2.(4) Immunogenicity and in vitro inhibition effect analysis: After select appropriate adjuvant, appropriate antigen dose ratio and confirmed no mutual competitive inhibition, we carried a series of experiment in mice, rabbits and monkeys to analysis the immunogenicity and in vitro inhibition effect of novel malaria vaccine PfCP-2.9+PfCSP-2 /Alum+GLA. 24 BALB/c mice were randomized 4 groups, 6 mice in each group. BALB/c mice were immunized cervicodorsal subcutaneous 3 times with PfCP-2.9+PfCSP-2 /Alum+GLA, saline/Alum+GLA, PfCP-2.9+PfCSP-2/Alum and saline/Alum; 20 New Zealand rabbits were randomized 4 groups, 5 rabbits in each group. Rabbits were immunized cervicodorsal subcutaneous 3 times with PfCP-2.9/Alum+GLA, PfCSP-2 /Alum+GLA , PfCP-2.9+PfCSP-2/Alum+GLA, saline/Alum+GLA; 10 rhesus monkeys were randomized 2 groups, 5 monkeys in each group. Rhesus monkeys were immunized intramscularly 3 times with PfCP-2.9+PfCSP-2/Alum+CpG and PfCP-2.9+PfCSP-2/Alum, and take blood after every immune. The novel multi-stage malaria vaccine showed good immunogenicity in different animal models, inducing high level specific antibodies. The immune sera can recognize natural antigen, and show parasite growth inhibition effect in vitro.(5) Preliminary observation of vaccine stability: In order to observe the stability of the novel malaria vaccine, we determined the effectiveness to induce animal immune response, namely half effective dose ED50. This method is take different time samples of multi-stage malaria vaccine preserved in 4℃and 37℃conditions, formulating five continious dilution, and immune BALB/c mice after the antigen mixed with Alum+GLA adjuvant. There were 10 BALB/c mice each group, and 5 males and 5 females. Mice were taken blood 4 weeks after every immune. Specific antibody levels of immune serum were detected by ELISA method and ED50 values were calculated. ED50 of different time is no significant deviation. The results demonstrate that the freeze-dried antigens is stable in 6 months at 4℃or in 7 days at 37℃at least.Summary: Developing a safe, effective malaria vaccine is one of the most possible solutions for malaria prevention. Based on extended research about malaria vaccine, we carried out the analysis of the solution structure of PfCP-2.9 and immunology and preclinical study of a multi-stage malaria vaccine based on PfCP-2.9 and PfCSP-2 antigen. The main results are as follows: (1) PfAMA1-III and PfMSP119 domains in chimeric protein remains its natural structure respectively; (2) There is no interaction between PfAMA1-III and PfMSP119 domains in chimeric protein; (3) We select the Alum+GLA for the adjuvant of multi-stage malaria vaccine; (4) Antigen dose ratio 1:1 of PfCP-2.9: PfCSP-2 is suitable for the next phase experiment; There is no mutual competitive inhibition between PfCP-2.9 and PfCSP-2; (5) The novel multi-stage malaria vaccine showed good immunogenicity and parasite growth inhibition effect in different animal models; (6) The freeze-dried antigens is stable in 6 months at 4℃or in 7 days at 37℃at least.