Patent Application: US-201314394964-A

Abstract:
a method for inducing protective immunity in a vertebrate host against malaria , by administering to the host a live rodent plasmodium organism and exploiting its cross - species protection potential . a method to use genetically engineered live rodent plasmodium organisms expressing antigens from different stages of the life cycle of human - infective plasmodium species to immunize vertebrate hosts against malaria . the invention further provides for the production of a vaccine composition , by suspending wild - type or genetically modified rodent plasmodium organisms in a suitable pharmaceutically acceptable carrier solution .

Description:
the present invention relates to an alternative approach to malaria vaccination that combines unprecedented versatility with high efficacy , whilst ensuring complete safety . we have demonstrated that ( i ) rodent malaria sporozoites ( spz ) can be used as a whole - organism pre - erythrocytic vaccine capable of generating a strong immune response because they are able to successfully infect and develop in human liver cells ; ( ii ) rodent malaria parasites are an extremely safe antigen delivery platform because they are completely unable to complete their life cycle inside human erythrocytes , which renders then unable to cause a disease - triggering human blood - stage infection ; ( iii ) rodent malaria parasites can induce a high level of cross - species protection against human plasmodium species because they possess conserved molecules that can be recognized by the human immune system iv ) genetically engineered rodent plasmodium parasites are highly immunogenic , being able to trigger specific immune responses against an engineered human plasmodium antigen capable of recognizing human plasmodium parasites and inhibiting human plasmodium infection . by rodent plasmodium organism or rodent plasmodium parasite is meant any member of the protozoan genus plasmodium whose natural host is a rodent , including the four known species , p . berghei , p . yoelii , p . vinckei and p . chabaudi . by human plasmodium organism or human plasmodium parasite is meant any member of the protozoan genus plasmodium known to cause human malaria , including p . falciparum , p . vivax , p . malariae , p . ovale and p . knowlesi . we monitored in parallel the in vitro infection of one mouse and two human hepatoma cell lines ( hepa 1 - 6 , hepg2 and huh7 ), and one human immortalized hepatocyte line ( hc04 ), and the ex vivo infection of mouse primary hepatocytes and human primary hepatocyte / fibroblast co - cultures by wild - type p . berghei , a rodent plasmodium organism . infection assessments by quantitative real - time pcr ( qrt - pcr ) and fluorescence microscopy showed that p . berghei traverses , invades and develops to similar extents in all in vitro systems studied and that it is able to invade and develop inside rodent and human primary hepatic cells ex vivo ( fig1 a , b , c , d ). in order to ascertain whether p . berghei is able to infect human liver cells in an in vivo context , we injected sporozoites into liver - humanized mice ( lh mice ) and monitored hepatic infection by confocal fluorescence microscopy . our results show that p . berghei can effectively infect human hepatocytes engrafted in the livers of the chimeric le mice . a comparison between infection of the human and mouse hepatocytes in the chimeric livers , reveals that the parasite is capable of similar development inside either type of cell ( fig1 e - g ). additionally , we employed a genetically engineered rodent p . berghei parasite where the endogenous cs has been replaced by that of p . falciparum ( pbcs pf ) [ 11 ]. we evaluated the hepatic infection behavior of this genetically modified parasite and showed that it retains the ability to infect human hepatic cells in vitro , as well primary hepatocytes as well as the livers of lh mice . overall , these results show that the rodent p . berghei parasite is fully competent to infect human hepatocytes , and that this competence is retained in the genetically engineered pbcs pf parasite , thereby fulfilling the immunity - generation condition for a malaria vaccine . ( ii ) rodent plasmodium parasites are unable to cause a human blood - stage infection that leads to pathology in order to be safe , a rodent plasmodium organism - based malaria vaccine must ensure that the immunizing parasite is unable to cause disease in humans . this requires that rodent plasmodium merozoites are unable to effectively invade and multiply inside human red blood cells ( rbc . in order to ascertain this , we employed a strategy based on the use of blood - humanized mice ( bh mice ), engrafted with defined proportions of human erythrocytes , which have been developed as models to evaluate drug efficacy against p . falciparum infection [ 12 ]. this system can be coupled with the use of the nuclear syto - 16 and the mouse - specific ter - 119 dyes to distinguish infected from non - infected cells and human from rodent erythrocytes , respectively , and thereby monitor infection of either type of cell by flow cytometry ( fig2 a 1 , 2 a 2 , 2 a 3 ). we started by infecting bh mice bearing different degrees of chimerism by transfusion of p . berghei - infected mouse blood and monitored the parasitemia in these mice at regular intervals , using non - chimeric mice as controls . our results show that while the syto - 16 + / ter - 116 + population increased to values ˜ 5 %, showing , as expected , infection of the mouse rbc ( m rbc ) population , the syto - 16 + / ter - 116 − population that would correspond to infected human rbcs ( h rbcs ) never surpassed 0 . 2 %, remaining within the range of the background levels observed for the non - humanized mice ( fig2 a 1 , 2 a 2 , 2 a 3 , 2 b 1 , 2 b 2 , 2 b 3 ). this result suggested that p . berghei is unable to infect h rbcs or that it may do so at very low levels . it is worth noting that this occurs in the context of a chimeric mouse , which contains a significant population of m rbcs that serve as an effective reservoir for the production of large numbers of merozoites . to further investigate the possibility of human rbc infection under these conditions , we analysed blood samples from these mice after staining with the nuclear dye dapi and with ter - 116 . we found very rare instances of dapi − / ter - 116 − cells , which indicated that a small degree of invasion of h rbcs could indeed occur . however , we were unable to find any h rbc bearing more than a single parasite nucleus , suggesting that p . berghei is unable to replicate inside the few h rbcs that it may invade ( fig2 e ). crucially , in vitro cultivation of flow cytometry - isolated infected h rbcs showed that these parasites are indeed incapable of completing their intra - erythrocytic life cycle in h rbcs ( fig2 c 1 , 2 c 2 , 2 d 1 , 2 d 2 ), rendering them safe for use in humans . similar results were obtained when infection was carried out with the pbcs pf parasite . since in these experiments infection was initiated with second - generation merozoites , obtained by transfusion of infected rbcs , we decided to investigate the behavior of the first - generation merozoites that are produced in the liver . to do this , we infected bh mice with sporozoites collected from the salivary glands of p . berghei - or pbcs pf - infected mosquitoes and , using non - chimeric mice as controls , we carried out the same type of analysis as described above . our results show that merozoites produced in the liver behave similarly to second - generation merozoites , showing that rodent p . berghei parasites are safe for use in humans and do not present the risks associated with inefficient attenuation of p . falciparum sporozoites . ( iii ) rodent plasmodium parasites have a high cross - species protection potential against human plasmodium parasites we evaluated serum samples from african malaria - infected individuals from cameroon and tanzania for the presence of antibodies against the p . berghei and p . falciparum cs proteins and for their ability to recognize spz from both these species . our results showed that while none of these samples contained antibodies against p . berghei cs ( fig3 a 1 , 3 a 2 ), 71 % of them were able to recognize both p . falciparum and p . berghei spz ( fig3 b 1 , 3 b 2 ). these data show that naturally acquired immunity against malaria includes an antibody response against conserved human plasmodium parasites and rodent plasmodium parasites epitopes on spz , besides the cs protein . overall , these results demonstrate that the use of rodent plasmodium parasites as a vaccination platform has the potential to raise an immune response against currently unknown conserved antigens . ( iv ) immunization with genetically modified rodent plasmodium parasites elicits specific highly effective protection against human plasmodium infection an additional advantage of our proposed vaccination method relies on the notion that we can enhance the intrinsic cross - species protection provided by rodent plasmodium parasites by introducing antigens of human plasmodium parasites through genetic modification , which will elicit highly effective specific immune responses . to establish the proof - of - concept of our proposed strategy , we employed the rodent pbcs pf parasite . we used immunofluorescence microscopy to confirm that pbcs pf expresses p . falciparum cs in liver cells , either ex vivo ( fig4 a ) or in vivo . ( fig4 b ). we then evaluated the immunogenicity of these transgenic parasites in rodent models of infection and determined the specificity of this response for the engineered p . falciparum cs antigen . c57bl / 6 mice were infected with pbcs pf sporozoites and subsequently treated with chloroquine to prevent the development of blood parasitemia and disease . five days after the initiation of chloroquine treatment , the mice were sacrificed and immune serum was obtained from collected blood . pre - immune serum from uninfected mice and serum from mice immunized with wild - type p . berghei were obtained and used as controls in these experiments . antibodies against p . falciparum cs in the serum were quantified by elisa ( fig5 a , 5 b ). our results show that mice immunized with the pbcs pf parasite produced significant amounts of this antibody , showing that immunization of rodents with pbcs pf elicited the generation of antibodies directed against p . falciparum cs , which are known to mediate protection against the human - infective parasite [ 13 ]. moreover , we demonstrated a clear cellular immune response against p . falciparum cs epitopes ( fig5 c ). finally , we showed that the serum of mice immunized with the genetically modified rodent pbcs pf can recognize and bind with high avidity to human plasmodium sporozoites ( fig5 d ). moreover , we showed that this immune serum is able to functionally inhibit the gliding motility ( fig5 e ) and hepatic cell invasion ( fig5 f ) of human plasmodium parasites . overall , our data show that the genetic modification of rodent plasmodium parasites can substantially increase the immunizing potential of these parasites against human plasmodium parasites . whole - organism approaches such as those provided by radiation attenuated ( ras ) and genetically attenuated ( gas ) sporozoites appear as very attractive alternatives to subunit - based pre - erythrocytic vaccination strategies , despite considerable technological challenges in terms of manufacturing , formulation , and delivery of such attenuated sporozoite vaccines . however , both these approaches pose undeniable safety concerns that arise from the fact that they are based on the attenuation of p . falciparum , the most deadly human malaria parasite . we propose an alternative strategy for the development of a pre - erythrocytic , whole - organism vaccine against malaria , based on the cross - species protection potential between rodent and human plasmodium parasites . such a vaccine may elicit cross - species protection by rodent antigens that may be immunogenic against their human plasmodium spp . counterparts . examples of cross - species protection induced by components other than cs following immunization with irradiated malaria sporozoites are available . this might be the case , for instance , of celtos , for cell - traversal protein for ookinetes and sporozoites , which has been recently identified as a target antigen for a pre - erythrocytic vaccine , based on its ability to induce protective immunity through humoral and cellular immune responses . celtos is highly conserved among the plasmodium species and immunization with pre - erythrocytic antigen celtos from p . falciparum has been shown to elicit cross - species protection against a heterologous challenge with p . berghei , suggesting that the reverse effect might also take place . besides their cross - protective potential , the immunogenicity of rodent plasmodium parasites can be enhanced by genetic engineering , effectively turning them into platforms for the delivery of immunogenic antigens of human - infective plasmodium species , capable of eliciting highly efficient specific immune responses . of course more than one antigen , already known or hitherto unknown , can be introduced , for either single or multiple stages of the plasmodium life cycle , and for a single or multiple species of human plasmodium organisms , because such redundancy may ensure an additional degree of protection against infection of parasitemia . in this invention , we demonstrate that the rodent parasite p . berghei can infect human hepatocytes whilst being unable to cause a human blood stage infection . these constitute two of the essential premises for a rodent - based parasite to be evaluated as a vaccination strategy . the third one is that a transgenic rodent plasmodium parasite may elicit a response against a challenge with a human - infective plasmodium species . to demonstrate this assumption , we employed a p . falciparum cs - expressing mutant of p . berghei ( pbcs pf ) and showed that it retains the main features of its wild - type counterpart whilst eliciting a specific protective response against a p . falciparum challenge . this establishes the proof - of - principle of our proposed strategy and opens further avenues for the design and production of other vaccine candidates based on the same principle . pbcspf itself has the drawback that it produces relatively low numbers of salivary gland sporozoites and lower hepatic infectivity than wild - type p . berghei . this is most likely due to the absence of the endogenous cs , coupled with an inappropriate conformation of the p . falciparum protein in this parasite . thus , several alternatives may be envisaged in order to improve the infection yields of pbcspf , such as the heterologous expression of p . falciparum cs in a neutral locus , under the control of the endogenous cs promoter , in addition rather than as a replacement of the endogenous protein . the use of rodent parasites as “ piggy - backs ” for human malaria genes can be extended to include antigens other than cs . among the obvious candidates , to generate a pre - erythrocytic immune response , are the thrombospondin - related adhesion protein ( trap ) and liver stage antigen 1 ( lsa - 1 ), both of which are expected to present immunogenic potential . in addition , sterile protective immunity against malaria is directed against a panel of novel p . falciparum antigens rather than one antigen in isolation . or strategy also allows for the inclusion of blood - stage antigens . apical membrane antigen i ( ama - i ), erythrocyte - binding antigen - 175 ( eba - 175 ), and merozoite surface protein 1 ( msp - 1 ) are prime candidates among these , given their established or proposed immunogenicity . when placed under the control of promoters , that enable their expression during the liver stages of infection , these antigens are expected to prime the host &# 39 ; s immune system against p . falciparum blood - stages , creating an additional layer of protection . moreover , this approach can be used to introduce a transmission blocking component in the pseudo - attenuated vaccine , by engineering gametocyte - specific genes , such as p48 / 45 , into the rodent plasmodium platform . this strategy can be extended beyond the development of immunity against p . falciparum , as one may envisage the inclusion of p . vivax antigens , such as cs , orthologs of the p . falciparum genes listed above , duffy - binding protein ( dbp ), or reticulocyte - binding proteins ( rbps ), in the immunogenic platform . 1 . hill a v . vaccines against malaria . philos trans r soc lond b biol sci 2011 ; 366 : 2806 - 14 . 2 . agnandji s t , lell b , soulanoudjingar s s , et al . first results of phase 3 trial of rts , s / as01 malaria vaccine in african children . n engl j med 2011 ; 365 : 1863 - 75 . 3 . prudencio m , rodriguez a and mota m m . the silent path to thousands of merozoites : the plasmodium liver stage . nat rev microbiol 2006 ; 4 : 849 - 56 . 4 . clyde d f , mccarthy v c , miller r m and hornick r b . specificity of protection of man immunized against sporozoite - 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